Methods of determining the suitability of cultured thymus tissue for implantation into humans and associated methods of use

Abstract

Methods and compositions for promoting donor-specific tolerance and immunocompetence to a recipient of a solid organ transplant, by implanting an allogeneic solid organ in a recipient in need of a solid organ transplant and further comprising surgical implantation of a tissue-engineered allogeneic cultured postnatal thymus tissue product in the recipient of a solid organ from a donor. Methods of producing an allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human; methods of culturing allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human and methods of using allogeneic cultured postnatal thymus tissue-derived product by implantation in a human subject.

Description

METHODS OF DETERMINING THE SUITABILITY OF CULTURED THYMUS TISSUE FOR IMPLANTATION INTO HUMANS AND ASSOCIATED METHODS OF USE FIELD OF INVENTION

[0001] Biomarkers useful for determining the viability and suitability of T cell-depleted cultured pediatric thymus tissue, also known as allogeneic cultured postnatal thymus tissue- derived product (or, sometimes “CTT"), for implantation and T cell reconstitution in subjects having thymus disorders, including congenital athymia and other immune system dysfunction due to thymus disorders. Methods and compositions for promoting donor-specific tolerance to allogeneic solid organ transplants in a recipient receiving an allogeneic solid organ transplant from a donor. BACKGROUND OF THE INVENTION

[0002] Organ transplantation requires the preparation and harvesting of a human solid organ from a donor and transplantation into the recipient. The major problem in solid organ transplantation is that the recipient is not tolerant of the donor. The recipient T cells will reject the organ and the recipient B cells will develop antibodies to the organ causing its eventual failure. The holy grail of solid organ transplantation is development of tolerance to the transplanted human organ by the recipient. More than 36,000 organ transplants are estimated to be performed per year in the U.S., and many more in in Europe, and other major countries. Itis further estimated that there are more than 120,000 patients on waiting lists in the U.S. for organ transplants. Demand for healthy organs significantly exceeds the supply of suitable organs. In 2018, approximately 10,000 donors were identified. See https: / / optn.transplant.hrsa.gov.

[0003] Transplant rejection is a substantial challenge in solid organ transplantation. Transplant rejection by both T cells and B cells can lead to significant complications in organ function or even to transplant failure. The 5-year graft survival, for example, for heart transplants is 77.7%, for kidney transplants is 78.6%, for liver transplants is 72.8%, and for lung transplants is 53.4%. Typically, this problem has been addressed, in part, through the matching of donors and recipients for major histocompatibility complex (MHC) antigens and by avoiding recipients with antibodies to the recipient tissue types. In addition, the use of immunosuppressive regimens to manage the immunological response underlying transplant rejection has improved. However, tolerance has not been achieved and the mean survival for many organs is only 10 years.

[0004] Preservation of organ viability prior to and during the implantation procedure is a second significant challenge. The removal, storage and transplantation of an organ may profoundly affect the internal structure and function of the organ and can influence significantly the degree to which the return of normal organ function is delayed or prevented after transplantation is completed.

[0005] The time period in which solid human organs may be effectively preserved varies by organ, with kidneys ranging from 24-36 hours, pancreas from 12-18 hours, liver from 8-12 hour and heart and lung from 4-6 hours. See https: / / unos.org / transplantation / matching-organs.

[0006] Organ injury occurs primarily as a result of ischemia and hypothermia, but may also be related to reperfusion of the organ ex vivo or during implantation.

[0007] Techniques for organ preservation, including ex vivo perfusion, are known in the art and serve to minimize organ damage and promote optimal graft survival and function.

[0008] The principal solid organs which have been the subject of transplantation procedures, include kidney, liver, heart, and lungs. Success in transplanting these solid organs has been achieved with varying degrees of success. The principal variability resides in the techniques that are used to interfere with immune-mediated graft rejection. Experience has shown that there is no one single immunosuppressive agent or technique that is useful in all settings involving solid organ transplantation. The limiting factor usually resides in the toxicity associated with each individual immunosuppressive agent. The toxicity associated with a given immunosuppressive agent may frequently hinder the normal functioning of the transplanted solid organ or of other organs such as the kidneys which can fail when calcineurin inhibitors are used to prevent rejection.

[0009] The toxicity drawbacks associated with known immunosuppressive agents normally used to prevent graft rejection in solid organ transplants presents a need to find new methods for preventing graft rejection of solid organ transplants.

[0010] The ability to discriminate between self and non-self antigens is central to the immune response. This discrimination results in self-tolerance. Autoimmunity develops when there has been a loss of self-tolerance. An unmet need exists in transplantation procedures to induce tolerance to solid organ transplants.

[0011] Preservation of thymus tissue viability prior to and during the implantation procedure for allogeneic cultured postnatal thymus tissue-derived product in subjects with thymus disorders, including congenital athymia and other immune system dysfunction due to thymus disorders is an important factor in the practice of the various aspects and embodiments of the present disclosure.

[0012] The thymus is necessary for development of T cells that can appropriately respond to foreign antigens and pathogens, while avoiding damaging self-reactivity.

[0013] The thymus is large in infancy due to its need to establish the initial T cell repertoire, but soon becomes homeostatic, followed by a slow process of involution that continues throughout adulthood. Work over the last two to three decades has established that while the overall output of adult thymus is decreased, the organ remains critical for producing T cells with novel specificities that can protect against infectious disease or cancer and repopulate the repertoire following immune insults such as radiation, chemotherapy, and some infections such as human immunodeficiency virus (HIV) (Gruver et al. 2007; Palmer et al. 2018; Sun et al. 2016; ‘Wickemeyer and Sekhsaria 2014). Thus, recent attention has been focused on identifying the mechanisms that govern age-related thymus involution and methods to facilitate thymus regeneration following immune injury in adults.

[0014] Age-related thymus involution in humans is characterized by loss of developing thymocytes and decreased numbers of thymic epithelial cells, with replacement of thymus parenchyma by adipose tissue. Determining whether the mechanisms driving these changes are thymus-intrinsic versus thymus-extrinsic is difficult to address using animal models due to constant trafficking to and from the thymus, and such questions are generally not possible to address in live humans.

[0015] Organ cultures of thymus tissue derived from young donors are useful for evaluating these questions, since in vitro culture of human thymus slices results in depletion of thymocytes, while generally maintaining the viability and function of the thymic epithelial and stromal cells. This is demonstrated by the ability of these slices to grow out monolayers (Markert et al. 1997b) and to facilitate T cell reconstitution when implanted into congenitally athymic recipients (Davies et al. 2017; Davis et al. 1997; Markert et al. 2004; Markert et al. 1999; Markert et al. 2007, Markert et al. 2010; Markert et al. 1997a; Markert et al. 2011; Markert et al. 2003). We hypothesized that the loss of thymocytes that occurs during thymic organ cultures could mimic acute and chronic involution and help to identify mechanisms that drive changes in the thymic microenvironment during aging.

[0016] Allogeneic cultured postnatal thymus tissue-derived product has been shown to be useful for the treatment of T cell immunodeficiency (primary immune deficiency) resulting from congenital athymia, for example in the treatment of complete DiGeorge Anomaly (cDGA) associated with 22q11.2 deletion and CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness) syndrome associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene and in athymic patients with forkhead box protein N1 (FOXN1) deficiency. Congenital athymia is a rare, fatal condition and currently has no drug treatment options utilizing regulatory approved drug products.

[0017] Experimental implantation of an allogeneic cultured postnatal thymus tissue-derived product that retains thymus epithelial cells (TECs) has been successfully applied to treat pediatric patients with congenital athymia (Markert ML, Devlin BH, McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol., 135(2): 236-46, Markert ML, et al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581; Markert ML, et al., 1999, “Transplantation of thymus tissue in complete DiGeorge syndrome,” N ¢ J Med 341(16):1180-1189 27).

[0018] In this reference, DiGeorge Syndrome is defined as a condition in which there are variable defects in the heart, thymus and parathyroid gland. Approximately 1% of infants with DiGeorge syndrome have athymia and hence cannot make naive T cells that mature and fight infection. These infants are said to have complete DiGeorge syndrome. Without intending to be inclusive, there are subgroups of children who meet the criteria of complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants with no syndromic or genetic defects. Congenital athymia may also be associated with mutations in the TBX1 or TBX2 genes.

[0019] Allogeneic cultured postnatal thymus tissue-derived product is a tissue-engineered product that is prepared, cultured and stored for up to 21 days (for example, a culturing regimen of about 6 to about 21 days) to produce partially T cell-depleted thymus tissue slices and which is differentiated from native thymus by a conditioning process. The conditioning regimen partially depletes the donor thymocytes from the cultured thymus tissue slices. Based on in vitro data (immunohistochemistry) a culture period between 6 and 21 days preserves the epithelial network as assessed using cytokeratin antibodies. The culturing is preferably done at 37°C ina 59% CO; incubator.

[0020] The culturing process significantly modifies the biological characteristics of the donor thymus tissue and constituent cells contained therein in the following manner to optimize the effective therapeutic properties of the allogeneic cultured postnatal thymus tissue-derived product slices. The culturing process assures that a defined composition of the cultured cells / tissue having the pre-requisite biological characteristics is obtained in a manner suitable for surgical implantation into a subject to enable reconstitution of the subject’s immune system.

[0021] The culturing process results in a loss of thymocytes and relative enrichment of TECs and other stromal cells in the donor thymus tissue slices. The culturing process further results in depletion of thymocytes and maintenance of TECs to enable reconstitution of the recipient’s immune system and allows tolerance to develop in the recipient to HLA antigens in the donor thymus. Overall, the culturing process is designed to deplete many of the thymocytes from the donor thymus tissue and to preserve the functional architecture of the thymic stroma (thymic epithelial cells and fibroblasts). Common lymphoid progenitors that develop from stem cells migrate to the thymus and enter the thymus as thymus settling progenitors.

[0022] The culturing process is described in W02019 / 165197A1, Cultured Thymus Tissue Transplantation Promotes Donor-Specific Tolerance to Allogeneic Solid Organ Transplants, Markert, M. L., which is hereby incorporated by reference in its entirety. The analysis of the suitability of allogeneic cultured postnatal thymus tissue-derived product slices is described in PCT / US2019 / 040275, which is incorporated by reference herein in its entirety.

[0023] The surgical administration of allogeneic, cultured postnatal thymus tissue-derived product (e.g., also known as “RVT-802") in athymic patients leads to a cascade of events resulting in the development of a functional immune system. Following surgical placement of allogeneic, cultured postnatal thymus tissue-derived product in a recipient, T cells are educated by donor TECs and recipient dendritic cells (DCs). Donor TECs in conjunction with recipient DCs enable tolerance to the implanted donor thymus tissue, which is implanted as cultured thymus tissue slices. This is the same tolerance induction as in a normal thymus. The donor TECs in conjunction with recipient DCs lead to tolerance to self.

[0024] Thymopoiesis in implanted donor thymus tissue has been documented by allograft biopsies and the presence of recipient naive T cells in the periphery (Markert ML, 2010,; Markert ML, et al., 2008, “Use of allograft biopsies to assess thymopoiesis after thymus transplantation,” J Immunol 180(9):6354-6364; Markert ML, et al., 2007, “Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants,” Blood 109(10):4539-454728), which are incorporated herein by reference.

[0025] Studies of children treated with investigational allogeneic, cultured postnatal thymus tissue-derived product show tolerance to donor major histocompatibility complex (MHC) by mixed lymphocyte reactions (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008, “Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277-281). In addition, the infants with congenital athymia, after allogeneic, cultured postnatal thymus tissue-derived transplantation, are able to control infections such as Epstein Barr virus (Markert ML, 2014, Thymus Transplantation. Stiehm’s Immune Deficiencies, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067.

[0026] Historically, allogeneic cultured postnatal thymus tissue-derived product release criteria has included histopathological evaluation of H&E and immunostained sections of tissue at the mid-point of the manufacturing process, which was later refined to days 6-210f the culture period. This histopathological evaluation has served as the potency assay, and has been performed as a qualitative analysis by a board-certified pathologist. Samples were prepared for evaluation either by freezing or formalin fixation prior to the tissue slice being sectioned and then fixed onto a slide. Samples prepared in this manner are stable over long periods of time, allowing for reanalysis to be performed.

[0027] The historical samples available from the 20+ years of development history can be linked to positive clinical outcomes, and thus provide a strong data set for development of a quantitative histology assay for evaluation of product quality.

[0028] A new digital histology assay was developed using scanned images of H&E slides from previous clinical lots and from experimental lots of allogeneic, cultured postnatal thymus-tissue derived product. These images were analyzed for development into a quantitative release assay. The digital histology assay is described more completely in PCT / US2019 / 040275.

[0029] Cellular migration in response to chemoattractant cytokines (chemokines) and other soluble molecules is a critical, but less intuitive, mechanism that regulates thymus function (Hu et al. 2015). Early thymocyte progenitors migrate from the bone marrow to the thymus under the influence of chemokine gradients. Chemokine gradients also influence their migration within the thymus. Early thymocyte progenitors and their CD4- / CD8- double negative (DN) progeny interact with cortical thymic epithelial cells, differentiate into CD4+ / CD8+ double positive (DP) thymocytes, and then are positively selected to differentiate into CD4+ or CD8+ single positive thymocytes (Lancaster 2018) that migrate to the thymic medulla. After self-reactive cells are deleted by negative selection, the resulting naive mature T cells are released into the periphery.

[0030] Notwithstanding the foregoing success of implanting allogeneic, cultured postnatal thymus-tissue derived product in children having congenital athymia, there is still a need to identify cultured thymus tissue that is viable, functional and suitable for implantation in order to achieve immune-reconstitution. SUMMARY OF THE INVENTION

[0031] Achieving donor-specific immune tolerance remains the ultimate immunologic goal in transplantation. Most of the current approaches focus on controlling peripheral mature donor- reactive T cells by depletion (e.g. alemtuzumab, thymoglobulin, etc.) or suppression (e.g. calcineurin inhibitors, basiliximab, etc.) without targeting the production of alloreactive T cells in thymus. However, even with the dramatic advancement of immunosuppressive drugs and new immunomodulatory regimens, transplant tolerance has not yet been consistently achieved.

[0032] The present inventors have shown that tolerance to solid organ transplants may be achieved through the implantation of allogeneic cultured postnatal thymus tissue-derived product (referred to herein also as “CTT” or as “RVT-802"), in a thymectomized recipient, to shorten the time period of use of post-transplantation immunosuppressive agents to prevent rejection of the transplanted organ. The removal of the recipient’s thymus and substitution of an allogeneic cultured postnatal thymus tissue-derived product results in reconstitution of the solid organ recipient’s immune system and tolerance to the recipient’s self as well as to the transplanted allogeneic solid organ.

[0033] Tolerance induction by surgical insertion of allogeneic cultured postnatal thymus tissue-derived product is similar to tolerance induction via donor dendritic cells (“DC”) in hematopoietic stem cell transplantation (Sharabi Y & Sachs DH, 1989, “Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen," J Exp Med 169(2):493-502; Manilay JO, Pearson DA, Sergio JJ, Swenson KG, & Sykes M, 1998, “Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen,” Transplantation 66(1):96-102.). A series of studies from Transplantation Biology Research Center (TBRC, Boston, MA) showed the crucial role of thymus in tolerance induction (Yamada K, ef al., 1997, “Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts,” J Exp Med 186(4):497-506) and tested thymus transplantation with tolerance induction in a large animal model (Yamada K, ez al., 2000, “Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients,” J Immunol 164(6):3079-3086; 5; Yamada K, et al., 2003, “Thymic transplantation in miniature swine: III. Induction of tolerance by transplantation of composite thymokidneys across fully major histocompatibility complex- mismatched barriers,” Transplantation 76(3):530-536; Nobori S, ef al., 2006, “Thymic rejuvenation and the induction of tolerance by adult thymic grafts," Proc Natl Acad Sci U S A 103(50):19081-19086. In their series of studies, this group successfully used HLA-Class II matched / Class I mismatched donor (thymus and kidney or heart) as thymus composite tissues (thymokidney and thymoheart) with 12 days of cyclosporine (“CsA”) for transplant tolerance induction. They claimed that non-vascularized thymus did not induce tolerance in their model. More precisely, however, non-vascularized thymus that was not cultured did not engraft long- term. As they indicated, the failure of engraftment of the uncultured thymus may have been due to ischemic injury in addition to alloimmunity (Yamada K, ez al., 2000). This elegant concept of generating vascularized thymus prior to transplantation to induce tolerance, would be difficult to translate to the clinic without using xenotransplantation. (Kwun, Jean, Li, Jie, Rouse, Clay, Park, Jae Berm, Farris, Alton B., Kuchibhatla, Maragatha, Turek, Joseph W. Knechtle, Stuart J. Kirk, Allan D. and Markert, M Louise, Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants, JCI Insight . 2020 Jun 4;5(11):129983. doi: 10.1172 / jci.insight. 129983).

[0034] The limitation of non-vascularized thymus transplantation can be overcome with a culture system as well as T cell depletion. Experimental transplantation of allogeneic cultured postnatal thymus tissue-derived product (CTT) that retains TECs has been successfully applied to treat pediatric patients with congenital athymia (Markert ML, Devlin BH, McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol., 135(2): 236-46; Markert ML, et al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581, Markert ML, et al., 1999, “Transplantation of thymus tissue in complete DiGeorge syndrome,” N Engl J Med 341(16):1180-1189 27).

[0035] In foregoing reference, DiGeorge Syndrome is defined as a condition in which there are variable defects in the heart, thymus and parathyroid gland. Approximately 1% of infants with DiGeorge syndrome have athymia and hence no T cells to fight infection. These infants are said to have complete DiGeorge syndrome. There are 4 subgroups of children who meet the criteria of complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants with no syndromic or genetic defects. In all four groups, the infants with athymia represent a very tiny group, possibly 1% of the total children carrying the diagnosis, such as the diagnosis of 22q11.2 deletion syndrome.

[0036] Thymopoiesis has been documented by allograft biopsies and the presence of recipient naive T cells in the periphery (Markert ML, 2010,; Markert ML, et al., 2008, “Use of allograft biopsies to assess thymopoiesis after thymus transplantation,” J Immunol 180(9).6354-6364; Markert ML, et al., 2007, “Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants,” Blood 109(10):4539-454728). Studies of children treated with investigational CTT show tolerance to donor MHC by mixed lymphocyte reactions (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008, “Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277-281). In addition, the infants with congenital athymia, after CTT implantation, are able to control infections such as Epstein Barr virus (Markert ML, 2014, Thymus Transplantation. Stiehm 's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067). Based on these data in humans with congenital athymia, it was determined that allogeneic cultured postnatal thymus tissue-derived product expressing the MHC of a solid organ donor after surgical insertion in the recipient will develop tolerance to both self and to the donor, while producing functional T cells that will protect the recipient from infection. Thymus Gland and Education of Thymocytes. (Kwun, Jean, Li, Jie, Rouse, Clay, Park, Jae Berm, Farris, Alton B., Kuchibhatla, Maragatha, Turek, Joseph W. Knechtle, Stuart J. Kirk, Allan D. and Markert, M Louise, Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants, JCI Insight . 2020 Jun 4;5(11):€129983. doi: 10.1172 / jci.insight. 129983.

[0037] The thymus gland normally is located on top of the heart. The thymus provides an essential microenvironment for T cell development and is critical to the establishment and maintenance of the adaptive immune system (Boehm T and Takahama Y, 2014, Thymic Development and Selection of T Lymphocytes. Heidelberg: Springer-Verlag). During post-natal development, the thymus educates hematopoietic stem cells migrating from the bone marrow to the thymus gland. The progenitor stem cells colonize the thymus thereby forming thymocytes. The thymocytes thereafter undergo a series of maturation steps. This is evidenced by the expression of a number of observable cell surface markers appearing on the thymocytes.

[0038] T cells are critical for the protection of the body from infections. T cells that develop in a normally functioning thymus develop a diverse set of T cell receptors (generally proteins on the surface of the cell), which enable the mature T cell to fight a wide variety of infections. During this education process the developing T cells are instructed by the thymus not to attack the body’s normal proteins, such as insulin or parathyroid hormone (which regulate glucose and calcium levels in the blood). These instructions are carried out under the influence of the autoimmune regulator gene (“AIRE gene).”

[0039] Briefly, the education process occurs in the normally functioning thymus gland. Thymocytes, present in the thymus gland, are formed from bone marrow stem cells. Thymocytes, are taught by thymus epithelial cells (“TECs”) and dendritic cells (“DCs”), located within the thymus, to not attack recipient major histocompatibility complex (MHC) proteins (antigens) such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA- DPBI1, HLA-DPA1 antigens. The HLA antigens have 2 proteins that hold a self-peptide in a groove. The self-peptide could be from a thyroid protein or an insulin peptide or almost any other protein expressed in the body. Thymocytes developing in the thymus form a T cell receptor (TCR) composed of two proteins that cross the membrane. The TCR is expressed on the cell surface of the T cells. Each T cell expresses many copies of its unique TCR. If the TCR binds too tightly to the self peptide: MHC on a dendritic cell, the dendritic cell provides a signal to make the T cell undergo apoptosis and die. This mechanism prevents the development of autoimmunity to self. The TEC can also provide a signal to thymocytes that they are binding too tightly. Lastly the DC can grab bits of membrane from the TEC and present the TEC self peptide: MHC to the developing thymocyte. If the thymocytes bind too tightly, the DC sends a signal so that the thymocyte undergoes apoptosis and dies. By these mechanisms, T cells that leave the thymus are not self-reactive. ~The T cells that leave the thymus are very variable and can recognize infections but they do not attack proteins of the body.

[0040] The two major constituents of thymus are epithelium and thymocytes that are produced in the thymus in the following manner. Cells derived from bone marrow stem cells, common lymphoid progenitors (“CLPs”), migrate to the thymus as early thymic progenitors. The CLPs enter the thymus in response to signals (chemokines) produced by the thymus epithelium and endothelium. In the thymus the CLPs differentiate into thymocytes and proliferate. Thymocytes develop a unique T-cell receptor (“TCR”) that is expressed on the cell surface. Thymocytes also begin to express the T cell molecules CD3, CD4 and CD8. A vast diversity of T cells develop rendering the cells capable of responding to infections throughout the life of the recipient. Mixed lymphocyte reactions show tolerance of the recipient T cells in children who are treated with cultured thymus tissue (RVT-802) to thymus donor MHC.

[0041] Self-reactive recipient thymocytes are deleted prior to exit from the thymus. This occurs by interaction of recipient thymocytes and recipient DCs that migrate to the thymus. Apoptosis is induced in recipient thymocytes that bind too tightly to the DCs as a measure to protect the body from autoimmune disease. After completion of this process, the thymocytes exit the thymus. The new circulating T cells, i.e., recent thymus emigrants, express the markers CD31, CD45RA and CD62L. After a few weeks, the CD31 marker is no longer expressed. The T cells expressing CD45RA and CD62L are called naive T cells. These recipient T cells proliferate normally in response to mitogens. They protect the recipient from infection without having autoreactivity to self. Allogeneic cultured postnatal thymus tissue-derived product.

[0042] Allogeneic cultured postnatal thymus tissue-derived product has been shown to be useful for the treatment of T cell immunodeficiency (primary immune deficiency) resulting from congenital athymia. T cell immunodeficiency due to athymia is associated with congenital disorders which prevent the development of a functional thymus, such as complete DiGeorge Anomaly (cDGA) associated with 22q11.2 deletion and CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness) syndrome associated with mutations in the cd” (chromodomain-helicase-DNA-binding protein 7) gene and in athymic patients with forkhead box protein N1 (FOXN1) deficiency. Other genetic defects causing athymia include TBX1, TBX2, PAX and semaphorine 3E (SEMA3E), and Bernstock, Joshua D, Totten, AH, and Atkinson, T. Prescott, "Recurrent microdeletions at chromosome 2p11.2,” Bernstock, Joshua D, Totten, AH, and Atkinson, T. Prescott, JACI 145:358-367. Congenital athymia is a rare fatal condition and currently has no drug treatment options utilizing regulatory approved drug products. If left untreated and no therapeutic reconstitution of the child’s immune system occurs, the primary immunodeficiency due to congenital athymia is fatal, with almost all infants dying before the age of three years, most often by severe infections.

[0043] Allogeneic cultured postnatal thymus tissue-derived product is a tissue-engineered product. Based on disclosures in this specification and Examples, CTT is expected to be useful for the development of tolerance in a recipient receiving a transplanted solid organ.

[0044] As described more fully in this specification and Examples, the surgical administration of allogeneic, cultured postnatal thymus tissue-derived product (e.g., “RVT-802") in athymic patients leads to a cascade of events resulting in the development of a functional immune system. Following surgical placement of allogeneic, cultured postnatal thymus tissue-derived product (e.g., RVT-802) in the recipient, T cells are educated by donor TECs and recipient DCs. Donor TECs in conjunction with recipient DCs enable tolerance to the implanted donor thymus tissue, which is implanted as cultured thymus tissue slices. This is the same tolerance induction as in a normal thymus. The recipient TECs in conjunction with recipient DCs lead to tolerance to self as described in this specification.

[0045] This complex process has been shown clinically to lead to > 70% survival in patients with congenital athymia receiving allogeneic, cultured postnatal thymus tissue derived product (e.g. RVT-802) due to the recipient’s capacity to fight infections (Markert ML, Devlin BH, Alexieff MJ, Li J, McCarthy EA, Gupton SE, et al., 2007, “Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants,” Blood, 109(10): 4539-47; Markert ML, Devlin BH, McCarthy EA. Thymus transplantation. 2010, Clin. Immunol. 135(2): 236-46). The recipients are able to control viral infections such as Epstein-Barr virus that would have been fatal prior to CTT. (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008). Overview of tolerance induction in solid organ transplant in combination with implantation of CITT

[0046] In accordance with the description, figures, examples and claims of the present specification, the inventor has demonstrated that CTT induces donor-specific tolerance in a rat heart transplantation model. The experiments reported herein used comparable CTT implantation methods that have been used clinically in subjects with congenital athymia, such as subjects afflicted by cDGA. cDGA infants have essentially no naive T cells prior to surgical placement of CTT. Following surgical placement of CTT, the infants developed naive T cells approximately 6 months after the surgical procedure. (Markert ML, ef al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581; Markert ML, Devlin BH, & McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol 135(2):236-246). The studies of tolerance induction in a rat model were based on the results of investigations of transplantation of allogeneic postnatal cultured thymus tissue- derived product (CTT) from 1993 to 2017 in athymic infants with complete DiGeorge anomaly. Favorable results were obtained in the reported studies of surgical placement of CTT in infants having congenital athymia. The published results showed an overall survival rate of 71% (61 / 86) (essentially all the deaths occurred in the first 9-12 months; median 11.7 years, range 1.2 to 25 years at the time of this assessment) in this otherwise fatal condition (Markert, ML, et al., 2010). Biopsies of the implanted cultured thymus tissue have demonstrated thymopoiesis on immunohistochemistry (Markert, ML, et al., 2008). Flow cytometry and spectratyping have shown development of a diverse T cell repertoire. Mixed lymphocyte reactions show tolerance of the recipient T cells to thymus donor antigen presenting cells (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008).

[0047] Importantly, the recipients of CTT are able to control viral infections such as Epstein— Barr virus that would have been fatal prior to CTT (Markert, ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067). Based on these human data showing tolerance to unmatched thymus MHC antigens, implantation of CTT in a rat model was evaluated using the same methods used clinically, for its ability to induce donor-specific tolerance in a rat heart transplantation model. These studies showed that transplanting unmatched hearts along with donor CTT expressing the heart donor’s MHC class I and class II antigens (with initial T cell depletion by anti CDS and immunosuppression with cyclosporine) induces tolerance to the antigens of the donor heart while preserving alloreactivity toward other MHC antigens.

[0048] The present invention substantiates donor thymus co-transplantation with solid organs as a method of tolerance induction with regard to the transplanted solid organ in the recipient. The patient groups that would most benefit from the procedure is adults with heart failure as well as infants needing heart transplants. Since postnatal thymic tissue is present and could be removed from deceased infants, and the recipient thymus is routinely removed from infants undergoing heart transplantation, no additional procedure aside from cultured thymus tissue implantation (CTT) would be needed to transfer this approach to the clinic. Similar transplants may also be performed in adults. Overview of Preparation of allogeneic, cultured postnatal thymus-tissue derived product

[0049] Allogeneic, cultured postnatal thymus-tissue derived product is prepared, cultured and stored for up to 21 days (for example,a culturing regimen of about 6 days to about 21 days), and, on the day of implantation, placed in individual sterile cups for transport to the operating room, as described in more detail herein.

[0050] The CTT (cultured thymus tissue) is aseptically processed and cultured under current Good Manufacturing Practices (“cGMP”), for example, cGMPs established by the U.S. Food & Drug Administration (“FDA”), to produce partially T cell-depleted thymus tissue slices. CTT is differentiated from native thymus by a conditioning process described in detail below. CTT effects the normal positive and negative selection process of developing T cells in the thymus after implantation, enabling the T cells to be tolerant to both the donor thymus and the donor solid organ transplant plus recipient tissues. In addition, these T cells can recognize foreign antigens in the context of recipient major histocompatibility (MHC) proteins so as to fight infection.

[0051] The route of administration is by surgical implantation of CTT, in the manner described below. A single administration is typically 1,000 to 22,000 mm? of CTT surface area per recipient body surface area (“BSA”) in m?. The surface area is the total of all the surface areas of all cultured tissue slices. The individual CTT slices are implanted in a single administration surgical procedure.

[0052] Surgical implantation of allogeneic cultured postnatal thymus tissue-derived product in athymic patients leads to a cascade of events resulting in the development of a functional immune system. (Markert ML, 2007, Markert ML, et al., 2010; Markert ML, Devlin BH, McCarthy EA. Chapter 84 Thymic reconstitution. 2013. In: Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM, editors. Clinical Immunology (Fourth Edition). London; pp. 1032- 8).

[0053] Recipient CLPs of the bone marrow migrate to the thymus allograft, enter as early thymic progenitors and there develop into recipient T cells. The donor thymus graft provides a microenvironment in which the recipient thymocytes develop a broad repertoire of TCRs capable of recognizing pathogens.

[0054] Migration of recipient DCs to the donor thymus depletes self-reactive recipient thymocytes that would attack the recipients tissues after the new T cells leave the thymus and enter the circulation. Genetically-recipient naive T cells are readily detectable in the circulation approximately 5-12 months after administration. These recipient T cells have diverse TCR repertoires and proliferate normally in response to mitogens. They protect the recipient from infection without having autoreactivity to self.

[0055] Recipient bone marrow CLPs migrate to the thymus allograft where they develop into recipient T cells. Negative selection by recipient DCs that have migrated to the donor thymus results in tolerance to the recipient MHC antigens. Inmunohistochemical evidence of thymopoiesis is observed in biopsies of the implanted cultured thymus tissue taken within approximately 2-3 months of transplantation. The thymopoiesis reflects the ability of the T cells to defend against and control infection, and prevent autoimmune disease.

[0056] Naive T cells are detected in the circulation 5-12 months post-transplantation, resulting in the ability to defend against and control infection, and the prevention of autoimmune disease.

[0057] Implantation of cultured thymus tissue was first shown to be beneficial in treating primary immune deficiency resulting from congenital athymia associated with conditions such as complete DiGeorge anomaly (cDGA) or forkhead box protein N1 (FOXN1) deficiency. It was discovered that replacement of defective thymus tissue with normal thymus tissue after culture (e.g. CTT and RVT-802) may also obviate the lack of tolerance observed in recipients of transplanted solid organs.

[0058] The non-clinical and clinical work underlying the treatment of congenital athymia through placement of cultured thymus tissue led to the realization that placement of CTT (e.g., RVT-802) in patients may permit the development of tolerance to a transplanted solid organ. Specifically, placement of CTT will reconstitute an immune system and induce tolerance to the donor organ if the subject is first thymectomized and immunosuppressed prior to the implantation of the CTT that expresses the MHC of the donor organ.

[0059] Measurement of the expression and distribution of certain markers associated with the cellular components of the thymus establish a phenotype following ex vivo culturing of thymus tissue. The culturing conditions described in this specification and Examples support the observation of in vivo thymopoiesis following placement of CTT in an athymic subject.

[0060] Importantly, after the surgical placement of CTT in an athymic recipient, the development of naive T cells and the presence of a broad range of TCR-variable regions provides clear evidence that culturing of thymus tissue can foster the development of a functional endogenous T cell population. In addition, the expression of key regulatory and structural genes was noted in thymus tissue during culturing. Circulating naive (CD45RA+CD62L+) T cells can be first detected 3-5 months after surgical insertion of CTT. These observations have been noted in the treatment of patients with complete DiGeorge Anomaly (Markert ML, 2010; Markert ML. 2013).

[0061] The nonclinical data described in the literature for thymus tissue implantation aligns with the robust clinical efficacy of implanting allogeneic cultured postnatal thymus tissue and supports its use in humans. (Markert ML, Watson TJ, Kaplan I, Hale LP, Haynes BF, 1997, “The human thymic microenvironment during organ culture,” Clin Immunol Immunopathol. Jan, 82(1):2 6-36; Hong R, Schulte-Wissermann H, Jarrett-Toth E, Horowitz SD, Manning DD, 1979, “Transplantation of cultured thymic fragments. II. Results in nude mice,” J Exp Med., 149(2): 398-415. Li B, Li J, Hsieh CS, Hale LP, Li YJ, Devlin BH, Markert ML, 2009, “Characterization of cultured thymus tissue used for transplantation with emphasis on promiscuous expression of thyroid tissue-specific genes,” Immunol Res. 2009; 44 (1-3):71-83; Li B, Li J, Devlin BH, Markert ML, 2011, “Thymic microenvironment reconstitution after postnatal human thymus transplantation,” Clin Immunol., Sep, 1403); 244-59).

[0062] Complete DiGeorge Anomaly patients may have defects in three glands that develop in the neck in the young embryo, the heart, the thymus and the parathyroid gland. Normally the heart and thymus descend into the chest and the parathyroid gland regulates calcium levels, and remain in the neck. Treatment of cDGA subjects with CTT led to the survival rates at two years of age of 75% compared to a survival rate of 6% in patients treated by other modalities (unpublished data). As noted above, almost all deaths are in the first year prior to development of naive T cells. (Markert, et al., 2010). Of note, the CTT implantation does not affect the problems of the heart and the parathyroid gland that must be managed separately.

[0063] An aspect of the present disclosure provides methods for the surgical placement of allogeneic cultured postnatal thymus tissue-derived product in a recipient to induce tolerance to a solid organ transplant in an immunologically normal recipient. Such methods comprise, consist of, or consist essentially of, removal of the thymus gland in an immunocompetent recipient followed by depleting the recipient’s T cells with an induction immunosuppressive regimen, comprising one or more immunosuppressive agent, such as with one or more antibody and / or one or more calcineurin inhibitor. The induction immunosuppressive regimen is administered in a therapeutically effective amount to deplete mature T cells in the subject and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ. A suitable solid human organ and a thymus gland from a deceased donor is obtained and the solid organ is transplanted into the recipient. A maintenance immunosuppressive regimen is administered for a period of time to suppress transplant rejection. The thymus gland from the deceased donor is subjected to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days), to aseptically process the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices thereby comprising the allogeneic cultured postnatal thymus tissue-derived product. The partially T-cell depleted donor thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei. The allogeneic cultured postnatal thymus tissue-derived product is then surgically placed in the recipient, typically in the quadriceps muscle of the thigh. The allogeneic cultured postnatal thymus tissue-derived product enables the recipient to develop naive T cells after implantation. All new T cells that develop are genetically recipient and are tolerant to both the recipient and to the donor. The dosage of thymus tissue slices is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m? Following implantation, the allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the subject.

[0064] Taking as an example a heart transplant, the donor would be a deceased donor. The thymus would be removed from the donor at the same time that the heart is removed. A heart transplantation is performed immediately with induction immunosuppression to decrease T cell numbers and suppress the remaining recipient T cells preventing them from attacking the donor heart. The donor thymus is processed to form human allogeneic cultured postnatal thymus tissue-derived product that can be used for implantation to induce tolerance after a period of at least about 6 days to about 21 days of conditioning. As a precaution, approximately half of the allogeneic cultured postnatal thymus tissue-derived product can be cryopreserved after conditioning, so that if there was a problem with later rejection of the heart necessitating the administration of high doses of steroids or other immunosuppressive agents to treat the rejection, and whereby the very high doses of steroid damage the allogeneic cultured postnatal thymus tissue-derived product, the cryopreserved allogeneic cultured postnatal thymus tissue-derived product would be available to implant after the rejection episode was controlled.

[0065] Importantly, after implantation of allogeneic cultured postnatal thymus tissue-derived product, immune tolerance is maintained even in the presence of infections. With other approaches such as co-stimulatory blockade, viral infections can led to loss of tolerance, because approximately a third of CD8 T cells have alloreactivity. When the immune system is activated to fight an infection, the alloreactive CD8 T cells start to reject the solid organ transplant. In contrast, when using thymus tissue processed into allogeneic cultured postnatal thymus tissue- derived product to induce tolerance, potentially alloreactive T cells against the donor are deleted through the process of negative selection in the thymus.

[0066] In an embodiment, the donor thymus tissue matches the HLA alleles in the donor organ that are not in the recipient. All new T cells that develop are genetically recipient and are tolerant to both the recipient and to the donor.

[0067] In another aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant, the method comprising the steps of’ (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing both a suitable solid human organ and a thymus gland from a deceased donor; (d) transplanting the solid human organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing an allogeneic cultured postnatal thymus tissue-derived product, wherein the allogeneic cultured postnatal thymus tissue-derived product is obtained from suitable thymus tissue of the solid organ donor; wherein the donor thymus tissue is subjected to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days) to produce allogeneic cultured postnatal thymus tissue- derived product; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the partially T-cell depleted donor thymus tissue slices show areas positive for cytokeratin (CK) (using antibody AE1 / AE3) scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei; and (g) implanting the allogeneic cultured postnatal thymus tissue-derived product into the recipient after about 6 to about 21 days of conditioning regimen, wherein the dosage of thymus tissue slices is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m?, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0068] In an embodiment, a method of promoting donor-specific tolerance to an allogeneic heart transplant in a recipient in need of a deceased donor heart is provided. The method comprises the following steps: (a) obtaining a suitable human heart from a deceased donor for transplantation; (b) removing the deceased donor thymus at the same time as the heart is obtained for conditioning into allogeneic cultured postnatal thymus tissue-derived product; wherein the donor thymus matches the HLA alleles in the donor transplanted organ; (c) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete and / or suppress the recipient’s T cells wherein the one or more immunosuppressive agents comprises glucocorticoids administered at the induction of anesthesia and after reperfusion; (d) transplanting the heart into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents selected from the group consisting of a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor and an anti-thymocyte globulin for a period of time sufficient to prevent or suppress transplant rejection of the heart; (f) between day 6 and 21 providing an allogeneic cultured postnatal thymus tissue- derived product, wherein the allogeneic cultured postnatal thymus tissue-derived product is obtained from the donor thymus tissue, wherein the donor thymus tissue is subjected to a conditioning regimen for a period of from about 6 to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the partially T-cell depleted donor thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei; (g) implanting a portion of the allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dosage of thymus tissue slices is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m?, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient; and (h) cryopreserving a portion of the allogeneic cultured postnatal thymus tissue-derived product to be used in the recipient in the event that there is an early rejection episode requiring high doses of steroids that would damage the portion of allogeneic cultured postnatal thymus tissue-derived product that was implanted in step (g).

[0069] In an embodiment, a method of promoting donor-specific tolerance to an allogeneic heart transplant in a recipient in need of a deceased donor heart is provided. The method comprises the following steps: (a) obtaining a suitable solid human heart from a deceased donor for transplantation; (b) removing the deceased donor thymus at the same time as the heart is obtained for conditioning into allogeneic cultured postnatal thymus tissue-derived product; wherein the donor thymus matches the HLA alleles in the donor transplanted organ; (c) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete and / or suppress the recipient’s T cells wherein the one or more immunosuppressive agents comprises glucocorticoids administered at the induction of anesthesia and after reperfusion; (d) surgically removing the heart and thymus of the recipient; (e) transplanting the donor human heart into the recipient; (f) treating the recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents selected from the group consisting of a calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor and an anti-thymocyte globulin for a period of time sufficient to prevent or suppress transplant rejection of the heart; wherein, if the post-operative condition of the recipient is too unstable to allow weaning of the glucocorticoids and safely implanting the allogeneic cultured postnatal thymus tissue-derived product in the recipient, the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved to be implanted at a later time when the recipient is stable, wherein the donor thymus tissue is subjected to a conditioning regimen for a period of from about 6 to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the partially T-cell depleted donor thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei; (h) implanting a portion of the allogeneic cultured postnatal thymus tissue-derived product into the recipient after the patient is stable, wherein the dosage of thymus tissue slices is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m?, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient; and (i) cryopreserving a portion of the allogeneic cultured postnatal thymus tissue-derived product to be used in the recipient in the event that there is a rejection episode requiring high doses of steroids that would damage the portion of allogeneic cultured postnatal thymus tissue- derived product that was implanted in step (h).

[0070] In another aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; ® providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period from about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted thymus tissue slices, wherein the thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei upon completion of the conditioning regimen; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0071] A fourth aspect of the present disclosure provides a method for promoting donor- specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (¢) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period from about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted thymus tissue slices, wherein the thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei upon completion of the conditioning regimen; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 — 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0072] In an embodiment of the foregoing aspects of the present disclosure, the allogeneic cultured postnatal thymus tissue-derived product, wherein the thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK 14 stains in a lacy pattern, and that >90% of nuclei are intact.

[0073] In an aspect of the present disclosure there is provided an allogeneic cultured postnatal thymus tissue-derived product for implantation into a subject undergoing a solid organ transplant prepared by obtaining suitable thymus tissue from a donor wherein the donor thymus tissue is subjected to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days); further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; wherein the donor thymus tissue slices show, between days 5 and 9 post-harvest, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue and presence of intact nuclei; recovering the partially T- cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product.

[0074] In an embodiment of the foregoing aspects of the present disclosure, the thymus, on the day of harvest from the donor, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK 14 stains in a lacy pattern, and that >90% of nuclei are intact.

[0075] In an embodiment of the foregoing aspects of the present disclosure, the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved.

[0076] In an embodiment, the cryopreserved allogeneic cultured postnatal thymus tissue- derived product is maintained in liquid nitrogen for future use.

[0077] In another embodiment, the cryopreserved allogeneic cultured postnatal thymus tissue- derived product is maintained in a cryopreserved tissue bank.

[0078] In an embodiment, the allogeneic cultured postnatal thymus tissue-derived product is prepared from suitable thymus tissue from a donor comprising HLA alleles matched to HLA alleles in a proposed recipient that are not present in the solid organ transplant.

[0079] In an embodiment the HLA alleles are: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA- DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA- DPB1, and HLA-DPA1.

[0080] In an aspect of the present disclosure there is provided a cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by method comprising the steps of: (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA- DRB3, HLA-DRB4, HLA-DRBS, HLA-DQA1, HLA- DPB1, HLA-DPA1: (c) subjecting the thymus tissue to a conditioning regimen for a period from about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; further wherein the donor thymus tissue slices show, on days 6 to 21, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei upon completion of the conditioning regimen; (d) harvesting the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (e) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and ® maintaining the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue- derived product bank.

[0081] In an embodiment, the thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattem, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.

[0082] In an embodiment, the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen is held for future use by the recipient.

[0083] In an aspect of the present disclosure, there is provided a method of preparing the donor thymus for implanting into a recipient subject. Such methods comprise, consist of, or consist essentially of culturing the donor thymus for up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, or up to about 21 days, and then surgically placing the cultured thymus tissue into the recipient, as further described herein. A culture period between about 6 and about 21 days results in good function. For successful transplantation of cryopreserved thymus tissue, the tissue is typically cultured for about 6 to about 21 days, and then cryopreserved.

[0084] In an aspect of the present disclosure, there is provided an allogeneic cultured postnatal thymus tissue-derived product (CTT; RVT-802) for implantation into a subject undergoing a solid organ transplant manufactured by the method of subjecting thymus tissue from a suitable donor to a conditioning regimen for a period up to 21 days (for example, a conditioning regimen of about 6 days to about 21 days); wherein the conditioning regimen for the allogeneic cultured postnatal thymus tissue-derived product comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, wherein the thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei.

[0085] In an embodiment, the donor thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK14 stains in a lacy pattern, and that >90% of nuclei are intact.

[0086] In an embodiment of the foregoing aspects and embodiments, the recipient’s thymus is obtained by surgery.

[0087] In an embodiment of the foregoing aspects and embodiments, the recipient’s thymus is obtained by robotic surgery.

[0088] In an embodiment of the foregoing aspects and embodiments, the recipient’s thymus is obtained by thorascopic surgery.

[0089] In an embodiment of the foregoing aspects and embodiments, the solid organ is a portion of a whole organ.

[0090] In an embodiment of the foregoing aspects and embodiments, the method of the first to fourth aspects further comprises the step of cryopreserving peripheral blood mononuclear cells from the deceased donor for future use in a mixed lymphocyte reaction to demonstrate cellular tolerance.

[0091] In an embodiment of the foregoing aspects and embodiments, the mixed lymphocyte reaction to demonstrate cellular tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor following the implantation of allogeneic cultured postnatal thymus tissue-derived product in accordance the implantation procedure of CTT in this specification.

[0092] In an embodiment of the foregoing aspects and embodiments, the mixed lymphocyte reaction to demonstrate cellular tolerance is performed with peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor about 6 to 12 months following the implantation of allogeneic cultured postnatal thymus tissue-derived product.

[0093] In an embodiment of the foregoing aspects and embodiments, the mixed lymphocyte reaction to demonstrate cellular tolerance is performed with peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor after naive T cells constitute about 10% of total T cells in the recipient.

[0094] In an embodiment of the foregoing aspects and embodiments, the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis in the subject within 12 months following the implantation of allogeneic cultured postnatal thymus tissue-derived product.

[0095] In an embodiment of the foregoing aspects and embodiments, the development of tolerance is determined by a mixed lymphocyte reaction performed with cryopreserved peripheral blood mononuclear cells from the deceased donor and T cells from the recipient.

[0096] In an embodiment of the foregoing aspects and embodiments, humoral tolerance is determined by the development of humoral immunity and the absence of donor reactive antibodies.

[0097] In an embodiment of the foregoing aspects and embodiments, the solid organ transplant is a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a heart / lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, an abdominal wall transplant, a craniofacial transplant, a scalp transplant, a penile transplant, a uterus transplant, a unilateral or bilateral upper limb transplant, a unilateral vascularized composite allograft, or combination thereof

[0098] In an embodiment of the foregoing aspects and embodiments, the method further comprises evaluating the recipient for HLA-Class I and HLA-Class II panel reactive antibodies (“PRA”) score prior to transplanting the solid organ.

[0099] In an embodiment of the foregoing aspects and embodiments, the solid organ transplant is a heart transplant.

[00100] In an embodiment of the foregoing aspects and embodiments, the solid organ transplant is a pediatric heart transplant.

[00101] In an embodiment of the foregoing aspects and embodiments, the solid organ transplant is an adult heart transplant.

[00102] In an embodiment of the foregoing aspects and embodiments, the method further comprises evaluating the recipient for HLA-Class I and HLA-Class II panel reactive antibodies (“PRA”) score prior to transplanting the solid organ.

[00103] In an embodiment of the foregoing aspects and embodiments, recipients with HLA antibodies are cross-matched with potential donors.

[00104] In an embodiment of the foregoing aspects and embodiments, recipients with HLA antibodies are virtually cross-matched with UNET.

[00105] In an embodiment of the foregoing aspects and embodiments, if a PRA score of >20% virtual cross-match is recorded, the method will further comprise the step of performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient.

[00106] In an embodiment of the foregoing aspects and embodiments, if a PRA score of >70% virtual cross-match is recorded, the method will further comprise the step of performing an actual prospective donor cross-match and performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient. Typically, transplants are not performed under these circumstance because of poor success rates.

[00107] In an embodiment of the foregoing aspects and embodiments, the method further comprises the step of evaluating recipients with HLA antibodies by cross-matching virtually with UNET.

[00108] In an embodiment of the foregoing aspects and embodiments, the method further comprises performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient if the HLA panel reactive antibodies have a score >20%.

[00109] In an embodiment of the foregoing aspects and embodiments, the method further comprises performing an actual prospective donor cross-match and performing plasmapheresis in the operating room at the time of solid organ transplant in the recipient if the HLA panel reactive antibodies have a score >70%.

[00110] In an embodiment of the foregoing aspects and embodiments, the solid organ is HLA- matched, in for instance, from a living related donor of the kidney, partial liver and partial intestine transplants to the recipient.

[00111] In an embodiment of the foregoing aspects and embodiments, the solid organ is HLA- mismatched.

[00112] In an embodiment of the foregoing aspects and embodiments, the solid organ is HLA matched. In another embodiment, the HLA match is determined by typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRBS, HLA- DQAI1, HLA-DPBI1, HLA-DPALI in the donor and the recipient.

[00113] In an embodiment of the foregoing aspects and embodiments, the solid organ transplants are ABO compatible.

[00114] In an embodiment of the foregoing aspects and embodiments, the solid organ is HLA- mismatched. In an embodiment, HLA-mismatched is determined by typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, HLA-DPA1 in the donor and the recipient.

[00115] In an embodiment of the foregoing aspects and embodiments, the cultured thymus tissue slices are surgically implanted into the quadriceps thigh muscle of the subject.

[00116] In an embodiment of the foregoing aspects and embodiments, the cultured thymus tissue slices are surgically implanted into the body of the subject in an area other than the quadriceps.

[00117] In an embodiment of the foregoing aspects and embodiments, a portion of the allogeneic cultured postnatal thymus tissue-derived product is surgically implanted into the quadriceps thigh muscle of the recipient.

[00118] In an embodiment of the foregoing aspects and embodiments, wherein the remaining portion of the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved in liquid nitrogen for future transplantation.

[00119] In an embodiment of the foregoing aspects and embodiments, the conditioning regimen is for a period of about 6 days to about 21 days.

[00120] In an embodiment of the foregoing aspects and embodiments, the conditioning period of the donor thymus tissue is about 5 days to about 21 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days, or about 21 days.

[00121] It will be appreciated by the person of ordinary skill in the art that there are numerous potential induction immunosuppressive regimens and maintenance immunosuppressive regimens known in the art, and that a suitable induction and maintenance immunosuppressive agent may be selected by the person of skill in the art without undue burden. The following illustrative induction immunosuppressive regimens and maintenance immunosuppressive regimens are exemplary of the practice of the methods of the first to the fourth aspects of the invention and support the inventions as claimed.

[00122] In an embodiment of the foregoing aspects and embodiments, the induction immunosuppressive regiment comprises an induction immunosuppressive agent selected from the group of glucocorticoid, anti-thymocyte globulin (rabbit), anti-thymocyte globulin (equine), and alemtuzimab.

[00123] In an embodiment of the foregoing aspects and embodiments, the ATG is antithymocyte globulin (rabbit).

[00124] In an embodiment of the foregoing aspects and embodiments, the induction immunosuppressive regimen comprises administration of a glucocorticoid. In an embodiment, the glucocorticoid comprises methylprednisolone. In another embodiment, the glucocorticoid is methylprednisolone sodium succinate. In a further embodiment, methylprednisolone sodium succinate is administered intravenously at no greater than 4 mg / kg / day.

[00125] In an embodiment of the foregoing aspects and embodiments, the induction immunosuppressive regimen comprises rabbit-derived anti-thymocyte globulin. In another embodiment, the rabbit-derived anti-thymocyte globulin is administered intravenously in a dose of about 1.5 mg / kg. In a further embodiment, the anti-thymocyte globulin is administered daily for four days. In another embodiment the ATG is equine derived ATG.

[00126] In an embodiment of the foregoing aspects and embodiments, the induction immunosuppressive regimen comprises basiliximab. In another embodiment, the basiliximab is administered at a dose of 10 mg intravenously for recipients less than 35 kg in body weight. In another embodiment, the basiliximab is administered at a dose of 20 mg intravenously for recipients more than 35 kg in body weight.

[00127] In an embodiment of the foregoing aspects and embodiments, the second immunosuppressive regimen comprises one or more immunosuppressive agent selected from the group consisting of a glucocorticoid, calcineurin inhibitor, an inosine monophosphate dehydrogenase inhibitor, azathioprine, and anti-thymocyte globulin (“ATG”).

[00128] In an embodiment of the foregoing aspects and embodiments, the immunosuppressive agent of the maintenance immunosuppressive regiment is anti-thymocyte globulin (ATG).

[00129] In an embodiment of the foregoing aspects and embodiments, the ATG is administered intravenously at a dose of about 1.5 mg / kg for a period of 3-14 days starting with administration in the operating room.

[00130] In an embodiment of the foregoing aspects and embodiments, the anti-thymocyte globulin is administered daily for 3-14 days at about 15 mg / kg / day by intravenous administration.

[00131] In an embodiment of the foregoing aspects and embodiments, the first immunosuppressive regimen comprises alemtuzumab.

[00132] In an embodiment of the foregoing aspects and embodiments, the alemtuzumab is administered at a dose of about 0.25 mg / kg for 4 days intravenously for recipients less than 35 kg in body weight. In another, embodiment the alemtuzumab is administered at a dose of about 3 to 20 mg for 4 days intravenously for recipients more than 35 kg in body weight.

[00133] In an embodiment of the foregoing aspects and embodiments, the second immunosuppressive regimen comprises one or more immunosuppressive agent selected from the group consisting of a calcineurin inhibitor, and inosine monophosphate dehydrogenase inhibitor, or azathioprine.

[00134] In an embodiment of the foregoing aspects and embodiments, the immunosuppressive agent of the maintenance immunosuppressive regimen the immunosuppressive agent is a calcineurin inhibitor. In an embodiment, the immunosuppressive agent of the maintenance immunosuppressive regimen the immunosuppressive agent is an inosine monophosphate dehydrogenase inhibitor.

[00135] In an embodiment of the foregoing aspects and embodiments, the immunosuppressive regimen comprises an inosine monophosphate dehydrogenase inhibitor, for example, mycophenolate mofetil. In an embodiment, mycophenolate mofetil is administered intravenously in a dose of about 15 to about 25 mg / kg. In an embodiment, the mycophenolate mofetil is administered intravenously two to three times a day.

[00136] In an embodiment of the foregoing aspects and embodiments, inosine monophosphate dehydrogenase inhibitor is mycophenolic acid. In another embodiment, the mycophenolic acid is administered at a dose of about 25 to about 50 mg / kg in 2 or 3 divided doses.

[00137] In an embodiment of the foregoing aspects and embodiments, the mycophenolic acid is administered at a dose of about for children about 400 mg / m?*dose twice daily with a maximum dose 720 mg, or BSA 1.19 to 1.59 m? about 540 mg twice daily, or for BSA > 1.58m? about 720 mg twice daily.

[00138] In an embodiment of the foregoing aspects and embodiments, the mycophenolate mofetil is administered for children at a dose of about 15 to about 25 mg / kg / dose twice a day or for adults about 1500 mg orally or intravenously twice daily and adjusted for a WBC of >3500.

[00139] In an embodiment of the foregoing aspects and embodiments, the second immunosuppressive regimen may further comprise a glucocorticoid selected from the group consisting of methylprednisolone, prednisone and prednisolone. In an embodiment, the dose of glucocorticoid is kept below 4mg / kg / day.

[00140] In an embodiment of the foregoing aspects and embodiments, the glucocorticoid is administered in a tapered dosage reduction, as described elsewhere in the present disclosure.

[00141] In an embodiment of the foregoing aspects and embodiments, the calcineurin inhibitor is tacrolimus. In another embodiment, the calcineurin inhibitor is cyclosporine A.

[00142] In an embodiment of the foregoing aspects and embodiments, the administration of the second immunosuppressant regimen is weaned after naive T cells reach 10% of total T cells. In yet another embodiment, the second immunosuppressant regimen is weaned after implantation of allogeneic cultured postnatal thymus tissue-derived product.

[00143] To evaluate how thymus production and / or release of chemokines and other soluble molecules may regulate these processes of cellular migration changes as thymocytes are depleted during preparation of donor thymus tissue for CTT, we screened conditioned media from human thymus organ cultures for the presence of 200 soluble molecules using antibody microarrays.

[00144] Expression of selected potentially mechanistically important candidate molecules was validated using additional thymic organ cultures and compared with a panel of well- characterized human thymus tissues obtained from donors ranging from age 5 days to 78 years. Through this analysis we identified certain potentially important biomarkers of thymocyte content.

[00145] A potentially important biomarker for thymocyte content of cultured thymus slices is L- selectin. Another important biomarker for thymic epithelial cell viability and function relies on the secretion of the chemokine CCL21 6Ckine.

[00146] Numerous additional potential biomarkers are set forth in Figs. 50 and 56. Of particular interest are the biomarkers set forth on Fig. 50, namely L-selectin, CCL21, CXCL16, M-CSF, galectin-7, CCL11, IL-16 and CXCL12. Also, of particular interest, are biomarkers set forth on Fig. 56, particularly biomarkers having a P-value of less than 0.05. These biomarkers would include: L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, CCL20 (MIP-3a), IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR (CD87), MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM. NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

[00147] The data reported in the Examples and in the literature support that the following biomarkers are indicative of TEC function: CCL21 (6Ckine, CXCL16, Osteopontin (OPN), CCL11, uPAR (CD87) and CXCL12. CXCL16: This chemokine has been shown to be produced by TECs (Bunting 2011).

[00148] Osteopontin (OPN; encoded by the SPP1 gene): This cytokine is increased during thymic stress and increased levels are associated with thymic atrophy (decrease in thymocyte number), decrease thymocyte numbers is a desirable state for cultured thymus (Wang 2009; Gridley 2013). OPN is required to make corticosteroids, which have been well-established to induce thymocyte apoptosis.

[00149] CCL11 (Eotaxin): This chemokine is made by medullary TE (Bunting 2011). It was initially named for its ability to attract eosinophils and we have shown that eosinophil infiltrates may be prominent in thymus tissues with active thymopoiesis (Flores 1999). However, eotaxin has also been shown to serve as a chemoattractant for both double-positive (DP) and single- positive (SP) human thymocytes (Bunting 2011).

[00150] uPAR (CD87; encoded by the PLAUR gene): Urokinase receptor, also known as urokinase plasminogen activator receptor, is expressed in both soluble and membrane-bound forms based on alternative splicing. It aids in local degradation of extracellular matrix. It has been shown to be expressed in human thymus and interestingly, by migrating epidermal keratinocytes (EK) at the edge of a wound (Loughner 2016). This latter characteristic is most interesting since TE cells mirror EK in expression of many genes (Patel 1995). Progressive increases in secretion by cultured thymus slices may reflect the activation of TE and thus be a marker for TE outgrowth following implantation.

[00151] CXCL12 (SDF-1a): This chemokine had an expression pattern that was different than the other analytes, in that it was only detected during the final 1 / 3 of the culturing period. First detectable on days 13-15, it rose linearly (in the In plots) to a much higher level over the next week in culture. CXCL12 has been documented to be produced by subcapsular cortical and medullary TE (Bunting 2011; Hernandez-Lopez 2002; Zaitseva 2002), but also can be made by thymic fibroblasts and endothelial cells present within the thymus. CXCL12 has been shown to recruit B cells and antigen-presenting cells (APC) to the thymus (Weiss 2003), which is expected to be important in generation of full thymic function. It is also involved in localization of thymocyte subsets within the thymus and it enhances thymocyte proliferation to IL-7 (Hernandez-Lopez 2002). Of note, antibodies that neutralize CXCL12 have been shown to decrease thymopoiesis in human thymus organ cultures in vitro and addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez 2002).

[00152] Additional biomarkers that may reflect thymocyte presence include L-selectin. This molecule is expressed at high levels on developing and naive T cells. It is released from the cell surface when thymocytes are cultured. Usually rapidly re-expressed when shed by healthy cells in vivo (Fitzhugh 2008), progressively decreased levels of shedding likely reflects the progressive loss of thymocyte viability because they normally do not re-express this molecule on their surface during culture (A. Macintyre, unpublished data).

[00153] Another biomarker that may reflect thymocyte presence is IL-16. This cytokine was included since its pattern (as decribed below) corresponded with that hypothesized for thymocytes. This biomarker is known to be made by lymphocytes.

[00154] Yet another biomarker that may reflect thymocyte presence is MIF. This chemokine was included since its pattern corresponded with that hypothesized for thymocytes.

[00155] Still another biomarker that may reflect thymocyte presence is CCL20 (MIP-3a): This chemokine was included since its pattern corresponded with that hypothesized for thymocytes.

[00156] Another biomarker that may reflect thymocycte presence is IGFBP-1. IGFBP2 - 6 are known to be expressed by thymic epithelium, which does not express IGFBP-1 (Gosteli-Peter 1994; Ketcha 1999).

[00157] Other biomarkers that showed initially high levels that then decreased with time, a hypothesized characteristic of thymocyte-derived biomarkers, which, based on the data in the Examples and the literature, correlate with the presence of viable thymocytes: L-selectin, IL-16, MIF. CCL20 (MIP-3a), and IGFBP-1. Decreases in these biomarkers over time is consistent with our more qualitative observations that T cells are depleted while the thymus tissue slices are in culture.

[00158] CCL21 has been shown to be expressed by thymic epithelial cells (Lkhagvasuren et al. 2013) and to be functionally important due to its chemotactic activity for thymocyte precursors (Liu et al. 2005), as well as for thymocyte migration within the thymus (Hu et al. 2015). This chemotactic property for thymocytes may be a critical determinant for successful immune reconstitution of the recipients following implantation of cultured thymus tissue, as described herein.

[00159] The results reported here should also be broadly applicable toward understanding mechanisms of age-related thymus involution as well as for understanding mechanisms involved in immune reconstitution of athymic recipients via implantation of cultured thymus tissue.

[00160] In the Examples and on Fig. 56, it is reported that at least 127 different analytes can be detected in spent / conditioned culture medium obtained from cultured human thymus tissue slices and that 42 of these analytes show progressive increases or decreases with culture time. Of these analytes, the amount of soluble L-selectin released was validated as a non-destructive marker for residual content of viable thymocytes in cultured thymus. Expression and / or secretion of the chemokines CCL21, CXCL16, CXCL12 and CCL11 by thymic epithelium was demonstrated to increase as thymocytes decrease, both during thymus organ culture # vitro and in vivo in un- manipulated thymus tissues obtained from healthy donors across the lifespan. Similarly, the expression or secretion of L-selectin, M-CSF, galectin-7 and IL-16 were observed to decrease in the thymus organ culturing medium during the course of the culturing process. These findings are directly relevant for understanding mechanisms of age-related thymus involution, as well as qualities of human thymus tissues that facilitate immune reconstitution. Parallels between the changes seen during culture of infant thymus and those associated with aging suggest that cultured human infant thymus also provides a model that may be useful for studying mechanisms that mediate age-related thymic involution.

[00161] To our knowledge, this is the first large screen of soluble molecules produced and released or secreted by the human thymus as thymocytes are depleted. Many of the 42 analytes that were found to have significantly increased or decreased release into media as cultures progressed are cytokines and / or chemokines that have previously been shown to be produced by cell types present within the thymus. Others are novel in this respect. While the experimental work focused primarily on release of L-selectin as a marker for thymocyte content and on CCL21 as a marker for TEC viability and function, the data reported from the antibody microarray screening can be used to identify additional analytes from Fig. 56 that can identify novel pathways that govern acute atrophy, chronic involution and / or regeneration of human thymus in vivo. These would include in particular the biomarkers set forth on Fig. 50. For example, some analytes that met pre-specified selection criteria were not considered further since they are known to be expressed or released during cell injury and / or hypoxic stress and their levels may reflect culture-associated tissue damage and inflammatory responses rather than thymus-specific biology. Additional biomarkers noted in the culture supernatant from cultured thymus tissue slices include CC25(TECK), osteopontin (OPN), uPAR (CD87), MIF, CCL20 (MIP-3a) and IGFBP-1. Study of additional analytes whose release may reflect the viability and / or activation of critical cell types in human thymus may lead to clinically and mechanistically important insights, particularly regarding responses to thymocyte loss.

[00162] CCL25 (TECK): Although detectable levels of TECK were only present in a few samples of supernatant late in the culture period of 2 of the 3 thymus cultures examined, it is of interest since this chemokine has been shown to be chemotactic for thymocytes (Liu 2005). It is known to be expressed by thymic dendritic cells (DC) and by both FoxN1+ and FoxN1- TE cells (Bunting 2011). However, its activity does not appear to be critical for thymus development based on studies of mice in which CCRY, the sole receptor for this chemokine, was deleted (Wurbel 2001).

[00163] Based on the microarray studies, it was determined that soluble L-selectin levels in conditioned culture media could serve as a potential biomarker of thymocyte presence and viability in cultured thymus slices. This is very plausible, since expression of L-selectin is limited to hematopoietic cells and is shed constitutively as well as during migration (Hafezi- Moghadam et al. 2001), then is usually rapidly re-expressed by healthy cells in vivo (Fitzhugh et al. 2008).

[00164] The experimental results show that the loss of L-selectin release temporally correlates with thymocyte death, as indicated by loss of thymocyte membrane integrity via histology and CD3 immunohistochemistry, as well as lack of characteristic thymocyte proliferation by Ki-67 immunohistochemistry.

[00165] Being able to non-destructively monitor thymocyte content of cultured human thymus slices is important to identify the most appropriate harvest time points for experimental studies. This can also provide clinically important information, since opening developmental niches for colonization by recipient thymocytes through depletion of donor thymocytes is believed to be critical for successful immune reconstitution of athymic patients via thymic implantation. Taken together, the studies presented in the Examples suggest that decreased L-selectin release is a useful biomarker for monitoring thymocyte depletion in cultured thymus slices.

[00166] Biomarkers that reflect the presence and function of thymic epithelium also can provide critical mechanistic information. Studies set forth in the Examples focused on CCL21, since the microarray screen indicated that this chemokine began to be secreted into the media at high levels soon after the start of culturing. CCL21 was previously shown to be expressed by thymic epithelium and to be chemotactic for thymocytes and their precursors (Liu et al. 2005). Our studies showed that expression of CCL21 could also be readily quantitated by enzyme immunoassay. Immunohistochemistry confirmed CCL21 expression by TECs in cultured as well as non-cultured thymus, with strongest expression in the medullary and subcapsular cortical thymic epithelium.

[00167] It is also important to note that CCL21 immunoreactivity of thymus slices did not necessarily increase as CCL21 secretion increased during culture. This may reflect that the additional CCL21 produced is secreted rather than being retained in the cytoplasm where it can be detected via immunohistochemistry. CCL21 is a transcriptionally regulated, high turnover molecule with a short half-life (Dudal et al. 2015), so the positive immunochistochemical reactivity observed represents cells that are actively producing this chemokine. That production of CCL21 markedly increases as thymocytes decrease in both cultured thymus and non- manipulated aging thymus suggests that thymic epithelial cells can sense thymocyte content and react in a homeostatic attempt to counteract thymocyte loss.

[00168] The identification of CCL21 as a secreted biomarker that reflects the viability and function of TECs is also important clinically with regards to thymus transplantation. Most established methods that can assess the quality of tissue to be implanted (e.g. flow cytometry, immunohistochemistry, gene expression analysis) destroy the samples during analysis. In addition to decreasing the amount of tissue available for eventual implantation, such results are subject to sampling error since the slice(s) tested is not part of the slices that are eventually implanted. As shown in Fig. 53C, assay of pooled spent media for CCL21 can integrate across all slices in a lot derived from any given thymus donor, providing a non-destructive picture of overall lot quality.

[00169] The chemokine CXCL12 (SDF-1a) differed from most other analytes in the screening, in that it became detectable in conditioned media relatively late during the culturing period (Fig. 50H). First detectable on days 13-15, it rose linearly (in the In plots) to a much higher level over the next week in culture. CXCL12 has been documented to be produced by subcapsular cortical and medullary TECs, but may also be made by thymic fibroblasts and endothelial cells present within the thymus (Bunting et al. 2011; Hernandez-Lopez et al. 2002; Zaitseva et al. 2002). CXCL12 recruits B cells and antigen-presenting cells to the thymus (Weiss et al. 2013), which is expected to be important in generation of full thymic function. CXCL12 is also involved in localization of thymocyte subsets within the thymus and it enhances thymocyte proliferation to IL-7 (Hernandez-Lopez et al. 2002). Of note, antibodies that neutralize CXCL12 have been shown to decrease thymopoiesis in human thymus organ cultures in vitro and addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez et al. 2002). The later timing of CXCL12 secretion during in vitro thymic organ cultures shown here, combined with its increased expression i vivo in thymus derived from donors >18 years, compared with younger, suggest that expression of this chemokine is induced by more long-standing depletion of thymocytes than is required to induced secretion of CCL21.

[00170] The screening data suggest that other chemokines, including CXCL16 and CCL11, are likely biomarkers for assessing the viability and function of cultured thymus, since they also increase as thymocytes are lost during thymus organ culture. Both CXCL16 and CCL11 have previously been shown to be made by TECs (Bunting et al. 2011).

[00171] CCL11 was originally named eotaxin for its ability to attract eosinophils. We previously showed that eosinophil infiltrates may be prominent adjacent to thymus tissues with active thymopoiesis (Flores et al. 1999), although CCL11 levels were not directly measured in those studies. However, CCL11 was subsequently also shown to serve as a chemoattractant for both double-positive and single-positive human thymocytes (Bunting et al. 2011). Evidence for a specific role of CXCL16 in thymopoiesis is less clear.

[00172] The studies presented in the Examples validate the use of L-selectin as a biomarker for the presence of viable thymocytes and CCL21 as a biomarker for TEC viability that may also be predictive of efficient immune reconstitution if thymocyte precursors are made available. Production of CCL21 is typically low when thymopoiesis is robust and is markedly induced when thymopoiesis becomes compromised, in both cultured thymus slices and non-cultured thymus tissues from older adults. Interestingly, after normalization to either TEC area or active cortical area, thymus tissues from younger donors (< 18 years) continued to express more CD3epsilon and CD1A mRNAs and less keratin8 (KRT8) and keratin14 (KRT14) mRNAs than thymus from older adults. This suggests that thymopoiesis and TEC maintenance may potentially be more efficient in these younger donors. Further, for both normalization methods, the production of CCL21 and CXCL12 are markedly increased for tissues derived from donors > 18 years, a timeframe when TEC content and active thymopoiesis is decreased compared to younger donors. That expression of CCL21 and CXCL12 both increase as numbers of thymocytes decrease in both in vitro in thymus organ cultures and in vivo during aging raises the possibility that induction of these chemokines is part of homeostatic mechanisms that attempt to counter the decreased thymocyte numbers through enhanced recruitment of T cell precursors. Increased secretion of thymocyte-attracting chemokines by thymocyte-depleted cultured thymus slices would be expected to enhance their colonization and ability to result in immunoreconstitution, as is observed when such slices are implanted into athymic infant recipients (Markert et al. 2008). In contrast, abundant secretion of CCL21 and CXCL12 may provide less benefit during aging, if the availability of thymocyte precursors or other critical aspects of the thymic microenvironment are limiting.

[00173] Together, these studies show that organ cultures of thymus derived from pediatric donors can be used to model at least some aspects of age-related thymic involution in humans, particularly those that are more directly related to loss of thymocytes. However, it is clear that T cell-depleted cultured pediatric thymus must differ substantially from aged adult thymus in other ways, since implantation of T cell-depleted cultured pediatric thymus into athymic recipients results in immune reconstitution and protection from infections, whereas aged adults with involuted thymus are more vulnerable to infections than younger adults with more robust thymus function. Palmer, S, Albergante L, Blackburn CC, Newman TJ. Thymic involution and rising disease incidence with age. Proc Natl Acad Sci USA 11colk:1883-1888, 2018.

[00174] The results presented here in Fig. 56 provide a rich source of additional molecules and pathways as potential biomarkers to model some aspects of human thymus aging in vitro using cultured infant thymus. This is important, since infant thymus is typically more readily available for research given the need to remove a portion of thymus from most infants to properly expose the operative field for corrective cardiac surgery. Adult thymus tissue is typically less readily available, since it is generally not necessary to remove thymus tissue to provide access for many types of cardiac surgery common in adults. Furthermore, any adult thymus tissue removed is not typically made available for research since it appears less organoid and grossly resembles fat. However, adult thymus tissue would potentially be cultured and used to co-transplant with a solid organ if tolerance is desired in adult solid organ recipients. The biomarkers also are useful in determining the suitability, functionality and viability of allogeneic, cultured postnatal thymus tissue-derived product derived from adult donors.

[00175] Thymus production of the thymocyte chemoattractants CCL21, CXCL16, CXCL12 and CCL11 increase as thymocyte content decreases. This suggests that thymocyte loss may activate homeostatic mechanisms that attempt to counteract potential atrophy, although ultimately unsuccessfully in the setting of aging, future studies to more fully elucidate these mechanisms will be useful for understanding and potentially reversing mechanisms that drive age-related thymus involution and may help to enhance thymus-driven immune reconstitution at all ages.

[00176] In an aspect of the present disclosure, there is provided a method of producing an allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of subjecting donor thymus to a conditioning regimen for a period from about 6 to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; further comprising detecting increasing levels of CCL21 in the thymus organ medium during the course of the conditioning regimen; and recovering the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product suitable for implantation.

[00177] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises the step of cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen for future implantation.

[00178] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises detecting decreasing levels of L-selectin in the thymus organ medium during the course of the conditioning regimen.

[00179] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting one or more of CCL21, CXCL12, CXCL16 or CCL11 in the thymus organ medium during the course of the conditioning regimen.

[00180] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting increasing levels of one or more of CXCL12, CXCL16 or CCL11 in the thymus organ medium during the course of the conditioning regimen.

[00181] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting increasing levels of CXCL12 in the thymus organ medium during the course of the conditioning regimen.

[00182] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting increasing CXCL16 in the thymus organ medium during the course of the conditioning regimen.

[00183] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting increasing levels of CCL11 in the thymus organ medium during the course of the conditioning regimen.

[00184] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting one or more of M-CSF, galectin-7 or IL-16 in the thymus organ medium during the course of the conditioning regimen.

[00185] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting decreasing levels of one or more of M-CSF, galectin-7 or IL-16 in the thymus organ medium during the course of the conditioning regimen.

[00186] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting decreasing levels of M-CSF in the thymus organ medium during the course of the conditioning regimen.

[00187] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting decreasing levels of galectin-7 in the thymus organ medium during the course of the conditioning regimen.

[00188] In an embodiment of the aspects and embodiments of the present disclosure, the method comprises detecting decreasing levels of IL-16 in the thymus organ medium during the course of the conditioning regimen.

[00189] In an embodiment of the aspects and embodiments of the present disclosure, the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.

[00190] In an embodiment of the aspects and embodiments of the present disclosure, the levels of CCL21 approximate the levels in Fig. SOE, and / or the levels of L-selectin approximate the levels in Fig. 50A, and / or the levels of M-CSF approximate the levels in Fig. 50B, and / or the levels of galectin-7 approximate the levels in Fig. 50C, and / or the levels of IL-16 approximate the levels in Fig. 50D, and / or the levels of CXCL16 approximate the levels in Fig. 50F, and / or wherein the levels of CCL11 approximate the levels in Fig. 50G, and / or the levels of CXL21 approximate the levels in Fig. SOH.

[00191] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises the step of determining in the donor thymus tissue slices between days 6 and 21, preferably between days 6 and 9 of the conditioning regimen areas positive for keratin AET1 / AES3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

[00192] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises detecting the level of at least one marker in the thymus organ medium during the culturing regimen, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or detecting at least eight markers in the thymus organ medium during the conditioning regimen, selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[00193] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises detecting the level of at least one marker in the thymus organ medium during the culturing regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF- 15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

[00194] In an aspect of the present disclosure, there is provided a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11 in the thymus organ medium.

[00195] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is L-selectin, and wherein the levels of L-selectin in the thymus organ medium decrease over time, i.e., during the course of the course of the conditioning regimen.

[00196] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is M-CSF, and wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen.

[00197] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is galectin-7, and wherein the levels of galectin-7 in the thymus organ medium decrease during the course of the conditioning regimen.

[00198] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is IL-16, and wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.

[00199] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is CCL21, and wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.

[00200] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is CXCL12, and wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.

[00201] In an embodiment of the aspects and embodiments of the present disclosure, the at least one marker is CXCL16, and wherein the levels of CXCL16 in the thymus organ medium increase during the course of the conditioning regimen.

[00202] In an embodiment of the aspects and embodiments of the present disclosure of the present disclosure, the at least one marker is CCL11, and wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

[00203] In an aspect of the present disclosure, there is provided a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF- AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

[00204] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises the step of determining in the donor thymus tissue slices between days 6 and 21 of the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK 14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

[00205] In an embodiment of the aspects and embodiments of the present disclosure, the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.

[00206] In an embodiment of the aspects and embodiments of the present disclosure, the levels of CCL21 approximate the levels in Fig. 50E, and / or the levels of L-selectin approximate the levels in Fig. 50A, and / or the levels of M-CSF approximate the levels in Fig. 50B, and / or the levels of galectin-7 approximate the levels in Fig. 50C, and / or the levels of IL-16 approximate the levels in Fig. 50D, and / or the levels of CXCL16 approximate the levels in Fig. 50F, and / or wherein the levels of CCL11 approximate the levels in Fig. 50G, and / or the levels of CX1.21 approximate the levels in Fig. 50H.

[00207] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen, or the levels of at least two, at least three, at least four, at least five, at least six, at least seven, or detecting at least eight markers, selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11, and wherein the levels of L-selectin, M-CSF, galectin-7, IL-16 decrease in the thymus organ medium during the course of the conditioning regimen, and further wherein the levels of CCL21, CXCL12, CXCL16, and CCL11 increase in the thymus organ medium during the course of the conditioning regimen.

[00208] In an aspect of the invention, there is a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from the markers in Fig. 56.

[00209] In an embodiment of the aspects and embodiments of the present disclosure, there is a method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

[00210] In an aspect of the present disclosure, there is a method of treating a thymic disorder, the improvement comprising implanting into a subject having a thymic disorder allogeneic cultured postnatal thymus tissue-derived slices subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[00211] In another aspect of the present disclosure, there is a method of treating thymic disorders, the improvement comprising implanting into a subject having a thymic disorder allogeneic cultured postnatal thymus tissue-derived slices subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF- AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

[00212] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is L-selectin, and wherein the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.

[00213] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is M-CSF, and wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen,

[00214] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is galectin-7, and wherein the levels of galectin-7 in the thymus organ medium decrease during the course of the conditioning regimen.

[00215] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is IL-16, and wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.

[00216] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is CCL21, and wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.

[00217] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is CXCL12, and wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.

[00218] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is CXCL16, and wherein the levels of CXCL16 in the thymus organ medium increase during the course of the conditioning regimen.

[00219] In an embodiment of the aspects and embodiments of the present disclosure, at least one marker is CCL11, and wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

[00220] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises the step of determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK 14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

[00221] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is congenital athymia associated with complete DiGeorge syndrome, 22q11.2 deletion, CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness), mutations in the CHD7 (chromodomain-helicase- DNA-binding protein 7) gene or forkhead box protein N1 (FOXNT1) deficiency.

[00222] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is thymic involution. In another embodiment, thymic disorder is congenital athymia associated with mutations in the TBX-1 or TBX-2 gene.

[00223] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is related to paired box 1 (PAX1), semaphorine 3E (SEMA3E) and recurrent microdeletions at chromosome 2p11.2.

[00224] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is associated with a thymoma. In still a further embodiment the thymoma is either non- malignant or malignant.

[00225] In an embodiment of the aspects and embodiments of the present disclosure, the thymic disorder is associated with myasthenia gravis (MG), pure red cell aplasia and hypogammaglobulinemia.

[00226] In an aspect of the present disclosure there is provided a method for providing immune- competence in a human subject, the improvement comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00227] In an aspect of the present disclosure, there is provided a method for providing immune-competence in a human subject, the improvement comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00228] In an aspect of the present disclosure, there is provided a method for providing immune-competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HL A-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00229] For better understanding of the development of tolerance in this model, one can refer to Fig 38 which has a primate model of this procedure that will provide data to support a human study for generating donor-specific tolerance. The experiment in Fig 38 has 3 monkeys. One is the thymus and heart donor (information in the left hand columns). The second is the thymus and heart recipient (information in the middle column on the 27d page. This column has the STAGE of the experiment). The third is the control (information in the right hand columns on the 37 page). Please note that the original spreadsheet had the procedures for all three animals one page wide by many pages long. Because the spread sheet was wider than the width allowed, each row of the spread sheet was divided into 3 pages. The table continues in groups of 3 panels for many weeks and several stages.

[00230] In an aspect of the present disclosure, there is provided a method for providing immune-competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HL A-Class I and II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regiment is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP- 1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00231] In an aspect of the present disclosure, there I provided a method for providing immune- competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching both HLA-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00232] In an aspect of the present disclosure, there is provided a method for providing immune-competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching both HLA-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP- 1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

[00233] In an aspect of the present disclosure, there is provided a method of promoting donor- specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant, the method comprising the following steps: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing both a suitable solid human organ and a thymus gland from a deceased donor; (d) transplanting the solid human organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing an allogeneic cultured postnatal thymus tissue-derived product, wherein the allogeneic cultured postnatal thymus tissue-derived product is subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product slices; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL- 16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; and (g) implanting the allogeneic cultured postnatal thymus tissue-derived product into the recipient after about 6 days to about 21 days of conditioning regimen, wherein the dosage of thymus tissue slices is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m?, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[00234] In an aspect of the present disclosure, there is provided a method of promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant, the method comprising the following steps: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing both a suitable solid human organ and a thymus gland from a deceased donor; (d) transplanting the solid human organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing an allogeneic cultured postnatal thymus tissue-derived product, wherein the allogeneic cultured postnatal thymus tissue-derived product is subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product slices; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; and (g) implanting the allogeneic cultured postnatal thymus tissue-derived product into the recipient after about 6 days to about 21 days of conditioning regimen, wherein the dosage of thymus tissue slices is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[00235] In an aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient's T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA- Class I and HLA-Class II alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product, and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient

[00236] In an embodiment of the aspects and embodiments of the present disclosure, permissive mismatches for HLA-DP can be allowed (Pidala J et al 2014 Blood 124:2596-2606). In addition, nonpermissive mismatches for HLA-DPB1 can be allowed if there is sufficient numerical functional distance (Crivello P et al 2016 Blood 128:120-129).

[00237] In an aspect of the present invention, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA- Class I and HLA-Class II alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP- 3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP- 3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRGI1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels for the marker in Fig. 56; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient

[00238] In an embodiment of the aspects and embodiments of the present disclosure, about one-half of the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product is transplanted into the recipient and the remainder is cryopreserved for future use.

[00239] In an embodiment of the aspects and embodiments of the present disclosure, step (h) is performed about one month or more after the transplantation of the solid organ.

[00240] In an aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (¢) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M- CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2 and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[00241] In an aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (¢) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 7; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[00242] In an aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by a method comprising the steps of: (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRBS, HLA-DQA1, HLA-DPB1, HLA-DPA1: (c) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; (d) detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL- 16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (e) retrieving the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (f) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (g) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

[00243] In an aspect of the present disclosure, there is a cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by a method comprising the steps of’ (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRBS, HLA-DQA1, HLA- DPB1, HLA-DPA1: (c) subjecting the thymus tissue to a conditioning regimen for a period from about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; (d) detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 7; (e) retrieving the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (f) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (g) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

[00244] In an aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of a about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regiment selected from the markers L-selectin, M- CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymus organ medium, wherein the level of the marker in the thymus organ medium is decreased if the marker is L- selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCLI11: (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[00245] In an aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by a method comprising the steps of: (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRBS, HLA-DQA1, HLA- DPB1, HLA-DPAI: (c) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; (d) detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, M-CSF, galectin-7, IL- 16, CCL21, CXCL12, CXCL16, or CCL11 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (e) retrieving the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (f) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (g) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

[00246] In an aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient's T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-BI, CEACAM-1, IL-1b, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels for the marker in Fig. 56; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient

[00247] In an embodiment of the aspects and embodiments of the present disclosure, the solid organ transplant is a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a heart / lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, an abdominal wall transplant, a craniofacial transplant, a scalp transplant, a penile transplant, a uterus transplant, a unilateral or bilateral upper limb transplant, a unilateral vascularized composite allograft, or combination thereof.

[00248] In an embodiment of the aspects and embodiments of the present disclosure, the solid organ transplant is a heart transplant, or a pediatric heart transplant, or an adult heart transplant.

[00249] In an embodiment of the aspects and embodiments of the present disclosure, the conditioning regimen is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.

[00250] In an aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of: (a) obtaining suitable thymus tissue from a human donor; (b) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; wherein levels of L-selectin, and / or M-CSF and / or galectin-7 and / or IL-16 in the thymus organ medium decrease during the course of the conditioning regimen; further wherein levels of CCL21 and / or CXCL12 and / or CXCL16 and / or CCL11 in the thymus organ medium increase during the course of the conditioning regimen; (c) harvesting the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (d) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (¢) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank

[00251] In an embodiment of the aspects and embodiments of the present disclosure, the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen

[00252] In an embodiment of the aspects and embodiments of the present disclosure, the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.

[00253] In an embodiment of the aspects and embodiments of the present disclosure, the levels of one or more of M-CSF, galectin-7, and IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.

[00254] In an embodiment of the aspects and embodiments of the present disclosure, the levels of one or more of CCL21, CXCL12, CXCL16, and CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

[00255] In an embodiment of the aspects and embodiments of the present disclosure, the method further comprises the step of determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK 14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

[00256] In an aspect of the present disclosure, there is provided a kit for performing the methods of any of the foregoing aspects and embodiments, together with instructions for use in determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human.

[00257] In an embodiment of the aspects and embodiments of the present disclosure, the kit comprises at least one antibody that specifically binds marker L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[00258] In an embodiment of the aspects and embodiments of the present disclosure, the kit comprises one or more antibody that specifically finds a marker set forth in Fig. 56.

[00259] In an aspect of the present disclosure, there is provided a kit for determining whether cryopreserved allogeneic cultured postnatal thymus tissue-derived product cultured in accordance with any one of the foregoing aspects and embodiments is suitable for implantation into a human together with instructions for use

[00260] In an embodiment of the aspects and embodiments of the present disclosure, the kit comprises at least one antibody that specifically binds marker L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[00261] It is further appreciated that certain features described herein, which are, for clarity, described in the context of different aspects of the present disclosure and / or in separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single aspect of the present disclosure and / or in a single embodiment, can also be provided separately or in any suitable sub- combination. BRIEF DESCRIPTION OF THE DRAWINGS

[00262] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[00263] Fig. 1 describes the manner in which allogeneic, cultured postnatal thymus tissue- derived product (e.g, CTT, RVT-802) provides for immune reconstitution in congenital athymia following implantation.

[00264] Fig. 2 shows a schematic of the steps for reconstituting the immune system in a rat, as described elsewhere in Example 5, by removing the thymus in an immunologically normal Lewis rat, administering an antibody to kill the recipient rat’s T cells, implanting cultured neonatal thymus tissue from a donor rat into the recipient rat, administering an immunosuppressive agent for about 4 months and evaluating T cell development in the recipient rat. Of note, all rats in the treatment group had over 10% naive T cells prior to stopping the cyclosporine.

[00265] Fig. 3 shows the development of naive T cells in two experimental recipient rats of Example 5 (rising lines on right) versus two controls rat not receiving a thymus tissue implant (thick lines at baseline).

[00266] Fig. 4 shows a schematic of the manufacturing process for harvesting a thymus from a donor, culturing thin slices of the donor thymus tissue made with a hand microtome for up to 21 days and implanting the cultured thymus tissue in the quadriceps muscle of the recipient.

[00267] Fig. SA shows a schematic showing the slicing of thymus tissue for characterization testing, as discussed in section

[00766] . Fig.5B is a figure showing slices of thymus tissue on cellulose filters on surgical sponges in a tissue culture dish as is used for culture of the thymus.

[00268] Figs. 6A-H depict histology testing of thymus tissue slices from a lot (MFG-056) of cultured thymus tissue on day 5, 9, 12 and 21 after harvest of the thymus from a donor. Hematoxylin and eosin-stained slices (left panels) and their corresponding reactivity with a cocktail of the anti-cytokeratin antibodies AE1 / AE3 (right panels; brown color denotes positive reactivity) are shown at day 5 (Fig. 6A, Fig. 6B), day 9 (Fig. 6C, Fig. 6D), day 12 (Fig. 6E, Fig. 6F), and day 21 (Fig 6G, Fig. 6H), respectively. Bars in the lower left of each panel represent 100 pm. Panels with H&E show progression depletion of T cells with time. Fig. 6E and Fig. 6F are predominantly epithelial cells. Condensation of the epithelium of the subcapsular cortex occurs as the thymocytes are depleted with time. Similar condensation occurs in medullary areas of the thymus. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00269] Figs. 7A and 7B depict the histology of thymus tissue slices on day 0 of the time course in a scale of 5 mm (Fig. 9A) and 100 um (Fig. 7B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 um) on day 0. This is normal thymus. At this time the cortex and medulla both have large numbers of thymocytes with dark blue nuclei contributing to the overall dark blue appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00270] Figs. 8A and 8B are images from H&E stained slide that depict the histology of thymus tissue slices on day 5 of the time course in a scale of 5 mm (Fig. 8A) and 100 pm (Fig. 8B), respectively. Progression of depletion of the thymocytes results in a more eosinophil (pink) appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00271] Figs. 9A and Fig. 9B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 9A) and 100 um (Fig. 9B), respectively. We see progressive depletion of thymocytes. The higher magnification shows numerous eosinophilic cell bodies lacking nuclei which are diagnostic of necrotic cells that have undergone karyolysis (dissolution of the nuclei). This degree of necrosis is expected at this time in culture. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00272] Fig. 10A and Fig. 10B depict H&E staining of thymus tissue slices on day 21 of the time course in a scale of 5 mm (Fig. 10A) and 100 um (Fig. 10B), respectively. Note the preservation of the overall architecture of the tissue including in Fig. 10B the subcapsular cortex, cortical region and medullary region containing numerous Hassall bodies. The small dark cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00273] Figs. 11A-E depict representative thymus slices which were immuno-stained with a cocktail of anti-cytokeratin antibodies (AE1 / AE3). Fig. 11A. Day 0; Fig. 11B. Day 5; Fig. 11C. Day 9; Fig. 11D. Day 12; and Fig. 11E. Day 21. The structure of the thymic epithelial network remains intact as the culture progresses. Bar represents 400 um. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00274] Figs. 12A and 12B depict the histology of thymus tissue slides after exposure to forced degradation conditions of 10X PBS. Fig. 12A depicts the cortex at day 9 after exposure to forced degradation conditions. Fig. 12B depicts the cortex at day 21 after exposure to forced degradation conditions. In Fig. 12A the smear of blue is DNA released from cells. The majority of cells show evidence of degradation although small foci of cells with intact nuclei can be identified. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00275] Fig. 13 depicts H&E stained histology sections for clinical sample MLM247. This is Day 0 of culture. The bar is 200 um. This is a frozen section from day 0. Because this was frozen, the tissue looks different from paraffin embedded formalin fixed tissue on day 0. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00276] Fig. 14 Frozen section, H&E stained histology sections for clinical sample MLM219. This is a frozen section so the tissue looks different from paraffin embedded formalin fixed tissue that was cultured and presented above. Nevertheless, the important histologic characteristics of thymocyte depletion and robust viability of TEC are well represented. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00277] Fig. 15 is a photograph of freshly harvested thymus tissue.

[00278] Fig. 16 is a schematic describing the harvesting, culturing, implantation and biopsy of the implantation of CTT under the rat kidney capsule, as presented in Example 5.

[00279] Figs. 17A-D present photographs of the harvesting of thymus tissue from 3-day old F1 (LWxDA) rats that was cut into four pieces as described in Example 5 (Fig. 17A). A photograph of thymus pieces cultured on sterile mixed cellulose ester filters with thymus organ medium for 5-7 days in a 37°C COz incubator, as described in Example 5 (Fig. 17B). Fig. 17Cisa photograph of CTT implanted under the kidney capsule of an LW rat. Fig. 17D is a photograph of a thymus graft harvested at 6 months after implantation. Arrows indicate the CTT under the kidney capsule.

[00280] Figs. 18A-D are photographs depicting the histologic appearance of fresh thymus tissue (top frames) and CTT (bottom frames) at 100x magnification. Fig. 18A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5. Fig. 18B shows the typical lacey pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin compared with fresh thymus tissue (top frame), as described in Example 5. Fig 18C shows fresh thymus tissue (top frame) and CTT depleted of T cells (bottom frame) when stained for Ki-67. Fig. 18D shows fresh thymus tissue stained for CD3 (top frame) and CTT thymus tissue cultured for 5 days and then stained for CD3 (bottom frame). The brown stain noted in the CD3 stained CTT (Fig. 18D, bottom frame), likely represents some viable cells plus the detritus of dead T cells that have not washed out of the tissue.

[00281] Figs. 19A-D are photographs depicting the histologic appearance of fresh thymus tissue (top frames) and CTT (bottom frames) at 600x magnification. Fig. 19A shows a comparison of medullary differentiation in H&E stained fresh thymus tissue (top frame) and CTT cultured for 5 days (bottom frame), as described in Example 5. Fig. 19B shows the typical lacey pattern observable in CTT cultured for 5 days (bottom frame) when stained for cytokeratin compared with fresh thymus tissue (top frame), as described in Example 5. Fig 19C shows fresh thymus tissue (top frame) and CTT depleted of T cells (bottom frame) when stained for Ki-67. Fig. 19D shows fresh thymus tissue stained for DC3 (top frame) and CTT thymus tissue cultured for 5 days and then stained for CD3 (bottom frame). The brown stain noted in the CD3 stained CTT (Fig. 19D, bottom frame), likely represents some viable cells plus the detritus of dead T cells that have not washed out of the tissue.

[00282] Fig. 20 is a schematic of the experimental design of the experiment reported in Example 5. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00283] Fig. 21 shows repopulating recipient-type T cells are seen in the lower right quadrant after CTT imallogernicplantation. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4:5(11).

[00284] Fig. 22A and Fig. 22B show implanted thymus explanted at 8.5 month after implantation showing positive cytokeratin staining (Fig. 22A), as well as T cell staining similar to native thymus (Fig. 22B). Original magnification x 400. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00285] Fig. 23 shows plots of significantly increased numbers of circulating CD4 and CD8 T cells compared to control animals without implantation of CTT. It also shows significantly increased numbers of naive CD4 and naive CD8 T cells in the cultured thymus tissue implantation (CTT) group compared to the control group that did not receive CTT and significantly increased numbers of CD4 and CDS recent thymus emigrants (RTE) in the cultured thymus tissue imsplantation (CTT) group compared to the control group that did not receive CTT. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00286] Fig. 24A shows immunohistologic analysis of implanted CTT explanted on day 180 showing normal thymus histology under the capsule of the kidney (right hand side of Figure 24A). Fig. 24B shows the explanted graft on H&E. Strains for viable T cells (CD3), T cell proliferation (Ki67), and cytokeratin (detected by a rabbit polyclonal antibody) are shown. In the panel stained for cytokeratin, a lacy pattern is seen with Hassall body formation (arrow) on TECs. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00287] Fig. 25 shows survival percentages of LW rats after thymectomy and immunosuppression with DA heart transplants with CTT (solid triangles, blue line) and without CTT (upside down triangles, red lines) transplants (CTT). The LW rats with CTT are tolerant; the LW rats without CTT are immunodeficient and thus do not reject the DA heart. The control shows complete rejection of DA heart transplants in LW unmanipulated rats (open squares). LW control animals also did not reject an LW cardiac graft (open circle with horizontal line) (n=9). This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00288] Figs. 26A and 26B are photographs of transplanted allografts (DA hearts) from animals implanted with (Fig. 26A) and without (Fig. 26B) CTT showing mononuclear cell infiltration with no signs of rejection by 2004 International Society for Heart &L Lung Transplantation (ISHLT) depicted in Fig. 26C (the solid blue squares and solid red triangles). This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00289] Fig. 27 is a plot of BN heart graft survival in the neck percentage animal survival vs. graft survival days in LW rats with CTT (that were immunocompetent and rejected the cervical allogeneic BN heart) and control LW animals without CTT (that were immunodeficient because of lack of thymus and could not reject the cervical BN heart) inserted vs. BN control (LW rat rejecting a cervical BN heart) and syngeneic controls (LW rats do not reject cervical LW hearts). This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00290] Fig. 28A and Fig. 28B are photographs of BN heart tissue with (Fig. 28A) and without (Fig. 28B) CTT insertions at 11 and 46 days, respectively. These pictures are the basis of the data in Fig. 27 and Fig. 29. The heart in Fig. 28A is not rejected because of tolerance. The heart in Fig. 28B is not rejected because of immunodeficiency from lack of thymus. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00291] Fig. 29 shows rejection grading of the cervical BN hearts. Syngeneic LW hearts placed into LW rats (open circles) were not rejected. BN hearts placed into LW rats (filled circles) were rejected. BN hearts placed in LW rats who received CTT were rejected (filled squares). BN hearts placed in LW rats who did not receive CTT were weakly rejected (shaded triangles) in 2 rats and not rejected by the other three rats. These data show that the rats with CTT were able to strongly reject 3% party hearts even while they accepted DA hearts (Fig. 26C) as the CTT expressed DA. The rats without CTT were immunodeficient and didn’t reject either the DA (Fig. 26C) or the BN hearts. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4:5(11).

[00292] Fig. 30A and Fig. 30B are photographs of BN hearts in which rats received or did not receive CTT insertions compared to LW and DA hearts, respectively. In Fig. 30A, after immunosuppression was removed and the BN heart transplanted, the BN heart was quickly rejected and thus is very large because of all the inflammation. The LW heart is normal sized for the heart pumping blood through the body. The DA heart is small as it was placed in the abdomen and didn’t need to pump blood. In Fig. 30B, the rat is immunodeficient and cannot reject either the BN or DA heart after immunosuppression is removed. This figure appears in Kwun, J. et al, JCI Insight (2020) Jun 4:5(11).

[00293] Fig. 31A and Fig. 31B are plots of rejection grading for explanted cervical BN hearts from rats with and without CTT insertions vs. BN controls and syngeneic control rats. Fig. 31A depicts quantification of inflammatory cells in the primary abdominal DA cardiac allograft. The syngeneic control shows that LW rats do not reject LW hearts. The DA control shows that LW rats do reject DA hearts. The CTT group does not reject the DA heart because of tolerance. The group without CTT doesn’t reject the DA heart because of immunodeficiency from lack of a thymus. Fig. 31B depicts quantification of inflammatory cells in secondary cervical BN cardiac allografts. The syngeneic control shows that LW rats do not reject LW hearts. The BN control shows that LW rats do reject BN hearts. The CTT group rejects the BN heart because it is immunocompetent. The group without CTT doesn’t reject the BN heart because of immunodeficiency from lack of a thymus. Fig. 31C shows DA and BN heart rats that were harvested from the LW recipients along with the native LW heart at the time of the cervical BN heart rejection. The lower right panel shows the T cells (brown) in the BN heart leading to its rejection. Fig. 31D shows T cell infiltration in the LW, DA, and BN hearts from control animals without insertion of CTT. There is no T cell infiltration because the animals are immunodeficient. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00294] Fig. 32A to Fig. 32C: Humoral tolerance after CTT. Fig. 32A shows representative histogram plots for post-transplant donor-specific alloantibody (anti-DA and anti-BN antibodies) measured by T cell flow crossmatch. The upper left panel of Fig. 32A (DA control) shows the development of anti-DA antibody (thick line) in a normal LW rat after receiving a heterotopic abdominal DA heart transplant. The upper middle panel of Fig. 32A shows lack of anti DA antibody in the LW rats that received CTT; this indicates tolerance. The upper right panel shows no response by the LW rats without CTT; this reflects the immunodeficiency of the rats after thymectomy and T cell depletion without receipt of a donor thymus. The lower left panel of Fig. 32A shows normal anti BN antibody formed by a normal LW rat receiving a cervical BN heart. The lower middle panel of Fig. 32A shows a normal response of the LW rats with CTT against BN after receiving a cervical BN heart transplant, showing immunocompetence and ability to reject 3 party. The lower right panel of Fig. 32A shows that there is no response of the LW rats without CTT against BN after having received a cervical BN heart transplant, showing immune-incompetence and lack of ability to reject 3 party. Fig. 32B shows levels of anti-DA antibody after primary DA heart transplantation. The LW rats with CTT from an LWxDA thymus donor do not make anti-DA antibody after a DA heart transplant because they are tolerant to DA. The LW rats without CTT do not make anti-DA antibody after DA heart transplantation because they are immunodeficient. Fig. 32C shows levels of anti-BN antibody after secondary cervical BN heart transplantation. The LW rats with CTT from an LWxDA donor make antibodies against BN showing immunocompetence against 3™ party. The LW rats without CTT do not make antibodies against BN showing immunoincompetence. This figure appears in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11).

[00295] Figs. 33A-J present photomicrographs of immunohistochemical assessment of fresh and cultured non-human primate (NHP) thymus tissue from an 8-month old NHP. The top row is NHP thymus on the day of harvest and the bottom row is NHP thymus after culture for 12 days. The tissue was stained with hematoxylin and eosin (Fig. 33A and Fig. 33F), CD3 (Fig. 33B and Fig. 33G), pan cytokeratin (CK) antibody AE1 / AE3 (Fig. 33C and Fig. 33H), Ki-67 (Fig. 33D and Fig. 331), and CK14 (Fig. 33E and Fig. 33J). All pictures are at 20X magnification.

[00296] Figs. 34A-P present analysis of cryopreserved cultured thymus tissue from an 8-month- old non-human primate (NHP) after 12 days of culture. The top row is cytokeratin at harvest (Fig. 34A), day 6 of culture (Fig. 34B), day 12 of culture (Fig. 34C), and after 12 days of culture followed by 35 days cryopreservation then thawing for the photo (Fig. 34D). The cytokeratin (AE1 / AE3) in Fig. 34D resembles the cytokeratin in Fig. 34C. The second row shows CK14 staining with the same time points in Fig. 34E (harvest), Fig. 34F (day 6 of culture), Fig. 34G (day 12 of culture) and Fig. 34H (after 12 days of culture followed by 35 days of cryopreservation then thawing), respectively. The CK14 in Fig. 34H is very similar to that in panel Fig. 34G. The third row shows CD3 staining in Fig. 341, Fig. 34J, Fig. 34K and Fig. 34L at the same time points, which has the expected loss of viable T cells through time. Panel Fig. 34L is similar to panel Fig. 34K in having very few T cells. The fourth row Fig. 34M, Fig. 34N, Fig. 340 and Fig. 34P shows Ki-67 staining of proliferating T cells at the same time points. Staining with Ki-67 is absent by day 6 (Fig. 34N), as the T cells have mainly died. This figure shows the ability to cryopreserve non-human primate thymus similarly to how cultured thymus tissue will be cryopreserved for patients. All pictures are at 40X magnification.

[00297] Fig. 35 presents a schematic diagram of the experimental transplantation strategy using maximally MHC-mismatched CM V-free rhesus macaques. Recipient animals (Y) undergo complete thymectomy. Donor animals (X) donate both cultured thymic tissue and a heart placed in a heterotopic position into recipient Y (first Tx and second Tx). Immunosuppressive drugs are then withdrawn and donor-specific tolerance is demonstrated by 1) continued beating of the donor heart, ii) tolerance to the donor in nMLR with reactivity against third party and iii) rejection of skin transplant from 3™ party donor animal Z (third Tx).

[00298] Fig. 36 shows graphs of results from flow cytometry experiments that describe the general gating strategy for identifying recent thymic emigrants (RTEs). Nonhuman primate peripheral blood mononuclear cells are collected and analyzed using polychromatic flow cytometry. The first step is identification of single cells in the first panel top row. The “Singlets” are used to identify the lymphocytes (low SSC, side scatter, and high CD45) in the second panel in top row. The CD3 T cells in the lymphocytes are identified in the 3rd panel, in top row. The CD4 and CDS cells are gated off the CD3 cells as shown in the 4th panel of the top row. In the bottom row, 1st panel, CD28 and CD95 are used to identify, using the CD4 gate, the CD4+ naive subset, the central memory subset and the effector memory subset. In the 2nd panel of the bottom row, CD31 is used to show the percentage of CD4+ naive cells that are RTEs. The 3rd and fourth panels on the bottom row show the same approach to RTEs but for CDS naive T cells. As can be seen, the RTEs are 99.6% and 95.2% of the CD4 and CD8 naive subsets.

[00299] Fig. 37 presents photomicrographic images and a graph of CCL21 assessment in cultured infant thymus. CCL21 is produced at high levels by cultured infant thymus. Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on day 16 of culture is shown in the left panels (upper left panel, 2X magnification; lower left panel, 20X magnification). A corresponding time course measuring daily CCL21 secretion into culture media is shown on the right for 3 infant thymus cultures (R&D Systems Duo-Set ELISA). Thus, the cultured thymus tissue can produce a functionally important biomolecule, CCL21, the chemokine responsible for attracting immature thymocyte precursors to the thymus.

[00300] Fig. 38 presents an outline of the experimental design of Example 8 directed to an assessment of successful engraftment of cultured thymic tissue followed by tolerance to matched heart and rejection of unmatched skin in a CMV-free NHP model. In particular, after thymectomy of the recipient (week 1 of Stage 2) and confirmation of complete thymectomy, the recipient is T cell depleted and started on immunosuppression with tacrolimus. Unmatched cultured donor thymus tissue from an unrelated NHP is engrafted into the recipient at week 3 of Stage 3. A biopsy is done of the thymus graft at week 10 of Stage 3 to evaluate for thymopoiesis. After naive T cells develop a few months later, the recipient should be tolerant to the donor. The recipient is then given a heterotopic heart transplant from the thymus donor (Week 4 of Stage 4). Immunosuppression is weaned off. The beating of the heart is followed (demonstrating tolerance). And a mixed lymphocyte reaction at week 10 of Stage 4 in done to show tolerance to cryopreserved donor cells and rejection of third party cells. Stage 5 is used if more time is needed for naive T cells to develop. Stage 6 is used if tolerance didn’t develop. Recipient thymus would be transplanted into the recipient NHP to prove that the thymus tissue transplant procedure is working in the NHP. It is helpful to know how Fig.38 was formatted. The experiment has 3 monkeys. Please note that the original spreadsheet had the procedures for all three animals in a document one page wide by many pages long. Because the spread sheet was wider than the width allowed in this patent application, each row of the spread sheet was divided into 3 pages. The first monkey is the thymus and heart donor; procedures on this monkey are in the left hand columns on the 1st, 4th, 7th etc pages. The second monkey is the thymus and heart recipient; information in the middle columns are the 2nd, 5th, 8th etc pages). The third monkey is the control; information in the right hand columns are on the 31, 6th, gth etc pages).

[00301] Fig. 39 presents an outline of the experimental design of Example 9 which is the same as that in Example 8 except that an additional immunosuppression medication is added, mycophenylate mofetil (MMF). The drug MMF is used routinely in heart transplantation. This study will assess if there is any detrimental effect of MMF on the cultured thymus tissue transplant.

[00302] Figs. 40A-D presents photomicrographs of slices of fresh thymus, dO of culture, showing thymic architecture. In Figs. 40A-B, hematoxylin and eosin (H&E) staining shows well-defined cortical and lighter-staining medullary areas, as expected for normal pediatric thymus. Fig. 40C shows immunohistochemistry with a cocktail of pan-cytokeratin antibodies (AE1 / AE3) that together detect all types of epithelial cells demonstrates that thymic epithelial cells are present beneath the capsule and in a light lacy network in both cortex and medulla (brown staining shows positive antibody reaction). Arrows in Fig. 40B and Fig. 40C point to a Hassall body. Fig. 40D shows Cytokeratin 14 (CK 14) antibody staining (brown). CK14 antibody reacts with thymic epithelial cells in the sub-capsular cortex and in the medulla, as well as with scattered thymic epithelial cells in the cortex. The dotted line highlights an area of medulla that is surrounded by cortex. SCC denotes sub-capsular cortex, Cor denotes cortex, and M denotes medulla. Scale bar in Fig. 40A represents 1 mm; scale bars in Figs. 40B-D represent 500 um.

[00303] Figs. 41A-D present photomicrographs showing examples of Hassall bodies in cultured thymic slices. The histologic appearance of Hassall bodies is shown on day 0 (Figs. 41A-B) and day 9 (Figs. 41C-D) of culture. Fig. 41A and Fig. 41C show hematoxylin and eosin (H&E) staining; Fig. 41B and Fig. 41D show reactivity with pan-cytokeratin (AE1 / AE3) antibodies (brown color indicates a positive reaction). Arrowheads in Figs. 41A-D point out representative Hassall bodies, which appear less prominent on H&E-stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall bodies can still be readily identified by careful examination or by using immunohistochemistry. Scale bar in Figs. 41A-D represents 100 um,

[00304] Figs. 42A-D presents photomicrographs showing the architecture of cultured thymus, day 7. Hematoxylin and eosin (H&E) staining in Figs. 42A-B shows marked depletion of thymocytes, although some cortical areas (Cor) still contain large numbers of thymocytes with retained nuclei. Pan-cytokeratin (AE1 / AE3) in Fig. 42C and cytokeratin 14 (CK14) immunohistochemistry in Fig. 42D show condensation of the thymic epithelium in the subcapsular cortex (SCC) and in the medulla (M). Brown color in Figs. 42C-D indicates a positive reaction with antibody. Scale bar represents 1 mm in Fig. 42A and 500 pum in Figs. 42B-D.

[00305] Figs. 43A-D present photomicrographs showing the architecture of cultured thymus, day 9. Figs. 43A-B show hematoxylin and eosin (H&E) staining. Few if any live T cells or thymic epithelial cells are present in the pale-staining area in Fig. 43A that is enclosed by the dotted line, which is almost completely necrotic (Necr). Most nuclei formerly present in this region have been degraded via karyolysis. Other areas where the nuclei from residual thymocytes have not been completely degraded continue to stain dark blue with hematoxylin. Arrow in Fig. 43B points to a Hassall body. Fig. 43C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown); Fig. 43D shows cytokeratin 14 (CK 14) immunoreactivity (brown). Scale bar represents 1 mm in Fig. 43A and 500 um in Figs. 43B-D.

[00306] Figs. 44A-D presents photomicrographs showing the architecture of cultured thymus, day 12. Figs. 44A-B show hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Some areas retain architecture characteristic of normal uncultured thymus with cortical-like areas (Cor) that stain more basophilic (blue) and medullary-like areas (M), although with greatly decreased thymocyte cellularity. Fig. 44C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown); Fig. 44D shows cytokeratin 14 (CK 14) immunoreactivity (brown). At this time point, the sub-capsular cortex (SCC) has thickened and epithelial cells appear more prominent due to the decreased numbers of thymocytes present. Arrows point to representative Hassall bodies. Bar represents 1 mm in Fig. 44A and 500 um in Figs. 44B-D.

[00307] Figs. 45A-D presents photomicrographs showing the architecture of cultured thymus, day 20. Figs. 45A-B shows hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Large groups of residual thymocytes are rare, although scattered cells with nuclear characteristics of thymocytes are evident. Fig. 45C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown). Much of the epithelium present in formerly medullary areas (M) is condensed due to loss of medullary thymocytes, but scattered epithelial cells indicative of a residual light, lacy, three-dimensional network of thymic epithelial cells remain. In Fig. 45D, cytokeratin 14 (CK 14) immunohistochemistry (brown) highlights former medullary areas and the subcapsular cortex. Arrows point to representative Hassall bodies. Scale bar represents 1 mm in Fig. 45A and 500 um in Figs. 45B-D.

[00308] Figs. 46A-B present photomicrographs showing examples of intact nuclei in thymus slices. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Fig. 46A) and in the medulla on day 21 (Fig. 46B). Hematoxylin and eosin stain; scale bar represents 50 um.

[00309] Figs. 47A-E present photomicrographs showing a comparison of the thymic epithelial network of cultured thymus tissue at different time points. Fig. 47A shows day 0, Fig. 47B shows day 5, Fig. 47C shows day 9, Fig. 47D shows day 12, and Fig. 47E shows day 21. Although there are time point-related differences in thymocyte depletion and the amount of necrosis such that the tissue become less basophilic (blue) with time, the structure of the thymic epithelial network (brown) remains intact as the culture progresses. Both cortical and medullary epithelium may condense as intervening thymocytes are depleted. Brown color indicates a positive reaction with a cocktail of anti-cytokeratin antibodies (AE1 / AE3); hematoxylin counterstain. Scale bar represents 400 um.

[00310] Figs. 48A-K presents photomicrographs showing examples of CD3 immunohistochemistry in thymus slices as a function of time in culture. Figs. 48A-B shows that on day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with CD3 antibody. Higher magnification (Fig. 48B) shows pale blue nuclei surrounded by a ring of brown immunoreactivity, consistent with membrane expression of CD3. In Figs. 48C-E, tissue still shows extensive reactivity with CD3 antibody on day 7. However, Figs. 48D-E shows that when viewed under higher magnification, the majority of the immunoreaction (brown) is associated with debris from dead thymocytes, as most brown foci lack evidence of nuclei (Fig. 48D). Small foci of cells that demonstrate intact nuclei and membrane staining (arrows) can still be identified in areas away from the debris (Fig. 49E). As cultures progress through day 9 (Figs. 48F-G), day 12 (Figs. 48H-I), and day 21 (Figs. 48J-K), reactivity with thymocyte cellular debris remains strong, making it difficult to reliably detect potentially intact cells amidst the debris. The slices shown are all from a single lot that is representative of multiple lots examined at these time points. Scale bar represents 500 um in Fig. 48A, Fig. 48C, Fig. 48F, Fig. 48H, and Fig. 487, and 50 um in Fig. 48B, Fig. 48D, Fig. 48E, Fig. 48G, Fig, 481, and Fig, 48K.

[00311] Figs. 49A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points. Figs. 49A-B shows that on day 0, the nuclei of the majority of immature T cells in the cortex (Cor) react strongly with antibody specific for Ki-67. Higher magnification (Fig. 49B) shows strong positive reactivity with the nuclei of cortical thymocytes (brown), whereas only rare lymphocytes in the medulla (M) react with Ki-67 antibody. Figs. 49C-E show that by day 7, the nuclei of most thymocytes that remain in cortical areas are small with indistinct nuclear borders consistent with apoptosis, and they fail to react with Ki-67-specific antibody. The cells that react with antibody (Fig. 49E, arrows) have larger nuclei, suggesting that they are thymic epithelial cells. A similar lack of Ki-67 labeling of residual thymocyte nuclei is seen on days 9 (Figs. 49F- G), 12 (Figs. 49H-I) and 21 (Figs. 49J-K). Scale bar represents 500 um in Fig. 49A, Fig. 49C, Fig. 49E, Fig. 49G, and Fig. 491 and 50 pm in Fig. 49B, Fig. 49D, Fig 49F, Fig. 4950H, and Fig. 49]

[00312] Figs. 50A to Fig. S0H shows plots of selected soluble molecules detected in conditioned media from human thymus organ cultures. Fig. 50A is a plot of L-selectin in Ln / pg versus days in culture; Fig. 50B is a plot of M-CSF in Ln / pg versus days in culture; Fig. 50C is a plot of galectin-7 in Ln / pg versus days in culture; Fig. 1D is a plot of IL-16 in Ln / pg versus days in culture; Fig. SOE is a plot of CCL21 in Ln / pg versus days in culture; Fig. SOF is a plot of CXCL16 in Ln / pg versus days in culture; Fig. 50G is a plot of CCL11 in Ln / pg versus days in culture; and Fig. 50H is a plot of CXCL12 in Ln / pg versus days in culture.

[00313] Fig. 51 shows plots assessing thymocyte content in cultured slices of human thymus in pg / ml from days 1 to 21 of the culturing process.

[00314] Fig. 52 shows photos of the immunohistochemical assessment of viable thymocytes in cultured slices of human thymus tissue as a function of time. Fig. 52A is a photo of the immunohistochemistry with anti-CD3 antibodies that identify cells as T lineage and with anti-Ki- 67 antibody that identifies proliferating cells, demonstrates the rapid loss of thymocyte viability early in culture. Fig. 52B is a photo on day 0 depicting the plasma membranes of essentially all immature T cells in the cortex and the medulla appear strongly reactive with anti-CD3 in a membrane pattern. Fig. 52C depicts immunohistochemistry using antibody specific for the Ki- 67 proliferation marker shows abundant reactivity with cortical thymocytes on day. Fig. 52D shows histology of cultured thymus on day 9, using hematoxylin and eosin stain. The decreased basophila (blue color) is indicative of loss of donor thymocytes during culture; Fig. 52E depicts after several days of culture, the majority of the brown color is due to the anucleate, but still immunoreactive, debris that remains after dead thymocytes undergo karyolysis / nuclear dissolution. Fig. 52F depicts thymocyte death that occurs during organ culture results in Ki-67 immunoreactivity only with larger cells morphologically consistent with TE cells at later time points during culture.

[00315] Fig. 53A shows a plot of CCL21 levels in pg / ml of conditioned media from cultured thymus tissue versus the number of days in culture. Fig. 53B is an illustration of the slicing procedure for thymus tissue to be subjected to conditioning. Fig. 53C is a plot of the secretion of CCL21by slice of thymus organ tissue cultures as a function of time.

[00316] Figs. 54A to Fig. 54D are photographs depicting the immunoreactivity in cultured and non-cultured thymus tissue. Fig. 54A is a photograph of the medullary region, but also includes TECS scattered throughout the cortex at Day 0 of culturing; Fig. 54B is as photograph of the medullary region, but also includes TECS scattered throughout the image at Day 16 of culturing; Fig. 54C is a photograph of TECs in medullary regions as well as in scattered TECs in cortical areas on Day 0 of culturing. Fig. 54D is a photograph of TECs in medullary regions as well as in scattered TECs in cortical areas on Day 16 of culturing.

[00317] Figs. 55A to Fig. 55F are plots of the expression of selected mRNAs in thymus tissue from across the lifespan. The relative amounts of the target mRNAs present in FFPE sections of thymus tissue were quantitated using the QuantiGene assay (Thermo Fisher) in accordance with the manufacturer’s directions. Data for each target mRNA is presented normalized to GAPDH (“Not adjusted”), then further normalized to the % area containing thymic epithelium (“Adjusted by TE”) or to the % area containing CD1a-positive cortical thymocytes (“Adjusted by Cor”). Data shown was obtained from 47 samples of thymus, derived from donors ranging in age from 5 days to 78 years. Fig. 55A and 55B are thymus tissues obtained from donors < 18 years (n = 25) showed higher expression of mRNAs encoding the T cell marker CD3e and the cortical thymocyte marker CD1a (Fig. 55A, B) relative to GAPDH when compared to donors older than 18 years, respectively. Fig. 55C and 55D are photos depicting mRNAs encoding cytokeratins 8 (KRT8) and 14 (KRT14) were decreased relative to GAPDH in donors < 18 years compared with older adults (Fig. 55C, D), respectively. Fig. 55E is a photo depicting unadjusted CCL21 gene expression which was consistently low relative to GAPDH in thymus from donors < 18 years of age. Fig. 55F is a photo depicting unadjusted CXCL21 gene expression which was consistently low relative to GAPDH in thymus from donors < 18 years of age.

[00318] Fig. 56 is a table of proteins present in spent media of thymic organ cultures, as determined by multiplex antibody arrays.

[00319] Figs. 57A to 57C are photographs of morphological measurements of thymus tissues. Areas included in each measurement were outlined using the “pen tool” provided by the ImageScope software (Aperio Technologies, Leica Biosystems imaging, Inc.) in accordance with the manufacturer’s instructions. Fig. 57A shows the total area of thymus tissue on the H&E- stained slide outlined in green. The proportion of this area that contains lymphocytes is further outlined in aqua. Fig. 57B is a photo showing areas containing thymic epithelium (“TE area”) outlined in yellow on the section reacted with AE1 / AE3 cocktail to identify pan-cytokeratins. Fig. 57C is a photo showing Areas containing immature thymocytes (“cortical area”) outlined in red on the section reacted with CD1a antibody. The thymus shown was derived from a 32 year old female at the time of aortic valve replacement surgery. Scale bar = 4 mm for each panel. In panels of Fig. 57B and 57C, brown color indicates a positive reaction with antibody.

[00320] Figs. 58A to 58C are photographs depicting the few viable thymocytes in thymus tissue slices cultured for 21 days. Fig. 58A is a photo depicting thymus slices containing few intact thymocytes on day 21 of culture, as indicated by the marked lack of basophilia (blue color) in H&E sections. Fig. 58B is a photo depicting cultured thymus tissue slices in which most of the strong brown immunoreaction seen with CD3 immunohistochemistry is associated with anucleate cellular debris, although dead thymocytes that exhibit nuclear and cytoplasmic staining characteristic of necrotic cells that have not yet undergone karyolysis (inset) are not uncommon. Fig. 58C is a photo depicting Ki-67 immunoreactivity on day 21 is limited to cells with larger nuclei that are characteristic of thymic epithelial cells. Bar represents 300 um in the main panels and 50 um in the insets.

[00321] Figs. 59A to 59C are graphs showing characteristics of human thymus tissues used for gene expression analysis. Fig. 59A depicts the age and sex distribution of the thymus tissues studied, with the lower black-filled circles designating females, upper open circles designating males, and the middle gray circle designating the one donor of unknown sex. Fig. 59B plots the % area containing thymic epithelial cells as a function of age for this panel of thymus tissues. Fig, 59C plots the % area with active thymopoiesis as defined by CD1a-positive thymocytes as a function of age for this panel of thymus tissues.

[00322] Fig. 60 is a photograph of freshly harvested thymus tissue.

[00323] Figs. 61A and 61B depict the histology of thymus tissue slides after exposure to forced degradation conditions of 10X PBS. Fig. 61A depicts the cortex at day 9 after exposure to forced degradation conditions. Fig. 61B depicts the cortex at day 21 after exposure to forced degradation conditions. In Fig. 61A the smear of blue is DNA released from cells. The majority of cells show evidence of degradation although small foci of cells with intact nuclei can be identified. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00324] Fig. 62A shows a schematic showing the slicing of thymus tissue for characterization testing, as discussed in section

[00520] . Fig.62B is a figure showing slices of thymus tissue on cellulose filters on surgical sponges in a tissue culture dish as is used for culture of the thymus.

[00325] Figs. 63A to 63H depict histology testing of thymus tissue slices from a lot (MFG-056) of cultured thymus tissue on day 5, 9, 12 and 21 after harvest of the thymus from a donor. Hematoxylin and eosin-stained slices (left panels) and their corresponding reactivity with a cocktail of the anti-cytokeratin antibodies AE1 / AE3 (right panels; brown color denotes positive reactivity) are shown at day 5 (Fig. 63A, Fig. 63B), day 9 (Fig. 63C, Fig. 63D), day 12 (Fig. 63E, Fig. 63F), and day 21 (Fig 63G, Fig. 63H), respectively. Bars in the lower left of each panel represent 100 pm. Panels with H&E show progression depletion of T cells with time. Fig. 14E and Fig. 63F are predominantly epithelial cells. Condensation of the epithelium of the subcapsular cortex occurs as the thymocytes are depleted with time. Similar condensation occurs in medullary areas of the thymus. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00326] Figs. 64A and 64B depict the histology of thymus tissue slices on day 0 of the time course in a scale of 5 mm (Fig. 64A) and 100 um (Fig. 64B), respectively. This shows the thymus and thymocytes at low power (bar 5 mm) and high power (bar 100 um) on day 0. This is normal thymus. At this time the cortex and medulla both have large numbers of thymocytes with dark blue nuclei contributing to the overall dark blue appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00327] Figs. 65A and 65B are images from H&E stained slide that depict the histology of thymus tissue slices on day 5 of the time course in a scale of 5 mm (Fig. 65A) and 100 um (Fig. 65B), respectively. Progression of depletion of the thymocytes results in a more eosinophilic (pink) appearance of the tissue. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00328] Figs. 66A and Fig. 66B depict H&E staining of the thymus tissue slices on day 12 of the time course in a scale of 5 mm (Fig. 66A) and 100 um (Fig. 66B), respectively. We see progressive depletion of thymocytes. The higher magnification shows numerous eosinophilic cell bodies lacking nuclei which are diagnostic of necrotic cells that have undergone karyolysis (dissolution of the nuclei). This degree of necrosis is expected at this time in culture. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00329] Fig. 67A and Fig. 67B depict H&E staining of thymus tissue slices on day 21 of the time course in a scale of 5 mm (Fig. 67A) and 100 um (Fig. 67B), respectively. Note the preservation of the overall architecture of the tissue including in Fig. 67B the subcapsular cortex, cortical region and medullary region containing numerous Hassall bodies. The small dark cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00330] Figs. 68A-E depict representative thymus slices which were immuno-stained with a cocktail of anti-cytokeratin antibodies (AE1 / AE3). Fig. 68A. Day 0; Fig. 68B. Day 5; Fig. 68C. Day 9; Fig. 68D. Day 12; and Fig. 68E. Day 21. The structure of the thymic epithelial network remains intact as the culture progresses. Bar represents 400 um. Photo by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[00331] Fig. 69 presents photomicrographic images and a graph of CCL21 assessment in cultured infant thymus. CCL21 is produced at high levels by cultured infant thymus. Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on day 16 of culture is shown in the left panels (upper left panel, 2X magnification; lower left panel, 20X magnification). A corresponding time course measuring daily CCL21 secretion into culture media is shown on the right for 3 infant thymus cultures (R&D Systems Duo-Set ELISA). Thus, the cultured thymus tissue can produce a functionally important biomolecule, CCL21, the chemokine responsible for attracting immature thymocyte precursors to the thymus.

[00332] Figs. 70A to 70D presents photomicrographs of slices of fresh thymus, dO of culture, showing thymic architecture. In Figs. 70A-B, hematoxylin and eosin (H&E) staining shows well-defined cortical and lighter-staining medullary areas, as expected for normal pediatric thymus. Fig. 70C shows immunohistochemistry with a cocktail of pan-cytokeratin antibodies (AE1 / AE3) that together detect all types of epithelial cells demonstrates that thymic epithelial cells are present beneath the capsule and in a light lacy network in both cortex and medulla (brown staining shows positive antibody reaction). Arrows in Fig. 70B and Fig. 70C point to a Hassall body. Fig. 70D shows Cytokeratin 14 (CK14) antibody staining (brown). CK14 antibody reacts with thymic epithelial cells in the sub-capsular cortex and in the medulla, as well as with scattered thymic epithelial cells in the cortex. The dotted line highlights an area of medulla that is surrounded by cortex. SCC denotes sub-capsular cortex, Cor denotes cortex, and M denotes medulla. Scale bar in Fig. 70A represents 1 mm; scale bars in Figs. 70B-D represent 500 um.

[00333] Figs. 71A to 71D present photomicrographs showing examples of Hassall bodies in cultured thymic slices. The histologic appearance of Hassall bodies is shown on day 0 (Figs. 71A-B) and day 9 (Figs. 71C-D) of culture. Fig. 71A and Fig. 71C show hematoxylin and eosin (H&E) staining; Fig. 71B and Fig. 71D show reactivity with pan-cytokeratin (AE1 / AE3) antibodies (brown color indicates a positive reaction). Arrowheads in Figs. 71A-D point out representative Hassall bodies, which appear less prominent on H&E-stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall bodies can still be readily identified by careful examination or by using immunohistochemistry. Scale bar in Figs. 71A-D represents 100 um.

[00334] Figs. 72A to 72D presents photomicrographs showing the architecture of cultured thymus, day 7. Hematoxylin and eosin (H&E) staining in Figs. 72A-B shows marked depletion of thymocytes, although some cortical areas (Cor) still contain large numbers of thymocytes with retained nuclei. Pan-cytokeratin (AE1 / AE3) in Fig. 72C and cytokeratin 14 (CK14) immunohistochemistry in Fig. 72D show condensation of the thymic epithelium in the subcapsular cortex (SCC) and in the medulla (M). Brown color in Figs. 72C-D indicates a positive reaction with antibody. Scale bar represents 1 mm in Fig. 23 and 500 um in Figs. 72B- D.

[00335] Figs. 73A to 73D present photomicrographs showing the architecture of cultured thymus, day 9. Figs. 73A-B show hematoxylin and eosin (H&E) staining. Few if any live T cells or thymic epithelial cells are present in the pale-staining area in Fig. 73A that is enclosed by the dotted line, which is almost completely necrotic (Necr). Most nuclei formerly present in this region have been degraded via karyolysis. Other areas where the nuclei from residual thymocytes have not been completely degraded continue to stain dark blue with hematoxylin. Arrow in Fig. 73B points to a Hassall body. Fig. 73C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown); Fig. 73D shows cytokeratin 14 (CK 14) immunoreactivity (brown). Scale bar represents 1 mm in Fig. 73A and 500 um in Figs. 73B-D.

[00336] Figs. 74A to 74D presents photomicrographs showing the architecture of cultured thymus, day 12. Figs. 74A-B show hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Some areas retain architecture characteristic of normal uncultured thymus with cortical-like areas (Cor) that stain more basophilic (blue) and medullary-like areas (M), although with greatly decreased thymocyte cellularity. Fig. 74C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown); Fig. 74D shows cytokeratin 14 (CK 14) immunoreactivity (brown). At this time point, the sub-capsular cortex (SCC) has thickened and epithelial cells appear more prominent due to the decreased numbers of thymocytes present. Arrows point to representative Hassall bodies. Bar represents 1 mm in Fig. 74A and 500 um in Figs. 74B-D.

[00337] Figs. 75A-B shows hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have either been lost from the tissue or have died and their nuclei have been dissolved, making the tissue more eosinophilic (pink). Large groups of residual thymocytes are rare, although scattered cells with nuclear characteristics of thymocytes are evident. Fig. 75C shows pan-cytokeratin (AE1 / AE3) immunoreactivity (brown). Much of the epithelium present in formerly medullary areas (M) is condensed due to loss of medullary thymocytes, but scattered epithelial cells indicative of a residual light, lacy, three-dimensional network of thymic epithelial cells remain. In Fig. 75D, cytokeratin 14 (CK 14) immunochistochemistry (brown) highlights former medullary areas and the subcapsular cortex. Arrows point to representative Hassall bodies. Scale bar represents 1 mm in Fig. 75A and 500 um in Figs. 75B-D.

[00338] Figs. 76A-B present photomicrographs showing examples of intact nuclei in thymus slices. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Fig. 76A) and in the medulla on day 21 (Fig. 76B). Hematoxylin and eosin stain; scale bar represents 50 um.

[00339] Figs. 77A to 77E present photomicrographs showing a comparison of the thymic epithelial network of cultured thymus tissue at different time points. Fig. 77A shows day 0, Fig. 77B shows day 5, Fig. 77C shows day 9, Fig. 77D shows day 12, and Fig. 77E shows day 21. Although there are time point-related differences in thymocyte depletion and the amount of necrosis such that the tissue become less basophilic (blue) with time, the structure of the thymic epithelial network (brown) remains intact as the culture progresses. Both cortical and medullary epithelium may condense as intervening thymocytes are depleted. Brown color indicates a positive reaction with a cocktail of anti-cytokeratin antibodies (AE1 / AE3); hematoxylin counterstain. Scale bar represents 400 um.

[00340] Figs. 78A-K presents photomicrographs showing examples of CD3 immunohistochemistry in thymus slices as a function of time in culture. Figs. 78A-B shows that on day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with CD3 antibody. Higher magnification (Fig. 78B) shows pale blue nuclei surrounded by a ring of brown immunoreactivity, consistent with membrane expression of CD3. In Figs. 78C-E, tissue still shows extensive reactivity with CD3 antibody on day 7. However, Figs. 78D-E shows that when viewed under higher magnification, the majority of the immunoreaction (brown) is associated with debris from dead thymocytes, as most brown foci lack evidence of nuclei (Fig. 78D). Small foci of cells that demonstrate intact nuclei and membrane staining (arrows) can still be identified in areas away from the debris (Fig. 78E). As cultures progress through day 9 (Figs. 78F-G), day 12 (Figs. 78H-I), and day 21 (Figs. 78J-K), reactivity with thymocyte cellular debris remains strong, making it difficult to reliably detect potentially intact cells amidst the debris. The slices shown are all from a single lot that is representative of multiple lots examined at these time points. Scale bar represents 500 um in Fig. 784A, Fig. 78C, Fig. 78F, Fig. 78H, and Fig. 78], and 50 um in Fig, 78B, Fig, 78D, Fig. TE, Fig. 78G, Fig. 78L and Fig. 78K.

[00341] Figs. 79A-K presents photomicrographs showing examples of Ki-67 immunohistochemistry in thymus slices as a function of time in culture. The slices shown are all from a single lot that is representative of multiple lots examined at similar time points. Figs. 79A-B shows that on day 0, the nuclei of the majority of immature T cells in the cortex (Cor) react strongly with antibody specific for Ki-67. Higher magnification (Fig. 79B) shows strong positive reactivity with the nuclei of cortical thymocytes (brown), whereas only rare lymphocytes in the medulla (M) react with Ki-67 antibody. Figs. 79C-E show that by day 7, the nuclei of most thymocytes that remain in cortical areas are small with indistinct nuclear borders consistent with apoptosis, and they fail to react with Ki-67-specific antibody. The cells that react with antibody (Fig. 79E, arrows) have larger nuclei, suggesting that they are thymic epithelial cells. A similar lack of Ki-67 labeling of residual thymocyte nuclei is seen on days 9 (Figs. 79F- G), 12 (Figs. 79H-I) and 21 (Figs. 79J-K). Scale bar represents 500 pm in Fig. 794, Fig. 79C, Fig. 79E, Fig. 79G, and Fig. 791 and 50 um in Fig. 79B, Fig. 79D, Fig. 79F, Fig. 79H, and Fig. 797.

[00342] Fig 80 is a plot of uPAR detected in conditioned media from human thymus organ cultures.

[00343] Fig. 81 is a plot of OPN detected in conditioned media from human thymus organ cultures.

[00344] Fig. 82 is a plot of MIP3a detected in conditioned media from human thymus organ cultures.

[00345] Fig. 83 is a plot of IGFBP-1 detected in conditioned media from human thymus organ cultures.

[00346] Fig. 84 is a plot of MIF detected in conditioned media from human thymus organ cultures.

[00347] Fig. 85 is a scatterplot of CCL21 Concentration in Spent Media vs. Day.

[00348] Fig. 86 is a boxplot of CCL21 Concentration in Spent Media vs. Day.

[00349] Fig. 87 is a graph of Figure 4: CCL21 Levels (pg / mL) in Forced Degradation Study — Lot MFG-053 and Lot-054.

[00350] Fig. 88 is a graph of CCL21 Levels (pg / mL) in Forced Degradation Study — Lot MFG- 066.

[00351] Fig. 89 is a scatterplot of CXCL16 Concentration in Spent Media vs. Day.

[00352] Fig. 90 is a boxplot of CXCL16 Concentration in Spent Media vs. Day.

[00353] Fig. 91 is a scatterplot of CXCL16 Levels (pg / mL) in Forced Degradation Study — Lot MFG-053 and Lot MFG-054.

[00354] Fig. 92 is a scatterplot of CXCL21 Levels in Forced Degradation Study — Lot MFG- 066.

[00355] Fig. 93 is a scatterplot of L-Selectin Concentration in Spent Media vs. Day.

[00356] Fig. 94 is a boxplot of L-Selectin Concentration in Spent Media vs. Day.

[00357] Fig. 95 is a scatterplot of L-Selectin Levels (pg / mL) in Forced Degradation Study — Lot MFG-053 and Lot MFG-054.

[00358] Fig. 96 is a scatterplot of L-Selectin Levels in Forced Degradation Study — Lot MFG- 066.

[00359] Fig. 97 is a scatterplot of uPAR Concentration in Spent Media vs. Day.

[00360] Fig. 98 is a boxplot of uPAR Concentration in Spent Media vs. Day.

[00361] Fig. 99 is a scatterplot of uPAR Levels (pg / mL) in Forced Degradation Study — Lot MFG-053 and Lot MFG-054.

[00362] Fig. 100 is a scatterplot of uPAR Levels in Forced Degradation Study — Lot MFG-066.

[00363] Fig. 101 is a scatterplot of CXCL16 vs. CCL21.

[00364] Fig. 102 is a quadratic regression model of CCL21 vs. uPAR.

[00365] Fig. 103 is a quadratic regression model of CXCL16 vs. uPAR.

[00366] Fig. 104 is a scatterplot of CCL11 Concentration in Spent Media vs. Day.

[00367] Fig. 105 is a boxplot of CCL11 Concentration in Spendt Media vs. Day.

[00368] Fig. 106 is a linear regression model of CCL11 Concentration in Spent Media vs. Day.

[00369] Fig. 107 is a plot of CCL11 Levels (pg / mL) in Forced Degradation Study — Lot MFG- 053 and Lot MFG-054.

[00370] Fig. 108 is alot of CCL11 Levels in Forced Degradation Study-Lot MFG-066.

[00371] Fig. 109 is a scatterplot of OPN Concentration in Spent Media vs. Day.

[00372] Fig. 110 is a boxplot of OPN Concentration in spent Media vs. Day.

[00373] Fig. 111 is a quadratic regression model of OPN Concentration in Spent Media vs. Day.

[00374] Fig. 112 is a plot of OPN Levels (pg / mL) in Forced Degradation Study-Lot MFG-053 and Lot MFC-054.

[00375] Fig. 113 is a plot of OPN Levels in Forced Degradation Study-Lot MFG-066.

[00376] Fig. 114 is a scatterplot of CXCL12 Concentration in Spent Media vs. Day.

[00377] Fig. 115 is a boxplot of CXCL12 Concentration in Spent Media vs. Day.

[00378] Fig. 116 is a fitted line plot of CXCL12 Concentration in Spent Media vs. Day.

[00379] Fig. 117 is a scatterplot of CXCL12 Levels (pg / mL) in Forced Degradation Study-Lot MFG-053 and Lot MFG-054.

[00380] Fig. 118 is a scatterplot of CXCL12 Levels in Forced Degradation Study—Lot MFG- 066.

[00381] Fig. 119 is a scatterplot of CCL20 Concentration in Spent Media vs. Day.

[00382] Fig. 120 is a boxplot of CCL20 Concentration in Spent Media vs. Day.

[00383] Fig. 121 is a cubic regression model of CCL20 Concentration in Spent media vs. Day.

[00384] Fig. 122 is a scatterplot of CCL20 Levels (pg / mL) in Forced Degradation Study-Lot MFG-053 and Lot MFG-054.

[00385] Fig. 123 is a scatterplot of CCL20 Levels in Forced Degradation Study-Lot MFG-066.

[00386] Fig. 124 is a scatterplot of IL-16 Concentration in Spent Media vs. Day.

[00387] Fig. 125 is a boxplot of IL-16 Concentration in Spent Media vs. Day.

[00388] Fig. 126 is a fitted line plot of IL-16 Concentration in Spent Media vs. Day.

[00389] Fig. 127 is a scatterplot of IL-16 Levels (pg / mL) in Forced Degradation Study — Lot MFG-053 and Lot MFG-054.

[00390] Fig. 128 is a scatterplot of IL-16 Levels in Forced Degradation Study-Lot MFG-066.

[00391] Fig. 129 is a scatterplot of IGFBP-1 Concentration in Spent Media vs. Day.

[00392] Fig. 130 is a boxplot of IGFBP-1 Concentration in Spent Media vs. Day.

[00393] Fig. 131 is a fitted line plot of IGFBP-1 Concentration in Spent Media vs. Day.

[00394] Fig. 132 is a scatterplot of IGFBP-1 Levels (pg / mL) in Forced Degradation Study-Lot MFG-053.

[00395] Fig. 133 is a scatterplot of OGFBP-1 Levels in Forced Degradation Study-Lot MFG- 066.

[00396] Fig. 134 is a scatterplot of MIF Concentration in Spent Media vs. Day.

[00397] Fig. 135 is a boxplot of MIF Concentration in Spent Media vs. Day.

[00398] Fig. 136 is a linear regression model of MIF Concentration in Spent Media vs. Day.

[00399] Fig. 137 is a scatterplot of MIF Levels (pg / mL) in Forced Degradation Study-Lot- MFG-053 and Lot MFG-054.

[00400] Fig. 138 is a scatterplot of MIF Levels in Forced Degradation Study-Lot MFG-066.

[00401] Fig. 139 is a scatterplot of CCL25 Concentration in Spent Media vs. Day.

[00402] Fig. 140 is a boxplot of CCL25 Concentration in Spent Media vs. Day.

[00403] Fig. 141 is a linear regression model plot of MIF Concentration in Spent Media vs. Day.

[00404] Fig. 142 is a scatterplot of CCL25 Levels (pg / mL) in Forced Degradation Study-Lot- 053 and Lot-054.

[00405] Fig. 143 is a scatterplot of CCL25 Levels in Forced Degradation Study-Lot MFG-066. DETAILED DESCRIPTION OF THE INVENTION

[00406] The titles, headings and subheadings provided herein should not be interpreted as limiting the various aspects of the disclosure. Accordingly, the terms defined below are more fully defined by reference to the specification in its entirety. All references cited herein are incorporated by reference in their entirety.

[00407] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only.

[00408] It is further noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list 1s not to the exclusion of other like items that can be substituted or added to the listed items.

[00409] The instant invention is most clearly understood with reference to the following definitions:

[00410] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of + / - 10%. As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

[00411] As used herein, the term "animal" includes, but is not limited to, humans and non- human vertebrates such as wild, domestic, and farm animals. The animal can also be referred to as a "subject."

[00412] As used herein, the term “biomarkers” refer to the substances listed in Fig. 56. Itis to be further understood that references to “decreased” and “increased” levels with respect to the appearance of the biomarkers in the thymus organ medium refers to increased measurements or decreasaed measurements of the particular biomarker over the time course of the conditioning regimen.

[00413] As used here, "biocompatible" refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal.

[00414] "Chronic transplant rejection" generally occurs in humans within several months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[00415] As used herein, the terms "comprising" (and any form of comprising, such as "comprise", "comprises", and "comprised"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Additionally, a term that is used in conjunction with the term "comprising" is also understood to be able to be used in conjunction with the term "consisting of' or "consisting essentially of."

[00416] As used herein, a "graft" refers to a tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect. The tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", "autologous transplant", "autologous implant" and "autologous graft". A graft from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A graft from an individual to his identical twin is referred to herein as an "isograft", a "syngeneic transplant", a "syngeneic implant" or a "syngeneic graft". A "xenograft", "xenogeneic transplant" or "xenogeneic implant" refers to a graft from one individual to another of a different species.

[00417] As used herein, the term "HLA matched" refers to a donor recipient pair in which none of the HLA antigens are mismatched between the donor and recipient. HLA matching in the methods of the invention comprise: HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRBI1, HLA- DQB1, HLA-DQAI, HLA-DPB1, and HLA-DPAI.

[00418] As used herein, the term "HLA mismatched" refers to matching in a donor and recipient HLA antigens, typically with respect to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQBI, HLA-DQA1, HLA-DPBI, and HLA-DPA1wherein an HLA mismatch between the donor and recipient occurs. In some cases, one haplotype is matched and the other is mismatched. This situation is frequently found with organs from living or deceased donors. An HLA mismatch in donor-recipient pairs results in an increased risk of graft rejection relative to HLA- matched pairs.

[00419] As background to the foregoing definitions, HLA antigens correspond to “human leukocyte antigens,” which are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells. They are also known as “major histocompatibility complex antigens.” Thus the MHC or HLA antigens are target molecules that are recognized by T-cells as being “self” or “non-self.” If the HLA antigens are derived from the same source of hematopoietic stem cells as the immune effector cells they are considered “self.” If, the HLA antigens are derived from another source of hematopoietic reconstituting cells, they are considered “non-self.”

[00420] Two main classes of HLA antigens are recognized: HLA-Class I and HLA-Class II. HLA-Class I antigens (A, B, and C in humans) render each cell recognizable as "self." HLA- Class II antigens (DRB1, DPB1, DPA1, DQBI1, and DQA1 in humans) are involved in reactions between lymphocytes and antigen presenting cells. Both classes of HLA antigens have been implicated as targets of rejection of transplanted organs.

[00421] HLA genes are clustered on human chromosome position 6p21. This cluster of genes encodes the six classical transplantation HLA genes. The segment of 6p21 also encodes genes encoding proteins having important roles in the regulation of the immune system and other fundamental molecular and cellular processes. The complete cluster measures roughly 3.6 Mb, with at least 224 gene loci. As a result of the clustering certain "haplotypes" occur (the set of alleles present on a single chromosome). The haplotypes inherited from one parent tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. HLA matching is used to identify the recipient’s haplotypes and help in identifying suitable matching donors. Certain haplotypes are more prevalent than others and they vary in frequency in different racial and ethnic groups.

[00422] As used herein, the phrase "in need thereof’ means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof.

[00423] As used herein, the phrase "integer from X to Y" means any integer that includes the endpoints. For example, the phrase "integer from X to Y" means 1, 2, 3, 4, or 5.

[00424] As used herein, the term "mammal" means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.

[00425] As used herein the term "organ" refers to a solid vascularized organ that performs a specific function or group of functions within an organism. The term organ includes, but is not limited to heart, lung, kidney, liver, pancreas, skin, uterus, bone, cartilage, small or large bowel, bladder, brain, breast, blood vessels, esophagus, fallopian tube, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limb including upper and lower, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, uterus.

[00426] As used herein, the terms "prevent", "preventing" and "prevention" refer to the administration of therapy to an individual who may ultimately manifest at least one symptom of a disease, disorder, or condition, but who has not yet done so, to reduce the chance that the individual will develop the symptom of the disease, disorder, or condition over a given period of time. Such a reduction may be reflected, for example, in a delayed onset of the at least one symptom of the disease, disorder, or condition in the patient.

[00427] As used herein, the terms "subject," "individual" or "patient," used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.

[00428] As used herein, the phrase "therapeutically effective amount" means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. The therapeutic effect is dependent upon the disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and / or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.

[00429] The term "tissue" as used herein refers to any type of tissue in human or animals, and includes, but is not limited to, vascular tissue, skin tissue, hepatic tissue, pancreatic tissue, neural tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue including bone and cartilage, adipose tissue, connective tissue including tendons and ligaments, amniotic tissue, chorionic tissue, dura, pericardia, muscle tissue, glandular tissue, facial tissue, ophthalmic tissue.

[00430] “Tissue bank” in the context of the present disclosure refers to long-term storage of cryopreserved allogeneic cultured postnatal thymus tissue-derived product stored under liquid nitrogen. General guidance for establishment of a repository for allogeneic cultured postnatal thymus tissue-derived product may be drawn from Guidance for Industry. Current Good Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps) available at https: / / www.fda.gov / downloads / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInfor mation / Guidances / Tissue / UCM285223 pdf.

[00431] "Tissue engineer(ing)(-ed)" refers to the process of generating tissues ex vivo for use in tissue replacement or reconstruction. Tissue engineering is an example of "regenerative medicine" which encompasses approaches to the repair or replacement of tissues and organs by incorporation of cells, genes or other biological building blocks, along with bioengineered materials and technologies.

[00432] The term "transplant rejection” encompasses both acute and chronic transplant rejection. "Acute rejection” is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by infiltration of the transplant tissue by immune cells of the recipient, which carry out their effector function and destroy the transplant tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or suppressed with immunosuppressive drugs such as rapamycin, cyclosporin A, anti- CD40L monoclonal antibody and the like.

[00433] As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic measures wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease.

[00434] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Ranges are approximate and may vary by more than an integer.

[00435] Units, prefixes, and symbols are denoted in their Systéme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Measured values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[00436] It is further appreciated that certain features described herein, which are, for clarity, described in the context of separate embodiments, can also be provided in combination ina single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. Harvesting of Donor Thymus Tissue.

[00437] Donor thymus tissue may be may be discarded during post-natal heart operations and may be used for CTT with informed consent of the donor's family. The removal of some thymus may be necessary to reveal the operative site. Thus, a portion of the thymus may be removed during the heart operation in post-natal heart surgeries, due to the nature of the surgical procedure.

[00438] During postnatal heart surgeries a portion of the thymus tissue may be discarded during the surgical procedure. For all heart surgeries, whether or not the thymus is being screened for transplantation, the surgeon places the thymus tissue that has been discarded into a sterile container.

[00439] Thymus tissue donors for thymus tissue transplantation in infants with complete DiGeorge syndrome have been infants under nine months of age. The drug substance allogeneic cultured postnatal thymus tissue-derived product is manufactured by processing and culturing the discarded thymus tissue as described herein.

[00440] Consent for use of the thymus in cultured thymus tissue implantation may be obtained before or after the thymus is harvested. However, consent allowing blood to be obtained from the infant prior to undergoing bypass is necessary and is always obtained prior to the surgery. This blood sample is used for donor screening.

[00441] The discarded thymus tissue is placed into a sterile container. Routine testing is done on the donor and donor’s birth mother in accordance with FDA guidelines for tissue transplantation. Tissue type matching is not required in the surgical procedures described herein, but such tissue type matching can be performed in certain situations.

[00442] Tissue may be processed immediately or stored refrigerated overnight for next day processing. If the thymus tissue is to be stored overnight, the tissue is aseptically added to thymus organ medium (“TOM medium described below) sufficient to completely cover the thymus tissue in the original container. The container with the thymus is placed in refrigerator until ready to process the next day. Overview of Conditioning of Thymus Tissue

[00443] The conditioning regimen depletes the donor thymocytes from the cultured thymus tissue slices. Based on in vitro data (immunohistochemistry) a culture period between 12 and 21 days preserves the epithelial network as assessed using cytokeratin antibodies. The culturing is preferably done at 37°C in a 5% CO: incubator.

[00444] For successful culture, the thymus tissue is preferably sliced and put on Millipore® cellulose or equivalent filters and placed on surgical sponges in tissue culture dishes. The medium comprises the thymus organ medium (TOM) and is changed daily.

[00445] The thymus on receipt is assessed by pathology. The test for identity must show >50% of areas positive for keratin in lacy staining pattern. The test for potency must show Hassall bodies; it must also show CK14 staining in lacy pattern. The test for viability must show >90% intact nuclei observed in sections. The lot release for the tissue is done on one day between day 5 and day 21 (inclusive) and is performed by pathology. For identity, areas on tissue between days 5 and 21 must be positive for keratin, AE1 / AE3. For potency, the cultured thymus tissue between days 5 and 21 must show cytokeratin CK 14 staining scattered throughout, and there must be at least one Hassall body identified. For viability, the cultured thymus tissue between days 5 and 21 must show intact nuclei.

[00446] In an embodiment, the thymus tissue slices are conditioned for about 12 days and then cryopreserved. In another embodiment all of the thymus tissue slices are conditioned for about 12 days, then about half are implanted in the recipient and the remaining thymus tissue slices are cryopreserved for future use.

[00447] Within 24 hours of harvest, the thymus is cut into thin slices. The slices are held in culture for 12-21 days. This culturing process, as described in detail below, depletes viable donor T cells and ultimately enables the surgically implanted tissue slices to reconstitute the athymic subject’s immune system, albeit at a immunologically effective level, although most subjects will have T cell counts below the 10th percentile for age. The culturing process, as outlined below, significantly modifies the biological characteristics of the donor thymus tissue and constituent cells contained therein in the following manner to optimize the effective therapeutic properties of the CTT slices.

[00448] The culturing process assures that a defined composition of the cultured cells / tissue having the pre-requisite biological characteristics is obtained in a manner suitable for surgical implantation into a subject to enable reconstitution of the subject’s immune system.

[00449] The culturing process results in a loss of thymocytes and relative enrichment of thymic epithelial cells and other stromal cells in the donor thymus tissue slices.

[00450] The culturing process further results in depletion of thymocytes and maintenance of TECs to enable reconstitution of the recipient’s immune system and allows tolerance to develop in the recipient to HLA antigens in the donor thymus.

[00451] Overall, the manufacturing process is designed to deplete thymocytes from the donor thymus tissue and to preserve the functional architecture of the thymic stroma (thymic epithelial cells and fibroblasts).

[00452] In an embodiment, processed donor thymus tissue is an engineered thymus tissue product capable of inducing tolerance to the thymus tissue types (HLA antigens) in a subject in need thereof following a surgical implantation procedure.

[00453] To keep the sliced thymus tissue viable, the thymus sections are placed on Millipore cellulose filters and surgical sponges inserted into medium-containing tissue culture dishes. The culture medium in each tissue culture dish is replaced daily from the day of harvest from the donor to the day of implantation (day 12 to day 21).

[00454] The culturing of donor thymus tissue depletes thymocytes in such processed tissue which minimizes the risk of graft versus host disease (“GvHD”), which could be highly problematic in profoundly immunodeficient subjects following a thymectomy.

[00455] During the first few days in culture, many thymocytes “fall out” of the tissue slices into the culture medium and are discarded during media changes. As culturing continues during the culturing period, donor thymocytes continue to die but their cellular remnants are retained within the CTT slices.

[00456] Without being bound by theory, the presence of these non-viable thymocytes and their remnants that lack nuclei are hypothesized to be important for the intended function of the tissue- engineered product, because they help to preserve the open pockets in the three-dimensional network of thymic epithelial cells that is necessary for the entry of recipient bone marrow stem cells post-treatment. The importance of having “space” for the entering bone marrow stem cells is supported by experience with patient DIG003 in Markert, 1999 (See list of reference infra). The patient described in the foregoing reference had been given a very large dose of steroids (40 mg / kg / day x 3 days of methylprednisolone) 35 days after CTT implantation, which led to apoptosis of the thymocytes and condensation of the epithelium. No naive T cells ever developed, and the patient succumbed to infection. At autopsy, the inserted thymus was a mass of viable epithelium with no space between the epithelial cells for thymocytes to enter.

[00457] During the culturing period, HLA typing is performed to see if the patient (recipient) and donor tissue share any HLA alleles. Anti-HLA antibody testing is performed in the recipient to determine if the recipient has any antibodies against HLA antigens in the thymus. If the recipient has antibodies targeting the donor’s MHC, another thymus would be sought. The donor and the mother of the donor are checked for infection per the FDA guidance document “Guidance for Industry. Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / Ps)” and more recent Guidance documents. The tissue is processed aseptically under the Code of Federal Regulations (CFR) 1271 subpart D “Current Good Tissue Practice.”

[00458] Following review of the batch records and QC testing the tissue is released from manufacturing and provided to the surgical team for implantation, the tissue is surgically implanted into the recipient, as described in this specification.

[00459] Cultured thymus tissue is produced in a process more completely described below and in the Examples set forth in this specification.

[00460] In summary, the culturing process of the harvested thymus tissue significantly modifies the biological characteristics of the donor tissue and constituent cells contained therein in the following manners: loss of donor thymocytes and enrichment of thymic epithelial cells and other stromal cells, and depletion of donor thymocytes modifies the physiologic functions of the tissue (e.g., secretion of cytokines and growth factors) as well as its structural properties.

[00461] During the first few days in culture, many thymocytes “fall out” of the tissue slices into the culture medium and are discarded during media changes.

[00462] Manipulations that occur during the manufacturing process result in changes in the gross and histologic appearance of the resulting cells contained in the finished product as compared with the source or starting material obtained from the donor.

[00463] Thymus tissue during the first few days of culturing appears red due to residual blood on and within the tissue. See, e.g., Fig. 15.

[00464] Between days 5 to 21 viable tissue is observed minus the blood contamination that was evident on day 1.

[00465] During the remaining days of culturing the depth of the tissue decreases as thymocytes are depleted. The decrease in density of thymocytes in the tissue is documented by immunohistochemistry and described in detail below.

[00466] On day 0 following harvesting of the discarded thymus tissue the tissue is densely populated with viable thymocytes embedded in a stroma that contains thymic epithelial cells and fibroblasts. AE1 / AE3 and CK14 staining confirm the presence of cytokeratin (CK)-positive thymic epithelial cells, which are characteristic of normal thymus. The thymic epithelial cells form a lacy three-dimensional network with delicate processes that surround neighboring thymocytes.

[00467] During the culturing process, slices of thymus are cultured, as described below. Numerous thymocytes are washed out of the tissue, especially over the first 3 days. This depletion can be identified histologically as early as day 2 by H&E stains that show decreased thymocyte density, particularly in medullary areas. The majority of the thymocytes that remain in the tissue show nuclear changes consistent with apoptosis and / or necrosis or demonstrate karyolysis (complete loss of nuclei). Many of these dead thymocytes and their cellular remnants remain present throughout the thymus tissue and are believed to prevent total collapse of the space between epithelial cells.

[00468] Some epithelial condensation can be seen in external areas, such as in the subcapsular cortex where loss of thymocytes has led the epithelial cell network to collapse. These condensed subcapsular cortical epithelial cells can form linear arrays several cell layers thick, which may add to the mechanical strength of the slices. Some medullary epithelium may also condense to form patches of contiguous epithelial cells.

[00469] Death of thymocytes continues as the culture progresses, with retention of necrotic thymocyte debris within the tissue. Further condensation of medullary and subcapsular cortical epithelium is minimal between approximately days 7 and 19 of culture.

[00470] Areas with epithelial architecture similar to normal thymus can still be observed late in culture for each thymus using the AE1 / AE3 stains. For tissues cultured longer, the epithelial architecture of cortex and medulla can still be readily discerned; for instance, Hassall bodies remain in the medullary areas. The degree of thymocyte depletion, however, results in a substantially different overall histologic appearance on H&E compared to that of normal thymus at time points later than day 0. Detailed Culturing of Thymus Tissue.

[00471] The general procedure in preparing allogeneic cultured thymus tissue-derived product is that thymus tissue for infants with complete DiGeorge syndrome is obtained as discarded tissue from infants under the age of 9 months undergoing cardiac surgery, as described previously. For solid organ transplantation, discarded thymus will be obtained from individuals up to 50 years of age. Use of the thymus tissue will depend on whether it meets criteria for use as set forth in this specification.

[00472] The thymus tissue is aseptically processed and cultured under cGMP conditions to produce partially T cell-depleted thymus tissue slices.

[00473] The manufacture of cultured thymus tissue (CTT) consists of the following general steps: receipt and processing of incoming thymus tissue, slicing, culturing, media changes, dose calculation, packaging and transporting the thymus to the operating room. In addition, incoming thymus tissue is tested for acceptability and in-process and release testing is conducted on thymus tissue slices.

[00474] In an embodiment, the thymus tissue slicing process entails using sterile, single-use scissors and forceps to cut off a piece of thymus tissue. The operator removes the capsule of the thymus with forceps and scissors and places the capsule in the lid of the plate for disposal later.

[00475] The piece of thymus tissue is placed in the single-use tissue slicer using forceps. The top of the slicer (e.g., Stadie-Riggs hand microtome (Thomas Scientific, Swedesboro, NJ)), is placed onto the middle portion of the slicer and tightened in place. The operator runs the blade through the tissue piece to cut off a slice. The slices are approximately 0.5 to 1 mm thick. Approximately 50-90% of the filter space is filled without overlap of the tissue slices.

[00476] Typically, three in-process pieces are cut off of the tissue at the beginning of slicing, all of which are roughly 3x3mm. One is sent for histology, and two are retained. Thymocytes flow freely into the medium as the thymus is sliced.

[00477] In an embodiment, the filter and thymus slices are transferred to a gelatin surgical sponge saturated with TOM in a tissue culture dish. The TOM wicks up wetting the Millipore filters, keeping the tissue moist. Two filters are placed per sponge and 2 sponges are used per tissue culture dish. The process of slicing pieces of thymus is repeated until the required number of slices has been prepared. Culture dishes are labeled with an operation number, dish number, and ISBT barcode label. Completed dishes are placed in a humidified incubator at 37°C with 5% COs.

[00478] The tissue engineered drug substance comprises thymus tissue slices after they have been placed in a culture dish in media and cultured for about 6 days to about 21 days, as described below. The tissue-engineered drug product comprises the thymus tissue slices after transfer into a drug product container. No other processing is conducted to create the drug product from the drug substance; the only processing of drug substance to create drug product is transfer of slices into the leak proof container and corresponding media change.

[00479] The culturing of thymus tissue slices is more specifically set forth in the following paragraphs.

[00480] In an embodiment, thymus tissue is obtained from the operating room as discarded tissue from infants aged 9 months and under undergoing cardiac surgery. The tissue is then placed in a sterile specimen cup with a screw-cap top by the surgical team and transported to a GMP facility under ambient conditions for processing. The sterile specimen container in which the thymus is received is labeled, including a barcode, with the donor’s name and medical record number. The donor screening group gives the thymus a unique identifier (thymuses are numbered consecutively) and a unique medical record number. For manufacturing, each tissue has an operation number, and a unique label. All identifiers are recorded on a “Confidential Thymus Donor Form” that is maintained separately from the batch record and kept confidential.

[00481] In an embodiment, the drug substance container closure system may be a cell culture dish with lid. One slice of thymus tissue is placed on a filter and two filters are placed on each gelatin sponge in thymus organ media in the dish. Four slices are placed in each culture dish and the dishes are stored in the incubator, with daily media changes, until ready for release.

[00482] In an embodiment, the culture dishes can be obtained from Corning. The dishes may be sterile, non-pyrogenic Falcon® 100 mm polystyrene cell culture dishes (product #353003). The dishes are cleaned by vacuum-gas plasma treatment and sterilized by gamma irradiation. The dimensions of the dish are 89.43 mm OD. x 19.18 mm.

[00483] In an exemplary embodiment, the Surgifoam® sponge may be manufactured by Ethicon and it meets the requirements for Absorbable Gelatin Sponge, USP. A suitable sponge is a sterile, water-insoluble, malleable, porcine gelatin absorbable sponge that is intended for hemostatic use. An illustrative example of the mixed cellulose esters filter is manufactured by Millipore (product # SMWP 02500). The 25 mm hydrophilic membrane has a 5.0 um pore size. It is made of biologically inert mixtures of cellulose acetate and cellulose nitrate. The filter is sterilized by ethylene oxide prior to use.

[00484] After release and acceptance of the donor thymus into the processing laboratory, the thymus is cut into thin slices, which are placed on sterile filter papers that are put on the surgical sponges in sterile culture dishes. If the tissue is not processed immediately, it is stored in thymus organ media (TOM), as described below, at 2 - 8 °C for up to 24 hours after harvest from the donor before processing is initiated. TOM consists of Ham's F-12 culture media, HEPES buffer, L-glutamine and heat-inactivated fetal bovine serum (FBS).

[00485] In an embodiment, processing occurs in an ISO 5 space of a biological safety cabinet (BSC) in an ISO 7 manufacturing clean room. Only one lot of thymus tissue from a single thymus is processed in the BSC at any time. The BSC is cleaned before use. The thymus is tested for appearance by visual inspection and weighed. The thymus is then placed in a 150-mm tissue culture dish in TOM. The capsule of the thymus is removed with sterile, single-use forceps and scissors. Tissue pieces are taken for testing and as retained samples. The incoming thymus tissue is tested for identity by histology. Donor eligibility is also confirmed. Processing continues prior to receipt of histologic results and all donor screening results.

[00486] The acceptance criteria for donor screening is that all donor eligibility requirements must be met. Donor screening is required per 21 CFR 1271 to protect the safety of the thymus tissue implant recipient. This screening minimizes the risk of disease transmission from donor to recipient. Thymus Organ Media (TOM)

[00487] The medium is made with ingredients approved for use in humans which are unlikely to cause allergic reactions, whenever such reagents are available.

[00488] All reagents must be tracked such that all ingredients can be identified after implantation if any problems develop.

[00489] Fetal Bovine Serum (FBS) must be manufactured using US material because of the concern of Creutzfeldt-Jakob Disease. Information on each lot must be sent to the FDA prior to use.

[00490] Medium must be tested for bacterial, fungal, and mycoplasmal contamination prior to use.

[00491] In an embodiment, the following materials are used to prepare TOM:

[00492] HAMS F12, Gibco #11765-054 (or case 11765-062), 500 ml bottles or equivalent source.

[00493] HEPES, Gibco #15630-080 or equivalent, IM solution, 100 ml bottles. Final concentration 25 mM.

[00494] L-Glutamine, Gibco #25030-081 or equivalent source, (stock 200 mM).

[00495] Fetal Bovine Serum, Gibco, #16140 (Heat Inactivated) or #10082-147 (heat inactivated, certified).

[00496] In an embodiment FBS that is HI may be used in the following manner:

[00497] FBS must be heat inactivated at 56°C for 30 min.

[00498] To decrease the likelihood of contamination of the medium, medium must be divided into aliquots and no aliquot should be used more than once.

[00499] Aliquots of remaining FBS may be stored frozen (-20°C) in 25ml aliquots for research use.

[00500] In an embodiment, TOM may be prepared in the following manner.

[00501] Thaw fetal bovine serum overnight in the refrigerator, or at 37°C with frequent gentle swirling.

[00502] If non-Heat inactivated fetal bovine serum is used, heat inactivate at 56°C for 30 minutes.

[00503] Put all media components together into 4 liter flask if making 4 liters at a time, stir for 3-5 minutes with stir bar on magnetic stir plate on medium speed (no frothing).

[00504] Sterilize using the 0.2 micron filter units.

[00505] In an embodiment, sterilization of the TOM preparation may be performed in the following manner. Dispense 1 liter TOM into one liter flask. Measure 80 ml TOM in a disposable sterile cylinder. Pour the 80 ml TOM into a 150 ml Corning filter sterilization unit. Attach house vacuum to filter and filter sterilize per manufacturer’s directions. Remove the filter unit from the container and discard. Cap the collection bottle with the sterile cap (provided with the unit). Label with TOM Lot No. Test one aliquot for bacterial culture with anaerobes; fungal culture, other; and Mycoplasma culture. Test one aliquot for endotoxin. Store all TOM aliquots in the -20° C freezer upright.

[00506] TOM media may be released for use if: LAL result must equal to or less than 2 EU / ml for samples diluted 20 fold for testing or 1 EU / ml for samples diluted 10 fold for testing; all culture results must be negative for growth.

[00507] A BSC must be used for the filtering and dispensing the medium.

[00508] TOM is tested for sterility and endotoxin before release. TOM is not released for culturing a donor thymus until after the 14-day sterility testing acceptance criterion has been met. Once prepared, TOM is stored at -20°C until thawed, at which point it may be stored for use in the refrigerator for up to two weeks.

[00509] In an embodiment, the 14-day sterility testing, may, for example, be conducted using the BacT / ALERT culture system. The BacT / ALERT (BioMerieux, Durham, NC) is a commercially available culture system that can be used to test samples using an automated microbial detection system.

[00510] All in-process and drug substance cultures are incubated for 14 days or reported immediately if the product becomes positive. For positive tests, the organism(s) are identified and their antibiotic sensitivities determined. Culture bottles containing medium for aerobic growth and bottles containing medium for anaerobic growth are inoculated with samples to be tested on day 1, day 7 and the day of release. All bottles are incubated for 14 days at 35-37°C.

[00511] FBS may be obtained from GIBCO brand, Life Technologies. The FBS is prepared by an aseptic, validated process. FBS meets USDA requirements for abattoir-sourced animals, traceability and country of origin. All fetal blood is collected from fetuses derived from healthy dams that have passed pre- and post-mortem certified veterinary inspection. All FBS are traceable by date and location of collection. FBS collected and processed in the United States is from USDA approved and inspected slaughter establishments. The United States is recognized by the USDA as being free of foot and mouth disease and rinderpest. To qualify the supplier, FBS is tested for pH, osmolality, endotoxin, total protein and identity before use

[00512] Completed dishes are placed in a humidified incubator at 37°C with 5% COz. Each lot of thymus tissue is stored in a separate incubator. After the thymus slices have been placed in the incubator, particle sampling and personnel monitoring is conducted.

[00513] Thymus slices are cultured for up to 21 days (for example a conditioning regimen of about 6 days to about 21 days), and during culture the medium is changed daily. These thymus slices are considered the drug substance. During the culture period, many thymocytes are washed out of the thymus tissue slices or the thymocytes undergo apoptosis while preserving the thymic stroma. All manufacturing steps are conducted using sterile, disposable equipment and supplies. Media is aspirated by pipette from the culture dish and pooled into a sterile collection container for in-process testing. Ten (10) mL of fresh thymus organ media is then gently dispensed to each culture dish in a rinsing manner over the tissue slices. After the media change is completed, samples are taken from the pooled media for sterility and histology, if needed. Particle sampling and personnel monitoring are completed and line clearance is performed.

[00514] The medium is changed daily.

[00515] The slices are cultured for up to 21 days (for example a conditioning regimen of about 6 days to about 21 days).

[00516] In-process testing is conducted to provide insight into the process and product quality and to help ensure the safety and quality of the final drug product. In-Process Testing

[00517] Samples are collected for sterility in-process testing on day 1 and day 7. Samples are collected for mycoplasma in-process testing on day 7. Samples are collected for in-process histology testing between days 5 and 9. The dose is determined on the day prior to release. Gram stain, BacT, mycoplasma and endotoxin are tested on the day of release.

[00518] The Gram stain is a bacteriological laboratory technique used to differentiate bacterial species into two groups, Gram-positive and Gram-negative. Gram stain is tested on pooled spent culture medium from the culture dishes. The method uses a staining technique to determine the classification based on the physical properties of the cell wall. This method is used to make a preliminary morphologic identification or to establish whether there are significant numbers of bacteria in a clinical specimen. Staining is conducted either manually or using an automated stainer. It has been demonstrated that the two different staining methods showed no qualitative differences that would impact culture results.

[00519] Histology testing is performed prior to implantation, and includes in an embodiment at a minimum: (1) determination that areas positive for keratin AE1 / AE3 are scattered throughout tissue; (2) at least 1 Hassall body is microscopically identified; (3) CK14 staining of the tissue slices is scattered throughout the thymus tissue; and (4) intact nuclei are microscopically observed. In an embodiment, histology testing is performed between about days 6 to about 21 days)The presence of Hassall bodies and intact nuclei as well as successful CK 14 staining are indicative of normal healthy thymus tissue that has been cultured.

[00520] Culture time is an important process parameter. As noted, culturing is performed for up to 21 days.

[00521] Testing of thymus samples in culture prior to implantation is conducted to confirm whether histology results generated in culture are representative of histology testing of historical specimens of cultured thymus tissue. Based on the observations made by the pathologist for the samples discussed in the Examples, the histologic appearance of the tissue slices at day 5 reflects what is observed at each of the later time points (day 9, 12 and 21) of culturing. In an embodiment testing of thymus tissue may be performed at various timer periods and intervals between days 5 and 21 of culturing. For example, the testing may be performed during the conditioning regimen which is for a period of five days, or six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days , or 18 days or 19 days, or 20 days, or 21days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days. In an embodiment, the conditioning regimen may be anytime between about 6 days and about 21 days prior to implantation of the cultured thymus tissue.

[00522] Histologic examination of any one slice corroborates the conclusion regarding acceptability of the entire lot. The relevant characteristics of any one slice from a thymus reflect those of the entire thymus, supporting the continued use of a single slice of tissue for histology testing.

[00523] Forced degradation testing indicated as indicated in Figs. 12A and12B demonstrated that the cultured thymus tissue product is not easily degraded and is most sensitive to freeze / thaw as well as changes in osmolarity. Other conditions tested during forced degradation showed little to no effect on the cultured thymus tissue product. Control of Cultured Thymus Product Drug Substance

[00524] Acceptance criteria for incoming thymus tissue product include the tests identified in Table 1 below. Table 1. Incoming Thymus Tissue Attribute Day of Test | Test Parameter | Acceptance Criteria Process Step: Incoming Thymus Tissue Safety Day 0 Donor Screening | Donor eligibility requirements are met per 21 CFR 1271 | Identity | Day 0 | Visual Inspection | o Container intact Label accurate Pink to dark red, black marks may be present > 50% of areas positive for keratin in | Day 0 | Histology lacy staining pattern Hassall bodies identified CK 14 staining in lacy pattern > 00% intact nuclei observed in sections Quality Day 0 Weight > 3 grams

[00525] Abbreviations: CK, cytokeratin; EU, endotoxin unit, USP, United States Pharmacopeia. A thymus tissue is processed prior to obtaining all donor screening results.

[00526] Generally, the acceptance criterion for weight is greater than or equal to 3 grams. This is the minimal thymus weight that is accepted to ensure sufficient material is available for proper dosing of the final product. The acceptance criterion is based on experience in processing thymus tissue.

[00527] Acceptance criteria for in-process testing is identified in Table 2 below. Table 2. In-process Testing: | Potency Days 5-9 Histology | * Areas positive for keratin AE1 / AE3 scattered throughout tissue on days 5-9 o Atleast 1 Hassall body identified ) o CK 14 staining scattered throughout tissue | * Intact nuclei observed

[00528] Acceptance criteria for cultured thymus tissue drug substance testing is identified in Table 3 below. Table 3. Cultured Thymus Tissue Drug Substance Testing Attribute Day of Test | Test Parameter | Acceptance Criteria Process Step: Drug Substance Release Testing | Visual inspection | © No evidence of tampering or damage to | Appearance containers e | Day of release Attribute Day of Test | Test Parameter | Acceptance Criteria ® Yellow to brown slices of tissue with varying thickness and shape | Identity | Days 5-9 | Histology | Thymus tissue identity is confirn | Barcode Day of release [ strength | Day before | Dose | Identity | Days 5-9 | Histology Thymus tissue identity is confirmed by histology on day 1 and at midpoint (days 5- | 9). Day of | Barcode A barcode is used for tracking of the tissue | release throughout the processing and the barcode is confirmed at release. Strength Day before | Dose 1000 — 22,000 mm? of thymus tissue / release recipient body surface area in m? Safety Day of Endotoxin | <5 EU / kg body weight / hr ra) ada TIRD «<Q&~) Endotoxin | <5 EUkg! (USP <85>) Sterility No growth | Mycoplasma | Negative for the presence of mycoplasma | Gram stain | Negative

[00529] The acceptance criterion for identity is that thymus tissue identity is confirmed by histology on day 1 and at the midpoint (days 5-9). A barcode is used for tracking of the tissue throughout the processing and the barcode is confirmed at release to verify the correct identity of the product. Histology by Immunochemistry

[00530] The histology method is a standard method used by hospitals for all tissue types, as is known by a person of skill in the art.

[00531] Samples of the product are fixed in 10% formalin and transported to the laboratory. Containers are labeled with a coded identifier instead of the patient’s name to protect patient privacy, along a medical record number. Upon arrival in pathology, the specimens are assigned a unique pathology accession number, and barcoded. The subsequent blocks, slides, and paperwork are all barcoded with this pathology accession number.

[00532] After the specimen is received in the lab, the formalin-fixed tissue is grossly examined, and a written gross description of the material is prepared that will become part of the final report. The formalin-fixed tissue is then processed and embedded into a paraffin block by standard methodology on an automated processor. Sections are cut from the paraffin block and the following stains are performed by ASCP-certified histotechnologists:

[00533] Hematoxylin & eosin.

[00534] Cytokeratin AE1 / AE3 immunohistochemistry.

[00535] Cytokeratin 14 immunohistochemistry.

[00536] CD3 immunohistochemistry.

[00537] Ki-67 immunohistochemistry.

[00538] During performance of the foregoing immunohistochemistry tests, appropriate control slides are also tested and reviewed. All control slides and internal controls demonstrate the expected immunoreactive patterns. The incoming thymus sample also serves as a control for tissue slices that have been in culture for about 6 days to about 21 days, when samples are tested as part of potency testing. The incoming thymus sample appears as a typical thymus sample and then changes occur to the tissue slices while they are cultured and then tested after about 6 days to about 21 days in culture. After about 6 days to about 21 days in culture, the sample must show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue and presence of intact nuclei.

[00539] Slides are interpreted by a pathologist certified in Anatomic Pathology, with additional experience in the histologic evaluation of thymic tissue. The final report is issued by the pathologist and the report documents the results.

[00540] Acceptance criteria for cultured thymus tissue drug substance testing is identified in Table 4 below. Table 4. Cultured Thymus Tissue Drug Substance Release Testing Process Step: Drug Product Release Testing Identity Day of Visual inspection | * No evidence of tampering or damage to release containers 1 Ndi maririirioom inant ham sai ih | * Yellow to brown slices of tissue with varying thickness and shape, adhered to | round white filter paper | Days 5-9 | Histology | Barcode Day of release Thymus tissue identity is confirmed by histology on day 1 and at midpoint (days 5- | 9). A barcode is used for tracking of the tissue | throughout the processing and the barcode is confirmed at release.

[00541] The cultured thymus tissue must be free of microorganisms. In the sterility test performed on day 1 and day 7, there should be no growth of microorganisms. Mycoplasma should be negative upon testing on day 7. The sterility test should be gram stain negative.

[00542] Product sterility is maintained using appropriate controls including aseptic technique; employing a training program and verifying the qualification of operators; utilizing appropriate clean room qualification procedures; employing establish clean media fill procedures and utilizing ready-to-use sterilized apparatus or apparatus sterilized utilizing validated sterilization cycles.

[00543] The containers of processed thymus tissue are visually examined for damage. Tissue slices normally exhibit a yellow to reddish brown appearance with varying thickness and shape.

[00544] Thymus tissue identity is confirmed by histology on day 1 and at midpoint (days 5-9).

[00545] A barcode is used for tracking of the tissue throughout the processing and the barcode is confirmed at release.

[00546] The dosage (area) is 1,000 — 22,000 mm? of thymus tissue / recipient body surface area inm2. Dose is controlled by the surface area of slices released to the operating room as appropriate for the patient’s body surface area.

[00547] The acceptable dose range is defined as 1,000 — 22,000 mm? of thymus tissue per recipient body surface area (BSA) in m2. The area of the thymus tissue is determined by photograph using software analysis (PAX-it Image Analysis Software). BSA is determined using the patient’s height in cm and weight in kg. The DuBois and DuBois formula is used to calculate the BSA:

[00548] BSA=0.007184 x [height (cm)]>7> x [weight (kg)]**%*.

[00549] The cultured thymus tissue is tested for endotoxin. The specification is < 5 EU / kg body weight / hr.

[00550] Endotoxin testing may be performed, for example, by using the Endosafe PTS system. The cartridges used with the Endosafe PTS use a chromogenic kinetic Limulus Amebocyte Lysate (LAL) test. Each cartridge contains precise amounts of LAL reagent, chromogenic substrate and control standard endotoxin. Test sample is pipetted into four sample reservoirs. The instrument draws and mixes the sample with LAL reagent in two channels (sample channels) and with the LAL reagent and positive product control in the other two (spike channels). The sample is incubated then combined with the chromogenic substrate. After mixing, the optical density of the wells is measured and compared to a standard curve archived in the instrument. The instrument measures the reaction time in each channel. The archived standard curve specific for each batch of cartridges is constructed using the log of the reaction time versus the log of the endotoxin standard concentration. The sample and spike values are calculated by interpolation off the standard curve using the reaction time. This testing meets the requirements of United States Pharmacopeia (USP).

[00551] Testing for mycoplasma may be performed in the following manner. A sample of the pooled media is removed from the plates on day 7 and tested before product release.

[00552] In the event of a positive culture during manufacturing, the lot will be discarded and will not be administered. In the event of a positive culture after clinical product administration, the patient's attending physician and sponsor will treat the patient appropriately. A positive culture requires that the species of the contaminating organism be identified and its antibiotic sensitivity determined. The attending physician will institute antibiotic therapy for the thymus recipient, if indicated.

[00553] The drug product undergoes similar visual inspections and histology testing before use.

[00554] After the thymus tissue slices have been cultured for up to 21 days, the slices are transferred into drug product containers for transport to the operating room. Once received in the operating room, the slices are inserted into the thigh muscle of the recipient patient

[00555] The container should be intact with no visible damage and the thymus tissue slices should appear as yellow to reddish-brown slices of tissue with varying thickness and shape. The tissue slices are visually examined to confirm that these acceptance criteria are met. Cryopreservation and thawing of allogeneic cultured post-natal thymus tissue derived product

[00556] Cryopreservation of the allogeneic cultured post-natal thymus tissue derived product may be performed in the following manner.

[00557] A cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by method comprising the steps of:

[00558] (a) obtaining suitable thymus tissue from a donor;

[00559] (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRBI, HLA-DQBI1, HLA- DRB3, HLA-DRB4, HLA-DRBS5, HLA-DQA1, HLA- DPB1, HLA-DPA1:

[00560] (c) subjecting the thymus tissue to a conditioning regimen for a period from about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; further wherein the donor thymus tissue slices show, on about 6 days to about 21 days, areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK 14 staining scattered throughout the tissue and presence of intact nuclei upon completion of the conditioning regimen;

[00561] (d) harvesting the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product;

[00562] (e) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and

[00563] (f) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank. In an embodiment, the cryopreserved allogeneic cultured postnatal thymus tissue- derived product of claim 66, wherein the thymus, on the day of harvest, demonstrates that >50% of areas are positive for keratin in a lacy staining pattern, that Hassall bodies are present, that CK 14 stains in a lacy pattern, and that >90% of nuclei are intact.

[00564] In an embodiment, the donor thymus is sliced and divided into roughly two equal portions, putting each slice with its cellulose filter in a separate cryovial (Nunc tube). The filter is folded in half to insert it into the tube. About 1 to about 1.5 ml of freezing medium [sterile filtered 90% heat inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] at room temperature is added to cover the tissue. The sterile cap of the cryovial is replaced on the tube. Put all the tubes in a Biocision Cool Cell or equivalent container which is at room temperature. Any empty slots in the CoolCell should be filled with tubes that have 1 ml of freezing media. The tubes are placed overnight in a -80°C freezer. Alternatively place each tissue plus filter in a 5ml CryoELITE tissue Vial (Wheaton). Put 3 to 5 ml of freezing medium at room temperature to cover the tissue. Putin a Styrofoam box and put in -80° freezer overnight. The vials are then transferred to the vapor phase of a liquid nitrogen freezer. Alternatively a controlled rate freezer can be used to bring the temperature of the cryovials to liquid nitrogen temperature.

[00565] To recover the tissue, remove the cryovial or the CryoELITE tissue Vial from the liquid nitrogen freezer. Thaw the thymic pieces in the vial rapidly with a swirling motion in a 37°C water bath. The tube is sprayed with 70% ethanol and then is placed in the Biological Safety Cabinet (BSC). The thymic tissue and filter are removed from either the Nunc cryovial or the CryoELITE tissue Vial using forceps. The tissue and filter are placed in a 50 ml conical tube containing 20ml of 49°C TOM. Up to 5 filters can be placed in each 50 ml conical tube that contains 20 ml of 4°C TOM. Immediately transfer the 5 filters with tissue to a fresh conical tube with 20 ml of 49°C TOM media and put at 4°C for 15 minutes. Repeat the wash 3 times. Keeping the tissue at 4°C, transfer each piece to its own 120 ml Starplex container with 5 mls 4° TOM. All containers are brought to the surgical suite in a temperature-controlled container with a cold pack in it. The Starplex containers with the tissue are brought into the operating room. The tissue on the filters are transferred to the sterile field into a tissue culture dish with approximately 2 ml of sterile saline. The scrub nurse removes the tissue from the filter paper by scrapping or pulling with forceps. The scrub nurse places the tissue, in an amorphous pile, back on the filter paper. The tissue culture dish with approximately 4 filters and the tissue is transferred to the operative site where the surgeon can easily access the tissue. The tissue is placed in the quadriceps muscles similarly to the procedure with CTT (RVT-802). The Cryo- CTT resembles the CTT in that it is partially T-cell depleted, the thymus tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the tissue, slices contain at least one Hassall body, CK14 staining is scattered throughout the tissue and presence of intact nuclei. Transplantation of CTT

[00566] In an embodiment, unmatched thymus tissue slices from the donor are cultured for about 6 days to about 21 days. On the day of the solid organ transplantation steroids are usually given at the induction of anesthesia. For heart or lung transplants, the thymus of the recipient is surgically removed at the time of the solid organ transplant. For other organ transplants the thymectomy may be done prior to the day of transplantation or on that day. The thymectomy method would be surgical, thorascopic or robotic. At the end of the surgery after reperfusion, the recipient is given more steroids prior to receiving equine anti-thymocyte globulin (e.g., rabbit anti-thymocyte globulin) over 3 to 7 days to kill most of the residual T cells (and NK cells) in the recipient or alemtuzumab over 4 days to kill the T, B and NK cells. Administration of an immunosuppressant (such as cyclosporine or tacrolimus) and mycophenylate is then started until T cells develop and show greater than 10% naive T cells. It may take 6 to 12 months for the naive T cells to increase to this number. Cultured thymus tissue is processed for the thymus of the donor of the solid organ. Half of the CTT can be implanted into the quadriceps muscle between about 6 days to about 21 days. The other half of the thymus will be cryopreserved for future use of the recipient. The immunosuppressive regimens will suppress any remaining T cells until naive T cells are released by the cultured thymus tissue slices implanted in the recipient and the recipient meets criteria for weaning off the maintenance immunosuppression regimen. (Over 10% naive T cells are needed to wean the immunosuppression.) Thymectomy Protocol

[00567] The patient is taken to the operating room and is placed under general anesthesia by endotracheal tube.

[00568] The chest and abdomen are prepped and draped in a sterile fashion.

[00569] The patient undergoes a full sternotomy through a skin incision of approximately 4 cm.

[00570] Both pleural spaces are entered to guarantee a complete resection.

[00571] The phrenic nerves are visualized on both sides and care is taken to not compromise them.

[00572] The thymus is identified and carefully dissected away from the pleural investment of the lung, starting with the inferior horns and extending to the superior horns.

[00573] A complete thymectomy is performed.

[00574] Hemostasis is attained within the mediastinum.

[00575] Chest tube placement. One chest tube is always inserted (into the mediastinum). If a single pleural space is entered during the operation, the chest tube is continued from the mediastinum into that pleural space. If both pleural spaces are entered, a second chest tube is used in a similar fashion from the mediastinum to the other pleural space.

[00576] Drain size. #15 Blake drains are used for infants up to 2 years of age. #19 Blake drains are used for children 2 years and older.

[00577] Sternum closure: In neonates or infants, 0-Ticron sutures are used to close the sternum. At about 1-2 year of age, #1 sternal wires are used. At about 2-5 years of age, #4 sternal wires are used.

[00578] The fascia, subcutaneous tissue and skin are closed with running absorbable suture.

[00579] A skin wound vac is placed on the sternum.

[00580] The patient is extubated in the operating room.

[00581] A sponge, instrument and needle counts are taken and must be correct at the end of the case. Surgical implantation of allogeneic cultured postnatal thymus tissue-derived product.

[00582] Allogeneic cultured postnatal thymus tissue-derived product should be implanted in accordance with the following instructions. Implantation of thymus tissue into the thigh requires a healthy bed of muscle tissue. Preparation for implantation procedure

[00583] The maximum and minimum dosage of planned implanted allogeneic cultured postnatal thymus tissue-derived product should be calculated for each individual patient. Properly identify the intended recipient prior to administration.

[00584] Under sterile conditions within a laminar flow hood, the tissue slices on the filter papers that are on surgical sponges in medium are removed from the tissue culture dishes and placed in 120 ml sterile cups with 20 ml medium, packaged to maintain sterility, and delivered to the operating room or packaged for shipment. Tissue slices are not removed from the individual containers until ready to be used. Verify the product expiration date and time.

[00585] Always handle allogeneic cultured postnatal thymus tissue-derived product (tissue slices) using strict sterile technique. Inspect each container for leaks or evidence of damage. Do not use if there is evidence of contamination. Outside the sterile field, unpack allogeneic cultured postnatal thymus tissue-derived product containers from the shipping box. Remove racks containing polypropylene containers from the outer bag. When ready, a team member outside the sterile field, but adjacent to the sterile prep table, will open and remove the cap from each container, one at a time. Each open container is then held by the team member outside the sterile field extending his / her arm over the sterile field without touching the sterile field.

[00586] The sterile field team member will use a pair of forceps to remove the individual tissue slice with its filter paper from the container and place it in a sterile tissue culture dish containing approximately 2 ml preservative-free saline on the sterile prep table. Four tissue slices with the filter papers taken from four containers are placed in one sterile tissue culture dish that is on the sterile field in front of the sterile field team member. Using sterile forceps, the sterile field team member then peels the tissue slice away from the filter paper using two pairs of forceps, one of which holds the filter in place while the other pulls the tissue or scrapes the tissue into a pile. The tissue removed from each filter paper is than put on that filter paper in a pile in the middle of the filter paper. The sterile tissue culture dish is then transferred to the sterile field. The next set of four allogeneic cultured postnatal thymus tissue-derived product containers will then be processed the same way while the surgeon is implanting the first 4 slices. When the surgeon finishes implanting the first four slices, the next dish with 4 pieces of tissue is put in the surgical field and the initial tissue culture dish is returned to the sterile field in front of the sterile field team member for loading the 3™ set of four tissue slices. Continue this cycle until all the desired tissue is implanted. All of the tissue slices are not transferred at the beginning to avoid contamination from the air in the operating room. Surgical procedure

[00587] Step 1. Skin opening.

[00588] After induction of general anesthesia, make a vertical skin incision (typically ~5 cm in length) over one of the anterior thigh compartment. Note: The size of the incision and the use of one or both legs for the implantation procedure is determined by the size of the patient, planned amount of implanted tissue, and his / her muscle mass. If all or most of the tissue can be implanted in one leg, then only one leg should be used.

[00589] Step 2. Open the fascia to expose the anterior compartment muscles.

[00590] Step 3. Muscle spreading and implantation.

[00591] Separate the muscle using a tonsil clamp or similar instrument along the natural furrows of the quadriceps muscle. The allogeneic cultured postnatal thymus tissue-derived product individual thymus slices should be implanted without cutting muscle tissue. Place individual tissue slices in “pockets” approximately 1 cm apart and approximately 1 cm in depth within the quadriceps muscle along the natural furrows. Depending on the size of the patient, the surgeon may place approximately 6-7 slices into 6 to 7 pockets along each furrow. Individual allogeneic cultured postnatal thymus tissue-derived product slices may be cut in half prior to implantation depending on the mass of tissue on each filter. A thick slice of tissue that had fully covered the filter paper should be cut in half for optimal vascularization of each tissue. Implant as much required tissue within each anterior compartment up to the maximum planned dose.

[00592] Step 4. Muscle closure.

[00593] Close the muscle with a single suture over the site where the thymus tissue was implanted to prevent muscle reopening and graft coming out of muscle. Ensure that the implanted tissue is entirely covered by muscle tissue with no exposed thymus tissue prior to closing the incision.

[00594] Step 5. Repeat Steps 3-4 for each allogeneic cultured postnatal thymus tissue-derived product tissue slice up to the maximum intended dose.

[00595] Step 6. Incision closing.

[00596] Confirm hemostasis. Close the skin incision with two layers of absorbable sutures and apply standard dressing such as wound closure strips or skin glue. Leave the fascia open to allow room for the muscle compartment swelling. An occlusive dressing may be used to prevent contamination. Post-operative surgical / medical management.

[00597] Use mild analgesics as needed. Monitor for signs of infection or dehiscence.

[00598] If the donor is a living related donor as for lung, kidney, intestine, or partial liver, a portion of that solid organ donors’ thymus may be adequate for culture and implantation. The pathology criteria listed above for the day of harvest and until implantation would need to be met.

[00599] Cryopreserved-cultured thymus tissue may be available from a 3™ party donor. However, the 3™ party donor must express all the recipient HLA alleles that are not expressed by the solid organ donor. This includes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA- DQAL1, HLA-DPBI, and HLA-DPAI1. Mismatching in HLA-DP alleles is acceptable if the mismatch is “permissive.” In other alleles minor mismatching is allowed, e.g., HLA-A*01:02 into a recipient carrying HLA-A*01:01, in other words, the second field (after the colon) can be different, the first field (before the colon) must be identical. Human Heart Transplant Procedures

[00600] Eligibility of a subject is determined based on a number of criteria including: Poor 12 to 24 month prognosis without cardiac transplantation despite current maximum supportive therapies; congenital or acquired heart disease with failure to thrive as defined by UNOS criteria; symptoms of advanced heart failure in the setting of congenital or acquired heart disease refractory to medical therapy; abnormal hemodynamics or increasing pulmonary vascular resistance; inoperable structural heart disease; symptomatic arrhythmias not amenable to medication or device therapy or poor exercise tolerance.

[00601] A number of absolute and relative contraindications to heart transplant surgery are assessed, including, for example, reversible renal dysfunction unless the subject is a candidate for heart / kidney transplant; irreversible liver disease unless candidate for heart / liver transplant; irreversible pulmonary dysfunction, use of non-conventional mechanical ventilator support (i.e. high frequency ventilator, maximal settings of CMV) or fixed pulmonary hypertension (TPG>15) unless the subject is a candidate for a heart-lung transplant; Diabetes mellitus with microvascular disease; or active, uncontrolled seizure disorder.

[00602] Other contraindications may include other diseases limiting long term survival and rehabilitation following a heart transplant; substance abuse, morbid obesity, malignancies; active psychiatric disorder; and other reasons such as documented medical non-compliance.

[00603] Relative contraindications include active infections; cognitive dysfunction; inadequate vascular access and significant allosensitization.

[00604] Donor and recipient histocompatibility management is also considered. Patients are evaluated for panel reactive antibodies (“PRA”) for pre-formed HLA antibodies that the recipient may have formed in response to a “sensitizing event” such as prior transfusions or major surgeries using bypass / blood products or homograft materials.

[00605] An assessment is made of the recipient’s presensitization history, for example, previous blood transfusions; number, dates; previous surgeries; previous pregnancies; immunization history and IVIG administration (with dates).

[00606] Patients with an excessively high HLA-Class I or Class-II PRA or those undergoing repeat transplant may be candidates for desensitization strategies. Specific strategies will be individualized to the potential recipient and may include use of plasmapheresis, IVIG, rituximab and / or bortezomib. Significant antibodies are those that remain present after a 1:16 dilution.

[00607] Pre-sensitized patients may require an actual prospective cross-match with potential donors. Patients with a history of HLA antibodies will undergo virtual cross-match in UNET at the time a transplant is being considered.

[00608] Pre-sensitized patients may require an actual prospective cross-match with potential donors via the following general guidelines. All patients with a history of HLA antibodies will undergo virtual cross-match in UNET at the time an offer is being considered.

[00609] If the cPRA < 20%: virtual cross-match in UNET, no additional measures are needed; proceed with routine retrospective donor cross-match and routine immunosuppression.

[00610] If cPRA > 20%: virtual cross-match in UNET, recipient receives plasmapheresis in the operating room (“OR”) at the time of transplant.

[00611] If cPRA > 70%: virtual cross-match, consider obtaining an actual prospective donor cross-match if time allows; administer plasmapheresis in the OR. Consider placement of a pheresis catheter in the OR for post-op continuation of pheresis.

[00612] Ongoing antibody reduction interventions are determined by donor cross-match, DSA’s and clinical course.

[00613] For all pre-sensitized patients, blood will be sent for donor-specific antibodies within the first two weeks post-transplant and repeated as clinically indicated. Routine testing for DSA will be done on all post-transplant patients at least every 6 months post-transplant and as needed if any clinical concerns.

[00614] Standard blood product transfusion protocols are followed both pre- and post- transplantation. Immunosuppression Management

[00615] Immunosuppression management is determined based on the solid organ to be transplanted. Pediatric Heart Transplants

[00616] All patients will receive induction therapy with basiliximab or anti-thymocyte globulin based on their clinical situation and risk factors. The type of induction therapy used will be determined prior to transplant (First Inmunosuppression Regimen drug therapy).

[00617] In an embodiment, pre-transplant / induction therapy typically comprises administration of:

[00618] Mycophenolate mofetil (CellCept®) 25 mg / kg IV prior to the operating room.

[00619] Methylprednisolone 10 mg / kg IV on induction (max dose 500 mg).

[00620] Methylprednisolone 10 mg / kg IV on release of x-clamp (max dose 500 mg).

[00621] ATG (antithymocyte globulin) 1.5 mg / kg IV on release of x-clamp.

[00622] Alternatively use Basiliximab (Simulect®), dosed by weight of recipient.

[00623] <35 kg, 10 mg on release of x-clamp and 2nd dose 4 days later.

[00624] >35 kg, 20 mg on release of x-clamp and 2nd dose 4 days later.

[00625] In an embodiment pre-transplant induction immunosuppressive therapy for heart and CTT implant candidates may comprise:

[00626] Basiliximab (Simulect®)—(if cannot give ATG).

[00627] Dosing :

[00628] <35kg: Initial dose: 10 mg IV administered preoperatively by anesthesia on induction of anesthesia (after stable and methylprednisolone given).

[00629] Second dose: 10 mg IV administered 4 days after transplantation; hold second dose if complications occur (including severe hypersensitivity reactions or graft loss)

[00630] >35kg: Initial dose: 20 mg IV administered preoperatively by anesthesia on induction of anesthesia when stable.

[00631] Second dose: 20 mg IV administered 4 days after transplantation; hold second dose if complications occur (including severe hypersensitivity reactions or graft loss.

[00632] Administration: IV infusion over 20-30 minutes. Half-life children 1-11 years: 9.5 days, adolescents 12-16 years: 9.1 days, adults: 7.2 days.

[00633] In an embodiment, antithymocyte globulin (ATG, rabbit derived, Thymoglobulin®), may be given according to the following dosing schedule for heart transplant subjects:

[00634] 1.5mg / kg / day for 3 to 7 days based on lymphocyte count and markers and platelet count. The first dose is given after organ reperfusion on release of cross-clamp after second dose of steroids (intra-operatively).

[00635] Continued daily based on above parameters, will be determined by transplant MD.

[00636] Administration: Slow IV infusion over 6 hours for first dose, subsequent doses can be given over 4 hours as tolerated.

[00637] In an embodiment, in addition to routine preoperative / intraoperative methylprednisolone for CPB / pump cases, patients may receive a dose of methylprednisolone (SoluMedrol) 10mg / kg (max dose 500mg) IV to be administered by anesthesia on induction of anesthesia before basiliximab and then a second dose of 10mg / kg (max 500mg) on reperfusion (before ATG). Post-operative Maintenance Immunosuppression (Maintenance im...

Claims

Claims ‘What is claimed is:

1. A method of producing an allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of subjecting donor thymus to a conditioning regimen for a period from about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; further comprising detecting increasing levels of CCL21 in the thymus organ medium during the course of the conditioning regimen; and recovering the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product suitable for implantation. 2% The method according to claim 1, further comprising the step of cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen for future implantation.

3. The method according to claim 1 or 2, further comprising detecting decreasing levels of L-selectin in the thymus organ medium during the course of the conditioning regimen.

4. The method according to any one of claims 1 to 3, further comprising detecting decreasing levels of one or more of M-CSF, galectin-7 or IL-16 in the thymus organ medium during the course of the conditioning regimen. 5 The method according to claim 4, wherein M-CSF decreases in the thymus organ medium during the course of the conditioning regimen.

6. The method according to claim 4, wherein galectin-7 decreases in the thymus organ medium during the course of the conditioning regimen.

7. The method according to claim 4, wherein IL-16 decreases in the thymus organ medium during the course of the conditioning regimen.

8. The method according to any one of claims 1 to 7, further comprising detecting increasing levels of one or more of CXCL12, CXCL16 or CCL11 in the thymus organ medium during the course of the conditioning regimen.

9. The method according to claim 8, wherein CXCL12 increases in the thymus organ medium during the course of the conditioning regimen.

10. The method according to claim 8, wherein CXCL16 increases in the thymus organ medium during the course of the conditioning regimen.

11. The method according to claim 8, wherein CCL11 increases in the thymus organ medium during the course of the conditioning regimen.

12. The method according to any one of claims 1-11, wherein the conditioning regimen is for a period of six days, or seven days, or eight days, or nine days, or 10 days, or 11 days, or 12 days, or 13 days, or 14 days, or 15 days, or 16 days, or 17 days, or 18 days or 19 days, or 20 days, or 21days; or for a period of 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, or 6 to 7 days, or 6 to 8 days, or 6 to 9 days, or 6 to 10 days, or 6 to 11 days, or 6 to 12 days, or 6 to 21 days, or 7 to 21 days, or 8 to 21 days, or 9 to 21 days, or 10 to 21 days, or 11 to 21 days, or 12 to 21 days, 13 to 21 days or 14 to 21 days, or 15 to 21 days, or 16 to 21 days or 17 to 21 days or 18 to 21 days, or 19 to 21 days, or 20 to 21 days.

13. The method according to claim 8, wherein the levels of CCL21 approximate the levels in Fig. 50E.

14. The method according to claim 8, wherein the levels of L-selectin approximate the levels in Fig. S50A.

15. The method according to claim 8, wherein the levels of M-CSF approximate the levels in Fig. 50B.

16. The method according to claim 8, wherein the levels of galectin-7 approximate the levels in Fig. 50C.

17. The method according to claim 8, wherein the levels of IL-16 approximate the levels in Fig. 50D.

18. The method according to claim 8, wherein the levels of CXCL16 approximate the levels in Fig. SOF.

19. The method according to claim 8, wherein the levels of CCL11 approximate the levels in Fig. 50G.

20. The method according to claim 8, wherein the levels of CXL21 approximate the levels in Fig. 50H.

21. A method of producing an allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of subjecting donor thymus to a conditioning regimen for a period from about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; further comprising detecting levels of at least one biomarker selected from L-selectin, CXCL16, M- CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL- 6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK- 1 and ANG-1in the thymus organ medium during the course of the conditioning regimen; and recovering the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product suitable for implantation.

22. The method of any one of the preceding claims, further comprising the step of: determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK 14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

23. A method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1. 24, The method according to claim 24, wherein the at least one marker is selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

25. The method according to claim 23 or 24, wherein the at least one marker is L-selectin.

26. The method according to claim 25, wherein the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.

27. The method according to claim 23 or 24, wherein the at least one marker is M-CSF.

28. The method according to claim 27, wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen. 29, The method according to claim 23 or 24, wherein the at least one marker is galectin-7.

30. The method according to claim 29, wherein the levels of galectin- 7 in the thymus organ medium decrease during the course of the conditioning regimen.

31. The method according to claim 23 or 24, wherein the at least one marker is IL-16.

32. The method according to claim 31, wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen 33. The method according to claim 23 or 24, wherein the at least one marker is CCL21.

34. The method according to claim 33, wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.

35. The method according to claim 23 or 24, wherein the at least one marker is CXCL12.

36. The method according to claim 35, wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.

37. The method according to claim 23 or 24, wherein the at least one marker is CXCL16.

38. The method according to claim 37, wherein the levels of CXCL16 lin the thymus organ medium increase during the course of the conditioning regimen.

39. The method according to claim 23 or 24, wherein the at least one marker is CCL11.

40. The method according to claim 39, wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

41. The method of any one of preceding claims 23 to 40, further comprising the step of: determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

42. A method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers in Fig.

56.

43. A method for determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human, the method comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-BI, CEACAM-1, IL-1b, DKK-1 and ANG-1.

44. The method according to any one of claims 23 to 43, wherein detecting the level of at least one marker in the thymus organ medium during the culturing regimen, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or detecting at least eight markers in the thymus organ medium during the conditioning regimen, are selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11 45. Ina method of treating a thymic disorder in a subject, the improvement comprising implanting into the subject having a thymic disorder allogeneic cultured postnatal thymus tissue- derived slices subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; and detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

46. The method according to claim 45, wherein the at least one marker is selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

47. The method according to claim 45 or 46, wherein the at least one marker is L-selectin.

48. The method according to claim 47, wherein the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.

49. The method according to claim 45 or 46, wherein the at least one marker is M-CSF.

50. The method according to claim 49, wherein the levels of M-CSF in the thymus organ medium decrease during the course of the conditioning regimen.

51. The method according to claim 45 or 46, wherein the at least one marker is galectin-7.

52. The method according to claim 51, wherein the levels of galectin- 7 in the thymus organ medium decrease during the course of the conditioning regimen.

53. The method according to claim 45 or 46, wherein the at least one marker is IL-16.

54. The method according to claim 53, wherein the levels of IL-16 in the thymus organ medium decrease during the course of the conditioning regimen 55. The method according to claim 45 or 46, wherein the at least one marker is CCL21.

56. The method according to claim 55, wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.

57. The method according to claim 45 or 46, wherein the at least one marker is CXCL12.

58. The method according to claim 57, wherein the levels of CXCL12 in the thymus organ medium increase during the course of the conditioning regimen.

59. The method according to claim 45 or 46, wherein the at least one marker is CXCL16.

60. The method according to claim 59, wherein the levels of CXCL16 in the thymus organ medium increase during the course of the conditioning regimen.

61. The method according to claim 45 or 46, wherein the at least one marker is CCL11.

62. The method according to claim 61, wherein the levels of CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

63. The method of any one of preceding claims 45 to 62, further comprising the step of: determining in the donor thymus tissue slices during the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

64. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with complete DiGeorge syndrome.

65. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with 22q11.2 deletion.

66. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with CHARGE (coloboma, heart defect, choanal atresia, growth or mental retardation, genital hypoplasia and ear anomalies or deafness).

67. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene.

68. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with forkhead box protein N1 (FOXN1) deficiency.

69. The method of any one of claims 45 to 63, wherein the thymic disorder is congenital athymia associated with mutations in the TBX-1 or TBX-2 gene.

70. The method of any one of claims 45 to 63, wherein the thymic disorder is age-related thymic involution.

71. The method of any one of claims 45 to 63, wherein the thymic disorder is associated with a thymoma.

72. The method of claim 71, wherein the thymoma is malignant.

73. The method of claim 71, wherein the thymoma is non-malignant.

74. The method of any one of claims 45 to 63, wherein the thymic disorder is associated with myasthenia gravis (MG), pure red cell aplasia and hypogammaglobulinemia.

75. Ina method for providing immune-competence in a human subject, the improvement comprising the steps of conditioning donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers from L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP- 1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

76. The method according to claim 75, wherein the at least one marker is selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11, and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

77. A method for providing immune-competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HL A-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regiment is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

78. The method according to claim 77, wherein the at least one marker is selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

79. A method for providing immune-competence in a human subject undergoing a solid organ transplant, the method comprising the following steps of removing the thymus of the human subject; obtaining thymus tissue from a donor matching HL A-Class I and HLA-Class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymus tissue slices in a thymus organ medium for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

80. The method according to claim 79, wherein the at least one marker is selected from L- selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein the level is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; implanting the solid organ; and implanting the partially T-cell depleted donor thymus tissue slices into the human subject.

81. A method of promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased donor, in a recipient in need of a solid organ transplant, the method comprising the following steps: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing both a suitable solid human organ and a thymus gland from a deceased donor; (d) transplanting the solid human organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing an allogeneic cultured postnatal thymus tissue-derived product, wherein the allogeneic cultured postnatal thymus tissue-derived product is subjected to a conditioning regimen in a thymus organ medium for a period of about 6 days to about 21 days to produce allogeneic cultured postnatal thymus tissue-derived product slices; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in the thymus organ medium to produce partially T-cell depleted donor thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, [CAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; and (g) implanting the allogeneic cultured postnatal thymus tissue-derived product into the recipient after up to 21 days of conditioning regimen, wherein the dosage of thymus tissue slices is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

82. The method according to claim 81, wherein the at least one marker is selected L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; and (g) implanting the allogeneic cultured postnatal thymus tissue-derived product into the recipient after up to 21 days of conditioning regimen, wherein the dosage of thymus tissue slices is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m?, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

83. A method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a living human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product was processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA- Class I and HLA-Class II alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices; detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen is selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP- 3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP- 3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in for the marker in Fig. 7; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product, and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient. 84, The method according to claim 83, wherein the at least one marker is selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product, and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

85. The method according to claim 83 or 84, wherein HLA matching permits minor variations in amino acid sequence of the alleles in the second field.

86. The method according to 83 or 84, wherein HLA matching permit mismatches for HLA- DP alleles in the second field.

87. The method according to claim 83 or 84, wherein about one-half of the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product is transplanted into the recipient and the remainder is cryopreserved for future use.

88. The method according to claim 83 or 84, wherein step (h) is performed about one month or more after the transplantation of the solid organ.

89. A method for promoting donor-specific tolerance to an allogeneic solid organ transplant obtained from a deceased human donor, in a human recipient in need of a solid organ transplant, the method comprising the steps of: (a) removal of the thymus of the recipient; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agent to deplete the recipient’s T cells and / or to suppress the recipient’s T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor expressing HLA alleles matched to HLA alleles in the recipient that are not present in the solid organ transplant; wherein the donor thymus tissue was subjected to a conditioning regimen for a period of about 6 days to about 21 days; further wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T-cell depleted thymus tissue slices, detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 56; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue- derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

90. The method according to claim 89, wherein the at least one marker is selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymus organ medium; wherein the level of the marker in the thymus organ medium is decreased if the marker is L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dosage of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1,000 - 22,000 mm? of thymus tissue surface area / recipient body surface area in m2, and further wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

91. A cryopreserved allogeneic cultured postnatal thymus tissue-derived product, prepared by a method comprising the steps of: (a) obtaining suitable thymus tissue from a donor; (b) typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB 1, HLA-DQB 1, HLA- DRB3, HLA-DRB4, HLA-DRBS, HLA-DQA1, HLA- DPB1, HLA-DPA1: (c) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; (d) detecting the level of at least one marker in the thymus organ medium during the course of the conditioning regimen selected from the markers L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1; wherein the level of the marker in the thymus organ medium is increased or decreased in accordance with the levels in Fig. 7; (e) retrieving the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (f) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (g) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

92. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product according to claim 91, wherein the at least one marker is selected from L-selectin, M-CSF, galectin-7, or IL-1 or increased if the marker is CCL21, CXCL12, CXCL16, or CCL11; (e) retrieving the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (f) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (g) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue- derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

93. The method according to any one of claims 77-89, wherein the solid organ transplant is a heart transplant, a kidney transplant, a liver transplant, a lung transplant, a heart / lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, an abdominal wall transplant, a craniofacial transplant, a scalp transplant, a penile transplant, a uterus transplant, a unilateral or bilateral upper limb transplant, a unilateral vascularized composite allograft, or combination thereof 94. The method according to claim 93, wherein the solid organ transplant is a heart transplant, 95. The method according to claim 93, wherein the heart transplant is a pediatric heart transplant, 96. The method according to claim 93, wherein the heart transplant is an adult heart transplant.

97. The method according to any one of the preceding claims, wherein the conditioning regimen is 5 days.

98. The method according to any one of the preceding claims, wherein the conditioning regimen is 6 days.

99. The method according to any one of the preceding claims, wherein the conditioning regimen is 7 days.

100. The method according to any one of the preceding claims, wherein the conditioning regimen is 8 days.

101. The method according to any one of the preceding claims, wherein the conditioning regimen is 9 days.

102. The method according to any one of the preceding claims, wherein the conditioning regimen is 10 days.

103. The method according to any one of the preceding claims, wherein the conditioning regimen is 11 days.

104. The method according to any one of the preceding claims, wherein the conditioning regimen is 12 days.

105. The method according to any one of the preceding claims, wherein the conditioning regimen is 13 days.

106. The method according to any one of the preceding claims, wherein the conditioning regimen is 14 days.

107. The method according to any one of the preceding claims, wherein the conditioning regimen is 15 days.

108. The method according to any one of the preceding claims, wherein the conditioning regimen is 16 days.

109. The method according to any one of the preceding claims, wherein the conditioning regimen is 17 days.

110. The method according to any one of the preceding claims, wherein the conditioning regimen is 18 days.

111. The method according to any one of the preceding claims, wherein the conditioning regimen is 19 days.

112. The method according to any one of the preceding claims, wherein the conditioning regimen is 20 days.

113. The method according to any one of the preceding claims, wherein the conditioning regimen is 21 days.

114. A cryopreserved allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of: (a) obtaining suitable thymus tissue from a human donor; (b) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; wherein levels of L-selectin, and / or M-CSF and / or galectin-7 and / or IL-16 in the thymus organ medium decrease during the course of the conditioning regimen; further wherein levels of CCL21 and / or CXCL12 and / or CXCL16 and / or CCL11 in the thymus organ medium increase during the course of the conditioning regimen; (c) harvesting the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (d) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (¢) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue- derived product bank.

115. A cryopreserved allogeneic cultured postnatal thymus tissue-derived product suitable for implantation into a human, comprising the steps of: (a) obtaining suitable thymus tissue from a human donor; (b) subjecting the thymus tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymus tissue comprises aseptically processing the donor thymus tissue in a thymus organ medium to produce partially T- cell depleted donor thymus tissue slices; wherein levels of L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP- 1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 in the thymus organ medium increase or decrease during the course of the conditioning regimen in accordance with the levels in Fig. 56; (c) harvesting the partially T-cell depleted donor thymus tissue slices as allogeneic cultured postnatal thymus tissue-derived product; (d) cryopreserving the allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen; and (¢) maintaining the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue- derived product bank.

116. The cryopreserved allogeneic cultured postnatal thymus tissue-derived product of claim 114 or 115, wherein the levels of CCL21 in the thymus organ medium increase during the course of the conditioning regimen.

117. The cryopreserved allogeneic cryopreserved cultured postnatal thymus tissue-derived product of claim 114 to 116, wherein the levels of L-selectin in the thymus organ medium decrease during the course of the conditioning regimen.

118. The cryopreserved allogeneic cryopreserved cultured postnatal thymus tissue-derived product of claim 114 to 117, wherein the levels of one or more of M-CSF, galectin-7, and IL-16 in the thymus organ medium decrease during the course of the conditioning regimen.

119. The cryopreserved allogeneic cryopreserved cultured postnatal thymus tissue-derived product of claim 114 to 118, wherein the levels of one or more of, CXCL12, CXCL16, and CCL11 in the thymus organ medium increase during the course of the conditioning regimen.

120. The cryopreserved allogeneic cryopreserved cultured postnatal thymus tissue-derived product of any one of preceding claims 114 to 119, further comprising the step of: determining in the donor thymus tissue slices between about 6 days to about 21 days of the conditioning regimen areas positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices the presence of at least one Hassall body, CK 14 staining scattered throughout the donor thymus tissue slices and the presence of intact nuclei.

121. A kit for performing the methods of any one of the preceding claims, together with instructions for use in determining whether allogeneic cultured postnatal thymus tissue-derived product is suitable for implantation into a human.

122. A kit for performing the method of any one of the preceding claims, wherein the kit comprises at least one antibody that specifically binds marker L-selectin, CXCL16, M-CSF, CCL21 / (Ckin), galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL- 12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

123. A kit according to claim 122, wherein the antibody specifically binds to L-selectin, M- CSE, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

124. A kit for determining whether cryopreserved allogeneic cultured postnatal thymus tissue- derived product cultured in accordance with any one of claims 115 to 120 is suitable for implantation into a human together with instructions for use.

125. A kit according to claim 110, wherein the kit comprises at least one antibody that specifically binds marker L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP- 1b, PIGF, PF4, CCL11 / Eotaxin, HVEM, IGFBP-6, IL-6R. IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1.

126. The kit according to claim 125, wherein the marker is selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

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