Method for determining the suitability of cultured thymic tissue for implantation into a human and related methods of use

Abstract

Methods and compositions for promoting donor - specific tolerance and immune competence in a recipient of a solid organ graft, which are effected by: implanting an allogeneic solid organ in a recipient in need of a solid organ transplant, and further comprising surgically implanting a tissue - engineered allogeneic cultured post - natal thymic tissue product in the recipient of the solid organ from the donor. A method for producing an allogeneic cultured post - natal thymic tissue - derived product suitable for implantation into a human; a method for culturing an allogeneic cultured post - natal thymic tissue - derived product suitable for implantation into a human; and a method for using an allogeneic cultured post - natal thymic tissue - derived product by implanting into a human subject.

Description

Technical Field

[0001] Cultured pediatric thymic tissue that can be used to determine T cell exhaustion (also known as allogeneic cultured postnatal thymic tissue-derived product (or sometimes "CTT")), which is used as a biomarker for the viability and suitability of implantation and T cell reconstitution in subjects with thymic disorders, including congenital athymia and other immune system dysfunctions caused by thymic disorders. Methods and compositions for promoting donor-specific tolerance to allogeneic solid organ grafts in recipients receiving allogeneic solid organ grafts from a donor. Background Art

[0002] Organ transplantation requires the preparation and harvesting of human solid organs from a donor and transplantation into a recipient. The major problem with solid organ transplantation is recipient intolerance to the donor. Recipient T cells will reject the organ, and recipient B cells will produce antibodies against the organ, leading to eventual organ failure. The essence of solid organ transplantation is to generate recipient tolerance to the transplanted human organ. It is estimated that more than 36,000 organ transplants are performed annually in the United States, and the number of organ transplants in Europe and other major countries is even higher. It is further estimated that there are more than 120,000 patients on the organ transplant waiting list in the United States. The demand for healthy organs far 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 major challenge in solid organ transplantation. Transplant rejection by both T cells and B cells can lead to serious complications of organ function or even transplant failure. For example, the 5-year transplant survival rate is 77.7% for heart transplants, 78.6% for kidney transplants, 72.8% for liver transplants, and 53.4% for lung transplants. Generally, this problem has been partially addressed by matching the major histocompatibility complex (MHC) antigens of the donor and recipient and by avoiding recipients with antibodies against the recipient tissue type. In addition, the use of immunosuppressive regimens to manage the immunological response behind transplant rejection has also been improved. However, tolerance has not been achieved, and the average survival period of many organs is only 10 years.

[0004] Maintaining organ viability before and during the implantation procedure is a second major challenge. The removal, storage, and transplantation of an organ can profoundly affect the internal structure and function of the organ and can significantly affect the extent to which normal organ function recovery is delayed or prevented after transplantation is completed.

[0005] The period of time during which an intact human organ can be effectively preserved varies depending on the organ, with kidneys in the range of 24 - 36 hours, pancreas in the range of 12 - 18 hours, liver in the range of 8 - 12 hours, and heart and lungs in the range of 4 - 6 hours. See https: / / unos.org / transplantation / matching-organs .

[0006] Organ injury occurs primarily due to ischemia and hypothermia, but may also be associated with reperfusion of the organ during ex vivo or implantation.

[0007] Techniques for organ preservation (including ex vivo perfusion) are known in the art and are used to minimize organ injury and promote optimal graft survival and function.

[0008] The major solid organs that have been the subject of transplantation procedures include the kidney, liver, heart, and lungs. Varying degrees of success have been achieved in successfully transplanting these solid organs. The major variability lies in the techniques used to interfere with immune-mediated transplant rejection. Experience has shown that no single immunosuppressant or technique can be used in all settings involving solid organ transplantation. The limiting factor is often the toxicity associated with each individual immunosuppressant. The toxicity associated with a given immunosuppressant may often impede the normal function of the transplanted solid organ or other organs such as the kidney, which may fail when calcineurin inhibitors are used to prevent rejection.

[0009] The toxic drawbacks associated with known immunosuppressants commonly used to prevent transplant rejection in solid organ grafts have created a need to find new ways to prevent transplant rejection in solid organ grafts.

[0010] The ability to distinguish self from non-self antigens is key to the immune response. This discrimination leads to self-tolerance. When self-tolerance is lost, autoimmunity results. There is an unmet need to induce tolerance to solid organ grafts in transplantation procedures.

[0011] For allogeneic cultured postnatal thymic tissue-derived products in subjects with thymic disorders (including congenital athymia and other immune system dysfunctions due to thymic disorders), maintaining the viability of the thymic tissue before and during the implantation procedure is an important factor in various aspects and embodiments of practicing the present disclosure.

[0012] The thymus is necessary for the production of T cells that can appropriately respond to foreign antigens and pathogens while avoiding damage to self-reactive ones.

[0013] The thymus is large in infancy (due to its need to establish an initial T cell repertoire), but quickly becomes quiescent and then undergoes a slow process of continuous involution throughout adulthood. Work over the past two to three decades has established that, although the total output of the adult thymus is reduced, the organ remains crucial for generating T cells with novel specificities that can protect against infectious diseases or cancer and for repopulating the repertoire after 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 focused on identifying mechanisms that govern age-related thymic involution in adults and methods to promote thymic regeneration after immune injury.

[0014] Age-related thymic involution in humans is characterized by a loss of thymocyte production and a reduction in the number of thymic epithelial cells, with the thymic parenchyma being replaced by adipose tissue. Determining whether the mechanisms driving these changes are intrinsic or extrinsic to the thymus has proven difficult to resolve using animal models (due to the constant trafficking to and from the thymus) and is generally not possible to address in living humans.

[0015] Organ cultures of thymic tissue from younger donors can be used to evaluate these issues because in vitro cultures of human thymic slices result in the depletion of thymocytes while largely maintaining the viability and function of thymic epithelial and stromal cells. This is demonstrated by the ability of these slices to grow as a monolayer (Markert et al., 1997b) and to promote T cell reconstitution when implanted into 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 culture could mimic acute and chronic involution and contribute to the identification of mechanisms that drive changes in the thymic microenvironment during aging.

[0016] It has been shown that allogeneic cultured postnatal thymus tissue-derived products can be used to treat T cell immunodeficiencies (primary immunodeficiencies) caused by congenital athymia, such as for the treatment of complete DiGeorge anomaly (cDGA) associated with 22q11.2 deletion and CHARGE (coloboma, heart defects, 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, as well as athymic patients with a deficiency in forkhead box protein N1 (FOXN1). Congenital athymia is a rare and fatal condition, and there are currently no pharmaceutical treatment options using regulatory-approved drug products.

[0017] Experimental implantation of allogeneic cultured postnatal thymus tissue-derived products that retain thymic 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 c J Med 341(16):1180-1189 27).

[0018] In that reference, DiGeorge syndrome was defined as a condition with variable defects in the heart, thymus, and parathyroid glands. Approximately 1% of infants with DiGeorge syndrome have athymia and are thus unable to produce mature and infection-resistant naive T cells. These infants are said to have complete DiGeorge syndrome. Not meant to be inclusive, there are subgroups of children who meet the criteria for complete DiGeorge syndrome, 22q11.2 deletion syndrome CHARGE, infants of diabetic mothers, and infants without symptoms or genetic defects. Congenital athymia may also be associated with mutations in the TBX1 or TBX2 genes.

[0019] A product derived from cultured postnatal thymic tissue of allogeneic origin is a tissue-engineered product that is prepared, cultured, and stored for up to 21 days (e.g., a culture protocol of about 6 to about 21 days) to produce a partially T cell-depleted thymic tissue slice, and the product is differentiated from natural thymus by a conditioning process. The conditioning protocol partially depletes donor thymocytes from the cultured thymic tissue slice. Based on in vitro data (immunohistochemistry), the epithelial network is preserved during the culture period between 6 and 21 days, as evaluated using cytokeratin antibodies. The culture is preferably carried out in a 5% CO2 incubator at 37°C.

[0020] The culturing process significantly alters the biological characteristics of the donor thymic tissue and the constituent cells contained therein in the following manner to optimize the effective therapeutic properties of the slices of the product derived from allogeneically cultured postnatal thymic tissue. The culturing process ensures the acquisition of a defined composition of cultured cells / tissue with prerequisite biological characteristics in a manner suitable for surgical implantation into a subject to enable the reconstitution of the subject's immune system.

[0021] The culturing process results in the loss of thymocytes and the relative enrichment of TECs and other stromal cells in the donor thymic tissue slice. The culturing process further results in thymocyte depletion and the maintenance of TECs to enable the reconstitution of the recipient's immune system and allows for the generation of tolerance to HLA antigens in the donor thymus in the recipient. Overall, the culturing process is designed to deplete many thymocytes from the donor thymic tissue and 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 thymic seeding progenitors.

[0022] The culturing process is described in WO2019 / 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 slices of the product derived from allogeneically cultured postnatal thymic tissue is described in PCT / US2019 / 040275, which is incorporated by reference herein.

[0023] Surgical administration of an allogeneic cultured postnatal thymic tissue-derived product (e.g., also known as “RVT-802”) in athymic patients leads to a series of events that result in the development of a functional immune system. After surgical placement of the allogeneic cultured postnatal thymic tissue-derived product in a recipient, T cells are educated by donor TECs and recipient dendritic cells (DCs). The association of donor TECs with recipient DCs enables tolerance to the implanted donor thymic tissue that is implanted as cultured thymic tissue slices. This is the same as tolerance induction in the normal thymus. The association of donor TECs with recipient DCs results in tolerance to self.

[0024] Thymopoiesis in implanted donor thymic tissue has been demonstrated by allograft biopsy 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 hereby incorporated by reference.

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

[0026] Historically, the release criteria for postnatal thymus tissue-derived products cultured allogeneically have included histopathological evaluation of H&E and immunohistochemical tissue sections at intermediate points in the production process (which were later refined to days 6-21 of the culture period). This histopathological evaluation served as a potency assay and was performed as a qualitative analysis by board-certified pathologists. Samples for evaluation were prepared by freezing or formalin fixation, followed by sectioning of the tissue and then mounting onto slides. Samples prepared in this way were stable over long periods of time, allowing for reanalysis.

[0027] Historical samples obtained from over 20 years of development can be correlated with positive clinical outcomes and thus provide a powerful dataset for the development of quantitative histological assays for evaluating product quality.

[0028] A novel digital histological assay was developed using scanned images from previous clinical batches and experimental batches of postnatal thymus tissue-derived products cultured allogeneically. These images were analyzed to develop a quantitative assay. The digital histological assay is more fully described in PCT / US2019 / 040275.

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

[0030] Although there have been the aforementioned successes in implanting allogeneic culture-derived postnatal thymic tissue products in children with congenital athymia, there remains a need to identify cultured thymic tissue that is viable, functional, and suitable for implantation to achieve immune reconstitution. Summary of the Invention

[0031] Achieving donor-specific immune tolerance remains the ultimate immunological goal in transplantation. Most current methods focus on controlling peripheral mature donor-reactive T cells by depletion (e.g., alemtuzumab, thymoglobulin, etc.) or inhibition (e.g., calcineurin inhibitors, basiliximab, etc.), without targeting the generation of alloreactive T cells in the thymus. However, even with the great progress made in immunosuppressive drugs and novel immunomodulatory regimens, transplantation tolerance has not been consistently achieved.

[0032] The present inventors have shown that tolerance to solid organ grafts can be achieved by implanting an allogeneic culture-derived postnatal thymic tissue product (also referred to herein as "CTT" or "RVT-802") in thymectomized recipients to shorten the use of immunosuppressive agents after transplantation, thereby preventing rejection of the transplanted organ. Removing the recipient's thymus and replacing it with an allogeneic culture-derived postnatal thymic tissue product enables the reconstitution of the immune system of the solid organ recipient and induces tolerance to the recipient's own as well as the transplanted allogeneic solid organ.

[0033] Tolerance induction by surgical insertion of postnatal thymic tissue-derived products cultured allogeneically is similar to tolerance induction via donor dendritic cells (“DCs”) in hematopoietic stem cell transplantation (Sharabi Y and 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 and 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 conducted by the Transplantation Biology Research Center (TBRC, Boston, Massachusetts) have shown the crucial role of the thymus in tolerance induction (Yamada K et 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 thymic transplantation with tolerance induction has been tested in large animal models (Yamada K et 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 et 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, the group successfully used HLA class II-matched / class I-mismatched donors (thymus and kidney or heart) as thymic composite tissues (thymokidney and thymocardium), in which 12-day cyclosporine (“CsA”) was used for graft tolerance induction.They claim that the non-vascularized thymus does not induce tolerance in their model. However, more precisely, uncultured non-vascularized thymus cannot be transplanted long-term. As they pointed out, the failure of uncultured thymus transplantation may be due to ischemic injury other than allogeneic immunity (Yamada K et al., 2000). Without using xenotransplantation, this ingenious concept of generating vascularized thymus before transplantation to induce tolerance will be difficult to translate into the clinic. (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. June 4, 2020; 5(11):e129983. doi:10.1172 / jci.insight.129983).

[0034] The limitations of non-vascularized thymus transplantation can be overcome by culture systems as well as T cell depletion. Experimental transplantation of allogeneic cultured postnatal thymus tissue-derived products (CTT) that retain 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):1180-118927).

[0035] In the foregoing reference, DiGeorge syndrome is defined as a condition with variable defects in the heart, thymus, and parathyroid glands. Approximately 1% of infants with DiGeorge syndrome have athymia and, thus, no T cells to fight infection. These infants are said to have complete DiGeorge syndrome. There are four subgroups of children who meet the criteria for complete DiGeorge syndrome, 22q11.2 deletion syndrome CHARGE, infants of diabetic mothers, and infants without symptoms or genetic defects. In all four groups, the infants with athymia represent only a small group, perhaps 1% of the total number of children carrying a diagnosis such as a diagnosis of 22q11.2 deletion syndrome.

[0036] Thymopoiesis has been demonstrated 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 have shown that the mixed lymphocyte reaction shows tolerance to donor MHC (Chinn IK, Devlin BH, Li YJ and Markert ML, 2008, “Long - term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277 - 281). In addition, after CTT implantation, infants with congenital athymia are able to control infections such as Epstein - Barr virus (Markert ML, 2014, Thymus Transplantation. Edited by Stiehm’s Immune Deficiences, Sullivan KE and Stiehm ER (Academic Press), 1st edition, pp. 1059 - 1067). Based on these data in people with congenital athymia, it was determined that a product derived from allogeneic cultured postnatal thymic tissue expressing the MHC of the solid organ donor would be tolerant to both self and donor while generating functional T cells that would protect the recipient from infection.

[0037] Domestication of the thymus and 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. June 4, 2020; 5(11):e129983. doi:10.1172 / jci.insight.129983.)

[0038] The thymus is typically located at the top of the heart. The thymus provides the necessary microenvironment for T cell development and is essential for 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 postnatal development, the thymus domesticates hematopoietic stem cells to migrate from the bone marrow to the thymus. Progenitor stem cells colonize the thymus and give rise to thymocytes. Thereafter, thymocytes undergo a series of maturation steps. This is demonstrated by the expression of a variety of observable cell surface markers that appear on thymocytes.

[0039] T cells are essential for protecting the body from infection. T cells developed in a normally functioning thymus develop a wide variety of T cell receptors (usually proteins on the cell surface), which enables mature T cells to resist a wide variety of infections. During this domestication process, the thymus instructs developing T cells not to attack normal body proteins, such as insulin or parathyroid hormone (which regulate the levels of glucose and calcium in the blood). These instructions are carried out under the influence of the autoimmune regulator gene (“AIRE gene”).

[0040] In short, the process of domestication occurs in a normally functioning thymus. Thymocytes present in the thymus are formed from bone marrow stem cells. Thymocytes are taught by thymic epithelial cells ("TEC") and dendritic cells ("DC") located within the thymus not to attack the recipient's major histocompatibility complex (MHC) proteins (antigens), such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, HLA-DPA1 antigens. HLA antigens have two proteins that hold self-peptides in a groove. The self-peptides can be from thyroid proteins or insulin peptides or almost any other protein expressed in the body. Thymocytes developing in the thymus form a T cell receptor (TCR) consisting of two transmembrane proteins. The TCR is expressed on the cell surface of T cells. Each T cell expresses many copies of its unique TCR. If the TCR binds too tightly to the self-peptide:MHC on the dendritic cell, the dendritic cell provides a signal for the T cell to undergo apoptosis and die. This mechanism prevents autoimmunity against self. The TEC can also provide signals to thymocytes that they are binding too tightly. Finally, the DC can capture a portion of the membrane from the TEC and present the TEC self-peptide:MHC to the developing thymocytes. If the thymocytes bind too tightly, the DC sends a signal for the thymocytes to undergo apoptosis and die. By these mechanisms, T cells leaving the thymus are not self-reactive. T cells leaving the thymus vary greatly and can recognize infections, but they do not attack the body's proteins.

[0041] The two main components of the thymus are epithelial cells and thymocytes that are produced in the thymus in the following manner. Cells derived from bone marrow stem cells (common lymphoid progenitors ("CLP")) migrate to the thymus to serve as early thymic progenitors. The CLP enter the thymus in response to signals (chemokines) produced by thymic epithelial cells and endothelial cells. In the thymus, the CLP differentiate into thymocytes and proliferate. Thymocytes produce 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 wide diversity of T cells are produced, enabling the cells to respond to infections throughout the recipient's life. The mixed lymphocyte reaction shows tolerance of recipient T cells to the MHC of the thymic donor in children treated with cultured thymic tissue (RVT-802).

[0042] Autoreactive recipient thymocytes are deleted before leaving the thymus. This occurs through the interaction of recipient thymocytes with recipient DCs that migrate into the thymus. As a measure to protect the body from autoimmune diseases, apoptosis is induced in recipient thymocytes that bind too tightly to DCs. After this process is completed, the thymocytes leave the thymus. Newly circulating T cells (i.e., thymic emigrants) express the markers CD31, CD45RA, and CD62L. After several weeks, the CD31 marker is no longer expressed. T cells expressing CD45RA and CD62L are called naive T cells. These recipient T cells normally proliferate in response to mitogens. They can protect the recipient from infection without being autoreactive to self.

[0043] Allogeneic cultured postnatal thymus tissue-derived product.

[0044] It has been shown that allogeneic cultured postnatal thymus tissue-derived products can be used to treat T cell immunodeficiencies (primary immunodeficiencies) caused by congenital athymia. T cell immunodeficiencies caused by athymia are associated with congenital disorders that prevent functional thymus development, such as complete DiGeorge anomaly (cDGA) associated with 22q11.2 deletion and CHARGE (coloboma, heart defects, choanal atresia, retarded 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, as well as athymic patients with forkhead box protein N1 (FOXN1) deficiency. Other genetic defects causing athymia include TBX1, TBX2, PAX1, and semaphorin 3E (SEMA3E), as well as 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 and fatal condition, and there are currently no pharmaceutical treatment options using regulatory-approved drug products. If untreated and without therapeutic reconstitution of the child's immune system, the major immunodeficiency caused by congenital athymia is fatal, and almost all infants die before the age of three, most often due to severe infections.

[0045] Allogeneic cultured postnatal thymus tissue-derived products are tissue-engineered products. Based on the disclosures in this specification and the examples, it is expected that CTT can be used to induce tolerance in recipients of transplanted solid organs.

[0046] As more fully described in this specification and the examples, surgical administration of an allogeneic culture-derived product from postnatal thymic tissue (e.g., “RVT-802”) in athymic patients results in a series of events that lead to the development of a functional immune system. After surgical placement of the allogeneic culture-derived product from postnatal thymic tissue (e.g., RVT-802) in a recipient, T cells are educated by donor TECs and recipient DCs. The association of donor TECs with recipient DCs enables tolerance to the implanted donor thymic tissue that is implanted as cultured thymic tissue slices. This is the same as tolerance induction in the normal thymus. As described in this specification, the association of recipient TECs with recipient DCs leads to tolerance to self.

[0047] This complex process has been clinically shown to result in a survival rate >70% in patients with congenital athymia who receive an allogeneic culture-derived product from postnatal thymic tissue (e.g., RVT-802) (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). Recipients are able to control viral infections that may be fatal prior to CTT, such as Epstein-Barr virus (Chinn IK, Devlin BH, Li YJ and Markert ML, 2008).

[0048] Overview of the combination of tolerance induction in solid organ grafts and CTT implantation

[0049] Based on the description, drawings, examples, and claims of this specification, the inventors have demonstrated that CTT induces donor-specific tolerance in a rat heart transplantation model. The experiments reported herein used a comparable CTT transplantation method that has been clinically used in subjects with congenital athymia, such as subjects afflicted with cDGA. Prior to surgical placement of CTT, cDGA infants essentially had no naive T cells. After surgical placement of CTT, the infants developed naive T cells approximately 6 months after the surgical procedure. (Markert ML et al., 2004, “Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome,” Blood 104(8):2574-2581; Markert ML, Devlin BH, and McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol 135(2):236-246). The studies on inducing tolerance in the rat model were based on the results of studies on transplantation of allogeneic postnatal cultured thymus tissue-derived products (CTT) in athymic infants with complete DiGeorge anomaly from 1993 to 2017. Favorable results were obtained in reported studies of surgical placement of CTT in infants with congenital athymia. The published results showed that in this otherwise fatal condition, the overall survival rate was 71% (61 / 86) (essentially all deaths occurred in the first 9-12 months at the time of this assessment; median 11.7 years, range from 1.2 years to 25 years) (Markert, ML et al., 2010). Biopsies of implanted cultured thymus tissue have immunohistochemically demonstrated thymopoiesis (Markert, ML et al., 2008). Flow cytometry and spectral typing have shown the development of a diverse T cell repertoire. Mixed lymphocyte reactions have shown tolerance of recipient T cells to antigen-presenting cells of the thymic donor (Chinn IK, Devlin BH, Li YJ, and Markert ML, 2008).

[0050] Importantly, recipients of CTT are able to control potentially lethal viral infections prior to CTT, such as Epstein-Barr virus (Markert, ML, 2014, Thymus Transplantation. In Stiehm’s Immune Deficiences, edited by Sullivan KE and Stiehm ER (Academic Press), 1st Edition, pp. 1059-1067). Based on these human data showing tolerance to mismatched thymic MHC antigens, the ability of CTT to induce donor-specific tolerance in a rat cardiac transplantation model was evaluated using the same methods used clinically for the engraftment of CTT in the rat model. These studies showed that transplantation of a mismatched heart together with donor CTT expressing MHC class I and class II antigens of the heart donor (by initially depleting T cells with anti-CD5 and immunosuppressing with cyclosporine) induced tolerance to the antigens of the donor heart while maintaining alloreactivity to other MHC antigens.

[0051] The present invention demonstrates co-transplantation of donor thymus with a solid organ as a method for tolerance induction to the transplanted solid organ in a recipient. The patient population that would benefit most from the procedure is adults with heart failure and infants in need of a heart transplant. Since postnatal thymic tissue exists and can be removed from a deceased infant and the recipient thymus is routinely removed from infants undergoing heart transplantation, no additional procedure is required to transfer the method to the clinic other than cultured thymic tissue transplantation (CTT). Similar transplants can also be performed in adults.

[0052] Overview of the preparation of allogeneic cultured postnatal thymic tissue-derived products

[0053] Allogeneic cultured postnatal thymic tissue-derived products are prepared, cultured, and stored for up to 21 days (e.g., a culture protocol of about 6 days to about 21 days), and on the day of implantation, they are placed in a separate sterile cup for transport to the operating room, as described in more detail herein.

[0054] CTT (cultured thymic tissue) is aseptically processed and cultured under current Good Manufacturing Practices (“cGMP”) (e.g., cGMP established by the U.S. Food and Drug Administration (“FDA”)) to produce partially T cell-depleted thymic tissue slices. CTT is distinct from native thymus by the conditioning process described in detail below. CTT affects the normal positive and negative selection processes of developing T cells in the thymus after implantation, enabling the T cells to be tolerant to both the donor thymus and donor solid organ grafts as well as recipient tissues. In addition, these T cells can recognize foreign antigens in the context of the recipient's major histocompatibility (MHC) proteins in order to resist infection.

[0055] The route of administration is by surgically implanting CTT in the manner described below. A single administration is typically 1000 to 22000 mm 2 of CTT per recipient body surface area (“BSA”). The surface area is the sum of the surface areas of all the cultured tissue slices. Individual CTT slices are implanted in a single administration surgical procedure. 2

[0056] Surgical implantation of an allogeneic cultured postnatal thymic tissue-derived product in athymic patients results in a series of events that lead to 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 (4th edition). London; pp. 1032-8).

[0057] The recipient's bone marrow CLPs migrate to the donor thymic graft, enter as early thymic progenitors and develop into recipient T cells there. The donor thymic graft provides the microenvironment in which recipient thymocytes generate a broad TCR repertoire capable of recognizing pathogens.

[0058] Recipient DCs migrate to the donor thymus to deplete self-reactive recipient thymocytes, which, after new T cells leave the thymus and enter the circulation, will attack the recipient's tissues. Genetically received naive T cells can be readily detected in the circulation approximately 5 - 12 months after administration. These recipient T cells have a diverse TCR repertoire and proliferate normally in response to mitogens. They can protect the recipient from infection without being autoreactive to self.

[0059] Recipient bone marrow CLPs migrate to the thymic allograft, where they develop into recipient T cells. Negative selection of recipient DCs that have migrated to the donor thymus results in tolerance to recipient MHC antigens. Immunohistochemical signs of thymopoiesis are observed in biopsies of implanted cultured thymic tissue obtained approximately 2 - 3 months after transplantation. Thymopoiesis reflects the ability of T cells to defend against and control infection as well as prevent autoimmune diseases.

[0060] Naive T cells are detected in the circulation 5 - 12 months after transplantation, resulting in the ability to defend against and control infection as well as prevent autoimmune diseases.

[0061] It was first shown that implantation of cultured thymic tissue is beneficial in treating primary immunodeficiencies caused by congenital athymia, which is associated with conditions such as complete DiGeorge anomaly (cDGA) or forkhead box protein N1 (FOXN1) deficiency. It was found that replacing defective thymic tissue with cultured normal thymic tissue (e.g., CTT and RVT - 802) can also eliminate the lack of tolerance observed in transplanted solid organ recipients.

[0062] Pre-clinical and clinical work underlying the treatment of congenital athymia by placement of cultured thymic tissue led to the realization that placement of CTT (e.g., RVT - 802) in patients may allow for the development of tolerance to transplanted solid organs. Specifically, if a subject is first thymectomized and immunosuppressed before implantation of CTT expressing the MHC of the donor organ, placement of CTT will reconstitute the immune system and induce tolerance to the donor organ.

[0063] Measurement of the expression and distribution of certain markers associated with the cellular components of the thymus established the phenotype after in vitro culturing of thymic tissue. The culture conditions described in this specification and the examples support in vivo thymopoiesis observed after placement of CTT in athymic subjects.

[0064] Importantly, after placement of CTT surgery in athymic recipients, the development of naive T cells and the presence of a broad TCR variable region provide clear evidence that culturing of thymic tissue can promote the development of a functional endogenous T cell population. In addition, expression of key regulatory and structural genes was noted in thymic tissue during the culturing process. Circulating naive (CD45RA+CD62L+) T cells could first be 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).

[0065] The non-clinical data on thymic tissue implantation described in the literature are consistent with the robust clinical efficacy of implanting allogeneic cultured postnatal thymic tissue and support 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. January; 82(1):26-36; 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., September; 140(3):244-59).

[0066] Patients with complete DiGeorge anomaly are defective in three glands that develop in the neck, heart, thymus, and parathyroids of the embryo. Normally, the heart and thymus descend into the chest, and the parathyroids regulate calcium levels and remain in the neck. Treatment of cDGA subjects with CTT resulted in a 75% survival rate at two years of age, while the survival rate of subjects treated otherwise was 6% (unpublished data). As noted above, almost all deaths occurred within one year before the development of naive T cells. (Markert et al., 2010). Notably, CTT implantation does not affect the issues of the heart and parathyroids that must be managed separately.

[0067] One aspect of the present disclosure provides a method for surgically placing an allogeneic cultured postnatal thymic tissue-derived product in a recipient to induce tolerance to a solid organ graft in an immunologically normal recipient. Such methods comprise, consist of, or consist essentially of: removing the thymus in an immunocompetent recipient, followed by depleting the recipient's T cells with an induction immunosuppressive regimen comprising one or more immunosuppressive agents, such as one or more antibodies and / or one or more calcineurin inhibitors. The induction immunosuppressive regimen is administered in a therapeutically effective amount so as to deplete mature T cells in the subject and / or inhibit T cell rejection of the transplanted solid organ by the recipient. A suitable solid human organ and thymus are obtained from a deceased donor, and the solid organ is transplanted into the recipient. A maintenance immunosuppressive regimen is administered for a period of time to inhibit transplant rejection. A conditioning regimen for the thymus from the deceased donor is carried out for a period of up to 21 days (e.g., a conditioning regimen of about 6 days to about 21 days) to aseptically process the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice, thereby comprising the allogeneic cultured postnatal thymic tissue-derived product. The partially T cell-depleted donor thymic tissue slice shows areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei. The allogeneic cultured postnatal thymic tissue-derived product is then surgically placed in the recipient, typically in the quadriceps of the thigh. The allogeneic cultured postnatal thymic tissue-derived product enables the recipient to develop naive T cells after implantation. All newly developed T cells are genetically accepted and are tolerant to both the recipient and the donor. The dose of the thymic tissue slice is about 1000 - 22000 mm 2 of thymic tissue surface area / m 2 recipient body surface area. After implantation, the allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the subject.

[0068] For example, in the case of a heart transplant, the donor would be a deceased donor. The thymus would be removed from the donor at the same time as the heart is removed. The heart transplant would be performed immediately, and induction immunosuppression would be carried out to reduce the number of T cells and inhibit the remaining recipient T cells from attacking the donor heart. The donor thymus is processed to form a human allogeneic cultured postnatal thymic tissue-derived product, which can be used for implantation to induce tolerance after conditioning for a period of at least about 6 days to about 21 days. As a precautionary measure, approximately half of the allogeneic cultured postnatal thymic tissue-derived product can be cryopreserved after conditioning, such that if a heart rejection problem occurs later and high doses of steroids or other immunosuppressants are required to treat the rejection, and the very high doses of steroids damage the allogeneic cultured postnatal thymic tissue-derived product, the cryopreserved allogeneic cultured postnatal thymic tissue-derived product can be implanted after the rejection episode has been controlled.

[0069] Importantly, immune tolerance can be maintained even in the presence of infection after implantation of the allogeneic cultured postnatal thymic tissue-derived product. Using other methods such as co-stimulation blockade, viral infection can lead to loss of tolerance because approximately one-third of CD8 T cells are alloreactive. When the immune system is activated to fight an infection, alloreactive CD8 T cells start to reject solid organ grafts. In contrast, when tolerance is induced using thymic tissue processed into an allogeneic cultured postnatal thymic tissue-derived product, potential alloreactive T cells against the donor are deleted through the process of negative selection in the thymus.

[0070] In one embodiment, the donor thymic tissue is matched to the HLA alleles in the donor organ that are not present in the recipient. All newly developed T cells are genetically accepted and are tolerant to both the recipient and the donor.

[0071] In another aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased donor in a recipient in need of a solid organ transplant, the method comprising the steps of:

[0072] (a) removing the thymus of the recipient;

[0073] (b) treating the recipient with an induction immunosuppression regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0074] (c) providing a suitable solid human organ and thymus from a deceased donor;

[0075] (d) Transplant the solid human organ into the recipient;

[0076] (e) Treat the recipient with a maintenance immunosuppressive regimen;

[0077] (f) Provide an allogeneic cultured postnatal thymic tissue-derived product, wherein the allogeneic cultured postnatal thymic tissue-derived product is obtained from suitable thymic tissue of the solid organ donor; wherein the donor thymic tissue is subjected to a conditioning protocol for a period of up to 21 days (e.g., a conditioning protocol of about 6 days to about 21 days) to produce the allogeneic cultured postnatal thymic tissue-derived product; further, wherein the conditioning protocol for the donor thymic tissue includes aseptic treatment of the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; wherein the partially T cell-depleted donor thymic tissue slice shows regions positive for cytokeratin (CK) (using antibody AE1 / AE3) dispersed throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining dispersed throughout the tissue, and the presence of intact nuclei; and

[0078] (g) At about 6 to about 21 days after the conditioning protocol, implant the allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the thymic tissue slice is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0079] In one embodiment, a method for promoting donor-specific tolerance to an allogeneic heart graft in a recipient in need of a deceased donor heart is provided. The method comprises the steps of:

[0080] (a) Obtain a suitable human heart from the deceased donor for transplantation;

[0081] (b) Remove the deceased donor thymus at the time of obtaining the heart

[0082] for conditioning into an allogeneic cultured postnatal thymic tissue-derived product; wherein the donor thymus is HLA allele-matched to the donor transplant organ;

[0083] (c) Treat the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete and / or inhibit the recipient's T cells, wherein the one or more immunosuppressive agents include glucocorticoids administered at the time of induction of anesthesia and after reperfusion;

[0084] (d) Transplant the heart into the recipient;

[0085] (e) Treat the recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents for a period sufficient to prevent or inhibit transplant rejection of the heart, the one or more immunosuppressive agents selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulin;

[0086] (f) Between day 6 and day 21, provide an allogeneic cultured postnatal thymic tissue-derived product, wherein the allogeneic cultured postnatal thymic tissue-derived product is obtained from the donor thymic tissue; wherein the donor thymic tissue is subjected to a conditioning protocol for a period of about 6 days to about 21 days to produce an allogeneic cultured postnatal thymic tissue-derived product; further, wherein the conditioning protocol for the donor thymic tissue comprises aseptic processing of the donor thymic tissue in thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; wherein the partially T cell-depleted donor thymic tissue slice shows areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei;

[0087] (g) Implant a portion of the allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the thymic tissue slice is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient; and

[0088] (h) Cryopreserve a portion of the allogeneic cultured postnatal thymic tissue-derived product for use in the recipient in the event of an early rejection episode, such that a high dose of steroids is required, which high dose of steroids would compromise the portion of the allogeneic cultured postnatal thymic tissue-derived product implanted in step (g).

[0089] In one embodiment, a method of promoting donor-specific tolerance to an allogeneic heart graft in a recipient in need of a deceased donor heart is provided. The method comprises the steps of:

[0090] (a) Obtain a suitable solid human heart from the deceased donor for transplantation;

[0091] (b) Removing the thymus of the deceased donor at the time of obtaining the heart for conditioning into an allogeneic cultured postnatal thymus tissue-derived product; wherein the donor thymus is HLA allele-matched to the donor transplant organ;

[0092] (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 include glucocorticoids administered at the time of induction of anesthesia and after reperfusion;

[0093] (d) Surgically removing the heart and thymus of the recipient;

[0094] (e) Transplanting the donor human heart into the recipient;

[0095] (f) Treating the recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents for a period sufficient to prevent or inhibit transplant rejection of the heart, the one or more immunosuppressive agents selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulin;

[0096] Wherein, if the recipient's postoperative condition is too unstable to discard glucocorticoids and the allogeneic cultured postnatal thymus tissue-derived product cannot be safely implanted in the recipient, the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved for implantation at a later time when the recipient is stable, wherein the conditioning protocol for the donor thymus tissue lasts for a period of about 6 days to about 21 days to produce an allogeneic cultured postnatal thymus tissue-derived product; further, wherein the conditioning protocol for the donor thymus tissue includes aseptic treatment of the donor thymus tissue in thymic organ culture medium to produce a partially T cell-depleted donor thymus tissue slice; wherein the partially T cell-depleted donor thymus tissue slice shows regions positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei;

[0097] (h) Implanting a portion of the allogeneic cultured postnatal thymus tissue-derived product into the recipient after the patient is stable, wherein the dose of the thymus tissue slice is about 1000 - 22000 mm 2 thymus tissue surface area / m 2the recipient's body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient; and (i) cryopreserving a portion of the allogeneic cultured postnatal thymic tissue-derived product for use in the recipient in the event of a rejection episode such that high doses of steroids are required, the high doses of steroids being detrimental to the portion of the allogeneic cultured postnatal thymic tissue-derived product implanted in step (h).

[0098] In another aspect of the present disclosure, a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a living donor in a human recipient in need of a solid organ transplant is provided, the method comprising the steps of:

[0099] (a) removing the recipient's thymus;

[0100] (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0101] (c) providing a suitable solid organ from a living donor;

[0102] (d) transplanting the solid organ into the recipient;

[0103] (e) treating the recipient with a maintenance immunosuppressive regimen;

[0104] (f) providing a cryopreserved allogeneic cultured postnatal thymic tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymic tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is processed from thymic tissue from a thymic donor that expresses HLA alleles that match the HLA alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning regimen for the donor thymic tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning regimen for the donor thymic tissue comprises aseptic treatment of the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice, wherein after completion of the conditioning regimen, the thymic tissue slice shows areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei;

[0105] (g) thawing the cryopreserved allogeneic cultured postnatal thymic tissue-derived product; and

[0106] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is about 1000 - 22000 mm 2 Thymic tissue surface area / m 2 Recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0107] The fourth aspect of the present disclosure provides a method for promoting donor - specific tolerance to an allogeneic solid organ graft obtained from a deceased human donor in a human recipient in need of a solid organ transplant, the method comprising the following steps:

[0108] (a) Removing the thymus of the recipient;

[0109] (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0110] (c) Providing a suitable solid organ from a living donor;

[0111] (d) Transplanting the solid organ into the recipient;

[0112] (e) Treating the recipient with a maintenance immunosuppressive regimen;

[0113] (f) Providing a cryopreserved allogeneic cultured postnatal thymic tissue-derived product held in a cryopreserved allogeneic cultured postnatal thymic tissue-derived product bank; wherein the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is processed from thymic tissue from a thymic donor expressing HLA alleles that match the HLA alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning protocol for the donor thymic tissue lasts for a period of about 6 days to about 21 days; further, wherein the conditioning protocol for the donor thymic tissue comprises aseptically processing the donor thymic tissue in a thymic organ culture medium to produce a partially T cell - depleted thymic tissue slice, wherein after completion of the conditioning protocol, the thymic tissue slice shows areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei;

[0114] (g) Thawing the cryopreserved allogeneic cultured postnatal thymic tissue-derived product; and

[0115] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1000 - 22000 mm 2 Thymus tissue surface area / m 2 Recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0116] In one embodiment of the foregoing aspect of the present disclosure, an allogeneic cultured postnatal thymus tissue-derived product, wherein the thymus shows >50% of the area positive for keratin with a lacy staining pattern, the presence of Hassall's corpuscles, CK14 staining in a lacy pattern, and >90% of the nuclei are intact on the day of harvest.

[0117] In one aspect of the present disclosure, there is provided an allogeneic cultured postnatal thymus tissue-derived product for implantation into a subject undergoing solid organ transplantation, the product being prepared by: obtaining suitable thymus tissue from a donor, wherein the donor thymus tissue is subjected to a conditioning protocol for a period of up to 21 days (e.g., a conditioning protocol of about 6 days to about 21 days); further, wherein the conditioning protocol for the donor thymus tissue includes aseptic treatment of the donor thymus tissue in a thymus organ culture medium to produce a partially T cell-depleted donor thymus tissue slice; wherein the donor thymus tissue slice shows areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei between 5 and 9 days after harvest; and recovering the partially T cell-depleted donor thymus tissue slice as an allogeneic cultured postnatal thymus tissue-derived product.

[0118] In one embodiment of the foregoing aspect of the present disclosure, the thymus shows >50% of the area positive for keratin with a lacy staining pattern, the presence of Hassall's corpuscles, CK14 staining in a lacy pattern, and >90% of the nuclei are intact on the day of harvest from the donor.

[0119] In one embodiment of the foregoing aspect of the present disclosure, the allogeneic cultured postnatal thymus tissue-derived product is cryopreserved.

[0120] In one embodiment, the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is maintained in liquid nitrogen for future use.

[0121] In another embodiment, the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is maintained in a cryopreserved tissue bank.

[0122] In one embodiment, the allogeneic cultured postnatal thymic tissue-derived product is prepared from suitable thymic tissue from a donor, the donor comprising HLA alleles that match the HLA alleles in the proposed recipient that are not present in the solid organ graft.

[0123] In one 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.

[0124] In one aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymic tissue-derived product, which is prepared by a method comprising the following steps:

[0125] (a) Obtaining suitable thymic tissue from a donor;

[0126] (b) Typing the following HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1;

[0127] (c) Subjecting the thymic tissue to a conditioning protocol for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically processing the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; further, wherein after completion of the conditioning protocol, the donor thymic tissue slice shows areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei on days 6 to 21;

[0128] (d) Harvesting the partially T cell-depleted donor thymic tissue slice as an allogeneic cultured postnatal thymic tissue-derived product;

[0129] (e) Cryopreserving the allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen; and

[0130] (f) Maintain the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymus tissue-derived product bank.

[0131] In one embodiment, on the day of harvest, the thymus exhibits >50% of the area being positive for keratin with a lacy staining pattern, the presence of Hassall's corpuscles, CK14 staining being a lacy pattern, and >90% of the nuclei being intact.

[0132] In one embodiment, store the cryopreserved allogeneic cultured postnatal thymus tissue-derived product in liquid nitrogen for future use by the recipient.

[0133] In one aspect of the present disclosure, there is provided a method of preparing a donor thymus for implantation 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 in the recipient, as further described herein. A culture period between about 6 days and about 21 days results in good function. For successful transplantation of cryopreserved thymus tissue, the tissue is typically cultured for about 6 days to about 21 days and then cryopreserved.

[0134] In one 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 solid organ transplantation, the product being produced by the following method: subjecting thymus tissue from a suitable donor to a conditioning protocol for a period of up to 21 days (e.g., a conditioning protocol of about 6 days to about 21 days); wherein the conditioning protocol for the allogeneic cultured postnatal thymus tissue-derived product comprises aseptically treating the donor thymus tissue in thymic organ medium to produce a partially T cell-depleted thymic tissue slice, wherein the thymic tissue slice shows areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei.

[0135] In one embodiment, on the day of harvest, the donor thymus exhibits >50% of the area being positive for keratin with a lacy staining pattern, the presence of Hassall's corpuscles, CK14 staining being a lacy pattern, and >90% of the nuclei being intact.

[0136] In one or more embodiments of the foregoing aspect, the thymus of the subject is obtained surgically.

[0137] In one or more embodiments of the foregoing aspect, the thymus of the subject is obtained by robotic surgery.

[0138] In one or more embodiments of the foregoing aspect, the thymus of the subject is obtained by thoracoscopic surgery.

[0139] In one or more embodiments of the foregoing aspect, the solid organ is part of an entire organ.

[0140] In one or more embodiments of the foregoing aspect, the methods of the first to fourth aspects further comprise the step of cryopreserving peripheral blood mononuclear cells from the deceased donor for future use in a mixed lymphocyte reaction for demonstrating cellular tolerance.

[0141] In one or more embodiments of the foregoing aspect, after implanting an allogeneic cultured postnatal thymus tissue-derived product according to the CTT implantation procedure in this specification, a mixed lymphocyte reaction for demonstrating cellular tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor.

[0142] In one or more embodiments of the foregoing aspect, about 6 to 12 months after implanting an allogeneic cultured postnatal thymus tissue-derived product, a mixed lymphocyte reaction for demonstrating cellular tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor.

[0143] In one or more embodiments of the foregoing aspect, after naive T cells constitute about 10% of the total T cells in the recipient, a mixed lymphocyte reaction for demonstrating cellular tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor.

[0144] In one or more embodiments of the foregoing aspect, within 12 months after implanting an allogeneic cultured postnatal thymus tissue-derived product, the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis in the subject.

[0145] In one or more embodiments of the foregoing aspect, the generation of tolerance is determined by a mixed lymphocyte reaction using cryopreserved peripheral blood mononuclear cells from the deceased donor and naive T cells from the recipient.

[0146] In one or more embodiments of the foregoing aspects, humoral tolerance is determined by the generation of humoral immunity and the absence of donor-reactive antibodies.

[0147] In one or more embodiments of the foregoing aspects, the solid organ graft is a heart graft, a kidney graft, a liver graft, a lung graft, a heart / lung graft, a pancreas graft, an intestine graft, a stomach graft, an abdominal wall graft, a craniofacial graft, a scalp graft, a penile graft, a uterine graft, a unilateral or bilateral upper limb graft, a unilateral vascularized composite allograft, or a combination thereof.

[0148] In one or more embodiments of the foregoing aspects, the method further comprises evaluating the recipient's HLA class I or HLA class II panel reactive antibody ("PRA") score prior to transplantation of the solid organ.

[0149] In one or more embodiments of the foregoing aspects, the solid organ graft is a heart graft.

[0150] In one or more embodiments of the foregoing aspects, the solid organ graft is a pediatric heart graft.

[0151] In one or more embodiments of the foregoing aspects, the solid organ graft is an adult heart graft.

[0152] In one or more embodiments of the foregoing aspects, the method further comprises evaluating the recipient's HLA class I or HLA class II panel reactive antibody ("PRA") score prior to transplantation of the solid organ.

[0153] In one or more embodiments of the foregoing aspects, a recipient with HLA antibodies is crossmatched with a potential donor.

[0154] In one or more embodiments of the foregoing aspects, a recipient with HLA antibodies is virtually crossmatched with UNET.

[0155] In one or more embodiments of the foregoing aspects, if a virtual crossmatch with a PRA score > 20% is recorded, the method will further comprise the step of performing plasmapheresis in the operating room during solid organ transplantation in the recipient.

[0156] In one or more embodiments of the foregoing aspects, if a virtual crossmatch with a PRA score > 70% is recorded, the method will further comprise the steps of performing an actual prospective donor crossmatch and performing plasmapheresis in the operating room during solid organ transplantation in the recipient. Typically, transplantation is not performed in this case due to the low success rate.

[0157] In one or more embodiments of the foregoing aspect, the method further comprises the step of evaluating a recipient with HLA antibodies by virtual crossmatching with UNET.

[0158] In one or more embodiments of the foregoing aspect, the method further comprises: if the score of HLA panel reactive antibodies > 20%, performing plasmapheresis in the operating room when performing solid organ transplantation in the recipient.

[0159] In one or more embodiments of the foregoing aspect, the method further comprises: if the score of HLA panel reactive antibodies > 70%, performing an actual prospective donor crossmatch and performing plasmapheresis in the operating room when performing solid organ transplantation in the recipient.

[0160] In one or more embodiments of the foregoing aspect, for example, in life-related donors to recipients such as kidney grafts, partial liver grafts, and partial intestine grafts, the solid organ is HLA-matched.

[0161] In one or more embodiments of the foregoing aspect, the solid organ is HLA-mismatched.

[0162] In one or more embodiments of the foregoing aspect, the solid organ is HLA-matched. In another embodiment, HLA matching is determined by typing the following HLA alleles in the donor and the recipient: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1.

[0163] In one or more embodiments of the foregoing aspect, the solid organ graft is ABO-compatible.

[0164] In one or more embodiments of the foregoing aspect, the solid organ is HLA-mismatched. In one embodiment, HLA mismatch 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.

[0165] In one or more embodiments of the foregoing aspect, surgically implanting cultured thymic tissue slices into the quadriceps femoris thigh muscle of the subject.

[0166] In one or more embodiments of the foregoing aspect, surgically implanting cultured thymic tissue slices into an area of the subject other than the quadriceps femoris.

[0167] In one or more embodiments of the foregoing aspect, a portion of the allogeneically cultured postnatal thymic tissue-derived product is surgically implanted into the quadriceps femoris thigh muscle of the recipient.

[0168] In one or more embodiments of the foregoing aspect, the remaining portion of the allogeneically cultured postnatal thymic tissue-derived product is cryopreserved in liquid nitrogen for future transplantation.

[0169] In one or more embodiments of the foregoing aspect, the conditioning regimen lasts for a period of about 6 days to about 21 days.

[0170] In one or more embodiments of the foregoing aspect, the conditioning period of the donor thymic 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.

[0171] Those of ordinary skill in the art will understand that there are many potential induction immunosuppressive regimens and maintenance immunosuppressive regimens known in the art, and those skilled in the art can select suitable induction immunosuppressants and maintenance immunosuppressants without undue burden. The following exemplary induction immunosuppressive regimens and maintenance immunosuppressive regimens are practical examples of the methods of the first to fourth aspects of the present invention and support the claimed invention.

[0172] In one or more embodiments of the foregoing aspect, the induction immunosuppressive regimen includes induction immunosuppressants selected from the group consisting of glucocorticoids, antithymocyte globulin (rabbit), antithymocyte globulin (horse), and alemtuzumab.

[0173] In one or more embodiments of the foregoing aspect, the ATG is antithymocyte globulin (rabbit).

[0174] In one or more embodiments of the foregoing aspect, the induction immunosuppressive regimen includes the administration of glucocorticoids. In one embodiment, the glucocorticoid includes methylprednisolone. In another embodiment, the glucocorticoid is methylprednisolone sodium succinate. In additional embodiments, methylprednisolone sodium succinate is administered intravenously at no greater than 4 mg / kg / day.

[0175] In one or more embodiments of the foregoing aspect, the induction immunosuppression regimen includes rabbit - derived antithymocyte globulin. In another embodiment, the rabbit - derived antithymocyte globulin is administered intravenously at a dose of about 1.5 mg / kg. In a further embodiment, the antithymocyte globulin is administered daily for four days. In another embodiment, the ATG is horse - derived ATG.

[0176] In one or more embodiments of the foregoing aspect, the induction immunosuppression regimen includes basiliximab. In another embodiment, for recipients weighing less than 35 kg, basiliximab is administered intravenously at a dose of 10 mg. In another embodiment, for recipients weighing more than 35 kg, basiliximab is administered intravenously at a dose of 20 mg.

[0177] In one or more embodiments of the foregoing aspect, the second immunosuppression regimen includes one or more immunosuppressants selected from the group consisting of: glucocorticoids, calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, azathioprine, and antithymocyte globulin (“ATG”).

[0178] In one or more embodiments of the foregoing aspect, the immunosuppressant of the maintenance immunosuppression regimen is antithymocyte globulin (ATG).

[0179] In one or more embodiments of the foregoing aspect, starting from administration in the operating room, the ATG is administered intravenously at a dose of about 1.5 mg / kg for a period of 3 - 14 days.

[0180] In one or more embodiments of the foregoing aspect, the antithymocyte globulin is administered daily at about 15 mg / kg / day by intravenous administration for 3 - 14 days.

[0181] In one or more embodiments of the foregoing aspect, the first immunosuppression regimen includes alemtuzumab.

[0182] In one or more embodiments of the foregoing aspect, for recipients weighing less than 35 kg, alemtuzumab is administered intravenously at a dose of about 0.25 mg / kg for 4 days. In another embodiment, for recipients weighing more than 35 kg, alemtuzumab is administered intravenously at a dose of about 3 to 20 mg for 4 days.

[0183] In one or more embodiments of the foregoing aspect, the second immunosuppression regimen includes one or more immunosuppressants selected from the group consisting of: calcineurin inhibitors and inosine monophosphate dehydrogenase inhibitors or azathioprine.

[0184] In one or more embodiments of the foregoing aspect, the immunosuppressant of the maintenance immunosuppression regimen is a calcineurin inhibitor.

[0185] In one embodiment, the immunosuppressant of the maintenance immunosuppression regimen is an inosine monophosphate dehydrogenase inhibitor.

[0186] In one or more embodiments of the foregoing aspect, the immunosuppression regimen includes an inosine monophosphate dehydrogenase inhibitor, such as mycophenolate mofetil. In one embodiment, mycophenolate mofetil is administered intravenously at a dose of about 15 to about 25 mg / kg. In one embodiment, mycophenolate mofetil is administered intravenously two to three times a day.

[0187] In one or more embodiments of the foregoing aspect, the inosine monophosphate dehydrogenase inhibitor is mycophenolic acid. In another embodiment, mycophenolic acid is administered in 2 or 3 divided doses at a dose of about 25 to about 50 mg / kg.

[0188] In one or more embodiments of the foregoing aspect, mycophenolic acid is administered to children at a dose of about 400 mg / m 2 / dose twice daily, with a maximum dose of 720 mg; or for a BSA of 1.19 to 1.59 m 2 , about 540 mg is administered twice daily; or for a BSA > 1.58 m 2 , about 720 mg is administered twice daily.

[0189] In one or more embodiments of the foregoing aspect, mycophenolate mofetil is administered to children at a dose of about 15 to about 25 mg / kg / dose twice daily, or to adults at a dose of about 1500 mg twice daily orally or intravenously, and is adjusted for WBC > 3500.

[0190] In one or more embodiments of the foregoing aspect, the second immunosuppression regimen may further include a glucocorticoid selected from the group consisting of methylprednisolone, prednisone, and prednisolone. In one embodiment, the dose of the glucocorticoid is kept below 4 mg / kg / day.

[0191] In one or more embodiments of the foregoing aspect, as described elsewhere in the present disclosure, the glucocorticoid is administered at a gradually decreasing dose.

[0192] In one or more embodiments of the foregoing aspect, the calcineurin inhibitor is tacrolimus. In another embodiment, the calcineurin inhibitor is cyclosporine A.

[0193] In one or more embodiments of the foregoing aspect, after naive T cells reach 10% of total T cells, administration of the second immunosuppressive regimen is discontinued. In yet another embodiment, after implantation of an allogeneic cultured postnatal thymic tissue-derived product, the second immunosuppressive regimen is discontinued.

[0194] To evaluate how thymic production and / or release of chemokines and other soluble molecules during depletion of thymocytes in donor thymic tissue for CTT regulate these processes of cell migration changes, we screened conditioned media from human thymic organ cultures for the presence of 200 soluble molecules using antibody microarrays.

[0195] Expression of selected potentially mechanistically important candidate molecules was verified using additional thymic organ cultures and compared to a well-characterized set of human thymic tissues obtained from donors ranging in age from 5 days to 78 years. By this analysis, we identified certain potentially important biomarkers of thymocyte content.

[0196] A potentially important biomarker of thymocyte content in cultured thymic slices is L-selectin. Another important biomarker of thymic epithelial cell viability and function depends on secretion of the chemokine CCL21 6Ckine.

[0197] Multiple additional potential biomarkers are shown in Figure 50 and 56 Of particular interest are the biomarkers shown in Figure 50 namely CCL21, CXCL16, M-CSF, galectin-7, CCL11, IL-16, and CXCL12. Additionally, of particular interest are the biomarkers shown in Figure 56 especially those with a P value of less than 0.05. These biomarkers will 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 / SDF-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.

[0198] Data reported in the examples and in the literature support the following biomarkers as indicators 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).

[0199] Osteopontin (OPN; encoded by the SPP1 gene): This cytokine increases during thymic stress, and increased levels are associated with thymic atrophy (decrease in thymocyte number), which is the desired state for cultured thymus (Wang 2009; Gridley 2013). OPN is required for the production of corticosteroids, and it is well established that corticosteroids induce thymocyte apoptosis.

[0200] CCL11 (eotaxin): This chemokine is produced by medullary TECs (Bunting 2011). It was originally named for its ability to attract eosinophils, and we have shown that eosinophil infiltration may be prominent in thymic tissue with active thymopoiesis (Flores 1999). However, it has also been shown that eotaxin acts as a chemoattractant for double-positive (DP) and single-positive (SP) human thymocytes (Bunting 2011).

[0201] uPAR (CD87; encoded by the PLAUR gene): The urokinase receptor (also known as the urokinase plasminogen activator receptor) is expressed in soluble and membrane-bound forms based on alternative splicing. It contributes to the local degradation of the extracellular matrix. It has been shown to be expressed in the human thymus, and interestingly, it is expressed by epidermal keratinocytes (EKs) at the migrating wound edge (Loughner 2016). This latter feature is of particular interest because TE cells are very similar to EKs in terms of the expression of many genes (Patel 1995). A gradual increase in secretion in cultured thymic slices may reflect the activation of TEs and is thus a marker of TE growth after implantation.

[0202] CXCL12 (SDF-1a): This chemokine has an expression pattern that is different from other analytes as it is only detected during the last third of the culture period. It is first detected on days 13 - 15 and it linearly increases (in the ln plot) to much higher levels during the following week of culture. CXCL12 has been shown to be produced by subcapsular cortex and medullary TE (Bunting 2011; Hernandez-Lopez 2002; Zaitseva 2002), but can also be produced by thymic fibroblasts and endothelial cells present within the thymus. CXCL12 has been shown to recruit B cells and antigen presenting cells (APCs) to the thymus (Weiss 2003), which is expected to be important for the generation of full thymic function. It is also involved in the positioning of thymocyte subsets within the thymus and it enhances thymocyte proliferation in response to IL-7 (Hernandez-Lopez 2002). Notably, antibodies that neutralize CXCL12 have been shown to reduce thymopoiesis in in vitro human thymic organ cultures and the addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez 2002).

[0203] Other biomarkers that can reflect the presence of thymocytes include L-selectin. This molecule is expressed at high levels on developing and naive T cells. When thymocytes are cultured, it is released from the cell surface. It is normally rapidly re-expressed when shed by healthy cells in the body (Fitzhugh 2008), and a gradually decreasing low shedding level may reflect a progressive loss of thymocyte viability as they generally do not re-express this molecule on their surface during culture (A. Macintyre, unpublished data).

[0204] Another biomarker that may reflect the presence of thymocytes is IL-16. This cytokine is included because its pattern (described below) is consistent with the hypothesized pattern for thymocytes. This biomarker is known to be produced by lymphocytes.

[0205] Yet another biomarker that may reflect the presence of thymocytes is MIF. This chemokine is included because its pattern is consistent with the hypothesized pattern for thymocytes.

[0206] Still another biomarker that may reflect the presence of thymocytes is CCL20 (MIP-3a): This chemokine is included because its pattern is consistent with the hypothesized pattern for thymocytes.

[0207] Another biomarker that may reflect the presence of thymocytes is IGFBP-1. IGFBP2 - 6 are known to be expressed by thymic epithelium that does not express IGFBP-1 (Gosteli-Peter 1994; Ketcha 1999).

[0208] Other biomarkers initially showing high levels and then decreasing over time (hypothetical characteristics of thymocyte-derived biomarkers) associated with the presence of live thymocytes based on data in the examples and the literature: L-selectin, IL-16, MIF, CCL20 (MIP-3a), and IGFBP-1. The decrease in these biomarkers over time is consistent with our more qualitative observations of T cell depletion when culturing thymic tissue slices.

[0209] CCL21 has been shown to be expressed by thymic epithelial cells (Lkhagvasuren et al., 2013) and is 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 of thymocytes may be a crucial determinant for the successful immune reconstitution of recipients after implantation of cultured thymic tissue as described herein.

[0210] The results reported herein should also be widely applicable to understanding the mechanisms of age-related thymic involution and to understanding the mechanisms involved in immune reconstitution of athymic recipients via implantation of cultured thymic tissue.

[0211] In the examples and on Figure 56 it was reported that at least 127 different analytes could be detected in spent / conditioned media obtained from cultured human thymic tissue slices, and 42 of these analytes showed a gradual increase or decrease over the culture time. Among these analytes, the amount of soluble L-selectin released was verified as a non-destructive biomarker for the residual content of live thymocytes in cultured thymus. During in vitro thymic organ culture and in vivo in unmanipulated thymic tissue obtained from healthy donors throughout the life cycle, the expression and / or secretion of the chemokines CCL21, CXCL16, CXCL12, and CCL11 by thymic epithelium all showed an increase with the decrease in thymocytes. Similarly, during the culture process, a decrease in the expression or secretion of L-selectin, M-CSF, galectin-7, and IL-16 in thymic organ media was observed. These findings are directly relevant to understanding the mechanisms of age-related thymic involution and to the quality of human thymic tissue for promoting immune reconstitution. The contrast between the changes observed during infant thymus culture and those associated with aging indicates that cultured human infant thymus also provides a model that can be used to study the mechanisms mediating age-related thymic involution.

[0212] To our knowledge, this is the first large-scale screen of soluble molecules produced and released or secreted by the human thymus upon thymocyte depletion. Many of the 42 analytes found to be significantly increased or decreased in release into the culture medium over the course of the culture are cytokines and / or chemokines that have previously been shown to be produced by cell types present within the thymus. Other analytes are novel in this regard. While the experimental work has mainly focused on the release of L-selectin as a marker of thymocyte content and CCL21 as a marker of TEC viability and function, the data reported from the antibody microarray screen can be used to identify additional analytes from Figure 56 that may identify novel pathways that govern acute atrophy, chronic degeneration, and / or regeneration of the human thymus in vivo. These will specifically include Figure 50 the biomarkers listed therein. For example, some analytes that met the pre-specified selection criteria were not further considered because they are known to be expressed or released during cell injury and / or hypoxic stress, and their levels may reflect culture-related tissue damage and inflammatory responses rather than thymus-specific biology. Additional biomarkers noted in the culture supernatants from cultured thymic tissue slices include CC25 (TECK), osteopontin (OPN), uPAR (CD87), MIF, CCL20 (MIP-3a), and IGFBP-1. Investigation of additional analytes whose release may reflect the viability and / or activation of crucial cell types in the human thymus may lead to clinically and mechanistically important insights, particularly regarding the response to thymocyte loss.

[0213] CCL25 (TECK): Although detectable levels of TECK were present in only a few supernatant samples during the later stages of the culture period in 2 of the 3 thymic cultures examined, it was of interest because this chemokine has been shown to be chemotactic for thymocytes (Liu 2005). It is known to be expressed by thymic dendritic cells (DC) as well as both FoxN1+ and FoxN1-TE cells (Bunting 2011). However, based on studies in mice, its activity appears not to be essential for thymic development, in which the sole receptor for this chemokine, CCR9, was deleted (Wurbel 2001).

[0214] Based on microarray studies, it was determined that soluble L-selectin levels in conditioned medium can serve as a potential biomarker for the presence and viability of thymocytes in cultured thymic slices. This is highly plausible because the expression of L-selectin is restricted to hematopoietic cells and is shed constitutively as well as during migration (Hafezi-Moghadam et al., 2001), and is then typically rapidly re-expressed by healthy cells in vivo (Fitzhugh et al., 2008).

[0215] The experimental results showed that the loss of L-selectin release was temporally associated with thymocyte death, as indicated by the loss of thymocyte membrane integrity measured by histology and CD3 immunohistochemistry and the lack of characteristic thymocyte proliferation measured by Ki-67 immunohistochemistry.

[0216] The ability to non-destructively monitor the thymocyte content of cultured human thymic slices is important for identifying the most appropriate harvest time point for experimental studies. This could also provide clinically important information, as it is believed that opening up the developmental niche for recipient thymocyte colonization by depleting donor thymocytes is crucial for successful immune reconstitution of athymic patients via thymic implantation. In summary, the studies presented in the examples showed that reduced L-selectin release is a useful biomarker for monitoring thymocyte depletion in cultured thymic slices.

[0217] Biomarkers reflecting the presence and function of thymic epithelium can also provide crucial mechanistic information. The studies described in the examples focused on CCL21 because microarray screening showed that this chemokine began to be secreted into the culture medium at high levels shortly after the start of culture. It has previously been shown that CCL21 is expressed by thymic epithelium and is chemotactic for thymocytes and their precursors (Liu et al., 2005). Our studies showed that the expression of CCL21 could also be easily quantified by enzyme immunoassay. Immunohistochemistry confirmed CCL21 expression in TECs in both cultured and non-cultured thymus, with the strongest expression in the medulla and subcapsular cortical thymic epithelium.

[0218] It is also important to note that the CCL21 immunoreactivity of thymic slices does not necessarily increase as CCL21 secretion increases during culture. This may reflect that additional CCL21 produced is secreted rather than retained in the cytoplasm where it can be detected by immunohistochemistry. CCL21 is a transcriptionally regulated, high-turnover molecule with a short half-life (Dudal et al., 2015), so the observed positive immunohistochemical reactivity represents cells that are actively producing this chemokine. The increase in production of this CCL21 in both cultured thymus and unmanipulated aged thymus as thymocytes decrease suggests that thymic epithelial cells can sense thymocyte content and respond in a homeostatic attempt to counter thymocyte loss.

[0219] The identification of CCL21 as a secreted biomarker that reflects the viability and function of TECs is also important clinically for thymus transplantation. Most established methods for assessing the quality of the tissue to be implanted (e.g., flow cytometry, immunohistochemistry, gene expression analysis) destroy the sample during the analysis. In addition to reducing the amount of tissue available for the final implant, such results are also affected by sampling error, since the sections tested are not part of the sections that will be finally implanted. As Figure 53 shown in

[0220] C, the assay of conditioned medium pooled from CCL21 can be integrated across all sections in a batch from any given thymus donor, providing a non-destructive picture of the overall batch quality. Figure 50 The chemokine CXCL12 (SDF-1α) differs from most of the other analytes in the screen in that it becomes detectable in the conditioned medium relatively late during the culture period (

[0221] H). First detectable at days 13 - 15, it rises linearly (in a ln plot) to much higher levels during the following week of culture. CXCL12 has been shown to be produced by subcapsular cortex and medullary TECs, but also by thymic fibroblasts and endothelial cells upon intrathymic manipulation (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 for the generation of full thymic function. CXCL12 is also involved in the positioning of thymocyte subsets within the thymus, and it enhances thymocyte proliferation in response to IL-7 (Hernandez-Lopez et al., 2002). Notably, antibodies that neutralize CXCL12 have been shown to reduce thymopoiesis in in vitro human thymic organ cultures, and the addition of CXCL12 increases thymopoiesis in these cultures (Hernandez-Lopez et al., 2002). The later timing of CXCL12 secretion during the in vitro thymic organ culture process shown here combined with its increased in vivo expression in thymuses from donors >18 years old compared to younger donors suggests that the expression of this chemokine is induced by a more long-lived thymocyte depletion compared to that required for the induction of CCL21 secretion. The screening data suggest that other chemokines, including CXCL16 and CCL11, may be biomarkers for assessing the viability and function of cultured thymus, since they also increase during thymic organ culture with the loss of thymocytes. Both CXCL16 and CCL11 have previously been shown to be produced by TECs (Bunting et al., 2011).

[0222] CCL11 was originally named eosinophil chemotactic factor because of its ability to attract eosinophils. We previously showed that eosinophil infiltration may be prominent adjacent to thymic tissue with active thymopoiesis (Flores et al., 1999), although CCL11 levels were not directly measured in these studies. However, it was subsequently also shown that CCL11 acts as a chemoattractant for double-positive and single-positive human thymocytes (Bunting et al., 2011). Evidence for the specific role of CXCL16 in thymopoiesis is less clear.

[0223] 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, which can also predict efficient immune reconstitution if thymocyte precursors become available. In both cultured thymic slices and non-cultured thymic tissue from older adults, production of CCL21 is generally low when thymopoiesis is robust and is significantly induced when thymopoiesis is impaired. Interestingly, after normalizing to the TEC area or the active cortical area, thymic tissue from younger donors (≤18 years) continues to express more CD3ε and CD1A mRNA and less keratin 8 (KRT8) and keratin 14 (KRT14) mRNA compared to thymus from older adults. This suggests that thymopoiesis and TEC maintenance may be more efficient in these younger donors. In addition, for both normalization methods, production of CCL21 and CXCL12 is significantly increased for tissue from donors >18 years (a time range with reduced TEC content and active thymopoiesis) compared to younger donors. Expression of both CCL21 and CXCL12 in vitro in thymic organ culture and in vivo during aging increases with decreasing thymocyte numbers, raising the possibility that induction of these chemokines is part of a homeostatic mechanism to counteract thymocyte depletion by enhancing T cell precursor recruitment. Cultured thymic slices depleted of thymocytes are expected to enhance their ability to colonize and effect immune reconstitution by increasing secretion of thymocyte-attracting chemokines, as observed when such slices are implanted into athymic infant recipients (Markert et al., 2008). In contrast, if the availability of thymocyte precursors or other crucial aspects of the thymic microenvironment are limited, the substantial secretion of CCL21 and CXCL12 may provide less benefit during aging.

[0224] In summary, these studies show that organ cultures of thymus from pediatric donors can be used to model at least some aspects of age-related thymic involution in humans, particularly those more directly related to thymocyte loss. However, it is clear that cultured pediatric thymus depleted of T cells must be very different in other respects from the thymus of aged adults, because implanting cultured pediatric thymus depleted of T cells into athymic recipients results in immune reconstitution and protection from infection, whereas aged adults with involuted thymus are more susceptible to infection compared to younger adults with more robust thymic 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.

[0225] The results presented herein Figure 56 provide a rich source of additional molecules and pathways to use the cultured infant thymus model to model some aspects of the aging human thymus in vitro. This is important because, given the need to remove a portion of the thymus from most infants for cardiac corrective surgery to adequately expose the surgical field, infant thymus is generally more readily available for study. Adult thymic tissue is generally less accessible because removal of thymic tissue is not usually required for many types of cardiac surgery common in adults to allow access. In addition, any removed adult thymic tissue is generally not available for study because it appears less organ-like and very similar to fat. However, if tolerance is needed in adult solid organ recipients, adult thymic tissue may be cultured and used for co-transplantation with solid organs. Biomarkers can also be used to determine the suitability, functionality, and viability of allogeneic cultured postnatal thymic tissue-derived products from adult donors.

[0226] The thymic production of thymocyte chemokines CCL21, CXCL16, CXCL12, and CCL11 increases with the decrease in thymocyte content. This suggests that thymocyte loss may activate homeostatic mechanisms that attempt to counter potential atrophy, although ultimately not successfully in the aging context. Future studies that more comprehensively elucidate these mechanisms will be useful for understanding and potentially reversing the mechanisms driving age-related thymic involution and could contribute to enhancing thymus-driven immune reconstitution at all ages.

[0227] In one aspect of the present disclosure, there is provided a method for generating an allogeneic cultured postnatal thymic tissue-derived product suitable for implantation into a human, the method comprising the steps of: subjecting a donor thymus to a conditioning protocol for a period of about 6 to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically processing the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; further comprising detecting an increased level of CCL21 in the thymic organ culture medium during the course of the conditioning protocol; and recovering the partially T cell-depleted donor thymic tissue slice as an allogeneic cultured postnatal thymic tissue-derived product suitable for implantation.

[0228] In one or more embodiments of the aspect of the present disclosure, the method further comprises the step of cryopreserving the allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen for future implantation.

[0229] In one or more embodiments of the aspect of the present disclosure, the method further comprises detecting a decreased level of L-selectin in the thymic organ culture medium during the course of the conditioning protocol.

[0230] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting one or more of CCL21, CXCL12, CXCL16 or CCL11 in the thymic organ culture medium during the course of the conditioning protocol.

[0231] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting an increased level of one or more of CXCL12, CXCL16 or CCL11 in the thymic organ culture medium during the course of the conditioning protocol.

[0232] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting an increased level of CXCL12 in the thymic organ culture medium during the course of the conditioning protocol.

[0233] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting an increasing CXCL16 in the thymic organ culture medium during the course of the conditioning protocol.

[0234] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting an increased level of CCL11 in the thymic organ culture medium during the course of the conditioning protocol.

[0235] In one or more embodiments of the aspect of the present disclosure, the method comprises detecting one or more of M-CSF, galectin-7 or IL-16 in the thymic organ culture medium during the course of the conditioning protocol.

[0236] In one or more embodiments of aspects of the present disclosure, the method includes detecting a reduced level of one or more of M-CSF, galectin-7, or IL-16 in the thymic organ culture medium during the conditioning regimen.

[0237] In one or more embodiments of aspects of the present disclosure, the method includes detecting a reduced level of M-CSF in the thymic organ culture medium during the conditioning regimen.

[0238] In one or more embodiments of aspects of the present disclosure, the method includes detecting a reduced level of galectin-7 in the thymic organ culture medium during the conditioning regimen.

[0239] In one or more embodiments of aspects of the present disclosure, the method includes detecting a reduced level of IL-16 in the thymic organ culture medium during the conditioning regimen.

[0240] In one or more embodiments of aspects of the present disclosure, the conditioning regimen lasts 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 21 days; or lasts 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.

[0241] In one or more embodiments of aspects of the present disclosure, the level of CCL21 is approximate Figure 50 to the level in E, and / or the level of L-selectin is approximate Figure 50 to the level in A, and / or the level of M-CSF is approximate Figure 50 to the level in B, and / or the level of galectin-7 is approximate Figure 50 to the level in C, and / or the level of IL-16 is approximate Figure 50 to the level in D, and / or the level of CXCL16 is approximate Figure 50 to the level in F, and / or wherein the level of CCL11 is approximate Figure 50 to the level in G, and / or the level of CXL21 is approximate Figure 50 to the level in H.

[0242] In one or more embodiments of aspects of the present disclosure, the method further comprises the steps of: determining, between day 6 and day 21 of the conditioning regimen, preferably between day 6 and day 9, in the donor thymic tissue sections, regions that are positive for cytokeratin AE1 / AE3, the presence of at least one Hassall's corpuscle, CK14 staining throughout the donor thymic tissue sections, and the presence of intact nuclei, which are dispersed throughout the donor thymic tissue sections.

[0243] In one or more embodiments of aspects of the present disclosure, the method further comprises detecting the level of at least one marker in the thymic organ culture medium during the culture regimen, or detecting at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight markers selected from the following in the thymic organ culture medium during the conditioning regimen: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[0244] In one or more embodiments of aspects of the present disclosure, the method further comprises detecting the level of at least one marker selected from the following in the thymic organ culture medium during the culture regimen: 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 / SDF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGFR, 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.

[0245] In one aspect of the present disclosure, there is provided a method for determining whether a postnatal thymic tissue-derived product cultured allogeneically is suitable for implantation into a human, the method comprising the steps of: conditioning donor thymic tissue slices in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker selected from the following in the thymic organ culture medium: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[0246] In one or more embodiments of the aspect of the present disclosure, the at least one marker is L-selectin, and wherein the level of L-selectin in the thymic organ culture medium decreases over time, i.e., during the course of the conditioning protocol.

[0247] In one or more embodiments of the aspect of the present disclosure, the at least one marker is M-CSF, and wherein the level of M-CSF in the thymic organ culture medium decreases during the course of the conditioning protocol.

[0248] In one or more embodiments of the aspect of the present disclosure, the at least one marker is galectin-7, and wherein the level of galectin-7 in the thymic organ culture medium decreases during the course of the conditioning protocol.

[0249] In one or more embodiments of the aspect of the present disclosure, the at least one marker is IL-16, and wherein the level of IL-16 in the thymic organ culture medium decreases during the course of the conditioning protocol.

[0250] In one or more embodiments of the aspect of the present disclosure, the at least one marker is CCL21, and wherein the level of CCL21 in the thymic organ culture medium increases during the course of the conditioning protocol.

[0251] In one or more embodiments of the aspect of the present disclosure, the at least one marker is CXCL12, and wherein the level of CXCL12 in the thymic organ culture medium increases during the course of the conditioning protocol.

[0252] In one or more embodiments of the aspect of the present disclosure, the at least one marker is CXCL16, and wherein the level of CXCL16 in the thymic organ culture medium increases during the course of the conditioning protocol.

[0253] In one or more embodiments of aspects of the present disclosure, the at least one marker is CCL11, and wherein the level of CCL11 in the thymic organ culture medium increases during the conditioning protocol.

[0254] In one aspect of the present disclosure, there is provided a method for determining whether a postnatal thymic tissue-derived product cultured allogeneically is suitable for implantation into a human, the method comprising the steps of: conditioning a donor thymic tissue slice in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: 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 / SDF-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.

[0255] In one or more embodiments of aspects of the present disclosure, the method further comprises the steps of: determining, between day 6 and day 21 of the conditioning protocol, in the donor thymic tissue slice, regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymic tissue slice, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue slice, and the presence of intact nuclei.

[0256] In one or more embodiments of aspects of the present disclosure, the conditioning regimen lasts 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 21 days; or lasts 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.

[0257] In one or more embodiments of aspects of the present disclosure, the level of CCL21 is approximate Figure 50 to the level in E, and / or the level of L - selectin is approximate Figure 50 to the level in A, and / or the level of M - CSF is approximate Figure 50 to the level in B, and / or the level of galectin - 7 is approximate Figure 50 to the level in C, and / or the level of IL - 16 is approximate Figure 50 to the level in D, and / or the level of CXCL16 is approximate Figure 50 to the level in F, and / or the level of CCL11 is approximate Figure 50 to the level in G, and / or the level of CXL21 is approximate Figure 50 to the level in H.

[0258] In one or more embodiments of aspects of the present disclosure, the method further includes detecting the level of at least one marker in the thymic organ culture medium during the course of the conditioning regimen, or detecting at least two, at least three, at least four, at least five, at least six, at least seven or 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 thymic organ culture medium during the course of the conditioning regimen, and further wherein CCL21, CXCL12, CXCL16 and CCL11 increase in the thymic organ culture medium during the course of the conditioning regimen.

[0259] In one aspect of the present invention, there is provided a method for determining whether a product derived from allogeneic cultured postnatal thymic tissue is suitable for implantation into a human, the method comprising the steps of: conditioning donor thymic tissue slices in thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker selected from Figure 56 in the thymic organ culture medium.

[0260] In one or more embodiments of the aspects of the present disclosure, there is provided a method for determining whether a product derived from allogeneic cultured postnatal thymic tissue is suitable for implantation into a human, the method comprising the steps of: conditioning donor thymic tissue slices in thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker selected from the following during the course of the conditioning protocol: 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 / SDF-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.

[0261] In one aspect of the present disclosure, a method of treating a thymic disorder is provided, the improvement comprising implanting into a subject having a thymic disorder slices of allogeneic cultured postnatal thymic tissue origin, and subjecting the slices to a conditioning protocol in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker selected from: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[0262] In another aspect of the present disclosure, a method of treating a thymic disorder is provided, the improvement comprising implanting into a subject having a thymic disorder slices of allogeneic cultured postnatal thymic tissue origin, and subjecting the slices to a conditioning protocol in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; and detecting the level of at least one marker 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 / SDF-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 during the course of the conditioning protocol.

[0263] In one or more embodiments of the aspects of the present disclosure, at least one marker is L-selectin, and wherein the level of L-selectin in the thymic organ culture medium decreases during the course of the conditioning protocol.

[0264] In one or more embodiments of the aspects of the present disclosure, at least one marker is M-CSF, and wherein the level of M-CSF in the thymic organ culture medium decreases during the course of the conditioning protocol.

[0265] In one or more embodiments of aspects of the present disclosure, at least one marker is galectin-7, and wherein the level of galectin-7 in the thymic organ culture medium decreases during the conditioning regimen.

[0266] In one or more embodiments of aspects of the present disclosure, at least one marker is IL-16, and wherein the level of IL-16 in the thymic organ culture medium decreases during the conditioning regimen.

[0267] In one or more embodiments of aspects of the present disclosure, at least one marker is CCL21, and wherein the level of CCL21 in the thymic organ culture medium increases during the conditioning regimen.

[0268] In one or more embodiments of aspects of the present disclosure, at least one marker is CXCL12, and wherein the level of CXCL12 in the thymic organ culture medium increases during the conditioning regimen.

[0269] In one or more embodiments of aspects of the present disclosure, at least one marker is CXCL16, and wherein the level of CXCL16 in the thymic organ culture medium increases during the conditioning regimen.

[0270] In one or more embodiments of aspects of the present disclosure, at least one marker is CCL11, and wherein the level of CCL11 in the thymic organ culture medium increases during the conditioning regimen.

[0271] In one or more embodiments of aspects of the present disclosure, the method further comprises the steps of: during the conditioning regimen, determining in the donor thymic tissue section regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymic tissue section, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue section, and the presence of intact nuclei.

[0272] In one or more embodiments of aspects of the present disclosure, the thymic disorder is congenital athymia associated with complete DiGeorge syndrome, 22q11.2 deletion, CHARGE (coloboma, heart defects, 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 (FOXN1) deficiency.

[0273] In one or more embodiments of aspects of the present disclosure, the thymic disorder is thymic involution. In another embodiment, the thymic disorder is congenital athymia associated with mutations in the TBX-1 or TBX-2 gene.

[0274] In one or more embodiments of aspects of the present disclosure, the thymic disorder is associated with paired box 1 (PAX1), semaphorin 3E (SEMA3E), and a recurrent microdeletion at chromosome 2p11.2.

[0275] In one or more embodiments of aspects of the present disclosure, the thymic disorder is associated with thymoma. In further embodiments, the thymoma is non-malignant or malignant.

[0276] In one or more embodiments of aspects of the present disclosure, the thymic disorder is associated with myasthenia gravis (MG), pure red cell aplasia, and hypogammaglobulinemia.

[0277] In one aspect of the present disclosure, a method for providing immunocompetence in a human subject is provided, the improvement comprising the steps of: conditioning donor thymic tissue slices in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce partially T cell-depleted donor thymic tissue slices; detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7, or IL-1, the level is decreased, or if the marker is CCL21, CXCL12, CXCL16, or CCL11, the level is increased; and implanting the partially T cell-depleted donor thymic tissue slices into the human subject.

[0278] In one aspect of the present disclosure, a method for providing immune competence in a human subject is provided, the improvement comprising the steps of: conditioning donor thymic tissue slices in thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; detecting, during the course of the conditioning protocol, the level of at least one marker selected from the group consisting of markers from the following in the thymic organ culture medium: 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 / SDF-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 thymic tissue slice into the human subject.

[0279] In one aspect of the present disclosure, a method for providing immune competence in a human subject undergoing a solid organ graft is provided, the method comprising the steps of: removing the thymus of the human subject; obtaining thymic tissue from a donor matched for HLA class I and HLA class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymic tissue slices in thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice; detecting, during the course of the conditioning protocol, the level of at least one marker selected from the group consisting of the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymic organ culture medium; wherein if the marker is L-selectin, M-CSF, galectin-7, or IL-1, the level is decreased, or if the marker is CCL21, CXCL12, CXCL16, or CCL11, the level is increased; implanting the solid organ; and implanting the partially T cell-depleted donor thymic tissue slice into the human subject.

[0280] To better understand the generation of tolerance in this model, reference can be made to Figure 38 , which has a primate model of this procedure, and the model will provide data to support human studies on the generation of donor-specific tolerance. Figure 38 The experiment in has three monkeys. One is the thymus and heart donor (information in the left column). The second is the thymus and heart recipient (information in the middle column on page 2. This column has the stages of the experiment). The third is the control (information in the right column on page 3). Note that the original spreadsheet has a one-page width × multiple-page length procedure for all three animals. Since the spreadsheet is wider than the allowed width, each row of the spreadsheet is divided into 3 pages. The table continues for many weeks and several stages in groups of 3 small figures.

[0281] In one aspect of the present disclosure, a method for providing immune competence in a human subject undergoing a solid organ graft is provided, the method comprising the steps of: removing the thymus of the human subject; obtaining thymic tissue from a donor matched for HLA-I and class II alleles in the solid organ; slicing the donor thymus; conditioning the donor thymic tissue slices in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically treating the donor thymic tissue in the thymic organ culture medium to produce partially T cell-depleted donor thymic tissue slices; detecting, during the course of the conditioning protocol, the level of at least one marker selected from the following in the thymic organ culture medium: 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 thymic tissue slices into the human subject.

[0282] In one aspect of the present disclosure, a method for providing immune competence in a human subject undergoing an organ transplant is provided, the method comprising the steps of: removing the thymus of the human subject; obtaining thymic tissue from a donor who is HLA-class I and HLA-class II allele-matched to the solid organ; slicing the donor thymus; conditioning the donor thymic tissue slices in a thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises subjecting the donor thymic tissue to aseptic treatment in the thymic organ culture medium to produce partially T cell-depleted donor thymic tissue slices; detecting, during the course of the conditioning protocol, the level of at least one marker selected from the following in the thymic organ culture medium: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16 or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7 or IL-1, the level is decreased, or if the marker is CCL21, CXCL12, CXCL16 or CCL11, the level is increased; implanting the solid organ; and implanting the partially T cell-depleted donor thymic tissue slices into the human subject.

[0283] In one aspect of the present disclosure, there is provided a method for providing immune competence in a human subject undergoing an allogeneic solid organ graft, the method comprising the steps of: removing the thymus of the human subject; obtaining thymic tissue from a donor who is HLA class I and HLA class II allele matched to the solid organ; slicing the donor thymus; conditioning the sliced donor thymic tissue in thymic organ culture medium for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptic treatment of the donor thymic tissue in the thymic organ culture medium to produce a partially T cell depleted donor thymic tissue slice; detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: 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 / SDF-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 thymic tissue slice into the human subject.

[0284] In one aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased donor in a recipient in need of solid organ transplantation, the method comprising the steps of:

[0285] (a) removing the thymus of the recipient;

[0286] (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0287] (c) providing a suitable solid human organ and thymus from a deceased donor;

[0288] (d) transplanting the solid human organ into the recipient;

[0289] (e) treating the recipient with a maintenance immunosuppressive regimen;

[0290] (f) Providing a product derived from allogeneically cultured postnatal thymic tissue, wherein the product derived from allogeneically cultured postnatal thymic tissue is subjected to a conditioning protocol in a thymic organ culture medium for a period of about 6 days to about 21 days to produce sections of the product derived from allogeneically cultured postnatal thymic tissue; further, wherein the conditioning protocol for the donor thymic tissue includes aseptically treating the donor thymic tissue in the thymic organ culture medium to produce partially T cell-depleted donor thymic tissue sections; during the course of the conditioning protocol, detecting the level of at least one marker selected from the following in the thymic organ culture medium: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7, or IL-1, the level of the marker in the thymic organ culture medium is decreased, or if the marker is CCL21, CXCL12, CXCL16, or CCL11, the level of the marker in the thymic organ culture medium is increased; and

[0291] (g) About 6 days to about 21 days after the conditioning protocol, implanting the product derived from allogeneically cultured postnatal thymic tissue into the recipient, wherein the dose of the thymic tissue sections is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted product derived from allogeneically cultured postnatal thymic tissue induces thymopoiesis and tolerance in the recipient.

[0292] In one aspect of the present disclosure, a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased donor in a recipient in need of a solid organ transplant is provided, the method comprising the steps of:

[0293] (a) Removing the thymus of the recipient;

[0294] (b) Treating the recipient with an induction immunosuppressive protocol comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0295] (c) Providing a suitable solid human organ and thymus from a deceased donor;

[0296] (d) Transplanting the solid human organ into the recipient;

[0297] (e) Treating the recipient with a maintenance immunosuppressive protocol;

[0298] (f) Provide a product derived from allogeneically cultured postnatal thymic tissue, wherein the product derived from allogeneically cultured postnatal thymic tissue is subjected to a conditioning protocol in a thymic organ culture medium for a period of about 6 days to about 21 days to produce sections of the product derived from allogeneically cultured postnatal thymic tissue; further, wherein the conditioning protocol for the donor thymic tissue includes aseptically processing the donor thymic tissue in the thymic organ culture medium to produce partially T cell-depleted sections of the donor thymic tissue; during the course of the conditioning protocol, detecting the level of at least one marker selected from the following in the thymic organ culture medium: 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 / SDF-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

[0299] (g) About 6 days to about 21 days after the conditioning protocol, implant the product derived from allogeneically cultured postnatal thymic tissue into the recipient, wherein the dose of the thymic tissue sections is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted product derived from allogeneically cultured postnatal thymic tissue induces thymopoiesis and tolerance in the recipient.

[0300] In one aspect of the present disclosure, a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a living human donor in a human recipient in need of a solid organ transplant is provided, the method comprising the steps of:

[0301] (a) Removing the thymus of the recipient;

[0302] (b) Treating the recipient with an induction immunosuppressive protocol comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0303] (c) Providing a suitable solid organ from a living human donor;

[0304] (d) Transplant the solid organ into the recipient;

[0305] (e) Treat the recipient with a maintenance immunosuppressive regimen;

[0306] (f) Provide a cryopreserved allogeneic cultured postnatal thymic tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymic tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is processed from thymic tissue from a thymic donor who expresses HLA alleles that match the HLA class I and HLA class II alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning regimen for the donor thymic tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning regimen for the donor thymic tissue includes aseptic treatment of the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice; detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning regimen: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7, or IL-1, the level of the marker in the thymic organ culture medium is decreased, or if the marker is CCL21, CXCL12, CXCL16, or CCL11, the level of the marker in the thymic organ culture medium is increased;

[0307] (g) Thaw the cryopreserved allogeneic cultured postnatal thymic tissue-derived product; and

[0308] (h) Implant the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient

[0309] In one or more embodiments of aspects of the present disclosure, permissive mismatches of HLA-DP may be allowed (Pidala J et al., 2014 Blood 124:2596-2606). Additionally, non-permissive mismatches of HLA-DPB1 may be allowed if there are sufficient numbers of functional distances (Crivello P et al., 2016 Blood 128:120-129).

[0310] In one aspect of the invention, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a living donor in a human recipient in need of a solid organ transplant, the method comprising the steps of:

[0311] (a) removing the thymus of the recipient;

[0312] (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0313] (c) providing a suitable solid organ from a living donor;

[0314] (d) transplanting the solid organ into the recipient;

[0315] (e) treating the recipient with a maintenance immunosuppressive regimen;

[0316] (f) Providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymus tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor who expresses HLA alleles that match the HLA class I and HLA class II alleles in the recipient and are not present in the solid organ graft; wherein the conditioning protocol for the donor thymus tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning protocol for the donor thymus tissue includes aseptically processing the donor thymus tissue in a thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice; detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: 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 / SDF-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 thymic organ culture medium; wherein the level of the marker in the thymic organ culture medium increases or decreases according to Figure 56 the level of the marker described in

[0317] (g) Thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and

[0318] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient

[0319] In one or more embodiments of aspects of the present disclosure, approximately half of the thawed cryopreserved allogeneic postnatal thymic tissue-derived product is transplanted into the recipient and the remainder is cryopreserved for future use.

[0320] In one or more embodiments of aspects of the present disclosure, step (h) is performed about one month or more after transplantation of the solid organ.

[0321] In one aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased human donor in a human recipient in need of solid organ transplantation, the method comprising the steps of:

[0322] (a) Removing the recipient's thymus;

[0323] (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0324] (c) Providing a suitable solid organ from a living donor;

[0325] (d) Transplanting the solid organ into the recipient;

[0326] (e) Treating the recipient with a maintenance immunosuppressive regimen;

[0327] (f) Providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymus tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor, the thymus donor expressing HLA alleles that match the HLA alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning protocol for the donor thymus tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning protocol for the donor thymus tissue includes aseptic treatment of the donor thymus tissue in thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice, and detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16 or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7 or IL-1, the level of the marker in the thymic organ culture medium is decreased, or if the marker is CCL21, CXCL12, CXCL16 or CCL11, the level of the marker in the thymic organ culture medium is increased;

[0328] (g) Thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and

[0329] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0330] In one aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased human donor in a human recipient in need of a solid organ transplant, the method comprising the steps of:

[0331] (a) Removing the thymus of the recipient;

[0332] (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0333] (c) Provide a suitable solid organ from a living donor;

[0334] (d) Transplant the solid organ into the recipient;

[0335] (e) Treat the recipient with a maintenance immunosuppressive regimen;

[0336] (f) Provide a cryopreserved allogeneic cultured postnatal thymic tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymic tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is processed from thymic tissue from a thymic donor who expresses HLA alleles that match the HLA alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning regimen for the donor thymic tissue is for a period of about 6 days to about 21 days; further, wherein the conditioning regimen for the donor thymic tissue includes aseptic processing of the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice, and detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning regimen: 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 thymic organ culture medium increases or decreases according to the level in Figure 7;

[0337] (g) Thaw the cryopreserved allogeneic cultured postnatal thymic tissue-derived product; and

[0338] (h) Implant the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is about 1000 - 22000 mm 2 thymic tissue surface area / m 2the recipient's body surface area, and further wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0339] In one aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymic tissue-derived product, which is prepared by a method comprising the following steps:

[0340] (a) Obtaining suitable thymic tissue from a donor;

[0341] (b) Typing the following HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1;

[0342] (c) Subjecting the thymic tissue to a conditioning protocol for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically processing the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice;

[0343] (d) Detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16 or CCL11; wherein if the marker is L-selectin, M-CSF, galectin-7 or IL-1, the level of the marker in the thymic organ culture medium is decreased, or if the marker is CCL21, CXCL12, CXCL16 or CCL11, the level of the marker in the thymic organ culture medium is increased;

[0344] (e) Recovering the partially T cell-depleted donor thymic tissue slice as an allogeneic cultured postnatal thymic tissue-derived product;

[0345] (f) Cryopreserving the allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen; and

[0346] (g) Maintaining the cryopreserved allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen in a cryopreserved allogeneic cultured postnatal thymic tissue-derived product bank.

[0347] In one aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymic tissue-derived product, which is prepared by a method comprising the following steps:

[0348] (a) Obtain suitable thymic tissue from a donor;

[0349] (b) Type the following HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1;

[0350] (c) Subject the thymic tissue to a conditioning protocol for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptic treatment of the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted donor thymic tissue slice;

[0351] (d) Detect the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: 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 / SDF-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 thymic organ culture medium increases or decreases according to the level in Figure 7;

[0352] (e) Recover the partially T cell-depleted donor thymic tissue slice as a product for use as a source of allogeneically cultured postnatal thymic tissue;

[0353] (f) Cryopreserve the product for use as a source of allogeneically cultured postnatal thymic tissue in liquid nitrogen; and

[0354] (g) Maintain the cryopreserved product for use as a source of allogeneically cultured postnatal thymic tissue in liquid nitrogen in a cryopreserved product bank for use as a source of allogeneically cultured postnatal thymic tissue.

[0355] In one aspect of the present disclosure, there is provided a method for promoting donor - specific tolerance to an allogeneic solid organ graft obtained from a deceased human donor in a human recipient in need of solid organ transplantation, the method comprising the steps of:

[0356] (a) Removing the thymus of the recipient;

[0357] (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T - cells and / or inhibit the recipient's T - cells from rejecting the transplanted solid organ;

[0358] (c) Providing a suitable solid organ from a living donor;

[0359] (d) Transplanting the solid organ into the recipient;

[0360] (e) Treating the recipient with a maintenance immunosuppressive regimen;

[0361] (f) Providing a cryopreserved allogeneic cultured post - natal thymic tissue - derived product maintained in a cryopreserved bank of allogeneic cultured post - natal thymic tissue - derived products; wherein the cryopreserved allogeneic cultured post - natal thymic tissue - derived product is processed from thymic tissue from a thymic donor expressing HLA alleles that match the HLA alleles in the recipient and that are not present in the solid organ graft; wherein the conditioning protocol for the donor thymic tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning protocol for the donor thymic tissue comprises aseptic treatment of the donor thymic tissue in thymic organ culture medium to produce a partially T - cell - depleted thymic tissue slice, and during the course of the conditioning protocol, detecting the level of at least one marker selected from the following in the thymic organ culture medium: the markers L - selectin, M - CSF, galectin - 7, IL - 16, CCL21, CXCL12, CXCL16, or CCL11 in the thymic organ culture medium; wherein if the marker is L - selectin, M - CSF, galectin - 7, or IL - 1, the level of the marker in the thymic organ culture medium is decreased, or if the marker is CCL21, CXCL12, CXCL16, or CCL11, the level of the marker in the thymic organ culture medium is increased;

[0362] (g) Thawing the cryopreserved allogeneic cultured post - natal thymic tissue - derived product; and

[0363] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is about 1000 - 22000 mm 2 Surface area of thymic tissue / m 2 Recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

[0364] In one aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymic tissue-derived product, which is prepared by a method comprising the following steps:

[0365] (a) Obtaining suitable thymic tissue from a donor;

[0366] (b) Typing the following HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1;

[0367] (c) Subjecting the thymic tissue to a conditioning protocol for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymic tissue comprises aseptically processing the donor thymic tissue in thymic organ medium to produce a partially T cell-depleted donor thymic tissue slice;

[0368] (d) Detecting the level of at least one marker selected from the following in the thymic organ medium during the course of the conditioning protocol: the markers L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16 or CCL11 in the thymic organ medium; wherein if the marker is L-selectin, M-CSF, galectin-7 or IL-1, the level of the marker in the thymic organ medium is decreased, or if the marker is CCL21, CXCL12, CXCL16 or CCL11, the level of the marker in the thymic organ medium is increased;

[0369] (e) Recovering the partially T cell-depleted donor thymic tissue slice as an allogeneic cultured postnatal thymic tissue-derived product;

[0370] (f) Cryopreserving the allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen; and

[0371] (g) Maintaining the cryopreserved allogeneic culture of postnatal thymus tissue-derived product in liquid nitrogen in a cryopreserved allogeneic culture of postnatal thymus tissue-derived product bank.

[0372] In one aspect of the present disclosure, there is provided a method for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a deceased human donor in a human recipient in need of solid organ transplantation, the method comprising the steps of:

[0373] (a) Removing the thymus of the recipient;

[0374] (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ;

[0375] (c) Providing a suitable solid organ from a living human donor;

[0376] (d) Transplanting the solid organ into the recipient;

[0377] (e) Treating the recipient with a maintenance immunosuppressive regimen;

[0378] (f) Providing a cryopreserved allogeneic cultured postnatal thymus tissue-derived product maintained in a cryopreserved bank of allogeneic cultured postnatal thymus tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor who expresses HLA alleles that match the HLA alleles in the recipient and are not present in the solid organ graft; wherein the conditioning protocol for the donor thymus tissue is carried out for a period of about 6 days to about 21 days; further, wherein the conditioning protocol for the donor thymus tissue includes aseptically processing the donor thymus tissue in thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice, and detecting the level of at least one marker selected from the following in the thymic organ culture medium during the course of the conditioning protocol: 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 / SDF-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 thymic organ culture medium increases or decreases according to Figure 56 the level of the marker described in

[0379] (g) Thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and

[0380] (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymus tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is about 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymus tissue-derived product induces thymopoiesis and tolerance in the recipient

[0381] In one or more embodiments of aspects of the present disclosure, the solid organ graft is a heart graft, a kidney graft, a liver graft, a lung graft, a heart / lung graft, a pancreas graft, an intestine graft, a stomach graft, an abdominal wall graft, a craniofacial graft, a scalp graft, a penile graft, a uterine graft, a unilateral or bilateral upper limb graft, a unilateral vascularized composite allograft, or a combination thereof.

[0382] In one or more embodiments of aspects of the present disclosure, the solid organ graft is a heart graft, or a pediatric heart graft, or an adult heart graft.

[0383] In one or more embodiments of aspects of the present disclosure, the conditioning regimen lasts 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 21 days; or lasts 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.

[0384] In one aspect of the present disclosure, there is provided a cryopreserved allogeneic cultured postnatal thymic tissue-derived product suitable for implantation into a human, comprising the following steps:

[0385] (a) Obtaining suitable thymic tissue from a human donor;

[0386] (b) Subjecting the thymic tissue to a conditioning regimen for a period of about 6 days to about 21 days; wherein the conditioning regimen for the donor thymic tissue comprises aseptically treating the donor thymic tissue in a thymic organ culture medium to produce a partially T cell-depleted thymic tissue slice; wherein the levels of L-selectin and / or M-CSF and / or galectin-7 and / or IL-16 in the thymic organ culture medium decrease during the course of the conditioning regimen; further, wherein the levels of CCL21 and / or CXCL12 and / or CXCL16 and / or CCL11 in the thymic organ culture medium increase during the course of the conditioning regimen;

[0387] (c) Harvesting the partially T cell-depleted donor thymic tissue slice as an allogeneic cultured postnatal thymic tissue-derived product;

[0388] (d) cryopreserving the allogeneically cultured postnatal thymic tissue-derived product in liquid nitrogen; and

[0389] (e) maintaining the cryopreserved allogeneically cultured postnatal thymic tissue-derived product in liquid nitrogen in a cryopreserved allogeneically cultured postnatal thymic tissue-derived product bank.

[0390] In one or more embodiments of aspects of the present disclosure, the level of CCL21 in the thymic organ culture medium increases during the conditioning regimen.

[0391] In one or more embodiments of aspects of the present disclosure, the level of L-selectin in the thymic organ culture medium decreases during the conditioning regimen.

[0392] In one or more embodiments of aspects of the present disclosure, the level of one or more of M-CSF, galectin-7, and IL-16 in the thymic organ culture medium decreases during the conditioning regimen.

[0393] In one or more embodiments of aspects of the present disclosure, the level of one or more of CCL21, CXCL12, CXCL16, and CCL11 in the thymic organ culture medium increases during the conditioning regimen.

[0394] In one or more embodiments of aspects of the present disclosure, the method further comprises the steps of: during the conditioning regimen, determining in the donor thymic tissue section regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymic tissue section, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue section, and the presence of intact nuclei.

[0395] In one aspect of the present disclosure, there is provided a kit for performing the method of any one of the foregoing aspects and embodiments, together with instructions for use in determining whether an allogeneically cultured postnatal thymic tissue-derived product is suitable for implantation into a human.

[0396] In one or more embodiments of aspects of the present disclosure, the kit comprises at least one antibody that specifically binds to the marker L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[0397] In one or more embodiments of aspects of the present disclosure, the kit comprises antibodies that specifically bind to Figure 56 one or more of the markers shown.

[0398] In one aspect of the present disclosure, there is provided a kit for determining whether a cryopreserved allogeneic culture-derived postnatal thymic tissue product cultured according to any one of the foregoing aspects and embodiments is suitable for implantation into a human, together with instructions for use.

[0399] In one or more embodiments of the aspects of the present disclosure, the kit comprises at least one antibody that specifically binds to a marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[0400] It should be further understood that certain features described herein in the context of different aspects and / or separate embodiments of the present disclosure for clarity may also be provided in combination in a single embodiment. Conversely, the various features described in the context of a single aspect and / or a single embodiment of the present disclosure for brevity may also be provided separately or in any suitable sub-combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0401] To more fully understand the principles disclosed herein and their advantages, reference is made to the following description in conjunction with the accompanying drawings, in which:

[0402] Figure 1 The manner in which an allogeneic culture-derived postnatal thymic tissue product (e.g., CTT, RVT-802) provides immune reconstitution in congenital athymia after implantation is described.

[0403] Figure 2 A schematic diagram of the steps for reconstituting the immune system in rats as described elsewhere in Example 5 is shown: removing the thymus in immunocompetent Lewis rats, administering an antibody to kill T cells in the recipient rats, implanting cultured neonatal thymic tissue from donor rats into the recipient rats, administering an immunosuppressant for approximately 4 months, and evaluating T cell development in the recipient rats. Notably, all rats in the treatment group had more than 10% naive T cells before cyclosporine was discontinued.

[0404] Figure 3 The development of naive T cells in two experimental recipient rats (ascending lines on the right) of Example 5 and two control rats (thick lines at baseline) that did not receive thymic tissue implants is shown.

[0405] Figure 4 A schematic diagram of the production process for harvesting thymus from a donor, culturing thin slices of donor thymic tissue obtained with a manual slicing mechanism for up to 21 days, and implanting the cultured thymic tissue into the quadriceps muscle of a recipient is shown.

[0406] Figure 5AShows a schematic diagram that depicts a section of thymic tissue used to characterize a test, as discussed in section

[00766] . Figure 5B Is a diagram of a section of thymic tissue on a cellulose filter membrane on a surgical sponge in a tissue culture dish such as is used for culturing thymus.

[0407] Figure 6A -H depicts histological tests of sections of thymic tissue from a batch (MFG-056) of cultured thymic tissue at days 5, 9, 12, and 21 after harvesting the thymus from a donor. At day 5 ( Figure 6A , Figure 6B ), day 9 ( Figure 6C , Figure 6D ), day 12 ( Figure 6E , Figure 6F ), and day 21 ( Figure 6G , Figure 6H ) are shown hematoxylin and eosin stained sections (left insets) and their corresponding reactivity with a mixture of anti-cytokeratin antibodies AE1 / AE3 (right insets; brown indicates positive reactivity), respectively. The bar in the lower left of each inset represents 100 μm. The insets with H&E show the depletion of T cells over time. Figure 6E And Figure 6F Are mainly epithelial cells. Condensation of the subcapsular cortical epithelium occurs as thymocytes are depleted over time. A similar condensation occurs in the medullary region of the thymus. Photographed by Laura P. Hale, MD, PhD, Department of Pathology, Duke University.

[0408] Figure 7A And Figure 7B Depict the histology of thymic tissue sections at day 0 of the time course at scales of 5 mm ( Figure 9A ) and 100 μm ( Figure 7B ), respectively. This shows the thymus and thymocytes at low magnification (scale bar 5 mm) and high magnification (scale bar 100 μm) at day 0. This is a normal thymus. At this time, both the cortex and medulla have a large number of thymocytes, and the dark blue nuclei contribute to the overall dark blue appearance of the tissue. Photographed by MD, PhD, Department of Pathology, Duke University.

[0409] Figure 8A And Figure 8B Are images from H&E stained slides that depict at scales of 5 mm ( Figure 8A ) and 100 μm ( Figure 8B)'s scale depicts the histology of a thymus tissue section on day 5 of the time course. The progression of thymocyte depletion results in a more eosinophilic (pink) appearance of the tissue. Photographed by MD, PhD, Department of Pathology, Duke University.

[0410] Figure 9A and Figure 9B depict the H&E staining of thymus tissue sections on day 12 of the time course at scales of 5 mm ( Figure 9A ) and 100 μm ( Figure 9B ), respectively. We see the progressive depletion of thymocytes. Higher magnification shows numerous eosinophilic cell bodies lacking nuclei, which can diagnose necrotic cells that have undergone karyolysis (dissolution of the nucleus). This degree of necrosis is expected at this time in culture. Photographed by MD, PhD, Department of Pathology, Duke University.

[0411] Figure 10A and Figure 10B depict the H&E staining of thymus tissue sections on day 21 of the time course at scales of 5 mm ( Figure 10A ) and 100 μm ( Figure 10B ), respectively. Note the preservation of the overall structure of the tissue, which contains a subcapsular cortex, cortical regions, and medullary regions with numerous Hassall's corpuscles in Figure 10B . The smaller, darker cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photographed by MD, PhD, Department of Pathology, Duke University.

[0412] Figure 11A -E depicts representative thymus sections immunostained with a mixture of anti-cytokeratin antibodies (AE1 / AE3). Figure 11A . Day 0; Figure 11B . Day 5; Figure 11C . Day 9; Figure 11D . Day 12; and Figure 11E . Day 21. As the culture progresses, the structure of the thymic epithelial network remains Complete . Scale bar represents 400 μm. Photographed by MD, PhD, Department of Pathology, Duke University.

[0413] Figure 12A and 12B depict the histology of thymus tissue slides after exposure to forced degradation conditions with 10X PBS. Figure 12A depicts the cortex on day 9 after exposure to forced degradation conditions. Figure 12B depicts the cortex on day 21 after exposure to forced degradation conditions. At Figure 12AIn it, the blue smear is DNA released from cells. Although small foci of cells with intact nuclei can be identified, most cells show signs of degradation. Photographed by M.D., Ph.D. of the Department of Pathology, Duke University.

[0414] Figure 13 Depicts a histological section of H&E staining of clinical sample MLM247. This is day 0 of culture. The scale bar is 200 um. This is a frozen section from day 0. Since this section has been frozen, the tissue looks different from the formalin-fixed paraffin-embedded tissue at day 0. Photographed by M.D., Ph.D. of the Department of Pathology, Duke University.

[0415] Figure 14 Frozen section of clinical sample MLM219, which is a histological section of H&E staining. This is a frozen section, so the tissue looks different from the formalin-fixed paraffin-embedded tissue cultured and presented above. However, the important histological features of thymocyte depletion and the robust viability of TECs have been well demonstrated. Photographed by M.D., Ph.D. of the Department of Pathology, Duke University.

[0416] Figure 15 Is a photograph of freshly harvested thymic tissue.

[0417] Figure 16 Is a schematic diagram depicting the harvest, culture, implantation, and implantation biopsy of CTT under the renal capsule of rats as presented in Example 5.

[0418] Figure 17A -D presents a photograph of thymic tissue harvested from 3-day-old F1 (LWxDA) rats as described in Example 5, and the thymic tissue is cut into four pieces ( Figure 17A ). A photograph of thymic slices cultured on a sterile mixed cellulose ester filter membrane with thymic organ medium for 5 - 7 days in a 37°C CO2 incubator as described in Example 5 ( Figure 17B ). Figure 17C Is a photograph of CTT implanted under the renal capsule of LW rats. Figure 17D Is a photograph of a thymic graft harvested 6 months after implantation. The arrow indicates the CTT under the renal capsule.

[0419] Figure 18A -D is a photograph depicting the histological appearance of fresh thymic tissue (top frame) and CTT (bottom frame) at 100x magnification. Figure 18A Shows a comparison of medullary differentiation in H&E-stained fresh thymic tissue (top frame) and CTT cultured for 5 days (bottom frame) as described in Example 5. Figure 18BShows the typical lace-like pattern observable in CTT cultured for 5 days (bottom frame) compared to fresh thymic tissue (top frame) when stained for cytokeratin as described in Example 5. Figure 18C Shows fresh thymic tissue (top frame) and T cell-depleted CTT (bottom frame) when stained for Ki-67. Figure 18D Shows fresh thymic tissue stained for CD3 (top frame) and CTT thymic tissue cultured for 5 days and then stained for CD3 (bottom frame). The brown staining noted in the CD3-stained CTT ( Figure 18D , bottom frame) may represent debris of some live cells as well as dead T cells that have not been washed out of the tissue.

[0420] Figure 19A -D is a photograph depicting the histological appearance of fresh thymic tissue (top frame) and CTT (bottom frame) at 600x magnification. Figure 19A Shows a comparison of medullary differentiation in H&E-stained fresh thymic tissue (top frame) and CTT cultured for 5 days (bottom frame) as described in Example 5. Figure 19B Shows the typical lace-like pattern observable in CTT cultured for 5 days (bottom frame) compared to fresh thymic tissue (top frame) when stained for cytokeratin as described in Example 5. Figure 19C Shows fresh thymic tissue (top frame) and T cell-depleted CTT (bottom frame) when stained for Ki-67. Figure 19D Shows fresh thymic tissue stained for DC3 (top frame) and CTT thymic tissue cultured for 5 days and then stained for CD3 (bottom frame). The brown staining noted in the CD3-stained CTT ( Figure 19D , bottom frame) may represent debris of some live cells as well as dead T cells that have not been washed out of the tissue.

[0421] Figure 20 Is a schematic diagram of the experimental design of the experiment reported in Example 5. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0422] Figure 21 Shows that repopulated recipient-type T cells were seen in the lower right quadrant after CTT allotransplantation. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0423] Figure 22A and Figure 22BShows an implanted thymus removed at 8.5 months post-implantation, which shows positive cytokeratin staining ( Figure 22A ), and T cell staining similar to that of native thymus ( Figure 22B ). Original magnification x400. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0424] Figure 23 Shows a figure with a significant increase in the number of circulating CD4 and CD8 T cells compared to control animals without implanted CTT. The figure also shows a significant increase in the number of naive CD4 and naive CD8 T cells in the CTT group compared to the control group without cultured thymus tissue implantation (CTT), and a significant increase in the number of CD4 and CD8 recent thymic emigrants (RTE) in the CTT group compared to the control group without cultured thymus tissue implantation (CTT). This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0425] Figure 24 shows an immunohistological analysis of an implanted CTT removed on day 180, which shows normal thymic histology under the renal capsule ( Figure 24A right hand side). Figure 24B Shows the removed graft on H&E. Staining for live T cells (CD3), T cell proliferation (Ki67), and cytokeratin (detected by rabbit polyclonal antibody) is shown. In the inset stained for cytokeratin, a lacy pattern is observed with Hassall's corpuscle formation on TEC (arrow). This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0426] Figure 25 Shows the percentage of survival of LW rats undergoing DA heart transplantation after thymectomy and immunosuppression in the presence of grafts (CTT) with CTT (solid triangles, blue line) and without CTT (inverted triangles, red line). LW rats with CTT were tolerant; LW rats without CTT were immunodeficient and thus did not reject the DA heart. The control shows complete rejection of the DA heart graft in unmanipulated LW rats (open squares). LW control animals also did not reject LW heart grafts (open circles with horizontal line) (n = 9). This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0427] Figure 26A and 26BPhotographs of transplanted allografts (DA hearts) from animals implanted with CTT (26A) and non-implanted animals (26B) showing mononuclear cell infiltration, Figure 26C do not depict signs of rejection (blue solid and red solid triangles) of the International Society for Heart & Lung Transplantation (ISHLT) in 2004. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0428] Figure 27 Graph of BN heart graft survival as a percentage of animals surviving in the neck versus the number of days of graft survival in LW rats implanted with CTT (which are immunocompetent and reject cervical allogeneic BN hearts), non-implanted control LW animals (which are immunodeficient due to thymic deficiency and cannot reject cervical BN hearts), BN controls (LW rats that reject cervical BN hearts), and syngeneic controls (LW rats that do not reject cervical LW hearts). This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0429] Figure 28A and Figure 28B are photographs of BN heart tissue on day 11 and day 46 with CTT inserted ( Figure 28A ) and without CTT inserted ( Figure 28B ), respectively. These pictures are the basis of the data in Figure 27 and Figure 29 . Due to tolerance, the heart in Figure 28A was not rejected. Due to immunodeficiency caused by thymic deficiency, the heart in Figure 28B was not rejected. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0430] Figure 29 Shows the rejection grades of cervical BN hearts. Syngeneic LW hearts (hollow circles) placed in LW rats were not rejected. BN hearts (solid circles) placed in LW rats were rejected. BN hearts placed in LW rats that received CTT were rejected (solid squares). BN hearts placed in LW rats that did not receive CTT were weakly rejected in 2 rats (shaded triangles) and not rejected by the other three rats. These data show that even rats with CTT accept DA hearts ( Figure 26C)DA expressed as CTT can also strongly reject the hearts of third parties. Rats without CTT are immunodeficient and neither reject DA hearts ( Figure 26C ) nor reject BN hearts. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0431] Figure 30A and Figure 30B are photos of BN hearts from rats that received or did not receive CTT insertion compared to LW and DA hearts respectively. In Figure 30A , after removing immunosuppression and transplanting the BN heart, due to all the inflammation, the BN heart was rapidly rejected and thus very large. The LW heart is of normal size and is used for the heart to pump blood through the body. The DA heart is small because it is placed in the abdomen and does not need to pump blood. In Figure 30B , the rats are immunodeficient and cannot reject the heart BN or DA heart after removing immunosuppression. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0432] Figure 31A and Figure 31B are graphs of the rejection grades of removed cervical BN hearts from rats with and without CTT insertion, as well as BN controls and syngeneic control rats. Figure 31A Depicts the quantification of inflammatory cells in a primary abdominal DA heart allograft. The syngeneic control shows that LW rats do not reject LW hearts. The DA control shows that LW rats do reject DA hearts. Due to tolerance, the CTT group does not reject DA hearts. Due to immunodeficiency caused by thymic deficiency, the group without CTT does not reject DA hearts. Figure 31B Depicts the quantification of inflammatory cells in a secondary cervical BN heart allograft. 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 due to its immune competence. Due to immunodeficiency caused by thymic deficiency, the group without CTT does not reject BN hearts. Figure 31C Shows DA and BN heart rats harvested from LW recipients together with native LW hearts at the time of cervical BN heart rejection. The lower right small figure shows the T cells (brown) in the BN heart that caused its rejection. Figure 31D Shows T cell infiltration in LW hearts, DA hearts, and BN hearts from control animals without CTT insertion. Since the animals are immunodeficient, there is no T cell infiltration. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0433] Figures 32A to 32C : Humoral tolerance after CTT. Figure 32A Shows representative histograms of donor-specific alloantibodies (anti-DA antibody and anti-BN antibody) after transplantation measured by T cell flow crossmatch. Figure 32A The upper left inset of () (DA control) shows the production of anti-DA antibody (thick line) in normal LW rats after receiving an ectopic abdominal DA heart transplant. Figure 32A The middle upper inset of () shows the lack of anti-DA antibody in LW rats that received CTT; this indicates tolerance. The upper right inset shows that LW rats without CTT were non-responsive; this reflects the immunodeficiency of rats that did not receive donor thymus after thymectomy and T cell depletion. Figure 32A The lower left inset of () shows normal anti-BN antibody formed by normal LW rats that received a cervical BN heart. Figure 32A The middle lower inset of () shows the normal response of LW rats with CTT to BN after receiving a cervical BN heart transplant, which shows immunocompetence and the ability to reject a third party. Figure 32A The lower right inset of () shows that LW rats without CTT were non-responsive to BN after receiving a cervical BN heart transplant, which shows insufficient immunocompetence and lack of the ability to reject a third party. Figure 32B Shows the level of anti-DA antibody after the first DA heart transplant. LW rats with CTT from an LWxDA thymic donor did not produce anti-DA antibody after DA heart transplant because they were tolerant to DA. LW rats without CTT did not produce anti-DA antibody after DA heart transplant because they had immunodeficiency. Figure 32C Shows the level of anti-BN antibody after the second cervical BN heart transplant. LW rats with CTT from an LWxDA donor produced antibodies against BN, which shows immunocompetence against a third party. LW rats without CTT did not produce antibodies against BN, which shows insufficient immunocompetence. This figure was published in Kwun, J. et al., JCI Insight (2020) January 4; 5(11).

[0434] Figure 33 A-J present micrographs of immunohistochemical evaluation of fresh and cultured NHP thymic tissue from 8-month-old non-human primates (NHP). The top row is NHP thymus on the day of harvest, and the bottom row is NHP thymus after 12 days of culture. The tissue was stained with hematoxylin and eosin ( Figure 33 A and Figure 33 F), CD3 ( Figure 33 B and Figure 33 G), pancytokeratin (CK) antibody AE1 / AE3 ( Figure 33 C and Figure 33 H), Ki-67 (Figure 33 D and Figure 33 I), and CK14( Figure 33 E and Figure 33 J) staining. All images were taken at 20X magnification.

[0435] Figure 34A -P presents an analysis of cryopreserved cultured thymic tissue from 8-month-old non-human primates (NHPs) after 12 days of culture. The top row is at the time of harvest ( Figure 34A ), at day 6 of culture ( Figure 34B ), at day 12 of culture ( Figure 34C ), and at 35 days of cryopreservation followed by thawing for imaging 12 days after culture ( Figure 34D ) of cytokeratin. Figure 34D The cytokeratin (AE1 / AE3) in Figure 34C is similar to the cytokeratin in Figure 34E (harvest), Figure 34F (day 6 of culture), Figure 34G (day 12 of culture), and Figure 34H (35 days of cryopreservation followed by thawing 12 days after culture) shows CK14 staining. Figure 34H The CK14 in Figure 34G is very similar to the CK14 in inset Figure 34I , Figure 34J , Figure 34K and Figure 34L shows CD3 staining at the same time points, with the expected loss of viable T cells over time. Inset Figure 34L has very few T cells similar to inset Figure 34K . The fourth row Figure 34M , Figure 34N , Figure 34O and Figure 34P shows Ki-67 staining of proliferating T cells at the same time points. Absence of Ki-67 staining by day 6 ( Figure 34N ) because most of the T cells have died. This figure shows the ability to cryopreserve non-human primate thymus in a manner similar to that which would be used to cryopreserve cultured thymic tissue for a patient. All images were taken at 40X magnification.

[0436] Figure 35Schematic of an experimental transplantation strategy using CMV-free rhesus monkeys with maximal MHC mismatch. Recipient animals (Y) undergo total thymectomy. Donor animals (X) donate both cultured thymic tissue and a heart that is placed into recipient Y at an ectopic location (first Tx and second Tx). Immunosuppressive drugs are then withdrawn, and donor-specific tolerance is demonstrated by: i) continued beating of the donor heart, ii) tolerance to the donor and reactivity to a third party in the nMLR, and iii) rejection of a skin graft (third Tx) from a third-party donor animal Z.

[0437] Figure 36 Graphs showing results from flow cytometry experiments that depict a general gating strategy for identifying thymic recent thymic emigrants (RTE). Peripheral blood mononuclear cells from non-human primates are collected and analyzed using multi-color flow cytometry. The first step is the identification of single cells in the top row first panel. "Singlets" are used to identify lymphocytes (low SSC, side scatter, and high CD45) in the second panel of the top row. CD3 T cells among the lymphocytes are identified in the third panel of the top row. CD4 and CD8 cells are gated separately from CD3 cells, as shown in the fourth panel of the top row. In the first panel of the bottom row, using the CD4 gate, CD4+ naive subsets, central memory subsets, and effector memory subsets are identified using CD28 and CD95. In the second panel of the bottom row, CD31 is used to show the percentage of CD4+ naive cells that are RTE. The third and fourth panels of the bottom row show the same method as for RTE, but for CD8 naive T cells. As can be seen, RTE account for 99.6% and 95.2% of the CD4 and CD8 naive subsets.

[0438] Figure 37 Micrograph images and graphs of CCL21 assessment in cultured infant thymus are presented. CCL21 is produced at high levels by cultured infant thymus. Immunohistochemical reactivity with a 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). The corresponding time course of daily CCL21 secretion into the culture medium for 3 infant thymus cultures is shown on the right (R&D Systems Duo-Set ELISA). Thus, cultured thymic tissue can produce the functionally important biomolecule CCL21, a chemokine responsible for attracting immature thymocyte precursors to the thymus.

[0439] Figure 38Presents a summary of the experimental design of Example 8, which involves evaluating the successful transplantation of cultured thymic tissue in a CMV-free NHP model, followed by tolerance to a matched heart and rejection of a mismatched skin. Specifically, after thymectomy of the recipient (week 1 of stage 2) and confirmation of complete thymectomy, the recipient is T cell depleted and immunosuppression with tacrolimus is initiated. In week 3 of stage 3, unmatched cultured donor thymic tissue from an unrelated NHP is transplanted into the recipient. A biopsy of the thymic graft is performed in week 10 of stage 3 to evaluate thymopoiesis. After the development of naive T cells several months later, the recipient should be tolerant to the donor. Then an orthotopic heart transplant from the thymic donor is given to the recipient (week 4 of stage 4). Immunosuppression is discontinued. The pulsation of the heart is tracked (demonstrating tolerance). Additionally, a mixed lymphocyte reaction is performed in week 10 of stage 4 to show tolerance to cryopreserved donor cells and rejection of third-party cells. If more time is required for naive T cell development, stage 5 is used. If tolerance is not generated, stage 6 is used. The recipient thymus will be transplanted into the recipient NHP to demonstrate the effectiveness of the thymic tissue transplantation procedure in NHP.

[0440] Understand Figure 38 How to format is helpful. The experiment has 3 monkeys. Note that the original spreadsheet has procedures for all three animals in a one-page wide × multi-page long document. Since the spreadsheet is wider than the width allowed in this application, each row of the spreadsheet is divided into 3 pages. The first monkey is the thymus and heart donor; the procedures on this monkey are in the left column on pages 1, 4, 7, etc. The second monkey is the thymus and heart recipient; the information in the middle column is on pages 2, 5, 8, etc.). The third monkey is the control; the information in the right column is on pages 3, 6, 9, etc.).

[0441] Figure 39 Presents a summary of the experimental design of Example 9, which is the same as the summary of the experimental design in Example 8 except for the addition of the additional immunosuppressive drug mycophenolate mofetil (MMF). The drug MMF is commonly used in heart transplantation. This study will evaluate whether MMF has any adverse effects on cultured thymic tissue grafts.

[0442] Figure 40 A-D present photomicrographs of fresh thymic sections cultured at d0, which show thymic structure. In Figure 40 A-B, hematoxylin and eosin (H&E) staining shows a distinct cortical region and a lighter-staining medullary region, as expected for a normal pediatric thymus. Figure 40Panel C shows immunohistochemistry with a mixture of pancytokeratin antibodies (AE1 / AE3) that co-detect all types of epithelial cells, demonstrating the presence of thymic epithelial cells beneath the capsule and in a delicate lacy network in the cortex and medulla (brown staining indicates a positive antibody reaction). Figure 40 B and Figure 40 The arrows in C point to Hassall's corpuscles. Figure 40 Panel D shows cytokeratin 14 (CK14) antibody staining (brown). The CK14 antibody reacts with thymic epithelial cells in the subcapsular cortex and medulla, as well as scattered thymic epithelial cells in the cortex. The dashed line highlights the medullary area surrounded by the cortex. SCC indicates the subcapsular cortex, Cor indicates the cortex, and M indicates the medulla. Figure 40 The scale bar in A represents 1 mm; Figure 40 The scale bars in B - D represent 500 μm.

[0443] Figure 41 Panels A - D present photomicrographs showing examples of Hassall's corpuscles in cultured thymic sections. The histological appearance of Hassall's corpuscles is shown at day 0 of culture ( Figure 41 A - B) and day 9 of culture ( Figure 41 C - D). Figure 41 A and Figure 41 C show hematoxylin and eosin (H&E) staining; Figure 41 B and Figure 41 D show reactivity with pancytokeratin (AE1 / AE3) antibody (brown indicates a positive reaction). Figure 41 The arrowheads in A - D point to representative Hassall's corpuscles, which appear less prominent on H&E - stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall's corpuscles can still be easily identified by careful examination or by using immunohistochemistry. Figure 41 The scale bars in A - D represent 100 μm.

[0444] Figure 42 Panels A - D present photomicrographs showing the structure of the thymus at day 7 of culture. Figure 42 Hematoxylin and eosin (H&E) staining in A - B shows significant depletion of thymocytes, but some cortical areas (Cor) still contain a large number of thymocytes with preserved nuclei. Figure 42 Pancytokeratin (AE1 / AE3) in C and Figure 42 Cytokeratin 14 (CK14) immunohistochemistry in D shows condensation of thymic epithelium in the subcapsular cortex (SCC) and medulla (M). Figure 42 Brown in C - D indicates a positive reaction with the antibody. The scale bar Figure 42 in A represents 1 mm, and in Figure 42500 μm is represented in B-D.

[0445] Figure 43 A-D present micrographs showing the structure of the thymus cultured for 9 days. Figure 43 A-B shows hematoxylin and eosin (H&E) staining. In Figure 43 the pale-stained area surrounded by the dashed line in A, there are few viable T cells or thymic epithelial cells, if any, and it is almost completely necrotic (Necr). Most of the nuclei that originally existed in this area have been degraded by karyolysis. Other areas where the nuclei of residual thymocytes are not completely degraded continue to be stained dark blue by hematoxylin. Figure 43 The arrow in B points to Hassall's corpuscles. Figure 43 C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 43 D shows cytokeratin 14 (CK14) immunoreactivity (brown). The scale bar represents 1 mm in Figure 43 A and 500 μm in Figure 43 B-D.

[0446] Figure 44 A-D present micrographs showing the structure of the thymus cultured for 12 days. Figure 44 A-B shows hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have been lost from the tissue or have died, and their nuclei have lysed, making the tissue more eosinophilic (pink). Some areas retain the structural characteristics of normal uncultured thymus, with a cortex-like area (Cor) and a medulla-like area (M) that stain more basophilic (blue), but with a greatly reduced thymocyte cell composition. Figure 44 C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 44 D shows cytokeratin 14 (CK14) immunoreactivity (brown). At this time, the subcapsular cortex (SCC) has thickened and the epithelial cells appear more prominent due to the reduced number of thymocytes present. The arrow points to a representative Hassall's corpuscle. The scale bar represents 1 mm in Figure 44 A and 500 μm in Figure 44 B-D.

[0447] Figure 45 A-D present micrographs showing the structure of the thymus cultured for 20 days. Figure 45 A-B shows hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have been lost from the tissue or have died, and their nuclei have lysed, making the tissue more eosinophilic (pink). Large groups of residual thymocytes are rare, but scattered cells with nuclear characteristics of thymocytes are evident.Figure 45 Panel C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown). Due to the loss of medullary thymocytes, many epithelia that were originally present in the medullary region (M) are condensed, but the remaining shallow lace-like three-dimensional network of epithelial cells indicative of thymic epithelial cells remains. In Figure 45 Panel D, cytokeratin 14 (CK14) immunohistochemistry (brown) highlights the premedullary region and the subcapsular cortex. Arrows point to representative Hassall's corpuscles. Scale bars are indicated in Figure 45 Panel A as 1 mm and in Figure 45 Panels B–D as 500 μm.

[0448] Figure 46 Panels A–B present photomicrographs showing examples of intact nuclei in thymic sections. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex at day 9 ( Figure 46 Panel A) and in the medulla at day 21 ( Figure 46 Panel B). Hematoxylin and eosin staining; scale bar represents 50 μm.

[0449] Figure 47 Panels A–E present photomicrographs showing a comparison of the thymic epithelial network of thymic tissues cultured at different time points. Figure 47 Panel A shows day 0, Figure 47 Panel B shows day 5, Figure 47 Panel C shows day 9, Figure 47 Panel D shows day 12, and Figure 47 Panel E shows day 21. Although there are time point-related differences in thymocyte depletion and necrosis such that the tissue becomes less basophilic (blue) over time, the structure of the thymic epithelial network (brown) remains intact as the culture progresses. As the intervening thymocytes are depleted, both cortical and medullary epithelia may condense. Brown indicates a positive reaction with a mixture of anti-cytokeratin antibodies (AE1 / AE3); counterstained with hematoxylin. Scale bar represents 400 μm.

[0450] Figure 48 Panels A–K present photomicrographs showing examples of CD3 immunohistochemistry in thymic sections over culture time. Figure 48 Panels A–B show that, at day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with the CD3 antibody. Higher magnification ( Figure 48 Panel B) shows pale blue nuclei surrounded by a brown immunoreactive ring, which is consistent with membrane expression of CD3. In Figure 48 Panels C–E, the tissue still shows extensive reactivity with the CD3 antibody at day 7. However, Figure 48D-E shows that when observed at higher magnification, most immunoreactivity (brown) is associated with debris from dead thymocytes, as most brown foci lack signs of nuclei ( Figure 48 D). Small foci of cells showing intact nuclear and membrane staining (arrows) can still be identified in areas away from the debris ( Figure 49 E). As the culture progresses to day 9 ( Figure 48 F-G), day 12 ( Figure 48 H-I), and day 21 ( Figure 48 J-K), reactivity with thymocyte debris remains strong, making it difficult to reliably detect potentially intact cells among the debris. All sections shown are from a single batch representative of multiple batches examined at these time points. Scale bars represent 500 μm in Figure 48 A, Figure 48 C, Figure 48 F, Figure 48 H, and Figure 48 J, and 50 μm in Figure 48 B, Figure 48 D, Figure 48 E, Figure 48 G, Figure 48 I, and Figure 48 K.

[0451] Figure 49 A-K present photomicrographs showing examples of the variation in Ki-67 immunohistochemistry in thymic sections over culture time. All sections shown are from a single batch representative of multiple batches examined at similar time points. Figure 49 A-B shows that at day 0, the nuclei of most immature T cells in the cortex (Cor) strongly react with an antibody specific for Ki-67. Higher magnification ( Figure 49 B) shows a strong positive reaction with the nuclei of cortical thymocytes (brown), while only rare lymphocytes in the medulla (M) react with the Ki-67 antibody. Figure 49 C-E shows that by day 7, the nuclei of most thymocytes remaining in the cortical area are small, with unclear nuclear boundaries consistent with apoptosis, and they do not react with the Ki-67-specific antibody. Cells reacting with the antibody ( Figure 49 E, arrow) have larger nuclei, indicating that they are thymic epithelial cells. A similar lack of Ki-67 labeling of residual thymic nuclei is observed at day 9 ( Figure 49 F-G), day 12 ( Figure 49 H-I), and day 21 ( Figure 49 J-K). Scale bars represent 500 μm in Figure 49 A, Figure 49 C, Figure 49 E, Figure 49 G, and Figure 49In I, it represents 500 μm, and in Figure 49 B, Figure 49 D, Figure 49 F, Figure 49 H and Figure 49[[ J, it represents 50 μm.

[0452] ​ A through ​ H show graphs of selected soluble molecules detected in conditioned medium from human thymic organ cultures. ​ A is a graph of L-selectin (Ln / pg) versus days of culture; ​ B is a graph of M-CSF (Ln / pg) versus days of culture; ​ C is a graph of galectin-7 (Ln / pg) versus days of culture; ​ D is a graph of IL-16 (Ln / pg) versus days of culture; ​ E is a graph of CCL21 (Ln / pg) versus days of culture; ​ F is a graph of CXCL16 (Ln / pg) versus days of culture; ​ G is a graph of CCL11 (Ln / pg) versus days of culture; and ​ H is a graph of CXCL12 (Ln / pg) versus days of culture.

[0453] ​ Show graphs of thymocyte content (pg / ml) in cultured sections of human thymus evaluated from day 1 to day 21 of the culture process.

[0454] ​ Show photomicrographs of immunohistochemical evaluations of viable thymocytes over time in cultured sections of human thymic tissue. ​ A is an immunohistochemical photomicrograph identifying cells as of the T lineage with an anti-CD3 antibody and proliferating cells with an anti-Ki-67 antibody, showing the rapid loss of thymocyte viability in the early stages of culture. ​ B is a photomicrograph at day 0, depicting the plasma membranes of essentially all immature T cells in the cortex and medulla appearing to strongly react with the anti-CD3 in a membrane pattern. Figure 52 C depicts immunohistochemistry using an antibody specific for the Ki-67 proliferation marker showing abundant reactivity with cortical thymocytes at day 0. Figure 52 D shows the histology of the thymus cultured at day 9, stained with hematoxylin and eosin. Reduced basophils (blue) indicate the loss of donor thymocytes during the culture process; Figure 52 E depicts that after several days of culture, most of the brown color is due to anucleate but still immunoreactive debris remaining after dead thymocytes undergo karyolysis / dissolution of the nucleus.Figure 52 F depicts thymocyte death that occurs during organ culture, resulting in Ki-67 immunoreactivity only with larger cells that are morphologically consistent with TE cells at later time points during the culture.

[0455] Figure 53 A shows a graph of CCL21 levels (pg / ml) in the conditioned medium from cultured thymic tissue relative to the number of days in culture. Figure 53 B is a diagram of the sectioning procedure for conditioning thymic tissue. Figure 53 C is a graph of the change in CCL21 secretion over time in sections of thymic organ cultures.

[0456] Figure 54 A to Figure 54 D is a photograph depicting immunoreactivity in cultured and non-cultured thymic tissue. Figure 54 A is a photograph of the medullary region on day 0 of culture, but also includes TECs scattered throughout the cortex; Figure 54 B is a photograph of the medullary region on day 16 of culture, but also includes TECs scattered throughout the image; Figure 54 C is a photograph of TECs in the medullary region and TECs scattered in the cortical region on day 0 of culture. Figure 54 D is a photograph of TECs in the medullary region and TECs scattered in the cortical region on day 16 of culture.

[0457] Figure 55 A to Figure 55 A to F are graphs of the expression of selected mRNAs in thymic tissue throughout the life cycle. The relative amounts of target mRNAs present in FFPE sections of thymic tissue were quantified using the QuantiGene assay (Thermo Fisher) according to the manufacturer's instructions. Data for each target mRNA are presented as normalized against GAPDH ("unadjusted"), and then further normalized against the area % containing thymic epithelium ("adjusted by TE") or the area % containing CD1a-positive cortical thymocytes ("adjusted by Cor"). The data shown were obtained from 47 thymic samples from donors ranging in age from 5 days to 78 years. Figure 55 A and 55B are respectively: Thymic tissue obtained from donors ≤18 years old (n = 25) showed higher expression of mRNAs encoding the T cell marker CD3ε and the cortical thymocyte marker CD1a relative to GAPDH when compared to donors over 18 years old ( Figure 55 A, B). Figure 55 C and 55D are photographs respectively depicting reduced expression of mRNAs encoding cytokeratin 8 (KRT8) and 14 (KRT14) relative to GAPDH in donors ≤18 years old compared to older adults (Figure 55 C, D). Figure 55 E is a photograph depicting unadjusted CCL21 gene expression, which is consistently lower relative to GAPDH in the thymus from donors ≤18 years old. Figure 55 F is a photograph depicting unadjusted CXCL21 gene expression, which is consistently lower relative to GAPDH in the thymus from donors ≤18 years old.

[0458] Figure 56 is a table of proteins present in the spent medium of thymic organ cultures, as determined by a multiplex antibody array.

[0459] Figure 57 A through 57C are photographs of morphological measurements of thymic tissue. Using the "pen tool" provided by ImageScope software (Aperio Technologies, Leica Biosystems imaging, Inc.), the areas included in each measurement were outlined according to the manufacturer's instructions. Figure 57 A shows the total area of thymic tissue on an H&E-stained slide outlined in green. The portion of this area containing lymphocytes is further outlined in light green. Figure 57 B is a photograph showing the area containing thymic epithelium ("TE area") outlined in yellow on a section, which reacts with an AE1 / AE3 mixture to identify pan-cytokeratin. Figure 57 C is a photograph showing the area containing immature thymocytes ("cortical area") outlined in red on a section, which reacts with a CD1a antibody. The shown thymus was from a 32-year-old female at the time of aortic valve replacement surgery. The scale bar for each inset = 4 mm. In Figure 57 the insets of B and 57C, brown indicates a positive reaction with the antibody.

[0460] Figure 58 A through 58C are photographs depicting a few live thymocytes in thymic tissue sections cultured for 21 days. Figure 58 A is a photograph depicting a thymic section containing a few intact thymocytes on day 21 of culture, as indicated by the apparent lack of basophils (blue) in the H&E section. Figure 58 B is a photograph of a cultured thymic tissue section in which most of the strongly brown immunoreactivity seen with CD3 immunohistochemistry is associated with anuclear cell debris, but dead thymocytes showing characteristic nuclear and cytoplasmic staining of necrotic cells that have not yet undergone karyolysis (inset) are not uncommon. Figure 58C is a photograph depicting Ki-67 immunoreactivity limited to cells with larger nuclei characteristic of thymic epithelial cells on day 21. The scale bar represents 300 μm in the main micrograph and 50 μm in the inset.

[0461] Figure 59 A through 59C are graphs showing the characteristics of human thymic tissue for gene expression analysis. Figure 59 A depicts the age and sex distribution of the thymic tissue studied, where the lower solid black circles indicate females, the upper open circles indicate males, and the middle gray circle indicates a donor of unknown sex. Figure 59 B plots the percentage of area containing thymic epithelial cells against the age of this group of thymic tissue. Figure 59 C plots the percentage of area with active thymopoiesis, as defined by CD1a-positive thymocytes, against the age of this group of thymic tissue.

[0462] Figure 60 is a photograph of freshly harvested thymic tissue.

[0463] Figure 61 A and 61B depict the histology of thymic tissue slides after exposure to forced degradation conditions with 10X PBS. Figure 61 A depicts the cortex on day 9 after exposure to forced degradation conditions. Figure 61 B depicts the cortex on day 21 after exposure to forced degradation conditions. In Figure 61 A, the blue smears are DNA released from cells. Although small foci of cells with intact nuclei can be identified, most cells show signs of degradation. Photographed by MD, PhD, Department of Pathology, Duke University.

[0464] Figure 62A shows a schematic diagram that shows a section of thymic tissue used to characterize the test, as discussed in section

[00520] . Figure 62B is a diagram of a section of thymic tissue on a cellulose filter on a surgical sponge in a tissue culture dish such as used for culturing thymus.

[0465] Figure 63 A through 63H depict histological tests of sections of thymic tissue from a batch (MFG-056) of cultured thymic tissue on days 5, 9, 12, and 21 after harvesting the thymus from the donor. On day 5 ( Figure 63 A, Figure 63 B), day 9 ( Figure 63 C, Figure 63 D), day 12 ( Figure 63 E, Figure 63 F), and day 21 ( Figure 63 G, Figure 63H) Sections stained with hematoxylin and eosin (left inset) and their corresponding reactivity with a mixture of anti-cytokeratin antibodies AE1 / AE3 are shown separately (right inset; brown indicates positive reactivity). The bar in the lower left of each inset represents 100 μm. The inset with H&E shows the depletion of T cells over time. Figure 14 E and Figure 63 F are mainly epithelial cells. Condensation of the subcapsular cortical epithelium occurs as thymocytes are depleted over time. Similar condensation occurs in the medullary region of the thymus. Photographed by MD, PhD, Department of Pathology, Duke University.

[0466] Figure 64A and Figure 64B depict the histology of thymic tissue sections on day 0 of the time course at scales of 5 mm ( Figure 64A ) and 100 μm ( Figure 64B ), respectively. This shows the thymus and thymocytes at low magnification (scale bar 5 mm) and high magnification (scale bar 100 μm) on day 0. This is a normal thymus. At this time, both the cortex and medulla have a large number of thymocytes, and the dark blue nuclei contribute to the overall dark blue appearance of the tissue. Photographed by MD, PhD, Department of Pathology, Duke University.

[0467] Figure 65A and Figure 65B are images from H&E-stained slides that depict the histology of thymic tissue sections on day 5 of the time course at scales of 5 mm ( Figure 65A ) and 100 μm ( Figure 65B ), respectively. The progression of thymocyte depletion results in a more eosinophilic (pink) appearance of the tissue. Photographed by MD, PhD, Department of Pathology, Duke University.

[0468] Figure 66A and Figure 66B depict the H&E staining of thymic tissue sections on day 12 of the time course at scales of 5 mm ( Figure 66A ) and 100 μm ( Figure 66B ), respectively. We see the progressive depletion of thymocytes. Higher magnification shows many eosinophilic cell bodies lacking nuclei, which can be diagnosed as necrotic cells that have undergone karyolysis (dissolution of the nucleus). This degree of necrosis is expected at this time in culture. Photographed by MD, PhD, Department of Pathology, Duke University.

[0469] Figure 67A and Figure 67B depict the H&E staining of thymic tissue sections on day 21 of the time course at scales of 5 mm ( Figure 67A ) and 100 μm ( Figure 67B ), respectively. Note that the overall structure of the tissue is preserved. At Figure 67BIt contains a subcapsular cortex, cortical area, and medullary area containing numerous Hassall corpuscles. The smaller, darker cells are mostly necrotic thymocytes that have not yet undergone karyolysis. Photographed by MD, PhD, Department of Pathology, Duke University.

[0470] Figure 68 A - E depict representative thymic sections immunostained with a mixture of anti - cytokeratin antibodies (AE1 / AE3). Figure 68 A. Day 0; Figure 68 B. Day 5; Figure 68 C. Day 9; Figure 68 D. Day 12; and Figure 68 E. Day 21. As the culture progresses, the structure of the thymic epithelial network remains Complete . Scale bar represents 400 μm. Photographed by MD, PhD, Department of Pathology, Duke University.

[0471] Figure 69 Presents micrograph images and graphs of CCL21 assessment in cultured infant thymus. CCL21 is produced by cultured infant thymus at high levels. Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on day 16 of culture is shown in the left inset (upper left inset, 2X magnification; lower left inset, 20X magnification). The corresponding time course of daily CCL21 secretion into the culture medium of 3 infant thymus cultures is shown on the right (R&D Systems Duo - Set ELISA). Thus, cultured thymic tissue can produce the functionally important biomolecule CCL21, a chemokine responsible for attracting immature thymocyte precursors to the thymus.

[0472] Figure 70 A through 70D present micrographs of fresh thymic sections at culture d0, which show thymic structure. In Figure 70 A - B, hematoxylin and eosin (H&E) staining shows a clear cortical area and a lighter - stained medullary area, as expected for a normal pediatric thymus. Figure 70 C shows that immunohistochemistry with a mixture of pancytokeratin antibodies (AE1 / AE3) that co - detect all types of epithelial cells demonstrates the presence of thymic epithelial cells beneath the capsule and in a shallow lacy network in the cortex and medulla (brown staining shows positive antibody reaction). Figure 70 B and Figure 70 The arrows in C point to Hassall corpuscles. Figure 70Panel D shows cytokeratin 14 (CK14) antibody staining (brown). The CK14 antibody reacts with thymic epithelial cells in the subcapsular cortex and medulla and scattered thymic epithelial cells in the cortex. The dotted line highlights the medullary area surrounded by the cortex. SCC indicates the subcapsular cortex, Cor indicates the cortex, and M indicates the medulla. Figure 70 The scale bar in A represents 1 mm; Figure 70 The scale bars in B - D represent 500 μm.

[0473] Figure 71 Panels A to D show photomicrographs demonstrating examples of Hassall's corpuscles in cultured thymic sections. The histological appearance of Hassall's corpuscles is shown at day 0 of culture ( Figure 71 A - B) and day 9 of culture ( Figure 71 C - D). Figure 71 Panel A and Figure 71 Panel C show hematoxylin and eosin (H&E) staining; Figure 71 Panel B and Figure 71 Panel D show reactivity with the pancytokeratin (AE1 / AE3) antibody (positive reaction indicated by brown). Figure 71 The arrows in A - D point to representative Hassall's corpuscles, which are less prominent on H&E - stained sections of cultured thymus due to depletion and necrosis of surrounding thymocytes. However, Hassall's corpuscles can still be easily identified by careful examination or by using immunohistochemistry. Figure 71 The scale bars in A - D represent 100 μm.

[0474] Figure 72 Panels A to D show photomicrographs demonstrating the structure of the thymus at day 7 of culture. Figure 72 Hematoxylin and eosin (H&E) staining in A - B shows significant depletion of thymocytes, but some cortical areas (Cor) still contain a large number of thymocytes with preserved nuclei. Figure 72 Pancytokeratin (AE1 / AE3) in Panel C and Figure 72 Cytokeratin 14 (CK14) immunohistochemistry in Panel D shows condensation of thymic epithelium in the subcapsular cortex (SCC) and medulla (M). Figure 72 The brown color in C - D indicates a positive reaction with the antibody. The scale bar represents 1 mm in Figure 23 and 500 μm in Figure 72 B - D.

[0475] Figure 73 Panels A to D show photomicrographs demonstrating the structure of the thymus at day 9 of culture. Figure 73 Panels A - B show hematoxylin and eosin (H&E) staining. At Figure 73In A, there are few viable T cells or thymic epithelial cells (if any) in the pale-stained area surrounded by the dashed line, which is almost completely necrotic (Necr). Most of the nuclei that originally existed in this area have been degraded by karyolysis. Other areas where the nuclei of the remaining thymocytes are not completely degraded continue to be stained dark blue by hematoxylin. Figure 73 The arrow in B points to Hassall's corpuscles. Figure 73 C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 73 D shows cytokeratin 14 (CK14) immunoreactivity (brown). The scale bar is Figure 73 shown as 1 mm in A, and Figure 73 shown as 500 μm in B - D.

[0476] Figure 74 A to D present micrographs showing the structure of the thymus cultured for 12 days. Figure 74 A - B show hematoxylin and eosin (H&E) staining. At this time point, many thymocytes have been lost from the tissue or have died, and their nuclei have lysed, making the tissue more eosinophilic (pink). Some areas retain the structural characteristics of the normal uncultured thymus, which has a cortex-like area (Cor) and a medulla-like area (M) that stain more basophilic (blue), but with a greatly reduced thymocyte cell composition. Figure 74 C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 74 D shows cytokeratin 14 (CK14) immunoreactivity (brown). At this time, the subcapsular cortex (SCC) has thickened and the epithelial cells appear more prominent due to the reduced number of thymocytes present. The arrow points to a representative Hassall's corpuscle. The scale bar is Figure 74 shown as 1 mm in A, and Figure 74 shown as 500 μm in B - D.

[0477] Figure 75 A - B show hematoxylin and eosin (H&E) staining. At this time point, most thymocytes have been lost from the tissue or have died, and their nuclei have lysed, making the tissue more eosinophilic (pink). Large clusters of remaining thymocytes are rare, but scattered cells with the nuclear characteristics of thymocytes are evident. Figure 75 C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown). Due to the loss of medullary thymocytes, many of the epithelia that originally existed in the medulla area (M) have condensed, but scattered epithelial cells indicate that the remaining shallow lace-like three-dimensional network of thymic epithelial cells remains. In Figure 75In D, cytokeratin 14 (CK14) immunohistochemistry (brown) highlights the premedullary region and the subcapsular cortex. Arrows point to representative Hassall's corpuscles. Scale bars represent 1 mm in Figure 75 A and 500 μm in Figure 75 B–D.

[0478] Figure 76 A–B present micrographs showing examples of intact nuclei in thymic sections. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex at day 9 ( Figure 76 A) and in the medulla at day 21 ( Figure 76 B). Hematoxylin and eosin staining; scale bar represents 50 μm.

[0479] Figure 77 A–E present micrographs showing a comparison of the thymic epithelial network of thymic tissue cultured at different time points. Figure 77 A shows day 0, Figure 77 B shows day 5, Figure 77 C shows day 9, Figure 77 D shows day 12, and Figure 77 E shows day 21. Although there are time point-related differences in the amount of thymocyte depletion and necrosis, such that the tissue becomes less basophilic (blue) over time, the structure of the thymic epithelial network (brown) remains intact as culture proceeds. As the intervening thymocytes are depleted, both cortical and medullary epithelia may condense. Brown indicates a positive reaction with a mixture of anti-cytokeratin antibodies (AE1 / AE3); hematoxylin counterstain. Scale bar represents 400 μm.

[0480] Figure 78 A–K present micrographs showing examples of CD3 immunohistochemistry in thymic sections over culture time. Figure 78 A–B show that, at day 0, essentially all immature T cells in the cortex and more mature cells in the medulla react strongly with the CD3 antibody. Higher magnification ( Figure 78 B) shows pale blue nuclei surrounded by a brown immunoreactive ring, which is consistent with membrane expression of CD3. In Figure 78 C–E, the tissue still shows extensive reactivity with the CD3 antibody at day 7. However, Figure 78 D–E show that, when viewed at higher magnification, most immunoreactivity (brown) is associated with debris from dead thymocytes, as most brown foci lack signs of nuclei ( Figure 78 D). Small foci of cells showing intact nuclei and membrane staining (arrows) can still be identified in areas away from the debris ( Figure 78 E). As culture proceeds to day 9 ( Figure 78F - G), day 12( Figure 78 H - I) and day 21( Figure 78 J - K), the reactivity with thymocyte debris remained strong, making it difficult to reliably detect potentially intact cells among the debris. All the sections shown were from a single batch representative of multiple batches examined at these time points. Scale bars are shown as Figure 78 A, Figure 78 C, Figure 78 F, Figure 78 H and Figure 78 J represent 500 μm, and in Figure 78 B, Figure 78 D, Figure 78 E, Figure 78 G, Figure 78 I and Figure 78 K represent 50 μm.

[0481] Figure 79 A - K present photomicrographs showing examples of the change in Ki - 67 immunohistochemistry in thymic sections over culture time. All the sections shown were from a single batch representative of multiple batches examined at similar time points. Figure 79 A - B show that, at day 0, the nuclei of most immature T cells in the cortex (Cor) strongly reacted with an antibody specific for Ki - 67. Higher magnification( Figure 79 B) shows a strong positive reaction with the nuclei of cortical thymocytes (brown), while only rare lymphocytes in the medulla (M) reacted with the Ki - 67 antibody. Figure 79 C - E show that, by day 7, the nuclei of most thymocytes remaining in the cortical area were small, with unclear nuclear boundaries consistent with apoptosis, and they did not react with the Ki - 67 - specific antibody. The cells that reacted with the antibody( Figure 79 E, arrow) had larger nuclei, indicating that they were thymic epithelial cells. At day 9( Figure 79 F - G), day 12( Figure 79 H - I) and day 21( Figure 79 J - K), a similar lack of Ki - 67 labeling of residual thymic nuclei was observed. Scale bars are shown as Figure 79 A, Figure 79 C, Figure 79 E, Figure 79 G and Figure 79 I represent 500 μm, and in Figure 79 B, Figure 79 D, Figure 79 F, Figure 79 H and Figure 79 J represent 50 μm.

[0482] Figure 80Graph of uPAR detected in the conditioned medium from human thymic organ cultures.

[0483] Figure 81 Graph of OPN detected in the conditioned medium from human thymic organ cultures.

[0484] Figure 82 Graph of MIP3a detected in the conditioned medium from human thymic organ cultures.

[0485] Figure 83 Graph of IGFBP-1 detected in the conditioned medium from human thymic organ cultures.

[0486] Figure 84 Graph of MIF detected in the conditioned medium from human thymic organ cultures.

[0487] Figure 85 Scatter plot of CCL21 concentration in the spent medium versus the number of days.

[0488] Figure 86 Box plot of CCL21 concentration in the spent medium versus the number of days.

[0489] Figure 87 Is Figure 4 Graph of: Forced degradation study - CCL21 levels (pg / mL) in Batch MFG-053 and Batch - 054.

[0490] Figure 88 Graph of CCL21 levels (pg / mL) in Forced degradation study - Batch MFG-066.

[0491] Figure 89 Scatter plot of CXCL16 concentration in the spent medium versus the number of days.

[0492] Figure 90 Box plot of CXCL16 concentration in the spent medium versus the number of days.

[0493] Figure 91 Scatter plot of CXCL16 levels (pg / mL) in Forced degradation study - Batch MFG-053 and Batch MFG-054.

[0494] Figure 92 Scatter plot of CXCL21 levels in Forced degradation study - Batch MFG-066.

[0495] Figure 93 Scatter plot of L-selectin concentration in the spent medium versus the number of days.

[0496] Figure 94It is a box plot of the L-selectin concentration in the used culture medium relative to the number of days.

[0497] Figure 95 It is a scatter plot of the L-selectin levels (pg / mL) in the forced degradation studies - Batch MFG-053 and Batch MFG-054.

[0498] Figure 96 It is a scatter plot of the L-selectin levels in the forced degradation study - Batch MFG-066.

[0499] Figure 97 It is a scatter plot of the uPAR concentration in the used culture medium relative to the number of days.

[0500] Figure 98 It is a box plot of the uPAR concentration in the used culture medium relative to the number of days.

[0501] Figure 99 It is a scatter plot of the uPAR levels (pg / mL) in the forced degradation studies - Batch MFG-053 and Batch MFG-054.

[0502] Figure 100 It is a scatter plot of the uPAR levels in the forced degradation study - Batch MFG-066.

[0503] Figure 101 It is a scatter plot of CXCL16 relative to CCL21.

[0504] Figure 102 It is a quadratic regression model of CCL21 relative to uPAR.

[0505] Figure 103 It is a quadratic regression model of CXCL16 relative to uPAR.

[0506] Figure 104 It is a scatter plot of the CCL11 concentration in the used culture medium relative to the number of days.

[0507] Figure 105 It is a box plot of the CCL11 concentration in the used culture medium relative to the number of days.

[0508] Figure 106 It is a linear regression model of the CCL11 concentration in the used culture medium relative to the number of days.

[0509] Figure 107 It is a graph of the CCL11 levels (pg / mL) in the forced degradation studies - Batch MFG-053 and Batch MFG-054.

[0510] Figure 108 It is a graph of the CCL11 levels in the forced degradation study - Batch MFG-066.

[0511] Figure 109 It is a scatter plot of the OPN concentration in the spent medium relative to the number of days.

[0512] Figure 110 It is a box plot of the OPN concentration in the spent medium relative to the number of days.

[0513] Figure 111 It is a quadratic regression model of the OPN concentration in the spent medium relative to the number of days.

[0514] Figure 112 It is a graph of the OPN levels (pg / mL) in the forced degradation studies - Lot MFG - 053 and Lot MFC - 054.

[0515] Figure 113 It is a graph of the OPN levels in the forced degradation study - Lot MFG - 066.

[0516] Figure 114 It is a scatter plot of the CXCL12 concentration in the spent medium relative to the number of days.

[0517] Figure 115 It is a box plot of the CXCL12 concentration in the spent medium relative to the number of days.

[0518] Figure 116 It is a fitted line graph of the CXCL12 concentration in the spent medium relative to the number of days.

[0519] Figure 117 It is a scatter plot of the CXCL12 levels (pg / mL) in the forced degradation studies - Lot MFG - 053 and Lot MFG - 054.

[0520] Figure 118 It is a scatter plot of the CXCL12 levels in the forced degradation study - Lot MFG - 066.

[0521] Figure 119 It is a scatter plot of the CCL20 concentration in the spent medium relative to the number of days.

[0522] Figure 120 It is a box plot of the CCL20 concentration in the spent medium relative to the number of days.

[0523] Figure 121 It is a cubic regression model of the CCL20 concentration in the spent medium relative to the number of days.

[0524] Figure 122 It is a scatter plot of the CCL20 levels (pg / mL) in the forced degradation studies - Lot MFG - 053 and Lot MFG - 054.

[0525] Figure 123 It is a scatter plot of the CCL20 levels in the forced degradation study - batch MFG - 066.

[0526] Figure 124 It is a scatter plot of the IL - 16 concentration in the used culture medium versus the number of days.

[0527] Figure 125 It is a box plot of the IL - 16 concentration in the used culture medium versus the number of days.

[0528] Figure 126 It is a fitted line plot of the IL - 16 concentration in the used culture medium versus the number of days.

[0529] Figure 127 It is a scatter plot of the IL - 16 levels (pg / mL) in the forced degradation studies - batch MFG - 053 and batch MFG - 054.

[0530] Figure 128 It is a scatter plot of the IL - 16 levels in the forced degradation study - batch MFG - 066.

[0531] Figure 129 It is a scatter plot of the IGFBP - 1 concentration in the used culture medium versus the number of days.

[0532] Figure 130 It is a box plot of the IGFBP - 1 concentration in the used culture medium versus the number of days.

[0533] Figure 131 It is a fitted line plot of the IGFBP - 1 concentration in the used culture medium versus the number of days.

[0534] Figure 132 It is a scatter plot of the IGFBP - 1 levels (pg / mL) in the forced degradation study - batch MFG - 053.

[0535] Figure 133 It is a scatter plot of the OGFBP - 1 levels in the forced degradation study - batch MFG - 066.

[0536] Figure 134 It is a scatter plot of the MIF concentration in the used culture medium versus the number of days.

[0537] Figure 135 It is a box plot of the MIF concentration in the used culture medium versus the number of days.

[0538] Figure 136 It is a linear regression model of the MIF concentration in the used culture medium versus the number of days.

[0539] Figure 137Is a scatter plot of MIF levels (pg / mL) in the forced degradation studies - Batch - MFG - 053 and Batch MFG - 054.

[0540] Figure 138 Is a scatter plot of MIF levels in the forced degradation study - Batch MFG - 066.

[0541] Figure 139 Is a scatter plot of the CCL25 concentration in the used culture medium relative to the number of days.

[0542] Figure 140 Is a box plot of the CCL25 concentration in the used culture medium relative to the number of days.

[0543] Figure 141 Is a linear regression model plot of the MIF concentration in the used culture medium relative to the number of days.

[0544] Figure 142 Is a scatter plot of the CCL25 levels (pg / mL) in the forced degradation studies - Batch - 053 and Batch - 054.

[0545] Figure 143 Is a scatter plot of the CCL25 levels in the forced degradation study - Batch MFG - 066. Detailed implementation

[0546] The names, titles, and subtitles provided herein should not be construed as limiting the various aspects of the present disclosure. Thus, the terms defined below can be more comprehensively defined by referring to the specification as a whole. All references cited herein are incorporated by reference in their entirety.

[0547] Unless otherwise defined, scientific and technical terms used herein will have the meanings commonly understood by those of ordinary skill in the art. Additionally, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. In this application, unless otherwise stated, the use of "or" means "and / or". In the context of multiple dependent claims, the use of "or" only refers to more than one of the alternatives independent of the preceding claims or dependent claims.

[0548] It should also be 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 clearly limited to one referent. As used herein, the term "comprising" and its grammatical variants are intended to be non - restrictive, such that the recitation of items in a list does not preclude other similar items that may be substituted or added to the listed items.

[0549] The present invention is most clearly understood with reference to the following definitions:

[0550] The term "about" is used herein to mean approximately, within a range, generally or around. When the term "about" is used in connection with a numerical range, it modifies the range by extending the boundaries above and below the recited numerical values. Generally speaking, the term "about" is used herein to modify a numerical value by a variation of + / - 10% above and below the stated value. As used herein, the term about refers to a numerical value, including, for example, integers, fractions and percentages, whether or not expressly stated. The term about generally refers to a range of numerical values (e.g., + / - 5% to 10% of the recited range) that are considered by one of ordinary skill in the art to be 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 term modifies all of the values or ranges provided in the list. In some cases, the term about may include a numerical value rounded to the nearest significant digit.

[0551] As used herein, the term "animal" includes, but is not limited to, human and non-human vertebrates, such as wild animals, domestic animals and farm animals. An animal may also be referred to as a "subject".

[0552] As used herein, the term "biomarker" refers to Figure 56 the substances listed in. It should be further understood that references to "reduced" and "increased" levels with respect to the occurrence of a biomarker in the thymic organ culture medium refer to an increased measurement or a reduced measurement of a particular biomarker over the course of a conditioning regimen.

[0553] As used herein, "biocompatible" refers to any material that does not cause an adverse response in a mammal when implanted therein.

[0554] "Chronic transplant rejection" typically occurs in humans within months to years after transplantation, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ grafts.

[0555] As used herein, the term "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, unrecited elements or method steps. Additionally, terms used in conjunction with the term "comprising" are also understood to be capable of being used in conjunction with the terms "consisting of" or "consisting essentially of".

[0556] As used herein, a "graft" refers to a tissue or organ that is implanted into an individual, generally to replace, correct, or otherwise overcome a defect. The tissue or organ may be composed of cells originating from the same individual; such a 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 their identical twin is referred to herein as an "isograft, syngeneic transplant, syngeneic implant, or syngeneic graft". A "xenograft, xenogeneic transplant, or xenogeneic implant" refers to a graft from one individual to another individual of a different species.

[0557] As used herein, the term "HLA match" refers to a donor-recipient pair in which there are no HLA antigen mismatches between the donor and the recipient. HLA matching in the methods of the present invention includes the HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

[0558] As used herein, the term "HLA mismatch" refers to a match in HLA antigens between a donor and a recipient, typically with respect to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1, where an HLA mismatch occurs between the donor and the recipient. In some cases, one haplotype is matched while the other is mismatched. This situation is often found in organs from living or deceased donors. An HLA mismatch in a donor-recipient pair results in an increased risk of transplant rejection relative to an HLA-matched pair.

[0559] As background to the foregoing definition, HLA antigens correspond to "human leukocyte antigens", which are protein molecules expressed on the cell surface that confer a unique antigenic identity to these cells. The antigens are also referred to as "major histocompatibility complex antigens". Thus, MHC or HLA antigens are target molecules that are recognized by T cells as "self" or "non-self". An HLA antigen is considered "self" if it is derived from the same hematopoietic stem cell source as the immune effector cell. If the HLA antigen is derived from another source of hematopoietic reconstituting cells, it is considered "non-self".

[0560] Two major classes of HLA antigens have been identified: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) enable each cell to be recognized as "self". HLA class II antigens (DRB1, DPB1, DPA1, DQB1, and DQA1 in humans) are involved in the reaction between lymphocytes and antigen-presenting cells. Both classes of HLA antigens have been implicated as targets of transplant organ rejection.

[0561] The HLA genes are clustered on human chromosome 6p21. This gene cluster encodes six classical transplantation HLA genes. The region of 6p21 also encodes genes that encode proteins having important roles in immune system regulation and other fundamental molecular and cellular processes. The entire cluster measures approximately 3.6 Mb and contains at least 224 loci. As a result of the clustering, certain "haplotypes" (sets of alleles present on a single chromosome) occur. Haplotypes inherited from one parent tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, and some alleles tend to associate together. HLA matching is used to identify the recipient's haplotype and to help identify a suitable matched donor. Certain haplotypes are more common than others and have different frequencies in different racial and ethnic groups.

[0562] As used herein, the phrase "in need thereof" refers to a subject that has been identified as in need of a particular method or treatment. In some embodiments, the identification can be by any diagnostic means. A subject may be in need thereof in any of the methods and treatments described herein.

[0563] As used herein, the phrase "an integer from X to Y" means any integer including the endpoints. For example, the phrase "an integer from X to Y" represents 1, 2, 3, 4, or 5.

[0564] As used herein, the term "mammal" means a rodent (i.e., mouse, rat, or guinea pig), monkey, cat, dog, cow, horse, pig, or human. In some embodiments, the mammal is a human.

[0565] As used herein, the term "organ" refers to a vascularized organ that is an entity performing a specific function or group of functions within a living organism. The term organ includes but is not limited to the heart, lungs, kidneys, liver, pancreas, skin, uterus, bone, cartilage, small or large intestine, bladder, brain, mammary gland, blood vessel, esophagus, fallopian tube, gallbladder, ovary, pancreas, prostate, placenta, spinal cord, limb (including upper and lower limbs), spleen, stomach, testis, thymus, thyroid, trachea, ureter, urethra, uterus.

[0566] As used herein, the terms "prevent", "preventing", and "prevention" refer to administering a therapy to an individual who may ultimately exhibit at least one symptom of a disease, disorder, or condition but who has not yet exhibited said symptom, to reduce the chance that the individual will develop the symptoms of the disease, disorder, or condition within a given time period. Such reduction can be reflected, for example, as a delay in the onset of at least one symptom of the patient's disease, disorder, or condition.

[0567] As used herein, the terms "subject", "individual", or "patient" used interchangeably mean any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates such as humans.

[0568] As used herein, the phrase "therapeutically effective amount" means the amount of an active compound or agent that elicits a biological or medical response sought by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human. The therapeutic effect depends on the disorder being treated or the desired biological effect. Thus, the therapeutic effect can be to reduce the severity of the symptoms associated with the disorder and / or inhibit the progression (partially or completely) of the disorder, or to ameliorate, cure, prevent, or eliminate the disorder or side effects. The amount required to elicit a therapeutic response can be determined based on the age, health status, body size, and gender of the subject. The optimal amount can also be determined based on monitoring the subject's response to the treatment.

[0569] As used herein, the term "tissue" refers to any type of tissue in a human or animal, and includes but is not limited to vascular tissue, skin tissue, liver tissue, pancreatic tissue, nerve tissue, urogenital tissue, gastrointestinal tissue, skeletal tissue (including bone and cartilage), adipose tissue, connective tissue (including tendons and ligaments), amniotic tissue, chorionic tissue, dura mater, pericardium, muscle tissue, glandular tissue, facial tissue, ophthalmic tissue.

[0570] In the context of the present disclosure, a "tissue bank" refers to the long-term storage of cryopreserved allogeneic cultured postnatal thymic tissue-derived products stored under liquid nitrogen. General guidelines for establishing a repository of allogeneic cultured postnatal thymic tissue-derived products can be obtained from the Industry Guidance available at https: / / www.fda.gov / downloads / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Tissue / UCM285223.pdf.Current Good Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps).

[0571] "Tissue engineering" refers to the generation of tissue ex vivo for use in the process of tissue replacement or reconstruction. Tissue engineering is an example of "regenerative medicine", which encompasses methods for repairing or replacing tissues and organs by incorporating cells, genes, or other biological components together with bioengineered materials and technologies.

[0572] The term "transplant rejection" encompasses both acute transplant rejection 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 the infiltration of the recipient's immune cells into the transplanted tissue, which perform their effector functions and destroy the transplanted tissue. The onset of acute rejection is rapid and typically occurs within a few weeks after transplantation in humans. Generally, acute rejection can be inhibited or prevented with immunosuppressive drugs such as rapamycin, cyclosporine A, anti-CD40L monoclonal antibodies, etc.

[0573] As used herein, the terms "treat", "treated" or "treating" mean both therapeutic treatment and prophylactic measures, wherein the purpose is to slow down (mitigate) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For example, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms; reduction in the extent of a condition, disorder or disease; stabilization (i.e., non-worsening) of the state of a condition, disorder or disease; delay in the onset of or slowdown in the progression of a condition, disorder or disease; improvement in the state of a condition, disorder or disease or remission (whether partial or total), whether detectable or not; improvement in at least one measurable physical parameter, not necessarily discernible by the patient; or promotion or improvement of a condition, disorder or disease.

[0574] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the recited range, and, when appropriate, their fractions (such as one-tenth and one-hundredth of an integer). The ranges are approximate values and may differ by more than an integer.

[0575] Units, prefixes and symbols are expressed in their recognized form in the International System of Units (SI). Numerical ranges include the numbers defining the said range. Measured values should be understood as approximate values, taking into account significant figures and errors associated with the measurements.

[0576] It should be further understood that certain features described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, different features described in the context of a single embodiment for sake of brevity may also be provided separately or in any suitable sub-combination.

[0577] Harvesting of donor thymic tissue.

[0578] Donor thymic tissue can be discarded during postnatal cardiac surgery and can be used for CTT with the informed consent of the donor's family. It may be necessary to remove some thymus to expose the surgical site. Thus, due to the nature of the surgical procedure, during postnatal cardiac surgery, a portion of the thymus can be removed during the cardiac surgery.

[0579] During postnatal cardiac surgery, a portion of the thymic tissue can be discarded during the surgical procedure. For all cardiac surgeries, regardless of whether the thymus is screened for transplantation, the surgeon places the discarded thymic tissue in a sterile container.

[0580] The thymic tissue donor for thymic tissue transplantation in infants with complete DiGeorge syndrome is an infant under nine months of age. As described herein, the active pharmaceutical ingredient allogeneic cultured postnatal thymic tissue-derived product is produced by processing and culturing discarded thymic tissue.

[0581] Consent to use the thymus in cultured thymic tissue transplantation can be obtained before or after harvesting the thymus. However, consent to obtain blood from the infant before undergoing bypass surgery is required and is always obtained prior to the surgery. This blood sample is used for donor screening.

[0582] The discarded thymic tissue is placed in a sterile container. Routine testing of the donor and the biological mother of the donor is performed according to FDA tissue transplantation guidelines. Tissue type matching is not required in the surgical procedures described herein, but can be performed in certain cases.

[0583] The tissue can be processed immediately or stored refrigerated overnight for processing the next day. If the thymic tissue is stored overnight, the tissue is aseptically added to thymic organ medium (the "TOM" medium described below) sufficient to completely cover the thymic tissue in the original container. The container with the thymus is placed in a refrigerator until ready for processing the next day.

[0584] Overview of Conditioning of Thymic Tissue

[0585] The conditioning protocol depletes donor thymocytes from cultured thymic tissue slices. Based on in vitro data (immunohistochemistry), the epithelial network is preserved during a culture period between 12 and 21 days, as evaluated using cytokeratin antibodies. Culturing is preferably performed in a 5% CO2 incubator at 37°C.

[0586] For successful culturing, preferably the thymic tissue slices are placed on cellulose or equivalent filter membranes and placed on a surgical sponge in a tissue culture dish. The medium includes thymic organ medium (TOM) and is changed daily.

[0587] Evaluate the thymus at the time of receipt by pathology. The identity test must show >50% of the area positive for keratin with a lacy staining pattern. The potency test must show Hassall corpuscles; it must also show CK14 staining in a lacy pattern. The viability test must show >90% of intact nuclei observed in the sections. Batch release of the tissue is done on one day (inclusive of the end points) between day 5 and day 21 and is done by pathology. For identity, areas on the tissue between day 5 and day 21 must be positive for keratin AE1 / AE3. For potency, the cultured thymus tissue between day 5 and day 21 must show scattered cytokeratin CK14 staining and at least one Hassall corpuscle must be identified. For viability, the cultured thymus tissue between day 5 and day 21 must show intact nuclei.

[0588] In one embodiment, the thymus tissue sections are conditioned for about 12 days and then cryopreserved. In another embodiment, all thymus tissue sections are conditioned for about 12 days, then about half of the thymus tissue sections are implanted in recipients, and the remaining thymus tissue sections are cryopreserved for future use.

[0589] Within 24 hours of harvest, the thymus is sectioned. The sections are cultured for 12 - 21 days. As described in detail below, this culturing process depletes live donor T cells and ultimately enables surgically implanted tissue sections to reconstitute the immune system of athymic subjects, although at immunologically effective levels, most subjects will have T cell counts below the 10th percentile for age. The culturing process outlined below significantly alters the biological characteristics of the donor thymus tissue and the constituent cells contained therein to optimize the effective therapeutic properties of the CTT sections.

[0590] The culturing process ensures a defined composition of cultured cells / tissue with the prerequisite biological characteristics in a manner suitable for surgical implantation into a subject to enable reconstitution of the subject's immune system.

[0591] The culturing process results in the loss of thymocytes and a relative enrichment of thymic epithelial cells and other stromal cells in the donor thymus tissue sections.

[0592] The culturing process further results in thymocyte depletion and maintenance of TECs to enable reconstitution of the recipient's immune system and allows for tolerance to HLA antigens in the donor thymus in the recipient.

[0593] Overall, the production process is designed to deplete thymocytes from the donor thymus tissue and preserve the functional architecture of the thymic stroma (thymic epithelial cells and fibroblasts).

[0594] In one embodiment, the processed donor thymic tissue is an engineered thymic tissue product capable of inducing tolerance to the thymic tissue type (HLA antigens) in a subject in need following a surgical implantation procedure.

[0595] To keep the thymic tissue slices viable, the thymic slices were placed on a Millipore cellulose filter membrane, and a surgical sponge was inserted into a tissue culture dish containing a culture medium. The culture medium in each tissue culture dish was replaced daily from the date of harvest from the donor to the date of implantation (days 12 to 21).

[0596] Culturing of the donor thymic tissue depleted the thymocytes in this processed tissue, which minimized the risk of graft-versus-host disease (“GvHD”), which can be a significant problem in subjects with severe immunodeficiency following thymectomy.

[0597] During the first few days of culture, many thymocytes “fell out” of the tissue slices into the culture medium and were discarded during the culture medium replacement process. As culturing continued during the culture period, the donor thymocytes continued to die, but their cellular remnants remained within the CTT slices.

[0598] Without being bound by theory, it is hypothesized that the presence of these non-viable thymocytes and their anucleate remnants is important for the intended function of the tissue-engineered product because they help preserve the open pockets within the three-dimensional network of thymic epithelial cells, which are essential for the entry of post-treatment recipient bone marrow stem cells. The experience of patient DIG003 in Markert, 1999 (see the reference list below) supports the importance of having “space” for the entry of bone marrow stem cells. Thirty-five days after CTT transplantation, a very high dose of steroids (40 mg / kg / day x methylprednisolone for 3 days) was given to the patient described in the aforementioned reference, which led to thymocyte apoptosis and epithelial condensation. No naive T cells were produced, and the patient died of infection. At autopsy, the implanted thymus was a mass of live epithelium with no space between the epithelial cells for thymocyte entry.

[0599] During the culture period, HLA typing is performed to see if the patient (recipient) and the donor tissue share any HLA alleles. An anti-HLA antibody test is performed in the recipient to determine if the recipient has any antibodies against the HLA antigens in the thymus. If the recipient has antibodies targeting the donor MHC, another thymus will be sought. The donor and the donor's mother are checked for infections according to the FDA guidance document "Guidance for Industry. Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / P)" and more recent guidance documents. The tissue is aseptically processed according to Title 21 of the Code of Federal Regulations (CFR), Subpart D, "Current Good Tissue Practice".

[0600] After reviewing the batch records and QC tests, the tissue is released from production and provided to the surgical team for transplantation. As described in this instruction, the tissue is surgically implanted into the recipient.

[0601] Cultured thymic tissue is generated in the process more fully described in the examples set forth below and in this specification.

[0602] In summary, the culturing process of the harvested thymic tissue significantly alters the biological characteristics of the donor tissue and the constituent cells contained therein as follows: loss of donor thymocytes and enrichment of thymic epithelial cells and other stromal cells, and depletion of donor thymocytes alters the tissue's physiological functions (e.g., secretion of cytokines and growth factors) and its structural properties.

[0603] During the first few days of culture, many thymocytes "fall out" of the tissue sections into the culture medium and are discarded during medium changes.

[0604] Compared to the source or raw material obtained from the donor, the manipulations that occur during the production process result in changes in the gross and histological appearance of the resulting cells contained in the final product.

[0605] Due to residual blood on and within the tissue, the thymic tissue appears red during the first few days of culture. See, for example Figure 15 .

[0606] Viable tissue is observed between days 5 and 21, minus the blood contamination apparent on day 1.

[0607] Over the remaining days of culture, as thymocytes were depleted, the depth of the tissue decreased. The reduction in thymocyte density in the tissue was demonstrated by immunohistochemistry and is described in detail below.

[0608] On day 0 after harvesting the discarded thymic tissue, viable thymocytes were densely clustered in the tissue, embedded in a stroma containing thymic epithelial cells and fibroblasts. AE1 / AE3 and CK14 staining confirmed the presence of cytokeratin (CK)-positive thymic epithelial cells characteristic of normal thymus. The thymic epithelial cells formed a lace-like three-dimensional network with elaborate processes surrounding adjacent thymocytes.

[0609] During the culture process, thymic slices were cultured as described below. A large number of thymocytes were washed out of the tissue, especially within the first 3 days. This depletion could be histologically identified as early as day 2 by H&E staining, which showed a decrease in thymocyte density, particularly in the medullary region. Most of the thymocytes remaining in the tissue showed nuclear changes consistent with apoptosis and / or necrosis, or karyolysis (complete loss of the nucleus). Many of these dead thymocytes and their cellular remnants remained throughout the thymic tissue and were believed to prevent complete collapse of the space between epithelial cells.

[0610] In the outer regions where loss of thymocytes led to collapse of the epithelial cell network (such as in the subcapsular cortex), some epithelial condensations could be seen. These condensed subcapsular cortical epithelial cells could form linear arrays several cell layers thick, which could increase the mechanical strength of the section. Some medullary epithelium might also condense to form patches of continuous epithelial cells.

[0611] As the culture proceeded, death of thymocytes continued and necrotic thymocyte debris remained within the tissue. Between approximately day 7 and day 19 of culture, further condensation of medullary and subcapsular cortical epithelium was minimal.

[0612] In the later stages of culture of each thymus stained with AE1 / AE3, regions with epithelial structures similar to those of normal thymus could still be observed. For tissues with longer culture times, the epithelial structures of the cortex and medulla could still be easily discerned; for example, Hassall's corpuscles remained in the medullary region. However, at time points after day 0, the degree of thymocyte depletion led to a significantly different overall histological appearance of the H&E compared to normal thymus.

[0613] Detailed culture of thymic tissue.

[0614] The general procedure for preparing allogeneic cultured thymic tissue-derived products is, as previously described, to obtain thymic tissue for infants with complete DiGeorge syndrome from discarded tissue of infants less than 9 months of age undergoing cardiac surgery. For solid organ transplantation, discarded thymus will be obtained from individuals no older than 50 years of age. The use of thymic tissue will depend on whether it meets the usage criteria set forth in this specification.

[0615] The thymic tissue is aseptically processed and cultured under cGMP conditions to produce partially T cell-depleted thymic tissue slices.

[0616] The production of cultured thymic tissue (CTT) consists of the following general steps: receiving and processing incoming thymic tissue, slicing, culturing, changing the medium, calculating the dose, packaging the thymus and transporting it to the operating room. In addition, incoming thymic tissue is subjected to acceptability testing and in-process testing, and thymic tissue slices are subjected to release approval testing.

[0617] In one embodiment, the thymic tissue slicing process requires using sterile disposable scissors and forceps to cut a piece of thymic tissue. The operator removes the capsule of the thymus with forceps and scissors and places the capsule on the lid of the plate for later processing.

[0618] A piece of thymic tissue is placed in a disposable tissue slicer using forceps. The top of the slicer (e.g., Stadie-Riggs manual slicer (Thomas Scientific, Swedesboro, NJ)) is placed on the middle part of the slicer and tightened in place. The operator moves the blade through the tissue block to cut slices. The slices are approximately 0.5 to 1 mm thick. Approximately 50%-90% of the filter membrane space is filled and the tissue slices do not overlap.

[0619] Typically, three in-process blocks, all approximately 3x3 mm, are cut from the tissue at the start of slicing. One is sent for histological examination and two are retained. When the thymus is sliced, thymocytes freely flow into the medium.

[0620] In one embodiment, the filter membrane and thymic slices are transferred to a gelatin surgical sponge saturated with TOM in a tissue culture dish. TOM wicks upward, wetting the Millipore filter membrane, thus keeping the tissue moist. Two filter membranes are placed in each sponge and 2 sponges are used per tissue culture dish. The process of slicing the thymic blocks is repeated until the desired number of slices is prepared. The culture dish is labeled with the operation number, culture dish number and ISBT barcode label. The completed culture dish is placed in a humidified incubator at 37°C with 5% CO2.

[0621] The tissue-engineered bulk drug product comprises thymic tissue slices that have been placed in a culture dish in a culture medium as described below and cultured for about 6 to about 21 days. The tissue-engineered finished drug product comprises thymic tissue slices transferred to a finished drug product container. No other processing is performed to produce the finished drug product from the bulk drug product; the only processing of the bulk drug product used to produce the finished drug product is transferring the slices to a leak-proof container and performing the appropriate culture medium replacement.

[0622] The culturing of the thymic tissue slices is described in more detail in the following paragraphs.

[0623] In one embodiment, thymic tissue is obtained from the operating room as discarded tissue from a 9-month-old infant who has undergone heart surgery. The tissue is then placed by the surgical team in a sterile specimen cup with a screw-cap top and transported to a GMP facility for processing under ambient conditions. The sterile specimen container containing the thymus is labeled with the donor's name and medical record number, including a barcode. The donor screening group assigns a unique identifier (sequentially numbering the thymus) and a unique medical record number to the thymus. For production, each tissue has a processing number and a unique label. All identifiers are recorded on a "Confidential Thymic Donor List", which is kept separate from the batch records and is confidential.

[0624] In one embodiment, the bulk drug product container closure system can be a cell culture dish with a lid. A slice of thymic tissue is placed on a filter membrane, and two filter membranes are placed on each gelatin sponge in the thymic organ culture medium in the culture dish. Four slices are placed in each culture dish, and the culture dishes are stored in an incubator with the culture medium changed daily until ready for release.

[0625] In one embodiment, the culture dishes can be obtained from Corning. The culture dishes can be sterile and pyrogen-free 100 mm polystyrene cell culture dishes (product number 353003). The culture dishes are cleaned by vacuum gas plasma treatment and sterilized by gamma irradiation. The dimensions of the culture dishes are 89.43 mm O.D. x 19.18 m.

[0626] In an exemplary embodiment, The sponges can be produced by Ethicon and meet the requirements of USP absorbable gelatin sponges. Suitable sponges are sterile, water-insoluble, extensible, absorbable porcine gelatin sponges, which are intended for hemostatic use. Illustrative examples of mixed cellulose ester filter membranes are produced by Millipore (product number SMWP02500). The 25 mm hydrophilic membrane has a pore size of 5.0 μm. It is made of a biocompatible mixture of cellulose acetate and cellulose nitrate. Before use, the filter membranes are sterilized with ethylene oxide.

[0627] After the donor thymus is released and received in the processing laboratory, the thymus is sliced thinly and placed on a sterile filter paper which is placed on a surgical sponge in a sterile Petri dish. If the tissue is not to be processed immediately, it is stored in thymus organ medium (TOM) at 2-8 °C for up to 24 hours after harvest from the donor and then processing is commenced. TOM consists of Ham's F-12 medium, HEPES buffer, L-glutamine and heat-inactivated fetal bovine serum (FBS).

[0628] In one embodiment, processing is carried out in the ISO 5 space of a biological safety cabinet (BSC) in an ISO 7 production cleanroom. At any time, only one batch of thymus tissue from a single thymus is processed in the BSC. The BSC must be cleaned before use. The appearance of the thymus is tested 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 using sterile disposable forceps and scissors. Tissue blocks are taken for testing and kept as retained samples. The identity of the incoming thymus tissue is tested by histology. Donor eligibility is also confirmed. Processing is continued until the histological results and all donor screening results are received.

[0629] The acceptance criteria for donor screening are that all donor eligibility requirements must be met. Donor screening is required under 21 CFR 1271 to protect the safety of the recipient of thymus tissue implantation. This screening minimizes the risk of disease transmission from the donor to the recipient.

[0630] Thymus Organ Medium (TOM)

[0631] The medium is made from components approved for use in humans and is less likely to cause an allergic reaction whenever such reagents are available.

[0632] All reagents must be tracked so that if any problems arise, all components can be identified after implantation.

[0633] Due to concerns about Creutzfeldt-Jakob Disease, fetal bovine serum (FBS) must be produced using US materials. Before use, the information of each batch must be sent to the FDA.

[0634] Before use, the medium must be tested for bacterial, fungal and mycoplasma contamination.

[0635] In one embodiment, TOM is prepared using the following materials:

[0636] HAMS F12, Gibco #11765-054 (or case 11765-062), 500 ml bottle or equivalent source.

[0637] HEPES, Gibco #15630 - 080 or equivalent, 1M solution, 100 ml bottle. Final concentration 25 mM.

[0638] L - Glutamine, Gibco #25030 - 081 or equivalent source (stock solution 200 mM).

[0639] Fetal bovine serum, Gibco, #16140 (heat - inactivated) or #10082 - 147 (heat - inactivated, certified).

[0640] In one embodiment, FBS can be used as HI as follows:

[0641] The FBS must be heat - inactivated at 56 °C for 30 min.

[0642] To reduce the likelihood of medium contamination, the medium must be aliquoted and the aliquots must not be used more than once.

[0643] The remaining FBS aliquots can be stored frozen (-20 °C) in 25 ml aliquots for research use.

[0644] In one embodiment, TOM can be prepared as follows.

[0645] Thaw fetal bovine serum in a refrigerator overnight or thaw at 37 °C with frequent gentle vortexing.

[0646] If using non - heat - inactivated fetal bovine serum, heat - inactivate at 56 °C for 30 minutes.

[0647] If preparing 4 liters at a time, place all medium components together in a 4 - liter flask and stir with a stir bar on a magnetic stirrer plate at medium speed (without foaming) for 3 - 5 minutes.

[0648] Sterilize using a 0.2 - micron filter unit.

[0649] In one embodiment, the TOM preparation can be sterilized as follows. Dispense 1 liter of TOM into a one - liter flask. Measure 80 ml of TOM in a disposable sterile graduated cylinder. Pour 80 ml of TOM into a 150 ml Corning filter sterilization unit. Attach a house vacuum device to the filter and sterilize the filter according to the manufacturer's instructions. Remove the filter unit from the container and discard. Cover the collection bottle with a sterile cap (provided with the unit). Label with the TOM lot number. Test one aliquot for anaerobic bacterial culture; fungal culture, others; and mycoplasma culture. Test one aliquot for endotoxin. Store all TOM aliquots in a -20 °C upright freezer.

[0650] TOM medium can be released for use under the following conditions: for samples tested at a 20-fold dilution, the LAL result must be equal to or less than 2 EU / ml; for samples tested at a 10-fold dilution, the LAL result must be equal to or less than 1 EU / ml; all culture results must be negative for growth.

[0651] A BSC must be used to filter and dispense the medium.

[0652] Before release, test the sterility and endotoxin of TOM. TOM will not be released for culturing donor thymus until the 14-day sterility test acceptance criteria have been met. After preparation, store TOM at -20 °C until thawed, at which time it can be stored in a refrigerator for up to two weeks.

[0653] In one embodiment, a 14-day sterility test can be performed, for example, using a BacT / ALERT culture system. BacT / ALERT (BioMerieux, Durham, NC) is a commercially available culture system that can be used to test samples using an automated microbial detection system.

[0654] Incubate all in-process and API cultures for 14 days, or report immediately if the product turns positive. For positive tests, one or more organisms can be identified and their antibiotic sensitivities determined. On day 1, day 7, and the day of release, inoculate culture bottles containing medium for aerobic growth and bottles containing medium for anaerobic growth with the sample to be tested. Incubate all bottles at 35 - 37 °C for 14 days.

[0655] FBS can be obtained from the GIBCO brand of Life Technologies. FBS is prepared by a sterile and validated process. FBS meets the USDA requirements for animals from slaughterhouse sources, traceability, and country of origin. All fetal blood is collected from fetuses of healthy mothers that have been certified by pre- and post-mortem veterinary inspections. All FBS can be traced by collection date and location. FBS collected and processed in the United States comes from USDA-approved and inspected slaughter establishments. The United States is recognized by the USDA as a country free of foot-and-mouth disease and rinderpest. To qualify a supplier, the pH, osmotic pressure, endotoxin, total protein, and identity of FBS must be tested before use

[0656] Place the completed culture dishes in a humidified incubator at 37 °C with 5% CO2. Each batch of thymic tissue is stored in a separate incubator. After the thymic slices have been placed in the incubator, perform particulate sampling and personnel monitoring.

[0657] Thymic slices are cultured for up to 21 days (e.g., a conditioning regimen of about 6 days to about 21 days), and the culture medium is changed daily during the culture. These thymic slices are considered the active pharmaceutical ingredient. During the culture period, many thymocytes are washed out from the thymic tissue slices, or the thymocytes undergo apoptosis while the thymic stroma is preserved. All production steps are carried out using sterile disposable equipment and supplies. The culture medium is aspirated from the culture dishes with a pipette and combined into a sterile collection container for in-process testing. Then ten (10) mL of fresh thymic organ culture medium is gently dispensed in a rinsing manner onto the tissue slices in each culture dish. After the culture medium change is completed, if necessary, samples are taken from the combined culture medium for sterility and histology. Particle sampling and personnel monitoring are completed and the pipeline is cleaned.

[0658] The culture medium is changed daily.

[0659] Thymic slices are cultured for up to 21 days (e.g., a conditioning regimen of about 6 days to about 21 days).

[0660] In-process testing is carried out to provide insights into the process and product quality and to help ensure the safety and quality of the final finished drug.

[0661] In-process testing

[0662] Samples are collected on Day 1 and Day 7 for in-process sterility testing. Samples are collected on Day 7 for in-process mycoplasma testing. Samples are collected between Day 5 and Day 9 for in-process histology testing. The dose is determined on the day before release. Gram stain, BacT, mycoplasma, and endotoxin are tested on the day of release.

[0663] Gram stain is a bacteriological laboratory technique used to distinguish bacterial species into two groups, Gram-positive and Gram-negative. Gram stain is tested on the combined used 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 for preliminary morphological identification or to determine the presence of a large number of bacteria in clinical specimens. Staining can be done manually or using an automatic stainer. Two different staining methods have been shown to have no qualitative differences that affect the culture results.

[0664] Histology testing is carried out before implantation and, in one embodiment, includes at least: (1) determining that areas positive for keratin AE1 / AE3 are scattered throughout the tissue; (2) microscopically identifying at least 1 Hassall's corpuscle; (3) CK14 staining of the tissue sections is scattered throughout the thymic tissue; and (4) intact nuclei are observed microscopically. In one embodiment, histology testing is carried out between about 6 and about 21 days. The presence of Hassall's corpuscles and intact nuclei and successful CK14 staining indicate that normal healthy thymic tissue has been cultured.

[0665] The culture time is an important process parameter. As noted, the culture is carried out for up to 21 days.

[0666] The thymus samples in culture are tested prior to implantation to confirm whether the histological results produced in culture represent the histological tests of a historical sample of the cultured thymus tissue. Based on the observations of the samples discussed in the examples by the pathologist, the histological appearance at day 5 of the tissue sections reflects the histological appearance observed at each subsequent time point in culture (days 9, 12, and 21). In one embodiment, the thymus tissue can be tested at different timing periods and intervals between day 5 and day 21 of the culture. For example, the test can be carried out during a conditioning regimen that lasts for five, or six, or seven, or eight, or nine, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21 days; 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 one embodiment, the conditioning regimen can be at any time between about 6 days and about 21 days prior to implanting the cultured thymus tissue.

[0667] The histological examination of any one section confirmed the conclusion regarding the acceptability of the entire batch. The relevant characteristics of any one section from the thymus reflect the characteristics of the entire thymus, which supports the continued use of a single tissue section for histological testing.

[0668] As Figure 12A and 12B indicated by the forced degradation tests, the cultured thymus tissue product is not easily degraded and is most sensitive to freeze / thaw and osmotic pressure changes. The other conditions tested during the forced degradation showed little effect on the cultured thymus tissue product.

[0669] Control of the cultured thymus product drug substance

[0670] The acceptance criteria for the incoming thymus tissue product include the tests identified in Table 1 below.

[0671] Table 1. Incoming thymus tissue

[0672]

[0673] Abbreviations: CK, cytokeratin; EU, endotoxin unit; USP, United States Pharmacopeia. Thymus tissue is processed before all donor screening results are obtained.

[0674] Generally, the acceptance criteria for weight are greater than or equal to 3 grams. This is the minimum acceptable thymus weight to ensure there is sufficient material for proper formulation of the final product. The acceptance criteria are based on experience in processing thymus tissue.

[0675] The acceptance criteria for tests during the process are identified in Table 2 below.

[0676] Table 2. Tests during the process:

[0677]

[0678]

[0679] The acceptance criteria for tests on the cultured thymus tissue drug substance are identified in Table 3 below.

[0680] Table 3. Tests on the cultured thymus tissue drug substance

[0681]

[0682]

[0683] The acceptance criteria for identity are that thymus tissue identity is confirmed histologically on Day 1 and at midpoint (Days 5 - 9). Barcodes are used to track the tissue throughout the process, and the barcode is confirmed at release to verify the correct identity of the product.

[0684] Immunohistochemical histology

[0685] The histological method is the standard method used by the hospital for all tissue types, as known to those skilled in the art.

[0686] Product samples are fixed in 10% formalin and transported to the laboratory. To protect patient privacy, the containers are labeled with coded identifiers instead of the patient's name and medical record number. After entering the pathology department, a unique pathology accession number is assigned to the specimen and barcoded. All subsequent blocks, slides, and paperwork are barcoded with this pathology accession number.

[0687] After receiving the specimen in the laboratory, the formalin-fixed tissue is grossly examined, and a written gross description of the material is prepared, which will become part of the final report. The formalin-fixed tissue is then processed on an automated processor by standard methods and embedded in paraffin blocks. Sections are cut from the paraffin blocks and stained by an ASCP-certified histotechnologist as follows:

[0688] Hematoxylin and eosin.

[0689] Cytokeratin AE1 / AE3 immunohistochemistry.

[0690] Cytokeratin 14 immunohistochemistry.

[0691] CD3 immunohistochemistry.

[0692] Ki-67 immunohistochemistry.

[0693] During the aforementioned immunohistochemistry tests, appropriate control slides were also tested and viewed. All control slides and internal controls demonstrated the expected immunoreactive patterns. When testing samples as part of the potency test, the incoming thymus samples also served as controls for tissue sections that had been cultured for approximately 6 days to approximately 21 days. The incoming thymus samples appeared as typical thymus samples and then changed on the tissue sections when cultured, and then were tested after being cultured for approximately 6 days to approximately 21 days. After being cultured for approximately 6 days to approximately 21 days, the samples must show areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the tissue, and the presence of intact nuclei.

[0694] The slides were interpreted by a pathologist certified in anatomic pathology who had additional experience in the histological evaluation of thymic tissue. The final report was issued by the pathologist, and the report documented the results.

[0695] The acceptance criteria for the cultured thymic tissue drug substance testing are identified in Table 4 below.

[0696] Table 4. Release Testing of Cultured Thymic Tissue Drug Substance

[0697]

[0698] The cultured thymic tissue must be free of microorganisms. In the sterility tests conducted on Day 1 and Day 7, no microorganisms should grow. At the test on Day 7, mycoplasma should be negative. The sterility test should be Gram stain negative.

[0699] Use appropriate controls including aseptic techniques to maintain product sterility; adopt a training program and verify the qualifications of operators; utilize appropriate cleanroom qualification procedures; adopt established clean media filling procedures, and utilize ready-to-use sterilization equipment or equipment sterilized using a validated sterilization cycle.

[0700] Visually inspect the containers of the processed thymic tissue for damage. The tissue sections typically exhibit a yellow to reddish-brown appearance with varying thicknesses and shapes.

[0701] Thymic tissue identity was histologically confirmed on day 1 and at the midpoint (days 5 - 9).

[0702] Barcodes were used to track the tissue throughout the process and the barcodes were confirmed at release.

[0703] The dose (area) was 1000 - 22000 mm 2 thymic tissue / m² recipient body surface area. The dose was controlled by the surface area of the sections released to the operating room to fit the patient's body surface area.

[0704] The acceptable dose range was defined as 1000 - 22000 mm 2 thymic tissue / m 2 recipient body surface area (BSA). The area of the thymic tissue was determined by photos using software analysis (PAX-it image analysis software). BSA was determined using the patient's height (cm) and weight (kg). BSA was calculated using the DuBois and DuBois formula:

[0705] BSA = 0.007184 × [height (cm)] 0.725 × [weight (kg)] 0.425 .

[0706] The endotoxin of the cultured thymic tissue was tested. The specification was ≤5 EU / kg body weight / hour.

[0707] The endotoxin test can be performed, for example, by using the Endosafe PTS system. The cartridges used with the Endosafe PTS use a chromogenic kinetic Limulus amoebocyte lysate (LAL) test. Each cartridge contains a precise amount of LAL reagent, chromogenic substrate, and control standard endotoxin. The test sample was pipetted into four sample reservoirs. The instrument aspirated the sample in two channels (sample channels) and mixed it with the LAL reagent, and in the other two channels (spiking channels) with the LAL reagent and positive product control. The sample was incubated and then combined with the chromogenic substrate. After mixing, the optical density of the wells was measured and compared with the standard curve archived in the instrument. The instrument measured the reaction time in each channel. An archived standard curve specific to each batch of cartridges was constructed using the logarithm of the reaction time and the logarithm of the endotoxin standard concentration. The sample and peak were calculated by interpolation from the standard curve using the reaction time. This test meets the requirements of the United States Pharmacopeia (USP).

[0708] The mycoplasma test can be performed as follows. Samples of the pooled medium were removed from the plates on day 7 and tested before product release.

[0709] In the event of a positive culture during production, the batch will be discarded and not managed. In the event of a positive culture after clinical product administration, the patient's attending physician and sponsor will appropriately treat the patient. A positive culture requires identification of the species of the contaminating organism and determination of its antibiotic sensitivity. If indicated, the attending physician will institute antibiotic therapy for thymus recipients.

[0710] The finished product undergoes similar visual inspection and histological testing before use.

[0711] After the thymus tissue slices have been cultured for up to 21 days, the slices are transferred to a finished product container for transport to the operating room. After receiving the slices in the operating room, they are inserted into the thigh muscle of the recipient patient.

[0712] The container should be intact without visible damage, and the thymus tissue slices should appear as yellow to reddish-brown tissue slices with varying thicknesses and shapes. Visually inspect the tissue slices to confirm that these acceptance criteria are met.

[0713] Cryopreservation and thawing of allogeneically cultured postnatal thymus tissue-derived products

[0714] Cryopreservation of allogeneically cultured postnatal thymus tissue-derived products can be performed as follows.

[0715] A cryopreserved allogeneically cultured postnatal thymus tissue-derived product prepared by a method comprising the following steps:

[0716] (a) Obtaining suitable thymus tissue from a donor;

[0717] (b) Typing the following HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, HLA-DPA1;

[0718] (c) Subjecting the thymus tissue to a conditioning protocol for a period of about 6 days to about 21 days; wherein the conditioning protocol for the donor thymus tissue comprises aseptic treatment of the donor thymus tissue in thymus organ culture medium to produce partially T cell-depleted donor thymus tissue slices; further, wherein after completion of the conditioning protocol, the donor thymus tissue slices show areas positive for cytokeratin AE1 / AE3 dispersed throughout the tissue, the presence of at least one Hassall's corpuscle, CK14 staining dispersed throughout the tissue, and the presence of intact nuclei at about 6 days to about 21 days.

[0719] (d) Harvest the partially T cell-depleted donor thymic tissue sections as a source product of allogeneically cultured postnatal thymic tissue;

[0720] (e) Cryopreserve the source product of allogeneically cultured postnatal thymic tissue in liquid nitrogen; and

[0721] (f) Maintain the cryopreserved source product of allogeneically cultured postnatal thymic tissue in liquid nitrogen in a cryopreserved source product library of allogeneically cultured postnatal thymic tissue. In one embodiment, the cryopreserved source product of allogeneically cultured postnatal thymic tissue as claimed in claim 66, wherein the thymus on the day of harvest exhibits >50% of the area positive for keratin with a lacy staining pattern, Hassall's corpuscles are present, CK14 staining is in a lacy pattern and >90% of the nuclei are intact.

[0722] In one embodiment, the donor thymus is sectioned and divided into two substantially equal parts, and each section with a cellulose filter membrane is placed in a separate cryovial (Nunc tube). The filter membrane is folded in half to insert it into the tube. Approximately 1 to approximately 1.5 ml of cryopreservation medium [sterile filtered 90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] is added at room temperature to cover the tissue. The sterile cap of the cryovial is replaced on the tube. All tubes are placed in a Biocision Cool Cell or equivalent container at room temperature. All empty slots in the CoolCell should be filled with tubes having 1 ml of cryopreservation medium. The tubes are placed in a -80 °C freezer overnight. Alternatively, each tissue plus filter membrane is placed in a 5 ml CryoELITE tissue vial (Wheaton). 3 to 5 ml of cryopreservation medium is placed at room temperature to cover the tissue. It is placed in a polystyrene foam box and placed in a -80 °C freezer overnight. Then the vials are transferred to the vapor phase of a liquid nitrogen freezer. Alternatively, a controlled rate freezer can be used to cool the cryovials to liquid nitrogen temperature.

[0723] To recover the tissue, remove the cryovial or CryoELITE tissue vial from the liquid nitrogen freezer. Rapidly thaw the thymic mass in the vial in a 37 °C water bath with vortex motion. Spray the tube with 70% ethanol and then place it in a biosafety cabinet (BSC). Use forceps to remove the thymic tissue and filter membrane from the Nunc cryovial or CryoELITE tissue vial. Place the tissue and filter membrane in a 50 ml conical tube containing 20 ml of 4 °C TOM. Up to 5 filter membranes can be placed in each 50 ml conical tube containing 20 ml of 4 °C TOM. Immediately transfer 5 filter membranes with tissue to a fresh conical tube with 20 ml of 4 °C TOM medium and place at 4 °C for 15 minutes. Repeat the wash 3 times. Keep the tissue at 4 °C and transfer each piece to its own 120 ml Starplex container with 5 ml of 4 °C TOM. Send all containers in a temperature-controlled container with a refrigerated bag to the operating room. Send the Starplex container with tissue to the operating room. Transfer the tissue on the filter membrane to the sterile area into a tissue culture dish with approximately 2 ml of sterile saline. The surgical assistant nurse removes the tissue from the filter paper by scraping or pulling with forceps. The surgical assistant nurse returns the tissue in an amorphous mass back to the filter paper. Transfer the tissue culture dish with approximately 4 filter membranes and the tissue to the surgical site where the surgeon can easily access the tissue. Place the tissue in the quadriceps muscle similarly to the procedure for CTT (RVT-802). The cryo-CTT is similar to CTT in that it is partially T cell depleted, the thymic tissue sections show areas positive for cytokeratin AE1 / AE3 scattered throughout the tissue, the sections contain at least one Hassall's corpuscle, CK14 staining is scattered throughout the tissue and intact nuclei are present.

[0724] Transplantation of CTT

[0725] In one embodiment, mismatched thymic tissue slices from a donor are cultured for about 6 days to about 21 days. On the day of solid organ transplantation, steroids are typically administered during induction of anesthesia. For heart or lung transplantation, the recipient's thymus should be surgically removed at the time of solid organ transplantation. For other organ transplants, thymectomy can be performed before or on the day of transplantation. The thymectomy method will be surgical, thoracoscopic, or robotic. At the end of the surgery after reperfusion, more steroids are administered to the recipient to kill most of the remaining T cells (and NK cells) in the recipient within 3 to 7 days before receiving horse antithymocyte globulin (e.g., rabbit antithymocyte globulin), or alemtuzumab is administered within 4 days to kill T cells, B cells, and NK cells. Then immunosuppressants (such as cyclosporine or tacrolimus) and mycophenolate are started until T cells develop and show more than 10% naive T cells. It may take 6 to 12 months for naive T cells to increase to this number. The cultured thymic tissue is processed as the thymus of the solid organ donor. Between about 6 days and about 21 days, half of the CTT can be implanted into the limb muscle. The other half of the thymus will be cryopreserved for future recipients. The immunosuppression regimen will suppress any remaining T cells until the cultured thymic tissue slices implanted in the recipient release naive T cells and the recipient meets the criteria to discontinue the maintenance immunosuppression regimen. (More than 10% naive T cells are required to discontinue immunosuppression.)

[0726] Thymectomy protocol

[0727] The patient is brought to the operating room and placed under general anesthesia via an endotracheal tube.

[0728] The chest and abdomen are prepared in a sterile manner and draped.

[0729] The patient undergoes a complete sternotomy through a skin incision of approximately 4 cm.

[0730] The two pleural cavities are entered to ensure complete resection.

[0731] The phrenic nerves are visualized on both sides and care is taken not to damage them.

[0732] The thymus is identified and carefully dissected from the pleural covering of the lungs, starting from the lower corner and extending to the upper corner.

[0733] A complete thymectomy is performed.

[0734] Hemostasis is achieved in the mediastinum.

[0735] Chest tube placement. A chest tube is always inserted (into the mediastinum). If a single pleural cavity is entered during surgery, the chest tube continues from the mediastinum into that pleural cavity. If two pleural cavities are entered, a second chest tube is used in a similar manner, from the mediastinum to the other pleural cavity.

[0736] Drain size. A #15 Blake drain is used for infants under 2 years of age. A #19 Blake drain is used for children 2 years of age and older.

[0737] Sternal closure: In neonates or infants, the sternum is closed using 0-Ticron suture. At approximately 1 - 2 years of age, a #1 sternal wire is used. At approximately 2 - 5 years of age, a #4 sternal wire is used.

[0738] The fascia, subcutaneous tissue, and skin are closed with continuous absorbable sutures.

[0739] A skin wound vacuum device is placed over the sternum.

[0740] The patient is extubated in the operating room.

[0741] Sponges, instruments, and needles are counted and must be correct at the end of the surgery.

[0742] Surgical implantation of a product derived from allogeneically cultured postnatal thymic tissue.

[0743] The product derived from allogeneically cultured postnatal thymic tissue should be implanted according to the following instructions. The thymic tissue is implanted into the thigh, which requires a healthy muscle tissue bed.

[0744] Preparation for the implantation procedure

[0745] The maximum and minimum doses of the product derived from allogeneically cultured postnatal thymic tissue to be implanted should be calculated for each individual patient. The intended recipient is correctly identified before administration.

[0746] Under aseptic conditions within a laminar flow hood, tissue sections on filter paper on a surgical sponge in a culture medium are removed from the tissue culture dish and placed in a 120 ml sterile cup with 20 ml of culture medium, packaged to maintain sterility, and delivered to the operating room, or packaged for shipment. Do not remove the tissue sections from the individual containers until ready for use. Verify the expiration date and time of the product.

[0747] Always handle allogeneically cultured postnatal thymic tissue-derived products (tissue slices) using strict aseptic technique. Inspect each container for signs of leakage or damage. Do not use if there are signs of contamination. Outside the sterile area, unload the containers of allogeneically cultured postnatal thymic tissue-derived products from the shipping box. Remove the rack containing the polypropylene containers from the outer bag. When ready, a team member outside the sterile area but adjacent to the aseptic preparation workbench will open and remove the lid from each container, one at a time. Then, to keep each open container: A team member outside the sterile area will extend his / her arm over the sterile area without touching the sterile area.

[0748] A team member in the sterile area 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 on the aseptic preparation workbench containing approximately 2 ml of preservative-free saline. Four tissue slices with filter paper will be removed from four containers and placed in one sterile tissue culture dish on the sterile area in front of the team member in the sterile area. Using sterile forceps, the team member in the sterile area will then use two pairs of forceps to peel the tissue slice from the filter paper, with one pair of forceps holding the filter membrane in place while the other pair pulls the tissue out or scrapes the tissue into a pile. Then the tissue removed from each filter paper will be piled up on that filter paper, in the middle of the filter paper. Then the sterile tissue culture dish will be transferred to the sterile area. Then, when the surgeon implants 4 slices, the next set of four containers of allogeneically cultured postnatal thymic tissue-derived products will be processed in the same manner. When the surgeon has completed implanting the first four slices, the next culture dish with 4 tissue slices will be placed in the surgical area and the original tissue culture dish will be returned to the sterile area in front of the team member in the sterile area to load the 3rd set of four tissue slices. Continue this cycle until all desired tissue has been implanted. Do not transfer all tissue slices at the beginning to avoid air contamination in the operating room.

[0749] Surgical Procedure

[0750] Step 1 . Skin Incision.

[0751] After induction of general anesthesia, make a vertical skin incision (usually about 5 cm long) over one of the anterior thigh compartments. Note: The size of the incision and whether one or both legs are used for the implant procedure is determined by the patient's body size, the amount of tissue planned for transplantation, and his / her muscle mass. If all or most of the tissue can be implanted into one leg, only one leg should be used.

[0752] Step 2 . Open the fascia to expose the anterior compartment muscles.

[0753] Step 3 . Muscle Dissection and Implantation.

[0754] Use a tonsil clamp or similar instrument to separate the muscle along the natural groove of the quadriceps muscle. Individual thymic slices of the allogeneic cultured postnatal thymic tissue-derived product should be implanted without cutting the muscle tissue. Place the individual tissue slices in "pockets" within the quadriceps muscle along the natural groove at approximately 1 cm intervals and approximately 1 cm deep. Depending on the patient's body size, the surgeon may place approximately 6 - 7 slices into 6 to 7 pockets along each groove. Depending on the quality of the tissue on each filter, slices of the allogeneic cultured postnatal thymic tissue-derived product can be cut in half before implantation. Thick tissue slices that completely cover the filter paper should be cut in half to optimize the vascularization of each tissue. Implant as much of the required tissue as possible in each anterior compartment until the maximum planned dose.

[0755] Step 4 . Muscle closure.

[0756] Close the muscle over the site of thymic tissue implantation with a single suture to prevent the muscle from reopening and the graft from falling out of the muscle. Before closing the incision, ensure that the implanted tissue is completely covered by muscle tissue and there is no exposed thymic tissue.

[0757] Step 5 . Repeat steps 3 - 4 for each tissue slice of the allogeneic cultured postnatal thymic tissue-derived product until the maximum expected dose.

[0758] Step 6 . Incision closure.

[0759] Confirm hemostasis. Close the skin incision with two layers of absorbable sutures and apply a standard dressing, such as a wound closure strip or skin glue. Keep the fascia open to allow space for muscle compartment swelling. Occlusive dressings can be used to prevent contamination.

[0760] Postoperative surgical / medical management.

[0761] Use mild painkillers as needed. Monitor for signs of infection or dehiscence.

[0762] If the donor is a living related donor for the lung, kidney, intestine, or partial liver, a portion of the thymus of the solid organ donor may be sufficient for culturing and implantation. The pathological criteria listed above regarding the day of harvest and until implantation need to be met.

[0763] Cryopreserved thymic tissue can be obtained from a third-party donor. However, the third-party donor must express all of 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-DQA1, HLA-DPB1, and HLA-DPA1. A mismatch in the HLA-DP alleles is acceptable if the mismatch is "permissible". In the case of the other alleles, a smaller mismatch is allowed, for example, 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 the same.

[0764] Human heart transplantation procedure

[0765] Subject eligibility is determined based on a number of criteria, including: having a poor 12 - 24 month prognosis without a heart transplant despite current maximal supportive therapy; having congenital or acquired heart disease as defined by the UNOS criteria and being unable to thrive; presenting with symptoms of advanced heart failure in the context of congenital or acquired heart disease that is refractory to medical therapy; having hemodynamic abnormalities or increased pulmonary vascular resistance; having inoperable structural heart disease; having symptomatic arrhythmias or poor exercise tolerance that are not amenable to medical therapy or device therapy.

[0766] A number of absolute and relative contraindications to heart transplantation surgery have been evaluated, including for example: reversible renal insufficiency, unless the subject is a candidate for heart / kidney transplantation; irreversible liver disease, unless the subject is a candidate for heart / liver transplantation; irreversible pulmonary dysfunction, using unconventional mechanical ventilator support (i.e., high-frequency ventilator, maximum settings for CMV) or fixed pulmonary hypertension (TPG > 15), unless the subject is a candidate for heart-lung transplantation; diabetes with microvascular disease;...

Claims

1. A method of producing an allogeneic cultured postnatal thymic tissue-derived product suitable for implantation into the philtrum, the method comprising the steps of: Cutting a donor thymus into donor thymus slices; Aseptically culturing the donor thymus slices in a thymic organ culture medium for 6 to 21 days to produce partially T cell-depleted donor thymic tissue slices; Detecting keratin AE1 / AE3, cytokeratin CK14, at least one Hassall's corpuscle, and intact nuclei in the partially T cell-depleted donor thymic tissue slices during the culturing step; Testing for sterility during the culturing step; Detecting the CCL21 level in the thymic organ culture medium during the culturing step; Comparing the CCL21 level in the thymic organ culture medium during the culturing step with a baseline level determined on day 0 of the culturing step; and If the CCL21 level has increased compared to the baseline level, and the partially T cell-depleted donor thymic tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the donor thymic tissue slices, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue slices, and the presence of intact nuclei, and the sterility test shows no microbial growth, then recovering the partially T cell-depleted donor thymic tissue slices as the allogeneic cultured postnatal thymic tissue-derived product suitable for implantation.

2. The method according to claim 1, further comprising the step of cryopreserving the allogeneic cultured postnatal thymic tissue-derived product in liquid nitrogen for future implantation.

3. The method according to claim 1, further comprising detecting the L-selectin level in the thymic organ culture medium during the culturing step, comparing the L-selectin level with an L-selectin baseline level determined on day 0 of the culturing step, wherein the L-selectin level is decreased compared to the L-selectin baseline level.

4. The method according to any one of claims 1-3, the method further comprising detecting the level of one or more of M-CSF, galectin-7, and IL-16 in the thymic organ culture medium during the culturing step, comparing the level of one or more of M-CSF, galectin-7, and IL-16 with the baseline level of the corresponding biomarker determined on day 0 of the culturing step, wherein the level of one or more of M-CSF, galectin-7, and IL-16 is decreased compared to the baseline level of the corresponding biomarker.

5. The method according to any one of claims 1-3, wherein the method further comprises detecting, during the culturing step, an increased level of one or more of CXCL12, CXCL16, and CCL11 in the thymic organ culture medium, comparing the level of one or more of CXCL12, CXCL16, and CCL11 with the baseline level of the corresponding biomarker determined on day 0 of the culturing step, wherein the level of one or more of CXCL12, CXCL16, and CCL11 is increased compared to the baseline level of the corresponding biomarker.

6. The method according to claim 1, wherein the culturing step 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 21 days.

7. The method according to claim 1 or 2, further comprising the steps of: detecting, during the culturing step, the level of at least one biomarker selected from the following in the thymic organ culture medium: L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, 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 comparing the level of the at least one biomarker with the baseline level of the corresponding biomarker determined on day 0 of the culturing step; and Wherein if the level of one or more of CXCL16, CCL11, CXCL12, GDNF, MIP-3b, GDF-15, LIGHT, uPAR, MIP-1b, PIGF, IGFBP-6, GCP-2, OPN, DKK-1, and ANG-1 is increased compared to the corresponding biomarker baseline level, or if the level of one or more of L-selectin, M-CSF, galectin-7, IL-16, MIF, CTACK, ICAM-1, PECAM-1, IL-2Rg, SCF R, PDGF-AA, MIP-3a, IL-2Ra, ICAM-3, IGFBP-1, BCMA, EGF R, PF4, HVEM, IL-6R, IL-12p40, RANTES, MICA, ALCAM, NRG1-B1, CEACAM-1, and IL-1b is decreased compared to the corresponding biomarker baseline level, then the partially T cell-depleted donor thymic tissue slices are recovered as the allogeneic cultured postnatal thymic tissue source product suitable for implantation.

8. A method for determining whether an allogeneic cultured postnatal thymic tissue source product is suitable for implantation into a human, the method comprising the following steps: Cutting a donor thymus into donor thymic slices; Sterile culturing the donor thymic slices in thymic organ medium for 6 to 21 days to produce partially T cell-depleted donor thymic tissue slices; Detecting keratin AE1 / AE3, cytokeratin CK14, at least one Hassall's corpuscle, and intact nuclei in the partially T cell-depleted donor thymic tissue slices during the culturing step; Testing for sterility during the culturing step; Detecting the level of CCL21 in the thymic organ medium during the culturing step and comparing the CCL21 level with the baseline level of CCL21 determined on day 0 of the culturing step; and If the CCL21 level is increased compared to the baseline level, and the partially T cell-depleted donor thymic tissue slices show areas positive for keratin AE1 / AE3 scattered throughout the donor thymic tissue slices, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue slices, and the presence of intact nuclei, and the sterility test shows no microbial growth, then determining that the allogeneic cultured postnatal thymic tissue source product is suitable for implantation into a human.

9. The method according to claim 8, further comprising the following steps: During the culturing step, the level of at least one biomarker selected from the following is detected in the thymic organ culture medium: L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, 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 the level of the at least one biomarker is compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step; wherein if the level of one or more of CXCL16, CCL11, CXCL12, GDNF, MIP-3b, GDF-15, LIGHT, uPAR, MIP-1b, PIGF, IGFBP-6, GCP-2, OPN, DKK-1, and ANG-1 increases compared to the baseline level of the corresponding biomarker, or if the level of one or more of L-selectin, M-CSF, galectin-7, IL-16, MIF, CTACK, ICAM-1, PECAM-1, IL-2Rg, SCF R, PDGF-AA, MIP-3a, IL-2Ra, ICAM-3, IGFBP-1, BCMA, EGF R, PF4, HVEM, IL-6R, IL-12p40, RANTES, MICA, ALCAM, NRG1-B1, CEACAM-1, and IL-1b decreases compared to the baseline level of the corresponding biomarker, then it is determined that the allogeneic cultured postnatal thymic tissue-derived product is suitable for implantation into a human.

10. The method according to claim 9, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16, and CCL11.

11. The method according to claim 9 or 10, wherein the at least one biomarker is L-selectin.

12. The method according to claim 9 or 10, wherein the at least one biomarker is M-CSF.

13. The method according to claim 9 or 10, wherein the at least one biomarker is galectin-7.

14. The method according to claim 9 or 10, wherein the at least one biomarker is IL-16.

15. The method according to claim 9 or 10, wherein the at least one biomarker is CXCL12.

16. The method according to claim 9 or 10, wherein the at least one biomarker is CXCL16.

17. The method according to claim 9 or 10, wherein the at least one biomarker is CCL11.

18. Use of at least one reagent for detecting the level of CCL21 in the preparation of a kit for treating thymic disorders in a subject, wherein the kit is for a method comprising: implanting into the subject a slice derived from allogeneic cultured postnatal thymic tissue, the slice being obtained by cutting a donor thymus into donor thymus slices, wherein the donor thymus slices have been aseptically cultured in thymic organ medium for a period of 6 to 21 days; wherein the culturing step results in a partially T cell-depleted donor thymic tissue slice, wherein the partially T cell-depleted donor thymic tissue slice shows regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymic tissue slice, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue slice, and the presence of intact nuclei, and sterility testing shows no microbial growth; and wherein the level of CCL21 in the thymic organ medium has been detected using the at least one reagent during the culturing step, and wherein the CCL21 level is increased compared to the baseline level of CCL21 determined on day 0 of the culturing step.

19. The use according to claim 18, further comprising: using at least one reagent for detecting the level of at least one biomarker selected from L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, 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 preparation of a kit for treating thymic disorders in a subject; wherein the level of at least one biomarker in the thymic organ medium is detected using the at least one reagent during the culturing step, and the level of the at least one biomarker is compared to the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

20. The use according to claim 19, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16 and CCL11.

21. The use according to claim 19 or 20, wherein the at least one biomarker is L-selectin.

22. The use according to claim 21, wherein the level of L-selectin in the thymic organ culture medium is decreased during the culturing step as compared to the baseline level of L-selectin determined on day 0 of the culturing step.

23. The use according to claim 19 or 20, wherein the at least one biomarker is M-CSF.

24. The use according to claim 23, wherein the level of M-CSF in the thymic organ culture medium is decreased during the culturing step as compared to the baseline level of M-CSF determined on day 0 of the culturing step.

25. The use according to claim 19 or 20, wherein the at least one biomarker is galectin-7.

26. The use according to claim 25, wherein the level of galectin-7 in the thymic organ culture medium is decreased during the culturing step as compared to the baseline level of galectin-7 determined on day 0 of the culturing step.

27. The use according to claim 19 or 20, wherein the at least one biomarker is IL-16.

28. The use according to claim 27, wherein the level of IL-16 in the thymic organ culture medium is decreased during the culturing step as compared to the baseline level of IL-16 determined on day 0 of the culturing step.

29. The use according to claim 19 or 20, wherein the at least one biomarker is CXCL12.

30. The use according to claim 29, wherein the level of CXCL12 in the thymic organ culture medium is increased during the culturing step as compared to the baseline level of CXCL12 determined on day 0 of the culturing step.

31. The use according to claim 19 or 20, wherein the at least one biomarker is CXCL16.

32. The use according to claim 31, wherein the level of CXCL16 in the thymic organ culture medium is increased during the culturing step as compared to the baseline level of CXCL16 determined on day 0 of the culturing step.

33. The use according to claim 19 or 20, wherein the at least one biomarker is CCL11.

34. The use according to claim 33, wherein the level of CCL11 in the thymic organ culture medium is increased during the culturing step as compared to the baseline level of CCL11 determined on day 0 of the culturing step.

35. The use according to claim 18, wherein the thymic disorder is congenital athymia associated with complete DiGeorge syndrome.

36. The use according to claim 18, wherein the thymic disorder is congenital athymia associated with 22q11.2 deletion.

37. The use according to claim 18, wherein the thymic disorder is congenital thymopathy associated with CHARGE (coloboma, heart defects, choanal atresia, retardation of growth or mental, genital hypoplasia, and ear anomalies or deafness).

38. The use according to claim 18, wherein the thymic disorder is congenital athymia associated with a mutation in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene.

39. The use according to claim 18, wherein the thymic disorder is congenital athymia associated with a deficiency of forkhead box protein N1 (FOXN1).

40. The use according to claim 18, wherein the thymic disorder is congenital athymia associated with a mutation in the TBX-1 or TBX-2 gene.

41. The use according to claim 18, wherein the thymic disorder is age-related thymic involution.

42. The use according to claim 18, wherein the thymic disorder is associated with thymoma.

43. The use according to claim 42, wherein the thymoma is malignant.

44. The use according to claim 42, wherein the thymoma is non-malignant.

45. The use according to claim 18, wherein the thymic disorder is associated with myasthenia gravis (MG), pure red cell aplasia, and hypogammaglobulinemia.

46. Use of at least one reagent for detecting the level of CCL21 in the preparation of a kit for providing immune competence in a human subject, wherein the kit is for a method comprising the steps of: Cutting a donor thymus into donor thymus slices; Sterilely culturing the donor thymus slices in a thymic organ culture medium for 6 to 21 days to produce partially T cell-depleted donor thymus tissue slices; Detecting keratin AE1 / AE3, cytokeratin CK14, at least one Hassall's corpuscle, and intact nuclei in the partially T cell-depleted donor thymus tissue slices during the culturing step, wherein the partially T cell-depleted donor thymus tissue slices show regions positive for keratin AE1 / AE3 scattered throughout the donor thymus tissue slices, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymus tissue slices, and the presence of intact nuclei; Testing for sterility during the culturing step, wherein testing for sterility shows no microbial growth; Detecting the level of CCL21 in the thymic organ culture medium using the at least one reagent during the culturing step and comparing the CCL21 level with a baseline level of CCL21 determined on day 0 of the culturing step, wherein the CCL21 level is increased compared to the baseline level; and Implanting the partially T cell-depleted donor thymus tissue slices into the human subject.

47. The use according to claim 46, which further comprises: At least one reagent for detecting the level of at least one biomarker selected from L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 is used for preparing a kit for providing immune competence in a human subject; Wherein the method further comprises the steps of: during the culturing step, using the at least one reagent to detect the level of at least one biomarker in the thymic organ culture medium, and comparing the level of the at least one biomarker with the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

48. The use according to claim 47, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16 and CCL11; wherein the level of one or more of L-selectin, M-CSF, galectin-7 and IL-16 is decreased compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step, or wherein the level of one or more of CXCL12, CXCL16 and CCL11 is increased compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

49. Use of at least one reagent for detecting the level of CCL21 in the preparation of a kit for providing immune competence in a human subject who has undergone solid organ transplantation, wherein the kit is for a method comprising the steps of: Removing the thymus of the human subject; Obtaining thymic tissue from a donor matched to the HLA class I and HLA class II alleles in the solid organ; Cutting the donor thymus into donor thymic slices; Sterile culturing the donor thymic slices in a thymic organ culture medium for a period of 6 to 21 days to produce a partially T cell-depleted donor thymic tissue slice, wherein the partially T cell-depleted donor thymic tissue slice shows regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymic tissue slice, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymic tissue slice, and the presence of intact nuclei, and sterility testing shows no microbial growth; During the culturing step, detecting the level of CCL21 in the thymic organ culture medium using the at least one reagent, and comparing the CCL21 level with the baseline level of CCL21 determined on day 0 of the culturing step, wherein the CCL21 level increases compared to the baseline level; Implanting the solid organ; and Implanting the partially T cell-depleted donor thymic tissue slices into the human subject.

50. The use according to claim 49, further comprising: Using at least one reagent for detecting the level of at least one biomarker selected from L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 for preparing a kit for providing immune competence in a human subject undergoing a solid organ graft; wherein the method further comprises the steps of: during the culturing step, detecting the level of at least one biomarker in the thymic organ culture medium using the at least one reagent, and comparing the level of the at least one biomarker with the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

51. The use according to claim 50, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16 and CCL11; wherein the level of one or more of L-selectin, M-CSF, galectin-7 and IL-16 is decreased compared to the baseline level of the corresponding biomarker determined on day 0 of the culturing step, or wherein the level of one or more of CXCL12, CXCL16 and CCL11 is increased compared to the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

52. Use of at least one reagent for detecting the level of CCL21 in the preparation of a kit for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a donor in a recipient in need of solid organ transplantation, wherein the kit is for a method comprising the steps of: (a) Removing the thymus of the recipient; (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ; (c) Provide suitable solid human organs and thymus from a donor; (d) Transplant the solid human organ into the recipient; (e) Treat the recipient with a maintenance immunosuppressive regimen; (f) Provide a partially T cell-depleted donor thymus tissue slice, which is obtained by cutting a donor thymus into donor thymus slices, wherein the donor thymus slices have been aseptically cultured in a thymus organ culture medium for a period of 6 to 21 days; wherein the culturing step produces a partially T cell-depleted donor thymus tissue slice, wherein the partially T cell-depleted donor thymus tissue slice shows regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymus tissue slice, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymus tissue slice, and the presence of intact nuclei, and sterility testing shows no microbial growth; and wherein the level of CCL21 in the thymus organ culture medium has been detected using the at least one reagent during the culturing step, and wherein the CCL21 level has been compared with the baseline level of CCL21 determined on day 0 of the culturing step, wherein the CCL21 level has increased compared to the baseline level; and (g) At most 21 days after the culturing step, implant the partially T cell-depleted donor thymic tissue slices into the recipient, wherein the dose of the thymic tissue slices is 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

53. The use according to claim 52, further comprising: Using at least one reagent for detecting the level of at least one biomarker selected from L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 for preparing a kit for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a donor in a recipient in need of solid organ transplantation; wherein the level of at least one biomarker in the thymus organ culture medium is detected using the at least one reagent during the culturing step, and the level of the at least one biomarker is compared with the corresponding baseline level of the biomarker determined on day 0 of the culturing step.

54. Use according to claim 53, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16 and CCL11; wherein the level of one or more of L-selectin, M-CSF, galectin-7 and IL-16 is reduced compared to the baseline level of the corresponding biomarker determined on day 0 of the culturing step, or wherein the level of one or more of CXCL12, CXCL16 or CCL11 is increased compared to the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

55. Use of at least one reagent for detecting the level of CCL21 in the preparation of a kit for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a human donor in a human recipient in need of solid organ transplantation, wherein the kit is for a method comprising the following steps: (a) Removing the thymus of the recipient; (b) Treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or inhibit the recipient's T cells from rejecting the transplanted solid organ; (c) Providing a suitable solid organ from a 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 bank of allogeneic cultured postnatal thymus tissue-derived products; wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived product is processed from thymus tissue from a thymus donor who expresses HLA alleles that match the HLA class I and HLA class II alleles in the recipient and that are not present in the solid organ graft; wherein the donor thymus is cut into donor thymus slices, and wherein the donor thymus slices have been aseptically cultured in thymus organ medium for a period of 6 to 21 days; wherein the culturing step results in partially T cell-depleted donor thymus tissue slices, wherein the partially T cell-depleted donor thymus tissue slices show regions positive for cytokeratin AE1 / AE3 scattered throughout the donor thymus tissue slices, the presence of at least one Hassall's corpuscle, CK14 staining scattered throughout the donor thymus tissue slices, and the presence of intact nuclei, and testing for sterility shows no microbial growth; and wherein the level of CCL21 in the thymus organ medium has been detected using the at least one reagent during the culturing step, and wherein the CCL21 level is increased compared to the baseline level of CCL21 determined on day 0 of the culturing step; (g) Thawing the cryopreserved allogeneic cultured postnatal thymus tissue-derived product; and (h) Implanting the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product into the recipient, wherein the dose of the cryopreserved allogeneic cultured postnatal thymic tissue-derived product is 1000 - 22000 mm 2 thymic tissue surface area / m 2 recipient body surface area, and further, wherein the implanted allogeneic cultured postnatal thymic tissue-derived product induces thymopoiesis and tolerance in the recipient.

56. Use according to claim 55, further comprising: At least one reagent for detecting the level of at least one biomarker selected from L-selectin, CXCL16, M-CSF, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1 and ANG-1 is used for preparing a kit for promoting donor-specific tolerance to an allogeneic solid organ graft obtained from a human donor in a human recipient in need of solid organ transplantation; Wherein during the culturing step, the level of at least one biomarker in the thymic organ culture medium is detected using the at least one reagent, and the level of the at least one biomarker is compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

57. The use according to claim 56, wherein the at least one biomarker is selected from L-selectin, M-CSF, galectin-7, IL-16, CXCL12, CXCL16 and CCL11; wherein the level of one or more of L-selectin, M-CSF, galectin-7 or IL-16 is decreased compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step, or wherein the level of one or more of CXCL12, CXCL16 or CCL11 is increased compared with the baseline level of the corresponding biomarker determined on day 0 of the culturing step.

58. The use according to any one of claims 55 to 57, wherein the HLA matching allows minor changes in the amino acid sequence of the allele in the second field.

59. The use according to any one of claims 55 to 57, wherein the HLA matching allows a mismatch of the HLA-DP allele in the second field.

60. The use according to any one of claims 55 to 57, wherein half of the thawed cryopreserved allogeneic cultured postnatal thymic tissue-derived product is transplanted into the recipient and the remaining part is cryopreserved for future use.

61. The use according to any one of claims 55 to 57, wherein step (h) is performed one month or more after transplantation of the solid organ.

62. The use according to claim 49 or 55, wherein the solid organ graft is a heart graft, a kidney graft, a liver graft, a lung graft, a heart / lung graft, a pancreas graft, an intestine graft, a stomach graft, an abdominal wall graft, a craniofacial graft, a scalp graft, a penile graft, a uterine graft, a unilateral or bilateral upper limb graft, a unilateral vascularized composite allograft, or a combination thereof.

63. The use according to claim 62, wherein the solid organ graft is a heart graft.

64. The use according to claim 62, wherein the heart graft is a pediatric heart graft.

65. The use according to claim 62, wherein the heart graft is an adult heart graft.

66. The method according to claim 1, wherein the culturing step is 6 days.

67. The method according to claim 1, wherein the culturing step is 7 days.

68. The method according to claim 1, wherein the culturing step is 8 days.

69. The method according to claim 1, wherein the culturing step is 9 days.

70. The method according to claim 1, wherein the culturing step is 10 days.

71. The method according to claim 1, wherein the culturing step is 11 days.

72. The method according to claim 1, wherein the culturing step is 12 days.

73. The method according to claim 1, wherein the culturing step is 13 days.

74. The method according to claim 1, wherein the culturing step is 14 days.

75. The method according to claim 1, wherein the culturing step is 15 days.

76. The method according to claim 1, wherein the culturing step is 16 days.

77. The method according to claim 1, wherein the culturing step is 17 days.

78. The method according to claim 1, wherein the culturing step is 18 days.

79. The method according to claim 1, wherein the culturing step is 19 days.

80. The method according to claim 1, wherein the culturing step is 20 days.

81. The method according to claim 1, wherein the culturing step is 21 days.

82. The use according to claim 52 or 55, wherein the donor is deceased.

83. The use according to claim 52 or 55, wherein the donor is alive.

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