Method for determining the suitability of cultured thymic tissue for transplantation into humans and related methods for the same application

Abstract

Methods and compositions for promoting donor-specific immune tolerance and immunocompetence in a solid organ transplant recipient by transplanting an allogeneic solid organ into a recipient in need of a solid organ transplant, the method and composition further comprising surgically transplanting a cultured tissue-engineered postnatal allogeneic thymic tissue product into the recipient of the donor-derived solid organ. Methods for producing cultured products derived from postnatal allogeneic thymic tissue suitable for transplantation into humans; methods for culturing products derived from postnatal allogeneic thymic tissue culture suitable for transplantation into humans; and methods for using products derived from cultured postnatal allogeneic thymic tissue by transplantation in human subjects.

Description

[Technical Field]

[0001] A biomarker useful for determining the viability and suitability of T-cell depleted cultured pediatric thymus tissue, also known as a culture product derived from postnatal allogeneic thymus tissue (or sometimes called "CTT"), for transplantation and T-cell rearrangement in subjects with thymic disorders (including congenital athymus and other immune system dysfunctions due to thymic disorders). A method and composition for promoting donor-specific immune tolerance to allogeneic solid organ transplantation in recipients receiving allogeneic solid organ grafts from donors. [Background technology]

[0002] Organ transplantation requires the preparation and harvesting of human solid organs from donors, as well as their transplantation into recipients. A major problem in solid organ transplantation is that recipients are not immune to the donor. The recipient's T cells reject the organ, and their B cells develop antibodies against it, ultimately leading to failure. The ultimate goal of solid organ transplantation is to develop immune tolerance in the recipient to the transplanted human organ. An estimated 36,000 organ transplants are performed annually in the United States, and even more in Europe and other major countries. Furthermore, it is estimated that there are over 120,000 patients on organ transplant waiting lists in the United States. The demand for healthy organs far exceeds the supply of suitable organs. In 2018, approximately 10,000 donors were found. See https: / / optn.transplant.hrsa.gov.

[0003] Graft rejection is a major issue in solid organ transplantation. Graft rejection by both T cells and B cells can cause serious complications related to organ function or even graft failure. Graft survival at five years is, for example, 77.7% for heart transplantation, 78.6% for kidney transplantation, 72.8% for liver transplantation, and 53.4% for lung transplantation. Usually, this problem is addressed, in part, by matching the major histocompatibility complex (MHC) antigens between the donor and recipient and by avoiding the recipient having antibodies against the recipient tissue type. Additionally, the use of immunosuppressive regimens to manage the immunological responses underlying graft rejection has improved. However, immune tolerance has not been achieved, and the average survival period for many organs is only 10 years.

[0004] Maintaining the viability of the organ before and during transplantation is a second major issue. The removal, preservation, and transplantation of the organ can have a significant impact on the internal structure and function of the organ and can have such a major impact that the recovery of normal organ function is delayed or inhibited after transplantation is complete.

[0005] The period for which a human solid organ can be effectively preserved varies by organ, being 24 - 36 hours for the kidney, 12 - 18 hours for the pancreas, 8 - 12 hours for the liver, and 4 - 6 hours for the heart and lungs. https: / / unos.org / transplantation / matching-organs See reference.

[0006] Organ damage occurs mainly as a result of ischemia and hypothermia but may also be associated with reperfusion of the organ ex vivo or during transplantation. <于

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

[0008] The major solid organs that are the targets of transplantation procedures include the kidney, liver, heart, and lungs. Transplantation of these solid organs has been achieved with varying degrees of success. The main variability lies in the techniques utilized to prevent immune-mediated graft rejection. Experience has shown that there is no single immunosuppressive agent or immunosuppressive technique that is useful in all situations, including solid organ transplantation. The limiting factor is usually the toxicity associated with each individual immunosuppressive agent. The toxicity associated with a particular immunosuppressive agent often inhibits the normal function of other organs, such as the kidney, which may become dysfunctional when a transplanted solid organ or a calcineurin inhibitor is used to prevent rejection.

[0009] The drawbacks of the toxicity associated with known immunosuppressive agents that are commonly used to prevent graft rejection in solid organ transplantation indicate the need to find new methods for preventing graft rejection in solid organ transplantation.

[0010] The ability to distinguish self-antigens from non-self-antigens is essential for the immune response. This discrimination leads to self-tolerance. Autoimmunity appears when self-tolerance is lost. There are still unmet needs for transplantation procedures that induce tolerance to solid organ transplants.

[0011] In subjects with thymic disorders, including congenital athymia and other immune system dysfunctions due to thymic disorders, maintenance of the viability of thymic tissue before and during transplantation procedures of products derived from postnatal allogeneic thymic tissue culture is an important factor in implementing various aspects and embodiments of the present disclosure.

[0012] The thymus is necessary for the development of T cells that can respond appropriately to foreign antigens and pathogens without compromising self-reactivity.

[0013] The thymus, large in childhood due to its need to establish the initial T cell repertoire, soon becomes homeostatic and then undergoes a slow regression process that continues throughout adulthood. Research over the past 20-30 years has established that, although overall production of the adult thymus declines, the organ remains important for producing novel, specific T cells that can protect against infection or cancer and regrow the repertoire after immune damage such as radiation, chemotherapy, and certain infections such as human immunodeficiency virus (HIV) (Gruver et al. 2007; Palmer et al. 2018; Sun et al. 2016; Wickemeyer and Sekhsaria 2014). Therefore, recent attention has focused on elucidating the mechanisms governing age-related thymic regression and on ways to promote thymic regeneration after immune damage in adults.

[0014] Age-related thymic regression in humans is characterized by the loss of thymic cell development and a decrease in the number of thymic epithelial cells, accompanied by replacement of the thymic parenchyma with adipose tissue. Determining whether the mechanisms driving these changes are endogenous or exogenous in the thymus is difficult to address using animal models due to the continuous transport into or out of the thymus, and such problems are generally impossible to address in living humans.

[0015] Organ cultures of thymic tissue derived from young donors are useful for evaluating such questions because in vitro culture of human thymic sections leads to thymocyte depletion, while generally maintaining the viability and function of thymic epithelial and stromal cells. This is demonstrated by the ability of these sections to proliferate monolayers (Markert et al. 1997b) and promote T cell rearrangement when transplanted into recipients of congenital athymia (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). The inventors hypothesized that the disappearance of thymocytes during thymic organ culture mimics acute and chronic regression, and that this could help elucidate the mechanisms governing changes in the thymic microenvironment during aging.

[0016] Products derived from cultured postnatal allogeneic thymic tissue have been shown to be useful in the treatment of T-cell immunodeficiency (primary immunodeficiency) resulting from congenital athymus, such as complete DiGeorgia syndrome (cDGA) associated with 22q11.2 deletion and CHARGE syndrome (deficiency, cardiac defects, posterior nostril atresia, developmental delay or intellectual disability, genital hypoplasia and ear abnormalities or hearing loss) associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene, as well as in patients with athymus due to forkhead box protein N1 (FOXN1) deficiency. Congenital athymus is a rare and fatal condition, and currently there are no drug treatment options using regulatory-approved formulations.

[0017] Experimental transplantation of products derived from postnatal allogeneic thymic tissue cultures retaining thymic epithelial cells (TECs) has been successfully applied to treat pediatric patients with congenital athymus (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 this reference, DiGorge syndrome is defined as a condition characterized by various defects in the heart, thymus, and parathyroid glands. Approximately 1% of infants with DiGorge syndrome have athymia, and are therefore unable to produce mature naive T cells that fight infection. These infants are described as having complete DiGorge syndrome. While not intended to be exhaustive, there are subgroups of children who meet the criteria for complete DiGorge syndrome, 22q11.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants who do not have a syndrome or genetic abnormality. Congenital athymia may also be associated with mutations in the TBX1 or TBX2 genes.

[0019] Products derived from cultured postnatal allogeneic thymic tissue are tissue engineering products prepared, cultured, and stored for up to 21 days (e.g., culture regimens of approximately 6 to 21 days) to create partially T-cell depleted thymic tissue sections, which are differentiated from native thymus by an adaptation process. The adaptation regimens partially deplete donor thymocytes from cultured thymic tissue sections. Based on in vitro data (immunohistochemistry), the 6-21 day culture period maintains an epithelial network as assessed using cytokeratin antibodies. This culture is preferably carried out in an incubator at 37°C and 5% CO2.

[0020] The culture process significantly alters the biological properties of donor thymocytes and their constituent cells by optimizing the effective therapeutic properties of product sections derived from postnatal allogeneic thymocyte tissue cultures, using the methods described below. This culture process ensures that a specific composition of cultured cells / tissues possessing the prerequisite biological characteristics is obtained in a manner suitable for surgical transplantation into the subject, enabling the reconstitution of the subject's immune system.

[0021] The culture process results in the disappearance of thymocytes and the relative enrichment of TECs and other stromal cells in donor-thym tissue sections. The culture process further enables the reconstitution of the recipient's immune system by resulting in thymocyte depletion and maintenance of TECs, allowing for the emergence of tolerance to HLA antigens in the donor-thym in the recipient. Overall, this culture process is designed to deplete most thymocytes from donor-thym tissue while maintaining the functional structure of the thymic stroma (thymic epithelial cells and fibroblasts). Common lymphoid progenitor cells, which originate from stem cells, migrate to the thymus and enter the thymus as thymic migrant progenitor cells.

[0022] This culture process is described in WO2019 / 165197A1 (Cultured Thymus Tissue Transplantation Promotes Donor-Specific Tolerance to Allogeneic Solid Organ Transplants, Markert, ML), and this reference is incorporated herein by reference in its entirety. An analysis of the suitability of product sections derived from postnatal allogeneic thymus tissue culture is described in PCT / US2019 / 040275, and this reference is incorporated herein by reference in its entirety.

[0023] Surgical administration of a product derived from cultured postnatal allogeneic thymus tissue (e.g., also known as "RVT-802") in patients with athymopathy results in a series of events that lead to the development of a functional immune system. Following the surgical introduction of the product derived from cultured postnatal allogeneic thymus tissue in the recipient, T cells are educated by donor TECs and recipient dendritic cells (DCs). The donor TECs, along with the recipient DCs, enable immune tolerance to the transplanted donor thymus tissue, which is transplanted as a cultured thymus tissue section. This is the same tolerance induction as in a normal thymus. The donor TECs, along with the recipient DCs, result in immune tolerance to the self.

[0024] Thymocyte proliferation in transplanted donor thymic tissue 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). These references are incorporated herein by reference.

[0025] Studies treating children with experimental products derived from cultured postnatal allogeneic thymus tissue have shown tolerance to the donor's major histocompatibility complex (MHC) through a mixed lymphocyte response (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008, “Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277-281). Furthermore, infants with congenital athymus can suppress infections such as Epstein-Barr virus after transplantation from cultured postnatal allogeneic thymus tissue (Markert ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiencies, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067).

[0026] Historically, the delivery criteria for culture products derived from postnatal allogeneic thymus tissue included histopathological evaluation of hematoxylin-eosin stained and immunostained sections of the tissue at an intermediate point in the manufacturing process; this was later revised to days 6–21 of the culture period. This histopathological evaluation serves as an efficacy assay and is performed as a qualitative analysis by a qualified pathologist. Samples are prepared for evaluation by either freezing or formalin fixation before the tissue is cut into thin sections and mounted on slides. Samples prepared in this manner are stable over long periods, allowing for re-analysis.

[0027] Historical samples available from a disease history spanning over 20 years can often be associated with favorable clinical outcomes, providing a robust dataset for the development of quantitative histological assays for product quality assessment.

[0028] A novel digital histological assay was developed using scanned images of hematoxylin-eosin-stained slides from multiple experimental lots of products derived from historical clinical studies and postnatal allogeneic thymic tissue cultures. These images were analyzed to develop a quantitative release assay method. This digital histological assay is described in detail in PCT / US2019 / 040275.

[0029] Cell migration in response to chemoattractant cytokines (chemokines) and other soluble molecules is an important, but not particularly innate, mechanism that controls thymic function (Hu et al. 2015). Early thymocyte progenitor cells migrate from the bone marrow to the thymus under the influence of a chemokine gradient. The chemokine gradient also influences their migration within the thymus. Early thymocyte progenitor cells and their CD4- / CD8- double-negative (DN) progeny interact with cortical thymic epithelial cells and differentiate into CD4+ / CD8+ double-positive (DP) thymocytes, which are then positively selected to differentiate into CD4+ or CD8+ single-positive thymocytes that migrate to the thymic medulla (Lancaster 2018). After autoreactive cells are eliminated by negative selection, the resulting naive mature T cells are released into the periphery.

[0030] Despite the aforementioned success in transplanting cultured products derived from allogeneic thymic tissue into children with congenital athymus after birth, there remains a need to identify viable, functional, and transplant-suitable cultured thymic tissue in order to achieve immune rearrangement. [Overview of the project]

[0031] Achieving donor-specific immune tolerance is the ultimate immunological goal in transplantation. Most current approaches focus on controlling peripheral mature donor-responsive T cells by depletion (e.g., alemtuzumab, thymoglobulin, etc.) or suppression (e.g., calcinulin inhibitors, basiliximab, etc.) rather than targeting allogeneic-responsive T cell production in the thymus. However, despite significant advances in immunosuppressants and novel immunomodulatory regimens, transplant tolerance remains unachieved.

[0032] The inventors have shown that tolerance to solid organ transplantation can be achieved in thymectomy recipients by transplanting a product derived from postnatal allogeneic thymic tissue culture (hereinafter also referred to as "CTT" or "RVT-802"), with the aim of shortening the duration of post-transplant immunosuppressant use to prevent transplanted organ rejection. Removal of the recipient's thymus and replacement with a culture product derived from postnatal allogeneic thymic tissue results in immune system rearrangement of the solid organ recipient, as well as self-tolerance and immune tolerance to the transplanted allogeneic solid organ in the recipient.

[0033] The induction of immune tolerance by surgical introduction of products derived from allogeneic thymic tissue cultures after birth is similar to tolerance induction via donor-dendritic cells ("DCs") in hematopoietic stem cell transplantation (Sharabi Y & Sachs DH, 1989, "Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen," J Exp Med 169(2):493-502; Manilay JO, Pearson DA, Sergio JJ, Swenson KG, & Sykes M, 1998, "Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen," Transplantation 66(1):96-102).A series of studies from the Transplantation Biology Research Center (TBRC), Boston, MA, have focused on immune 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 experimental thymic transplantation with tolerance induction 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 This miniature swine demonstrated the major role of the thymus in the following cases: "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 USA 103(50):19081-19086).In a series of studies, this research group successfully used HLA class II matched / class I mismatched donors (thymus and kidney or heart) as composite thymic tissue (thymokidney and thymcardiac) with 12 days of cyclosporine ("CsA") for transplant immune tolerance induction. They claimed that non-angiogenic thymus did not induce tolerance in their model. More precisely, however, uncultured non-angiogenic thymus did not engraft long-term. As they demonstrated, the poor engraftment of uncultured thymus was due to ischemic injury in addition to alloimmunity (Yamada K, et al., 2000). This sophisticated idea of ​​creating angiogenic thymus before transplantation to induce tolerance would be difficult to clinically apply without the use of xenotransplantation. (Kwun, Jean, Li, Jie, Rouse, Clay, Park, Jae Berm, Farris, Alton B., Kuchibhatla, Maragatha, Turek, Joseph W. Knechtle, Stuart J. Kirk, Allan D. and Markert, M Louise, Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants, JCI Insight. 2020 Jun 4;5(11):e129983. doi: 10.1172 / jci.insight.129983).

[0034] The limitations of non-angiogenic thymus transplantation can be overcome by culturing systems and T cell depletion. Experimental transplantation of products derived from postnatal allogeneic thymic tissue cultures that retain TEC (CTT) has been successfully applied to treat pediatric patients with congenital athymus (Markert ML, Devlin BH, McCarthy EA, 2010, "Thymus transplantation," Clin Immunol., 135(2): 236-46; Markert ML, et al., 2004, "Postnatal thymus transplantation with immunosuppression as treatment for DiGeorge syndrome," Blood 104(8):2574-2581; Markert ML, et al., 1999, "Transplantation of thymus tissue in complete DiGeorge syndrome," N Engl J Med 341(16):1180-1189 27).

[0035] The aforementioned reference defines DiGeorge syndrome as a condition characterized by various deficiencies in the heart, thymus, and parathyroid glands. Approximately 1% of infants with DiGeorge syndrome have athymia and therefore lack T cells to fight infection. These infants are described as having complete DiGeorge syndrome. There are four subgroups of children that meet the criteria for complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE, infants of diabetic mothers, and infants without syndrome or genetic abnormalities. In all four subgroups, athymic infants represent only a very small group, perhaps 1% of all diagnosed children, including those diagnosed with 22q11.2 deletion syndrome.

[0036] Thymocyte proliferation has been demonstrated by allograft biopsies and the presence of naive T cells in the peripheral recipient (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). A study of children treated with experimental CTT showed tolerance to donor MHC through a mixed lymphocyte response (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008, “Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly,” Clin Immunol 126(3):277-281). Furthermore, infants with congenital athymus can suppress infections such as Epstein-Barr virus after CTT transplantation (Markert ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067). Based on these data from humans with congenital athymus, it was determined that, after surgical introduction in recipients, products derived from postnatal allogeneic thymic tissue cultures expressing MHC from solid organ donors develop immune tolerance to both the self and the donor, while simultaneously producing functional T cells that protect the recipient from infection. Thymus Gland and Education of Thymocytes. (Kwun, Jean, Li, Jie, Rouse, Clay, Park, Jae Berm, Farris, Alton B., Kuchibhatla, Maragatha, Turek, Joseph W. Knechtle, Stuart J. Kirk, Allan D. and Markert, M Louise, Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants, JCI Insight . 2020 Jun 4;5(11):e129983. doi: 10.1172 / jci.insight.129983.

[0037] The thymus is typically located above the heart. It provides an essential microenvironment for T cell development and is crucial 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 educates hematopoietic stem cells migrating from the bone marrow. These progenitor stem cells form colonies in the thymus, thereby creating thymocytes. Thymocytes then undergo a series of maturation processes, evidenced by the expression of multiple observable cell surface markers on the thymocytes.

[0038] T cells are crucial for protecting the body from infection. Developing in a properly functioning thymus, T cells produce a diverse set of T cell receptors (generally cell surface proteins), which allows mature T cells to fight various infections. During this education process, developing T cells are instructed by the thymus not to attack the body's normal proteins, such as insulin or parathyroid hormone (which regulates blood glucose and calcium levels). These instructions are influenced by autoimmune regulatory genes ("AIRE genes").

[0039] Simply put, this education process takes place in a properly functioning thymus. Thymocytes reside in the thymus and are formed from bone marrow stem cells. Thymocytes are educated by thymic epithelial cells ("TECs") and dendritic cells ("DCs") localized in the thymus to prevent them from attacking recipient major histocompatibility complex (MHC) proteins (antigens) (such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1 antigens). HLA antigens have two proteins that hold self-peptides in a groove. These self-peptides can originate from thyroid proteins, insulin peptides, or most other proteins expressed in the body. Thymocytes developing in the thymus form a T cell receptor (TCR) composed of two proteins that cross the membrane. The TCR is expressed on the cell surface of T cells. Each T cell multi-copies its own TCR. If TCRs bind too strongly to their own peptides, i.e., MHC on dendritic cells, the dendritic cells signal the T cells to undergo apoptosis and die. This mechanism prevents the development of autoimmunity against one's own body. TECs can also signal to thymocytes that they are binding too strongly. Finally, DCs can bind to a portion of the membrane derived from TECs and present TEC's own peptides, i.e., MHC, to developing thymocytes. If the thymocytes bind too strongly, the DCs signal the thymocytes to undergo apoptosis and die. These mechanisms ensure that T cells leaving the thymus are not self-reactive. T cells leaving the thymus are highly diverse and capable of recognizing infection, but they do not attack the body's own proteins.

[0040] The two main components of the thymus are epithelium and thymocytes. Common lymphoid progenitor cells ("CLPs"), which are cells derived from bone marrow stem cells and produced in the thymus, migrate to the thymus as early thymic progenitor cells. CLPs enter the thymus in response to signals (chemokines) produced by the thymic epithelium and endothelium. In the thymus, CLPs differentiate and proliferate into thymocytes. Thymocytes develop intrinsic T cell receptors ("TCRs") expressed on their cell surface. Thymocytes also begin to express T cell molecules CD3, CD4, and CD8. A wide variety of T cells are developed, enabling T cells to respond to infection throughout the recipient's lifetime. The mixed lymphocyte response shows recipient T cell tolerance to thymic donor-MHC in children treated with cultured thymic tissue (RVT-802).

[0041] Autoreactive recipient thymocytes are removed before they leave the thymus. This occurs through the interaction between recipient thymocytes and recipient DCs migrating to the thymus. Apoptosis is induced in recipient thymocytes that are too strongly bound to DCs as a means of protecting the body from autoimmune disease. After the completion of this process, the thymocytes leave the thymus. The new circulating T cells, i.e., the new thymic excipients, express the markers CD31, CD45RA, and CD62L. After several weeks, the marker CD31 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 protect the recipient from infection without exhibiting autoreactivity towards their own cells.

[0042] Products derived from cultured postnatal allogeneic thymus tissue Products derived from cultured postnatal allogeneic thymic tissue have been shown to be useful in treating T-cell immunodeficiency (primary immunodeficiency) resulting from congenital athymus. T-cell immunodeficiency resulting from athymus is associated with congenital disorders that inhibit the development of the functional thymus (such as complete DiGeorgia anomaly (cDGA) associated with 22q11.2 deletion, CHARGE syndrome associated with mutations in the chd7 (chromodomain-helicase-DNA-binding protein 7) gene (deficiency, cardiac defects, posterior nostril atresia, developmental delay or intellectual disability, genital hypoplasia and ear abnormalities or hearing loss), and forkhead box protein N1 (FOXN1) deficiency). Other genetic defects that can cause athymia include TBX1, TBX2, PAX1, and semaphorin 3E (SEMA3E) (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 currently there are no drug treatment options using approved formulations. If left untreated and no therapeutic rearrangement occurs in the child's immune system, the primary immunodeficiency resulting from congenital athymia is fatal, with almost all infants dying before the age of three, mostly from severe infections.

[0043] Products derived from cultured postnatal allogeneic thymus tissue are tissue engineering products. Based on the disclosures herein and in the examples, CTT is expected to be useful in the development of immune tolerance in recipients of transplanted solid organs.

[0044] As will be described in more detail herein and in the examples, surgical administration of a product derived from cultured postnatal allogeneic thymic tissue (e.g., "RVT-802") to a patient with athymopathy results in a series of events that lead to the development of a functional immune system. Following the surgical introduction of the product derived from cultured postnatal allogeneic thymic tissue (e.g., RVT-802) in the recipient, T cells are educated by the donor's TECs and recipient's DCs. The donor's TECs, together with the recipient's DCs, enable immune tolerance to the transplanted donor-thymic tissue, which is transplanted as a cultured thymic tissue section. This is the same tolerance induction as in the normal thymus. The recipient's TECs, together with the recipient's DCs, result in self-tolerance as described herein.

[0045] This complex process, coupled with the recipient's ability to fight infection, has been clinically demonstrated to result in over 70% survival rates in patients with congenital athymus who receive cultured products derived from allogeneic thymic tissue (e.g., RVT-802) postnatally (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 can suppress viral infections, such as the Epstein-Barr virus, which would otherwise be fatal before CTT (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008).

[0046] Overview of immune tolerance induction in solid organ transplantation combined with CTT transplantation Through the description, drawings, examples, and claims herein, the inventors have demonstrated that CTT induces donor-specific tolerance in a rat heart transplant model. The experiments reported herein utilized CTT transplantation methods equivalent to those clinically used in subjects with congenital athymus, such as subjects with cDGA. Infants with cDGA essentially lack naive T cells prior to surgical introduction of CTT. After surgical introduction of CTT, 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, & McCarthy EA, 2010, “Thymus transplantation,” Clin Immunol 135(2):236-246). The study of tolerance induction in a rat model was based on the results of transplantation of cultured postnatal allogeneic thymic tissue product (CTT) in athymic infants with complete DiGeorgia anomaly from 1993 to 2017. Favorable results were obtained in the reported study of surgical induction of CTT in infants with congenital athymus. Published results showed a 71% overall survival rate (61 / 86) in this inherently fatal condition (essentially all deaths occurred in the first 9–12 months; median 11.7 years at this point in time, range 1.2–25 years) (Markert, ML, et al., 2010). Biopsies of transplanted cultured thymic tissue demonstrated thymocyte proliferation based on immunohistochemistry (Markert, ML, et al., 2008). Flow cytometry and spectratyping showed the development of a diverse T cell repertoire. Mixed lymphocyte reactions indicate immune tolerance of recipient T cells to thymic donor antigen-presenting cells (Chinn IK, Devlin BH, Li YJ, & Markert ML, 2008).

[0047] Importantly, CTT recipients can suppress viral infections, such as Epstein-Barr virus, which would otherwise be fatal (Markert, ML, 2014, Thymus Transplantation. Stiehm's Immune Deficiences, eds Sullivan KE & Stiehm ER (Academic Press), 1st Ed, pp 1059-1067). Based on this human data showing tolerance to incompatible thymic MHC antigens, CTT transplantation in rat models was evaluated using the same clinically employed methods to assess its ability to induce donor-specific tolerance in rat cardiac transplant models. These studies demonstrated that transplanting an incompatible heart with a donor-CTT expressing MHC class I and class II antigens from the cardiac donor (with initial T-cell depletion by anti-CD5 and immunosuppression with cyclosporine) induced immune tolerance to donor-cardiac antigens while preserving alloreactivity to other MHC antigens.

[0048] This invention embodies co-transplantation of a donor thymus with a solid organ as a method for inducing tolerance of transplanted solid organs in recipients. The patient group that will benefit most from this method is adults and infants with heart failure who require heart transplantation. Since thymic tissue exists after birth and can be retrieved from deceased infants, and recipient thymuses are routinely retrieved from infants receiving heart transplants, no additional procedures other than cultured thymic tissue transplantation (CTT) are required for the clinical application of this approach. Similar transplants are also performed in adults.

[0049] Overview of the preparation of products derived from cultured postnatal allogeneic thymus tissue As will be described in more detail herein, products derived from cultured postnatal allogeneic thymic tissue are prepared, cultured, and stored for up to 21 days (e.g., a culture regimen of approximately 6 to 21 days), and on the day of transplantation, are placed in individual sterile cups for transport to the operating room.

[0050] Cultured thymus tissue (CTT) is cultured under current Good Manufacturing Practices ("cGMP"), such as those established by the U.S. Food and Drug Administration ("FDA"), to create sections of partially T-cell depleted thymus tissue. The CTT is differentiated from native thymus through the adaptation process detailed below. The CTT influences the normal positive and negative selection processes that generate T cells in the post-transplant thymus, allowing the T cells to become immune to both donor-thymus and donor solid organ grafts, as well as recipient tissues. In addition, these T cells can recognize foreign antigens in association with the recipient's major histocompatibility complex (MHC) proteins to fight infection.

[0051] The route of administration is by surgical transplantation of CTT in the manner described below. A single dose is usually administered via m 2 The unit is 1,000 to 22,000 mm² per recipient body surface area ("BSA"). 2 This is the CTT surface area. The surface area is the sum of the total surface areas of all cultured tissue sections. Each CTT section is transplanted in a single surgical procedure.

[0052] Surgical transplantation of cultured products derived from allogeneic thymic tissue after birth in patients with athymia leads to a series of events that result in the development of a functional immune system. (Markert ML, 2007; Markert ML, et al., 2010; Markert ML, Devlin BH, McCarthy EA. Chapter 84 Thymic reconstitution. 2013. In: Fleisher TA, Shearer WT, Schroeder HW, Frew AJ, Weyand CM, editors. Clinical Immunology (Fourth Edition). London; pp. 1032-8).

[0053] Recipient CLP cells from the bone marrow migrate to the allogeneic thymic graft, invade as early thymic progenitor cells, and develop into recipient T cells. The donor thymic graft provides a microenvironment where recipient thymocytes develop a broad TCR repertoire that can recognize pathogens.

[0054] The migration of recipient DCs to the donor thymus depletes autoreactive recipient thymocytes that attack recipient tissues after new T cells leave the thymus and enter circulation. Naive T cells, which are genetically from the recipient, can be readily detected in circulation approximately 5–12 months after administration. These recipient T cells have a diverse TCR repertoire and normally proliferate in response to mitogens. They protect the recipient from infection without exhibiting autoreactivity against their own tissues.

[0055] Recipient bone marrow CLPs migrate to the allogeneic thymic graft, where they develop into recipient T cells. Negative selection by recipient DCs that have migrated to the donor thymus results in tolerance to recipient MHC antigens. Immunohistochemical evidence of thymic cell proliferation is observed in biopsies of transplanted cultured thymic tissue taken approximately 2-3 months after transplantation. Thymic cell proliferation reflects the ability of T cells to defend against and suppress infection, and to prevent autoimmune diseases.

[0056] Naive T cells are detected in circulation 5–12 months after transplantation and possess the ability to defend against and control infections and prevent autoimmune diseases.

[0057] Transplantation of cultured thymic tissue has been shown for the first time to be beneficial in treating primary immunodeficiency resulting from congenital athymus associated with conditions such as complete DiGeorgia anomaly (cDGA) or forkhead box protein N1 (FOXN1) deficiency. Replacing defective thymic tissue with normal thymic tissue after culture (e.g., CTT and RVT-802) has also been found to prevent a lack of immune tolerance in recipients of transplanted solid organs.

[0058] Nonclinical and clinical studies underlying the treatment of congenital athymia with the introduction of cultured thymic tissue have led to the recognition that the introduction of CTT (e.g., RVT-802) in patients may develop immune tolerance to transplanted solid organs. Specifically, the introduction of CTT may reconfigure the immune system and induce tolerance to the donor organ if the subject is immunosuppressed by thymectomy before transplantation of a CTT expressing the donor organ's MHC.

[0059] Measuring the expression and distribution of specific markers related to thymic cellular components establishes the phenotype after ex vivo culture of thymic tissue. The culture conditions described herein and in the examples support the observation of in vivo thymic cell proliferation after CTT induction in subjects with athymia.

[0060] Importantly, the development of naive T cells and the presence of diverse TCR variable regions after surgical induction of CTT in athymic patients provide clear evidence that culturing thymic tissue can promote the development of a functional endogenous T cell population. Furthermore, expression of major regulatory and structural genes was observed in cultured thymic tissue. Circulating naive (CD45RA+CD62L+) T cells may be detected for the first time 3–5 months after surgical induction of CTT. These findings have been observed in the treatment of patients with complete DiGeorgia malformation (Markert ML, 2010; Markert ML. 2013).

[0061] Nonclinical data described in the literature on thymic tissue transplantation are consistent with the robust clinical efficacy of transplanting cultured postnatal allogeneic 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. Jan; 82(1):2 6-36; Hong R, Schulte-Wissermann H, Jarrett-Toth E, Horowitz SD, Manning DD, 1979, ``Transplantation of cultured thymic fragments. II. Results in nude mice,” J Exp Med., 149(2): 398-415. Li B, Li J, Hsieh CS, Hale LP, Li YJ, Devlin BH, Markert ML, 2009, “Characterization of cultured thymus tissue used for transplantation with emphasis on promiscuous expression of thyroid tissue-specific genes,” Immunol Res. 2009; 44 (1-3):71-83; Li B, Li J, Devlin BH, Markert ML, 2011, “Thymic microenvironment reconstitution after postnatal human thymus transplantation,” Clin Immunol., Sep; 140(3): 244-59).

[0062] Patients with complete DiGeorgia anomaly have defects in three types of glands that develop in the early embryo: the cervix, heart, thymus, and parathyroid glands. Normally, the heart and thymus descend to the chest, while the parathyroid glands, which regulate calcium levels, remain in the cervix. Treatment with CTT in patients with cDGA resulted in a 75% survival rate at age 2 compared to a 6% survival rate in patients treated by other means (unpublished data). As mentioned above, most deaths occur in the first year before naive T cell development (Markert, et al., 2010). Notably, CTT transplantation does not affect cardiac and parathyroid disorders, which must be managed separately.

[0063] One aspect of this disclosure provides a method for the surgical introduction of a cultured product derived from postnatal allogeneic thymic tissue in an immunologically normal recipient to induce tolerance to solid organ transplantation in the recipient. Such a method comprises, or essentially comprises, the removal of the thymus in an immunocompetent recipient, followed by depletion of the recipient's T cells with an inducible immunosuppression regimen comprising one or more immunosuppressants (such as one or more antibodies and / or one or more calcineurin inhibitors). The inducible immunosuppression regimen is administered in a therapeutically effective dose to deplete mature T cells in the subject and / or to suppress the recipient's T cells from rejecting the transplanted solid organ. A suitable human solid organ and thymus are obtained from a deceased donor, and the solid organ is transplanted into the recipient. A maintenance immunosuppression regimen is administered for a period of time to suppress transplant rejection. Thymus tissue obtained from a deceased donor is subjected to an acclimatization regimen of up to 21 days (e.g., approximately 6 to 21 days) and the donor-thymus tissue is aseptically treated in thymic organ medium to partially deplete T cells, thereby creating donor-thymus tissue sections containing products derived from postnatal allogeneic thymic tissue cultures. Partially T-cell depleted donor-thymic tissue sections show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall's body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei. The products derived from postnatal allogeneic thymic tissue cultures are then surgically implanted in the recipient, usually in the quadriceps femoris muscle. The products derived from postnatal allogeneic thymic tissue cultures allow the recipient to develop naive T cells after transplantation. All newly developed T cells are genetically recipient-derived and tolerant to both the recipient and the donor. The dosage of thymic tissue sections is approximately 1,000 to 22,000 mm³. 2 Thymus tissue surface area / Recipient body surface area (m²) 2 (Units) Products derived from allogeneic thymic tissue cultures after transplantation and after birth induce thymocyte proliferation and tolerance in the subjects.

[0064] Taking heart transplantation as an example, the donor is a deceased donor. The thymus is removed from the donor at the same time as the heart. The heart transplant is performed immediately with induced immunosuppression to reduce the number of T cells and suppress the remaining recipient T cells, preventing them from attacking the donor heart. The donor thymus is processed to create a product derived from postnatal allogeneic thymic tissue culture that can be used for transplantation, and tolerance is induced after an acclimatization period of at least 6 to 21 days. As a precaution, approximately half of the product derived from postnatal allogeneic thymic tissue culture may be cryopreserved after acclimatization, so that if there is a problem of heart rejection after transplantation and high doses of steroids or other immunosuppressants are required to treat the rejection, and very high doses of steroids damage the product derived from postnatal allogeneic thymic tissue culture, the cryopreserved culture product derived from postnatal allogeneic thymic tissue can be used for transplantation after the occurrence of rejection has been controlled.

[0065] Importantly, after transplantation of products derived from postnatal allogeneic thymic tissue cultures, immune tolerance is maintained even in the presence of infection. Viral infection can lead to loss of tolerance through other approaches such as co-stimulatory inhibition, because approximately one-third of CD8 T cells are alloreactive. When the immune system is activated to fight infection, alloreactive CD8 T cells begin to reject the solid organ graft. In contrast, when thymic tissue treated with products derived from postnatal allogeneic thymic tissue cultures is used to induce tolerance, potentially alloreactive T cells to the donor are eliminated by a process of negative selection in the thymus.

[0066] In one embodiment, donor thymic tissue is matched to HLA alleles in the donor organ that are not present in the recipient. All new T cells that develop are genetically recipient and tolerant to both the recipient and the donor.

[0067] In another aspect of this disclosure, a method is provided for promoting donor-specific tolerance to allogeneic solid organ grafts obtained from a deceased donor in recipients requiring solid organ transplantation, the method being: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process to provide both suitable human solid organs and thymus derived from a deceased donor; (d) The process of transplanting a human solid organ into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a product derived from a postnatal allogeneic thymic tissue culture, wherein the product derived from the postnatal allogeneic thymic tissue culture is obtained from suitable thymic tissue of a solid organ donor; the donor-thymic tissue is subjected to an acclimatization regimen of up to 21 days (e.g., an acclimatization regimen of about 6 to about 21 days) to produce a culture product derived from the postnatal allogeneic thymic tissue; further, the acclimatization regimen for the donor-thymic tissue comprises aseptically treating the donor-thymic tissue in thymic organ medium to produce a partially T-cell depleted donor-thymic tissue section; the partially T-cell depleted donor-thymic tissue section shows areas positive for cytokeratin (CK) (using antibodies 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; and (g) A step in which, after an acclimatization regimen of approximately 6 to 21 days, the recipient is implanted with a product derived from postnatal allogeneic thymic tissue culture, wherein the dose of thymic tissue section is approximately 1,000 to 22,000 mm 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The unit is, and furthermore, the product derived from the transplanted postnatal allogeneic thymic tissue culture induces thymocyte proliferation and tolerance in the recipient, process, Includes.

[0068] In one embodiment, a method is provided to promote donor-specific tolerance to allogeneic heart grafts in recipients who require the heart of a deceased donor. The method is as follows: (a) The process of obtaining a suitable human heart from a deceased donor for transplantation; (b) A step of removing the thymus of a donor that died at the same time as obtaining the heart in order to adapt it to a product derived from an allogeneic thymic tissue culture after birth; a step of adapting the donor thymus to the HLA alleles in the donor transplant organ; (c) A step of treating a recipient with an induction immunosuppression regime comprising one or more immunosuppressants to deplete and / or suppress the recipient's T cells, wherein the one or more immunosuppressants comprises glucocorticoids administered during anesthesia induction and after reperfusion; (d) The process of transplanting the heart into the recipient; (e) The step of treating the recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressants selected from the group consisting of calcinurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulins for a period sufficient to prevent or suppress cardiac transplant rejection; (f) A step of providing a product derived from a postnatal allogeneic thymic tissue culture between days 6 and 21, wherein the product derived from the postnatal allogeneic thymic tissue culture is obtained from donor-thymic tissue, the donor-thymic tissue is subjected to an acclimatization regimen of approximately 6 to approximately 21 days to produce a product derived from cultured postnatal allogeneic thymic tissue; further, the acclimatization regimen for donor-thymic tissue includes aseptic treatment of the donor-thymic tissue in thymic organ medium to produce a partially T-cell depleted donor-thymic tissue section; the partially T-cell depleted donor-thymic tissue section shows regions positive for keratin 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; (g) A step of transplanting a portion of the product derived from allogeneic thymic tissue culture into a recipient after birth, wherein the dose of thymic tissue section is approximately 1,000 to 22,000 mm 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of production, and furthermore, a product derived from transplanted postnatal allogeneic thymic tissue culture induces thymocyte proliferation and immune tolerance in the recipient; and (h) If early rejection requiring high doses of steroids occurs, which may damage the portion of the product derived from postnatal allogeneic thymus tissue culture transplanted in step (g), the step of cryopreserving a portion of the culture product derived from postnatal allogeneic thymus tissue to be used in the recipient, Includes.

[0069] In one embodiment, a method is provided to promote donor-specific tolerance to allogeneic heart grafts in recipients who require the heart of a deceased donor. The method is as follows: (a) the process of obtaining a suitable human solid heart from a deceased donor for transplantation; (b) A step of removing the thymus of a donor that died at the same time as obtaining the heart in order to adapt it to a product derived from an allogeneic thymic tissue culture after birth; a step of adapting the donor thymus to the HLA alleles in the donor transplant organ; (c) A step of treating a recipient with an induction immunosuppression regime comprising one or more immunosuppressants to deplete and / or suppress the recipient's T cells, wherein the one or more immunosuppressants comprises glucocorticoids administered during anesthesia induction and after reperfusion; (d) The process of surgically removing the recipient's heart and thymus; (e) The process of transplanting the donor's human heart into the recipient; (f) A step of treating a recipient with a maintenance immunosuppressive regimen comprising one or more immunosuppressive agents selected from the group consisting of calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, and antithymocyte globulin for a sufficient period to prevent or suppress heart transplant rejection, where, if the recipient's postoperative condition is too unstable to discontinue glucocorticoids and products derived from postnatal allogeneic thymic tissue culture cannot be safely transplanted into the recipient, the products derived from postnatal allogeneic thymic tissue culture are cryopreserved and transplanted at a later time when the recipient is stable, and the donor thymic tissue is subjected to an acclimation regimen for about 6 to about 21 days to create products derived from cultured postnatal allogeneic thymic tissue; further, the acclimation regimen for the donor thymic tissue includes aseptically processing the donor thymic tissue in a thymic organ medium to create a partially T-cell depleted donor thymic tissue section; the step where the partially T-cell depleted donor thymic tissue section shows regions 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; (h) A step of transplanting a portion of the product derived from postnatal allogeneic thymic tissue culture into the recipient after the patient has stabilized, where the dosage of the thymic tissue section is about 1,000 - 22,000 mm 2 of thymic tissue surface area / recipient body surface area (m 2 units), and further the transplanted product derived from postnatal allogeneic thymic tissue culture induces thymocyte proliferation and immune tolerance in the recipient; and (i) A step of cryopreserving a portion of the product derived from allogeneic thymic tissue culture utilized in the recipient if there is an appearance of rejection that requires high-dose steroids that can cause damage to the portion of the product derived from postnatal allogeneic thymic tissue culture transplanted in step (h), comprising.

[0070] In another aspect of this disclosure, a method is provided to promote donor-specific tolerance to allogeneic solid organ grafts obtained from living human donors in human recipients requiring solid organ transplantation, the method being: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process for providing suitable solid organs derived from a living human donor; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a cryopreserved postnatal allogeneic thymic tissue-derived culture product maintained in a cryopreserved postnatal allogeneic thymic tissue-derived culture product bank, wherein the cryopreserved postnatal allogeneic thymic tissue-derived culture product is processed from thymic donor-derived thymic tissue expressing an HLA allele that matches the recipient's HLA allele not present in the solid organ graft; the donor-thymic tissue is subjected to a habituation regimen of approximately 6 to 21 days; and the habituation regimen for the donor-thymic tissue further comprises aseptically processing the donor-thymic tissue in thymic organ medium to prepare a section of partially T cell-depleted thymic tissue, wherein the thymic tissue section, upon completion of the habituation regimen, shows regions positive for scattered keratin AE1 / AE3 throughout the tissue, the presence of at least one Hassall body, scattered CK14 staining throughout the tissue, and the presence of intact nuclei; (g) Thawing products derived from cryopreserved cultured postnatal allogeneic thymus tissue; and (h) A step of transplanting a thawed product derived from cryopreserved cultured postnatal allogeneic thymus tissue into a recipient, wherein the dose of the product derived from cryopreserved cultured postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2The process involves a unit of production, in which the product derived from the transplanted postnatal allogeneic thymic tissue culture induces thymocyte proliferation and tolerance in the recipient. Includes.

[0071] A fourth aspect of this disclosure provides a method for promoting donor-specific tolerance to allogeneic solid organ grafts obtained from deceased human donors in human recipients requiring solid organ transplantation, the method being: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process of providing suitable solid organs derived from living human donors; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a culture product derived from cryopreserved postnatal allogeneic thymus tissue, maintained in a cryopreserved postnatal allogeneic thymus tissue culture product bank, wherein the culture product derived from cryopreserved postnatal allogeneic thymus tissue is processed from thymus tissue derived from a thymus donor expressing an HLA allele that matches the recipient's HLA allele not present in the solid organ graft; the donor-thymus tissue is subjected to a habituation regimen of approximately 6 to 21 days; and the habituation regimen for the donor-thymus tissue further comprises aseptically processing the donor-thymus tissue in thymus organ medium to produce a section of partially T-cell depleted thymus tissue, wherein the thymus tissue section, upon completion of the habituation regimen, shows regions positive for scattered keratin AE1 / AE3 throughout the tissue, the presence of at least one Hassall body, scattered CK14 staining throughout the tissue, and the presence of intact nuclei; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue; and (h) A step of transplanting a culture product derived from thawed, cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the product derived from the cryopreserved cultured postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a culture product derived from transplanted postnatal allogeneic thymic tissue inducing thymocyte proliferation and tolerance in the recipient. Includes.

[0072] In one embodiment of the various aspects described above, a culture product derived from postnatal allogeneic thymic tissue, wherein the thymus is positive for keratin in a lace-like staining pattern in more than 50% of its region on the day of collection, hassall bodies are present, CK14 stains in a lace-like pattern, and more than 90% of the nuclei are intact.

[0073] In one aspect of this disclosure, a culture product derived from postnatal allogeneic thymus tissue for transplantation into a recipient of a solid organ graft is provided, which is prepared by obtaining suitable thymus tissue from a donor, wherein the donor-thymus tissue is subjected to an acclimatization regimen of up to 21 days (e.g., an acclimatization regimen of approximately 6 to 21 days); further comprising aseptically treating the donor-thymus tissue in thymic organ medium to prepare partially T cell depleted donor-thymus tissue sections; the donor-thymus tissue sections, on days 5 to 9 after recovery, show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei; the partially T cell depleted donor-thymus tissue sections are recovered as a product derived from postnatal allogeneic thymus tissue culture.

[0074] In one embodiment of the various aspects described above, the thymus shows, on the day of retrieval from the donor, that more than 50% of the area is positive for keratin with a lace-like staining pattern, that Hassall bodies are present, that CK14 stains with a lace-like pattern, and that more than 90% of the nuclei are intact.

[0075] In one embodiment of the various aspects described above, the product derived from postnatal allogeneic thymic tissue culture is cryopreserved.

[0076] In one embodiment, culture products derived from cryopreserved postnatal allogeneic thymus tissue are maintained in liquid nitrogen for future use.

[0077] In another embodiment, culture products derived from cryopreserved postnatal allogeneic thymus tissue are maintained in a cryopreserved tissue bank.

[0078] In one embodiment, a culture product derived from postnatal allogeneic thymic tissue is prepared from suitable thymic tissue derived from a donor containing HLA alleles that match the recipient candidate's HLA alleles, which are not present in solid organ transplants.

[0079] 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.

[0080] In one aspect of this disclosure, (a) The process of obtaining suitable thymic tissue from a donor; (b) HLA alleles: The process of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1; (c) A step of subjecting thymic tissue to an acclimatization regimen of approximately 6 to 21 days, wherein the acclimatization regimen for donor-thymic tissue comprises aseptically treating donor-thymic tissue in thymic organ medium to produce donor-thymic tissue sections that are partially T cell depleted; further, the donor-thymic tissue sections exhibit, on days 6 to 21, regions positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, and the presence of CK14 staining and intact nuclei scattered throughout the tissue at the completion of the acclimatization regimen; (d) A step of collecting donor thymus tissue sections, which are partially T-cell depleted, as culture products derived from allogeneic thymus tissue after birth; (e) A step of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (f) A process of maintaining cryopreserved postnatal allogeneic thymic tissue culture products in liquid nitrogen in a bank of cryopreserved postnatal allogeneic thymic tissue culture products, The present invention provides culture products derived from cryopreserved postnatal allogeneic thymus tissue, prepared by a method comprising [a specific method].

[0081] In one embodiment, the thymus shows that on the day of retrieval from the donor, more than 50% of the area is positive for keratin with a lace-like staining pattern, Hassall bodies are present, CK14 stains with a lace-like pattern, and more than 90% of the nuclei are intact.

[0082] In one embodiment, a product derived from cryopreserved cultured postnatal allogeneic thymus tissue is stored in liquid nitrogen for future use by the recipient.

[0083] In one aspect of this disclosure, a method for preparing a donor thymus for transplantation into a recipient is provided. Such a method, as further described herein, comprises or essentially comprises 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 introducing the cultured thymic tissue into the recipient. A culture period of about 6 to about 21 days yields good function. To ensure successful transplantation of cryopreserved thymic tissue, the tissue is typically cultured for about 6 to about 21 days and then cryopreserved.

[0084] In one aspect of this disclosure, a culture product (CTT;RVT-802) derived from postnatal allogeneic thymus tissue is provided for transplantation into a recipient subject of solid organ grafts prepared by subjecting thymus tissue derived from a suitable donor to an acclimatization regimen of up to 21 days (e.g., approximately 6 to approximately 21 days), the acclimatization regimen for products derived from postnatal allogeneic thymus tissue culture comprising aseptically treating donor-thymus tissue in thymic organ medium to prepare partially T-cell depleted thymus tissue sections, the thymus tissue sections showing regions positive for keratin 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.

[0085] In one embodiment, the thymus shows that on the day of retrieval from the donor, more than 50% of the area is positive for keratin with a lace-like staining pattern, Hassall bodies are present, CK14 stains with a lace-like pattern, and more than 90% of the nuclei are intact.

[0086] In one embodiment of the various aspects and embodiments described above, the recipient thymus is obtained by surgical procedure.

[0087] In one embodiment of the multiple aspects and embodiments described above, the recipient thymus is obtained by robotic surgery.

[0088] In one embodiment of the various aspects and embodiments described above, the recipient thymus is obtained by thoracoscopic surgery.

[0089] In one embodiment of the various aspects and embodiments described above, the solid organ is part of the whole organ.

[0090] In one embodiment of the multiple aspects and embodiments described above, the methods of the first to fourth aspects further include the step of cryopreserving peripheral blood mononuclear cells derived from a deceased donor for future use in mixed lymphocyte reactions to demonstrate cellular immune tolerance.

[0091] In one embodiment of the various aspects and embodiments described herein, a mixed lymphocyte response to demonstrate cellular immune tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor, after transplantation of a postnatal allogeneic thymic tissue culture-derived product in accordance with the CTT transplantation procedure described herein.

[0092] In one embodiment of the various aspects and embodiments described above, a mixed lymphocyte response to demonstrate cellular immune tolerance is performed approximately 6 to 12 months after transplantation of allogeneic thymic tissue culture-derived products postnatally, using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor.

[0093] In one embodiment of the multiple aspects and embodiments described above, a mixed lymphocyte response to demonstrate cellular immune tolerance is performed using peripheral blood mononuclear cells from the recipient and cryopreserved peripheral blood mononuclear cells from the donor, after naive T cells constitute approximately 10% of the total T cells in the recipient.

[0094] In one embodiment of the multiple aspects and embodiments described above, the transplanted postnatal allogeneic thymic tissue culture product induces thymocyte proliferation in the subject within 12 months after transplantation of the postnatal allogeneic thymic tissue culture product.

[0095] In one embodiment of the multiple aspects and embodiments described above, the development of immune tolerance is determined by a mixed lymphocyte response performed using cryopreserved deceased donor-derived peripheral blood mononuclear cells and recipient-derived T cells.

[0096] In one embodiment of the various aspects and embodiments described above, humoral tolerance is determined by the development of humoral immunity and the absence of donor-reactive antibodies.

[0097] In one embodiment of the various aspects and embodiments described above, solid organ transplants include heart transplants, kidney transplants, liver transplants, lung transplants, heart / lung transplants, pancreas transplants, intestine transplants, stomach transplants, abdominal wall transplants, craniofacial transplants, scalp transplants, penile transplants, uterine transplants, unilateral or bilateral upper limb transplants, unilateral angiogenic composite allogeneic transplants, or combinations thereof.

[0098] In one embodiment of the various aspects and embodiments described above, the method further includes evaluating the recipient with respect to HLA class I and HLA class II panel-reactive antibody ("PRA") scores before transplanting a solid organ.

[0099] In one embodiment of the various aspects and embodiments described above, the solid organ graft is a heart graft.

[0100] In one embodiment of the various aspects and embodiments described above, the solid organ graft is a pediatric heart graft.

[0101] In one embodiment of the various aspects and embodiments described above, the solid organ graft is an adult heart graft.

[0102] In one embodiment of the various aspects and embodiments described above, the method further includes evaluating the recipient with respect to HLA class I and HLA class II panel-reactive antibody ("PRA") scores before transplanting a solid organ.

[0103] In one embodiment of the multiple aspects and embodiments described above, a recipient possessing HLA antibodies performs a crossmatch test against a potential donor.

[0104] In one embodiment of the multiple aspects and embodiments described above, recipients having HLA antibodies are virtually cross-matched in UNET.

[0105] In one embodiment of the various aspects and embodiments described above, if a PRA score of more than 20% virtual crossmatch is recorded, the method further includes the step of performing plasmapheresis in the operating room at the time of solid organ transplantation in the recipient.

[0106] In one embodiment of the multiple aspects and embodiments described above, if a PRA score of more than 70% virtual cross-matching is recorded, the method further includes the steps of performing cross-matching of the actual candidate donor and plasma exchange in the operating room at the time of solid organ transplantation in the recipient. Normally, transplantation is not performed under such circumstances due to the poor success rate.

[0107] One embodiment of the aforementioned aspects and embodiments, and the method, further includes the step of evaluating recipients having HLA antibodies by virtual cross-matching in UNET.

[0108] In one embodiment of the multiple aspects and embodiments described above, the method further includes performing plasma exchange in the operating room at the time of solid organ transplantation in the recipient if the HLA panel-reactive antibody has a score greater than 20%.

[0109] In one embodiment of the multiple aspects and embodiments described above, the method further includes performing cross-matching of actual candidate donors and plasma exchange in the operating room at the time of solid organ transplantation in the recipient, if the HLA panel-reactive antibody has a score greater than 70%.

[0110] In one embodiment of the various aspects and embodiments described above, the solid organ is an HLA-matched transplant to the recipient, such as a kidney, partial liver, and partial intestine from a living relative donor.

[0111] In one embodiment of the various aspects and embodiments described above, the solid organ is HLA-incompatible.

[0112] In one embodiment of the multiple aspects and embodiments described above, the solid organs are HLA-compatible. In another embodiment, HLA compatibility is determined in the donor and recipient by typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

[0113] In one embodiment of the various aspects and embodiments described above, the solid organ graft is ABO-compatible.

[0114] In one embodiment of the multiple aspects and embodiments described above, the solid organ is HLA-incompatible. In one embodiment, HLA-incompatible is determined by typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1 in the donor and recipient.

[0115] In one embodiment of the various aspects and embodiments described above, a cultured thymic tissue section is surgically transplanted into the target quadriceps femoris muscle.

[0116] In one of the aforementioned aspects and embodiments, cultured thymic tissue sections are surgically transplanted to a part of the body other than the quadriceps muscle.

[0117] In one embodiment of the multiple aspects and embodiments described above, a portion of the postnatal allogeneic thymic tissue culture-derived product is surgically transplanted into the quadriceps femoris muscle of the recipient.

[0118] In one embodiment of the various aspects and embodiments described above, the remaining portion of the postnatal allogeneic thymic tissue culture-derived product is cryopreserved in liquid nitrogen for future transplantation.

[0119] In one embodiment of the various aspects and embodiments described above, the duration of the acclimatization regimen is approximately 6 to 21 days.

[0120] In one embodiment of the various aspects and embodiments described above, the donor-thymus tissue acclimatization period is approximately 5 to 21 days, or approximately 5 days, or approximately 6 days, or approximately 7 days, or approximately 8 days, or approximately 9 days, or approximately 10 days, or approximately 11 days, or approximately 12 days, or approximately 13 days, or approximately 14 days, or approximately 15 days, or approximately 16 days, or approximately 17 days, or approximately 18 days, or approximately 19 days, or approximately 20 days, or approximately 21 days.

[0121] Those skilled in the art will understand that there are numerous possible induction and maintenance immunosuppression regimens known in the art, and that suitable induction and maintenance immunosuppressants can be easily selected by those skilled in the art. The specific induction and maintenance immunosuppression regimens described below are examples of methods of the first to fourth aspects of the present invention and support the present invention as claimed.

[0122] In one embodiment of the various aspects and embodiments described above, the inducible immunosuppression regimen comprises an inducible immunosuppressant selected from the group consisting of glucocorticoids, anti-thymocyte globulin (rabbit), anti-thymocyte globulin (horse), and alemtuzumab.

[0123] In one embodiment of the various aspects and embodiments described above, ATG is antithymocyte globulin (rabbit).

[0124] In one embodiment of the multiple aspects and embodiments described above, the inducing immunosuppression regimen includes the administration of a glucocorticoid. In one embodiment, the glucocorticoid includes methylprednisolone. In another embodiment, the glucocorticoid is methylprednisolone sodium succinate. In yet another embodiment, methylprednisolone sodium succinate is administered intravenously at a dose of 4 mg / kg / day or less.

[0125] In one embodiment of the various aspects and embodiments described above, the inducible immunosuppression regimen includes rabbit-derived antithymocyte globulin. In another embodiment, the rabbit-derived antithymocyte globulin is administered intravenously at a dose of approximately 1.5 mg / kg. In yet another embodiment, the antithymocyte globulin is administered daily for 4 days. In yet another embodiment, the ATG is horse-derived ATG.

[0126] In one embodiment of the various aspects and embodiments described above, the inducing immunosuppression regimen includes basiliximab. In another embodiment, basiliximab is administered intravenously at a dose of 10 mg to recipients weighing less than 35 kg. In yet another embodiment, basiliximab is administered intravenously at a dose of 20 mg to recipients weighing more than 35 kg.

[0127] In one embodiment of the various aspects and embodiments described above, the second immunosuppressive regimen comprises one or more immunosuppressants selected from the group consisting of glucocorticoids, calcinulin inhibitors, inosine monophosphate dehydrogenase inhibitors, azathioprine, and antithymocyte globulin ("ATG").

[0128] In one embodiment of the various aspects and embodiments described above, the immunosuppressant of the maintenance immunosuppression regimen is anti-thymocyte globulin (ATG).

[0129] In one embodiment of the various aspects and embodiments described above, ATG is administered intravenously at a dose of approximately 1.5 mg / kg for a period of 3 to 14 days, with administration initiated in the operating room.

[0130] In one embodiment of the various aspects and embodiments described above, antithymocyte globulin is administered intravenously at a dose of approximately 15 mg / kg / day daily for 3 to 14 days.

[0131] In one embodiment of the various aspects and embodiments described above, the first immunosuppressive regimen comprises alemtuzumab.

[0132] In one embodiment of the various aspects and embodiments described above, alemtuzumab is administered intravenously to recipients weighing less than 35 kg at a dose of approximately 0.25 mg / kg for 4 days. In another embodiment, alemtuzumab is administered intravenously to recipients weighing more than 35 kg at a dose of approximately 3 to 20 mg for 4 days.

[0133] In one embodiment of the multiple aspects and embodiments described above, the second immunosuppressive regimen comprises one or more immunosuppressants selected from the group consisting of calcinulin inhibitors and inosine monophosphate dehydrogenase inhibitors or azathioprine.

[0134] In one embodiment of the multiple aspects and embodiments described above, the immunosuppressant in the maintenance immunosuppression regimen is a calcinurin inhibitor. In one embodiment, the immunosuppressant in the maintenance immunosuppression regimen is an inosine monophosphate dehydrogenase inhibitor.

[0135] In one embodiment of the various aspects and embodiments described above, the immunosuppressive regimen comprises an inosine monophosphate dehydrogenase inhibitor, such as mycophenol mofetil. In one embodiment, mycophenol mofetil is administered intravenously at a dose of about 15 to about 25 mg / kg. In one embodiment, mycophenol mofetil is administered intravenously two to three times a day.

[0136] In one embodiment of the various aspects and embodiments described above, the inosine monophosphate dehydrogenase inhibitor is mycophenolic acid. In another embodiment, mycophenolic acid is administered in a dose of approximately 25 to approximately 50 mg / kg, in two or three divided doses.

[0137] In one embodiment of the various aspects and embodiments described above, mycophenolic acid is administered to children at a dose of approximately 400 mg / m². 2 / Administered twice daily at a maximum dose of 720 mg, or BSA is 1.19-1.59 m 2 For approximately 540 mg twice a day, or BSA is 1.58 mg. 2 In cases of over-the-counter illness, approximately 720 mg is administered twice a day.

[0138] In one embodiment of the various aspects and embodiments described above, mycophenol mofetil is administered orally or intravenously to children at a dose of approximately 15 to 25 mg / kg / dose twice daily, and to adults at approximately 1500 mg twice daily, with adjustments made if the WBC count exceeds 3500.

[0139] In one embodiment of the various aspects and embodiments described above, the second immunosuppressive regimen may further include a glucocorticoid selected from the group consisting of methylprednisolone, prednisone, and prednisolone. In one embodiment, the glucocorticoid dose is kept below 4 mg / kg / day.

[0140] In one embodiment of the various aspects and embodiments described above, glucocorticoids are administered in tapered doses, as described in other parts of this disclosure.

[0141] In one embodiment of the multiple aspects and embodiments described above, the calcinulin inhibitor is tacrolimus. In another embodiment, the calcinulin inhibitor is cyclosporine A.

[0142] In one embodiment of the various aspects and embodiments described above, the administration of the second immunosuppressant regimen is discontinued after naive T cells have reached 10% of the total T cells. In yet another embodiment, the second immunosuppressant regimen is discontinued after transplantation of a product derived from postnatal allogeneic thymic tissue culture.

[0143] To evaluate how the production and / or release of chemokines and other soluble molecules in the thymus controls these processes of cell migration changes when thymocytes are depleted during the preparation of donor thymic tissue for CTT, we screened conditioned media derived from human thymic organ cultures for the presence of 200 soluble molecules using antibody microarrays.

[0144] The expression of selected, potentially mechanistically important candidate molecules was validated using a separate thymic organ culture and compared to a panel of well-characterized human thymic tissue obtained from donors aged 5 days to 78 years. Through this analysis, we identified several potentially important biomarkers of thymocyte content.

[0145] One potentially important biomarker for thymocyte content is L-selectin. Another important biomarker for thymic epithelial cell viability and function depends on the secretion of the chemokine CCL21 6Ckine.

[0146] Numerous other potential biomarkers are shown in Figures 50 and 56. Of particular interest are the biomarkers shown in Figure 50, namely L-selectin, CCL21, CXCL16, M-CSF, galectin-7, CCL11, IL-16, and CXCL12. Of particular interest are the biomarkers shown in Figure 56, especially those with P values ​​less than 0.05. These biomarkers include L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, CCL20 (MIP-3a), IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR (CD87), MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1.

[0147] The data reported in the examples and references support the suggestion of TEC function in the following biomarkers: CCL21 (6Ckine), CXCL16, osteopontin (OPN), CCL11, uPAR (CD87), and CXCL12. CXCL16: This chemokine has been shown to be produced by TEC (Bunting 2011).

[0148] Osteopontin (OPN; encoded by the SPP1 gene): This cytokine increases during thymic stress, and increased levels are associated with thymic atrophy (a decrease in thymocyte count), a reduced thymocyte count being a favorable state for cultured thymus (Wang 2009; Gridley 2013). OPN is required for corticosteroid production and is well known to induce thymocyte apoptosis.

[0149] CCL11 (eotaxin): This chemokine is produced by medullary TE (Bunting 2011). It was initially named for its ability to attract eosinophils, and the inventors have shown that eosinophil infiltration is prominent in thymic tissue showing active thymocyte proliferation (Flores 1999). However, eotaxin has also been shown to act as a chemotactic for both double-positive (DP) and single-positive (SP) human thymocytes (Bunting 2011).

[0150] uPAR (CD87; coded by the PLAUR gene): The uroquinase receptor (also known as the uroquinase plasminogen activator receptor) is expressed in both soluble and membrane-bound forms based on alternative splicing. It assists in the local degradation of the extracellular matrix. While uPAR has been shown to be expressed in the human thymus, interestingly, it is also expressed by epidermal keratinocytes (EKs) migrating at the wound edge (Loughner 2016). This latter characteristic is most intriguing because TE cells mirror (act as a mirror) EKs in the expression of many genes (Patel 1995). A gradual increase in secretion in cultured thymic sections may reflect TE activation and is therefore a marker of TE proliferation after transplantation.

[0151] CXCL12 (SDF-1a): This chemokine exhibited a different expression pattern from other test substances, in that it was detected only during the last third of the culture period. It was first detectable on days 13–15 of culture, and it increased linearly (in the ln plot) to fairly high levels over the following weeks in culture. CXCL12 has been demonstrated to be produced by subcapsular cortical and medullary TE cells (Bunting 2011; Hernandez-Lopez 2002; Zaitseva 2002), but it may 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 thought to be important in the development of full thymic function. It is also involved in the localization of thymocyte subsets within the thymus, which promotes thymocyte proliferation in response to IL-7 (Hernandez-Lopez 2002). Notably, antibodies that neutralize CXCL12 have been shown to reduce thymocyte proliferation in in vitro human thymic organ cultures, while the addition of CXCL12 increases thymocyte proliferation in these cultures (Hernandez-Lopez 2002).

[0152] Another biomarker that may reflect the presence of thymocytes is L-selectin. This molecule is highly expressed in developing naive T cells. It is released from the cell surface when thymocytes are cultured. When released in vivo by healthy cells, it is usually rapidly reexpressed (Fitzhugh 2008), and a gradual decrease in release levels is likely to reflect a progressive loss of thymocyte viability, because they do not normally reexpress this molecule on their cell surface during culture (A. Macintyre, unpublished data).

[0153] Another biomarker that reflects the presence of thymocytes is IL-16. This cytokine was included because its pattern (as described below) corresponds to the pattern assumed for thymocytes. This biomarker is known to be produced by lymphocytes.

[0154] Another biomarker that reflects the presence of thymocytes is MIF. This chemokine was included because its pattern corresponds to the pattern assumed for thymocytes.

[0155] Another biomarker that reflects the presence of thymocytes is CCL20(MIP-3a): this chemokine was included because its pattern corresponds to the pattern assumed for thymocytes.

[0156] Another biomarker that reflects the presence of thymocytes is IGFBP-1. IGFBP2-6 are known to be expressed by thymic epithelium, which does not express IGFBP-1 (Gosteli-Peter 1994; Ketcha 1999).

[0157] Initially showing high levels and then decreasing over time (hypothetical characteristics of thymocyte-derived biomarkers), other biomarkers correlate with the presence of viable thymocytes, based on examples and references: L-selectin, IL-16, MIF, CCL20 (MIP-3a), and IGFBP-1. The decrease in these biomarkers over time is consistent with our more qualitative findings that T cells were depleted during the culture of thymocyte tissue sections.

[0158] CCL21 is expressed by thymic epithelial cells (Lkhagvasuren et al. 2013) and has been shown to be functionally important due to its chemotactic activity towards thymic progenitor cells (Liu et al. 2005) and its activity related to thymic cell migration within the thymus (Hu et al. 2015). This chemotactic property towards thymocytes is likely to be a major determinant of successful immunoconversion in recipients after cultured thymic tissue transplantation, as described herein.

[0159] The results described herein may be broadly applicable to understanding the mechanisms of age-related thymic involution and the mechanisms of immune reconstitution in athymic recipients by cultured thymic tissue transplantation.

[0160] Examples and Figure 56 illustrate that at least 127 different test substances can be detected in used / conditioned medium obtained from cultured human thymic tissue sections, and that 42 of these test substances show gradual increase or decrease throughout the culture time. Of these test substances, the amount of released soluble L-selectin was demonstrated as a non-destructive marker for the residual content of viable thymocytes in cultured thymus. The expression and / or secretion of chemokines CCL21, CXCL16, CXCL12, and CCL11 by thymic epithelium was demonstrated to increase as thymocytes decreased, both in vitro and in vivo during thymic organ culture in untampered thymic tissue obtained from lifelong healthy donors. Similarly, the expression or secretion of L-selectin, M-CSF, galectin-7, and IL-16 was observed to decrease in thymic organ culture medium throughout the course of the culture process. These findings are directly relevant to understanding the mechanisms of age-related thymic regression, as well as the qualities of human thymic tissue that promote immune rearrangement. The similarities between changes observed in infant thymus culture and age-related changes suggest that cultured human infant thymus also provides a useful model for studying the mechanisms mediating age-related thymic regression.

[0161] To the best of our knowledge, this is the first large-scale screening of soluble molecules produced, released, or secreted by the human thymus when thymocytes are depleted.42 Many of the test substances found to have significantly increased or decreased release into the culture medium as the culture progressed are cytokines and / or chemokines, which have been previously shown to be produced by cell types present within the thymus. Others are novel in this respect. Experimental studies have focused on the release of L-selectin as a marker for thymocyte content and CCL21 as a marker for TEC viability and function, but data revealed from antibody microarray screening can be used to identify further test substances from Figure 56 that may identify novel pathways governing acute atrophy, chronic regression, and / or in vivo regeneration of the human thymus. These may include biomarkers shown in particular in Figure 50. For example, several test substances that met the pre-identified selection criteria were not further considered. This is because they are known to be expressed or released in response to cell damage and / or hypoxic stress, and their levels may reflect culture-related tissue damage and inflammatory responses rather than thymus-specific biology. Other biomarkers found in the culture supernatant from cultured thymic tissue sections include CC25 (TECK), osteopontin (OPN), uPAR (CD87), MIF, CCL20 (MIP-3a), and IGFBP-1. Study of additional test substances whose release may reflect the viability and / or activation of important cell types in the human thymus may provide clinically and mechanistically important insights, particularly regarding responses to thymocyte loss.

[0162] CCL25(TECK): Detectable levels of TECK were present only in small amounts of supernatant samples in the later stages of the culture period in two of the three thymic cultures tested, but this chemokine is interesting because it has been shown to be chemotactic to thymocytes (Liu 2005). This is due to chemotaxis by thymic dendritic cells (DCs) and FoxN1 + and FoxN1 -It is known to be expressed by both TE cells (Bunting 2011). However, its activity is thought to be unimportant for thymic development, based on studies of mice lacking CCR9, the sole receptor for this chemokine (Wurbel 2001).

[0163] Based on microarray testing, it was determined that the level of soluble L-selectin in conditioned medium can be used as a potential biomarker for the presence and viability of thymocytes in cultured thymic sections. This is highly reasonable because L-selectin expression is localized to hematopoietic cells and is released constitutively and during migration (Hafezi-Moghadam et al. 2001), and is subsequently rapidly reexpressed in vivo by healthy cells (Fitzhugh et al. 2008).

[0164] The experimental results indicate that the absence of L-selectin release is temporally correlated with thymocyte death, as evidenced by the loss of membrane integrity in thymocytes by histology and CD3 immunohistochemistry, and the absence of characteristic thymocyte proliferation by Ki-67 immunohistochemistry.

[0165] The ability to non-destructively observe the thymocyte content of cultured human thymocyte sections is crucial for identifying the most appropriate harvesting time for experimental studies. This may also provide clinically important information, as making available the developmental niche for colonization by recipient thymocytes via donor-thymocyte depletion is considered important for good immune reconstitution in athymic patients after thymocyte transplantation. In summary, the tests shown in the examples suggest that reduced L-selectin release is a useful biomarker for tracking thymocyte depletion in cultured thymocyte sections.

[0166] Biomarkers that reflect the presence and function of thymic epithelium can also provide important mechanistic information. In the example studies, we focused on CCL21 because microarray screening showed that this chemokine begins to be secreted into the culture medium at high levels immediately after the start of culture. CCL21 has previously been shown to be expressed by thymic epithelium and to be chemotactic to thymocytes and their precursor cells (Liu et al. 2005). Our research has shown that CCL21 expression can also be easily quantified by enzyme immunoassay. Immunohistochemistry confirmed CCL21 expression by TEC in cultured and uncultured thymus, with the strongest expression observed in the medulla and subcapsular cortical thymic epithelium.

[0167] It is also important to note that the CCL21 immunoreactivity of thymic sections did not necessarily increase when CCL21 secretion increased during culture. This may reflect that the additional CCL21 produced is secreted rather than remaining in the cytoplasm, which can be detected by immunohistochemistry. CCL21 is a transcriptionally regulated, rapidly turnovering molecule with a short half-life (Dudal et al. 2015), and therefore, the observed immunohistochemically positive reactivity indicates cells that actively produce this chemokine. The fact that CCL21 production significantly increases when thymocytes decrease in both cultured and unmanipulated senescent thymuses suggests that thymic epithelial cells can sense thymocyte content and respond based on homeostasis to prevent thymocyte loss.

[0168] The identification of CCL21 as a secretory biomarker reflecting the viability and function of TECs is also clinically important in relation to thym transplantation. The most standard methods for assessing the quality of transplanted tissue (e.g., flow cytometry, immunohistochemistry, gene expression analysis) destroy the sample during analysis. In addition to reducing the amount of tissue available for final transplantation, such results are subject to sampling error because the section examined is not part of the section that will ultimately be transplanted. As shown in Figure 53C, assays of pooled spent media for CCL21 can be integrated across all sections in a lot derived from any given thym donor, thereby providing a non-destructive state of overall lot quality.

[0169] The chemokine CXCL12 (SDF-1α) differed from most other test substances in screening in that it became detectable in acclimatized medium relatively late in the culture period (Figure 50H). It was first detectable at days 13–15, and it increased linearly (in the ln plot) to considerably high levels over the following weeks in culture. CXCL12 has been demonstrated to be produced by subcapsular cortical and medullary TE cells, but it can also be produced by thymic fibroblasts and endothelial cells present in the thymus (Bunting et al. 2011; Hernandez-Lopez et al. 2002; Zaitseva et al. 2002). CXCL12 recruits B cells and antigen-presenting cells to the thymus (Weiss et al. 2013), which is thought to be important for the formation of full thymic function. CXCL12 is also involved in the localization of thymocyte subsets within the thymus, which promotes thymocyte proliferation in response to IL-7 (Hernandez-Lopez et al. 2002). Notably, antibodies neutralizing CXCL12 have been shown to reduce thymocyte proliferation in in vitro human thymic organ cultures, while the addition of CXCL12 increases thymocyte proliferation in these cultures (Hernandez-Lopez et al. 2002). The delayed timing of CXCL12 secretion in in vitro thymic organ cultures, observed here, combined with increased in vivo expression in donor-derived thymuses from individuals over 18 years of age compared to younger donors, suggests that the expression of this chemokine is induced by thymocyte depletion that lasts longer than is required for CCL21 secretion induction.

[0170] Screening data suggest that other chemokines, including CXCL16 and CL11, could serve as biomarkers for evaluating the viability and function of cultured thymuses, because they also increase when thymocytes are lost during thymic organ culture. Both CXCL16 and CL11 have been previously shown to be produced by TEC (Bunting et al. 2011).

[0171] CCL11 was initially named eotaxin due to its ability to attract eosinophils. We previously showed that eosinophil infiltration is prominent near thymic tissue with active thymocyte proliferation (Flores et al. 1999), although CCL11 levels were not directly measured in those studies. However, CCL11 has since been shown to act as a chemotactic for both bipolar and monopolar human thymocytes (Bunting et al. 2011). Evidence regarding the specific role of CXCL16 in thymocyte proliferation is less clear.

[0172] The experiments described in the examples verify the use of L-selectin as a biomarker for the presence of viable thymocytes, and the use of CCL21 as a biomarker for TEC viability, which can also predict efficient immune rearrangement when thymocyte progenitor cells become available. CCL21 production is typically low when thymocyte proliferation is stable and significantly induced when thymocyte proliferation declines, in both cultured thymic sections and uncultured thymic tissue from aged donors. Interestingly, after normalization to either the TEC region or the active cortical region, thymic tissue from younger donors (≤18 years) continued to express more CD3 epsilon and CD1A mRNA, as well as less keratin 8 (KRT8) and keratin 14 (KRT14) mRNA, compared to thymus from aged donors. This suggests that thymocyte proliferation and TEC maintenance are potentially more efficient in these younger donors. Furthermore, for both normalization methods, production of CCL21 and CXCL12 is significantly increased in tissues derived from donors over 18 years of age, a period when TEC content and active thymocyte proliferation are reduced compared to younger donors. The fact that the expression of both CCL21 and CXCL12 increases with decreasing thymocyte number in both in vitro thymic organ culture and in vivo aging suggests that the induction of these chemokines may be part of a homeostatic mechanism that prevents the decrease in thymocyte number by enhancing T cell progenitor mobilization. Increased secretion of thymocyte-attracting chemokines by thymocyte-depleted cultured thymic sections is expected to enhance their ability to induce colonization and immune rearrangement, as observed when such sections are transplanted into infant recipients with athymopathy (Markert et al. 2008). In contrast, abundant secretion of CCL21 and CXCL12 would not be as beneficial in aging if the availability of thymic progenitor cells or other important aspects of the thymic microenvironment were limited.

[0173] In summary, these studies demonstrate that organ culture of pediatric donor-derived thymuses can be used to model at least some aspects of age-related thymic regression in humans, particularly those directly related to thymocyte loss. However, it is clear that T-cell depleted cultured pediatric thymuses must otherwise be substantially different from aged thymuses. This is because transplantation of T-cell depleted cultured pediatric thymuses into athymic recipients results in immune rearrangement and protection from infection, while older adults with regressed thymuses are more vulnerable to infection than younger adults with more stable 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.

[0174] The results shown in Figure 56 of this specification provide a rich source of further molecules and pathways as potential biomarkers for modeling several aspects of human thymic aging in vitro using cultured infant thymus. This is important because infant thymus is usually more readily available for study, given that it is usually necessary to remove a portion of the thymus from most infants to adequately expose the surgical field for cardiac corrective surgery. Adult thymic tissue is not usually readily available because it is not usually necessary to remove thymic tissue to provide access to many cardiac surgeries common in adults. Furthermore, any excised adult thymic tissue is generally not used for study because it does not look very organoid and is macroscopically similar to fat. However, adult thymic tissue can potentially be cultured and used for co-transplantation with solid organs when tolerance in adult solid organ septics is desired. The biomarkers are also useful in determining the compatibility, functionality, and viability of culture products derived from postnatal allogeneic thymic tissue from adult donors.

[0175] The production of thymic cytochemical attractants CCL21, CXCL16, CXCL12, and CCL11 in the thymus increases as the thymocyte content decreases. This suggests that thymocyte loss activates homeostatic mechanisms that attempt to counteract potential atrophy, but ultimately fails in the context of aging. Future research to more fully elucidate these mechanisms will be useful in understanding the mechanisms causing age-related thymic regression and potentially reversing it, which will help promote thymic immune rearrangement at all ages.

[0176] In one aspect of this disclosure, a method is provided for creating culture products derived from postnatal allogeneic thymus tissue suitable for transplantation into humans, the method being: A step of subjecting donor-thymus tissue to an acclimatization regimen of approximately 6 to 21 days, wherein the acclimatization regimen for donor-thymus tissue comprises aseptically treating donor-thymus tissue in thymic organ medium to prepare donor-thymus tissue sections in which T cells are partially depleted; and further comprises detecting an increase in CCL21 levels in thymic organ medium during the course of the acclimatization regimen; and A process for collecting donor thymus tissue sections with partially depleted T cells as culture products derived from postnatal allogeneic thymus tissue suitable for transplantation. Includes.

[0177] In one embodiment of the various aspects and embodiments of the present disclosure, the method further includes the step of cryopreserving a culture product derived from postnatal allogeneic thymus tissue in liquid nitrogen for future transplantation.

[0178] In one embodiment of the various aspects and embodiments of the present disclosure, the method further includes detecting a decrease in L-selectin levels in thymic organ culture medium during the course of a habituation regimen.

[0179] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting one or more of CCL21, CXCL12, CXCL16, or CCL11 in thymic organ culture medium during the course of a habituation regimen.

[0180] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting an increase in the level of one or more CXCL12, CXCL16, or CCL11 in thymic organ culture medium during the course of a habituation regimen.

[0181] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting an increase in CXCL12 levels in thymic organ culture medium during the course of a habituation regimen.

[0182] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting an increase in CXCL16 levels in thymic organ culture medium during the course of a habituation regimen.

[0183] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting an increase in CCL11 levels in thymic organ culture medium during the course of a habituation regimen.

[0184] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting one or more M-CSF, galectin-7, or IL-16 in a thymic organ medium during the course of a habituation regimen.

[0185] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting a decrease in the level of one or more M-CSF, galectin-7, or IL-16 in thymic organ culture medium during the course of a habituation regimen.

[0186] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting a decrease in M-CSF levels in thymic organ culture medium during the course of a habituation regimen.

[0187] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting a decrease in galectin-7 levels in thymic organ culture medium during the course of a habituation regimen.

[0188] In one embodiment of the various aspects and embodiments of the present disclosure, the method includes detecting a decrease in IL-16 levels in thymic organ culture medium during the course of a habituation regimen.

[0189] In one embodiment of the various aspects and embodiments of this disclosure, the habituation regimen is a period of 5 days, or 6 days, or 7 days, or 8 days, or 9 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 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, and This refers to a period of 6-7 days, or 6-8 days, or 6-9 days, or 6-10 days, or 6-11 days, or 6-12 days, or 6-21 days, or 7-21 days, or 8-21 days, or 9-21 days, or 10-21 days, or 11-21 days, or 12-21 days, or 13-21 days, or 14-21 days, or 15-21 days, or 16-21 days, or 17-21 days, or 18-21 days, or 19-21 days, or 20-21 days.

[0190] In one embodiment of multiple aspects and embodiments of the present disclosure, the level of CCL21 approaches the level shown in Figure 50E, and / or the level of L-selectin approaches the level shown in Figure 50A, and / or the level of M-CSF approaches the level shown in Figure 50B, and / or the level of galectin-7 approaches the level shown in Figure 50C, and / or the level of IL-16 approaches the level shown in Figure 50D, and / or the level of CXCL16 approaches the level shown in Figure 50F, and / or here the level of CCL11 approaches the level shown in Figure 50G, and / or the level of CXL21 approaches the level shown in Figure 50H.

[0191] In one embodiment of the various aspects and embodiments of the present disclosure, the method further includes determining in the Donner-thymus tissue section, on days 6 to 21, preferably days 6 to 9, the presence of regions positive for keratin AE1 / AE3 scattered throughout the Donner-thymus tissue section, the presence of at least one Hassall body, and the presence of CK14 staining and intact nuclei scattered throughout the Donner-thymus tissue section.

[0192] In one embodiment of the various aspects and embodiments of the present disclosure, the method further comprises detecting in thymic organ medium the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11 during the culture regime period, or the level of at least two markers, or the level of at least three markers, or the level of at least four markers, or the level of at least five markers, or the level of at least six markers, or the level of at least seven markers, in thymic organ medium during the acclimatization regime period.

[0193] In one embodiment of the various aspects and embodiments of this disclosure, the method involves adding L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, and SCF in thymic organ medium during the culture regimen period. The method further includes detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-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.

[0194] In one aspect of this disclosure, a method is provided for determining whether a cultured product derived from allogeneic thymic tissue after birth is suitable for transplantation into a human, the method being: The process includes a step of acclimatizing donor-thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days; A habituation regimen for donor-thymic tissue involves aseptically treating donor-thymic tissue in thymic organ culture medium to create donor-thymic tissue sections in which T cells are partially depleted; and The process includes detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[0195] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is L-selectin, and the level of L-selectin in the thymic organ medium decreases over time, i.e., during the course of the acclimatization regimen.

[0196] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is M-CSF, and the level of M-CSF in the thymic organ medium decreases over time, i.e., during the course of the acclimatization regimen.

[0197] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is galectin-7, and the level of galectin-7 in the thymic organ medium decreases over time, i.e., during the course of the acclimatization regimen.

[0198] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is IL-16, and the level of IL-16 in the thymic organ medium decreases over time, i.e., during the course of the acclimatization regimen.

[0199] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CCL21, and the level of CCL21 in the thymic organ medium increases over time, i.e., during the course of the acclimatization regimen.

[0200] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CXCL12, and the level of CXCL12 in the thymic organ medium increases over time, i.e., during the course of the acclimatization regimen.

[0201] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CXCL16, and the level of CXCL16 in the thymic organ medium increases over time, i.e., during the course of the acclimatization regimen.

[0202] In one embodiment of the various aspects and embodiments of the present disclosure, at least one marker is CCL11, and the level of CCL11 in the thymic organ medium increases over time, i.e., during the course of the acclimatization regimen.

[0203] In one aspect of this disclosure, a method is provided for determining whether a cultured product derived from allogeneic thymic tissue after birth is suitable for transplantation into a human, the method being: The process includes a step of acclimatizing donor-thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days; A habituation regimen for donor-thymic tissue involves aseptically treating donor-thymic tissue in thymic organ culture medium to create donor-thymic tissue sections in which T cells are partially depleted; During the acclimatization 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 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF were added to the thymic organ medium. The process includes detecting the level of at least one marker selected from 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.

[0204] In one embodiment of the various aspects and embodiments of the present disclosure, the method further includes determining in the Donner-thymus tissue section, between days 6 and 21 of the acclimatization regimen, areas positive for keratin AE1 / AE3 scattered throughout the section, the presence of at least one Hassall body, and the presence of CK14 staining and intact nuclei scattered throughout the section.

[0205] In one embodiment of the various aspects and embodiments of this disclosure, the habituation regimen is a period of 5 days, or 6 days, or 7 days, or 8 days, or 9 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 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, and This refers to a period of 6-7 days, or 6-8 days, or 6-9 days, or 6-10 days, or 6-11 days, or 6-12 days, or 6-21 days, or 7-21 days, or 8-21 days, or 9-21 days, or 10-21 days, or 11-21 days, or 12-21 days, or 13-21 days, or 14-21 days, or 15-21 days, or 16-21 days, or 17-21 days, or 18-21 days, or 19-21 days, or 20-21 days.

[0206] In one embodiment of several aspects and embodiments of the present disclosure, the level of CCL21 approaches the level shown in Figure 50E, and / or the level of L-selectin approaches the level shown in Figure 50A, and / or the level of M-CSF approaches the level shown in Figure 50B, and / or the level of galectin-7 approaches the level shown in Figure 50C, and / or the level of IL-16 approaches the level shown in Figure 50D, and / or the level of CXCL16 approaches the level shown in Figure 50F, and / or the level of CCL11 approaches the level shown in Figure 50G, and / or the level of CXL21 approaches the level shown in Figure 50H.

[0207] In one embodiment of the various aspects and embodiments of the present disclosure, the method further comprises detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11, or the levels of at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight markers in thymic organ medium during the course of the habituation regimen, wherein the levels of L-selectin, M-CSF, galectin-7, and IL-16 decrease in the thymic organ medium during the course of the habituation regimen, and the levels of CCL21, CXCL12, CXCL16, and CCL11 increase in the thymic organ medium during the course of the habituation regimen.

[0208] In one aspect of the present invention, there exists a method for determining whether a cultured product derived from allogeneic thymic tissue after birth is suitable for transplantation into a human. The method includes a step of acclimatizing donor-thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days; A habituation regimen for donor-thymic tissue involves aseptically treating donor-thymic tissue in thymic organ culture medium to create donor-thymic tissue sections in which T cells are partially depleted; and The process includes detecting the level of at least one marker selected from the markers in Figure 56.

[0209] In several aspects and one embodiment of the present disclosure, there exists a method for determining whether a culture product derived from allogeneic thymic tissue after birth is suitable for transplantation into a human. The method includes a step of acclimatizing donor-thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days; The donor-thymus tissue adaptation regimen includes aseptically treating donor-thymus tissue in thymic organ medium to create donor-thymus tissue sections with partially depleted T cells; and 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 The process includes detecting the level of at least one marker selected from 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 thymic organ medium during the acclimatization regimen.

[0210] In one aspect of this disclosure, a method exists for treating thymic disorders, the improvement comprising transplanting cultured sections derived from postnatal allogeneic thymic tissue subjected to a habituation regimen in thymic organ medium for a period of approximately 6 to 21 days into a subject with a thymic disorder; the habituation regimen for donor-thymic tissue comprises aseptically treating donor-thymic tissue in thymic organ medium to create donor-thymic tissue sections in which T cells are partially depleted; and comprising detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, and CCL11.

[0211] In another aspect of this disclosure, there exists a method for treating thymic disorders, the improvement comprising transplanting cultured sections derived from postnatal allogeneic thymic tissue subjected to a habituation regimen in thymic organ medium for a period of approximately 6 to 21 days into a subject with a thymic disorder; the habituation regimen for donor-thymic tissue comprises aseptically treating donor-thymic tissue in thymic organ medium to create donor-thymic tissue sections in which T cells are partially depleted; and during the course of the habituation regimen, L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF This involves detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 in thymic organ medium.

[0212] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is L-selectin, and the level of L-selectin in the thymic organ medium decreases over the course of the acclimatization regimen.

[0213] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is M-CSF, and the M-CSF level in the thymic organ medium decreases over the course of the acclimatization regimen.

[0214] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is galectin-7, and the level of galectin-7 in the thymic organ medium decreases over the course of the acclimatization regimen.

[0215] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is IL-16, and the level of IL-16 in the thymic organ medium decreases over the course of the acclimatization regimen.

[0216] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CCL21, and the level of CCL21 in the thymic organ medium increases over time during the acclimatization regimen.

[0217] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CXCL12, and the level of CXCL12 in the thymic organ medium increases over the course of the acclimatization regimen.

[0218] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CXCL16, and the level of CXCL16 in the thymic organ medium increases over the course of the acclimatization regimen.

[0219] In one embodiment of several aspects and embodiments of the present disclosure, at least one marker is CCL11, and the level of CCL11 in the thymic organ medium increases over time during the acclimatization regimen.

[0220] In one embodiment of the various aspects and embodiments of the present disclosure, the method further includes determining in the Donner-thymus tissue section during the course of a habituation regime the presence of regions positive for keratin AE1 / AE3 scattered throughout the Donner-thymus tissue section, the presence of at least one Hassall body, and the presence of CK14 staining and intact nuclei scattered throughout the Donner-thymus tissue section.

[0221] In aspects and embodiments of the present disclosure, thymic disorders include complete DiGeorge syndrome, 22q11.2 deletion, CHARGE (deficiency, cardiac defects, posterior nasal atresia, developmental delay or intellectual disability, genital hypoplasia and ear abnormalities or hearing loss), congenital athymus associated with mutations in the CHD7 (chromodomain-helicase-DNA-binding protein 7) gene or forkhead box protein N1 (FOXN1) deficiency.

[0222] In one embodiment of several aspects and embodiments of this disclosure, the thymic disorder is thymic regression. In another embodiment, the thymic disorder is congenital athymus associated with mutations in the TBX-1 or TBX-2 gene.

[0223] In several aspects and one embodiment of the embodiments of this disclosure, thymic disorders are associated with paired box 1 (PAX1), semaphorin 3E (SEMA3E), and recurrent microdeletions of chromosome 2p11.2.

[0224] In some aspects and embodiments of the present disclosure, the thymic disorder is associated with a thymoma. In a further embodiment, the thymoma is either non-malignant or malignant.

[0225] In several aspects and one embodiment of the embodiments of this disclosure, thymic disorders are associated with myasthenia gravis (MG), pure red cell aplasia, and hypogammaglobulinemia.

[0226] In one aspect of this disclosure, a method for conferring immunity to human subjects is provided, the improvement comprising the step of acclimatizing donor-thymocyte tissue sections in thymic organ medium for a period of approximately 6 to 21 days, wherein the acclimatization regimen for donor-thymocyte tissue comprises the step of aseptically treating donor-thymocyte tissue in thymic organ medium to produce donor-thymocyte tissue sections in which T cells are partially depleted; Markers: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12 The procedure includes the steps of: detecting the level of at least one marker selected from CXCL16 or CCL11 in thymic organ medium during the course of a habituation regimen, wherein the level decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, or increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; and transplanting a partially T-cell depleted donor thymic tissue section into a human subject.

[0227] In one aspect of this disclosure, a method for conferring immunity to human subjects is provided, the improvement comprising the step of acclimatizing donor-thymocyte tissue sections in thymic organ medium for a period of approximately 6 to 21 days; the acclimatization regimen for donor-thymocyte tissue comprises the step of aseptically treating donor-thymocyte tissue in thymic organ medium to produce donor-thymocyte tissue sections in which T cells are partially depleted; 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 The method includes the steps of: detecting the level of at least one marker selected from the markers 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 thymic organ medium during the course of a habituation regimen; and transplanting a partially T-cell depleted donor thymic tissue section into a human subject.

[0228] In one aspect of this disclosure, a method is provided for conferring immunity to a human subject receiving a solid organ transplant, the method comprising the steps of: removing the thymus of the human subject; obtaining thymic tissue from a donor whose HLA class I and HLA class II alleles are matched in the solid organ; excising the donor thymus into sections; and habituating the donor thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days, wherein the habituation regimen for the donor thymic tissue comprises aseptically treating the donor thymic tissue in thymic organ culture medium to produce donor thymic tissue sections in which T cells are partially depleted; -A step of detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the course of a habituation regimen, wherein the level decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, or increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; transplanting a solid organ; and transplanting a partially T-cell depleted donor thymic tissue section into a human subject.

[0229] For a better understanding of the development of immune tolerance in this model, refer to Figure 38, which includes a primate model of this procedure that provides data supporting human trials for inducing donor-specific tolerance. The experiment in Figure 38 has three monkeys. One is the thymus and cardiac donor (information in the left column). The second is the thymus and cardiac recipient (information in the middle column on page 2; this column includes the experimental stage). The third is the control (information in the right column on page 3). Note that the original spreadsheet contained the procedure for all three animals and spanned multiple pages with a width of one page. Since the spreadsheet was wider than the allowable width, each row of the spreadsheet was divided into three pages. The table continues in groups of three panels for multiple weeks and multiple stages.

[0230] In one aspect of this disclosure, a method is provided for conferring immunity to a human subject receiving a solid organ transplant, the method comprising: the steps of: removing the thymus of a human subject; obtaining thymic tissue from a donor whose HLA class I and II alleles are matched in the solid organ; excising the donor thymus into sections; and habituating the donor thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days, wherein the habituation regimen for the donor thymic tissue comprises aseptically treating the donor thymic tissue in thymic organ culture medium to produce donor thymic tissue sections that are partially T-cell depleted; L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF The procedure includes the steps of: detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 in thymic organ medium during the course of a habituation regimen; transplanting a solid organ; and transplanting a donor thymic tissue section partially depleted of T cells into a human subject.

[0231] In one aspect of this disclosure, a method is provided for conferring immunity to a human subject receiving a solid organ transplant, the method comprising: the steps of: removing the thymus of a human subject; obtaining thymic tissue from a donor whose HLA class I and HLA class II alleles are matched in the solid organ; excising the donor thymus into sections; and habituating the donor thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days, wherein the habituation regimen for the donor thymic tissue comprises aseptically treating the donor thymic tissue in thymic organ culture medium to produce donor thymic tissue sections in which T cells are partially depleted; The procedure comprises the steps of: detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the period of a habituation regimen; the level decreasing if the marker is L-selectin, M-CSF, galectin-7, or IL-1, or increasing if the marker is CCL21, CXCL12, CXCL16, or CCL11; transplanting a solid organ; and transplanting a partially T-cell depleted donor thymic tissue section into a human subject.

[0232] In one aspect of this disclosure, a method is provided for conferring immunity to a human subject receiving a solid organ transplant, the method comprising: the steps of: removing the thymus of a human subject; obtaining thymic tissue from a donor whose solid organ is matched for both HLA class I and HLA class II alleles; excising the donor thymus into sections; and habituating the donor thymic tissue sections in thymic organ culture medium for a period of approximately 6 to 21 days, wherein the habituation regimen for the donor thymic tissue comprises aseptically treating the donor thymic tissue in thymic organ culture medium to produce donor thymic tissue sections that are partially T-cell depleted; markers: L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF The procedure includes the steps of: detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 in thymic organ medium during the course of a habituation regimen; transplanting a solid organ; and transplanting a donor thymic tissue section partially depleted of T cells into a human subject.

[0233] In one aspect of this disclosure, a method is provided to promote donor-specific tolerance to allogeneic solid organ grafts obtained from deceased donors in recipients requiring solid organ transplantation. The method includes the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process to provide both suitable human solid organs and thymus derived from a deceased donor; (d) The process of transplanting a human solid organ into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a culture product derived from postnatal allogeneic thymus tissue, wherein the culture product derived from postnatal allogeneic thymus tissue is subjected to an acclimatization regimen of approximately 6 to 21 days in thymic organ medium to prepare sections of the culture product derived from postnatal allogeneic thymus tissue; further, an acclimatization regimen for donor-thymus tissue includes aseptic treatment of the donor-thymus tissue in thymic organ medium to prepare donor-thymus tissue sections in which T cells are partially depleted; Marker: L-selection A step of detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the period of a habituation regimen; a step of decreasing the level of the marker in thymic organ medium if the marker is L-selectin, M-CSF, galectin-7, or IL-1, or increasing if the marker is CCL21, CXCL12, CXCL16, or CCL11; and (g) A step in which, after an acclimatization regimen of approximately 6 to 21 days, a culture product derived from postnatal allogeneic thymic tissue is transplanted into the recipient; the dose of thymic tissue sections is approximately 1,000 to 22,000 mm 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0234] In one aspect of this disclosure, a method is provided to promote donor-specific tolerance to allogeneic solid organ grafts obtained from deceased donors in recipients requiring solid organ transplantation. The method includes the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process to provide both suitable human solid organs and thymus derived from a deceased donor; (d) The process of transplanting a human solid organ into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a culture product derived from allogeneic thymic tissue after birth, Culture products derived from allogeneic thymic tissue after birth are subjected to an acclimatization regimen of approximately 6 to 21 days in thymic organ culture medium to prepare sections of culture products derived from allogeneic thymic tissue after birth; Furthermore, the habituation regimen for donor-thymus tissue includes the process of aseptically treating donor-thymus tissue in thymic organ culture medium to create donor-thymus tissue sections in which T cells are partially depleted; 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 A step of detecting the level of at least one marker selected from 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 thymic organ medium during the course of the acclimatization regimen; and (g) A step in which, after an acclimatization regimen of approximately 6 to 21 days, a culture product derived from postnatal allogeneic thymic tissue is transplanted into the recipient; the dose of thymic tissue sections is approximately 1,000 to 22,000 mm 2 Thymus tissue surface area / Recipient body surface area (m²) 2The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0235] In one aspect of this disclosure, a method is provided for promoting donor-specific immune tolerance to allogeneic solid organ grafts obtained from living human donors in human recipients requiring solid organ transplantation, the method comprising the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process for providing suitable solid organs derived from a living human donor; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing cryopreserved postnatal allogeneic thymus tissue-derived culture products maintained in a cryopreserved postnatal allogeneic thymus tissue-derived culture product bank, wherein the cryopreserved postnatal allogeneic thymus tissue-derived culture products are processed from thymus tissue from a thymus donor expressing HLA alleles that match recipient HLA class I and HLA class II alleles not present in the solid organ graft; the donor-thymus tissue is subjected to a habituation regimen for a period of approximately 6 to 21 days; and the habituation regimen for the donor-thymus tissue further prepares sections of partially T-cell depleted thymus tissue. A step comprising: a sterile treatment of donor thymic tissue in thymic organ medium; a step comprising: detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the course of a habituation regimen; a step comprising: the level of the marker in the thymic organ medium decreasing if the marker is L-selectin, M-CSF, galectin-7, or IL-1, and increasing if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue, and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0236] In several aspects and one embodiment of the present disclosure, tolerable HLA-DP mismatches may be accepted (Pidala J et al 2014 Blood 124:2596-2606). Furthermore, non-tolerable HLA-DPB1 mismatches may be accepted if there is a sufficient numerical functional distance (Crivello P et al 2016 Blood 128:120-129).

[0237] In one aspect of the present invention, a method is provided to promote donor-specific immune tolerance to allogeneic solid organ grafts obtained from a living human donor in human recipients requiring solid organ transplantation, the method comprising the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process for providing suitable solid organs derived from a living human donor; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing cryopreserved postnatal allogeneic thymic tissue-derived culture products maintained in a cryopreserved postnatal allogeneic thymic tissue-derived culture product bank, wherein the cryopreserved postnatal allogeneic thymic tissue-derived culture products are processed from thymic donor-derived thymic tissue expressing HLA alleles that match recipient HLA class I and HLA class II alleles not present in the solid organ graft; and the donor-thymic tissue is processed for approximately 6 to 21 days. During the period of days, the tissue is subjected to a habituation regimen; further, the habituation regimen for donor thymus tissue includes the step of aseptically treating the donor thymus tissue in thymic organ medium to prepare sections of partially T cell-depleted thymus tissue; markers in thymic organ medium: L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF A step of detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 in thymic organ medium during the course of a habituation regimen; the level of the marker in the thymic organ medium increases or decreases according to the level of the marker in Figure 56; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue; and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2a step, in units, further comprising culturing a product derived from transplanted postnatal allogeneic thymic tissue that induces thymocyte proliferation and tolerance in a recipient.

[0238] In one embodiment of the various aspects and embodiments of the present disclosure, approximately half of the thawed culture product derived from cryopreserved postnatal allogeneic thymic tissue is transplanted into the recipient, and the remainder is cryopreserved for future use.

[0239] In one embodiment of the various aspects and embodiments of the present disclosure, step (h) is performed at least about one month after solid organ transplantation.

[0240] 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 requiring solid organ transplantation, the method comprising the following steps: (a) removing the recipient's thymus; (b) treating the recipient with an induction immunosuppressive regimen comprising one or more immunosuppressive agents to deplete the recipient's T cells and / or to inhibit the recipient's T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) A step of providing culture products derived from cryopreserved postnatal allogeneic thymus tissue, maintained in a cryopreserved postnatal allogeneic thymus tissue culture product bank; the culture products derived from cryopreserved postnatal allogeneic thymus tissue are processed from thymus tissue derived from a thymus donor expressing an HLA allele that matches the recipient's HLA allele that is not present in the solid organ graft; the donor-thymus tissue is subjected to a habituation regimen for a period of approximately 6 to 21 days; and the habituation regimen for the donor-thymus tissue is further applied to create sections of partially T-cell depleted thymus tissue. A step comprising aseptically treating donor thymic tissue in glandular organ medium, detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymic organ medium during the course of a habituation regimen; wherein the level of the marker in the thymic organ medium decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, and increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue; and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0241] In one aspect of this disclosure, a method is provided to promote donor-specific tolerance to allogeneic solid organ grafts obtained from deceased human donors in human recipients requiring solid organ transplantation, the method comprising the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process for providing suitable solid organs derived from a living human donor; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step to provide culture products derived from cryopreserved postnatal allogeneic thymus tissue, maintained in a cryopreserved postnatal allogeneic thymus tissue culture product bank; the culture products derived from cryopreserved postnatal allogeneic thymus tissue are processed from thymus tissue derived from a thymus donor expressing an HLA allele that matches the recipient's HLA allele not present in the solid organ graft; the donor-thymus tissue is subjected to a habituation regimen for a period of approximately 6 to 21 days; and the habituation regimen for the donor-thymus tissue further comprises aseptically processing the donor-thymus tissue in thymus organ medium to produce sections of partially T-cell depleted thymus tissue. Markers: L-selectin, CXCL16, M-CSF, CCL21 / 6Ckine, galectin-7, MIF, GDNF, CTACK, MIP-3b, ICAM-1, PECAM-1, IL-2Rg, SCF A step of detecting the level of at least one marker selected from R, IL-16, GDF-15, PDGF-AA, CXCL12 / SDFF-1a, MIP-3a, IL-2Ra, ICAM-3, LIGHT, IGFBP-1, BCMA, EGF R, uPAR, MIP-1b, PIGF, PF4, CCL11 / eotaxin, HVEM, IGFBP-6, IL-6R, IL-12p40, RANTES, MICA, GCP-2, OPN, ALCAM, NRG1-B1, CEACAM-1, IL-1b, DKK-1, and ANG-1 in thymic organ medium during the course of a habituation regimen; a step of increasing or decreasing the level of the marker in thymic organ medium according to the levels in Figure 7; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue; and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0242] In one aspect of this disclosure, we provide a culture product derived from cryopreserved postnatal allogeneic thymus tissue, prepared by a method comprising the following steps: (a) The process of obtaining suitable thymic tissue from a donor; (b) HLA alleles: The process of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1; (c) A step of subjecting thymic tissue to an adaptation regimen for a period of approximately 6 to 21 days, wherein the adaptation regimen for donor-thymic tissue includes aseptically treating the donor-thymic tissue in thymic organ culture medium to produce partially T-cell depleted donor-thymic tissue sections; (d) A step of detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the course of a habituation regimen, wherein the level of the marker in the thymic organ medium decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, and increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; (e) A step of recovering donor thymus tissue sections with partially depleted T cells as culture products derived from postnatal allogeneic thymus tissue; (f) A step of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (g) A step of maintaining culture products derived from cryopreserved postnatal allogeneic thymus tissue in liquid nitrogen in a cryopreserved postnatal allogeneic thymus tissue culture product bank.

[0243] In one aspect of this disclosure, there exists a culture product derived from cryopreserved postnatal allogeneic thymus tissue, prepared by a method comprising the following steps: (a) The process of obtaining suitable thymic tissue from a donor; (b) HLA alleles: The process of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1; (c) A step of subjecting thymic tissue to an acclimatization regimen for a period of approximately 6 to 21 days, wherein the acclimatization regimen for donor-thymic tissue includes aseptically treating the donor-thymic tissue in thymic organ culture medium to produce donor-thymic tissue sections in which T cells are partially depleted; (d) 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 A step of detecting the level of at least one marker selected from 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 thymic organ medium during the course of a habituation regimen, wherein the level of the marker in the thymic organ medium increases or decreases according to the levels in Figure 7; (e) A step of recovering donor thymus tissue sections with partially depleted T cells as culture products derived from postnatal allogeneic thymus tissue; (f) A step of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (g) A step of maintaining culture products derived from cryopreserved postnatal allogeneic thymus tissue in liquid nitrogen in a cryopreserved postnatal allogeneic thymus tissue culture product bank.

[0244] In one aspect of this disclosure, a method is provided for promoting donor-specific tolerance to allogeneic solid organ grafts obtained from deceased human donors in human recipients requiring solid organ transplantation, the method comprising the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A process for providing suitable solid organs derived from a living human donor; (d) The process of transplanting solid organs into the recipient; (e) The process of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing culture products derived from cryopreserved postnatal allogeneic thymus tissue, maintained in a cryopreserved postnatal allogeneic thymus tissue culture product bank, wherein the culture products derived from cryopreserved postnatal allogeneic thymus tissue are processed from thymus tissue derived from a thymus donor expressing an HLA allele that matches the recipient's HLA allele that is not present in the solid organ graft; the donor-thymus tissue is subjected to a habituation regimen for a period of approximately 6 to 21 days; and the habituation regimen for the donor-thymus tissue is further subjected to a thymus organ culture to create sections of partially T-cell depleted thymus tissue. A step comprising aseptically processing donor thymic tissue in soil, detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in the thymic organ medium during the course of a habituation regimen; wherein the level of the marker in the thymic organ medium decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, and increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; (g) Thawing culture products derived from cryopreserved postnatal allogeneic thymus tissue; and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0245] In one aspect of this disclosure, a culture product derived from cryopreserved postnatal allogeneic thymus tissue is provided, prepared by a method comprising the following steps: (a) The process of obtaining suitable thymic tissue from a donor; (b) HLA alleles: The process of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1; (c) A step of subjecting thymic tissue to an adaptation regimen for a period of approximately 6 to 21 days, wherein the adaptation regimen for donor-thymic tissue includes aseptically treating the donor-thymic tissue in thymic organ culture medium to produce partially T-cell depleted donor-thymic tissue sections; (d) A step of detecting the level of at least one marker selected from L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11 in thymic organ medium during the course of a habituation regimen, wherein the level of the marker in thymic organ medium decreases if the marker is L-selectin, M-CSF, galectin-7, or IL-1, and increases if the marker is CCL21, CXCL12, CXCL16, or CCL11; (e) A step of recovering donor thymus tissue sections with partially depleted T cells as culture products derived from postnatal allogeneic thymus tissue; (f) A step of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (g) A step of maintaining culture products derived from cryopreserved postnatal allogeneic thymus tissue in liquid nitrogen in a cryopreserved postnatal allogeneic thymus tissue culture product bank.

[0246] In one aspect of this disclosure, a method is provided to promote donor-specific tolerance to allogeneic solid organ grafts obtained from deceased human donors in human recipients requiring solid organ transplantation, the method comprising the following steps: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants in order to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) providing a suitable solid organ derived from a living human donor; (d) transplanting the solid organ into the recipient; (e) treating the recipient with a maintenance immunosuppressive regimen; (f) providing a culture product derived from cryopreserved postnatal allogeneic thymic tissue maintained in a cryopreserved culture product bank of postnatal allogeneic thymic tissue, wherein the culture product derived from the cryopreserved allogeneic thymic tissue is processed from thymic tissue from a thymic donor expressing HLA alleles that match the HLA alleles of the recipient that are not present in the solid organ transplant graft; the donor thymic tissue is subjected to an acclimation regimen for a period of about 6 days to about 21 days; and further, the acclimation regimen for the donor thymic tissue includes aseptically processing the donor thymic tissue in a thymic organ medium to create sections of thymic tissue with partially depleted T cells, markers: 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, detecting the level of at least one marker in the thymic organ medium during the course of the acclimation regimen; the level of the marker in the thymic organ medium increasing or decreasing according to the level of the marker in FIG. 56; (g) thawing the culture product derived from the cryopreserved postnatal allogeneic thymic tissue; and (h) A step of transplanting a thawed culture product derived from cryopreserved postnatal allogeneic thymus tissue into a recipient, wherein the dose of the culture product derived from cryopreserved postnatal allogeneic thymus tissue is approximately 1,000 to 22,000 ml 2 Thymus tissue surface area / Recipient body surface area (m²) 2 The process involves a unit of thymocyte proliferation and tolerance in the recipient, further comprising a culture product derived from transplanted postnatal allogeneic thymic tissue.

[0247] In some aspects and embodiments of the present disclosure, solid organ transplants include heart transplants, kidney transplants, liver transplants, lung transplants, heart / lung transplants, pancreas transplants, intestine transplants, stomach transplants, abdominal wall transplants, craniofacial transplants, scalp transplants, penile transplants, uterine transplants, unilateral or bilateral upper limb transplants, unilateral angiogenic composite allogeneic transplants, or combinations thereof.

[0248] In some aspects and embodiments of the present disclosure, solid organ transplantation is a heart transplant, or a pediatric heart transplant, or an adult heart transplant.

[0249] In one embodiment of the various aspects and embodiments of this disclosure, the habituation regimen is a period of 5 days, or 6 days, or 7 days, or 8 days, or 9 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 5-6 days, or 5-7 days, or 5-8 days, or 5-9 days, or 5-10 days, and This refers to a period of 6-7 days, or 6-8 days, or 6-9 days, or 6-10 days, or 6-11 days, or 6-12 days, or 6-21 days, or 7-21 days, or 8-21 days, or 9-21 days, or 10-21 days, or 11-21 days, or 12-21 days, or 13-21 days, or 14-21 days, or 15-21 days, or 16-21 days, or 17-21 days, or 18-21 days, or 19-21 days, or 20-21 days.

[0250] In one aspect of this disclosure, we provide a culture product derived from cryopreserved postnatal allogeneic thymus tissue suitable for transplantation into humans, comprising the following steps: (a) The process of obtaining thymic tissue from a human donor; (b) A step of subjecting thymic tissue to a habituation regimen for a period of approximately 6 to 21 days, wherein the habituation regimen for donor-thymic tissue comprises aseptically treating the donor-thymic tissue in thymic organ medium to produce donor-thymic tissue sections in which T cells are partially depleted; the levels of L-selectin and / or M-CSF and / or galectin-7 and / or IL-16 in the thymic organ medium decrease during the course of the habituation regimen; and the levels of CCL21 and / or CXCL12 and / or CXCL16 and / or CCL11 in the thymic organ medium increase during the course of the habituation regimen; (c) A step of recovering donor thymus tissue sections with partially depleted T cells as culture products derived from allogeneic thymus tissue after birth; (d) A step of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (e) A step of maintaining culture products derived from cryopreserved postnatal allogeneic thymus tissue in liquid nitrogen in a cryopreserved postnatal allogeneic thymus tissue culture product bank.

[0251] In one embodiment of several aspects and embodiments of this disclosure, the CCL21 level in the thymic organ medium increases during the course of the acclimatization regimen.

[0252] In one embodiment of several aspects and embodiments of this disclosure, the level of L-selectin in the thymic organ medium decreases during the course of the acclimatization regimen.

[0253] In one embodiment of several aspects and embodiments of the present disclosure, the levels of one or more M-CSF, galectin-7, and IL-16 in the thymic organ medium decrease during the course of the acclimatization regimen.

[0254] In one embodiment of several aspects and embodiments of the present disclosure, the levels of one or more CCL21, CXCL12, CXCL16, and CCL11 in the thymic organ medium increase over time during the acclimatization regimen.

[0255] In one embodiment of several aspects and embodiments of the present disclosure, the method further includes determining in the Donner-thymus tissue section during a habituation regime period the presence of regions positive for keratin AE1 / AE3 scattered throughout the Donner-thymus tissue section, the presence of at least one Hassall body, and the presence of CK14 staining and intact nuclei scattered throughout the Donner-thymus tissue section.

[0256] In one aspect of this disclosure, a kit for carrying out any of the above-described aspects and embodiments is provided, along with instructions for use in determining whether a product derived from postnatal allogeneic thymic tissue culture is suitable for transplantation into humans.

[0257] In one embodiment of the various aspects and embodiments of this disclosure, the kit comprises at least one antibody that specifically binds to a marker: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[0258] In one embodiment of the various aspects and embodiments of this disclosure, the kit comprises one or more antibodies that specifically recognize the marker shown in Figure 56.

[0259] In one aspect of this disclosure, a kit is provided, along with instructions for use, for determining whether a culture product derived from allogeneic, cultured postnatal thymus tissue, cryopreserved according to any of the above aspects and embodiments, is suitable for transplantation into humans.

[0260] In one embodiment of the various aspects and embodiments of this disclosure, the kit comprises at least one antibody that specifically binds to a marker: L-selectin, M-CSF, galectin-7, IL-16, CCL21, CXCL12, CXCL16, or CCL11.

[0261] For clarity, it will be further understood that certain features described herein, as described in the context of different aspects of this disclosure and / or in individual embodiments, may also be provided in combination as a single embodiment. Conversely, for simplicity, various features described in the context of a single aspect of this disclosure and / or in a single embodiment may be provided individually or in any preferred secondary combination.

[0262] For a more complete understanding of the principles and advantages disclosed herein, the following description is provided in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0263] [Figure 1] This paper describes how culture products derived from allogeneic thymic tissue after birth (e.g., CTT, RVT-802) provide immune rearrangement in congenital athymus after transplantation. [Figure 2] A schematic diagram of the process for reconstitution of the immune system in rats, as described somewhere in Example 5, is shown, which involves removing the thymus from immunologically normal Lewis rats; administering antibodies to kill T cells in recipient rats; transplanting cultured thymic tissue from donor rat offspring into recipient rats; administering immunosuppressants for approximately 4 months; and evaluating T cell development in recipient rats. Notably, all rats in the treatment group had more than 10% naive T cells before discontinuation of cyclosporine. [Figure 3] This shows the development of naive T cells in two experimental recipient rats (rising line on the right) and two control rats that did not receive a thymic tissue graft (thick line in the baseline area) in Example 5. [Figure 4] A schematic diagram of the manufacturing process is shown, which involves harvesting the thymus from a donor, culturing thin sections of donor thymus tissue prepared with a manual microtome for up to 21 days, and transplanting the cultured thymus tissue into the recipient's quadriceps muscle. [Figure 5] Figure 5A shows a schematic representation of a thymic tissue section for characterization testing, as described in paragraph

[0539] . Figure 5B shows a thymic tissue section on a cellulose filter on a surgical sponge in a tissue culture dish, similar to those used for culturing thymus. [Figure 6] Figures 6A–H show histological examination of thymic tissue sections derived from a lot (MFG-056) of cultured thymic tissue at 5, 9, 12, and 21 days after thymic harvesting from a donor. Hematoxylin and eosin-stained sections (left panel), as well as their corresponding reactivity with a cocktail of anti-cytokeratin antibodies AE1 / AE3 (right panel; brown indicates a positive reaction), are shown for day 5 (Figures 6A, 6B), day 9 (Figures 6C, 6D), day 12 (Figures 6E, 6F), and day 21 (Figures 6G, 6H), respectively. The bar in the lower left of each panel represents 100 μm. The hematoxylin and eosin-stained panels show the progression of T cell depletion over time. Figures 6E and 6F are mainly epithelial cells. Epithelial aggregation of the subcapsular cortex occurs as thymocytes are depleted over time. Similar aggregation occurs in the medullary region of the thymus. The photograph is by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 7] Figures 7A and 7B show the histology of thymic tissue sections at day 0 of time-lapse observation, at scales of 5 mm (Figure 9A) and 100 μm (Figure 7B), respectively. This shows the thymus and thymocytes at day 0 at low magnification (bar, 5 mm) and high magnification (bar, 100 μm). This is a normal thymus. At this point, both the cortex and medulla have numerous thymocytes with dark blue nuclei, contributing to the overall dark blue appearance of the tissue. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 8]Figures 8A and 8B are images of hematoxylin and eosin-stained slides showing the histology of thymic tissue sections at 5 mm (Figure 8A) and 100 μm (Figure 8B) scales, respectively, on day 5 of time-course observation. Progression of thymic cell depletion results in a more eosinophilic (pink) appearance of the tissue. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 9] Figures 9A and 9B are images of hematoxylin and eosin-stained slides of thymic tissue sections observed 12 days into time, at scales of 5 mm (Figure 9A) and 100 μm (Figure 9B), respectively. Progressive thymic cell depletion is observed. High magnification reveals numerous eosinophilic cell bodies without nuclei, characteristic of necrotic cells that have undergone nuclear lysis (lysis of the cell nucleus). This degree of necrosis is expected at this stage of culture. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 10] Figures 10A and 10B are images of hematoxylin and eosin-stained slides showing the histology of thymic tissue sections at 5 mm (Figure 10A) and 100 μm (Figure 10B) scales, respectively, on day 21 of time-series observation. Note the preservation of the overall structure of the tissue, including the subcapsular cortex, cortical region, and medullary region, which contain numerous Hassar bodies, in Figure 10B. The dark, small cells are mostly necrotic thymocytes that have not yet undergone nuclear lysis. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 11] Figures 11A–E show representative thymic sections immunostained with a cocktail 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. The thymic epithelial network structure remains intact even as the culture progresses. Bars represent 400 μm. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 12]Figures 12A and 12B show histological images of thymic tissue slides after exposure to forced degradation conditions in 10X PBS. Figure 12A shows the cortex 9 days after exposure to forced degradation conditions. Figure 12B shows the cortex 21 days after exposure to forced degradation conditions. In Figure 12A, the blue smear represents DNA released from cells. Most cells show signs of degradation, although small cell foci with intact nuclei are identifiable. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 13] These are hematoxylin and eosin-stained tissue sections from the clinical specimen MLM247. This is culture day 0. The bar is 200 μm. This is a frozen section on day 0. Because it was frozen, the tissue looks different from formalin-fixed tissue embedded in paraffin on day 0. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 14] These are frozen sections of clinical specimen MLM219, stained with hematoxylin and eosin. Because these are frozen sections, the tissue looks different from cultured, paraffin-embedded, formalin-fixed tissue shown above. However, the major histological features of thymocyte depletion and stable TEC viability are clearly shown. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 15] This is a photograph of thymic tissue that was just collected. [Figure 16] This is a schematic diagram illustrating the collection, culture, transplantation, and biopsy of a rat kidney capsule CTT graft, as shown in Example 5. [Figure 17]Figures 17A-D show photographs of thymic tissue collected from 3-day-old F1 (LWxDA) rats, cut into four pieces, as described in Example 5 (Figure 17A). Figure 17B shows photographs of thymic tissue pieces cultured for 5-7 days in thymic organ medium on a sterile mixed cellulose ester filter in a 37°C CO2 incubator, as described in Example 5 (Figure 17B). Figure 17C is a photograph of the CTT transplanted under the kidney capsule of an LW rat. Figure 17D is a photograph of the thymic graft collected 6 months after transplantation. The arrow indicates the CTT under the kidney capsule. [Figure 18] Figures 18A-D are photographs showing histological images of fresh thymic tissue (upper frame) and CTT (lower frame) at 100x magnification. Figure 18A shows a comparison of medullary differentiation in fresh thymic tissue (upper frame) stained with hematoxylin and eosin, and CTT cultured for 5 days (lower frame), as described in Example 5. Figure 18B shows a typical lace-like pattern observable in CTT cultured for 5 days when stained for cytokeratin, compared to fresh thymic tissue (upper frame), as described in Example 5. Figure 18C shows fresh thymic tissue (upper frame) and T-cell depleted CTT (lower frame) stained for Ki-67. Figure 18D shows fresh thymic tissue (upper frame) stained for CD3 and CTT cultured for 5 days and then stained for CD3 (lower frame). The brown staining observed in CD3-stained CTTs (Figure 18D, bottom frame) likely indicates some living cells and remnants of dead T cells that were not washed away from the tissue. [Figure 19]Figures 19A-D are photographs showing histological images of fresh thymic tissue (upper frame) and CTT (lower frame) at 600x magnification. Figure 19A shows a comparison of medullary differentiation of fresh thymic tissue (upper frame) and CTT cultured for 5 days (lower frame) stained with hematoxylin and eosin, as described in Example 5. Figure 19B shows a typical lace-like pattern observable in CTT cultured for 5 days when stained for cytokeratin, compared to fresh thymic tissue (upper frame), as described in Example 5. Figure 19C shows fresh thymic tissue (upper frame) and T-cell depleted CTT (lower frame) stained for Ki-67. Figure 19D shows fresh thymic tissue (upper frame) stained for DC3 and CTT thymic tissue cultured for 5 days and stained for CD3 (lower frame). The brown staining observed in CD3-stained CTT (Figure 19D, lower frame) likely indicates remnants of dead T cells that were not washed away from living cells and tissues. [Figure 20] This is a schematic diagram of the experimental design reported in Example 5. This diagram is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 21] This figure shows recipient-type T cells that regrow after allogeneic transplantation of CTT, as observed in the lower right quadrant. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 22] Figures 22A and 22B show transplanted thymuses removed 8.5 months after transplantation, exhibiting cytokeratin-positive staining (Figure 22A) and T-cell staining similar to that of a native thymus (Figure 22B). The original magnification was 400x. These figures are published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 23]The figure shows a plot of significantly increased circulating CD4 and CD8 T cell numbers compared to control animals that did not receive CTT. It also shows significantly increased numbers of naive CD4 and naive CD8 T cells in the cultured thymus tissue transplant (CTT) group compared to the control group that did not receive CTT, and significantly increased numbers of new thymic export cells (RTEs) of CD4 and CD8 in the cultured thymus tissue transplant (CTT) group compared to the control group that did not receive CTT. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 24] Figure 24A shows immunohistochemical analysis of a transplanted CTT removed on day 80, showing normal thymic tissue beneath the renal capsule (right side of Figure 24A). Figure 24B shows hematoxylin and eosin-stained explants. Staining for viable T cells (CD3), T cell proliferation (Ki67), and cytokeratin (detected by rabbit polyclonal antibody) is shown. In the panel stained for cytokeratin, a lace-like pattern is observed along with Hassall's bodies formation in the TEC (arrows). This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 25] This figure shows the survival rate (%) after thymectomy and immunosuppression in LW rats with DA heart grafts in the presence of a CTT graft (solid triangle, blue line) and in the absence of a CTT graft (inverted triangle, red line). LW rats with CTTs are immunotolerant; LW rats without CTTs are immunodeficient and therefore do not reject the DA heart. The control shows complete rejection of the DA heart graft in LW non-operated rats (white square). LW control animals also did not reject the LW heart graft (white circle with horizontal line) (n=9). This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 26]Figures 26A and 26B are photographs of allogeneic grafts (DA hearts) from animals that received (Figure 26A) and did not receive (Figure 26B) a CTT, showing mononuclear cell infiltration without signs of rejection according to the 2004 International Society for Heart & Lung Transplantation (ISHLT) (blue-filled squares and red-filled triangles), as shown in Figure 26C. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 27] This figure plots animal survival rate versus graft survival days for cervical BN heart graft engraftment in LW rats with introduced CTT (immunonormal and rejected cervical allogeneic BN hearts) and control LW animals without introduced CTT (immunodeficient due to thymic deficiency and unable to reject cervical BN hearts), which were inserted into BN controls (LW rats that rejected cervical BN hearts) and syngeneic controls (LW rats that did not reject cervical LW hearts). This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 28] Figures 28A and 28B are photographs of BN cardiac tissue at 11 and 46 days, respectively, of BN cardiac tissue with CTT induction (Figure 28A) and BN cardiac tissue without CTT induction (Figure 28B). These photographs form the basis for the data in Figures 27 and 29. The heart in Figure 28A is not rejected due to immune tolerance. The heart in Figure 28B is not rejected due to immunodeficiency caused by thymic deletion. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 29]This shows the rejection evaluation of cervical BN hearts. Syngeneic LW hearts (white circles) transplanted into LW rats were not rejected. BN hearts (black circles) transplanted into LW rats were rejected. BN hearts (black squares) transplanted into LW rats that had undergone CTT were rejected. BN hearts (shaded triangles) transplanted into LW rats that had not undergone CTT were weakly rejected by two rats and not rejected by the other three rats. These data indicate that rats with CTT, when CTT expresses DA, were able to accept DA hearts while strongly rejecting third-party hearts (Figure 26C). Rats without CTT were immunodeficient and did not reject either DA (Figure 26C) or BN hearts. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 30] Figures 30A and 30B are photographs of BN hearts in rats that underwent or did not undergo CTT induction, compared to LW and DA hearts, respectively. In Figure 30A, after immunosuppression was removed and the BN heart was transplanted, the BN heart was rapidly rejected and was very large in size due to its overall inflammation. The LW heart is of normal size for a heart that pumps blood to the body. The DA heart is small because it was located in the abdomen and did not need to pump blood. In Figure 30B, the rats were immunodeficient and could not reject either the BN or DA heart after immunosuppression was removed. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 31]Figures 31A and 31B are plots of rejection evaluation of transplanted cervical BN hearts from CTT-introduced rats and non-CTT-introduced rats compared to BN control rats and syngeneic control rats. Figure 31A shows the quantification of inflammatory cells in the first abdominal DA heart allograft. Syngeneic controls show that LW rats do not reject the LW heart. DA controls show that LW rats reject the DA heart. The CTT group does not reject the DA heart due to tolerance. The non-CTT group does not reject the DA heart due to immunodeficiency caused by thymic deletion. Figure 31B shows the quantification of inflammatory cells in the second cervical BN heart allograft. Syngeneic controls show that LW rats do not reject the LW heart. BN controls show that LW rats reject the BN heart. The CTT group rejects the BN heart because it is immunonormal. The non-CTT group does not reject the BN heart due to immunodeficiency caused by thymic deletion. Figure 31C shows the hearts of DA and BN rats, as well as a native LW heart, recovered from LW recipients upon rejection of a cervical BN heart. The lower right panel shows the T cells (brown) in the BN heart that led to the rejection. Figure 31D shows T cell infiltration in LW, DA, and BN hearts from control animals that were not CTT-induced. These animals were immunodeficient and therefore did not show T cell infiltration. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 32]Figures 32A-32C: Humoral tolerance after CTT. Figure 32A shows representative histogram plots for post-transplant donor-specific alloantibodies (anti-DA and anti-BN antibodies) measured by T cell flow crossmatch. The upper left panel of Figure 32A (DA control) shows the development of anti-DA antibodies (thick line) in normal LW rats after receiving an ectopic abdominal DA heart graft. The upper center panel of Figure 32A shows the absence of anti-DA antibodies in LW rats that received CTT; this suggests immune tolerance. The upper right panel shows unresponsiveness by LW rats without CTT; this reflects immunodeficiency in rats after thymectomy and T cell depletion without donor-thymus reception. The lower left panel of Figure 32A shows normal anti-BN antibodies formed by normal LW rats receiving a BN heart in the neck. The lower central panel of Figure 32A shows the normal response of LW rats with CTT to BN after receiving a BN heart graft in the neck, demonstrating their immune capacity and ability to reject third parties. The lower right panel of Figure 32A shows the unresponsiveness of LW rats without CTT to BN after receiving a BN heart graft in the neck, demonstrating immunodeficiency and a lack of ability to reject third parties. Figure 32B shows the anti-DA antibody levels after the first DA heart transplant. LW rats with CTT derived from an LWxDA thymus donor do not produce anti-DA antibodies after DA heart transplant because they are tolerant to DA. LW rats without CTT do not produce anti-DA antibodies after DA heart transplant because they are immunodeficient. Figure 32C shows the anti-BN antibody levels after the second neck BN heart transplant. LW rats with CTT derived from an LWxDA donor produce antibodies against BN, demonstrating their immune capacity against third parties. LW rats lacking CTT do not produce antibodies against BN and exhibit immunodeficiency. This figure is published in Kwun, J. et al., JCI Insight (2020) Jun 4;5(11). [Figure 33]Figures 33A–J show micrographs of immunohistochemical evaluations of fresh and cultured non-human primate (NHP) thymus tissues from 8-month-old NHPs. The top row shows NHP thymus on the day of collection, and the bottom row shows NHP thymus after 12 days of culture. The tissues were stained with hematoxylin and eosin (Figures 33A and 33F), CD3 (Figures 33B and 33G), pancytokeratin (CK) antibodies AE1 / AE3 (Figures 33C and 33H), Ki-67 (Figures 33D and 33I), and CK14 (Figures 33E and 33J). All images are at 20x magnification. [Figure 34] Figures 34A–P show the analysis of cryopreserved cultured thymic tissue from 8-month-old non-human primates (NHPs) after 12 days of culture. The top row shows cytokeratin at harvest (Figure 34A), 6 days of culture (Figure 34B), 12 days of culture (Figure 34C), and after thawing for 12 days of culture followed by 35 days of cryopreservation and subsequent photography (Figure 34D). The cytokeratin (AE1 / AE3) in Figure 34D is similar to the cytokeratin in Figure 34C. The second row shows CK14 staining at the same point in time in Figure 34E (at harvest), Figure 34F (6 days of culture), Figure 34G (12 days of culture), and Figure 34H (after 12 days of culture, 35 days of cryopreservation, and then thawing). The CK14 in Figure 34H is very similar to that in the panel of Figure 34G. The third column shows CD3 staining in Figures 34I, 34J, 34K, and 34L at the same time point, which has the expected disappearance of viable T cells over time. The panel in Figure 34L is similar to the panel in Figure 34K, which contains only a small number of T cells. The fourth column shows Ki-67 staining of proliferating T cells at the same time point: Figures 34M, 34N, 34O, and 34P. Staining with Ki-67 disappears by day 6 (Figure 34N), when T cells are almost dead. This figure illustrates the cryopreservation potential of non-human primate thymus as well as how cultured thymic tissue is cryopreserved for patient use. All photographs are at 40x magnification. [Figure 35]This is a schematic diagram of an experimental transplantation strategy using maximally MHC-incompatible CMV-uninfected rhesus monkeys. The recipient animal (Y) undergoes a complete thymectomy. The donor animal (X) provides both cultured thymic tissue and a heart (first Tx and second Tx) to be introduced into recipient Y at an ectopic site. Immunosuppressants are then discontinued, and donor-specific tolerance is indicated by (i) continuous beating of the donor heart, (ii) tolerance to the donor in nMLRs that are responsive to a third party, and (iii) rejection of a skin graft from a third-party donor animal Z (third Tx). [Figure 36] This graph shows flow cytometry results illustrating a common gating strategy for identifying new thymic extrusion cells (RTEs). Peripheral blood mononuclear cells from non-human primates are collected and analyzed using multicolor flow cytometry. The first step is single-cell identification in the upper panel 1. "Singlet" is used to identify lymphocytes (low SSC, lateral scatter, and high CD45) in the upper panel 2. CD3 T cells in the lymphocytes are identified in the upper panel 3. CD4 and CD8 cells are separated from CD3 cells as shown in the upper panel 4. In the lower panel 1, CD28 and CD95 are used with the CD4 gate to identify the CD4+ naive subset, central memory subset, and effector memory subset in the lower panel 1. In the lower panel 2, CD31 is used to show the percentage of CD4+ naive cells that are RTEs. The third and fourth panels in the lower section show the same approach to RTE, but for CD8 naive T cells. As can be seen from the figures, RTE accounts for 99.6% of the CD4 naive subset and 95.2% of the CD8 naive subset. [Figure 37]Microscopic images and graphs of CCL21 evaluation in cultured infant thymus are shown. CCL21 is produced at high levels by cultured infant thymus. Immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) on ​​day 16 of culture is shown in the left panel (upper left panel, 2x magnification; lower left panel, 20x magnification). The corresponding time course of CCL21 secretion per day into the culture medium is shown on the right for three infant thymus cultures (R&D Systems Duo-Set ELISA). Thus, cultured thymic tissue can produce CCL21, a functionally important biomolecule that is a chemokine involved in attracting immature thymocyte progenitor cells to the thymus. [Figure 38]This outlines the experimental design of Example 8 for evaluating the successful engraftment of cultured thymic tissue, subsequent immune tolerance to a compatible heart, and rejection of incompatible skin in a CMV-uninfected NHP model. Specifically, after thymectomy of the recipient (stage 2, week 1) and confirmation of complete thymic removal, the recipient is depleted of T cells and immunosuppression is initiated with tacrolimus. Incompatible cultured donor-thymic tissue derived from unrelated NHP is transplanted into the recipient at stage 3, week 3. A biopsy of the thymic graft is performed at stage 3, week 10 to evaluate thymic cell proliferation. After naive T cells develop several months later, the recipient should be tolerant to the donor. The recipient is then ectopically transplanted with a heart derived from the thymic donor (stage 4, week 4). Immunosuppression is discontinued. Cardiac beating occurs subsequently (indicating tolerance). Furthermore, a mixed lymphocyte response is performed at week 10 of Stage 4 to demonstrate tolerance to cryopreserved donor cells and rejection of third-party cells. If more time is needed for naive T cells to develop, Stage 5 is used. If tolerance does not appear, Stage 6 is used. To demonstrate that the thymic tissue transplantation method is successful in NHP, recipient thymus may be transplanted into recipient NHP. It is useful to know how the format in Figure 38 was adjusted. The experiment uses three monkeys. Note that the original spreadsheet contained procedures for all three animals and spanned multiple pages with a width of one page. Since the spreadsheet was wider than the allowable width in this patent application, each row of the spreadsheet was divided into three pages. The first monkey is the thymus and heart donor; the procedures for this monkey are shown in the left column, such as pages 1, 4, and 7. The second monkey is the recipient of the thymus and heart; the information in the center column is described on pages 2, 5, and 8, etc. The third monkey is the control; the information in the right column is described on pages 3, 6, and 9, etc. [Figure 39]The experimental design for Example 9 is outlined below, and is identical to that of Example 8, except for the addition of the immunosuppressant mycophenol mofetil (MMF). The drug MMF is routinely used in heart transplantation. This study evaluates whether MMF has any adverse effects on cultured thymic tissue grafts. [Figure 40] Figures 40A–D are micrographs of fresh thymic sections from culture d0 showing thymic structure. In Figures 40A–B, hematoxylin and eosin (H&E) staining shows clearly identified cortical and lightly stained medullary regions, as expected for a normal pediatric thymus. Figure 40C shows immunohistochemistry with a pancytokeratin antibody (AE1 / AE3) cocktail that detects all types of epithelial cells together, demonstrating that thymic epithelial cells are present subcapsularly and in a bright, lacy network in both the cortex and medulla (brown staining indicates a positive antibody reaction). The arrows in Figures 40B and 40C point to Hassall bodies. Figure 40D shows antibody staining for cytokeratin 14 (CK14) (brown). The CK14 antibody reacts with thymic epithelial cells in the subcapsular cortex and medulla, as well as with scattered thymic epithelial cells in the cortex. The dotted line highlights the medullary region surrounded by the cortex. SCC stands for subcapsular cortex, Cor stands for cortex, and M stands for medulla. The scale bar in Figure 40A represents 1 mm; the scale bar in Figures 40B-D represents 500 μm. [Figure 41]Figures 41A–D are micrographs showing examples of Hassall's bodies in cultured thymic sections. Histological images of Hassall's bodies are shown on day 0 (Figures 41A–B) and day 9 (Figures 41C–D) of culture. Figures 41A and 41C show hematoxylin and eosin (H&E) staining; Figures 41B and 41D show reactivity with pancytokeratin (AE1 / AE3) antibodies (brown indicates a positive reaction). Arrowheads in Figures 41A–D point to representative Hassall's bodies, which are not very prominent in hematoxylin and eosin-stained sections of cultured thymus due to depletion and necrosis of surrounding thymic cells. However, Hassall's bodies can still be easily identified by careful examination or by using immunohistochemistry. Scale bars in Figures 41A–D represent 100 μm. [Figure 42] Figures 42A–D are micrographs showing the structure of cultured thymus on day 7. Hematoxylin and eosin (H&E) staining in Figures 42A–B shows significant thymic depletion, although some cortical regions (Cor) still contain numerous thymocytes with retained nuclei. Pancytokeratin (AE1 / AE3) immunohistochemistry in Figure 42C and cytokeratin 14 (CK14) immunohistochemistry in Figure 42D show aggregation of thymic epithelium in the subcapsular cortex (SCC) and medulla (M). Brown areas in Figures 42C–D indicate antibody-positive reactions. The scale bar in Figure 42A represents 1 mm, and in Figures 42B–D it represents 500 μm. [Figure 43]Figures 43A–D are micrographs showing the structure of cultured thymus on day 9. Figures 43A–B show hematoxylin and eosin (H&E) staining. Only a very small number of viable T cells or thymic epithelial cells, if present at all, are found in the faintly stained area enclosed by the dotted line in Figure 43A (this is almost completely necrotic (Necr)). Most of the nuclei previously present in this area have been degraded by nuclear lysis. Other areas where nuclei from residual thymocytes have not been completely degraded still stain dark blue with hematoxylin. The arrows in Figure 43B point to Hassall bodies. Figure 43C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 43D shows cytokeratin 14 (CK14) immunoreactivity (brown). The scale bar in Figure 43A represents 1 mm, and in Figures 43B–D it represents 500 μm. [Figure 44] Figures 44A–D are micrographs showing the structure of cultured thymus on day 12. Figures 44A–B show hematoxylin and eosin (H&E) staining. At this point, many thymocytes have disappeared from the tissue or died, their nuclei are lysed, and the tissue is more eosinophilic (pink). Some areas have a significantly reduced thymocyte cellularity but retain the structure characteristic of normal uncultured thymus, including cortical-like regions (Cor) and medullary-like regions (M) that stain more basophilic (blue). Figure 44C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 44D shows cytokeratin 14 (CK14) immunoreactivity (brown). At this point, the subcapsular cortex (SCC) is thickened, and epithelial cells are more prominent due to the reduced number of thymocytes present. Arrows point to representative Hassall bodies. The bar in Figure 44A represents 1 mm, while in Figures 44B to D it represents 500 μm. [Figure 45]Figures 45A–D are micrographs showing the structure of cultured thymus on day 20. Figures 45A–B show hematoxylin and eosin (H&E) staining. At this point, most thymocytes have disappeared from the tissue or died, their nuclei are lysed, and the tissue is more eosinophilic (pink). Scattered cells with nuclei characteristic of thymocytes are clearly visible, but large groups of residual thymocytes are rare. Figure 45C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown). Much of the epithelium previously present in the medullary region (M) aggregates due to the disappearance of medullary thymocytes, but scattered epithelial cells remain, showing a bright, lace-like three-dimensional network of residual thymic epithelial cells. In Figure 45D, cytokeratin 14 (CK14) immunohistochemistry (brown) highlights the former medullary region and subcapsular cortex. Arrows point to Hassall bodies. The bar in Figure 45A represents 1 mm, while in Figures 45B to D it represents 500 μm. [Figure 46] Figures 46A and 46B are micrographs showing examples of intact nuclei in thymic sections. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Figure 46A) and the medulla on day 21 (Figure 46B). Hematoxylin and eosin staining; scale bar represents 50 μm. [Figure 47] Figures 47A–E are micrographs showing a comparison of the thymic epithelial network in cultured thymic tissue at different time points. Figure 47A shows day 0, Figure 47B shows day 5, Figure 47C shows day 9, Figure 47D shows day 12, and Figure 47E shows day 21. There are time-dependent differences between thymocyte depletion and necrosis, so the tissue becomes less halophilic (blue) over time, while the thymic epithelial network (brown) structure remains intact even as the culture progresses. Both cortical and medullary epithelium can aggregate when intercalating thymocytes are depleted. Brown indicates a positive reaction with an anti-cytokeratin antibody (AE1 / AE3) cocktail; hematoxylin counterstaining. Scale bar represents 400 μm. [Figure 48]Figures 48A–K are micrographs showing examples of CD3 immunohistochemistry of thymic sections as a function of time in culture. Figures 48A–B show that on 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 48B) shows pale blue nuclei surrounded by a brown ring of immunoreactivity, consistent with CD3 membrane expression. In Figures 48C–E, the tissue still shows extensive reactivity with the CD3 antibody even on day 7. However, Figures 48D–E, when observed at high magnification, show that most of the immunoreactivity (brown) is related to the remnants of dead thymocytes, as most of the brown foci are anucleated (Figure 48D). Small cell foci showing intact nuclei and membrane staining (arrows) are still identified in areas away from the remnants (Figure 49E). As the culture progresses to day 9 (Figures 48F-G), day 12 (Figures 48H-I), and day 21 (Figures 48J-K), the reactivity of thymocytes with cell debris remains strong, making it difficult to accurately detect potentially intact cells among the debris. All sections shown are from a single lot, representative of multiple lots examined at these points in time. The scale bar in Figures 48A, 48C, 48F, 48H, and 48J represents 500 μm; in Figures 48B, 48D, 48E, 48G, 48I, and 48K, it represents 50 μm. [Figure 49]Figures 49A–K are micrographs showing examples of Ki-67 immunohistochemistry in thymic sections as a function of time in culture. All sections shown are from a single lot representative of multiple lots tested at similar time points. Figures 49A–B show that on day 0, the nuclei of most immature T cells in the cortex (Cor) react strongly with the Ki-67 specific antibody. High magnification (Figure 49B) shows strong positive reactivity with the nuclei of cortical thymocytes (brown), while only a very small number of lymphocytes in the medulla (M) react with the Ki-67 antibody. Figures 49C–E show that by day 7, the nuclei of most thymocytes remaining in the cortical region are small, have indistinct nuclear boundaries consistent with apoptosis, and are unable to react with the Ki-67 specific antibody. Cells that react with the antibody (Figure 49E, arrows) have large nuclei, suggesting they are thymic epithelial cells. Similar lack of Ki-67 labeling in residual thymocyte nuclei is observed on day 9 (Figures 49F-G), day 12 (Figures 49H-I), and day 21 (Figures 49J-K). Scale bars in Figures 49A, 49C, 49E, 49G, and 49I represent 500 μm; in Figures 49B, 49D, 49F, 4950H, and 49J, they represent 50 μm. [Figure 50] Figures 50A to 50H show plots of selected soluble molecules detected in conditioned medium from human thymic organ cultures. Figure 50A is a plot of Ln / pg versus culture days for L-selectin; Figure 50B is a plot of Ln / pg versus culture days for M-CSF; Figure 50C is a plot of Ln / pg versus culture days for galectin-7; Figure 50D is a plot of Ln / pg versus culture days for IL-16; Figure 50E is a plot of Ln / pg versus culture days for CCL21; Figure 50F is a plot of Ln / pg versus culture days for CXCL16; Figure 50G is a plot of Ln / pg versus culture days for CCL11; and Figure 50H is a plot of Ln / pg versus culture days for CXCL12. [Figure 51] This plot shows the thymocyte content (pg / ml) in human thymus culture sections from day 1 to day 21 of the culture process. [Figure 52]These are photographs of immunohistochemical evaluation of viable thymocytes in human thymic tissue culture sections as a function of time. Figure 52A is a photograph of immunohistochemistry using an anti-CD3 antibody that identifies cells as T cell lineages and an anti-Ki-67 antibody that identifies proliferating cells, showing a rapid decline in thymocyte viability in the early stages of culture. Figure 52B is a photograph of day 0 showing that the plasma membranes of essentially all immature T cells in the cortex and medulla are reactive with anti-CD3 in membrane patterns. Figure 52C shows immunohistochemistry using an antibody specific to the Ki-67 proliferative marker, showing widespread reactivity with cortical thymocytes at day 9. Figure 52D is a histological image of cultured thymus at day 9 using hematoxylin and eosin staining. A decrease in basophilia (blue) suggests donor-thymocyte disappearance during culture; Figure 52E shows that after several days of culture, the remaining cell fragments after dead thymocytes undergo nuclear lysis / fusion, although they are mostly brown because they are anucleated. Figure 52F shows thymocyte death, which occurs during organ culture and results in Ki-67 immunoreactivity with only larger cells morphologically consistent with TE cells at a later stage of culture. [Figure 53] Figure 53A shows a plot of CCL21 levels (pg / ml) in acclimatization medium from cultured thymic tissue against the number of culture days. Figure 53B is a schematic diagram of the sectioning process of thymic tissue subjected to acclimatization. Figure 53C is a plot of CCL21 secretion from sections of thymic organ tissue culture as a function of time. [Figure 54] Figures 54A to 54D are photographs showing immunoreactivity in cultured and uncultured thymic tissue. Figure 54A is a photograph of the medullary region on day 0 of culture, including TECs scattered throughout the cortex; Figure 54B is a photograph of the medullary region on day 16 of culture, including TECs scattered throughout the cortex; Figure 54C is a photograph of TECs in the medullary region and scattered in the cortical region on day 0 of culture. Figure 54D is a photograph of TECs in the medullary region and scattered in the cortical region on day 16 of culture. [Figure 55]Figures 55A–55F plot the expression of selected mRNAs in thymic tissue over overall survival. 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 were expressed normalized to GAPDH ("unadjusted"), and then further normalized to a % region containing thymic epithelium ("adjusted by TE") or a % region containing CD1a-positive cortical thymocytes ("adjusted by Cor"). The data presented were obtained from 47 different thymic samples from donors aged 5 days to 78 years. Figures 55A and 55B show thymic tissue obtained from donors aged 18 years or younger (n=25), which showed higher relative expression of mRNA encoding the T cell marker CD3ε and the cortical thymocyte marker CD1a for GAPDH (Figures 55A and B), respectively, compared to donors over 18 years of age. Figures 55C and 55D are photographs showing that mRNA encoding cytokeratin 8 (KRT8) and 14 (KRT14) was reduced for GAPDH in donors aged 18 years or younger compared to older donors (Figures 55C and D). Figure 55E is a photograph showing consistently low unadjusted CCL21 gene expression for GAPDH in thymuses from donors aged 18 years or younger. Figure 55F is a photograph showing consistently low unadjusted CXCL21 gene expression for GAPDH in thymuses from donors aged 18 years or younger. [Figure 56] This is a table of proteins present in used culture media of thymic organs, as determined by a multi-antibody array. [Figure 57]Figures 57A–57C are photographs of morphological measurements of thymic tissue. The regions included in each measurement were outlined using the "pen tool" provided by ImageScope software (Aperio Technologies, Leica Biosystems imaging, Inc.) according to the manufacturer's instructions. Figure 57A shows the entire region of thymic tissue on a hematoxylin and eosin-stained slide, outlined in green. The portion of this region containing lymphocytes is further outlined in light blue. Figure 57B is a photograph showing the region containing thymic epithelium ("TE region"), outlined in yellow, on a section reacted with the AE1 / AE3 cocktail, which identifies pancytokeratin. Figure 57C is a photograph showing the region containing immature thymocytes ("cortical region"), outlined in red, on a section reacted with CD1a antibody. The thymus shown was obtained from a 32-year-old woman during aortic valve replacement surgery. In each panel, the scale bar represents 4 mm. In panels 57B and 57C, brown indicates a positive reaction with the antibody. [Figure 58] Figures 58A–58C are photographs showing a small number of viable thymocytes in thymic tissue sections cultured for 21 days. Figure 58A is a photograph of a thymic section containing only a few intact thymocytes at day 21 of culture, as suggested by the marked disappearance of basophilic staining (blue) in hematoxylin and eosin-stained sections. Figure 58B is a photograph of a cultured thymic tissue section; here, dead thymocytes showing nuclear and cytoplasmic staining (inset), characteristic of necrotic cells that have not yet undergone nuclear lysis, are not uncommon, but the majority of the strong brown immunoreaction observed in CD3 immunohistochemistry relates to anucleated cell fragments. Figure 58C is a photograph showing that the Ki-67 immunoreactivity at day 21 is limited to cells with large nuclei, characteristic of thymic epithelial cells. Bars in the main panel represent 300 μm, and bars in the inset represent 50 μm. [Figure 59]Figures 59A–59C are graphs showing the characteristics of human thymic tissue used for gene expression analysis. Figure 59A shows the age and sex ratio of the thymic tissues tested, with black circles at the bottom representing females, white circles at the top representing males, and a gray circle in the center representing one donor of unknown sex. Figure 59B plots the % region containing thymic epithelial cells as a function of age for the thymic tissues of this panel. Figure 59C plots the % region showing active thymic cell proliferation, identified by CD1a-positive thymocytes, as a function of age for the thymic tissues of this panel. [Figure 60] This is a photograph of newly collected thymic tissue. [Figure 61] Figures 61A and 61B show histological images of thymic tissue slides after exposure to forced degradation conditions in 10X PBS. Figure 61A is the cortex 9 days after exposure to forced degradation conditions. Figure 61B is the cortex 21 days after exposure to forced degradation conditions. In Figure 61A, the blue smear represents DNA released from cells. Small cell foci with intact nuclei can be identified, but most cells show signs of degradation. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 62] Figure 62A shows a schematic representation of a thymic tissue section for characterization testing, as described in paragraph

[0378] . Figure 62B shows a thymic tissue section on a cellulose filter on a surgical sponge in a tissue culture dish, as is used for thymic culture. [Figure 63]Figures 63A–63H show histological examination of thymic tissue sections derived from a lot (MFG-056) of cultured thymic tissue at 5, 9, 12, and 21 days after thymic harvesting from donors. Hematoxylin and eosin-stained sections (left panel), as well as their corresponding reactions with a cocktail of anti-cytokeratin antibodies AE1 / AE3 (right panel; brown indicates a positive reaction), are shown for day 5 (Figures 63A, 63B), day 9 (Figures 63C, 63D), day 12 (Figures 63E, 63F), and day 21 (Figures 63G, 63H), respectively. The bar in the lower left of each panel represents 100 μm. The hematoxylin and eosin-stained panels show the progression of T cell depletion over time. Figures 14E and 63F are primarily epithelial cells. Epithelial aggregation in the subcapsular cortex occurs as thymocytes are depleted over time. Similar aggregation occurs in the medullary region of the thymus. The photograph is by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 64] Figures 64A and 64B show the histology of thymic tissue sections at day 0 of time-lapse observation, at scales of 5 mm (Figure 64A) and 100 μm (Figure 64B), respectively. This shows the thymus and thymocytes at day 0 at low magnification (bar, 5 mm) and high magnification (bar, 100 μm). This is a normal thymus. At this point, both the cortex and medulla have numerous thymocytes with dark blue nuclei, contributing to the overall dark blue appearance of the tissue. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 65] Figures 65A and 65B are images from hematoxylin and eosin-stained slides showing the histology of thymic tissue sections at 5 mm (Figure 65A) and 100 μm (Figure 65B) scales, respectively, on day 5 of time-course observation. Progression of thymic cell depletion results in a more eosinophilic (pink) appearance of the tissue. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 66]Figures 66A and 66B are images of hematoxylin and eosin-stained slides showing the histology of thymic tissue sections at 5 mm (Figure 66A) and 100 μm (Figure 66B) scales, respectively, at 12 days of time-course observation. Progression of thymic cell depletion is observed. High magnification reveals numerous eosinophilic cell bodies without nuclei, which is an indication of necrotic cells that have undergone nuclear lysis (lysis of the cell nucleus). This degree of necrosis is expected at this stage of culture. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 67] Figures 67A and 67B are images of hematoxylin and eosin-stained slides showing the histology of thymic tissue sections at 5 mm (Figure 67A) and 100 μm (Figure 67B) scales, respectively, at 21 days of time-series observation. Note the preservation of the overall structure of the tissue, including the subcapsular cortex, cortical region, and medullary region, which contain numerous Hassar bodies, in Figure 67B. The dark, small cells are mostly necrotic thymocytes that have not yet undergone nuclear lysis. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 68] Figures 68A–E show representative thymic sections immunostained with a cocktail of anti-cytokeratin antibodies (AE1 / AE3). Figure 68A, day 0; Figure 68B, day 5; Figure 68C, day 9; Figure 68D, day 12; and Figure 68E, day 21. The thymic epithelial network structure remains intact even as the culture progresses. Bars represent 400 μm. Photographs by Laura P. Hale (MD, PhD) of the Department of Pathology, Duke University. [Figure 69]Microscopic images and graphs of CCL21 evaluation in cultured infant thymus are shown. CCL21 is produced at high levels by cultured infant thymus. The immunohistochemical reactivity with CCL21 antibody (Ab) (brown staining) at 16 days of culture is shown in the left panel (upper left panel, 2x magnification; lower left panel, 20x magnification). The corresponding time course, measuring daily CCL21 secretion into the culture medium, is shown on the right for three infant thymus cultures (R&D Systems Duo-Set ELISA). Thus, cultured thymic tissue can produce CCL21, a functionally important biomolecule that is a chemokine involved in attracting immature thymocyte progenitor cells to the thymus. [Figure 70] Figures 70A–70D are micrographs of fresh thymic sections from culture d0 showing thymic structure. In Figures 70A–B, hematoxylin and eosin (H&E) staining shows clearly identified cortical and lightly stained medullary regions, as expected for a normal pediatric thymus. Figure 70C shows immunohistochemistry with a pancytokeratin antibody (AE1 / AE3) cocktail that detects all types of epithelial cells together, demonstrating that thymic epithelial cells are present subcapsularly and in a bright, lacy network in both the cortex and medulla (brown staining indicates a positive antibody reaction). Arrows in Figures 70B and 70C point to Hassall bodies. Figure 70D shows cytokeratin 14 (CK14) antibody staining (brown). The CK14 antibody reacts with thymic epithelial cells in the subcapsular cortex and medulla, as well as with thymic epithelial cells scattered within the cortex. The dotted line highlights the medullary region surrounded by the cortex. SCC represents the subcapsular cortex, Cor represents the cortex, and M represents the medulla. The scale bar in Figure 70A represents 1 mm; the scale bar in Figures 70B-D represents 500 μm. [Figure 71]Figures 71A–71D are micrographs showing examples of Hassall's bodies in cultured thymic sections. Histological images of Hassall's bodies are shown at day 0 (Figures 71A–B) and day 9 (Figures 71C–D) of culture. Figures 71A and 71C show hematoxylin and eosin (H&E) staining; Figures 71B and 71D show reactivity with pancytokeratin (AE1 / AE3) antibodies (brown indicates a positive reaction). Arrowheads in Figures 71A–D point to representative Hassall's bodies, which are not very prominent in hematoxylin and eosin-stained sections of cultured thymus due to depletion and necrosis of surrounding thymic cells. However, Hassall's bodies can still be easily identified by careful examination or by using immunohistochemistry. Scale bars in Figures 71A–D represent 100 μm. [Figure 72] Figures 72A–72D show micrographs illustrating the structure of cultured thymus on day 7. Hematoxylin and eosin (H&E) staining in Figures 72A–B shows significant thymic depletion, although some cortical regions (Cor) still contain numerous thymocytes with retained nuclei. Pancytokeratin (AE1 / AE3) immunohistochemistry in Figure 72C and cytokeratin 14 (CK14) immunohistochemistry in Figure 72D show aggregation of thymic epithelium in the subcapsular cortex (SCC) and medulla (M). Brown in Figures 72C–D indicates a positive reaction with the antibody. Bars in Figure 23 represent 1 mm and 500 μm in Figures 72B–D. [Figure 73] Figures 73A–73D are micrographs showing the structure of cultured thymus on day 9. Figures 73A–B show hematoxylin and eosin (H&E) staining. Very few surviving T cells or thymic epithelial cells, if present, are found in the faintly stained area (almost completely necrotic (Necr)) enclosed by the dotted line in Figure 73A. Most of the nuclei previously present in this area have been degraded by nuclear lysis. Other areas where nuclei from residual thymocytes have not been completely degraded still stain dark blue with hematoxylin. The arrows in Figure 73B point to Hassall bodies. Figure 73C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 73D shows cytokeratin 14 (CK14) immunoreactivity (brown). The bars in Figure 73A represent 1 mm, and in Figures 73B–D, they represent 500 μm. [Figure 74] Figures 74A–74D are micrographs showing the structure of cultured thymus on day 12. Figures 74A–B show hematoxylin and eosin (H&E) staining. At this point, many thymocytes have disappeared from the tissue or have died and their nuclei have been lysed, making the tissue more eosinophilic (pink). The cytocellularity of the thymocytes is significantly reduced, but some regions retain the structure characteristic of normal uncultured thymus, including cortical-like regions (Cor) and medullary-like regions (M) that stain more basophilic (blue). Figure 74C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown); Figure 74D shows cytokeratin 14 (CK14) immunoreactivity (brown). At this point, the subcapsular cortex (SCC) is thickened, and epithelial cells are more prominent due to the reduced number of thymocytes present. Arrows point to representative Hassall bodies. The bar in Figure 74A represents 1 mm, while in Figures 74B to D it represents 500 μm. [Figure 75] Figures 75A-B show hematoxylin and eosin (H&E) staining. At this point, most thymocytes have disappeared from the tissue or have died, their nuclei lysed, making the tissue more eosinophilic (pink). Scattered cells with characteristic thymocyte nuclei are clearly visible, but large groups of residual thymocytes are rare. Figure 75C shows pancytokeratin (AE1 / AE3) immunoreactivity (brown). Much of the epithelium previously present in the medullary region (M) is aggregated due to the disappearance of medullary thymocytes, but scattered epithelial cells remain, showing a bright, lacy, three-dimensional network of residual thymic epithelial cells. In Figure 75D, cytokeratin 14 (CK14) immunohistochemistry (brown) highlights the former medullary region and subcapsular cortex. Arrows point to Hassall bodies. Bars in Figure 75A represent 1 mm, and in Figures 75B-D, they represent 500 μm. [Figure 76] Figures 76A and 76B are micrographs showing examples of intact nuclei in thymic sections. Examples of intact thymic epithelial cell nuclei (arrows) are shown in the subcapsular cortex on day 9 (Figure 76A) and in the medulla on day 21 (Figure 76B). Hematoxylin and eosin staining; scale bar represents 50 μm. [Figure 77]Figures 77A–77E are micrographs showing a comparison of the thymic epithelial network in cultured thymic tissue at different time points. Figure 77A shows day 0, Figure 77B shows day 5, Figure 77C shows day 9, Figure 77D shows day 12, and Figure 77E shows day 21. There are time-dependent differences in thymocyte depletion and necrosis, so the tissue becomes less halophilic (blue) over time, while the thymic epithelial network (brown) structure remains intact even as the culture progresses. Both cortical and medullary epithelium can aggregate when intercalating thymocytes are depleted. Brown indicates a positive reaction with an anti-cytokeratin antibody (AE1 / AE3) cocktail; hematoxylin counterstaining. Scale bar represents 400 μm. [Figure 78] Figures 78A–K are micrographs showing examples of CD3 immunohistochemistry of thymic sections as a function of time in culture. Figures 78A–B show that on 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 78B) shows pale blue nuclei surrounded by brown immunoreactivity rings, consistent with CD3 membrane expression. In Figures 78C–E, the tissue still shows extensive reactivity with the CD3 antibody even on day 7. However, Figures 78D–E show that, when observed at high magnification, most of the brown foci lack nuclear evidence, indicating that the majority of the immunoreactivity (brown) is related to the remnants of dead thymocytes (Figure 78D). Smaller cell foci showing intact nuclei and membrane staining (arrows) can still be identified in areas away from the remnants (Figure 78E). As the culture progresses to day 9 (Figures 78F-G), day 12 (Figures 78H-I), and day 21 (Figures 78J-K), the reactivity of thymocytes with cell debris remains strong, making it difficult to accurately detect potentially intact cells among the debris. All sections shown are from a single lot, representative of multiple lots tested at these points in time. The scale bar in Figures 78A, 78C, 78F, 78H, and 78J represents 500 μm; in Figures 78B, 78D, 78E, 78G, 78I, and 78K, it represents 50 μm. [Figure 79]Figures 79A–K are micrographs showing examples of Ki-67 immunohistochemistry in thymic sections as a function of time in culture. All sections shown are from a single lot, representative of multiple lots tested at similar time points. Figures 79A–B show that on day 0, the nuclei of most immature T cells in the cortex (Cor) react strongly with the Ki-67 specific antibody. High magnification (Figure 79B) shows strong positive reactivity with the nuclei of cortical thymocytes (brown), while in the medulla (M), only a very small number of lymphocytes react with the Ki-67 antibody. Figures 79C–E show that by day 7, the nuclei of most thymocytes remaining in the cortical region are small, have indistinct nuclear boundaries consistent with apoptosis, and do not react with the Ki-67 specific antibody. Cells that react with the antibody (Figure 79E, arrows) have large nuclei, suggesting they are thymic epithelial cells. Similar lack of Ki-67 labeling in residual thymocyte nuclei is observed on day 9 (Figures 79F-G), day 12 (Figures 79H-I), and day 21 (Figures 79J-K). Scale bars in Figures 79A, 79C, 79E, 79G, and 79I represent 500 μm; in Figures 79B, 79D, 79F, 79H, and 79J, they represent 50 μm. [Figure 80] This is a plot of uPAR detected in conditioned medium from human thymus organ cultures. [Figure 81] This is a plot of OPN detected in conditioned medium from human thymic organ cultures. [Figure 82] This is a plot of MIP3a detected in conditioned medium from human thymic organ cultures. [Figure 83] This is a plot of IGFBP-1 detected in conditioned medium from human thymic organ cultures. [Figure 84] This is a plot of MIF detected in conditioned medium from human thymus organ cultures. [Figure 85] This is a scatter plot of CCL21 concentration in used culture media as a percentage of days. [Figure 86] This is a box plot of the CCL21 concentration in used culture medium against the number of days. [Figure 87]Figure 4 is a graph showing the CCL21 level (pg / mL) in the forced decomposition test (lots MFG-053 and 054). [Figure 88] This is a graph of the CCL21 level (pg / mL) in the forced decomposition test (lot MFG-066). [Figure 89] This is a scatter plot of CXCL16 concentration in used culture media as a percentage of days. [Figure 90] This is a box plot of CXCL16 concentration in used culture medium against the number of days. [Figure 91] This is a scatter plot of CXCL16 levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 92] This is a scatter plot of the CXCL21 level in the forced decomposition test (lot MFG-066). [Figure 93] This is a scatter plot of L-selectin concentration in used culture media as a percentage of days. [Figure 94] This is a box plot of L-selectin concentration in used culture medium against the number of days. [Figure 95] This is a scatter plot of L-selectin levels (pg / mL) in forced degradation tests (lots MFG-053 and MFG-054). [Figure 96] This is a scatter plot of L-selectin levels in the forced degradation test (lot MFG-066). [Figure 97] This is a scatter plot of uPAR concentration in used culture media as a percentage of days. [Figure 98] This is a box plot of uPAR concentration in used culture media against the number of days. [Figure 99] This is a scatter plot of uPAR levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 100] This is a scatter plot of uPAR levels in the forced decomposition test (lot MFG-066). [Figure 101] This is a scatter plot of CXCL16 versus CCL21. [Figure 102]This is a quadratic regression model of CCL21 versus uPAR. [Figure 103] This is a quadratic regression model of CXCL16 versus uPAR. [Figure 104] This is a scatter plot of CCL11 concentration in used culture media as a percentage of days. [Figure 105] This is a box plot of CCL11 concentration in used culture medium against the number of days. [Figure 106] This is a linear regression model of the CCL11 concentration in used culture medium against the number of days. [Figure 107] This is a plot of CCL11 levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 108] This lot (Lot MFG-066) is at CCL11 level in the forced disassembly test. [Figure 109] This is a scatter plot of OPN concentration in used culture media as a percentage of days. [Figure 110] This is a box plot of OPN concentration in used culture media against the number of days. [Figure 111] This is a quadratic regression model of the OPN concentration in used culture medium as a function of the number of days. [Figure 112] This is a plot of OPN levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFC-054). [Figure 113] This is a plot of OPN levels in a forced decomposition test (lot MFG-066). [Figure 114] This is a scatter plot of CXCL12 concentration in used culture media as a percentage of days. [Figure 115] This is a box plot of CXCL12 concentration in used culture medium against the number of days. [Figure 116] This is an approximation curve plot of the CXCL12 concentration in used culture medium against the number of days. [Figure 117] This is a scatter plot of CXCL12 levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 118]This is a scatter plot of CXCL12 levels in the forced decomposition test (lot MFG-066). [Figure 119] This is a scatter plot of CCL20 concentration in used culture media as a percentage of days. [Figure 120] This is a box plot of the CCL20 concentration in used culture medium against the number of days. [Figure 121] This is a third-order regression model of the CCL20 concentration in used culture medium against the number of days. [Figure 122] This is a scatter plot of CCL20 levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 123] This is a scatter plot of the CCL20 level in the forced decomposition test (lot MFG-066). [Figure 124] This is a scatter plot of IL-16 concentration in used culture media as a percentage of days. [Figure 125] This is a box plot of IL-16 concentration in used culture medium against the number of days. [Figure 126] This is an approximation curve plot of IL-16 concentration in used culture medium against the number of days. [Figure 127] This is a scatter plot of IL-16 levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 128] This is a scatter plot of IL-16 levels in the forced decomposition test (lot MFG-066). [Figure 129] This is a scatter plot of IGFBP-1 concentration in used culture media as a percentage of days. [Figure 130] This is a box plot of IGFBP-1 concentration in used culture medium against the number of days. [Figure 131] This is an approximation curve plot of IGFBP-1 concentration in used culture medium against the number of days. [Figure 132] This is a scatter plot of IGFBP-1 levels (pg / mL) in the forced decomposition test (lot MFG-053). [Figure 133] This is a scatter plot of OGFBP-1 levels in the forced decomposition test (lot MFG-066). [Figure 134] This is a scatter plot of MIF concentration in used culture media as a percentage of days. [Figure 135] This is a box plot of MIF concentration in used culture medium against the number of days. [Figure 136] This is a linear regression model of the MIF concentration in used culture medium against the number of days. [Figure 137] This is a scatter plot of MIF levels (pg / mL) in forced decomposition tests (lots MFG-053 and MFG-054). [Figure 138] This is a scatter plot of MIF levels in the forced decomposition test (lot MFG-066). [Figure 139] This is a scatter plot of CCL25 concentration in used culture media as a percentage of days. [Figure 140] This is a box plot of the CCL25 concentration in used culture medium against the number of days. [Figure 141] This is a linear regression model plot of the MIF concentration in used culture medium against the number of days. [Figure 142] This is a scatter plot of CCL25 levels (pg / mL) in forced decomposition tests (lots 053 and 054). [Figure 143] This is a scatter plot of the CCL25 level in the forced decomposition test (lot MFG-066). [Modes for carrying out the invention]

[0264] The title, main headings, and subheadings of this specification should not be construed as limiting the various aspects of this disclosure. Therefore, the terms defined below are defined in more detail in this specification as a whole. Any references cited herein are incorporated herein by reference in their entirety.

[0265] Unless otherwise specified, scientific and technical terms used herein have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms also include plural meanings, and plural terms also include singular meanings. In this specification, the use of "or" means "and / or" unless otherwise specified. In the context of a multiple-claim dependent clause, the use of "or" refers retrospectively to a preceding independent or dependent claim only if it is an alternative.

[0266] It should be further noted that, as used herein and in the accompanying claims, the singular forms "a," "an," and "the," as well as any singular form of any word, include multiple referents unless explicitly and obviously limited to a single referent. The terms "include" and their grammatical derivatives herein are intended to be non-restrictive, and therefore the enumeration of items in a list does not exclude other similar items that may be substituted or added to the enumerated items.

[0267] This invention is best understood by referring to the following definitions.

[0268] In this specification, the term "about" is used to mean approximately, within a range, roughly, or about. When the term "about" is used with a numerical range, it modifies that range by extending the boundary above and below the numerical value in the notation. Generally, in this specification, the term "about" is used to modify a numerical value above and below the value in the notation by a variance of + / - 10%. In this specification, the term "about" refers to numerical values, whether explicitly or unexpressed, for example, integers, ratios, and percentages (%). The term "about" generally refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the enumerated range) that a person skilled in the art would consider equivalent to an enumerated value (e.g., having the same function or result). When terms such as "at least" and "about" precede a list or range of numerical values, these terms modify all the values ​​or ranges provided by that list. In some cases, the term "about" may also include numerical values ​​rounded to the nearest significant digit.

[0269] As used herein, the term “animal” includes, but is not limited to, humans and non-human vertebrates (wild animals, domesticated animals, and livestock). Animals may also be referred to as “subjects.”

[0270] As used herein, the term “biomarch” refers to the substances listed in Figure 56. It should be further noted that references to “decreased” and “increased” levels regarding the status of biomarkers in thymic organ culture media refer to decreased or increased measurements of a particular biomarker over time in the habituation regimen.

[0271] As used herein, "biocompatibility" refers to any material that, when implanted in a mammal, does not induce an adverse response in that mammal.

[0272] Chronic graft rejection generally occurs in humans within months to years after engraftment, even in the presence of good immunosuppression for acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants.

[0273] As used herein, the terms “comprising” (and any variations of “comprising,” such as “comprise,” “comprises,” and “comprised,” “having” (and any variations of “having,” such as “have,” and “has,” “including,” and any variations of “including,” such as “includes,” and “include,” or “containing” (and any variations of “containing,” such as “contains,” and “contain,” “contain,” “contain”) are comprehensive or non-exclusive and do not exclude additional, non-enumerated elements or additional, non-enumerated steps of methods. Furthermore, it is understood that terms used with the term “comprising” may also be used with the terms “consisting of” or “consisting essentially of.”

[0274] As used herein, “graft” typically refers to a tissue or organ that is implanted into an individual to replace, correct, or overcome a defect. The tissue or organ may consist of cells derived from the same individual; this graft is referred to herein by the following paraphrasable terms: “autograft,” “autologous transplant,” “autologous implant,” and “autologous graft.” A graft derived from a genetically different individual of the same species is referred to herein by the following paraphrasable terms: “allograft,” “allogeneic transplant,” “allogeneic implant,” and “allogeneic graft.” In this specification, a transplant from one individual to its identical sibling is referred to as an "isograft," "syngeneic transplant," "syngeneic implant," or "syngeneic graft." A "xenograft," "xenogeneic transplant," or "xenogeneic implant" refers to a transplant from one individual to another individual of a different species.

[0275] As used herein, the term "HLA matching" means a donor-recipient pair in which neither HLA antigen is mismatched. HLA matching in the method of the present invention includes HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

[0276] As used herein, the term “HLA mismatch” refers to the matching of HLA antigens between donor and recipient, typically with respect to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1. HLA mismatch occurs between donor and recipient. In some cases, one haplotype matches while others do not. This situation is not uncommon in organs derived from living or deceased donors. HLA mismatch in donor-recipient pairs carries an increased risk of graft rejection compared to HLA-matched pairs.

[0277] As background to the above definition, HLA antigens correspond to "human leukocyte antigens," which are protein molecules expressed on the cell surface that confer antigen specificity to those cells. They are known as "major histocompatibility complex antigens." Therefore, MHC or HLA antigens are target molecules that are recognized by T cells as either "self" or "non-self." If HLA antigens originate from the same hematopoietic stem cell source as immune effector cells, they are considered "self." If HLA antigens originate from other hematopoietic reconstituted cell sources, they are considered "non-self."

[0278] Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) enable each cell to recognize itself 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 implied as targets for transplant organ rejection.

[0279] HLA genes form a cluster at position 6p21 of the human chromosome. The genes in this cluster code for six classic transplantable HLA genes. The 6p21 segment also codes for genes that code for proteins that play crucial roles in regulating the immune system and other fundamental molecular and cellular processes. The entire cluster is approximately 3.6 Mb in size and contains at least 224 loci. As a result of clustering, several "haplotypes" occur (sets of alleles present on a single chromosome). Haplotypes inherited from one parent tend to be inherited as a single group. The sets of alleles inherited from each parent form a haplotype, within which some alleles tend to cooperate. HLA matching is used to identify the recipient's haplotype and helps identify a suitable matched donor. Some haplotypes are more common than others, and their prevalence varies across different races and ethnicities.

[0280] As used herein, the expression “needs it” means that the subject is identified as having a need for it in relation to a particular method or treatment. In some embodiments, the identification may be made by diagnosis by any means. In any of the methods and treatments described herein, the subject may need it.

[0281] In this specification, the expression "an integer X to Y" means an integer that includes each of the endpoints. For example, the expression "an integer X to Y" means 1, 2, 3, 4, or 5.

[0282] As used herein, the term “mammal” means rodents (i.e., mice, rats, or guinea pigs), monkeys, cats, dogs, cattle, horses, pigs, or humans. In some embodiments, the mammal is a human.

[0283] As used herein, the term “organ” refers to a solid, angiogenic organ that performs a specific function or group of functions within an organism. The term “organ” includes, but is not limited to, the heart, lungs, kidneys, liver, pancreas, skin, uterus, bones, cartilage, small and large intestines, bladder, brain, breasts, blood vessels, esophagus, fallopian tubes, gallbladder, ovaries, pancreas, prostate, placenta, spinal cord, limbs including the upper and lower limbs, spleen, stomach, testes, thymus, thyroid, trachea, ureters, urethra, and uterus.

[0284] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to the management of treatment for an individual who may eventually develop at least one symptom of a disease, disorder, or condition, but who has not yet developed that symptom, in order to reduce the likelihood that the individual will develop symptoms of that disease, disorder, or condition within a given period of time. Such reduction may be reflected, for example, in the delay of the onset of at least one symptom of the disease, disorder, or condition in the patient.

[0285] As used herein, the synonymous terms “subject,” “individual,” or “patient” mean any animal, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or mammals such as primates (including humans).

[0286] As used herein, the expression “therapeutic dose” means the amount of an active compound or drug that elicits a biological or pharmacokinetic response in a tissue, system, animal, individual, or human, as attempted by researchers, veterinarians, physicians, or other clinicians. The therapeutic effect depends on the disorder being treated or the desired biological effect. Therefore, the therapeutic effect may be a reduction in the severity of symptoms associated with the disorder and / or prevention (partial or complete) of the progression of the disorder, or an improvement in treatment, cure, prevention, or elimination of the disorder or side effects. The amount required to elicit a therapeutic response can be determined based on the age, health, weight, and sex of the subject. The optimal amount can also be determined based on monitoring the subject's response to the treatment.

[0287] As used herein, the term “tissue” means any type of tissue in human or animal, including, but not limited to, vascular tissue, skin tissue, liver tissue, pancreatic tissue, nerve tissue, genitourinary 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, and ocular tissue.

[0288] In the context of this disclosure, “tissue bank” refers to the long-term storage of cryopreserved postnatal allogeneic thymic tissue cultures stored under liquid nitrogen. General guidelines for establishing storage locations for postnatal allogeneic thymic tissue cultures may refer to industry guidance. “Current Good Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps)” is available at https: / / www.fda.gov / downloads / BiologicsBloodVaccines / GuidanceComplianceRegulatoryInformation / Guidances / Tissue / UCM285223.pdf.

[0289] Tissue engineering refers to the process of forming tissue ex vivo for use in tissue replacement or reconstruction. Tissue engineering is an example of regenerative medicine and includes approaches to repair or replace tissues and organs by incorporating cells, genes or other biological components, along with materials and techniques created through biotechnology.

[0290] The term "graft rejection" includes both acute and chronic graft rejection. "Acute rejection" is a rejection reaction by the recipient's immune system when the transplanted tissue is immunologically exogenous. Acute rejection is characterized by infiltration of the transplanted tissue by recipient immune cells, which exert their effector function 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 blocked or suppressed with immunosuppressants such as rapamycin, cyclosporine A, and anti-CD40L monoclonal antibodies.

[0291] As used herein, the terms “treat,” “treated,” or “treating” mean both therapeutic and preventive measures, the purpose of which is to slow (reduce) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms; reduction of the severity of a condition, disorder, or disease; stabilization of the condition, disorder, or disease (i.e., no worsening); delay of onset or slowing of the progression of a condition, disorder, or disease; improvement or remission (partial or complete), whether detectable or undetectable, of a condition, disorder, or disease; improvement of at least one measurable physical parameter (not necessarily perceptible to the patient); or promotion of improvement of a condition, disorder, or disease.

[0292] Any concentration range, percentage (%) range, ratio range, or integer range described herein shall be understood to include any integer value within the enumerated range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified. The ranges are approximations and may vary beyond a certain integer value.

[0293] Units, prefixes, and symbols are presented in a format conforming to the International System of Units (SI). Numerical ranges encompass the numbers that define that range. Measurements are understood to be approximations, taking into account significant figures and measurement errors.

[0294] It will be further understood that certain features described herein are explained in the context of individual embodiments for clarity and may be provided in combination in a single embodiment. Conversely, various features may be explained in a single embodiment for simplicity and may be provided individually or in any preferred combination.

[0295] Donner - Thymus tissue collection The donor's thymus tissue may be discarded during cardiac surgery after birth, and may be used for CTT with the informed consent of the donor's family. Removal of part of the thymus may be necessary to expose the surgical site; that is, a portion of the thymus may be removed during cardiac surgery after birth due to the nature of the surgical procedure.

[0296] During cardiac surgery after birth, a portion of the thymic tissue may be discarded during the surgical procedure. In all cardiac surgeries, whether or not the thymus is screened for transplantation, the surgeon places the discarded thymic tissue in a sterile container.

[0297] Thymic tissue donors for thymic tissue transplantation in infants with complete DiGeorge syndrome are infants under 9 months of age. The culture product derived from postnatal allogeneic thymic tissue, which is the raw material for the formulation, is prepared by processing and culturing discarded thymic tissue as described herein.

[0298] Consent for the use of the thymus in transplantation of cultured thymic tissue may be obtained before or after the thymus is harvested. However, consent to allow blood collection from an infant before bypass surgery is required and must be obtained in all cases before the surgical procedure. This blood sample is used for donor screening.

[0299] The discarded thymic tissue is placed in a sterile container. Routine examinations are performed on the donor and the donor's birth mother in accordance with FDA guidelines for tissue transplantation. Tissue type matching is not required for the surgical procedures described herein, but such matching may be performed under certain circumstances.

[0300] The tissue may be processed immediately or refrigerated overnight for processing the following day. If thymic tissue is to be stored overnight, aseptically place the tissue in a sufficient amount of thymic culture medium (TOM medium, described later) to completely cover the thymic tissue in its original container. Keep the container with the thymus in the refrigerator until ready for processing the following day.

[0301] Overview of thymic tissue acclimatization The acclimatization regimen depletes donor thymocytes from cultured thymic tissue sections. Based on in vitro data (immunohistochemistry), the culture period of 12 to 21 days preserves the epithelial network, which is evaluated using cytokeratin antibodies. Culturing is preferably carried out at 37°C in a 5% CO2 incubator.

[0302] For good culture, thymic tissue is preferably sectioned and placed on a Millipore® cellulose filter or equivalent filter, then placed on a surgical sponge in a tissue culture dish. The culture medium consists of thymic organ medium (TOM) and is changed daily.

[0303] The thymus is evaluated pathologically upon acceptance. Identity testing requires that more than 50% of the tissue be keratin-positive with a lace-like staining pattern. Efficacy testing requires that Hassall's bodies be present; it should show CK14 staining with a lace-like pattern. Viability testing requires that more than 90% of the tissue observed in sections be intact. Lot release of tissue is performed comprehensively over one day from day 5 to day 21 and is conducted pathologically. Regarding identity, the tissue area from day 5 to day 21 must be positive for keratin AE1 / AE3. Regarding efficacy, cultured thymic tissue from day 5 to day 21 must show scattered cytokeratin CK14 staining throughout and at least one Hassall's body must be identifiable. Regarding viability, cultured thymic tissue from day 5 to day 21 must show intact nuclei.

[0304] In one embodiment, thymic tissue sections are acclimatized for about 12 days and then cryopreserved. In another embodiment, all thymic tissue sections are acclimatized for about 12 days, then about half are transplanted into the recipient, and the remaining thymic tissue sections are cryopreserved for future use.

[0305] Within 24 hours of collection, the thymus is sectioned into thin sections. The sections are kept in culture for 12–21 days. Although most subjects have a T cell count below the 10th percentile for their age, this culture process depletes the donor's viable T cells, as outlined below, ultimately allowing the surgically transplanted tissue sections to reconstitute the immune system of the athymic subject, albeit at an immunologically effective level. The culture process significantly modifies the biological properties of the donor thymus tissue and the constituent cells contained therein in the manner outlined below, optimizing the effective therapeutic properties of the CTT sections.

[0306] The culture process ensures that a specific composition of cultured cells / tissues possessing the prerequisite biological characteristics is obtained in a manner suitable for surgical transplantation into the subject, enabling the reconstitution of the subject's immune system.

[0307] The culture process results in the disappearance of thymocytes and the relative enrichment of thymic epithelial cells and other stromal cells in donor-thymic tissue sections.

[0308] The culture process further leads to thymocyte depletion and maintenance of TEC, which allows for the reconstitution of the recipient's immune system, and develops tolerance in the recipient to HLA antigens in the donor thymus.

[0309] Overall, the manufacturing process is designed to deplete thymocytes from donor thymic tissue while preserving the functional structure of the thymic stroma (thymic epithelial cells and fibroblasts).

[0310] In one embodiment, the processed donor thymus tissue is an engineered thymus tissue product that can induce tolerance to the type of thymus tissue (HLA antigen) in subjects who require it after a surgical transplantation procedure.

[0311] To maintain the viability of the sectioned thymic tissue, the thymic sections are placed on a Millipore cellulose filter and then on a surgical sponge inserted into a tissue culture dish containing culture medium. The culture medium in each tissue culture dish is changed daily from the day of donor collection to the day of transplantation (days 12 to 21).

[0312] Donor-thymic tissue culture depletes thymocytes in such treated tissues, minimizing the risk of graft-versus-host disease ("GvHD"), which can be a significant problem in severely immunocompromised patients after thymectomy.

[0313] During the first few days of culture, many thymocytes "detach" from the tissue section into the culture medium and are discarded during medium changes. As the culture continues throughout the culture period, donor thymocytes continue to die, but the remnants of these cells are retained within the CTT section.

[0314] Without being bound by theory, the presence of these non-viable thymocytes and their nuclear-deleted remnants is considered important for the intended function of the tissue engineering product; this is because they help maintain open pockets in the three-dimensional network of thymic epithelial cells necessary for the entry of bone marrow stem cells in the recipient after treatment. The importance of having “space” for the entering bone marrow stem cells is supported by the experience of patient DIG003 described in Markert, 1999 (see the references list below). This patient, described in the above literature, was administered a very high dose of steroids (methylprednisolone, 40 mg / kg / day, x3 days) 35 days after CTT transplantation, which resulted in thymic apoptosis and epithelial aggregation. No naive T cells developed at all, and the patient died from infection. At autopsy, the introduced thymus was a mass of viable epithelium, and there was no space between epithelial cells for thymocyte entry.

[0315] During the culture period, HLA typing is performed to determine whether the patient (recipient) and donor tissue share any HLA alleles. Anti-HLA antibody testing is performed on the recipient to determine whether the recipient has antibodies against any HLA antigens in the thymus. If the recipient has antibodies targeting the donor's MHC, another thymus is searched. The donor and their mother are tested for infection in accordance with the FDA guidance document "Guidance for Industry. Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / Ps)" and more recent guidance documents. The tissue is aseptically processed in accordance with Federal Regulations (CFR) 1271 Subcategory D "Current Good Tissue Practice".

[0316] Following batch record review and QC testing, the tissue is withdrawn from manufacturing and provided to the surgical team for transplantation. The tissue is surgically transplanted into the recipient as described herein.

[0317] Cultured thymic tissue is produced by a process described in more detail below and in the examples herein.

[0318] In summary, the culture process of harvested thymic tissue significantly alters the biological properties of the donor tissue and the constituent cells it contains in the following ways: the disappearance of donor thymocytes and the enrichment of thymic epithelial cells and other stromal cells, as well as the depletion of donor thymocytes, which alters the physiological function (e.g., secretion of cytokines and growth factors) and structural properties of the tissue.

[0319] During the first few days of culture, many thymocytes "detach" from the tissue section into the culture medium and are discarded during medium changes.

[0320] Operations performed during the manufacturing process alter the macroscopic and histological appearance of the resulting cells in the final product compared to the source or starting material obtained from the donor.

[0321] During the first few days of culture, the tissue appears red, due to residual blood on or within the tissue. See, for example, Figure 15.

[0322] Between days 5 and 21, viable tissue is observed, and the blood contamination that was prominent on day 1 disappears.

[0323] During the remaining days of culture, tissue depth decreases as thymocytes are depleted. The decrease in thymocyte density in the tissue is demonstrated by immunohistochemistry, which will be described in detail later.

[0324] Day 0 after collection of discarded thymic tissue, the tissue is densely packed with viable thymocytes embedded in the stroma, including thymic epithelial cells and fibroblasts. AE1 / AE3 and CK14 staining confirm the presence of cytokeratin (CK)-positive thymic epithelial cells, characteristic of a normal thymus. The thymic epithelial cells form a lace-like three-dimensional network with delicate projections surrounding adjacent thymic cells.

[0325] During the culture process, thymic sections are cultured as described below. Numerous thymocytes are washed away from the tissue, particularly during the first three days. This depletion can be histologically identified as early as day two by hematoxylin and eosin staining, which shows a decrease in thymocyte density, especially in the medullary region. The majority of thymocytes remaining in the tissue show nuclear changes consistent with apoptosis and / or necrosis, or nuclear lysis (complete loss of the nucleus). Many of these dead thymocytes and their cellular remnants are thought to remain throughout the thymic tissue, preventing the complete breakdown of spaces between epithelial cells.

[0326] Some degree of epithelial aggregation can be observed in external regions, such as the subcapsular cortex, where thymocyte loss has led to the breakdown of the epithelial cell network. These aggregated subcapsular cortical epithelial cells can form linear arrangements several cell layers thick, which can increase the mechanical strength of the section. Some medullary epithelium may also aggregate to form patches of adjacent epithelial cells.

[0327] Thymocyte death continues as the culture progresses, and necrotic thymocyte fragments are retained within the tissue. Further aggregation of the medulla and subcapsular cortical epithelium is minimal around days 7–19 of the culture.

[0328] Regions with epithelial structures similar to those of a normal thymus are still observable in the later stages of culture of each thymus using AE1 / AE3 staining. In longer-cultured tissues, the epithelial structures of the cortex and medulla are still readily identifiable; for example, Hassall's bodies remain in the medullary region. However, this degree of thymic depletion gives a substantially different overall histological picture in hematoxylin and eosin staining compared to that of a normal thymus at day 1 and beyond.

[0329] Details of thymic tissue culture The general procedure for preparing allogeneic thymic tissue cultures is, as described above, to obtain thymic tissue from infants with complete DiGeorge syndrome as discard tissue from infants under 9 months of age undergoing cardiac surgery. In the case of solid organ transplantation, discard thymus may be obtained from individuals up to 50 years of age. The use of thymic tissue will depend on whether it meets the criteria for use described herein.

[0330] Thymic tissue is aseptically treated and cultured under cGMP conditions to create thymic tissue sections with partially depleted T cells.

[0331] The production of cultured thymic tissue (CTT) generally consists of the following steps: receiving and processing of incoming thymic tissue, sectioning, culturing, medium change, dose calculation, packaging, and transport of the thymus to the operating room. Furthermore, incoming thymic tissue is tested for receptivity, and in-process and release tests are performed on thymic tissue sections.

[0332] In one embodiment, the thymic tissue sectioning process inevitably involves the use of sterile, disposable scissors and forceps to cut out a piece of thymic tissue. The worker removes the thymic capsule with the forceps and scissors and places the capsule on a plate lid for later disposal.

[0333] Place the thymic tissue sample on a disposable tissue slicer using forceps. Position the top of the slicer (e.g., Stadie-Riggs hand microtome (Thomas Scientific, Swedesboro, NJ)) over the center of the slicer to secure it in place. The operator moves the blade to cut the tissue sample into sections. The sections are approximately 0.5–1 mm thick. Fill approximately 50–90% of the filter surface with tissue sections so that they do not overlap.

[0334] Typically, three intra-process sections are cut from the tissue at the start of the dissection, each roughly 3x3 mm in size. One section is transferred for histological examination, and the remaining two are retained. Thymocytes are freely released into the culture medium once the thymus is dissected into sections.

[0335] In one embodiment, filters and thymic sections are transferred to gelatin surgical sponges saturated with TOM in tissue culture dishes. The TOM wets the Millipore filters by capillary action, keeping the tissue moist. Two filters are placed in each sponge, and two sponges are used for each tissue culture dish. The thymic sectioning process is repeated until the required number of sections are prepared. Culture dishes are labeled with the surgical number, dish number, and ISBT barcode label. Completed dishes are placed in a humidified incubator at 37°C containing 5% CO2.

[0336] The tissue-engineered formulation materials contain thymic tissue sections after being cultured in culture medium in a culture dish for approximately 6 to 21 days, as described below. The tissue-engineered drug product contains thymic tissue sections after being transferred to a pharmaceutical product container. No other processing is performed to create a drug from the formulation materials; the only processing of the formulation materials for drug creation is transferring the sections to a leak-proof container and changing the corresponding culture medium.

[0337] The culture of thymic tissue sections is described in more detail in the following paragraphs.

[0338] In one embodiment, thymic tissue is obtained from a 9-month-old infant undergoing cardiac surgery, as waste tissue from the operating room. The tissue is then placed in a sterile sample cup with a screw cap by the surgical team and transported to a GMP facility under ambient conditions for processing. The sterile sample container receiving the thymus is marked with the donor's name and medical record number, including a barcode. The donor screening group assigns a unique identifier (sequential numbering of thymuses) and a unique medical record number to each thymus. For manufacturing purposes, each tissue has a surgical number and a unique label. All identifiers are recorded on a "Confidential Thymus Donor Form" which is maintained separately from batch records and kept confidential.

[0339] In one embodiment, the sealed system for pharmaceutical raw material containers may be a cell culture dish with a lid. One thymic tissue section is placed on a filter, and two filters are placed on each gelatin sponge in the thymic organ medium of the dish. Four sections are placed in each culture dish, and the dishes are stored in an incubator until ready for release, with the medium being changed daily.

[0340] In one embodiment, the culture dish can be obtained from Corning. The dish may be a sterile, pyrogenically-free Falcon® 100mm polystyrene cell culture dish (product number #353003). The dish is cleaned by vacuum gas plasma treatment and sterilized by gamma irradiation. The dimensions of the dish are 89.43mm OD x 19.18mm.

[0341] In exemplary embodiments, the Surgifoam® sponge may be manufactured by Ethicon, which meets the requirements of the Absorbent Gelatin Sponge, USP. A suitable sponge is a sterile, water-insoluble, malleable porcine gelatin absorbent sponge intended for hemostatic applications. A specific example of a mixed cellulose ester filter is manufactured by Millipore (product number #SMWP02500). The 25 mm hydrophilic membrane has a pore size of 5.0 μm. It is made of a biologically inert mixture of cellulose acetate and cellulose nitrate. The filter is sterilized with ethylene oxide before use.

[0342] After donor thymus transfer to and reception in the processing laboratory, the thymus is cut into thin sections; these are placed on a sterile filter on a surgical sponge in a sterile culture dish. If the tissue is not to be processed immediately, it is stored in thymic organ medium (TOM) at 2-8°C for up to 24 hours after collection from the donor and before processing begins, as described below. TOM consists of Ham's F-12 medium, HEPES buffer, L-glutamine, and heat-inactivated fetal bovine serum (FBS).

[0343] In one embodiment, the processing is carried out in an ISO 5 space within a biological safety cabinet (BSC) in an ISO 7 manufacturing cleanroom. Only a single lot of thymic tissue derived from a single thymus is processed in the BSC in any case. The BSC is purified before use. The thymus is examined for appearance by visual inspection and weight measurement. The thymus is then placed in a 150 mm tissue culture dish in a TOM. The thymic capsule is removed with sterile, disposable forceps and scissors. Tissue samples are taken for test and preservation. The incoming thymic tissue is examined for identity by histology. Donor eligibility is also confirmed. The processing is continued until histological results and the results of all donor screening are obtained.

[0344] The criteria for passing donor screening is that all donor eligibility requirements must be met. Donor screening is required in accordance with 21 CFR 1271 to ensure the safety of thymic tissue transplant recipients. This screening minimizes the risk of disease transmission from donor to recipient.

[0345] Thymus organ medium (TOM) Culture media are always prepared using ingredients approved for human use that are unlikely to cause allergic reactions, whenever such reagents are available.

[0346] All reagents must be tracked so that all components can be identified after transplantation if any problems arise.

[0347] Fetal bovine serum (FBS) must be manufactured using U.S. materials due to concerns about Creutzfeldt-Jakob disease. Information on each lot must be sent to the FDA before use.

[0348] Culture media must be tested for bacterial, fungal, and mycoplasma contamination before use.

[0349] In one embodiment, TOM is prepared using the following materials:

[0350] HAMS F12, Gibco #11765-054 (or case #11765-062), 500ml bottle or equivalent source.

[0351] HEPES, Gibco #15630-080 or equivalent, 1M solution, 100ml bottle. Final concentration, 25mM.

[0352] L-glutamine, Gibco#25030-081 or equivalent source (stock, 200mM).

[0353] Fetal bovine serum, Gibco, #16140 (thermally inactivated) or #10082-147 (thermally inactivated, certified).

[0354] In one embodiment, thermoactivated (HI) fetal bovine serum (FBS) may be used in the following manner:

[0355] FBS must be thermally inactivated at 56°C for 30 minutes.

[0356] To reduce the possibility of contamination of the culture medium, it must be dispensed in aliquots; and no aliquot should be reused.

[0357] The remaining FBS aliquots may be stored frozen (-20°C) in 25 ml portions for research purposes.

[0358] In one embodiment, TOM may be prepared by the following method.

[0359] Thaw the fetal bovine serum overnight in the refrigerator, or thaw it at 37°C while frequently and gently swirling it around.

[0360] If using fetal bovine serum that has not been heat-inactivated, heat-inactivate it at 56°C for 30 minutes.

[0361] To prepare 4 liters at once, place all the culture medium components together in a 4-liter flask and stir on a magnetic stirring plate using a stirring rod at a moderate speed (without creating bubbles) for 3 to 5 minutes.

[0362] Sterilization is performed using a 0.2-micron filter unit.

[0363] In one embodiment, sterilization of the TOM preparation may be carried out by the following method: Dispense 1 liter of TOM into a 1 liter flask. Measure 80 ml of TOM into a disposable sterile cylinder. Pour 80 ml of TOM into a 150 ml Corning filter-sterilization unit. Attach a house vacuum according to the manufacturer's instructions and filter and sterilize by filtration. Remove and discard the filter unit from the container. Cover the sample bottle with a sterile cap (provided in the unit). Label with the TOM lot number. Test one sample for bacterial cultures related to anaerobic bacteria; fungal cultures, etc.; and mycoplasma cultures. Test one sample for endotoxins. Store all TOM samples upright in a freezer at -20°C.

[0364] TOM medium may be delivered for use if: the LAL result is less than 2 EU / ml for a 20-fold diluted sample for testing, or less than 1 EU / ml for a 10-fold diluted sample for testing; all culture results must be negative for growth.

[0365] BSC must be used for filtering and dispensing the culture medium.

[0366] TOM is tested for sterility and endotoxins before delivery. TOM is not delivered for donor-thymus culture until it meets the acceptance criteria in a 14-day sterility test. Once prepared, TOM is stored at -20°C until thawed, at which point it may be stored for use in the refrigerator for up to two weeks.

[0367] In one embodiment, a 14-day sterility test may be performed, for example, using a BacT / ALERT culture system. BacT / ALERT (BioMerieux, Durham, NC) is a commercially available culture system used to test samples using an automated microbial detection system.

[0368] All in-process cultures and formulation raw material cultures are incubated for 14 days or reported immediately if the product is positive. For positive testing, the organism(s) are identified and their antibiotic susceptibility is determined. The test sample is inoculated into culture bottles containing aerobic growth medium and bottles containing anaerobic growth medium on day 1, day 7, and the day of delivery. All bottles are incubated at 35-37°C for 14 days.

[0369] FBS may be obtained under the GIBCO brand from Life Technologies. FBS is prepared by a sterile and validated process. FBS meets USDA requirements regarding animals sourced from meat processing plants, traceability, and country of origin. All fetal blood is collected from fetuses derived from healthy mother animals that have passed veterinary certification inspections before and after slaughter. All FBS is traceable by date and place of collection. FBS collected and processed in the United States originates from USDA-approved and inspected slaughterhouses. The United States is certified by the USDA as free from foot-and-mouth disease and rinderpest. To qualify the source, FBS is tested for osmotic pressure, endotoxins, total protein, and identity before use.

[0370] The finished dishes are placed in a humidified incubator at 37°C containing 5% CO2. Thymic tissue from each lot is stored in a separate incubator. After the thymic sections are placed in the incubator, particle sampling and worker monitoring are performed.

[0371] Thymic sections are cultured for up to 21 days (e.g., an acclimatization regimen of approximately 6 to 21 days), with the culture medium changed daily during the culture period. These thymic sections are considered as raw materials for the formulation. During the culture period, while maintaining the thymic stroma, a large number of thymocytes are washed away from the thymic tissue sections, or the thymocytes undergo apoptosis. The entire manufacturing process is carried out using sterile, disposable equipment and consumables. The culture medium is aspirated from the culture dish by pipette and pooled into a sterile recovery container for in-process inspection. 10 mL of fresh thymic organ medium is then gently dispensed into each culture dish, rinsing over the tissue sections. After the medium change is complete, samples are taken from the pool medium for sterility testing and histological examination as needed. Particle sampling and operator monitoring are completed, and the production line is cleaned.

[0372] The culture medium is changed daily.

[0373] The sections are cultured for up to 21 days (for example, an acclimatization regimen of approximately 6 to 21 days).

[0374] In-process inspections are conducted to gain insights into process and product quality, and to support the assurance of safety and quality of final pharmaceutical products.

[0375] In-process inspection Samples are collected on days 1-7 for in-process sterility testing. Samples are collected on day 7 for in-process mycoplasma testing. Samples are collected on days 5-9 for in-process histological testing. The dosage is determined the day before delivery. Gram staining, BacT, mycoplasma, and endotoxin testing are performed on the day of delivery.

[0376] Gram staining is a bacteriological experimental technique used to classify bacterial species into two groups: Gram-positive and Gram-negative. Gram staining is performed on used culture media pooled from culture dishes. This method utilizes staining techniques to determine classification based on the physical characteristics of the cell wall. It is used for preliminary morphological identification or to determine whether a significant number of bacteria are present in a clinical specimen. Staining can be performed manually or using an automated staining machine. These two different staining methods have been shown to exhibit no qualitative differences that would affect culture results.

[0377] Histological examination is performed before transplantation, which in one embodiment includes at least: (1) determination of scattered keratin AE1 / AE3-positive areas throughout the tissue; (2) microscopic identification of at least one Hassall's body; (3) scattered CK14 staining of the tissue section throughout the thymic tissue; and (4) microscopic observation of intact nuclei. In one embodiment, histological examination is performed between approximately day 6 and day 21. The presence of Hassall's bodies and intact nuclei, as well as good CK14 staining, are indicators of normal and healthy cultured thymic tissue.

[0378] Culture time is a crucial process parameter. As mentioned above, the culture is carried out for a maximum of 21 days.

[0379] Testing of thymic specimens in culture prior to transplantation is performed to confirm whether the histological results obtained from the culture are representative of the histological examination of previous specimens of cultured thymic tissue. Based on the pathologist's observations of the specimens described in the examples, the histological features of the tissue sections on day 5 reflect those observed at later points in the culture (days 9, 12, and 21). In one embodiment, thymic tissue examinations may be performed at various points and time intervals between days 5 and 21 of the culture. For example, the test may be performed during the acclimatization regimen period, which may be a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; or 5-6, 5-7, 5-8, 5-9, or 5-10 days, The acclimatization period is 6-7 days, or 6-8 days, or 6-9 days, or 6-10 days, or 6-11 days, or 6-12 days, or 6-21 days, or 7-21 days, or 8-21 days, or 9-21 days, or 10-21 days, or 11-21 days, or 12-21 days, 13-21 days, or 14-21 days, or 15-21 days, or 16-21 days, or 17-21 days, or 18-21 days, or 19-21 days, or 20-21 days. In one embodiment, the acclimatization regimen may be at any point between approximately day 6 and day 21 prior to cultured thym tissue transplantation.

[0380] Histological examination of any one section supports conclusions regarding the acceptability of the entire lot. The characteristics of any one section derived from the thymus reflect the characteristics of the entire thymus and support the continued use of single tissue sections for histological examination.

[0381] Forced degradation tests, as shown in Figures 12A and 12B, demonstrate that cultured thymic tissue products are not readily degraded and are most sensitive to freeze / thaw and osmotic changes. Other conditions examined during forced degradation had little to no effect on the cultured thymic tissue products.

[0382] Management of raw materials for cultured thymic tissue preparations The criteria for determining whether incoming thymic tissue products are acceptable include the tests shown in Table 1 below.

[0383] [Table 1]

[0384] Abbreviations: CK, cytokeratin; EU, endotoxin unit; USP, United States Pharmacopeia. Thymic tissue is processed before all donor screening results.

[0385] Generally, the acceptance criterion for weight is 3 grams or more. This is the minimum acceptable thymic weight, ensuring that sufficient material is available for proper administration of the final product. The acceptance criterion is based on experience in processing thymic tissue.

[0386] The acceptance criteria for in-process inspections are specified in Table 2 below.

[0387] [Table 2]

[0388] The criteria for determining acceptance of cultured thymic tissue as a raw material for pharmaceutical preparations are specified in Table 3 below.

[0389] [Table 3]

[0390] The acceptance criteria for identity are that the identity of the thymic tissue is confirmed by histology on day 1 and at intermediate points (days 5-9). Barcodes are used to track the tissue throughout the entire process, and barcodes are checked at the time of delivery to verify the correct identity of the product.

[0391] Histology using immunochemistry The histological methods are standard procedures used by hospitals for all types of tissue, and are well known to those skilled in the art.

[0392] The product sample is fixed in 10% formalin and transported to the laboratory. The container is labeled with an encrypted identifier according to the medical record number, instead of the patient's name, to protect patient privacy. Upon arrival at pathology, the specimen is assigned a unique pathology accession number and barcoded. All subsequent blocks, slides, and documents are barcoded with this pathology accession number.

[0393] After the specimen is received in the laboratory, the formalin-fixed tissue is visually examined, and a written report of the macroscopic findings of the material, which will be part of the final report, is prepared. The formalin-fixed tissue is then processed in an automated processing system according to standard procedures and embedded in paraffin blocks. Sections are cut from the paraffin blocks, and the following staining is performed by an ASCP-certified tissue technician: Hematoxylin and eosin Cytokeratin AE1 / AE3 Immunohistochemistry Cytokeratin-14 Immunohistochemistry CD3 immunohistochemistry Ki-67 immunohistochemistry.

[0394] During the immunohistochemical testing described above, appropriate control slides are also tested and reviewed. All control slides and internal controls exhibit the expected immunoreaction pattern. The incoming thymus samples are also used as controls for tissue sections cultured for approximately 6 to 21 days if the samples are tested as part of an efficacy test. The incoming thymus samples appear like typical thymus samples, then changes occur in the tissue sections, during which time they are cultured, and then tested after approximately 6 to 21 days. After approximately 6 to 21 days of culture, the samples should show positive areas for keratin 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.

[0395] The slides will be analyzed by a certified pathologist with further experience in the histological evaluation of thymic tissue. A final report will be submitted by the pathologist, which will clearly state the results.

[0396] The criteria for determining whether a cultured thymic tissue preparation raw material is acceptable are specified in Table 4.

[0397] [Table 4]

[0398] Cultured thymic tissue must be free of microorganisms. Sterility tests performed on days 1 and 7 should show no microbial growth. Mycoplasma should be negative on day 7. Sterility tests should be Gram-negative.

[0399] Maintain product sterility using appropriate controls, including aseptic techniques; utilize training programs and verify worker qualifications; employ appropriate cleanroom certification procedures; use established clean medium filling procedures and use ready-to-use sterile instruments or instruments sterilized using validated sterilization cycles.

[0400] The containers of processed thymic tissue are visually inspected for damage. Tissue sections typically have a yellow to reddish-brown appearance with varying thicknesses and shapes.

[0401] The identity of the thymic tissue will be histologically confirmed on day 1 and at intermediate points (days 5-9).

[0402] Barcodes are used to track the organization throughout the entire process, and barcodes are verified upon handover.

[0403] The dosage (area) is 1,000 to 20,000 mm² of thymic tissue. 2 / 1m 2 This is the recipient's body surface area. The dosage is controlled by the surface area of ​​the section handed over to the operating room, so as to be appropriate for the patient's body surface area.

[0404] The acceptable dose range is 1m 2 1,000-20,000 mm² per recipient body surface area (BSA) 2 It is defined as the thymic tissue. The area of ​​the thymic tissue is determined by photographs using software analysis (PAX-it image analysis software). The BSA is determined using the patient's height (cm) and weight (kg). The DuBois-DuBois formula is used to calculate the BSA: BSA = 0.007184 × [Height (cm)] 0.725 × [Weight (kg)] 0.425

[0405] Cultured thymic tissue is tested for endotoxins. The standard is 5 EU / kg body weight / hour or less.

[0406] Endotoxin testing may be performed, for example, using the Endosafe PTS system. The cartridges used in the Endosafe PTS utilize the chromogenic limulus amebocyte lysate (LAL) test. Each cartridge contains precise amounts of LAL reagent, chromogenic substrate, and control standard endotoxin. The test sample is pipetted into four sample reservoirs. The instrument aspirates the sample and mixes it with the LAL reagent in two channels (sample channels) and with the LAL reagent and positive product control in the other two channels (spike channels). The sample is incubated and then mixed with the chromogenic substrate. After mixing, the optical density of the wells is measured and compared to a standard curve stored in the instrument. The instrument measures the reaction time in each channel. A storage standard curve specific to each batch of cartridges is created using the log value of the reaction time relative to the log value of the standard endotoxin concentration. Sample values ​​and spike values ​​are calculated by interpolation from a standard curve using reaction time. This test meets the requirements of the United States Pharmacopeia (USP).

[0407] Mycoplasma testing may be performed by the following method: Take a sample from the pooled culture medium on day 7 and test it before product delivery.

[0408] If a culture during manufacturing tests positive, discard that lot and do not administer the drug. If a culture tests positive after administration of the clinical drug, the patient's physician and the facility manager should treat the patient appropriately. A positive culture requires identification of the contaminating organism and determination of its antibiotic susceptibility. The attending physician should initiate antibiotic therapy for the thymus recipient if instructed.

[0409] The preparation undergoes similar visual and histological examinations before use.

[0410] After culturing the thymic tissue sections for up to 21 days, the sections are transferred to a medical container for transport to the operating room. Upon receipt in the operating room, the sections are introduced into the thigh muscle of the recipient patient.

[0411] The container should be free of visible damage and undamaged, and the thymic tissue sections should have a yellow to reddish-brown appearance with varying thicknesses and shapes. The tissue sections should be visually inspected to confirm that these acceptance criteria are met.

[0412] Cryopreservation and thawing of cultured thymic tissue from allogeneic and allogeneic thymus after birth. The cultured product of allogeneic thymic tissue after birth may be cryopreserved using the following method.

[0413] Culture products derived from cryopreserved postnatal allogeneic thymus tissue, prepared by a method comprising the following steps: (a) The process of obtaining suitable thymic tissue from a donor; (b) HLA alleles: The process of typing HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1; (c) A step of subjecting thymic tissue to an acclimatization regimen for a period of approximately 6 to 21 days, wherein the acclimatization regimen for donor-thymic tissue includes aseptically treating the donor-thymic tissue in thymic organ medium to produce donor-thymic tissue sections in which T cells are partially depleted; further, the donor-thymic tissue sections, upon completion of the acclimatization regimen on approximately 6 to 21 days, show areas positive for keratin AE1 / AE3 scattered throughout the tissue, the presence of at least one Hassall body, CK14 staining scattered throughout the tissue, and the presence of intact nuclei; (d) A step of recovering donor thymus tissue sections with partially depleted T cells as culture products derived from allogeneic thymus tissue after birth; (e) The process of cryopreserving culture products derived from allogeneic thymic tissue after birth in liquid nitrogen; and (f) A step of maintaining a culture product derived from cryopreserved postnatal allogeneic thymus tissue in liquid nitrogen in a cryopreserved allogeneic thymus tissue culture product bank. In one embodiment, a culture product derived from cryopreserved postnatal allogeneic thymus tissue according to claim 66, wherein the thymus is positive for keratin in a lace-like staining pattern in more than 50% of its area on the day of collection, hassall bodies are present, CK14 is stained in a lace-like pattern, and more than 90% of the nuclei are intact.

[0414] In one embodiment, the donor thymus is sectioned and roughly divided in half, and each section is placed in a separate cryovial (Nunc tube) with its cellulose filter. The filter is folded in half and inserted into the tube. Approximately 1 to 1.5 ml of frozen medium [90% sterile filtered heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)] is added at room temperature to cover the tissue. The sterile caps of the frozen vials are returned to the tubes and tightened. All tubes are placed in a Biocision CoolCell or equivalent container at room temperature. All empty slots in the CoolCell must be filled with tubes containing 1 ml of frozen medium. The tubes are placed in a -80°C freezer overnight. Alternatively, each tissue and filter is placed in a 5 ml CryoELITE tissue vial (Wheaton). 3 to 5 ml of frozen medium is added at room temperature to cover the tissue. Place the vial in a styrofoam box and leave it in a -80°C freezer overnight. Then, transfer the vial to the vapor phase of a liquid nitrogen freezer. Alternatively, you can use a freezing rate control freezer to bring the temperature of the frozen vial to liquid nitrogen temperature.

[0415] To restore the tissue, remove the frozen vial or CryoELITE tissue vial from the liquid nitrogen freezer. Rapidly thaw the thymus fragments in the vial by swirling the vial in a 37°C water bath. Spray the tube with 70% ethanol and then place the tube in a biosafety cabinet (BSC). Remove the thymus tissue and filters from the Nunc frozen vial or CryoELITE tissue vial using forceps. Place the tissue and filters into a 50 ml conical tube containing 20 ml of 4°C TOM medium. Up to five filters can be placed in a 50 ml conical tube containing 20 ml of 4°C TOM medium. Immediately transfer the five filters containing the tissue into a new conical tube containing 20 ml of 4°C TOM medium and leave at 4°C for 15 minutes. Repeat the washing three times. While maintaining the tissue at 4°C, transfer each piece to a 120ml Starplex container containing 5ml of 4°C TOM. Transport all containers to the operating room in a temperature-controlled container with a cooling pack. Bring the Starplex containers containing the tissue into the operating room. Transfer the tissue on the filter into a culture dish containing approximately 2ml of sterile saline in a sterile field. The operating room nurse detaches the tissue from the filter by scraping or pulling with forceps. The operating room nurse returns the tissue to the filter in an amorphous mass. Transfer the tissue culture dish containing approximately four filters and tissue to the operating room where the surgeon can easily access the tissue. Introduce the tissue into the quadriceps muscle using a procedure similar to CTT (RVT-802). Frozen CTT is similar to CTT in that it is partially T-cell depleted, and thymic tissue sections show areas positive for keratin AE1 / AE3 scattered throughout the tissue, sections contain at least one Hassar body, CK14 staining is scattered throughout the tissue, and intact nuclei are present.

[0416] CTT transplantation In one embodiment, sections of incompatible thymic tissue from a donor are cultured for approximately 6 to 21 days. On the day of solid organ transplantation, steroids are usually administered during induction of anesthesia. In the case of heart or lung transplantation, the recipient's thymus is surgically removed at the time of solid organ transplantation. In the case of other organ transplantation, thymectomy is performed the day before or on the day of transplantation. The thymectomy method may be surgical, thoracoscopic, or robotic. At the end of the post-reperfusion surgery, the recipient is given additional steroids before receiving equine anti-thymocyte globulin (e.g., rabbit anti-thymocyte globulin) for 3 to 7 days to kill most of the remaining T cells (and NK cells) in the recipient, or before receiving alemtuzumab for 4 days to kill T cells, B cells, and NK cells. Immunosuppressants (such as cyclosporine or tacrolimus) and mycophenol are then administered until T cells develop and more than 10% naive T cells are present. It will likely take 6–12 months for naive T cells to increase to this number. Cultured thymic tissue is processed from the thymus of solid organ donors. Half of the CTT can be transplanted into the quadriceps muscle between approximately day 6 and day 21. The remaining half of the thymus is cryopreserved for future use in recipients. The immunosuppressive regimen will suppress any remaining T cells until the cultured thymic tissue sections transplanted into the recipient release naive T cells and the recipient meets the criteria for discontinuing the maintenance immunosuppressive regimen. (More than 10% naive T cells are required for immunosuppression discontinuation.)

[0417] Thymectomy protocol The patient is transported to the operating room and placed under general anesthesia via an endotracheal tube. Prepare and cover the chest and abdomen in a sterile manner. The patient undergoes a complete sternotomy through a skin incision of approximately 4 cm. To ensure complete resection, access is made to both pleural cavities. Visualize the phrenic nerves on both sides and take care not to damage them. Identify the thymus and carefully detach it from the pleural capsule, starting from the inferior horn and extending to the superior horn. A complete thymectomy will be performed. Hemostasis is performed in the mediastinum. Insert the pleural tube. Always insert one pleural tube (into the mediastinum). When entering a single pleural cavity during surgery, the pleural tube extends from the mediastinum into that pleural cavity. When entering both pleural cavities, use a second pleural tube from the mediastinum to the other pleural cavity in the same manner.

[0418] Drain Size Use #15 BreakDrain for infants up to 2 years old. Use #19 BreakDrain for children over 2 years old.

[0419] Sternal closure: In neonates or infants, 0-Tyclon sutures are used to close the sternum. Around 1-2 years of age, #1 sternal wires are used. Around 2-5 years of age, #4 sternal wires are used. The fascia, subcutaneous tissue, and skin are closed by continuous suturing using absorbable sutures. Place the skin wound VAC on the sternum. The patient's tube is removed in the operating room. The sponges, instruments, and needles must be counted, and this count must be correct at the end of the surgery.

[0420] Surgical transplantation of cultured products derived from allogeneic thymic tissue after birth. Culture products derived from allogeneic thymic tissue after birth should be transplanted according to the following instructions. Thymic tissue transplantation to the thigh requires a healthy layer of muscle tissue.

[0421] Preparation for transplantation The maximum and minimum doses of cultured postnatal allogeneic thymus tissue intended for transplantation should be calculated individually for each patient. The intended recipient should be properly identified before administration.

[0422] Under sterile conditions in a laminar flow hood, remove tissue sections from the tissue culture dish, place them in a 120 ml sterile cup containing 20 ml of medium, pack them in a way that maintains sterility, and transport them to the operating room or pack them for shipment. Tissue sections should not be removed from their individual containers until ready for use. Verify the expiration date and time of the product.

[0423] Culture products (tissue sections) derived from allogeneic thymus tissue after birth are always handled using strict aseptic techniques. Each container is inspected for leaks or damage. Do not use any containers showing signs of contamination. Outside the sterile field, the containers of culture products derived from allogeneic thymus tissue after birth are unpacked from the shipping boxes. The rack containing the polypropylene containers is removed from the outer bag. When ready, a team member, outside the sterile field but next to the sterile preparation table, opens and removes the caps from each container one at a time. Each opened container is then held by another team member outside the sterile field, extending their arm over the sterile field without touching it.

[0424] A sterile field team member uses one forceps to remove individual tissue sections with their filters from the container and places them in a sterile tissue culture dish containing approximately 2 ml of preservative-free saline on a sterile preparation table. The four tissue sections, each with its filter removed from the four containers, are then placed in a sterile tissue culture dish located on the sterile field in front of the sterile field team member. Using sterile forceps, the sterile field team member then uses two forceps to detach the tissue sections from their filters, with one forceps holding the filter in place and the other pulling or scraping the tissue to form a clump. The tissue detached from each filter is then clumped in the center of the filter and placed on top of it. The sterile tissue culture dish is then moved to the sterile field. While the surgeon is transplanting the first four sections, a second set of four containers of culture products derived from postnatal allogeneic thymus tissue is then processed in the same manner. Once the surgeon has transplanted the first four sections, the next dish containing the four tissue sections is placed in the surgical field, and the first tissue culture dish is returned to the sterile field in front of the sterile field team member to place the third set of four tissue sections on it. This cycle is continued until all the desired tissues have been transplanted. Not all tissue sections are initially moved to avoid contamination from the air in the operating room.

[0425] Surgical treatment Step 1: Skin incision After induction of general anesthesia, a skin incision (usually about 5 cm long) is made in one of the anterior femoral segments. Note: The size of the incision for the transplant procedure and the use of one or both legs are determined by the patient's weight, the planned amount of tissue to be transplanted, and the patient's muscle mass. If all or most of the tissue can be transplanted into one leg, only one leg should be used.

[0426] Step 2: Open the fascia to expose the muscles of the anterior region.

[0427] Step 3: Muscle separation and transplantation.

[0428] Using a tonsil clamp or similar instrument, the muscle is separated along the natural groove of the quadriceps femoris muscle. Individual thymic sections of cultured product derived from postnatal allogeneic thymic tissue should be transplanted without cutting the muscle tissue. The individual tissue sections are placed in “pockets” within the quadriceps femoris muscle along the natural groove, approximately 1 cm apart and approximately 1 cm deep. Depending on the patient’s weight, the surgeon may place about 6-7 sections in 6-7 pockets along each groove. Individual sections of cultured product derived from postnatal allogeneic thymic tissue may be cut in half before transplantation, depending on the mass of tissue on each filter. Thick tissue sections that completely cover the filter should be cut in half to optimize angiogenesis in each tissue. Transplant as much tissue as needed into each anterior section up to the maximum planned dose.

[0429] Step 4: Muscle Closure To prevent the muscle from separating again and the graft from emerging from the muscle, the muscle at the thymic tissue graft site is closed with a single suture. Before closing the incision, ensure that the graft tissue is completely covered by muscle tissue and that there is no exposure of the thymic tissue.

[0430] Step 5 Repeat steps 3-4 for each tissue section of the culture product derived from postnatal allogeneic thymus tissue until the maximum planned dose is reached.

[0431] Step 6: Closing the incision Confirm hemostasis. Close the skin incision with two layers of absorbable sutures and apply a standard bandage such as wound closure tape or skin adhesive. Leave the fascia open to allow room for the muscle compartment to expand. A occlusive dressing may be used to prevent contamination.

[0432] Postoperative surgical management / medical management Use a mild pain reliever as needed. Monitor for signs of infection or dehiscence.

[0433] If the donor is a living relative donor of the lungs, kidneys, intestines, or partial liver, a portion of the thymus from that solid organ donor may be suitable for culture and transplantation. The pathological criteria listed above must be met with respect to the date of collection and the time to transplantation.

[0434] Cryopreserved cultured thymic tissue can also be obtained from third-party donors. However, third-party donors must express all recipient HLA alleles not expressed by solid organ donors. These include HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DQA1, HLA-DPB1, and HLA-DPA1. Mismatch in the HLA-DP allele may be accepted if the mismatch is "acceptable". For other alleles, minor mismatches are accepted, for example, HLA-A*01:02 into a recipient with HLA-A*01:01; in other words, the second field (after the colon) may be different, but the first field (before the colon) must be identical.

[0435] Human heart transplantation method Eligibility will be determined based on several criteria, including: poor prognosis of 12–24 months without heart transplantation despite maximum current supportive care; congenital or acquired heart disease with growth retardation as identified by UNOS criteria; symptoms of progressive heart failure in congenital or acquired heart disease that are refractory to medical treatment; abnormal hemodynamics or increased pulmonary vascular resistance; inoperable organic heart disease; symptomatic arrhythmias or poor exercise tolerance uns...

Claims

1. A method for producing allogeneic, cultured postnatal thymus tissue-derived products suitable for transplantation into humans, A process of providing donor thymus to an acclimatization regimen for a period of approximately 6 to 21 days, wherein the acclimatization regimen for donor thymus tissue includes aseptically treating the donor thymus tissue in thymic organ culture medium to produce donor thymus tissue sections in which T cells are partially depleted. A step of detecting the increase in the level of CCL21 in the thymic organ medium during the course of the acclimatization regimen, compared with the baseline level of CCL21 measured on day 0 of the acclimatization regimen. A step of detecting the decrease in one or more levels of L-selectin, M-CSF, galectin-7, or IL-16 in the thymic organ medium during the course of the acclimatization regimen, compared to the baseline level of one or more of L-selectin, M-CSF, galectin-7, or IL-16 measured on day 0 of the acclimatization regimen, and A method comprising the step of recovering a partially T-cell depleted donor thymus tissue section as a product derived from allogeneic, cultured postnatal thymus tissue suitable for transplantation.

2. The method according to claim 1, further comprising the step of detecting an increasing level of one or more CXCL12, CXCL16, and CCL11 in a thymic organ culture medium during the course of the acclimatization regime.

3. The acclimatization regimen is for a period of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; or 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, or 6-9 days, The method according to claim 1 or 2, or a period of 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.

4. The method according to claim 1 or 2, wherein the acclimatization regimen is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

5. The method according to any one of claims 1 to 4, further comprising the step of determining, in a donor-thymus tissue section in which T cells were partially depleted during the habituation regimen, a region positive for cytokeratin (CK) staining, the presence of at least one Hassall body, and the presence of intact nuclei of thymic epithelial cells and other stromal cells.

6. The method according to claim 5, wherein during the course of the acclimatization regimen, the donor-thymus tissue section shows a region positive for cytokeratin 14 (CK14) staining.

7. The method according to claim 5, wherein a donor thymus tissue section with partially depleted T cells shows regions positive for CK14 staining in a lace-like pattern.

8. The method according to any one of claims 1 to 7, further comprising the step of cryopreserving allogeneic cultured postnatal thymus tissue-derived products in liquid nitrogen for future transplantation.

9. The method according to claim 8, further comprising the step of typing HLA alleles: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DPB1, and HLA-DPA1.

10. The method according to claim 8 or 9, further comprising the step of maintaining cryopreserved

11. A product derived from allogeneic cultured postnatal thymus tissue, prepared by the method described in any one of claims 1 to 7, Compared to the baseline level of CCL21 measured on day 0 of the acclimatization regimen, the secretion of CCL21 increased in the thymic organ medium during the course of the acclimatization regimen. Compared to the baseline levels of one or more of L-selectin, M-CSF, galectin-7, or IL-16 measured on day 0 of the habituation regimen, the secretion of one or more of L-selectin, M-CSF, galectin-7, or IL-16 at levels that decrease in the thymic organ medium during the course of the habituation regimen, and Products derived from allogeneic, cultured postnatal thymus tissue, characterized by exhibiting regions positive for cytokeratin (CK) staining, the presence of at least one Hassall body, and the presence of intact nuclei of thymic epithelial cells and other stromal cells during the course of the acclimatization regimen.

12. A product derived from allogeneic cultured postnatal thymus tissue, prepared by the method described in any one of claims 8 to 10, Compared to the baseline level of CCL21 measured on day 0 of the acclimatization regimen, the secretion of CCL21 increased in the thymic organ medium during the course of the acclimatization regimen. Compared to the baseline levels of one or more of L-selectin, M-CSF, galectin-7, or IL-16 measured on day 0 of the habituation regimen, the secretion of one or more of L-selectin, M-CSF, galectin-7, or IL-16 at levels that decrease in the thymic organ medium during the course of the habituation regimen, and Products derived from allogeneic, cultured postnatal thymus tissue, characterized by exhibiting regions positive for cytokeratin (CK) staining, the presence of at least one Hassall body, and the presence of intact nuclei of thymic epithelial cells and other stromal cells during the course of the acclimatization regimen.

13. A method for determining whether a product derived from allogeneic, cultured postnatal thymus tissue is suitable for transplantation into humans, A process of acclimatizing donor thymic tissue sections in thymic organ culture medium over a period of approximately 6 to 21 days, wherein the acclimatization regimen for donor thymic tissue includes aseptically treating the donor thymic tissue in thymic organ culture medium to produce donor thymic tissue sections in which T cells are partially depleted. A step of detecting the increasing level of CCL21 in thymic organ culture medium during the course of the acclimatization regimen, compared to the baseline level of CCL21 measured on day 0 of the acclimatization regimen, and A method comprising the step of detecting the decrease in one or more levels of L-selectin, M-CSF, galectin-7, or IL-16 in thymic organ culture medium during the course of an acclimatization regimen, compared to the baseline level of one or more of the L-selectin, M-CSF, galectin-7, or IL-16 measured on day 0 of the acclimatization regimen.

14. The method according to claim 13, further comprising the step of determining, in a donor-thymus tissue section partially T-cell depleted during the habituation regimen period, a region positive for CK staining, the presence of at least one Hassall body, and the presence of intact nuclei of thymic epithelial cells and other stromal cells.

15. The method according to claim 13 or 14, wherein the acclimatization regimen is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

16. The method according to any one of claims 13 to 15, wherein during the course of the acclimatization regimen, the donor-thymus tissue section shows a region positive for cytokeratin 14 (CK14) staining.

17. The method according to claim 16, wherein a donor thymus tissue section with partially depleted T cells shows regions positive for CK14 staining in a lace-like pattern.

18. Use of an allogeneic cultured postnatal thymic tissue-derived product according to claim 11 or 12 for preparing a pharmacopoeia for treating thymic disorders in a subject.

19. The use according to claim 18, wherein the thymic disorder is complete DiGorge syndrome, 22q11.2 deficiency, CHARGE (deficiency, cardiac defect, posterior nasal atresia, developmental delay or intellectual disability, genital hypoplasia and ear abnormalities or hearing loss), a mutation in the chd7 (chromodomain-helicase-DNA binding protein 7) gene, forkhead box protein N1 (FOXN1) deficiency, or congenital athymus associated with a mutation in the TBX-1 or TBX-2 gene.

20. The use according to claim 18, wherein the thymic disorder is age-related thymic regression or is related to thymoma, myasthenia gravis (MG), pure red cell aplasia or hypogammaglobulinemia.

21. The use according to claim 20, wherein the thymoma is malignant or non-malignant.

22. A product derived from allogeneic cultured postnatal thymus tissue according to claim 11, for use in a method of conferring immunity to a human subject, The method described above includes the step of transplanting a donor thymus tissue section in which T cells are partially depleted into a human subject, and is derived from allogeneic cultured postnatal thymus tissue.

23. A product derived from allogeneic cultured postnatal thymus tissue according to claim 11, for use in a method of conferring immunity to human subjects undergoing solid organ transplantation, wherein the method is: The process of transplanting solid organs, and This includes a step of transplanting donor thymus tissue sections with partially depleted T cells into human subjects. Products derived from allogeneic, cultured postnatal thymus tissue.

24. A product derived from allogeneic cultured postnatal thymus tissue according to claim 11, for use in a method to promote donor-specific tolerance to allogeneic solid organ grafts obtained from a donor in recipients requiring solid organ transplantation, wherein the method is: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A step of providing both a suitable human solid organ and a thymus derived from a donor; (d) The process of transplanting a human solid organ into the recipient; (e) The step of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing a product derived from allogeneic cultured postnatal thymus tissue; and (g) A step of transplanting the recipient with the allogeneic cultured postnatal thymus tissue-derived product after an acclimatization regimen of up to 21 days, wherein the dose of the partially T-cell-depleted donor thymus tissue section is approximately 1,000 to 22,000 ml ２ Thymus tissue surface area / Recipient body surface area (m²) ２ The process involves a unit of transplanted allogeneic cultured postnatal thymus tissue-derived product inducing thymocyte proliferation and immune tolerance in the recipient. Products derived from allogeneic, allogeneic, cultured postnatal thymus tissue, including [specific component].

25. A product derived from allogeneic cultured postnatal thymus tissue according to claim 12, for use in a method to promote donor-specific immune tolerance to allogeneic solid organ grafts obtained from human donors in human recipients requiring solid organ transplantation, wherein the method is: (a) the step of removing the thymus of the recipient; (b) The step of treating the recipient with an inducible immunosuppressive regimen comprising one or more immunosuppressants to deplete the recipient's T cells and / or to suppress the recipient's T cells from rejecting the transplanted solid organ; (c) A step of providing suitable solid organs derived from human donors; (d) The process of transplanting solid organs into the recipient; (e) The step of treating the recipient with a maintenance immunosuppression regimen; (f) A step of providing cryopreserved allogeneic cultured postnatal thymus tissue-derived products maintained in a bank of cryopreserved allogeneic cultured postnatal thymus tissue-derived products, wherein the cryopreserved allogeneic cultured postnatal thymus tissue-derived products are processed from thymus tissue derived from a thymus donor expressing an HLA allele that matches an HLA allele in the recipient that is not present in the solid organ graft. (g) Thawing products derived from cryopreserved allogeneic cultured postnatal thymus tissue, and (h) A step of transplanting a thawed, cryopreserved, allogeneic, cultured postnatal thymus tissue-derived product into a recipient, wherein the dose of the cryopreserved, allogeneic, cultured postnatal thymus tissue-derived product is approximately 1,000 to 22,000 ml ２ Thymus tissue surface area / Recipient body surface area (m²) ２ The process involves a unit of transplanted, allogeneic, cultured postnatal thymus tissue-derived product inducing thymocyte proliferation and immune tolerance in the recipient. Products derived from allogeneic, allogeneic, cultured postnatal thymus tissue, including [specific component].

26. The allogeneic cultured postnatal thymus tissue product according to claim 25, wherein the cryopreserved allogeneic cultured postnatal thymus tissue product is processed from thymus tissue derived from a thymus donor expressing HLA alleles that match HLA class I and HLA class II alleles in the recipient that are not present in the solid organ graft.

27. The allogeneic cultured postnatal thymus tissue-derived product according to claim 25 or 26, wherein in the method described above, about half of the thawed, cryopreserved, allogeneic, cultured postnatal thymus tissue-derived product is transplanted into a recipient, and the remainder is cryopreserved for future use.

28. The allogeneic cultured postnatal thymus tissue-derived product according to claim 25 or 26, wherein step (h) is performed approximately one month or more after the transplantation of a solid organ.

29. Allogeneic cultured postnatal thymus tissue-derived product according to any one of claims 23 to 28, wherein the solid organ graft is a heart graft, kidney graft, liver graft, lung graft, heart / lung graft, pancreas graft, intestine graft, stomach graft, abdominal wall graft, craniofacial graft, scalp graft, penis graft, uterine graft, unilateral or bilateral upper limb graft, unilateral angiogenic composite allogeneic graft, or a combination thereof.

30. The allogeneic cultured postnatal thymic tissue product according to claim 29, wherein the heart graft is a pediatric heart graft or an adult heart graft.

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