Cellular compositions derived from deceased donors to promote graft tolerance, and manufacture and use thereof

Abstract

To provide compositions for promoting mixed chimerism in organ transplant recipients, and methods of manufacturing and using the same.SOLUTION: The invention provides cellular compositions that contain CD34+ cells derived from bone marrow of a deceased donor and CD3+ cells derived from non-bone marrow of the deceased donor. The compositions are useful in promoting mixed chimerism in recipients of solid organ transplants. The invention also provides methods of making and using such compositions. In certain embodiments, the invention further provides methods of analyzing and preparing blood and blood components from a deceased donor for use in compositions of the invention to promote mixed chimerism in solid organ transplant recipients.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 732,823, filed September 18, 2018; U.S. Provisional Application No. 62 / 732,827, filed September 18, 2018; U.S. Provisional Application No. 62 / 732,830, filed September 18, 2018; and U.S. Provisional Application No. 62 / 732,832, filed September 18, 2018, the contents of each of which are incorporated herein by reference.

[0002] The present invention relates generally to therapeutic compositions comprising cells derived from deceased donors, and methods of making and using the same. [Background technology]

[0003] In the United States, nearly 35,000 organ transplants are performed annually. The major complication of organ transplantation is rejection of the organ by the recipient's immune system. To avoid organ rejection, most transplant recipients must take immunosuppressant drugs for the rest of their lives. However, immunosuppressant therapy has its own set of risks, including increased risk of infection, cancer, hypertension and liver damage. Furthermore, immunosuppression does not guarantee that the recipient will tolerate the transplant.

[0004] By reconstituting the recipient's immune system to contain a mixture of donor- and recipient-derived cells, a condition called mixed chimerism, long-term transplant tolerance can be achieved without immunosuppression. Thus, establishing mixed chimerism in organ transplant recipients has become highly desirable.

[0005] Mixed chimerism can be achieved by providing donor blood cells, including hematopoietic stem and progenitor cells (HSPCs) as well as T cells, to the recipient. Unfortunately, the supply of blood cells from adult donors is limited due to the difficulty of obtaining HSPCs from living donors. HSPCs can be collected from living donors by surgical extraction, but the surgery generally requires general anesthesia, can be painful, and carries the risk of infection or damage to nerves and muscles. Alternatively, HSPCs can be collected non-surgically by administering agents to the donor that mobilize HSPCs from the bone marrow to the blood and collecting the cells from the peripheral blood by apheresis. However, apheresis can take up to six hours and may need to be repeated for several days to obtain sufficient amounts of HSPCs, posing the risk of infection and blood clotting. Furthermore, regardless of the method, donation of HSPCs does not provide any medical benefit to the donor, so many individuals are unwilling to donate HSPCs unless they have a direct family or personal relationship with the recipient. Summary of the Invention [Means for solving the problem]

[0006] The present invention recognizes that deceased donors represent a potential alternative source of HSPCs and T cells, and provides various approaches and techniques that address the challenges associated with obtaining these therapeutically beneficial cells from deceased donors. In particular, the present invention utilizes a number of different insights and discoveries associated with studying deceased donor tissues and bodily fluids, which are exploited herein to develop new manufacturing processes for the production of new engineered hematopoietic cell products derived from deceased donors. For example, new blood collection devices for collecting blood from deceased donors, CD34 T cells from the bone marrow of deceased donors, and novel methods for the production of hematopoietic cell products derived from deceased donors. +New technologies, products and approaches have been developed, including but not limited to the development of new methods for ex vivo extraction of cells, new methods for processing deceased donor blood and new assays for analyzing deceased donor blood. All of these insights and developments have resulted in new manufacturing processes for the production of new engineered hematopoietic cell products derived from deceased donors.

[0007] In certain embodiments, the present invention provides novel cellular compositions comprising HSPCs derived from the bone marrow of a deceased donor and T cells derived from sources other than the bone marrow of a deceased donor. Until the development of the manufacturing processes described herein, CD3 T cells derived from sources other than the bone marrow of a deceased donor (e.g., blood of a deceased donor) have been used. + Compositions comprising T cells have not previously been possible. In certain embodiments, bone marrow derived HSPCs are identified by expression of CD34 and T cells are identified by expression of CD3. The cellular composition comprises CD34 in an amount sufficient to promote the establishment of mixed chimerism in solid organ transplant recipients. + Cells and CD3 + Contains cells.

[0008] The compositions and methods of the present invention greatly improve the utility of stem cell transfer to support organ transplantation. First, they avoid the need to obtain HSPCs from living individuals, who are often unwilling to tolerate the side effects of a donation procedure without medical benefit. At the same time, the compositions and methods provided herein overcome some of the problems associated with obtaining HSPCs and T cells from deceased donors. Importantly, the CD34 + Cells and CD3 + Different sources are used to obtain cells so that the yield of each cell type is optimized. For example, rich bone marrow sources, such as the iliac crest or vertebral bodies, are CD34 + Acts as a source of cells, but CD3 +The cells are obtained from blood. Separate processing of HSPCs and T cells also allows the different sources to be subjected to specific protocols that facilitate the isolation of specific cell populations and preserve their function. For example, bone marrow can be treated with agents such as granulocyte colony stimulating factor (G-CSF), which mobilizes HSPCs but may not be beneficial for T cells. The present invention also provides methods for the analysis of HSPCs and T cells obtained from deceased donors to ensure that such populations of cells are suitable for use in the generation of cell products to be administered to living recipients.

[0009] In another aspect, the present invention provides a cell product for establishing mixed chimerism in solid organ transplant recipients. The product comprises 1×10 6 cells / kg of recipient body weight derived from the bone marrow of a deceased donor. 5 More than CD34 + 1 × 10 cells / kg of recipient body weight derived from cells and other than bone marrow (e.g., blood) of deceased donors 5 More than CD3 + Contains cells.

[0010] In another aspect, the present invention provides a method for establishing mixed chimerism in a solid organ transplant recipient, comprising administering to a subject who has received or will receive a solid organ transplant 1×10 IgG per kg of recipient body weight derived from deceased donor bone marrow. 5 More than CD34 + cells, and 1 × 10 per kg of recipient body weight derived from sources other than bone marrow of deceased donors. 5 More than CD3 + Providing a product containing the cells.

[0011] Cell products include CD34 + Cells and CD3 + Various amounts of each of the cells can be included. The amount can be defined as the number of cells per kg of recipient body weight. For example, the cell product can contain at least 1×10 cells per kg of recipient body weight. 5 , 2×10 5 , 5×105 , 1×10 6 , 2×10 6 , 4×10 6 , 1×10 7 , 2×10 7 , 4×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD34 + The cell product may contain at least 1 x 10 cells per kg of recipient body weight. 4 , 2×10 4 , 5×10 4 , 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD3 + It may contain cells.

[0012] The cell product should be at least 1 × 10 per kg of recipient body weight. 5 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 5 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD34 + Cells, at least 5 × 10 per kg of recipient body weight 5 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 5 × 10 per kg of recipient body weight6 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 8 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 8 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD34 + cells, or at least 5 × 10 per kg of recipient body weight 8 CD34 + The cell product may contain at least 1 x 10 cells per kg of recipient body weight. 5 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 5 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD3 + Cells, at least 5 × 10 per kg of recipient body weight 5 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 6 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 6 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 6 CD3 + Cells, at least 5 × 10 per kg of recipient body weight 6 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 7 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 7 CD3+ Cells, at least 4 x 10 per kg of recipient body weight 7 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 8 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 8 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD3 + cells, or at least 5 × 10 per kg of recipient body weight 8 CD3 + The cell product may contain approximately 1 x 10 cells per kg of recipient body weight. 5 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 5 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 1 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 5 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 1 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 1 × 10 per kg of recipient body weight 8 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 8 CD3 +Cells, approximately 4 × 10 per kg of recipient body weight 5 CD3 + cells, or approximately 5 × 10 per kg of recipient body weight. 8 CD3 + It may contain cells.

[0013] The bone marrow may be derived from any bone source, for example, the bone may be derived from the iliac crest or vertebral body.

[0014] The non-bone marrow may be any tissue or body fluid other than bone marrow. For example, the non-bone marrow may be blood, liver, lymph nodes, spleen or thymus. Preferably, the non-bone marrow is blood.

[0015] Deceased donors can be adults, children or fetuses.

[0016] CD34 + cells, CD3 + The CD34 cells or both may be HLA matched to the solid organ transplant recipient. + cells, CD3 + The cells or both may be HLA-mismatched to the solid organ transplant recipient. The donor and recipient may be HLA-matched at 6, 8, 10 or 12 alleles between the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR genes. The donor and recipient may be HLA-mismatched at 1, 2, 3, 4, 5, 6 or more alleles between the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR genes.

[0017] CD34 + Cells and CD3 + The cells can be provided in separate containers. + Cells and CD3 + The cells may be provided as a mixture in one or more common containers.

[0018] The cell product may contain a cryopreservation medium, which may include a cryoprotectant, such as DMSO having a molecular weight of about 40,000 Da. or dextran. The cryoprotectant may be present at a concentration of about 1%, 2%, 3%, 4%, 5%, 7.5% or 10%.

[0019] The solid organ may be any solid organ that can be transplanted by methods known in the art. For example, and without limitation, the solid organ may be a kidney, lung, pancreas, pancreatic islet cells, heart, intestine, colon, liver, skin, muscle, gums, eye, or tooth. Preferably, the solid organ is a kidney.

[0020] In another aspect, the present invention provides a method for preparing a cell product for establishing mixed chimerism in a solid organ transplant recipient, the method comprising: + Obtaining cells, CD3 cells derived from sources other than the bone marrow of deceased donors + and administering the resulting CD34 cells to solid organ transplant recipients. + Cells and derived CD3 + The method includes producing a cell product comprising the cells. The cell product may include one or more of the above characteristics. In certain embodiments, the non-bone marrow may be blood, liver, lymph nodes, spleen or thymus. Preferably, the non-bone marrow is blood.

[0021] The method comprises administering to a subject a subject, comprising administering to a subject a subject, + The method can include exsanguination of the deceased donor before obtaining the cells from the bone marrow. The method can include removing bone marrow from a portion of the bone of the deceased donor. For example, the bone marrow can be removed by aspiration or trepanation. The method including trepanation can include one or more additional steps to separate the bone marrow from the bone fragments. For example and without limitation, the bone marrow can be separated from the bone fragments by one or more of agitation, enzymatic disaggregation, washing and filtration.

[0022] The method can include treating bone marrow with an anticoagulant.For example and without limitation, the anticoagulant can be acenocoumarol, antithrombin III, apixaban, argatroban, atromentin, betrixaban, bivalirudin, brodifacoum, dabigatran, dalteparin, difenacoum, edoxaban, EDTA, enoxaparin, fondaparinux, heparin, idraparinux, phenindione, phenprocoumon, rivaroxaban or warfarin.The bone marrow can be treated with an anticoagulant before its removal from the part of bone, or it can be treated with an anticoagulant after its removal from the part of bone.

[0023] The method involves the determination of CD34 from bone marrow. + This can include treating the bone marrow with an agent that mobilizes cells, such as, but not limited to, CD34 cells from the bone marrow. + Agents that recruit cells include adenosine receptor antagonists, BIO5192, CCR1 antagonists, CCR2 antagonists, CXCR2 antagonists, CXCR4 antagonists, cyclophosphamide, defibrotide, EphA3-Fc, erythropoietin (EPO), glycosaminoglycan (GAG) mimetics, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene beta (GRO-beta), human growth hormone, IL-8, and macrophage inflammatory protein-1 alpha (MIP-1 alpha). , met-SDF-1 beta, NSC23766, parathyroid hormone, pertussis toxin, plerixafor, poly-[1-6]-D-glucopyranosyl-[1-3]-D-glucopyranose (PGG) glucan, Rac1 inhibitor, retinoic acid receptor agonist, SB290157, SDF-1 alpha peptide analog, stem cell factor (SCF), sulfated colominic acid, sulfated polysaccharide, T134, T140, thrombopoietin (TPO), TPO receptor agonist, VCAM-1 inhibitor, VLA-1 inhibitor, VLA-4 inhibitor or an analog or derivative of any of the foregoing. The bone marrow may be isolated from the bone marrow prior to its removal from the portion of the bone marrow. +The cells can be treated with agents that mobilize them or can be isolated from the bone marrow after their removal from the bone marrow. + It can be treated with agents that mobilize the cells.

[0024] CD34 + Obtaining cells can include depleting red blood cells, platelets, or both from bone marrow.For example, and without limitation, red blood cells and / or platelets can be depleted from bone marrow by buoyancy-activated cell separation, cell lysis, hetastarch sedimentation, immunomagnetic depletion, size-based centrifugation, or spin membrane filtration.Red blood cells and / or platelets can be depleted from bone marrow before its removal from the bone part, or red blood cells and / or platelets can be depleted from bone marrow after its removal from the bone part.

[0025] CD34 + The cells were obtained by isolating CD34 cells from the extracted bone marrow. + The method may further comprise immunoselecting the cells. + The cells are immunoselected following removal of bone marrow from a portion of the bone.

[0026] CD3 + Obtaining cells can include depleting red blood cells, platelets, or both from other than bone marrow, such as blood. For example and without limitation, red blood cells and / or platelets can be depleted from bone marrow by buoyancy-activated cell separation, cell lysis, hetastarch sedimentation, immunomagnetic depletion, size-based centrifugation, or spin membrane filtration.

[0027] CD3 + Obtaining cells is CD3 + Other methods of enriching for cells other than bone marrow may be included. + Positive selection of non-CD3 cells + This may include depletion of CD3 cells or a combination thereof. + Positive selection of cells was performed using a binding agent immobilized on a solid substrate, CD3 +The binding of one or more markers on the cells may include non-CD3 + Cell depletion was performed using a binding agent immobilized on a solid substrate, i.e., CD3 + It may involve binding of one or more markers that are not present in the CD3 cell. + Markers for positive selection of cells can include one or more of CD3, CD4 and CD8. CD3 + Markers for negative selection of cells can include one or more of CD10, CD14, CD15, CD33, CD41, CD71, CD209 and CD235. The binding agent can be an antibody. The solid substrate can be a bead or particle. CD3 + Obtaining cells can include preventing and / or removing clots, cell clumps, or both from outside of bone marrow, e.g., blood. Removal of clots and / or clumps can include filtration, e.g., filtering the blood, or treating the blood with an anticoagulant.

[0028] CD3 + Obtaining the cells can include separating blood into a cellular fraction and a plasma fraction.

[0029] The method involves the determination of the CD34 + Cells and derived CD3 + Alternatively, the method can include combining the obtained CD34 cells. + Cells and derived CD3 + This can include storing the cells in separate containers.

[0030] The method involves the determination of the CD34 + Cells and derived CD3 + This can include cryopreserving the cells. For example, the cells can be cryopreserved by the addition of a cryoprotectant, such as DMSO or dextran having a molecular weight of about 40,000 Da.

[0031] In another aspect, the invention provides a method of producing a cellular product from hematopoietic cells obtained from ex vivo bone marrow, comprising obtaining a sample comprising bone marrow from the body of a subject, obtaining hematopoietic cells from the sample after removing the sample from the body of the subject, and producing a cellular product containing the hematopoietic cells for administration to a solid organ transplant recipient.

[0032] Preferably, when the sample is obtained from the subject's body, the subject is deceased. The subject may be any mammal, such as a human or a primate. Preferably, the subject is a human.

[0033] The sample may be obtained from any bone in the subject's body. Preferably, the sample is obtained from the iliac crest or vertebral body, or both.

[0034] Hematopoietic cells can be a subset of hematopoietic cells that express one or more markers. For example, and without limitation, hematopoietic cells can be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells (T SCM ), naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + It may be a cell.

[0035] Hematopoietic cells can be obtained from the sample by any suitable means. For example, and without limitation, hematopoietic cells can be obtained by removing bone marrow from the sample by aspiration or trepanation. Methods that include trepanation can include one or more additional steps to separate the bone marrow from bone debris, such as those described above. Hematopoietic cells can be obtained by isolating the bone marrow with CD34 +This can be achieved by contacting the sample with an agent that mobilizes the cells.

[0036] The bone marrow removed from the sample can be further processed. For example, the bone marrow can be treated with an anticoagulant, such as those described above. The bone marrow cells can be separated from the bone debris, for example, by the methods described above. The bone marrow can be depleted of red blood cells, platelets, or both, for example, by the methods described above. CD34 + Cells can be immunoselected from bone marrow.

[0037] The method can include cryopreserving the cellular product. The cellular product can be cryopreserved using a cryoprotectant, such as those described above.

[0038] The cell product can provide a therapeutic benefit to a solid organ transplant recipient, for example, the cell product can promote the establishment of mixed chimerism in a solid organ transplant recipient.

[0039] The solid organ may be any solid organ, such as one of those described above.

[0040] Hematopoietic cells express CD34 + Cells and CD3 + As noted above with respect to cell-containing cellular products, the cells may be HLA-matched or HLA-mismatched to the solid organ transplant recipient.

[0041] In another aspect, the invention provides a method for assessing whether blood from a deceased donor is suitable for use in the manufacture of a product to be administered to a living recipient, the method comprising obtaining blood from the deceased donor, analyzing the components of the blood, and determining, based on the analysis of the components, whether the blood is suitable for use in the manufacture of a product to be administered to a living recipient.

[0042] In another aspect, the invention provides a method for assessing whether blood from a deceased donor is suitable for use in the manufacture of a product to be administered to a living recipient, the method comprising obtaining blood from the deceased donor, analyzing non-blood tissue components from the deceased donor, and determining whether the blood is suitable for use in the manufacture of a product to be administered to a living recipient based on the analysis of the components.

[0043] The non-blood tissue may be bone marrow, spleen, liver, lymph node or thymus.

[0044] The method can include analysis of multiple components.The method can include analysis of multiple blood components, multiple components from a non-blood tissue, or at least one blood component and at least one component from a non-blood tissue.

[0045] A component of blood or non-blood tissue may be a cell type or population of cells. For example, and without limitation, the cells may be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + It may be a cell.

[0046] The blood or non-blood tissue component may be a non-cellular component, such as a molecule. For example, and without limitation, the molecule may be a cytokine, a pro-inflammatory cytokine, an anti-inflammatory cytokine, a chemokine, an antibody, or an immunoglobulin.

[0047] Suitability of blood for use in the manufacture of a product to be administered to a living recipient can include analysis of T cells. For example, and without limitation, one or more of T cell activation, exhaustion, anergy, proliferation, viability, and apoptosis can be analyzed. Analysis of one or more of T cell activation, exhaustion, and anergy can include detection of one or more markers or receptors on the surface of the T cells.

[0048] Suitability of blood for use in the manufacture of a product to be administered to a living recipient can include analysis of HSPCs. For example, and without limitation, one or more of HSPC proliferation, HSPC viability, and HSPC apoptosis can be analyzed. HSPC proliferation and / or viability can be assayed in colony formation, long-term culture, or mouse model repopulation assays.

[0049] Suitability of blood for use in the manufacture of products to be administered to a living recipient may include analysis of cytokine or chemokine levels.

[0050] Analysis of blood or non-blood tissue components from a deceased donor can include comparison with the same components from a living donor.

[0051] If the blood is determined to be suitable for such use, the method can include using material from the blood in the manufacture of the product. The material from the blood can be a cell type or population of cells. For example, and without limitation, the cells can be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 +Cells or CD3 + The material from the blood may be a cell. The material from the blood may be a non-cellular component, such as a molecule. For example, and without limitation, the molecule may be a cytokine, a chemokine, an antibody, or an immunoglobulin. The method may include using a plurality of materials from the blood in the manufacture of the product.

[0052] The method can include the expansion of the cellular material for use in the manufacture of the product.

[0053] The method can include treating the blood to minimize or reduce damage to materials in the blood that can be used in the manufacture of products.

[0054] In one aspect, the invention provides multiple cell products derived from a single deceased donor for establishing mixed chimerism in multiple solid organ transplant recipients. Each product contains 1×10 cells / kg of recipient body weight. 5 More than CD34 + cells, and 1 × 10 per kg of recipient body weight 5 More than CD3 + Multiple products are derived from a single deceased donor. + The cells are derived from bone marrow and are CD3 + The cells are derived from sources other than bone marrow. The cell product can include any of the characteristics of a cell product described above.

[0055] In another aspect, the invention provides a method for establishing mixed chimerism in multiple solid organ transplant recipients, comprising administering 1×10 IgG per kg of recipient body weight to each of a plurality of subjects who have received or will receive a solid organ transplant. 5 More than CD34 + cells, and 1 × 10 per kg of recipient body weight 5 More than CD3 + The method includes providing a product containing CD34 cells. + The cells were derived from the bone marrow of a single deceased donor, and each product contained CD3 +The cells are derived from other than the bone marrow of a deceased donor.The method can include any of the features described above with respect to the method for establishing mixed chimerism in a solid organ transplant recipient.

[0056] Each product was administered at least 1 × 10 per kg of recipient body weight. 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 4×10 6 , 1×10 7 , 2×10 7 , 4×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD34 + Each product can contain at least 1 x 10 cells per kg of recipient body weight. 4 , 2×10 4 , 5×10 4 , 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 4×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD3 + It may contain cells.

[0057] In one aspect, the invention provides a method for separating blood from a deceased donor into cellular and non-cellular components and using the cellular component material to produce a product for establishing mixed chimerism in a solid organ transplant recipient. The product can have any of the characteristics of the cellular products described above.

[0058] The method can include treating the blood or a component of blood with an anticoagulant, such as those described above. Treatment with the anticoagulant can be before or after separation of the blood into cellular and non-cellular components.

[0059] The method can include depleting the blood or blood components of red blood cells, platelets, or both. Depletion of red blood cells and / or platelets can be performed by any of the methods described above. Depletion of red blood cells and / or platelets can be performed prior to separation of the blood into cellular and non-cellular components, or can be after separation.

[0060] The method can include removing clots and / or aggregates from the blood or blood components. The clots and / or aggregates can be removed by any of the methods described above. The clots and / or aggregates can be removed prior to separation of the blood into cellular and non-cellular components or they can be removed after separation.

[0061] The method includes the step of determining whether a particular type of hematopoietic cell is a hematopoietic cell, e.g., B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + This can include enriching the blood or components of the blood for cells. One or more cell types can be enriched by immunoselection.

[0062] The method can include analyzing the blood or a component of blood by any of the methods described above. The method can include determining whether the blood or a component of blood is suitable for use in manufacturing a product for administration to a living recipient or for establishing mixed chimerism in a solid organ transplant recipient.

[0063] The method may include expanding the cellular components of the blood.

[0064] In another aspect, the present invention provides an apparatus for the collection of blood from a deceased donor. In certain embodiments, the apparatus includes one or more receptacles coupled to a cooling system.

[0065] The apparatus can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more receptacles. Preferably, the receptacles have a combined volume of about 5 to about 10 liters. For example, each receptacle can have a volume of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 liters.

[0066] The cooling system is configured to cool the blood in the receiver to a target temperature. The target temperature may be about 2° C. to about 8° C. The cooling system may prevent the blood from freezing or ice crystals from forming in the blood. The cooling system may cool the blood to the target temperature within a period of time. For example, the cooling system may cool the blood to the target temperature in about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, or 10 hours or less.

[0067] The device may include a needle connected to one or more receptacles. The needle may be connected to the one or more receptacles by tubing.

[0068] The device can include a vacuum system configured to apply a vacuum to remove blood from the deceased donor. The vacuum system can be coupled to one or more of the needle, the tubing, and the receptacle.

[0069] In one aspect, the present invention provides a method of collecting blood from a deceased donor for use in the manufacture of a product for transfer to a living recipient. The method can include transferring the blood to an apparatus including one or more receivers connected to a cooling system. The apparatus can include any of the features described above.

[0070] The method can include inserting a needle into a blood vessel, artery or vein of the deceased donor.The method can include applying a vacuum to the blood vessel, artery or vein of the deceased donor.

[0071] The method can include contacting the blood with a stabilizing agent. The stabilizing agent can be an anticoagulant, such as those described above, or an osmotic stabilizing agent, such as It may be human serum albumin.

[0072] The method can include contacting the blood with a cryoprotectant, such as those described above. [Brief description of the drawings]

[0073] [Figure 1] FIG. 1 is an illustration of an outer cortical cutting trocar of a marrow extraction apparatus according to an embodiment of the present invention.

[0074] [Diagram 2] FIG. 2 is an illustration of an inner trocar of a pulp extraction device according to an embodiment of the present invention.

[0075] [Diagram 3] FIG. 3 illustrates a blood collection device according to an embodiment of the present invention.

[0076] [Figure 4] FIG. 4 illustrates a blood collection device according to an embodiment of the present invention.

[0077] [Diagram 5] FIG. 5 is a flow diagram illustrating a method of preparing a cellular composition according to an embodiment of the present invention.

[0078] [Figure 6] FIG. 6 is an expanded view of the tissue collection step from the flow diagram of FIG.

[0079] [Figure 7] FIG. 7 is an expanded view of the bone marrow preparation steps from the flow diagram of FIG.

[0080] [Figure 8] FIG. 8 is an expanded view of the blood preparation steps from the flow diagram of FIG.

[0081] [Figure 9] FIG. 9 is an expanded view of the buffer preparation step from the flow diagram of FIG.

[0082] [Figure 10] FIG. 10 is an expanded view of the CD34+ cell enrichment step from the flow diagram of FIG.

[0083] [Figure 11] FIG. 11 is an expanded view of the division step from the flow chart of FIG.

[0084] [Figure 12] FIG. 12 is an expanded view of the cryopreservation step from the flow diagram of FIG.

[0085] [Figure 13] FIG. 13 is a flow diagram illustrating a method of preparing a cellular composition according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] The major challenge to overcome in organ transplantation is to get the recipient's immune system to tolerate the donor's tissue. If the recipient's immune system detects the donated organ as foreign, it will attack the tissue, leading to transplant rejection. To avoid transplant rejection, most transplant recipients must take drugs to suppress the immune system, but such drugs increase the recipient's risk of infection and cancer. As an alternative means of preventing transplant rejection, transplantation of solid organs may be accompanied by the transfer of donor-derived blood cell precursors. Providing donor blood cells allows the recipient's immune system to be reconstituted to contain cells educated to recognize the organ as non-foreign tissue. As a result, the donated organ is not attacked and the recipient tolerates the transplant.

[0087] One strategy for reconstituting the recipient's immune system requires the complete replacement of the recipient's hematopoietic system with cells derived exclusively from the donor to achieve a state of complete chimerism. However, a risk associated with complete chimerism is that the completely donor-derived immune system may identify the recipient's tissues as foreign and attack them, a condition known as graft-versus-host disease (GVHD). See, for example, Sachet et al., Induction of Tolerance through Mixed Chimerism, ColdSpring HarbPerspect Med 2014;4:a015529, doi: 10.1101 / cshperspect.a015529, the contents of which are incorporated herein by reference. As a result, complete chimeric patients must undergo immunosuppressive therapy indefinitely.

[0088] Another strategy is to repopulate the recipient's immune system with a mixture of donor- and recipient-derived cells to achieve a state called mixed chimerism. Compared to complete chimerism, mixed chimerism is associated with a lower rate of GVHD. Furthermore, mixed chimerism regimes require lower doses of immunosuppressive therapy initially and allow complete cessation of immunosuppression after the stability of the recipient's mixed chimerism has been established. To date, induction of mixed chimerism is the only method to produce transplantation tolerance in humans without maintenance of immunosuppressive therapy.

[0089] Current methods of establishing mixed chimerism require the transfer of two distinct populations of hematopoietic cells from a donor. Mature T cells recognize the transplanted organ as "self" tissue and prevent the immune system from attacking it. Mature T cells express the cell surface marker CD3, and different subpopulations can be found in the blood and lymph nodes. CD3 + Although T cells promote immune tolerance after transfer, they have a finite life span and cannot regenerate themselves. As a result, the transfer of hematopoietic stem and progenitor cells (HSPCs), multipotent cells that can differentiate into T cells, is also necessary to allow for the continuous replenishment of the donor-derived T cell population. HSPCs express the cell surface marker CD34 and reside primarily in the bone marrow.

[0090] The sacrifices that living donors must make to support long-term transplant tolerance in transplant recipients without lifelong immunosuppressive therapy are great. Thus, living donors must be highly motivated by altruism to undertake the steps necessary to provide the tissues and cells necessary to support immunosuppression-free transplant tolerance. As a result, few individuals are willing to become living donors of solid organs and hematopoietic cells, especially if they have no family or personal relationship with the intended recipient.

[0091] In comparison, a significantly higher percentage of individuals have agreed to donate organs and blood cells after death. However, when a donor is dead, it is more difficult to obtain suitable hematopoietic cells for transfer to a living recipient. For example, tissues must be removed quickly from the deceased donor to avoid the deleterious effects of cytokines released during the process of brain death. However, HSPCs must be taken from the bone marrow of most deceased donors, while blood is the best source of T cells. Thus, the three types of tissues or body fluids of primary interest, solid organs, blood, and bone marrow, must be collected from the deceased donor. Furthermore, the three sources of cellular material must be removed from the body in an order that best preserves the function of each while ensuring that the entire procedure is completed as quickly as possible.

[0092] Another problem with using hematopoietic cells from deceased donors is the uncertainty of the suitability of such cells for transfer into living patients. In the case of T cells, the state of cell activation, exhaustion and anergy influence whether the cells can promote transplantation tolerance in the recipient, and these characteristics may vary depending on the donor's cause of death, premortem health, age, sex, lifestyle and other factors. Cell viability, proliferation capacity and apoptotic status are critical variables in determining the utility of HSPCs and T cells in products that promote mixed chimerism in the recipient.

[0093] The present invention provides preparative and analytical methods that overcome the difficulties of obtaining hematopoietic cells from deceased donors and using the cells to generate compositions suitable for transfer into living recipients. The compositions of the present invention promote the establishment of mixed chimerism in solid organ transplant recipients, thus enabling such recipients to develop long-term transplant tolerance without immunosuppressive therapy. As a result, the present invention unlocks the therapeutic potential of donated tissue from deceased donors to extend and improve the life span of patients in need of organ transplants.

[0094] Cell products derived from deceased donors for transfer into living recipients The present invention provides a cell product containing hematopoietic cells obtained from a deceased donor. All hematopoietic cells originate from HSPCs, multipotent cells that can differentiate into various specialized cells and replicate to generate new HSPCs. HSPCs differentiate from lymphoid or myeloid precursors. Lymphoid precursors generate lymphocytes and natural killer cells. Myeloid precursors generate cells of the myeloid and erythroid lineages, such as erythrocytes, platelets, basophils, neutrophils, eosinophils, monocytes, macrophages, and antigen-presenting cells, such as dendritic cells. In adults, most hematopoietic development occurs in the bone marrow, while maturation and activation of some lymphoid cells occurs in the spleen, thymus, and lymph nodes.

[0095] The cellular compositions of the present invention contain two populations of cells that enable donor HSPCs to develop into mature cells of the immune system in the recipient's body. One population is CD34 + CD34 is a cell surface marker expressed on stem cells that are not committed to either myeloid or lymphoid lineages and their immediate progeny, multipotent progenitor cells. As a result, CD34 expression is a valuable tool for identifying populations of cells that contain HSPCs. In adults, CD34 + The cells are found mostly in the bone marrow.

[0096] The cellular composition of the present invention comprises CD3 + CD3 also includes T-cell receptor (TCR)-associated markers. CD3 comprises a group of polypeptides that interact with two polypeptide chains of the T-cell receptor to form the T-cell receptor complex. The CD3 complex includes a gamma chain, a delta chain, and two epsilon chains. CD3 is expressed on the surface of mature T cells and is therefore useful as a marker for T cells. CD3 + The cells are abundant in the circulating blood.

[0097] To promote the establishment of mixed chimerism in recipients, cell products are CD34 + Cells and CD3 + Contains sufficient amounts of cells. The cell product is CD34+ Cells and CD3 + The cells can be contained in a specified amount. A useful unit of measure for the amount of cells in a product is the number of cells per kg of recipient body weight. For example, and without limitation, a cell product may contain at least 1×10 cells per kg of recipient body weight. 4 , 2×10 4 , 5×10 4 , 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 or 4×10 6 , 1×10 7 , 2×10 7 , 4×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD34 + For example and without limitation, the cell product can contain at least 1×10 cells per kg of recipient body weight. 4 , 2×10 4 , 5×10 4 , 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 or 5×10 8 CD3 + It may contain cells.

[0098] The cell product should be at least 1 × 10 per kg of recipient body weight. 5 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 5 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD34 +Cells, at least 5 × 10 per kg of recipient body weight 5 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 6 CD34 + Cells, at least 5 × 10 per kg of recipient body weight 6 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 7 CD34 + Cells, at least 1 x 10 per kg of recipient body weight 8 CD34 + Cells, at least 2 x 10 per kg of recipient body weight 8 CD34 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD34 + cells, or at least 5 × 10 per kg of recipient body weight 8 CD34 + The cell product may contain at least 1 x 10 cells per kg of recipient body weight. 5 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 5 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD3 + Cells, at least 5 × 10 per kg of recipient body weight 5 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 6 CD3 +Cells, at least 2 x 10 per kg of recipient body weight 6 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 6 CD3 + Cells, at least 5 × 10 per kg of recipient body weight 6 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 7 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 7 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 7 CD3 + Cells, at least 1 x 10 per kg of recipient body weight 8 CD3 + Cells, at least 2 x 10 per kg of recipient body weight 8 CD3 + Cells, at least 4 x 10 per kg of recipient body weight 5 CD3 + cells, or at least 5 × 10 per kg of recipient body weight 8 CD3 + The cell product may contain approximately 1 x 10 cells per kg of recipient body weight. 5 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 5 × 10 per kg of recipient body weight 5 CD3 + Cells, approximately 1 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 6 CD3 + Cells, approximately 5 × 10 per kg of recipient body weight 6 CD3 +Cells, approximately 1 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 7 CD3 + Cells, approximately 1 × 10 per kg of recipient body weight 8 CD3 + Cells, approximately 2 × 10 per kg of recipient body weight 8 CD3 + Cells, approximately 4 × 10 per kg of recipient body weight 5 CD3 + cells, or approximately 5 × 10 per kg of recipient body weight. 8 CD3 + It may contain cells.

[0099] Other concentrations are exemplified in US Pat. Nos. 9,504,717 and 9,561,253, the contents of each of which are incorporated herein by reference in their entirety.

[0100] Cell products include CD34 + The cells can contain a specified degree of purity. For example, the cell product can contain CD34 cells that are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% pure. + Other purities can be exemplified in U.S. Patent Nos. 9,504,717 and 9,561,253, the contents of each of which are incorporated herein by reference in their entirety.

[0101] CD34 + Cells and CD3 + The cells may be provided as a mixture in one or more containers. + Cells and CD3 + The cells can be provided in a separate container. Any commercially available container approved for holding cell products can be used.

[0102] The cell product can be provided frozen. As a result, the cell product can contain a cryoprotectant. Any cryoprotectant known in the art can be used. For example and without limitation, the cryoprotectant can be DMSO, dextran with an average molecular weight of 40 kDa, serum, such as bovine serum, albumin, such as human serum albumin, or cell culture medium. The cryoprotectant can be present at a specified concentration. For example, the cell product can contain about 1% DMSO, about 2% DMSO, about 5% DMSO, about 7.5% DMSO, about 10% DMSO, about 12.5% ​​DMSO, about 15% DMSO, or about 20% DMSO. The cell product can contain about 1% dextran, about 2% dextran, about 5% dextran, about 7.5% dextran, about 10% dextran, about 12.5% ​​dextran, about 15% dextran, or about 20% dextran. The cryoprotectant can be a commercially available freezing medium, such as the medium sold under the trade name CryoStor 10 by BioLife Solutions (Bothell, WA). Cryoprotection is discussed in U.S. Patent No. 9,504,717 and U.S. Patent No. 9,561,253, the contents of each of which are incorporated herein by reference in their entirety.

[0103] The cell product can contain an agent that enhances the engraftment or functional mobilization of hematopoietic cells in the recipient. The cell product can contain an agent that prevents a negative reaction of the recipient to the hematopoietic cells. For example, and without limitation, the pharmaceutical composition can contain a cytokine, a chemokine, a growth factor, an enzyme, an excipient, a carrier, an antibody or fragment thereof, a small molecule, a drug, an agonist, an antagonist, a matrix protein, or a complementary cell type.

[0104] In certain embodiments, the cell product contains an enzyme, a substrate, or both. For example, the cell product can contain one or more alpha 1,3-fucosyltransferases, fucose donors, or both. Fucosylation of HSPCs enhances binding to E-selectin and P-selectin, improving their ability to home to bone marrow. Examples of alpha 1,3-fucosyltransferases include alpha 1,3-fucosyltransferase IV, alpha 1,3-fucosyltransferase VI, and alpha 1,3-fucosyltransferase VII. The fucose donor can be GDP-fucose. Fucosylation of HSPCs is described in detail in U.S. Patent No. 7,776,591, the contents of which are incorporated herein by reference.

[0105] The cell product can contain a buffer. The cell product can be buffered to maintain a physiologically compatible pH. For example, the cell product can be buffered to a neutral pH, for example, about 6.0 to about 8.0.

[0106] The cell product may be provided in the form of a pharmaceutical composition comprising an isotonic excipient, prepared under sufficiently sterile conditions for administration to humans. The choice of cell excipient and any associated elements of the composition is regulated by the route and device to be used for administration. For general principles of drug formulation, see CellTherapy: Stem Cell Transplantation, Gene Therapy, and See Cellular Immunotherapy, by G. Morstyn & W. Sheridan. eds., Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000.

[0107] CD34 + cells, CD3 +The cells or both may be HLA matched or HLA mismatched to the recipient. Human leukocyte antigens (HLA), also called major histocompatibility complex (MHC) antigens, are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells. MHC / HLA antigens are target molecules recognized by T cells and natural killer (NK) cells as originating from the same source of hematopoietic stem cells as the immune effector cells ("self") or from a different source of hematopoietic reconstituting cells ("non-self"). 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) cause each cell to be recognized as "self", while HLA class II antigens (DR, DP and DQ in humans) are involved in the reaction between lymphocytes and antigen-presenting cells.

[0108] A key aspect of the HLA gene system is its polymorphism. Each gene exists in different alleles. The products of the alleles differ in one or more amino acids in the alpha and / or beta domain(s). An individual has two alleles of each gene, for a total of 12 alleles between the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR genes. An HLA-matched donor can match with a recipient at 6, 8, 10 or 12 alleles selected from any combination of the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR genes. The most important genes for HLA typing are HLA-A, HLA-B and HLA-DR, and thus donors and recipients can be matched at all six alleles of the HLA-A, HLA-B and HLA-DR genes. HLA mismatched donor can have mismatches at 1, 2, 3, 4, 5, 6 or more alleles between HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ and HLA-DR genes. HLA typing can be performed by any method known in the art. Examples of HLA typing methods include serological cytotoxicity, flow cytometry and DNA typing. Such methods are described, for example, in U.S. Patent No. 9,561,253, the contents of which are incorporated herein by reference.

[0109] HLA genes are clustered in a super locus located at chromosome 6p21. As a result, sets of alleles present on a single chromosome, or haplotypes, tend to be inherited as a group. Identifying a patient's haplotype can help predict the probability of finding a matching donor and provide assistance in developing search strategies. Haplotypes differ in how common they are in the general population, as well as in their frequency of occurrence in different racial and ethnic groups.

[0110] The production of cell products from deceased donors can include additional characterization of the product. For example, CD34 + Cells and / or CD3 + For the composition containing cells, one or more secreted molecules, such as cytokines or chemokines, can be profiled.Alternatively, or in addition, for the composition, the expression profile of cell surface markers, such as CD3, CD34 or CD45, can be profiled.Any suitable method can be used to characterize the composition, including one or more of the methods described below with respect to the analysis of blood from deceased donors.Cell products can also be tested for the presence of pathogens, such as mycoplasma or endotoxins, which clearly indicate the presence of pathogens.

[0111] Preparation of cell products from deceased donors for transfer into living recipients The present invention relates to CD34 derived from deceased donors. + Cells and CD3 + A method for preparing a cell product containing cells, comprising: + Cells and CD3 + Considering the predominant anatomical location of the cells, different tissues or body fluids are the preferred sources for the two cell types. Therefore, the present invention provides a method for isolating CD34 cells from the bone marrow of deceased donors. + The method of obtaining cells and the CD3 + A method for obtaining the cells is provided.

[0112] Bones include a hard outer layer called cortical or compact bone, and an inner spongy portion called cancellous bone, which contains bone marrow. Bone marrow can be obtained from the cancellous bone material of large bones, such as the pelvis, vertebrae, ribs, femur, tibia, and sternum. The preferred sources of bone marrow are the iliac crest of the pelvis and the vertebral bodies of the vertebrae.

[0113] The method involves the use of CD3+ cells from the body of a deceased donor, such as bone marrow and blood. +The method may include removal of the cell source in any order. Preferably, blood is removed first, i.e., exsanguination from the body, and then bone marrow is obtained. The method may include removal of a solid organ of interest, such as kidney, lung, pancreas, pancreatic islet cells, heart, intestine, colon, liver, skin, muscle, gums, eye, or tooth. CD3 cells from solid organs of interest, bone marrow, and blood, etc. + The sources of cells can be removed in any order. Preferably, the removal is in the following order: solid organ of interest, blood and bone marrow.

[0114] Bone marrow may be removed from the body of a deceased donor by any suitable method. In some methods, the bone marrow is removed by aspiration. Aspiration involves inserting a needle into the bone and pulling out the marrow. In some methods, the bone marrow is removed by trepanation. A trepan is a saw with a circular blade that cuts into the bone and extracts a cylindrical portion of the bone.

[0115] FIG. 1 is an illustration of an outer cortical cutting trocar 101 of a marrow extraction device according to an embodiment of the present invention. The outer trocar 101 includes a hollow shaft 113, which may be made of surgical steel. At the distal end of the outer trocar 101 is a cutting tip 115, such as a toothed saw. The cutting tip 115 may be moved manually, pneumatically, or electrically. The cutting tip 115 may be replaceable. At the proximal end of the outer trocar 101 is a removable pneumatic drive adapter 117. The pneumatic drive adapter 117 couples the outer trocar 101 with a pneumatic drive and may be removable from the outer trocar 101. The outer trocar 101 may include a suction adapter that couples the outer trocar 101 to a vacuum pump or other suction source to facilitate removal of the bone fragments. When the drive adapter 117 is removed, the suction adapter may be attached to the proximal end of the outer trocar 101 or it may be attached directly to the drive adapter 117.

[0116] FIG. 2 is an illustration of an inner trocar 201 of a marrow extraction device according to an embodiment of the present invention. The inner trocar 201 includes a shaft 223 having one or more deployable rongeur blades 219 disposed at a distal end. The rongeur blades can be disposed at different angles to achieve different annular cutting radii. Adjusting the cutting radius is beneficial for cutting into bones of different sizes. For example, a smaller cutting radius is required for removal of vertebral bodies, while a larger cutting radius is optimal for extracting bone segments from the femur or tibia. The inner trocar 201 also includes one or more handles 217 or wings at its proximal end. The handles 217 can be rotated by hand to allow manual extraction of bone segments. Alternatively or additionally, the inner trocar 201 can be coupled to a pneumatic drive for low speed operation.

[0117] The following sequence of steps may be used to remove a portion of bone from a deceased donor. The following sequence is illustrative only, and one of ordinary skill in the art will appreciate that other methods of bone removal are possible within the scope of the present invention. First, the cortical surface of the target bone is exposed by standard surgical procedures. Next, an outer cutting trocar 101 with a cutting tip 115 and a pneumatic drive adapter 117 that is placed in the chuck of a pneumatic drive is used to cut through the cortex of the bone into the medullary cavity. The pneumatic drive adapter 117 is then removed from the drive. A solid inner trocar 201 is then inserted through the shaft 113 of the outer trocar 101 and a rongeur blade 219 is positioned. The handle 217 is used to twist the rongeur blade 219 to break the medullary trabeculae. Alternatively, the inner trocar 201 may be engaged with a low speed pneumatic drive. The inner trocar 201 is then removed from the shaft 113, and a suction adapter is installed in the hollow trocar. Finally, a suction device is attached via a suction adapter and suction is applied to drain the bone marrow cavity into a collection bag. Bone fragments obtained by drilling generally contain bone fragments, so CD34 +The method of obtaining cells can include a procedure of separating bone marrow cells from bone fragments. Various methods can be used to isolate bone marrow cells from bone fragments, such as physical agitation, enzymatic disaggregation, washing and filtration. The method can include treating the bone fragments with one or more agents that mobilize hematopoietic cells, such as those listed above, to improve the yield of HSSCs from the bone fragments. The treatment can include immersing the bone fragments in a storage solution or liquid containing one or more mobilizing agents. The treatment can be performed in a storage container, such that the release of HSPCs occurs during the transportation or transport of the material to be extracted. Thus, the bone fragments can be extracted from a donor at a first site and transported to a recipient at a second site, and the mobilization of hematopoietic cells can occur, at least in part, while the tissue is in transit.

[0118] HSPCs are present in the bone marrow in large part as a result of molecular interactions with osteoblasts, stromal cells, and the extracellular matrix. In vivo, such interactions tether HSPCs to the bone marrow and prevent them from entering the circulation. During extraction of HSPCs, the same molecular interactions also mediate the expression of CD34 from other cell types and non-cellular material. + This can interfere with the isolation of CD34 cells for use in cell products to support organ transplants. + The method for preparing CD34 cells from bone marrow +This can include treating the bone marrow or bone portion with one or more agents that mobilize cells. Classes of agents that mobilize HSPCs from bone marrow include chemotherapeutic drugs, hematopoietic growth factors, chemokines, inhibitors of chemokine receptors, and inhibitors of integrins. For example and without limitation, mobilizing agents include adenosine receptor antagonists, BIO5192, CCR1 antagonists, CCR2 antagonists, CXCR2 antagonists, CXCR4 antagonists, cyclophosphamide, defibrotide, EphA3-Fc, erythropoietin (EPO), glycosaminoglycan (GAG) mimics, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene beta (GRO-beta), human growth hormone, IL-8, macrophage inflammatory protein-1 alpha (MIP -1 alpha), met-SDF-1 beta, NSC23766, parathyroid hormone, pertussis toxin, plerixafor, poly[1-6]-D-glucopyranosyl-[1-3]-D-glucopyranose (PGG) glucan, Rac1 inhibitors, retinoic acid receptor agonists, SB290157, SDF-1 alpha peptide analogs, stem cell factor (SCF), sulfated colominic acid, sulfated polysaccharides, T134, T140, thrombopoietin (TPO), TPO receptor agonists, VCAM-1 inhibitors, VLA-1 inhibitors, VLA-4 inhibitors or analogs or derivatives thereof.

[0119] Although bone marrow is the native environment for HSPCs, HSPCs represent only a small proportion of bone marrow cells. + These cells account for only about 1% of all nucleated cells in bone marrow. Therefore, the method of the present invention involves converting bone marrow extracts from deceased donors to CD34 + A step of enriching for the cells can be included.

[0120] One form of enrichment is to separate mononuclear cells from enucleated cells, such as red blood cells and platelets, and cells with multilobed nuclei, such as granulocytes, including neutrophils, basophils, and eosinophils. Several methods for enriching for isolating or enriching for mononuclear cells are known in the art. For example and without limitation, mononuclear cells can be isolated or enriched by buoyancy-activated cell separation, cell lysis, hetastarch sedimentation, immunomagnetic depletion, size-based centrifugation, and spin membrane filtration. A system for buoyancy-activated cell separation is commercially available from Cesca Therapeutics, Inc. (Rancho Cordova, CA), and is described, for example, in U.S. Patent No. 9,695,394, the contents of which are incorporated herein by reference. A system for size-based centrifugation is commercially available from Sepax Technologies, Inc. (Newark, DE). Systems for spinning membrane filtration are commercially available, such as the system sold under the trade name Lovo Automate Cell Processing System by Fresenius Kabi USA, LLC (Lake Zurich, IL).

[0121] CD34 + Cells can be purified based on the qualitative or quantitative expression of one or more cell surface markers. Examples of suitable cell surface markers include AC133, CD3, CD34, CD38, CD45 and Thy-1. CD34 + Cells can be purified based on the presence or absence of a marker, or on the expression level of a marker, e.g., high versus low. + Purification of cells from starting material and CD34 + Comparisons of marker expression, complete blood counts and / or mononuclear cell counts between materials enriched for cells can be included.

[0122] CD34 +The cells can be purified by selectively binding a suitable affinity reagent to CD34 or another marker. The affinity reagent can be an antibody, a full-length antibody, a fragment of an antibody, a naturally occurring antibody, a synthetic antibody, an engineered antibody, a full-length affibody, a fragment of an affibody, a full-length affilin, a fragment of an affilin, a full-length anticalin, a fragment of anticalin, a full-length avimer, a fragment of an avimer, a full-length DARPin, a fragment of a DARPin, a full-length finomer, a fragment of a finomer, a full-length Kunitz domain peptide, a fragment of a Kunitz domain peptide, a full-length monobody, a fragment of a monobody, a peptide, a polyamino acid, etc. The affinity reagent can be directly conjugated to the detection reagent and / or the purification reagent. The detection reagent and the purification reagent can be the same or they can be different. For example, the detection reagent and / or the purification reagent can be fluorescent, magnetic, etc. The detection reagent and / or the purification reagent can be magnetic particles for column purification. For example, magnetic column purification can be performed using Miltenyi columns, antibodies, buffers, preparations and reagents systems known to those skilled in the art. + and CD3 + Methods for affinity purification of hematopoietic cells, including cells, and analysis of purified populations are described, for example, in US Pat. Nos. 9,561,253; and 9,452,184, the contents of which are incorporated herein by reference.

[0123] CD34 + The cells can be isolated, enriched or purified by any method. For example, CD34 + The cells can be isolated, enriched or purified by column purification, flow cytometry, cell sorting or immunoadsorption column separation. + The cells are purified using an immunomagnetic column system such as that sold under the trade name CliniMACS by Miltenyi Biotec Inc. (Auburn, Calif.). +Methods for affinity purification of hematopoietic cells, including cells, and analysis of purified populations are described, for example, in U.S. Pat. No. 9,561,253; U.S. Pat. No. 9,452,184; Nget et al., Isolation of human and mouse hematopoietic stem cells, Methods Mol Biol. (2009) 506:13-21. doi: 10.1007 / 978-1-59745-409-4_2; and Spohn et al., Automated CD34 + cell isolation of peripheral blood stem cellapheresis product, Cytotherapy (2015) Oct;17(10):1465-71. doi:10.1016 / j.jcyt.2015.04.005, the contents of each of which are incorporated herein by reference. The method can include positive selection, negative selection, or both.

[0124] The method includes administering to a living recipient a CD34 antibody that is used in the manufacture of a product. + Cells and / or CD3 +Various other treatments of the bone marrow, blood or other tissue source that facilitate recovery of cells can be included. For example, blood, bone marrow or other tissue can be treated to remove clots and / or cell aggregates, i.e., clumps. Clots and clumps can be removed by any suitable method. Non-limiting examples of clot and clump removal include filtration, such as spin membrane filtration as described above; treatment with thrombolytic agents, such as alteplase, anistreplase, fungal kinase, recombinant tissue plasminogen activator, reteplase, streptokinase, tenecteplase and urokinase; ultrasound; and, e.g., as described in Khalil, et al., Rubbing Against Blood Clots Using Helical Robots: Modeling and In VitroExperimental Validation, IEEE Robotics and Automation Letters ( 2(2):927-934, April 2017, DOI: 10.1109 / LRA.2017.2654546 Mechanical friction is included, the contents of which are incorporated herein by reference.

[0125] Bone marrow, blood or other tissues can be treated with one or more anticoagulants to prevent or minimize clotting.For example and without limitation, anticoagulants include acenocoumarol, antithrombin III, apixaban, argatroban, atromentin, betrixaban, bivalirudin, brodifacoum, dabigatran, dalteparin, difenacoum, edoxaban, EDTA, enoxaparin, fondaparinux, heparin, idraparinux, phenindione, phenprocoumon, rivaroxaban and warfarin.Anticoagulants can be administered to the body of a deceased donor.Alternatively or additionally, bone marrow, blood or other tissues can be treated with anticoagulants after being removed from the body.

[0126] Bone marrow, blood or other tissues can be treated to deplete red blood cells and / or platelets. For example, and without limitation, red blood cells and / or platelets can be depleted by buoyancy-activated cell separation, cell lysis, hetastarch sedimentation, immunomagnetic depletion, size-based centrifugation or spin membrane filtration, as described above.

[0127] CD3 from blood + Methods for obtaining cells can include separating blood into different components, such as a cellular fraction and a plasma fraction, as detailed below.

[0128] CD3 + Obtaining cells is CD3 + Other methods of enriching blood or other non-bone marrow sources for cells may be included. Enrichment may include enrichment for CD3 + Positive selection of non-CD3 cells + For example and without limitation, depletion of CD3 + The cells are CD3 + Positive selection can be achieved by the use of antibodies or other agents that bind to cell surface markers, such as CD3, CD4, or CD8. For example and without limitation, non-CD3 + Depletion of cells CD3 + Depletion can be achieved by using antibody agents that bind to markers absent from the cell surface, such as CD10, CD14, CD15, CD33, CD41, CD71, CD209 or CD235. Positive selection or depletion can be achieved by binding antibodies conjugated to particles or beads to a subpopulation of cells and sorting the subpopulation of cells by methods known in the art, such as those described in U.S. Pat. No. 9,090,871; U.S. Patent Application Publication No. 2010 / 0310588; and International Patent Application Publication No. WO2017 / 005647, the contents of each of which are incorporated herein by reference.

[0129] CD34 for use in cell products + Cells and / or CD3 +During the preparation of cells, cells can be frozen, i.e., cryopreserved, at any stage. Cryopreservation can include the addition of one or more cryoprotectants, such as those described above with respect to the cell product of the present invention. Cryopreservation generally involves lowering the temperature of a cell-containing sample at a controlled rate. Cryopreservation can include thawing a cell-containing sample and washing the sample to remove one or more cryoprotectants. Methods and reagents for cryopreservation, including freezing, thawing and washing a sample, are known in the art and are described, for example, in U.S. Patent No. 9,561,253, the contents of which are incorporated herein by reference.

[0130] In some circumstances, CD34 is first obtained from a deceased donor + Cells and / or CD3 + The number of cells may be insufficient to generate a product to promote mixed chimerism in a living recipient. Therefore, the present method uses CD34 + Cells and / or CD3 + This can include expanding the cells ex vivo. Any desired cell type or population of cells can be expanded. For example, and without limitation, the expanded population can be HSPCs, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, megakaryocytes, myeloblasts, monoblasts, monocytes, macrophages, dendritic cells, CD34 + cells, CD3 + Cells or CD4 + The cells may be cultured in the presence of another cell type, such as feeder cells. Expansion may be before or after freezing. Expansion may include providing one or more growth factors and may include culturing the cells in the presence of another cell type, such as feeder cells. Methods for expanding hematopoietic cells are described, for example, in U.S. Patent No. 9,561,253, the contents of which are incorporated herein by reference.

[0131] CD34 + Cells and / or CD3 + Cells may be genetically modified ex vivo, for example in autologous transfer of donor cells, where genetic defects can be corrected using gene therapy. Methods of gene therapy are described, for example, in Mali, Delivery systems for gene therapy, Indian J Hum Genet. 2013Jan-Mar; 19(1): 3-8,doi: 10.4103 / 0971-6866.112870; Gennady Ermak (2015) FRONTMATTER. EmergingMedical Technologies, ISBN: 978-981-4675-80-2,doi.org / 10.1142 / 9789814675826_fmatter; and Bakhuraysah et al., Hematopoietic stemcell transplantation for multiple sclerosis: is it aclinical reality? StemCell Res Ther. 2016; 7:12, doi: 10.1186 / s13287-015-0272-1, and the the contents of each of which are incorporated herein by reference.

[0132] Ex vivo extraction of hematopoietic cells from bone marrow The present invention provides a method for the preparation of CD34+ cellular products from the bone marrow of deceased donors for use in the manufacture of cell products for transfer into living recipients. + Also provided is a method for extracting hematopoietic cells, such as adult CD34 cells. + The cells reside predominantly in the bone marrow, where they are anchored to other cells and to the extracellular matrix. + The cells are CD34 + They can be mobilized into the blood of living patients by treatment with drugs that disrupt the interactions between the cells and components of the bone marrow, but maximal mobilization takes several days. +Mobilization of cells slows any manufacturing process and, in the case of deceased donors, extends the exposure of the cells to cytokines and other factors released after brain death. The present invention utilizes CD34+ cells from the bone marrow of deceased donors by performing mobilization ex vivo. + The present invention provides a method for improving cell harvesting, which involves the use of CD34+ in a container while the bone marrow is being transported to a manufacturing facility. + The ability to extract the cells can speed up the manufacturing process.

[0133] Ex vivo extraction of hematopoietic cells from bone marrow expedites production of the cellular compositions of the invention. Extraction may involve the addition of one or more agents that facilitate the release of the cells of interest from the bone marrow while the bone sample is in the container, such as the CD34 + It involves contacting the bone sample with an agent that mobilizes the cells. Thus, this step can be performed during storage of the sample or during transport to a processing facility. Furthermore, since ex vivo extraction allows for rapid recovery of bone marrow cells, it improves the quality of the cells for use in the manufacture of products that promote mixed chimerism.

[0134] The method includes obtaining a bone sample from a deceased donor. The sample may be from any large bone that contains spongy material, such as the pelvis, vertebrae, ribs, femur, tibia, and sternum. Preferably, the sample is from the iliac crest of the pelvis and the vertebral body of the vertebrae.

[0135] The method further comprises obtaining bone marrow cells from the bone sample after it is removed from the body. The bone marrow cells can be obtained from the bone sample by any method. For example, the bone marrow cells can be obtained by trepanation or aspiration as described above. The sample obtained by trepanation can be treated to remove bone debris as described above.

[0136] The method includes the step of obtaining CD34 mobilizing agents from bone marrow, such as one of the mobilizing agents described above. + This can include treating the bone sample with an agent that mobilizes the cells.

[0137] The method can be used to obtain any type of hematopoietic cell. For example, and without limitation, the cells can be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + Preferably, the cells are HSPCs or CD34 + It is a cell.

[0138] The method can include one or more additional steps, such as those described above with respect to the preparation of the cell product. For example, the method can include any of the following: treating the hematopoietic cells with an anticoagulant, such as those described above; depleting the hematopoietic cells or red blood cells and / or platelets; and isolating the hematopoietic cells from CD34 cells. + Enriching hematopoietic cells for a cell type or population of cells, such as cells.

[0139] The method can include using hematopoietic cells extracted ex vivo from bone marrow, such as one of the cellular compositions described above, for use in the manufacture of a product for transfer into a living recipient.

[0140] The present invention relates to a method for the treatment of CD34 + The present invention also includes materials useful for ex vivo extraction of hematopoietic cells, such as CD34 cells, from the bone marrow of deceased donors. For example, the present invention provides methods for the extraction of hematopoietic cells, such as CD34 cells, from bone marrow, such as one of the mobilizing agents described above. + A receptacle is provided that is preloaded with one or more agents that mobilize cells. The invention also includes a system that includes a container that contains one or more such agents and one or more portions of bone derived from a deceased donor.

[0141] Materials and systems for ex vivo extraction of hematopoietic cells can be characterized by their ability to release specific cell types or populations of cells into suspension over time. For example, materials and systems can be used to extract CD34 cells from bone marrow in comparison to a reference preservation system. + Cellular release can be increased 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more.

[0142] Materials and systems for ex vivo extraction of hematopoietic cells can achieve release of specific cell types or populations of cells into suspension for defined periods of time, for example, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours or longer.

[0143] The materials and systems can achieve the release into suspension of specific cell types or populations of cells that have characteristics that improve the usefulness of the cells for the manufacture of products that promote mixed chimerism. For example, and without limitation, the cells can have superior viability, proliferation potential, marker expression, and / or apoptotic characteristics compared to cells from a reference preservation system.

[0144] Fractions of blood from deceased donors The present invention also provides a method for fractionating blood from a deceased donor into two or more components, and using one or more of the components in the manufacture of a product for transfer to a living recipient. Apheresis of blood from a living donor involves the separation of blood into different components, the isolation of one or more components, and the return of the remainder to circulation. For example, leukapheresis requires the isolation of leukapheresis from the donor's blood, and the return of the remaining cells and plasma to the donor's body. Apheresis methods and devices are known in the art and are described, for example, in U.S. Patent Application Publication No. 2002 / 0107469; U.S. Patent No. 5,607,579; U.S. Patent No. 9,364,600; and U.S. Patent No. 6,743,192, the contents of each of which are incorporated herein by reference.

[0145] The present invention provides a method in which a procedure similar to apheresis is used to separate components of blood from deceased donors. The advantage of using blood from deceased donors is that the unused portion of the blood does not need to be returned to the donor's body after isolation of the components of interest. For example, blood from deceased donors can be separated into a cellular fraction and a plasma fraction, or into two or more different cellular fractions. Blood can be fractionated sequentially. For example, after an initial separation into cellular and non-cellular fractions, CD3 + The cellular fraction can be further processed to enrich for specific cell types or populations of cells, such as CD3 + Any method known in the art can be used to enrich for cells, including any of the methods described above.

[0146] Separating blood from a deceased donor into different fractions is beneficial to obtain fractions enriched for specific components, e.g., cell types or populations of cells, for inclusion in the cell product of the present invention. The cells may be any type of hematopoietic cell, e.g., B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + In particular, the blood fraction may be a CD3 + It is useful to obtain a fraction enriched for CD34 cells. + It is also useful to obtain an enriched fraction for cells, which is the bone marrow-derived CD34 + It can be used to supplement cells.

[0147] Several insights of the present invention enable the use of fractionated blood from deceased donors in the manufacture of cellular products for transfer to living recipients. One is that the inventive devices and methods for collection of blood from deceased donors, as described below, allow the blood to be rapidly removed from the body, collected in one or more sterile receptacles, and refrigerated to a storage temperature that preserves the activity of the blood cells. The present invention provides a method for the preparation of CD34 cells, as described above, and a method for the preparation of a cellular product for transfer to a living recipient. + Cells and CD3 + The present invention also enables the use of fractionated blood from deceased donors by providing a method to enrich for a cell type of interest, such as cells. Another advancement that makes fractionated blood from deceased donors useful for the manufacture of therapeutic products is the ability to characterize the fractionation products by analytical methods described below to determine their usefulness as starting materials for such products.

[0148] Fractionation methods can be performed on blood supplemented with additional components from a deceased donor. For example, cells extracted from bone marrow or other tissues as described above can be combined with blood to enrich for the desired cell type or population of cells in the mixture. For example, and without limitation, enriched populations can include HSPCs, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, megakaryocytes, myeloblasts, monoblasts, monocytes, macrophages, dendritic cells, CD34 + cells, CD3 + Cells or CD4 + The cells extracted from bone marrow or other tissues may contain CD34 + Cells and CD3 + The supplemented blood may then be separated into fractions as described above.

[0149] Analysis of blood from deceased donors The invention also provides an analytical method for determining whether blood from a deceased donor is suitable for use in the manufacture of a product to be administered to a living recipient. The method may involve analysis of the blood itself, or may involve analysis of other tissues from the deceased donor. The analytical method may be combined with any of the preparation methods described above.

[0150] The method is useful for determining whether material in blood is suitable for use in the manufacture of a product for transfer to a living recipient. Preferably, the material from blood is a cell type or population of cells. For example, and without limitation, the cells may be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg ) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + The blood material may be a non-cellular component, such as a molecule. For example and without limitation, the molecule may be a cytokine, a chemokine, an antibody, or an immunoglobulin. The analytical method may be combined with any of the preparation methods described above. Alternatively, or in addition, the material from the blood may be a non-cellular component, such as a molecule. For example, and without limitation, the molecule may be a cytokine, a chemokine, an antibody, or an immunoglobulin.

[0151] To determine whether T cells promote mixed chimerism in the transplant recipient, analysis of several characteristics of T cells from the deceased donor is beneficial. For example, and without limitation, analysis of T cell activation, anergy, apoptosis, exhaustion, proliferation, viability and cytokine secretion provides information that can be used to assess whether the T cells can provide a therapeutic benefit to the living recipient.

[0152] T cells or CD3 from deceased donors +Characterization of cells can include analysis of cells in blood. Any suitable assay can be used. For example and without limitation, the method can include proliferation assays, DNA synthesis assays, apoptosis assays, colony formation assays, marker expression assays, etc. Methods for analyzing T cells are known in the art and are described in, for example, Brousseau, et al., eds., Manual of Immunological Methods, CRC Press,Boca Raton, 1998, ISBN9780849385582;Detrick, et al., eds., Manual of Molecular and Clinical LaboratoryImmunology, Eighth Edition, American Society forMicrobiology Press, WashingtonD.C., USA, 2016, ISBN-13: 978-1555818715 and Balakrishnan, et al., Practical Immunology: A Laboratory Manual, Lap Lambert Academic Publishing, 2017, ISBN-13: 978-3330352032, each of which The contents of which are incorporated herein by reference.

[0153] T cells or CD3 from deceased donors +Cell characterization can include the analysis of non-cellular components of blood. For example, and without limitation, the non-cellular components can be cytokines, such as pro- or anti-inflammatory cytokines, chemokines, antibodies, or immunoglobulins. The non-cellular components can be analyzed in serum or plasma from deceased donors. The non-cellular components can be analyzed by immunoassays, such as enzyme-linked immunoassays (ELISA), radioimmunoassays, immunoPCR, and immunoassay systems sold under the trade names Luminex xMAP by ThermoFisher Scientific (Waltham, MA); Meso Scale Discovery by Meso Scale Diagnostics (Rockville, MD); AlphaLISA and DELFIA by Perkin Elmer (Waltham, MA); Gyrolab by Gyros Protein Technologies (Uppsala, Sweden); and Erenna by MilliporeSigma (Burlington, MA). Other methods of analysis of non-cellular components are known in the art and are described, for example, in Brousseau, et al., eds., Manual of Immunological Methods, CRC Press,Boca Raton, 1998, ISBN9780849385582; Detrick, et al., eds., Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition, American Society for Microbiology Press, Washington D.C., USA, 2016, ISBN-13: 978-1555818715; and Balakrishnan, et al., Practical Immunology: A Laboratory Manual, Lap Lambert Academic Publishing, 2017, ISBN-13:978-3330352032, the contents of each of which are incorporated herein by reference.

[0154] The method involves the determination of HSPCs or CD34 in blood from deceased donors. + The assay may include an assay for whether the cells are suitable for use in the manufacture of a product for transfer to a living recipient. Properties such as HSPC proliferation, HSPC viability, and HSPC apoptosis may be assayed to determine whether HSPCs in blood can provide a therapeutic benefit and may be used to evaluate whether HSPCs can provide a therapeutic benefit to a living recipient. For example and without limitation, the assay may include a colony formation assay or a long-term culture assay that measures cell proliferation in response to exogenous growth factors. The assay may include an assay for the ability of HSPCs to repopulate a mouse model, such as a severe combined immunodeficient (SCID) mouse. These and other methods are known in the art and are described, for example, in Loring and Peterson, eds., Human Stem Cell Manual: A Laboratory Guide, Second Edition, Academic Press, 2012, ISBN: 978-0-12-385473-5; Stein, et al., eds., Human Stem Cell Technology and Biology: A Research Guide and Laboratory Manual, Wiley-Blackwell, 2011, ISBN 978-0-470-59545-9 ;Frisch and Calvi, Hematopoietic Stem Cell Cultures and Assays, Methods MolBiol. 2014;1130: 315-324, doi: 10.1007 / 978-1-62703-989-5_24;Dick JE, et al., Assay of human stem cells by repopulation of NOD / SCID mice, Stem Cells, 1997;15Suppl1:199-203; discussion 204-7, DOI: 10.1002 / stem.5530150826; and T Tatekawa, et.a., (2006) A novel direct competitive repopulation assay for human hematopoietic stemcells using NOD / SCID mice, Cytotherapy, 8:4, 390-398, DOI:10.1080 / 14653240600847191, the contents of each of which are incorporated herein by reference.

[0155] The analysis can include providing a report of blood or non-blood tissue characteristics from the deceased donor. For example, and without limitation, the report can include the percentage of cells having any of the following characteristics: marker expression, viability, proliferative capacity, apoptotic characteristics, anergy, exhaustion, activation, and cytokine secretion. The report can describe characteristics of subpopulations of cells. For example, and without limitation, the report can describe characteristics of CD34 + Cells or CD3 + These may include the percentage of cells expressing particular markers such as cells, proliferation potential, apoptotic characteristics, anergy, exhaustion, activation or cytokine secretion.

[0156] Determining whether the material in the blood is suitable for use in the manufacture of a product for transfer to a living recipient can include analysis of non-blood tissue from the deceased donor. For example and without limitation, the non-blood tissue can be bone marrow, spleen, liver, lymph nodes or thymus. The cellular components of the non-blood tissue can be analyzed, such as hematopoietic cells, such as those described above, or non-cellular components, such as molecules, such as those described above. Any analytical method can be used, including those described above.

[0157] The content and location of hematopoietic cells in the body varies during proliferation and development. For example, the spleen is a rich source of lymphocytes in adults, whereas the liver contains many lymphocytes in the developing fetus. Thus, the tissue source of hematopoietic cells can account for the age of the donor at the time of death.

[0158] The method can include the analysis of multiple components. The multiple components can be from the same source or from different sources. For example, the method can include the analysis of multiple cellular components of blood, multiple non-cellular components of blood, multiple components of non-blood tissue, or any combination of cellular blood components, non-cellular blood components, and non-blood tissue components.

[0159] To better determine whether components from the blood of a deceased donor are suitable for use in manufacturing a product for transfer to a living recipient, the analysis can include a comparison of one or more parameters from the tissue or fluid of the deceased donor with corresponding material from a living donor. The analysis can include a comparison of any of the characteristics described above. For example, and without limitation, the analysis can include a comparison of one or more of T cell activation, T cell anergy, T cell apoptosis, T cell exhaustion, T cell proliferation, T cell viability, T cell cytokine secretion, HSPC proliferation, HSPC viability, HSPC apoptosis, cytokine levels, chemokine levels, antibody levels, and immunoglobulin levels.

[0160] The determination of whether blood or blood components from a deceased donor are suitable for use in the manufacture of a product for transfer to a living recipient can depend on a comparison of one or more parameters between the deceased donor and the living donor. For example, if the value of one or more parameters from the deceased donor is comparable to the corresponding value from a living donor or a population of living donors, the blood or blood components from the deceased donor can be considered suitable for use in the manufacture of a product for transfer to a living recipient. The comparative analysis can include establishing a profile of characteristics for the living donor and donors at various time stages after brain death. Values ​​can be compared between the deceased donor and the living donor or population of living donors that meet one or more decision criteria. For example, the living donor or population of living donors can have one of the following: previously donated a solid organ; previously donated hematopoietic cells, such as HSPCs or T cells; or previously donated tissue that supported the establishment of mixed chimerism in the recipient. The living donor or population of living donors can be healthy, can be within a certain age range, can be of a certain gender, etc.

[0161] The method can include using one or more materials from the blood of the deceased donor in the manufacture of a product for transfer to a living recipient. The materials can be any of the materials described above, such as cellular or non-cellular components.

[0162] The method can include the expansion of a cell type or population of cells for use in the manufacture of a product for transfer into a living recipient. For example, the cells can be B cells, basophils, eosinophils, hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), lymphocytes, lymphoid progenitor cells, macrophages, mast cells, megakaryocytes, monocytes, myeloblasts, myeloid progenitor cells, natural killer (NK) cells, neutrophils, platelets / thrombocytes, T cells, T regulatory (T reg) cells, memory T cells, effector memory T cells, central memory T cells, stem memory T cells, naive T cells, cytotoxic T cells, gamma delta T cells, natural killer T cells, CD34 + cells, CD4 + Cells or CD3 + It may be a cell.

[0163] The method can include treating blood from a deceased donor to reduce damage to materials in the blood that can be used in the manufacture of products for transfer to a living recipient. For example, the method can include adding an anticoagulant, a buffer, a cryoprotectant, or a stabilizer to the blood. The stabilizer can prevent degradation of blood components or promote osmotic stability. For example, the stabilizer can be human serum albumin (HSA) or an electrolyte solution sold under the trade name Plasma-Lyte by Baxter International Inc. (Deerfield, IL).

[0164] The method can include cryopreserving the product. The product can be frozen at a suitable temperature for long-term storage, for example at about -150°C. The method can include gradually reducing the temperature of the product to the long-term storage temperature.

[0165] Apparatus and method for collection of blood from deceased donors - Patents.com The present invention also provides an apparatus and method for the collection of blood from deceased donors for use in the manufacture of products for transfer to living recipients. Blood must be processed rapidly to be used as a source of material for transfer to living recipients. In particular, it must be removed from the body promptly, preferably within a few hours, after brain death, prior to the release of cytokines that can alter the function of blood cells. Furthermore, blood must be rapidly cooled to an appropriate short-term storage temperature, typically 2-8°C, after removal from the body, but freezing should be avoided until the blood or blood products are ready for cryogenic storage. Total blood volume in an adult human should be 5 liters or greater, and CD34 + and CD3 + Collection of blood from deceased donors presents unique challenges, given the need for maximal recovery in order to obtain sufficient quantities of cells.

[0166] FIG. 3 shows a blood collection device 301 according to an embodiment of the present invention. The device 301 includes one or more receptacles 303a and 303b coupled to a cooling system. The receptacles can have a combined volume of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 liters, preferably about 5 to about 10 liters. The device can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more receptacles. The receptacles can be rigid and have a fixed shape or can be flexible and expandable. For example and without limitation, the receptacles can be cups, bottles, cylinders, bags, pouches, etc. The receptacles can be made of flexible or pliable plastic. In one embodiment, each receptacle is a rectangular prism measuring 25 cm by 20 cm by 2 cm and has a volume of 1 liter.

[0167] The device 301 can include one or more cannulas 305 that can be inserted into a vein or artery of a deceased donor. The cannulas can be connected to one or more receptacles 303a and 303b by connectors 307a, such as tubing. In a device 301 having multiple receptacles 303a and 303b, blood can be collected from receptacles 303a and 303b in parallel, sequentially, or some combination. In a device having receptacles arranged in sequence, receptacles 303a and 303b can be connected with connectors 307b. One or more of connectors 307a and 307b can include a one-way valve that prevents backflow of blood through the device 301, thereby minimizing contamination of the blood and maintaining its sterility.

[0168] 4 illustrates a blood collection device 401 according to an embodiment of the present invention. The device includes receptacles 403a, 403b, 403c, and 403d disposed in a refrigerated compartment 409. The refrigerated compartment 409 includes a coolant 411 that cools the blood after its removal from the donor. Any suitable coolant 411 can be used. For example, and without limitation, the coolant 411 can include ammonium chloride, ammonium nitrate, saline, calcium ammonium nitrate, diethylene glycol, hydroxyethyl cellulose, ice, propylene glycol silica gel, sodium polyacrylate, urea, or water.

[0169] The cooling system is configured to cool the blood in the receiver to a target temperature. The target temperature may be from about 2° C. to about 8° C. The cooling system may prevent the blood from freezing or from forming ice crystals in the blood. The cooling system may cool the blood to the target temperature within a specified time. For example, the cooling system may cool the blood to the target temperature in about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, or 10 hours or less.

[0170] The device can include a vacuum system configured to apply a vacuum to extract blood from the deceased donor.The vacuum system can be a pump.Vacuum pumps for blood collection are known in the art and are described, for example, in US Patent Application Publication No. 2010 / 0042015; and US Patent Application Publication No. 2008 / 0199949, the contents of each of which are incorporated herein by reference.The vacuum system can be connected to one or more of the needle, the tubing, and the receptacle.

[0171] The invention provides a method of removal of blood from a deceased donor using the device of the invention. The method includes transferring the blood to one or more receivers connected to a cooling system. The method can include inserting a needle into a blood vessel, artery or vein of the deceased donor. The method can include applying a vacuum to a blood vessel, artery or vein of the deceased donor. The method can include contacting the blood with a stabilizing agent or cryoprotectant, such as those described above.

[0172] Treating organ transplant recipients with cell products derived from deceased donors The cell products of the present invention can be provided to a solid organ transplant recipient. The cell products can be provided by any suitable means. For example and without limitation, CD34 + Cells and / or CD3 + The cells can be delivered to the recipient by injection using a needle, catheter, central venous catheter, etc. In some cases, the cells can be delivered intravascularly, intravenously, intraarterially, subcutaneously, intramuscularly, directly to bone, or through any source that allows hematopoietic cells to home to the appropriate site in the recipient so that they persist, regenerate, and differentiate in the recipient. + Cells and / or CD3 + The cells can be provided by injection. + Cells and / or CD3 +The cells can be provided in an inpatient setting or in an outpatient setting. Inpatient treatment requires admission to a hospital, where the patient may spend one or more nights in the hospital. Outpatient treatment does not require admission to a hospital, and can be performed in a non-hospital setting, such as a clinic, a doctor's office, at home, or elsewhere.

[0173] The composition of the present invention can be used in conjunction with any solid organ transplant.For example and without limitation, the solid organ can be kidney, lung, pancreas, pancreatic islet cells, heart, intestine, colon, liver, skin, muscle, gums, eye or tooth. Transplant can include cells from a complete organ, a part of an organ or tissue of an organ. The cell product can be provided before, during or after solid organ transplant.For example and without limitation, the cell product can be provided 1, 2, 3, 4, 5 or 6 days or 1, 2, 3 or 4 weeks before solid organ transplant, or it can be provided 1, 2, 3, 4, 5 or 6 days or 1, 2, 3 or 4 weeks after solid organ transplant.

[0174] To facilitate the establishment of mixed chimerism in the recipient, the recipient's immune system can be pretreated with the provision of the cellular product. For example, non-myeloablative conditioning can be used. In non-myeloablative conditioning, the recipient is exposed to drugs, antibodies, irradiation, or some combination thereof, at doses too low to eradicate all bone marrow cells. Generally, the conditioning regimen includes treatment with antithymocyte globulin (ATG), total lymphatic irradiation, and corticosteroids (e.g., prednisone) for about 10-12 days (e.g., about 11 days). The irradiation can be targeted to a specific location in the recipient's body. For example, the irradiation can be targeted to a tissue, organ, region of the body, or the entire body. The irradiation can be targeted to lymph nodes, spleen, or thymus, or any other area known to those skilled in the art. If multiple doses of irradiation are administered, the doses can be targeted to the same location or different locations. Non-myeloablative conditioning can include the use of T cell depleting agents, such as monoclonal antibodies or drugs, such as fludarabine. Regimens for non-myeloablative conditioning are known in the art and are described, for example, in U.S. Patent No. 9,561,253, the contents of which are incorporated herein by reference.

[0175] The method may include immunosuppressive therapy. Immunosuppressive therapy or immunosuppression involves the treatment of transplant recipients with agents that reduce the host immune system's response to donor cells that can lead to transplant rejection. Major immunosuppressive agents include calcineurin inhibitors, such as tacrolimus, cyclosporine A. Usually, adjuvant agents are combined with calcineurin inhibitors. Adjuvant agents include steroids, azathioprine, mycophenolic acid (MPA) agents, such as mycophenolate mofetil, mTOR inhibitors, such as sirolimus, and belatacept. The use of adjuvant agents allows clinicians to achieve sufficient immunosuppression while reducing the dose and toxicity of individual agents. Antibody-based therapy may use monoclonal (e.g., muromonab-CD3) or polyclonal antibodies, or anti-CD25 antibodies (e.g., basiliximab, daclizumab). Antibody-based therapy allows the avoidance or dose reduction of calcineurin inhibitors, possibly reducing the risk of nephrotoxicity. Regimens for immunosuppressive therapy are known in the art and are described, for example, in US Pat. No. 9,561,253, the contents of which are incorporated herein by reference.

[0176] Immunosuppression can also reduce the response of donor immune cells to recipient tissues, which can lead to GVHD. GVHD can be acute or chronic. Acute GVHD generally occurs during the first 3 months after transplantation and can affect the skin, intestine, or liver. Treatment for acute GVHD usually includes high-dose corticosteroids, such as prednisone. Chronic GVHD generally occurs after the first 3 months after transplantation and is the major source of late treatment-related complications. Chronic GVHD can cause functional disability and may require long-term immunosuppressive therapy.

[0177] Immunosuppressive therapy can be performed in multiple phases. For example, an immunosuppressive regimen can have an induction phase and a maintenance phase. The induction and maintenance phase strategies can use different pharmaceuticals, with dosages adjusted to achieve a target therapeutic level to enhance the establishment of mixed chimerism in the recipient.

[0178] After stable mixed chimerism is established in the recipient, immunosuppressive therapy can be discontinued. The chimeric state of the recipient can be monitored as described below and can be considered stable after a certain period of time, for example, 3 months, 6 months, 12 months, 18 months, 24 months or longer. Thus, immunosuppression for the recipient can be discontinued after a certain period of time, for example, 3 months, 6 months, 12 months, 18 months, 24 months or longer. Discontinuing immunosuppressive therapy can include tapering, i.e., gradually reducing the dosage or frequency of treatment.

[0179] As is known in the art, the determination of whether an individual is fully chimeric, mixed chimeric, or non-chimeric can be made by analysis of a hematopoietic cell sample, e.g., peripheral blood, bone marrow, etc., from the solid organ transplant recipient. The analysis can be performed by any convenient method of typing. The analysis can be performed by typing hematopoietic cells or subsets thereof, e.g., total mononuclear cells, T cells, B cells, CD56 + NK cells and CD15 + It can be performed on neutrophils. Chimerism can be assessed by PCR analysis of microsatellites. For example, commercial kits are available that distinguish polymorphisms in the length of short terminal repeat sequences of donor and host origin. Automated readers provide the percentage of donor-type cells based on a standard curve from a mixture of artificial donor and host cells.

[0180] A recipient can be classified as a complete chimera, mixed chimera, or non-chimera based on the percentage of cells derived from the donor. For example, a recipient can be considered as a complete chimera if it has at least 90%, at least 95%, at least 98% or at least 99% cells derived from the donor. A recipient can be considered as a mixed chimera if it has too few cells derived from the donor to be classified as a complete chimera, but has a percentage of cells derived from the donor that exceeds a certain threshold, such as at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 7.5%, at least 10% cells derived from the donor. A recipient can be considered as a non-chimera if the percentage of cells derived from the donor is below the threshold required to be classified as a mixed chimera.

[0181] Cell products derived from a single deceased donor for transfer into multiple living recipients The present invention also provides multiple cell products derived from a single deceased donor for transfer into multiple living recipients. + Cells and CD3 + The generation of multiple cell products containing cells allows the products to be transferred to different recipients. Thus, two or more solid organs from a deceased donor can be transplanted into different recipients, each of which contains the donor's CD34 pluripotent stem cells, promoting the establishment of mixed chimerism. + Cells and CD3 + For example, the products and methods of the present invention allow a single deceased donor to donate a kidney and CD34 cells to each of two recipients. + Cells and CD3 + As a result, the products and methods of the invention increase the number of patients who can benefit from organ transplantation procedures without the need for additional organ donors.

[0182] A single deceased donor can be the source of material for one, two, three, four, five or more cell products that promote mixed chimerism in a solid organ transplant recipient. Multiple cell products from a single deceased donor can include any element of the cell products described above, such as cell type, cell number, degree of HLA matching or mismatching, cell purity, cryoprotectants, stabilizers or transplant enhancers.

[0183] The present invention also provides a method for preparing multiple cell products from a single deceased donor for transfer into multiple living recipients. The method comprises extracting CD34 cells from the bone marrow of a deceased donor. + cells and from sources other than donor bone marrow + The method includes obtaining CD3 cells from sources other than bone marrow. + The method may include any of the elements described above with respect to ex vivo extraction of cells, fractionation of blood, or analysis of blood. For example, and without limitation, the method may include removal of bone marrow, separation of cells from bone debris, extraction of CD34 + Treatment with agents to mobilize cells, enucleation or separation of mononuclear cells from polynuclear cells, depletion of RBCs and platelets, CD34 + Cells and / or CD3 + This may include one or more of: enrichment of cells, removal of clots and / or cell aggregates, treatment with anticoagulants, cryopreservation, ex vivo expansion of cells, genetic modification of cells, and analysis of T cell activation, T cell anergy, T cell apoptosis, T cell exhaustion, T cell proliferation, T cell viability, T cell cytokine secretion, HSPC proliferation, HSPC viability, HSPC apoptosis, cytokine levels, chemokine levels, antibody levels, and / or immunoglobulin levels. EXAMPLES

[0184] Example 1 According to an embodiment of the present invention, CD34 derived from bone marrow of a deceased donor + CD3 derived from cells and blood of deceased donors +Methods for preparing cellular compositions containing cells are described below.

[0185] FIG. 1 illustrates a CD34 antibody derived from deceased donor bone marrow in accordance with an embodiment of the present invention. + CD3 derived from cells and blood of deceased donors + 1 is a flow diagram illustrating a method for preparing a cellular composition containing cells. The method includes a series of steps described in more detail below. The method includes the following steps: collection of tissue; preparation of bone marrow; preparation of blood; preparation of buffers; extraction of CD34 from bone marrow. + The steps are: enrichment of cells; dividing the combined material into individual doses; and cryopreservation. The order of steps described herein is for illustrative purposes only. One of ordinary skill in the art will understand that certain steps must be performed before others, but the method is not limited to a particular order of steps.

[0186] Figure 6 is an expanded view of the tissue collection step from the flow diagram of Figure 5. Blood, bone marrow and one or more solid organs of interest are harvested from the body of a deceased donor. Bone marrow is extracted by trepanation as described above. Blood is collected in one or more receptacles of the device of the invention as described above. Blood or other material from the body is tested for infectious disease markers (IDM).

[0187] Figure 7 is an expanded view of the bone marrow preparation steps from the flow diagram in Figure 5. The extracted bone marrow is passed through a sieve to remove bone debris and depleted of red blood cells by one of the methods described above. The bone marrow is also analyzed for cell count, sterility, viability, colony forming ability and expression of CD3, CD34 and CD45 markers. Viability is assayed by 7-aminoactinomycin D (7-AAD) exclusion and marker expression is analyzed by flow cytometry.

[0188] Figure 8 is an expanded view of the blood preparation steps from the flow diagram of Figure 5. Blood is depleted of red blood cells and platelets by one of the methods described above. Blood is also analyzed for cell count, sterility, viability, and expression of CD3, CD34, and CD45 markers. Viability is assayed by exclusion of 7-AAD, and marker expression is analyzed by flow cytometry.

[0189] Figure 9 is an expanded view of the buffer preparation step from the flow diagram of Figure 5. Two buffers are prepared, one containing HSA and the other containing a balanced electrolyte solution.

[0190] FIG. 10 is a flow chart of the CD34 + A close-up of the cell enrichment step. CD34 + A buffer is used with the cells obtained from the bone marrow preparation step to enrich for cells. The sample is purified by immunomagnetic selection to identify CD34 + The cells are further enriched for CD34 cells. Before or after spin membrane filtration, cell number, viability, colony forming ability and marker expression are analyzed as described above. As described above, cells from the blood preparation step are analyzed for cell number, viability, marker expression, T cell activation, T cell proliferation and T cell apoptosis. + Cells enriched for bone marrow-derived cells and CD3 + If both blood-derived cell containing cells are determined to be suitable for use in the cell product, the two cell fractions are combined.

[0191] Figure 11 is an expanded view of the splitting step from the flow diagram of Figure 5. The pooled cells are filtered to remove clumps and cell aggregates, i.e., clumps. The pooled cells are then split into two individual doses, each of which is transfected with CD34 to promote mixed chimerism in separate organ transplant recipients. + Cells and CD3 +Contains sufficient amount of cells. During this step, cell number, viability and marker expression are analyzed as described above. Cells can also be analyzed for sterility, endotoxin, mycoplasma, colony forming ability, release of cytokines such as IL-2.

[0192] Figure 12 is an expanded view of the cryopreservation step from the flow diagram of Figure 5. The doses can be further divided into aliquots or can be maintained in a single container. The doses are frozen in a freezer that reduces the temperature of the cell products to the target temperature at a controlled rate. After the cell products reach the target temperature, they are transferred to liquid nitrogen for long-term storage.

[0193] Example 2 CD34 + Cells and CD3 + An exemplary process for preparing the cell-containing cell product, MDR-104, is described below. The process provides two doses, each containing 100×10 cells per kg of recipient body weight from a single deceased donor. 6 of T cells and >4 × 10 per kg of recipient body weight 6 CD34 + Each dose supports the transplant of one kidney from a deceased donor into a different recipient. The product is available in 10 5 Also contains >500 colony forming units (CFU) per cell. IL-2 releasing and viable CD34 + Cells and CD3 + The product can be further characterized to determine the cellular percentage.

[0194] FIG. 13 is a flow diagram illustrating a method for preparing a cellular composition according to an embodiment of the invention. As described in more detail below, CD3 + Cells were prepared from whole blood with MDR-T treatment and were CD34 + was prepared from bone marrow treated with MDR-E and CD3 + Cells and HSPCs are combined with MDR-34T treatment.

[0195] CD3 required for the cellular composition of the final drug product + T cells and CD34 + Whole blood (WB) and bone marrow (BM) are collected as starting materials to supply HSPCs, respectively. To facilitate transport from the collection site to the manufacturing site, exsanguinated blood is collected into a single collection device or several smaller collection devices after systemic heparinization of deceased donors. The whole blood is subsequently processed using an apheresis machine for platelet and red blood cell depletion, and then purified to identify CD34 cells. + may be washed for additional platelet removal prior to or following the addition of selected manufacturing intermediates.

[0196] Bone marrow is harvested through one or both of two methods. One method uses standard bone marrow aspiration from the exposed iliac crest of a deceased donor. In the second method, marrow is harvested with a drilling orthopedic device capable of collecting cores of cortical and cancellous bone containing the BM. Bone marrow can also be harvested from the vertebral bodies of deceased donors. Bone marrow harvested by each of the two methods is placed into an appropriate shipping container that supports the transport of the harvested BM to the manufacturing site.

[0197] Isolated CD34 + The HSPCs are combined with leukapheresis blood products and separated as needed to meet the dose requirements of each recipient of the deceased donor kidney. Each dose is formulated and cryopreserved as a separate final drug product lot.

[0198] Whole blood collected from deceased donors will be used to identify CD3+ cells for each recipient of a deceased donor kidney. + To provide the required dose of CD34 T cells for each recipient of a donor kidney, + To provide the required dose of cells, CD34 + HPSCs are isolated from the harvested bone marrow.

[0199] Whole blood is collected from exsanguinated deceased donors. Exsanguination occurs after systemic heparinization and concomitant with a cold flush of preservation solution. Exsanguinated whole blood is drained from the deceased donor through two cannulas from the arterial and venous supply and connected to a collection device through a sterile connection for transport to the manufacturing site.

[0200] Bone marrow from deceased donors is collected by one or both of two independent methods: standard bone marrow aspiration and marrow recovery by a shaping device, the perforator. Bone marrow aspiration is performed through repeated aspiration using a heparinized syringe and a Jamshedie needle. The perforator allows for the taking of a core of bone marrow containing cancellous bone fragments.

[0201] Exsanguinated blood is required to arrive at the manufacturing site with minimal hemagglutination, red blood cell lysis and cell clumping, and with high white blood cell viability to facilitate downstream processing of red blood cell and platelet depletion. Whole blood is collected in a device that allows the blood to be maintained at a controlled temperature and transported in a sterile, stable carrier. The collection device is also compatible with closed system bioprocessing.

[0202] Bone marrow collected by standard aspiration or puncture is purified with minimal hemagglutination, red blood cell lysis, and cell aggregation, as well as red blood cell, platelet depletion, and CD34 + Total CD34 cells are required to arrive at the manufacturing site with high leukocyte viability to facilitate downstream processing of HSPC isolation. + Yield and recovery are tracked as extrapolated metrics. The total volume of bone marrow harvested is calculated for each method and extrapolated for each deceased donor.

[0203] Bone marrow harvested from deceased donors is CD34 + Before being used as starting material for hPSC isolation, they undergo downstream processing. Several methods and / or techniques can be used to establish the optimal method for isolation of hematopoietic marrow from cancellous bone debris and red blood cell depletion to obtain bone marrow mononuclear cells (BMMNCs).

[0204] The deceased donor's blood is removed prior to harvesting the bone marrow.

[0205] Incoming bone marrow collected by needle aspiration is diluted with buffer and filtered through a suitable filtration device to remove bone debris. To obtain maximum recovery of mononuclear cells (MNC), one or more of the following methods can be used: size-based centrifugation, hetastarch sedimentation, immunomagnetic depletion or lysis followed by a spin membrane filtration wash step, or buoyancy-activated cell separation.

[0206] The incoming bone marrow cores obtained using the perforator are processed by one or more of physical agitation, enzymatic disaggregation or washing and filtration to recover the hematopoietic marrow compartment embedded within the spongy core.

[0207] Incoming bone marrow is analyzed for complete blood counts, sterility, and leukocyte viability through flow cytometric analysis using cell surface specific antigens of target cell populations including, but not limited to, CD45, CD34, and CD3. Data from flow cytometric analysis is analyzed using CD34 as a reference value from a deceased donor. + Viability, CD34 + HSPC frequency and CD34 + Used to determine quantification of cell number.

[0208] Exsanguinated blood collected from deceased donors is processed by apheresis into a suitable starting material prior to formulation and cryopreservation.

[0209] Incoming whole blood received at the manufacturing facility is processed to enrich for peripheral blood mononuclear cells to achieve a suitable starting material for the MDR-104 manufacturing process. To reduce the volume and obtain a comparable leukapheresis intermediate material, the incoming whole blood starting material first undergoes apheresis. Filtration of the whole blood product is performed as needed to remove clots and cell aggregates from the incoming material as well as the resulting leukapheresis intermediate material. The resulting leukapheresis intermediate material is then subjected to CD3+ cell counts for each recipient of a deceased donor kidney.+ Used to formulate doses of T cells.

[0210] Incoming whole blood is analyzed for complete blood counts, sterility, and leukocyte viability through flow cytometry analysis using cell surface specific antigens of target cell populations, including but not limited to CD45, CD34, and CD3. The ability to meet T cell dose expectations and T cell recovery is calculated before and after apheresis.

[0211] The red blood cell-depleted intermediate material expresses CD34 + It is used for the immunoselection of HSPCs by first depleting platelets and then subjecting the RBC and platelet-reduced material to CD34 immunoselection.

[0212] The red blood cell depleted intermediate material resulting from the bone marrow preparation sub-process is further depleted of platelets using spin membrane filtration. The RBC and platelet depleted sample is then incubated with a CD34 capture reagent and washed using spin membrane filtration to remove unbound beads and capture the CD34 + Load into an immunomagnetic column system for immunoselection of HSPCs.

[0213] Incoming bone marrow is analyzed for complete blood counts, sterility, and leukocyte viability through flow cytometric analysis using cell surface specific antigens of target cell populations, including but not limited to CD45, CD34, and CD3. + Cell Dose Expectation and CD34 + The ability to meet harvest is calculated before and after immunoselection.

[0214] MDR-104 inhibits CD34 + HSPCs are CD3 + The combined product dose formulation is combined with the number of T cells required to meet the dose requirement. Initially, for each recipient of a deceased donor kidney, >4 × 10 6 CD34 + To meet the minimum dose requirement of cells / kg, MDR-E subprocesses were treated with sufficient CD34+ Divide into two separate bags based on cell count. Then, quantitate CD3 + T cells, 100 x 10 6 CD3 CD3 T cells / kg required + Add to each MDR-E preformulation bag to complete the T cell dose.

[0215] MDR-T34 is CD34 + 1 represents the sub-process step of combining the MDR-E derived from immune selection with the required T cell dose derived from the resulting leukapheresis of the whole blood preparation unit.

[0216] MDR-E process intermediates are viable CD34 + HSPCs were then quantified and then determined to be >4×10 6 of viable CD34 + Viable CD3 T cells are quantified from the leukapheresis process intermediate and 100 x 10 viable CD3 T cells are added to each MDR-E preproduction bag based on the T cell dose requirement for each patient. 6 cells / kg are added. Each preformulated intermediate then undergoes an additional spin membrane filtration step to further reduce the platelet concentration.

[0217] Platelet loading and platelet reduction efficiency are monitored before and after spin membrane filtration. + HSPCs and CD3 + T cell loss is also monitored before and after spin membrane filtration. Total cell counts and quantitative flow cytometry are used to monitor the frequency and viability of the target cell population. Sufficient T cells with expected functional characteristics and CD34 + Functional assays are used as needed to ensure that HSPCs are maintained at dose requirements.

[0218] The resulting preformulated process intermediate is combined with CryoStor 10 (CS10) in a 1:2 ratio to produce the drug, which is then dispensed into 3-4 final drug product containers immediately prior to cryopreservation. The preformulated MDR-E and T cells are combined with a cryoprotectant to produce the final product.

[0219] The preformulated MDR-T34 process intermediate for each recipient is combined with an equal volume of CS10 in a collection vessel. Each product is then split into final product vessels at a total volume of 50 mL each. The need for filtration is evaluated as needed prior to vessel closure and cryogenic storage. After completion and vessel closure of both MDR-104 products, the final drug products undergo controlled cryogenic storage using a controlled rate freezer before being transferred to vapor phase liquid N2 for long term storage at ≦-150°C.

[0220] Incorporation by Reference Throughout this disclosure, other documents are referenced and cited, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0221] Equivalent Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the scientific and patent references cited herein. The subject invention herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents. The present invention provides, for example, the following items. (Item 1) 1. A cell product for establishing mixed chimerism in a solid organ transplant recipient, comprising: 1 × 10 per kg of recipient body weight, derived from deceased donor bone marrow 5 More than CD34 + cells; and 1 x 10 per kg of recipient body weight derived from bone marrow other than that of a deceased donor5 More than CD3 + cell Cellular products including. (Item 2) 2. The cell product of item 1, wherein the bone marrow is derived from the iliac crest or vertebral body. (Item 3) 2. The cell product of item 1, wherein the non-bone marrow is selected from the group consisting of blood, liver, lymph nodes, spleen and thymus. (Item 4) 4. The cell product of item 3, wherein the other than bone marrow is blood. (Item 5) CD34 + Cells and CD3 + 2. The cell product of item 1, wherein the cells are HLA-matched to the solid organ transplant recipient. (Item 6) CD34 + Cells and CD3 + 2. The cell product of item 1, wherein the cells are HLA-mismatched to the solid organ transplant recipient. (Item 7) CD34 + Cells and CD3 + 2. The cell product of item 1, wherein the cells are provided in a separate container. (Item 8) CD34 + Cells and CD3 + 2. The cell product of item 1, wherein the cells are provided as a mixture in a common container. (Item 9) 2. The cell product of item 1, further comprising a cryopreservation medium. (Item 10) 10. The cell product of item 9, wherein the cryopreservation medium comprises at least one cryoprotectant selected from the group consisting of DMSO and dextran having a molecular weight of about 40,000 Da. (Item 11) 1. A method for establishing mixed chimerism in a solid organ transplant recipient, comprising administering to a subject who has received or will receive a solid organ transplant: 1 × 10 per kg of recipient body weight, derived from deceased donor bone marrow 5 More than CD34 + cells; and 1 × 10 per kg of recipient body weight derived from sources other than deceased donor bone marrow 5 More than CD3 + cell providing a product comprising: (Item 12) 12. The method of claim 11, wherein the bone marrow is derived from the iliac crest or vertebral body. (Item 13) 12. The method of claim 11, wherein the non-bone marrow is selected from the group consisting of blood, liver, lymph nodes, spleen and thymus. (Item 14) 14. The method of claim 13, wherein the other than bone marrow is blood. (Item 15) CD34 + Cells and CD3 + 12. The method of claim 11, wherein the cells are HLA-matched to the solid organ transplant recipient. (Item 16) CD34 + Cells and CD3 + 12. The method of claim 11, wherein the cells are HLA-mismatched to the solid organ transplant recipient. (Item 17) CD34 + Cells and CD3 + 12. The method of item 11, wherein the cells are provided in separate containers. (Item 18) CD34 + Cells and CD3 + 12. The method of claim 11, wherein the cells are provided as a mixture in a common container. (Item 19) 12. The method of claim 11, wherein the product further comprises a cryopreservation medium. (Item 20) 20. The method of claim 19, wherein the cryopreservation medium comprises at least one cryoprotectant selected from the group consisting of DMSO and dextran having a molecular weight of about 40,000 Da. (Item 21) 1. A method for preparing a cell product for establishing mixed chimerism in a solid organ transplant recipient, comprising: CD34 derived from deceased donor bone marrow + Obtaining cells; CD3 derived from sources other than the bone marrow of the deceased donor + Obtaining cells; and The CD34 obtained for administration to solid organ transplant recipients. + Cells and the resulting CD3 + Producing a cellular product comprising the cells. A method comprising: (Item 22) 22. The method of claim 21, wherein the non-bone marrow is selected from the group consisting of blood, liver, lymph nodes, spleen and thymus. (Item 23) 23. The method of claim 22, wherein the other than bone marrow is blood. (Item 24) CD34 + 24. The method of claim 23, wherein the deceased donor is exsanguinated prior to obtaining cells from the bone marrow. (Item 25) CD34 + 22. The method of claim 21, wherein obtaining cells comprises removing the bone marrow from a portion of a bone through aspiration or puncture. (Item 26) 26. The method of claim 25, wherein the portion of bone is derived from the iliac crest or vertebral body. (Item 27) 26. The method of claim 25, wherein the removed bone marrow is treated with an anticoagulant. (Item 28) 28. The method of claim 27, wherein the anticoagulant is selected from the group consisting of acenocoumarol, antithrombin III apixaban, argatroban, atromentin and betrixaban, bivalirudin, brodifacoum, dabigatran, dalteparin, difenacoum, edoxaban, enoxaparin, fondaparinux, heparin, idraparinux, phenindione, phenprocoumon, rivaroxaban and warfarin. (Item 29) The bone marrow is removed via puncture and the CD34 + 26. The method of claim 25, wherein the step of obtaining cells comprises isolating bone marrow cells from bone fragments. (Item 30) 30. The method of claim 29, wherein the separation step comprises one selected from the group consisting of stirring, enzymatic disaggregation, washing and filtering. (Item 31) CD34 + The step of obtaining cells comprises: + 26. The method of claim 25, comprising contacting the cells with an agent that mobilizes the cells. (Item 32) The agent is selected from the group consisting of adenosine receptor antagonists, BIO5192, CCR1 antagonists, CCR2 antagonists, CXCR2 antagonists, CXCR4 antagonists, cyclophosphamide, defibrotide, EphA3-Fc, erythropoietin (EPO), glycosaminoglycan (GAG) mimetics, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene beta (GRO-beta), human growth hormone, IL-8, macrophage inflammatory protein-1 alpha (MIP-1 alpha), met-SDF 32. The method of claim 31, wherein the therapeutic agent is selected from the group consisting of .DELTA.-1 beta, NSC23766, parathyroid hormone, pertussis toxin, plerixafor, poly-[1-6]-D-glucopyranosyl-[1-3]-D-glucopyranose (PGG) glucan, Rac1 inhibitors, retinoic acid receptor agonists, SB290157, SDF-1 alpha peptide analogs, stem cell factor (SCF), sulfated colominic acid, sulfated polysaccharides, T134, T140, thrombopoietin (TPO), TPO receptor agonists, VCAM-1 inhibitors, VLA-1 inhibitors, VLA-4 inhibitors and analogs and derivatives thereof. (Item 33) CD34 + 26. The method of claim 25, wherein obtaining cells comprises depleting red blood cells, platelets or both from the removed bone marrow. (Item 34) 34. The method of claim 33, wherein the depletion step comprises at least one selected from the group consisting of buoyancy-activated cell separation, cell lysis, hetastarch precipitation, immunomagnetic depletion, size-based centrifugation, and spin membrane filtration. (Item 35) CD34 + 26. The method of claim 25, wherein obtaining cells comprises immunoselecting CD34+ cells from the removed bone marrow. (Item 36) CD3 + 24. The method of claim 23, wherein obtaining cells comprises depleting red blood cells, platelets or both from the blood. (Item 37) 37. The method of claim 36, wherein the depletion step comprises at least one selected from the group consisting of buoyancy-activated cell separation, cell lysis, hetastarch precipitation, immunomagnetic depletion, size-based centrifugation, and spin membrane filtration. (Item 38) CD3 + 24. The method of claim 23, wherein obtaining cells comprises removing clots or cell aggregates from the blood. (Item 39) 40. The method of claim 38, wherein the removing step comprises filtering the blood. (Item 40) CD3 + 24. The method of claim 23, wherein obtaining the cells comprises separating the blood into a cellular fraction and a plasma fraction. (Item 41) The step of producing the cell product comprises: + Cells and the resulting CD3 + 22. The method of claim 21, comprising combining the cells. (Item 42) The step of producing the cell product comprises: + Cells and the resulting CD3 + 22. The method of claim 21, comprising cryopreserving the cells. (Item 43) 1. A method for producing a cell product from hematopoietic cells obtained ex vivo from bone marrow, comprising: Obtaining a sample comprising bone marrow from the body of a subject; obtaining hematopoietic cells from said sample after said sample is removed from said subject's body; and Producing a cell product comprising said hematopoietic cells for administration to a solid organ transplant recipient. A method comprising: (Item 44) 44. The cell product of item 43, wherein the sample is derived from the iliac crest or vertebral body. (Item 45) 44. The method of claim 43, wherein the subject is deceased when the sample is obtained from the subject's body. (Item 46) 46. ​​The method of claim 45, wherein the subject is a human. (Item 47) The hematopoietic cells are CD34 + 44. The method of claim 43, comprising hematopoietic cells. (Item 48) The step of obtaining hematopoietic cells comprises: + 44. The method of claim 43, comprising contacting the cells with an agent that mobilizes the cells. (Item 49) The agent is selected from the group consisting of adenosine receptor antagonists, BIO5192, CCR1 antagonists, CCR2 antagonists, CXCR2 antagonists, CXCR4 antagonists, cyclophosphamide, defibrotide, EphA3-Fc, erythropoietin (EPO), glycosaminoglycan (GAG) mimetics, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-regulated oncogene beta (GRO-beta), human growth hormone, IL-8, macrophage inflammatory protein-1 alpha (MIP-1 alpha), met-SDF 49. The method of claim 48, wherein the therapeutic agent is selected from the group consisting of: -1 beta, NSC23766, parathyroid hormone, pertussis toxin, plerixafor, poly-[1-6]-D-glucopyranosyl-[1-3]-D-glucopyranose (PGG) glucan, Rac1 inhibitors, retinoic acid receptor agonists, SB290157, SDF-1 alpha peptide analogs, stem cell factor (SCF), sulfated colominic acid, sulfated polysaccharides, T134, T140, thrombopoietin (TPO), TPO receptor agonists, VCAM-1 inhibitors, VLA-1 inhibitors, VLA-4 inhibitors and analogs and derivatives thereof. (Item 50) 44. The method of claim 43, wherein obtaining hematopoietic cells comprises removing bone marrow from the sample via aspiration or puncture. (Item 51) 51. The method of claim 50, wherein the removed bone marrow is treated with an anticoagulant. (Item 52) 52. The method of claim 51, wherein the anticoagulant is selected from the group consisting of acenocoumarol, antithrombin III apixaban, argatroban, atromentin and betrixaban, bivalirudin, brodifacoum, dabigatran, dalteparin, difenacoum, edoxaban, enoxaparin, fondaparinux, heparin, idraparinux, phenindione, phenprocoumon, rivaroxaban and warfarin. (Item 53) 51. The method of claim 50, wherein the bone marrow is removed via trephination and obtaining hematopoietic cells comprises separating bone marrow cells from bone debris. (Item 54) 54. The method of claim 53, wherein the separation step comprises one selected from the group consisting of agitation, enzymatic disaggregation, washing and filtration. (Item 55) 51. The method of claim 50, wherein obtaining hematopoietic cells comprises depleting red blood cells, platelets or both from the removed bone marrow. (Item 56) 56. The method of claim 55, wherein the depletion step comprises at least one selected from the group consisting of buoyancy-activated cell separation, cell lysis, hetastarch precipitation, immunomagnetic depletion, size-based centrifugation, and spin membrane filtration. (Item 57) The step of obtaining hematopoietic cells comprises isolating hematopoietic cells from the extracted bone marrow by CD34 + 51. The method of claim 50, comprising immunoselecting the cells. (Item 58) 44. The method of claim 43, further comprising the step of cryopreserving the cell product. (Item 59) 44. The method of claim 43, wherein the cell product promotes the establishment of mixed chimerism in a solid organ transplant recipient. (Item 60) 60. The method of item 59, wherein the solid organ is a kidney. (Item 61) 60. The method of claim 59, wherein the hematopoietic cells obtained are HLA-matched to the solid organ transplant recipient. (Item 62) 60. The method of claim 59, wherein the hematopoietic cells obtained are HLA-mismatched to the solid organ transplant recipient. (Item 63) 1. A method for assessing the suitability of blood from a deceased donor for use in the manufacture of a product to be administered to a living recipient, comprising: obtaining blood from a deceased donor; analyzing the components of the blood; and determining, based on the analysis of said components, whether said blood is suitable for use in the manufacture of a product to be administered to a living recipient. A method comprising: (Item 64) 64. The method of claim 63, wherein the component comprises a T cell. (Item 65) 65. The method of claim 64, wherein the analyzing step comprises analyzing T cell activation, T cell exhaustion, T cell anergy, T cell proliferation, T cell viability, or T cell apoptosis. (Item 66) 64. The method of claim 63, wherein the components comprise hematopoietic stem and progenitor cells (HSPCs). (Item 67) 67. The method of claim 66, wherein the analyzing step comprises analyzing HSPC proliferation, HSPC viability, or HSPC apoptosis. (Item 68) 64. The method of claim 63, wherein the components include non-cellular components. (Item 69) 69. The method of claim 68, wherein the non-cellular component comprises a cytokine or chemokine. (Item 70) 64. The method of claim 63, further comprising using material from said blood in the manufacture of said product if said blood is determined to be suitable for such use. (Item 71) 71. The method of claim 70, wherein the material comprises T cells. (Item 72) 71. The method of claim 70, wherein the material comprises HSPC. (Item 73) 1. A method for assessing the suitability of blood from a deceased donor for use in the manufacture of a product to be administered to a living recipient, comprising: obtaining blood from a deceased donor; Analyzing non-blood tissue components from the deceased donor; and determining, based on the analysis of said components, whether said blood is suitable for use in the manufacture of a product to be administered to a living recipient. A method comprising: (Item 74) 74. The method of claim 73, wherein the component comprises a T cell. (Item 75) 75. The method of claim 74, wherein the analyzing step comprises analyzing T cell activation, T cell exhaustion, and T cell anergy. (Item 76) 74. The method of claim 73, wherein the components include non-cellular components. (Item 77) 77. The method of claim 76, wherein the non-cellular component comprises a cytokine or chemokine. (Item 78) 74. The method of claim 73, wherein the non-blood tissue comprises bone marrow. (Item 79) and analyzing a second component, the second component being a component of the blood, the determining step also being based on the analysis of the second component. Item 74. The method according to item 73. (Item 80) using material from said blood in the manufacture of said product if said blood is determined to be suitable for such use. 74. The method of claim 73, further comprising: (Item 81) 81. The method of claim 80, wherein the material comprises T cells. (Item 82) 81. The method of claim 80, wherein the material comprises HSPC.

Claims

[Claim 1] The invention described in this specification.

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