Systems and methods for extraction and cryopreservation of bone marrow

Abstract

Methods, systems and compositions are provided for extracting bone marrow cells from bone obtained from a deceased donor, for preparing bone marrow for cryopreservation, and for obtaining desired cells from cryopreserved fresh bone marrow.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 091,890, filed on October 14, 2020; U.S. Provisional Application No. 63 / 110,571, filed on November 6, 2020; U.S. Provisional Application No. 63 / 130,255, filed on December 23, 2020; and U.S. Provisional Application No. 63 / 168,178, filed on March 30, 2021. The entire contents of each of the four priority applications are expressly incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with U.S. Government support under Contract No. 5R44AI129444 from the National Institutes of Health. Background Art

[0005] Bone marrow is currently collected from the brothers and sisters of HLA matching or the unrelated donor of best matching for clinical purposes.Other transplantation sources are also utilized now, including relatives or unrelated donors and umbilical cord blood (CB) of mismatched haploid matching.When being transplanted into the patient suffering from certain diseases, the hematopoietic stem cell (HSC) in the donor bone marrow is implanted in the patient, and immune and hematopoietic systems are reconstructed.Bone marrow is also a good source of mesenchymal stroma / stem cell (MSC), which is a multipotent progenitor cell of self-renewal, with the multi-lineage potential of being differentiated into the cell type (such as adipocyte, osteocyte and chondrocyte) of mesoderm origin.

[0006] Currently, bone marrow is typically collected by making a hole in the cortical bone with a trocar and then using a bone marrow aspiration needle and syringe to draw the marrow into a syringe. Multiple syringes are often required to extract enough marrow from the bone. The syringes are then removed from the sterile field, each connected to a collection bag containing an anticoagulant, and the marrow is pushed into the bag. This step is repeated multiple times, usually in two pelvic bones, and can result in contamination of the aspirate.

[0007] It is recognized that whole bone marrow (BM) can be obtained from deceased donors. However, a variety of obstacles have prevented the mainstream use of cadaveric bone marrow. An important obstacle is to find a streamlined process to controllably extract and preserve the marrow of deceased donors and the cell yield from the marrow. Another issue regarding the use of cadaveric bone relates to the cryopreservation and recovery of bone. Specifically, the issue relates to the quality of viable cells, such as HSC, that can be obtained from cryopreserved donor bone. Summary of the invention

[0008] Aspects of the present disclosure include a method for processing a biological sample containing cells or derivatives thereof, the method comprising: generating a first volume of the biological sample containing cells or derivatives thereof, wherein the first volume contains a first concentration of cells or derivatives thereof; generating a second volume of the biological sample containing cells or derivatives thereof, wherein the second volume is less than the first volume and contains a second concentration of cells, wherein the second concentration of cells differs from the first concentration of cells by no more than 30%; and cooling the first volume at a first cooling rate and cooling the second volume at a second cooling rate, wherein the first cooling rate is about the same as the second cooling rate; wherein the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw proliferation rate of the cells in the second volume by no more than 30%. In some embodiments, the first volume is contained in a first container, wherein the second volume is contained in a second container, and wherein the first container and the second container are exposed to a common temperature. In some embodiments, the second volume is less than 50% of the first volume. In some embodiments, the second volume is less than 40% of the first volume. In some embodiments, the second volume is less than 37.5% of the first volume. In some embodiments, the second volume is less than 35% of the first volume. In some embodiments, the second volume is less than 30% of the first volume. In some embodiments, the second volume is less than 20% of the first volume. In some embodiments, the second volume is less than 15% of the first volume. In some embodiments, the second volume is less than 10% of the first volume. In some embodiments, the second volume is less than 5% of the first volume. In some embodiments, the second volume is less than 1% of the first volume. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 30%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 25%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 20%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 15%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 13.6%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 10%. In some embodiments, the post-thaw viability of the cells in the first volume differs by no more than 5% from the post-thaw viability of the cells in the second volume. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs by no more than 25% from the post-thaw cell proliferation rate of the cells in the second volume.In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 20%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 15%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 13.6%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 10%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 5%. In some embodiments, the survival rate of the cells after thawing is at least 50%. In some embodiments, the proliferation rate after thawing of the cells is at least 1 CFU-GM / 10. 5Cells. In some embodiments, the first cooling rate and the second cooling rate include a superfreeze rate of about -0.1 ° C / min to about -5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a superfreeze rate of about -2.5 ° C / min to about -4 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a superfreeze rate of about -2.5 ° C / min to about -3.5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a subfreeze rate of about -1 ° C / min to about -2 ° C / min. In some embodiments, the post-thaw survival rate of the cell is at least 60%. In some embodiments, the post-thaw survival rate of the cell is at least 70%. In some embodiments, the post-thaw survival rate of the cell is at least 80%. In some embodiments, the post-thaw survival rate of the cell is at least 90%. In some embodiments, (c) occurs in one or more freezers. In some embodiments, the first container and the second container are disposed in a first freezer in one or more freezers. In some embodiments, the first container is contained in a first freezer in one or more freezers, and the second container is contained in a second freezer in one or more freezers. In some embodiments, one or more freezers include a static freezer. In some embodiments, the first freezer, the second freezer, or both are static freezers. The method of any of the preceding claims, wherein one or more freezers include a controlled rate freezer. In some embodiments, the first freezer, the second freezer, or both are controlled rate freezers. In some embodiments, one or more freezers are set at about -70°C to -90°C. In some embodiments, one or more freezers are set at less than -80°C. In some embodiments, one or more freezers are set at -86°C. In some embodiments, the second volume is placed directly in an insulated container so that each vial is adjacent to the insulating material of the insulated container. In some embodiments, the method further comprises arranging the first volume in a static freezer so that the first volume does not contact the wall of the one or more freezers. In some embodiments, the biological sample comprising cells or their derivatives in the first volume and the biological sample comprising cells or their derivatives in the second volume experience the same cooling rate. In some embodiments, the cell is a stem cell or an immune cell. In some embodiments, the stem cell includes hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), or both. In some embodiments, the biological sample includes one or more organs, blood, or both. In some embodiments, the immune cell includes T cells. In some embodiments, the blood is cord blood or peripheral blood.In some embodiments, the HSCs include CD34+ cells. In various embodiments, the method further comprises the step of transferring the first volume and the second volume to a long-term storage container, for example, a long-term storage container at less than -86°C.

[0009] Another aspect of the present disclosure includes a method for processing bone marrow or a derivative thereof, the method comprising: generating a first volume of bone marrow or a derivative thereof, wherein the first volume comprises a first concentration of bone marrow or a derivative thereof; generating a second volume of bone marrow or a derivative thereof, wherein the second volume is less than the first volume and comprises a second concentration, wherein the second concentration differs from the first concentration by no more than 30%; and cooling the first volume at a first cooling rate and cooling the second volume at a second cooling rate, wherein the first cooling rate is about the same as the second cooling rate; wherein the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 30%. In some embodiments, the first volume is contained in a first container, wherein the second volume is contained in a second container, and wherein the first container and the second container are exposed to a single / same temperature. In some embodiments, the second volume is less than 50% of the first volume. In some embodiments, the second volume is less than 40% of the first volume. In some embodiments, the second volume is less than 37.5% of the first volume. In some embodiments, the second volume is less than 35% of the first volume. In some embodiments, the second volume is less than 30% of the first volume. In some embodiments, the second volume is less than 20% of the first volume. In some embodiments, the second volume is less than 15% of the first volume. In some embodiments, the second volume is less than 10% of the first volume. In some embodiments, the second volume is less than 5% of the first volume. In some embodiments, the second volume is less than 1% of the first volume. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 30%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 25%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 20%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 15%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 13.6%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 10%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population in the first volume differs from the post-thaw survival rate of the bone marrow-derived cell population in the second volume by no more than 5%.In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 25%. In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 20%. In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 15%. In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 13.6%. In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 10%. In some embodiments, the post-thaw cell proliferation rate of the bone marrow-derived cell population in the first volume differs from the post-thaw proliferation rate of the bone marrow-derived cell population in the second volume by no more than 5%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population is at least 50%. In some embodiments, the post-thaw proliferation rate of the bone marrow-derived cell population is at least 1 CFU-GM / 10. 5cells. In some embodiments, the first cooling rate and the second cooling rate include a superfreezing rate of about -0.1 ° C / min to about -5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a superfreezing rate of about -2.5 ° C / min to about -4 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a superfreezing rate of about -2.5 ° C / min to about -3.5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a subfreezing rate of about -1 ° C / min to about -2 ° C / min. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population is at least 60%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population is at least 70%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population is at least 80%. In some embodiments, the post-thaw survival rate of the bone marrow-derived cell population is at least 90%. In some embodiments, (c) occurs in one or more freezers. In some embodiments, the first container and the second container are disposed in a first freezer in one or more freezers. In some embodiments, the first container is contained in a first freezer in one or more freezers, and the second container is contained in a second freezer in one or more freezers. In some embodiments, one or more freezers include a static freezer. In some embodiments, the first freezer, the second freezer, or both are static freezers. The method of any of the preceding claims, wherein one or more freezers include a controlled rate freezer. In some embodiments, the first freezer, the second freezer, or both are controlled rate freezers. In some embodiments, one or more freezers are set at about -70°C to -90°C. In some embodiments, one or more freezers are set at less than -80°C. In some embodiments, one or more freezers are set at -86°C. In some embodiments, the second volume is placed directly in an insulated container so that each vial is adjacent to the insulating material of the insulated container. In some embodiments, the method further comprises arranging the first volume in a static freezer so that the first volume does not contact the wall of the one or more freezers. In some embodiments, the biological sample of bone marrow or its derivatives in the first volume and the biological sample of bone marrow or its derivatives in the second volume experience the same cooling rate. In some embodiments, the bone marrow-derived cells are stem cells or immune cells. In some embodiments, the stem cells include hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), or both. In some embodiments, HSC includes CD34+ cells. In various embodiments, the method further includes the following steps: transferring the first volume and the second volume to a long-term storage container, for example, a long-term storage container below -86°C.

[0010] Another aspect of the present disclosure includes a method for processing MSCs, the method comprising: generating a first volume of MSCs, wherein the first volume comprises a first concentration of bone marrow or a derivative thereof; generating a second volume of MSCs, wherein the second volume is less than the first volume and comprises a second concentration, wherein the second concentration differs from the first concentration by no more than 30%; and cooling the first volume at a first cooling rate and cooling the second volume at a second cooling rate, wherein the first cooling rate is about the same as the second cooling rate; wherein the post-thaw cell proliferation rate of the MSCs in the first volume differs from the post-thaw proliferation rate of the MSCs in the second volume by no more than 30%. In some embodiments, the first volume is contained in a first container, wherein the second volume is contained in a second container, and wherein the first container and the second container are exposed to a single / same temperature. In some embodiments, the second volume is less than 50% of the first volume. In some embodiments, the second volume is less than 40% of the first volume. In some embodiments, the second volume is less than 37.5% of the first volume. In some embodiments, the second volume is less than 35% of the first volume. In some embodiments, the second volume is less than 30% of the first volume. In some embodiments, the second volume is less than 20% of the first volume. In some embodiments, the second volume is less than 15% of the first volume. In some embodiments, the second volume is less than 10% of the first volume. In some embodiments, the second volume is less than 5% of the first volume. In some embodiments, the second volume is less than 1% of the first volume. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 30%. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 25%. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 20%. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 15%. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 13.6%. In some embodiments, the survival rate of MSCs after thawing in the first volume differs from the survival rate of MSCs after thawing in the second volume by no more than 10%. In some embodiments, the post-thaw survival rate of the MSCs in the first volume differs from the post-thaw survival rate of the MSCs in the second volume by no more than 5%. In some embodiments, the post-thaw cell proliferation rate of the MSCs in the first volume differs from the post-thaw proliferation rate of the MSCs in the second volume by no more than 25%. In some embodiments, the post-thaw cell proliferation rate of the MSCs in the first volume differs from the post-thaw proliferation rate of the MSCs in the second volume by no more than 20%.In some embodiments, the cell proliferation rate of MSC after thawing in the first volume differs from the cell proliferation rate of MSC after thawing in the second volume by no more than 15%. In some embodiments, the cell proliferation rate of MSC after thawing in the first volume differs from the cell proliferation rate of MSC after thawing in the second volume by no more than 13.6%. In some embodiments, the cell proliferation rate of MSC after thawing in the first volume differs from the cell proliferation rate of MSC after thawing in the second volume by no more than 10%. In some embodiments, the cell proliferation rate of MSC after thawing in the first volume differs from the cell proliferation rate of MSC after thawing in the second volume by no more than 5%. In some embodiments, the survival rate of MSC after thawing is at least 50%. In some embodiments, the proliferation rate of MSC after thawing is at least 1 CFU-GM / 10. 5MSC. In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -0.1 ° C / min to about -5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -2.5 ° C / min to about -4 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -2.5 ° C / min to about -3.5 ° C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a sub-freezing rate of about -1 ° C / min to about -2 ° C / min. In some embodiments, the post-thaw survival rate of MSC is at least 60%. In some embodiments, the post-thaw survival rate of MSC is at least 70%. In some embodiments, the post-thaw survival rate of MSC is at least 80%. In some embodiments, the post-thaw survival rate of MSC is at least 90%. In some embodiments, (c) occurs in one or more freezers. In some embodiments, the first container and the second container are arranged in the first freezer in one or more freezers. In some embodiments, the first container is contained in a first freezer in one or more freezers, and the second container is contained in a second freezer in one or more freezers. In some embodiments, one or more freezers include a static freezer. In some embodiments, the first freezer, the second freezer, or both are static freezers. In some embodiments, one or more freezers include a controlled rate freezer. In some embodiments, the first freezer, the second freezer, or both are controlled rate freezers. In some embodiments, one or more freezers are set at about -70°C to -90°C. In some embodiments, one or more freezers are set at less than -80°C. In some embodiments, one or more freezers are set at -86°C. In some embodiments, the second volume is directly placed in an insulated container so that each vial is adjacent to the insulating material of the insulated container. In some embodiments, the method further comprises arranging the first volume in a static freezer so that the first volume does not contact the wall of the one or more freezers. In some embodiments, the MSCs in the first volume and the MSCs in the second volume experience the same cooling rate. In some embodiments, the MSCs are bone marrow-derived MSCs (BM-MSCs) or vertebral bone-adherent MSCs (vBA-MSCs).

[0011] Another aspect of the disclosure includes a method for processing a biological sample containing cells or derivatives thereof, the method comprising: generating a first volume of the biological sample containing cells or derivatives thereof, wherein the first volume contains a first concentration of cells or derivatives thereof; generating a second volume of the biological sample containing cells or derivatives thereof, wherein the second volume is less than the first volume and contains a second concentration of cells, wherein the second concentration of cells differs from the first concentration of cells by no more than 30%; generating a cell-specific freezing curve; cooling the first volume at a first cooling rate, wherein the first cooling rate is generated according to the freezing curve; and cooling the second volume at a second cooling rate, wherein the first cooling rate is generated according to the freezing curve; wherein the first cooling rate is about the same as the second cooling rate; and wherein a post-thaw cell proliferation rate of cells in the first volume differs from a post-thaw cell proliferation rate of cells in the second volume by no more than 30%. The method of claim 1, wherein the first volume is contained in a first container, wherein the second volume is contained in a second container, and wherein the first container and the second container are exposed to a single / same temperature. In some embodiments, the second volume is less than 50% of the first volume. In some embodiments, the second volume is less than 40% of the first volume. In some embodiments, the second volume is less than 37.5% of the first volume. In some embodiments, the second volume is less than 35% of the first volume. In some embodiments, the second volume is less than 30% of the first volume. In some embodiments, the second volume is less than 20% of the first volume. In some embodiments, the second volume is less than 15% of the first volume. In some embodiments, the second volume is less than 10% of the first volume. In some embodiments, the second volume is less than 5% of the first volume. In some embodiments, the second volume is less than 1% of the first volume. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 30%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 25%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 20%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 15%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 13.6%. In some embodiments, the post-thaw viability of the cells in the first volume differs from the post-thaw viability of the cells in the second volume by no more than 10%. In some embodiments, the post-thaw viability of the cells in the first volume differs from the post-thaw viability of the cells in the second volume by no more than 5%.In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 25%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 20%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 15%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 13.6%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 10%. In some embodiments, the cell proliferation rate after thawing of the cells in the first volume differs from the cell proliferation rate after thawing of the cells in the second volume by no more than 5%. In some embodiments, the post-thawing survival rate of the cells is at least 50%. In some embodiments, the post-thawing proliferation rate of the cells is at least 1 CFU-GM / 10. 5cells. In some embodiments, the post-thaw viability of the cells is at least 60%. In some embodiments, the post-thaw viability of the cells is at least 70%. In some embodiments, the post-thaw viability of the cells is at least 80%. In some embodiments, the post-thaw viability of the cells is at least 90%. In some embodiments, (c) occurs in one or more freezers. In some embodiments, the first container and the second container are arranged in the first freezer in one or more freezers. In some embodiments, the first container is contained in the first freezer in one or more freezers, and the second container is contained in the second freezer in one or more freezers. In some embodiments, one or more freezers include a static freezer. In some embodiments, the first freezer, the second freezer, or both are static freezers. The method of any of the preceding claims, wherein one or more freezers include a controlled-rate freezer. In some embodiments, the first freezer, the second freezer, or both are controlled-rate freezers. In some embodiments, one or more freezers are set at about -70°C to -90°C. In some embodiments, one or more freezers are set at less than -80°C. In some embodiments, one or more freezers are set at -86°C. In some embodiments, the second volume is placed directly in the insulated container so that each vial is in close proximity to the insulating material of the insulated container. In some embodiments, the method further comprises arranging the first volume in a static freezer so that the first volume does not contact the walls of one or more freezers. In some embodiments, the biological sample containing cells or their derivatives in the first volume and the biological sample containing cells or their derivatives in the second volume experience the same cooling rate. In some embodiments, the cells are stem cells or immune cells. In some embodiments, the stem cells include hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), or both. In some embodiments, the biological sample comprises one or more organs, blood, or both. In some embodiments, the immune cells include T cells. In some embodiments, the blood is umbilical cord blood or peripheral blood. In some embodiments, HSC includes CD34+ cells. In various embodiments, the method further comprises the following steps: transferring the first volume and the second volume to a long-term storage container, for example, a long-term storage container below -86°C.

[0012] Another aspect of the present disclosure includes a method for processing bone marrow or a derivative thereof, wherein the bone marrow or a derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally processing the bone into a bone fragment; extracting the bone marrow or a derivative thereof from the bone or bone fragment; and cryopreserving the bone marrow or a derivative thereof, wherein the cryopreservation comprises reducing the temperature of the bone marrow or a derivative thereof at a freezing rate greater than about -1°C / min in a static temperature freezer. In some embodiments, the cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -5°C / min until at least ice has nucleated in the freezing medium. In some embodiments, the cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -4°C / min until at least ice has nucleated in the freezing medium. In some embodiments, the cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -3.5°C / min until at least ice has nucleated in the freezing medium. In some embodiments, the cryopreservation includes cooling the bone marrow or its derivative at a subfreezing rate of about -1°C / min to about -2°C / min. In some embodiments, the superfreezing rate and the subfreezing rate are maintained without using a passive cooling box. In some embodiments, the cryopreservation includes arranging one or more aliquots of the bone marrow or its derivative in a static freezer so that the aliquots do not contact the wall of the static freezer. In various embodiments, in a static freezer, an aliquot of the bone marrow or its derivative is not directly stored above another aliquot of the bone marrow or its derivative. In some embodiments, the bone marrow or its derivative comprises a CD34+ cell population. In some embodiments, the CD34+ cell population comprises at least 70% of viable CD34+ cells after the bone marrow or its derivative is thawed. In some embodiments, the CD34+ cell population comprises at least 80% of viable CD34+ cells after the bone marrow or its derivative is thawed. In some embodiments, the static freezer is set at about -70°C to -90°C. In some embodiments, the static freezer is set at -86°C. In some embodiments, the static freezer is set at less than -80°C.

[0013] One aspect of the present disclosure includes a method for processing bone marrow or its derivatives, wherein the bone marrow or its derivatives are derived from a deceased donor, the method comprising: obtaining bone from a deceased donor; contacting the bone with a bleach solution for at least about 10 minutes to at least about 25 minutes, wherein the bone is immersed in the bleach solution; extracting bone marrow or its derivatives from the bone, wherein at least 90% of the CD34+ cells contained in the bone marrow or its derivatives are viable. In some embodiments, the bone marrow or its derivatives are contacted with the bleach solution for at least about 25 minutes. In some embodiments, the bleach solution contains 10% bleach. In some embodiments, the bone is a vertebral body. In some embodiments, the hydrogen peroxide is a 3% hydrogen peroxide solution. In some embodiments, the method further comprises transferring the bleached bone product from a container containing the bleach solution to a container containing the hydrogen peroxide solution. In some embodiments, the method further comprises agitating the bleached bone product in the hydrogen peroxide solution. In some embodiments, immersing the bleached bone product in a solution comprising hydrogen peroxide in c) comprises: immersing the bleached bone product in a container comprising a hydrogen peroxide solution; detecting foam or foam-like substances associated with the bleached bone product; and repeating i and / or ii until no foam or foam-like substances are detected. In some embodiments, the method further comprises manually removing soft tissue associated with the foam or foam-like substances in ii from the bleached bone product. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance the visibility of any foam or foam-like substances associated with the bleached bone product.

[0014] Another aspect of the present disclosure is a method for processing bone marrow or a derivative thereof, wherein the bone marrow or a derivative thereof is derived from a deceased donor, the method comprising: obtaining bone or bone fragments from a deceased donor, optionally processing the bone into bone fragments; mechanically grinding the bone or bone fragments in the presence of a grinding solution to produce a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 revolutions per minute ("RPM") for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises bone marrow or a derivative thereof, wherein the bone marrow or a derivative thereof comprises at least about 1,000 CD34+ cells / ml, 1,500 CD34+ cells / ml, 3,000 CD34+ cells / ml, 5,000 CD34+ cells / ml, 6,000 CD34+ cells / ml, 7,000 CD34+ cells / ml, 8,000 CD34+ cells / ml, 9,000 CD34+ cells / ml, 10,000 CD34+ cells / ml, 11,000 CD34+ cells / ml, 12,000 CD34+ cells / ml, 13,000 CD34+ cells / ml, 14,000 CD34+ cells / ml, 15,000 CD34+ cells / ml, 16,000 CD34+ cells / ml, 17,000 CD34+ cells / ml, 18,000 CD34+ cells / ml, 19,000 CD34+ cells / ml, 20,000 CD34+ cells / ml, 21,000 CD34+ cells / ml, 22,000 CD34+ cells / ml, 23,000 CD34+ cells / ml, 24,000 CD34+ cells / ml, 25,000 CD34+ cells / CD34+ cells / ml, 10,000 CD34+ cells / ml, 15,000 CD34+ cells / ml, 30,000 CD34+ cells / ml, 50,000 CD34+ cells / ml, 100,000 CD34+ cells / ml, 150,000 CD34+ cells / ml, 200,000 CD34+ cells / ml, 250,000 CD34+ cells / ml, 300,000 CD34+ cells / ml, 350,000 CD34+ cells / ml, 400,000 CD34+ cells / ml, 450,000 CD34+ cells / ml, 500,000 CD34+ cells / ml cells / ml, 550,000 CD34+ cells / ml, 600,000 CD34+ cells / ml, 650,000 CD34+ cells / ml, 700,000 CD34+ cells / ml, 750,000 CD34+ cells / ml, 800,000 CD34+ cells / ml, 850,000 CD34+ cells / ml, 900,000 CD34+ cells / ml, 950,000 CD34+ cells / ml, 1,000,000 CD34+ cells / ml, 1,050,000 CD34+ cells / ml, 1,100,000 CD34+ cells / ml, 1,150,000 1,200,000 CD34+ cells / ml, 1,250,000 CD34+ cells / ml, 1,300,000 CD34+ cells / ml, 1,350,000 CD34+ cells / ml, 1,400,000 CD34+ cells / ml, 1,450,000 CD34+ cells / ml, 1,500,000 CD34+ cells / ml, 1,550,000 CD34+ cells / ml, 1,600,000 CD34+ cells / ml, 1,650,000 CD34+ cells / ml, 1,700,000 CD34+ cells / ml, 1,750,000 CD34+ cells / ml, 1,800,000 CD34+ cells / ml, 1,850,000 CD34+ cells / ml, 1,900,000 CD34+ cells / ml, 1950,000 CD34+ cells / ml, 2,000,000 CD34+ cells / ml, 2,000,000 CD34+ cells / ml, 3,000,000 CD34+ cells / ml, 5,000,000 CD34+ cells / ml or 10,000,000 CD34+ cells / ml bone marrow or its derivatives or more. ,

[0015] In some embodiments, the method further comprises contacting the solution with a flushing medium and repeating c, and then removing the solution from the shaker. In some embodiments, the method further comprises repeating step c one or more times, and then removing the solution from the shaker. In some embodiments, at least about 1,500,000 CD34+ cells / ml bone marrow or its derivatives contain at least 85% viable CD34+ cells. In some embodiments, at least about 1,500,000 CD34+ cells / ml bone marrow or its derivatives contain at least 90% viable CD34+ cells.

[0016] Another aspect of the present disclosure includes a method for processing a CD34+ cell population obtained from bone marrow or a derivative thereof, wherein the bone marrow or a derivative thereof is derived from a deceased donor, the method comprising: obtaining bone or bone fragments from a deceased donor, optionally processing the bone into bone fragments; extracting bone marrow or a derivative thereof from the bone or bone fragments; and contacting the bone marrow or a derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises greater than about 3 U / ml of a nuclease; performing a CD34+ cell separation assay to produce a cell composition comprising a CD34+ cell population, wherein the composition comprising a CD34+ cell population comprises at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with the stabilization buffer. In some embodiments, at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with the stabilization buffer comprises at least 70% viable CD34+ cells. In some embodiments, at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with a stabilization buffer comprises at least 80% viable CD34+ cells. In some embodiments, at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with a stabilization buffer comprises at least 90% viable CD34+ cells. In some embodiments, the stabilization buffer comprises greater than about 5 U / ml of nuclease. In some embodiments, the stabilization buffer comprises greater than about 10 U / ml of nuclease. In some embodiments, the stabilization buffer comprises greater than about 15 U / ml of nuclease. In some embodiments, the stabilization buffer comprises about 20 U / ml of nuclease. In some embodiments, the stabilization buffer comprises greater than about 20 U / ml of nuclease. In some embodiments, the nuclease is or In some embodiments, the stabilization buffer further comprises an anticoagulant greater than about 5U / ml. In some embodiments, the stabilization buffer further comprises an anticoagulant greater than about 10U / ml. In some embodiments, the stabilization buffer further comprises an anticoagulant of about 10U / ml. In some embodiments, the anticoagulant is heparin. In some embodiments, the stabilization buffer further comprises human serum albumin (HSA). In some embodiments, the stabilization buffer comprises 0.5% HSA.

[0017] Another aspect of the present disclosure includes a stabilization buffer comprising: at least 5 U / ml of an anticoagulant; and greater than 3 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 5 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 10 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 15 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 20 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises about 20 U / ml of a nuclease. In some embodiments, the nuclease is or In some embodiments, the stabilization buffer further comprises an anticoagulant greater than about 10U / ml. In some embodiments, the stabilization buffer further comprises an anticoagulant of about 10U / ml. In some embodiments, the anticoagulant is heparin. In some embodiments, the stabilization buffer further comprises human serum albumin (HSA). In some embodiments, the stabilization buffer comprises 0.5% HSA.

[0018] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein.

[0019] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and description should be regarded as illustrative, not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the present invention are utilized and the accompanying drawings (also referred to herein as "drawings" and "figures"), in which:

[0021] Figure 1 is an illustration of a filtration system according to one feature of the present disclosure.

[0022] Figure 2 is an illustration of a sterile bag containing a bone marrow pellet processed according to the methods of the present disclosure.

[0023] Figure 3 yes Figure 2 View of a sterile bag with a clamp clamped around the bag to separate the fat from the bone marrow pellet.

[0024] Figure 4 An apparatus for separating a bone marrow pellet is shown.

[0025] Figure 5 is a perspective view of a cooling box according to one aspect of the present disclosure.

[0026] Figure 6 is a flow chart of a method according to the present disclosure.

[0027] Fig. 7A and Figure 7B are side and perspective views of an automated bone processing system according to one aspect of the present disclosure.

[0028] Fig. 8A and Figure 8B yes Fig. 7A and Figure 7B A perspective view of the bone cleaning station of the system shown in FIG.

[0029] Fig. 9A and Fig. 9B yes Fig. 7A and Figure 7B Perspective and front views of the bone grinding station of the system shown in .

[0030] Fig.10 yes Fig. 7A and Figure 7B A perspective view of a screening station of the system shown in FIG.

[0031] Figures 11A-11C Table 2 shows CD34+ cell survival as a function of warm and cold ischemia time with and without body cooling.

[0032] Figure 12A-12C is a table of CFU-total counts as a function of warm and cold ischemia time with and without body cooling.

[0033] Figure 13A-13C is a table of CFU-total counts as a function of warm and cold ischemia time with and without body cooling.

[0034] Fig.14 Shown is the location of the HPC, Marrow experimental cassette in one shelf of a -86°C Eppendorf Cryocube Model F740hi.

[0035] Fig.15 An exemplary alternative arrangement of cassette positions in one shelf of a -86°C Eppendorf Cryocube model F740hi is shown.

[0036] Fig.16Examples of graphs used to determine superfreezing / subfreezing cooling rates and nucleation temperatures are shown.

[0037] Figure 17A-17E It was shown that the formation of aggregates was prevented when bone marrow cells were processed from frozen samples with a stabilization buffer. Fig.17A Bone marrow cell slurry after antibody labeling is shown. The numerical numbers correspond to the buffer used. Bone marrow cell samples processed with stabilization buffer (4) showed the absence of aggregates. Fig. 17B The lack of entrapped aggregates after filtration in the bone marrow cell sample processed with the stabilization buffer is shown. Fig. 17C and Fig.17D Aggregate formation of bone marrow cells processed with CliniMACS buffer is shown ( Fig. 17C ), or the aggregates of bone marrow cells treated with stabilization buffer were absent ( Fig.17D ). Fig.17E It is shown that bone marrow cells processed with stabilization buffer exhibit increased survival yield and CD34 expression of bone marrow cells. The purity is greater than 60%, and greater than 60% CD34 cells are recovered. The ratio between CD3 count and CD34 count is 0.5% (e.g., 5 cells expressing CD3 / 100 cells expressing CD34).

[0038] Fig.18 An exemplary clean room diagram of clean room C in Example 9 is shown.

[0039] Fig.19 An exemplary cleanroom workflow for decontaminating VB as described in Example 9 is shown.

[0040] Fig. 20 An exemplary flow cytometry plot of human CD34+ in blood at 8 weeks is shown.

[0041] Fig.21 The gating strategy used to determine the phenotype of BM cells isolated from deceased and living donors is shown.

[0042] Fig. 22 Relative and absolute values ​​of CD45+ leukocytes, CD34+ HSPCs, and CD3+ T cells in BM of living donors versus deceased donors are shown. Bars represent mean + / - standard deviation.

[0043] Fig.23 Comparison of CFU potential between HPC bone marrow and living donor BM is shown.

[0044] Fig.24 Shows similar viability and numbers of CD34+ HSCs isolated from organ donor (OD) and living donor (LD) BM. Mean values ​​from 5 studies.

[0045] Fig.25 Shown are the levels of human CD45+ cells in the bone marrow of irradiated NSG mice 16 weeks after injection of CD34+ cells. Mock controls were bone marrow from untreated mice that were not irradiated. Cell surface CD45 expression was determined by flow cytometry. Thick bars represent the mean of N=5 (cord blood) or 10 (HPC bone marrow) mice. Standard deviations are shown by thin vertical lines.

[0046] Fig.26 Shown are the percentages of human CD45+ and CD34+ cells in the bone marrow (BM), peripheral blood (PB), and spleen 16 weeks after irradiation of NSG mice and transplantation with CD34+ cells.

[0047] Fig. 27 Secondary transplants are depicted. Levels of human CD45+ cells in the bone marrow of irradiated NSG mice 16 weeks after injection of total bone marrow from mice engrafted with CD34+ cells from the indicated donors. Mock controls were bone marrow from untreated mice that were not irradiated. Cell surface CD45 expression was determined by flow cytometry. Thick bars represent the mean of N=10 mice. Standard deviations are shown by thin vertical lines.

[0048] Fig.28 is an overall continuous manufacturing and process control flow diagram for producing cryopreserved bone marrow ("HPC bone marrow"), as described in Example 16.

[0049] The novel features of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments. DETAILED DESCRIPTION

[0050] introduction

[0051] The compositions, systems and methods disclosed herein provide the required supplement to existing bone marrow and stem cell sources. Specifically, the compositions, systems and methods disclosed herein provide the technology for separating, processing and using bone marrow and hematopoietic stem cells ("HSC") from human corpses. The unique features of the compositions, systems and methods described herein lead to improvements in the current state of the art. These features lead to significantly improved yields of functional bone marrow and HSC, alleviating the overall burden of resources usually required for separation, processing and use of bone marrow and HSC.

[0052] The compositions, systems and methods disclosed herein provide a departure from the isolation and processing techniques of bone marrow and HSC known in the art, particularly those used to process bone marrow and HSC from living donors. This disclosure describes various embodiments of processing techniques for producing the bone marrow and HSC compositions described herein.

[0053] As described below, various bones are taken out and prepared from dead donors for mechanical and enzymatic processing. The bone is then mechanically processed, wherein bone marrow and / or bone marrow-derived cells (e.g., HSC) are filtered to produce surviving bone marrow (and HSC contained in or near the bone marrow) compositions. The bone and cell compositions are further mechanically / enzymatically processed to produce surviving cells of optimal yield. These processing steps provide a departure from the current state of the art, resulting in improved cell compositions and processing methods thus.

[0054] The bone marrow is then further processed for immediate use or preservation. In some embodiments, the bone marrow is further processed to produce a cell composition comprising a specific HSC population (e.g., CD34+ cells). Optimized compositions, systems, and methods are described herein, providing improved bone marrow and HSC processing techniques and compositions.

[0055] These optimized compositions, systems and methods described herein provide unique solutions to current problems recognized by medical practitioners (e.g., immunology, regenerative medicine). Utilizing the optimized compositions, systems and methods described herein, bone marrow "banks" or reservoirs that will have more viable cells than current depots can be generated, and bone marrow depots generated using the compositions, systems and methods described herein will also have more diverse (e.g., different HLA phenotypes) bone marrow / HSC compositions, thereby generating a larger potential subject population that can benefit from bone marrow depots generated using the compositions, systems and methods described herein.

[0056] Aspects of the present disclosure provide a cryopreserved cell product that is divided into two volumes, a first volume (e.g., a cryopreservation bag) comprising a cell product for transplantation into an object in need, and a second volume serving as a substitute for the first volume. As used herein, a surrogate vial is typically a smaller volume of a cell product, and the surrogate can be thawed and measured, for example, cell viability (especially "functional viability" determined by post-thaw proliferation) as needed. The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using a surrogate, it is not necessary to thaw the first volume for determination, but to thaw when needed, for example, for transplantation into an object in need.

[0057] Without wishing to be bound by theory, for a given cell type, a specific optimal cooling rate is required so that the cell type or cell product survives cryopreservation. This optimal cooling rate balances the damage caused by intracellular ice formation (IIF) with the damage caused by the high solute concentration generated by extracellular ice formation. If the cells are cooled too quickly, damaging IIF is possible; if the cells are cooled too slowly, damaging solute effects are possible. In order for the surrogate vial to accurately represent the first volume, the cells should be frozen at approximately the same rate (i.e., the optimized rate) in the two volumes; this common rate results in equivalent survival and viability of the cells in the two volumes. Importantly, the second volume can be stored in the same long-term storage system as the first volume, and therefore will be exposed to the same conditions during the long-term storage duration, thereby facilitating the ability of the surrogate vial to accurately represent the cryopreservation bag containing cells for transplantation. These ultimately allow the second volume to be tested to at least determine whether the storage of the cryopreservation bag is properly maintained and it is not necessary to manipulate and test the cells of the first volume. More specifically, by assaying the surrogate vial, the cryopreservation bag does not need to be warmed and / or treated prior to its immediate use. This feature is particularly helpful to the outcome and health of the subject in two ways. First, the cells used for transplantation are thawed only when they are ready to be administered to the subject (and preferably at the site of administration), rather than being thawed approximately two weeks prior to use so that assays can be performed to ensure that the cell product is suitable for use; this two-week delay (during which the cells are kept at room temperature, on ice, or at 37°C, or worse, refrozen and returned to cold storage) may adversely affect their survival and utility once transplanted. Second, because the subject may undergo myeloablative conditioning prior to transplantation, the patient may be able to prevent myeloablative conditioning until the cell product has been assayed and determined to be suitable for use, which typically takes about two weeks; because of the surrogate vial, the subject begins myeloablative conditioning once a suitable product has been identified, thereby shortening the length of time the subject remains immunocompromised.

[0058] The present disclosure provides a method for ensuring that the two volumes are cooled at the same rate. At least based on the laws of physics, the smaller volume of a cell product will cool at a faster rate than the larger volume of a cell product. Thus, relative to a larger volume with a slower cooling rate, a smaller volume with a faster cooling rate should have an increased IIF. The method of the present disclosure promotes the equivalent rate of cell cooling between the first (larger) volume and the second (smaller) volume, so that each volume will have a similar amount of IFF, so that a surrogate bottle (smaller volume) will accurately represent the larger first volume. Without wishing to be bound by theory, the method of the present disclosure is based in part on using different types of containers that directly or indirectly hold the first volume or the second volume and / or placing the container in the same freezer (e.g., static temperature freezer), slowing the cooling rate of the second (smaller) volume to the rate that the first (larger) volume undergoes. Thus, the cells in the smaller volume (i.e., the second volume / surrogate vial) experience a similar cooling rate (e.g., about -1°C / minute) as the cells in the larger volume (i.e., the first volume / cryopreservation bag) when placed in the same static freezer (e.g., a -86°C static freezer). Importantly, to slow the cooling rate of the smaller second volume, the surrogate vial is placed directly in an insulated vial container (e.g., In a cryopreservation system, the cells in the surrogate vials are subjected to a freezing rate of about -1°C / minute when the container is placed in a static freezer below -80°C (e.g., a -86°C static freezer); without the use of an insulated vial container, the cells in the surrogate vials may be subjected to a freezing rate of about -10°C / minute, thereby potentially causing damage due to IIF. On the other hand, the larger first volume (cryopreservation bag) does not need to be placed directly in an insulated container, but rather the cells in the cryopreservation bag are preferably subjected to a freezing rate of about -1°C / minute when the bag is placed in a non-insulated (to avoid slowing the cooling rate of the bag) box and moved to a -86°C static freezer. "Directly placed" means that each vial is in close proximity to the insulating material of the insulated container. These manipulations allow the first and second volumes of cells to have approximately equal penetration of intracellular water into the extracellular space of the cells; this penetration increases intracellular solute concentrations and helps avoid the formation of (harmful) intracellular ice crystals and promotes extracellular ice formation (which is less harmful to cells). Once the first step of freezing (in a -86°C static freezer), the cryopreservation bag and surrogate vial are placed in the same long-term storage device (e.g., a liquid nitrogen storage tank) and positioned in approximately similar locations within the long-term storage device.

[0059] Thus, the methods of the present disclosure allow for the production of surrogate samples of cell products that are expected to accurately represent the portion of the cell product to be administered to a subject in need thereof, and provide a cell product that not only therapeutically benefits the subject, but also promotes the subject's outcome and health, in a manner that is not achievable when the cryopreserved cell product is contained solely in a single bag and without a surrogate vial.

[0060] Preparation of donor bone

[0061] The vertebral bodies and iliac bones represent the largest sustainable reserves of high quality red marrow. Recovery of marrow has been optimized using one or both sources, particularly by implementing the industrialized, scalable GMP processes disclosed herein. In some embodiments, completion of the processes disclosed herein results in cryopreservation of the final product in a standard blood bag at a target storage volume of 60-70 ml of total nucleated cells / ml at 100-150 million. In some cases, the production method provides a system in which a skilled tissue processing technician can process a series of donor bones within a 6 hour window to produce a meaningful amount of viable marrow.

[0062] In some embodiments, the donor bone is a vertebral body. However, it should be understood that the methods described herein can be used for iliac bones, combinations of vertebral bodies and iliac bones, or other bones suitable for extracting bone marrow and cells from bone marrow, even with donor bones of lower expected yield.

[0063] It should be understood that donor bone can be obtained according to a fixed protocol for clinical recovery. Bone can be recovered from consenting organ and tissue donors by a surgeon or trained organ procurement organization (OPO) personnel using osteotome and mallet. Vertebral segments must be carefully recovered, preferably from the thoracic and lumbar vertebrae. The bone segments are cut and removed by using osteotome and mallet. As much spinal cord as possible is removed. A licensed surgeon can supervise these steps to ensure effective recovery of the VB and prevent disease transmission and bacterial translocation.

[0064] The vertebral segments, once recovered, are swabbed for microbial culture testing and placed in sterile labeled bags with sterile pads, sponges or towels soaked with saline to ensure moisture retention during low-temperature transportation. These are then placed in a cooler between wet ice packs for transportation. Recovery of the VB must occur within a minimum warm ischemia time (≤8 hours). The start of transportation and processing must be completed within a minimum cold ischemia time (≤40 hours). The packages are finally transported to the processing facility.

[0065] The VB material is wrapped and double-bagged. The bags are placed in an insulated shipping box, with bags of wet ice surrounding them. The shipping box is sealed and sent to Ossium via medical courier. Upon arrival, the packaging is checked for compliance with protocol, and the vertebral temperature is measured to ensure compliance with shipping requirements.

[0066] The process of preparing donor bone can occur shortly after obtaining bone from a deceased donor, or can occur after the donor bone has been transported to a processing facility under a cryogenic environment. Because the donor bone may be subjected to prolonged ischemia during recovery and transportation to the processing facility, care must be taken to track the length and type of ischemia, e.g., warm ischemia and cold ischemia. As described in more detail herein, bones that have undergone warm ischemia and / or cold ischemia for a predetermined period are suitable for obtaining a meaningful number of viable bone marrow cells.

[0067] During processing of the donor bone, the bone is cleaned in an ISO-5 (class 100) environment (biosafety cabinet) and an ISO-7 (class 10,000) background (clean room), with particular attention paid to sterilizing the bag containing the donor bone, for example by spraying with 70% isopropyl alcohol. In one embodiment, cleaning is performed manually by using a scalpel, osteotome, and chisel. When processing vertebrae, a spinal column segment comprising multiple vertebrae will typically be provided. In a typical case, the spinal column segment extends from T8 to L5, ten vertebral bodies. During the initial cleaning of the spinal column segment, when enough soft tissue has been removed to visualize the pedicles, the bone is removed by using a tissue processing band saw or bone saw, such as a Stryker System 6 Saw (Stryker, Kalamazoo, MI), or with a Figure 1 A to Figure 1 The hand tool shown in D removes the pedicle. Special care is taken to avoid damaging the cortical bone and possibly exposing the cancellous bone to ensure that the hypoxic cancellous marrow remains protected throughout the debridement process. The anterior element of the vertebral body containing the cancellous material is retained, while the pedicle and posterior elements are discarded.

[0068] The vertebral bodies (VB) are separated at the intervertebral disc using a boning knife or a tissue processing band saw. The intervertebral disc and soft tissue remaining on each vertebral body are removed with a scalpel, scissors and / or osteotome, leaving a clean, isolated VB. In the case of donor iliac crests, the soft tissue can be removed with a chisel and scalpel, again with special attention to ensure that the cortical bone is not destroyed. Any anatomical pathology or damage to the bone is noted and recorded as part of the batch record of the bone marrow ultimately obtained from the bone. Damaged bone is discarded during the recovery process.

[0069] In some cases, cadaveric bone is "pre-processed" to reduce contaminants carried by the cadaveric bone, which risks transferring contamination to the facility where the bone is processed. In these cases, two technicians perform different aspects of pre-processing. The first technician opens the package containing the collected cadaveric bone, preferably contained in a sealed inner bag. The second technician, wearing sterile gloves, removes the cadaveric bone from the package and places the tissue in a first (rinse) basin. The second technician vigorously scrubs all surfaces of the cadaveric bone in or over the rinse basin for about 3 minutes with about 4% chlorhexidine gluconate solution. The first technician, wearing sterile gloves, pours sterile saline on the scrubbed cadaveric bone, and the overflow is captured in the rinse basin. Pour enough saline on the cadaveric bone to rinse all its surfaces. The rinsed cadaveric bone is then placed on a sterile cloth adjacent to the rinse basin. The saline rinse can be repeated as necessary. Pour alcohol, such as 70% isopropyl alcohol, on the cadaveric bone. Pour enough alcohol on the cadaveric bone to contact all its surfaces. The alcohol overflow is captured in the rinse basin. The cadaveric bone is placed in an open container, sprayed with alcohol, and the open container and bone are then transferred to a hood where the bone can be further processed.

[0070] Aspects of the present disclosure include a method for processing bone marrow or its derivatives, wherein the bone marrow or its derivatives are derived from a deceased donor, the method comprising: obtaining bone from a deceased donor; contacting the bone with a bleach solution for at least about 10 minutes to at least about 25 minutes, wherein the bone is immersed in the bleach solution; extracting bone marrow or its derivatives from the bone, wherein at least 90% of the CD34+ cells contained in the bone marrow or its derivatives are viable. In some embodiments, the bone marrow or its derivatives are contacted with the bleach solution for at least about 25 minutes. In some embodiments, the bleach solution contains 10% bleach. In some embodiments, the bone is a vertebral body. In some embodiments, the hydrogen peroxide is a 3% hydrogen peroxide solution. In some embodiments, the method further comprises transferring the bleached bone product from a container containing the bleach solution to a container containing the hydrogen peroxide solution. In some embodiments, the method further comprises a step of agitating the bleached bone product in the hydrogen peroxide solution. In some embodiments, immersing the bleached bone product in a solution comprising hydrogen peroxide comprises: immersing the bleached bone product in a container comprising a hydrogen peroxide solution; detecting foam or foam-like objects associated with the bleached bone product; and repeating the immersion until no foam or foam-like objects are detected. In some embodiments, the method further comprises manually removing soft tissue associated with the foam or foam-like objects from the bleached bone product. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance the visibility of any foam or foam-like objects associated with the bleached bone product.

[0071] In some embodiments, the bone is agitated (eg, shaken) in the bleach solution.

[0072] The pre-processed or non-pre-processed VB is placed in a sterile bag and immersed in about 10% bleach solution (0.5% sodium hypochlorite in sterile water) to produce a concentration of 5,000 ppm free chlorine for a predetermined period of time, typically about 5 minutes to about 25 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes or more and any length of time therebetween. Bleach has a broad spectrum of antimicrobial activity, does not leave toxic residues, is not affected by water hardness and is fast acting.

[0073] Bone marrow from each set of VBs processed at different durations of bleach treatment can be tested by flow cytometry to assess the viability of cells isolated from the bone marrow. As illustrated in Table 4, VB soaking for greater than 10 minutes did not produce a significant difference in cell viability compared to when the VB was soaked for up to 25 minutes. However, without wishing to be bound by theory, an increase in bleaching time improves the final product. For example, increasing the period of time that the VB is soaked in bleach allows the bleach to fill the pores or cracks of the VB and further decontaminate or sterilize the VB.

[0074] The bleach solution can be about 5% bleach to about 15% bleach, for example, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or about 15% bleach. In some embodiments, the bleach treatment includes using 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more percentage of bleach. In some embodiments, the bleach treatment comprises contacting the VB with the bleach for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or longer duration. In some embodiments, the VB is immersed in the bleach solution for at least about 10 minutes to at least about 25 minutes, for example, about 10 to about 12 minutes, about 11 to about 14 minutes, about 13 to about 16 minutes, about 15 to about 18 minutes, about 17 to 20 minutes, about 19 to 22 minutes, or about 21 to 25 minutes and any interval therebetween. In some embodiments, the survival rate of bone marrow cells isolated from the bleach-treated VB is not reduced at any duration of the bleach treatment described herein compared to bone marrow cells isolated from VBs that have not been treated with bleach. In some embodiments, the survival rate of bone marrow cells isolated from VBs treated under bleach treatment for 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or more durations is not reduced or is reduced by less than 3% compared to the survival rate of bone marrow cells isolated from VBs treated under 10 minutes of bleach treatment. In some embodiments, the survival rate of bone marrow cells isolated from VBs treated for greater than 10 minutes is reduced by less than 2% compared to the survival rate of bone marrow cells isolated from VBs treated under 10 minutes of bleach treatment. In some embodiments, the survival rate of bone marrow cells isolated from VBs treated for greater than 10 minutes is reduced by less than 1% compared to the survival rate of bone marrow cells isolated from VBs treated under 10 minutes of bleach treatment.

[0075] Interestingly, the bleach treatment provided surface sterilization of the bone, but did not penetrate into the compartment containing the BM. Thus, the bleach treatment disclosed herein does not substantially reduce the yield of viable cells obtained from the BM.

[0076] In some embodiments, the percentage of viable CD34+ cells contained in the bone marrow or its derivative extracted from the bone immersed in the bleach is at least about 80% to about 95%. In some embodiments, the percentage of viable CD34+ cells contained in the bone marrow or its derivative extracted from the bone immersed in the bleach is at least about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the percentage of viable CD34+ cells contained in the bone marrow or its derivative extracted from the bone immersed in the bleach is at least about 80%, about 85%, about 90% or about 95%. In some embodiments, the percentage of viable CD34+ cells contained in the bone marrow or its derivative extracted from the bone immersed in the bleach is at least about 80%, about 85% or about 90%. In some embodiments, the bone marrow or derivative thereof extracted from a bone immersed in a bleaching agent comprises a percentage of viable CD34+ cells of at most about 85%, about 90%, or about 95%.

[0077] At the end of the bleaching period, the bones were transferred to another sterile bag and immersed in 3% hydrogen peroxide (H 2 O 2 ) solution. In some cases, H 2 O 2 Solution contains PLASMA-LYTE TM (Multi-Electrolyte Injection, a sterile, pyrogen-free, isotonic solution that is a crystalloid essential source of water and electrolyte balance for cells, obtained from Baxter Healthcare, Ltd.). 2 O 2 Solution contains PLASMA-LYTE TM and human serum albumin (HSA), which is a stabilizer and storage agent (it can be 2 O 2 The bag was closed and shaken briefly to ensure that the entire surface of the bone was in contact with the solution. Most living cells contain catalase, which catalyzes H 2 O 2 Decompose into H 2 O and O 2 When H 2 O 2This decomposition shows as foam or foam when solution contacts soft tissue but not contact bone.Can observe foam level, as the indication of the amount of remaining soft tissue on bone.This observation can be carried out manually by treatment personnel, or in another embodiment, by automated processor.Automated processor comprises visual device such as camera, and target recognition software (it can determine the foam level in bag).The interpolation of inert contrast dye can help treatment personnel or automated processor to detect foam level.If observe any foam or foam, then bone is returned for further processing and removes all remaining soft tissue from bone.Once VB or ilium have been cleared of all soft tissues, bone is transferred to new sterile bag.This bag is filled with 1L of PLASMA-LYTE TM , or other suitable sterile, nonpyrogenic, isotonic solution. The bag is closed and briefly shaken to ensure that the entire bone is completely soaked with PLASMA-LYTE. TM touch.

[0078] In some embodiments, the method further comprises the step of agitating the bleached bone product in a hydrogen peroxide solution. In some embodiments, immersing the bleached bone product in a solution comprising hydrogen peroxide comprises: immersing the bleached bone product in a container comprising a hydrogen peroxide solution; detecting foam or foam-like substances associated with the bleached bone product; and repeating the immersion until no foam or foam-like substances are detected. In some embodiments, the method further comprises manually removing soft tissue associated with the foam or foam-like substances from the bleached bone product. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance the visibility of any foam or foam-like substances associated with the bleached bone product.

[0079] The bleaching step and the hydrogen peroxide step can be repeated multiple times.

[0080] Without wishing to be bound by theory, it is believed that H 2 O 2 Not only does the solution help to sterilize the surface of the bone, it helps break down any residual bleach into salt, oxygen, and water.

[0081] After surface sterilization, the cadaveric bone may be rinsed with water, saline, or a cryoprotectant solution.The surface sterilized cadaveric bone may then be placed in a closed container containing a cryoprotectant solution and the pressure reduced.

[0082] Cryoprotectants penetrate into cadaver bones

[0083] The cadaveric bone can be contacted with the cryoprotectant solution for a sufficient length of time and under conditions to allow the cryoprotectant solution to penetrate into the cadaveric bone. Methods for cryopreserving bones are described below and in other sections. In some cases, conditions sufficient to allow penetration of the cryoprotectant solution include infiltration of the cadaveric bone with a vacuum, as disclosed in PCT / US2021 / 042064, the contents of which are incorporated herein by reference in their entirety. In other cases, the cadaveric bone is immersed in the cryoprotectant solution and no vacuum is used.

[0084] An aspect of the present disclosure is a method for cryopreserving cadaveric bone using a vacuum to assist the penetration of a cryoprotectant into the cadaveric bone. The method comprises the steps of: (a) placing the cadaveric bone in a closed container containing a cryoprotectant solution; (b) reducing the pressure in the closed container, and optionally maintaining the closed container under reduced pressure, to remove at least a portion of the water present in the cadaveric bone; (c) increasing the pressure in the closed container and maintaining the closed container under the increased pressure to allow the cryoprotectant solution to penetrate into the cadaveric bone; (d) removing the cadaveric bone from the closed container; and (e) cooling the cadaveric bone to a temperature of at least below 0°C, thereby cryopreserving the cadaveric bone.

[0085] Surprisingly, by immersing the cadaveric bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cryoprotectant penetrated the cadaveric bone significantly more rapidly than would have occurred through passive diffusion. Figure 2 and Figure 4 A and Figure 4 B, and Figure 3 and Figure 5 This efficient infiltration of the cryoprotectant contributes to reduced ice crystal formation during freezing of cadaveric bone and the ultimate extraction of viable bone marrow cells with replicative potential.

[0086] Steps (b) and (c) may occur only once, or steps (b) and (c) may be repeated at least once, at least twice, at least four times, at least five times, or at least six times. In some embodiments, repeated decompression and pressurization may increase the penetration of the cryoprotectant into the cadaveric bone. See, e.g., PCT / US2021 / 042064. Figure 5 In other embodiments, there is sufficient infiltration of the cryoprotectant into the cadaveric bone following a single cycle of decompression and pressurization.

[0087] In various embodiments, cadaveric bone (eg, vertebral body) is bisected, quartered, or more divided prior to vacuum-assisted infiltration of the cryoprotectant.

[0088] The reduced pressure in the closed container can be any pressure value of about -400mmHg to about -800mmHg. The pressure requirement should be enough to remove at least a portion of the water present in the cadaver bone. The reduced pressure in the closed container can have following values: about -400mmHg, -425mmHg, -450mmHg, -475mmHg, -500mmHg, -525mmHg, -550mmHg, -575mmHg, -600mmHg, -625mmHg, -650mmHg, -675mmHg, -700mmHg, -725mmHg, -750mmHg, -775mmHg or -800mmHg. In some embodiments, the reduced pressure in the closed container is about -400mmHg to about -500mmHg.

[0089] In some embodiments, once the pressure in the closed container begins to decrease, the closed container needs about 1 minute to about 10 minutes to reach the desired decompression. As an example, the closed container may need to be less than 1 minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or about 10 minutes and any time length therebetween (e.g., fractions of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds and any seconds therebetween) to reach the desired decompression. In some embodiments, the cadaver bone quickly (e.g., about 1 second to about 1 minute) reaches the desired decompression.

[0090] In some embodiments, once decompression has been reached, cadaveric bone is maintained under decompression. Cadaveric bone can be maintained less than 1 minute to about 50 minutes. As an example, closed container is maintained under decompression less than 1 minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes or about 50 minutes and any time length therebetween (e.g., 1 minute fraction, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds and any seconds therebetween). In some embodiments, the cadaveric bone is not maintained under reduced pressure for any measurable time, and instead, the method proceeds to step (c) by increasing the pressure in the closed container.

[0091] In step (c), the pressure of the closed container is increased until the pressure is about 0 mmHg to about 760 mmHg. In other words, the pressure is increased to as high as standard atmospheric temperature. The exact boost can be any amount within a specific range, for example, 0 mmHg, 50 mmHg, 100 mmHg, 150 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 650 mmHg, 700 mmHg or 750 mmHg. However, the boost must be high enough to allow the cryoprotectant solution to penetrate into the cadaver bone.

[0092] The closed container can be kept less than about 2 hours under boost. As an example, less than 1 hour, less than half an hour, about half an hour or shorter time. In some embodiments, the closed container is kept for 10 minutes under boost. The duration of the closed container under boost must be long enough to allow the cryoprotectant solution to infiltrate the corpse bone. As an example, the closed container is kept for about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes or about 30 minutes under boost, and any time length therebetween (for example, 1 minute fraction, for example, about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds and any seconds therebetween).

[0093] The closed container and the cryoprotectant contained therein can be at room temperature. Alternatively, the closed container and the cryoprotectant contained therein can be below room temperature, for example, as low as 4° C. The closed container and the cryoprotectant contained therein can be above room temperature, for example, up to 37° C.

[0094] Any suitable cryoprotectant may be used in the cryoprotectant solution. Examples of cryoprotectants include dimethyl sulfoxide (also known as DMSO, C 2 H 6In some embodiments, the cryoprotectant is DMSO. The cryoprotectant solution can comprise from about 5% DMSO to about 100% DMSO, for example, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% DMSO. %, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% DMSO. The cryoprotectant solution may contain about 10% DMSO. The cryoprotectant solution may contain about 20% DMSO. In some embodiments, the cryoprotectant solution may comprise about 40% DMSO or 60% DMSO. In some embodiments, a higher percentage of cryoprotectant is preferred, for example, a percentage twice the equivalent cell suspension value to help drive osmotic infiltration.

[0095] The cryoprotectant solution can have water or saline as a base. In some embodiments, the saline is isotonic to human tissue. In embodiments, the saline is a 0.9% saline solution. Any commercially available saline solution can be used: sodium chloride solution, PBS, HEPES, Ringer's solution, or lactate. The saline can be 0.9% sodium chloride.

[0096] The cryoprotectant solution may also contain a protein. As an example, the protein may be human albumin (e.g., HSA) or a component of human platelet lysate. An example of a commercially available human platelet lysate product is Stemulate TM (From Regentec).

[0097] In some embodiments, the cryoprotectant solution comprises about 10% protein, eg, 10% human platelet lysate or 10% albumin.

[0098] In one example, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0099] In another example, the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0100] In yet another example, the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0101] In another example, the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0102] In another example, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.

[0103] In any of the above aspects, the method may include the steps of: introducing a compressed gas (e.g., nitrogen, xenon, CO 2 , Argon, H 2 S or helium), gases released by sublimation (e.g., CO 2 ) or by evaporation of a gas (e.g., nitrogen from liquid nitrogen) to increase the pressure in the closed container containing the cryoprotectant to above 760 mmHg, thereby allowing the gas to penetrate into the cadaveric bone. In an embodiment, the gas is CO 2 , for example, compressed CO 2 In some embodiments, the gas is nitrogen, for example, compressed nitrogen. When the gas is compressed, the time required for the gas to penetrate into the vertebral body is less than that required for gas obtained by sublimation.

[0104] Alternatively, in any of the above aspects, rather than placing the cadaver bone in a closed container containing a cryoprotectant solution, the cadaver bone is placed in a closed container lacking a cryoprotectant solution. In these alternative aspects, the method includes the steps of: introducing a compressed gas (e.g., nitrogen, xenon, CO 2 , Argon, H 2 S or helium), gases released by sublimation (e.g., CO 2) or by evaporation of a gas (e.g., nitrogen from liquid nitrogen), the pressure in the closed container (lacking a cryoprotectant solution) is increased to above 760 mmHg, so that the gas penetrates into the cadaveric bone. Any of the methods disclosed herein may be adapted to include the following initial steps: placing the cadaveric bone in a closed container lacking a cryoprotectant solution, and increasing the pressure in the closed container to above 760 mmHg by introducing a compressed gas, a gas released by sublimation, or a gas provided by evaporation; in a subsequent step, the cryoprotectant solution is added to the closed container. In an embodiment, the gas is CO 2 , for example, compressed CO 2 In some embodiments, the gas is nitrogen, eg, compressed nitrogen.

[0105] Without wishing to be bound by theory, the evaporation of the membrane may be accomplished by introducing compressed gas (e.g., nitrogen, xenon, CO 2 , Argon, H 2 S or helium), gases released by sublimation (e.g., CO 2 ) or by providing a gas through evaporation (e.g., nitrogen from liquid nitrogen) to increase the pressure in the closed container, promoting penetration of the cryoprotectant solution into the cadaveric bone.

[0106] In some cases, the closed container comprises a solid material, such as metal, plastic or other polymers. In some cases, the closed container comprises a foam material, such as Styro foam.

[0107] In an alternative aspect, the cryoprotectant is infiltrated into the cadaveric bone without the use of a vacuum. Here, an intact vertebral body, a vertebral body that has been bisected, quartered, or more divided, is immersed in a cryoprotectant solution for a period of time and under conditions sufficient to allow the cryoprotectant solution to infiltrate the cadaveric bone.

[0108] The bone or bone fragment is placed in (e.g., immersed in) a cryoprotectant solution and incubated at about 4°C for 1 hour. In some embodiments, the incubation period is about 1 hour to about 3 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours or about 3 hours.

[0109] Any suitable cryoprotectant may be used in the cryoprotectant solution. Examples of cryoprotectants include dimethyl sulfoxide (also known as DMSO, C 2 H 6OS and ME2SO); 1,2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; formamide; ethanediol, butane 2,3 diol; hydroxyethyl starch (HES); dextran; sucrose; trehalose; lactose; raffinose; ribitol; mannitol; and polyvinyl pyrrolidone (PVP). In some embodiments, the cryoprotectant is DMSO. The cryoprotectant solution can comprise from about 5% DMSO to about 100% DMSO, for example, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% DMSO. %, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% DMSO. The cryoprotectant solution may contain about 20% DMSO. In some embodiments, the cryoprotectant solution may contain about 40% DMSO or 60% DMSO. In some embodiments, higher percentages of cryoprotectant are preferred, for example, twice the percentage of an equivalent cell suspension value to help drive osmotic infiltration.

[0110] The cryoprotectant solution can have water or saline as a base. In some embodiments, the saline is isotonic to human tissue. In embodiments, the saline is a 0.9% saline solution. Any commercially available saline solution can be used: sodium chloride solution, PBS, HEPES, Ringer's solution, or lactate. The saline can be 0.9% sodium chloride.

[0111] The cryoprotectant solution may also contain a protein. As an example, the protein may be human albumin (e.g., HSA) or a component of human platelet lysate. An example of a commercially available human platelet lysate product is Stemulate TM (From Regentec).

[0112] In some embodiments, the cryoprotectant solution comprises about 10% protein, eg, 10% human platelet lysate or 10% albumin.

[0113] In one example, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0114] In another example, the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0115] In yet another example, the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0116] In another example, the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0117] In another example, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.

[0118] Two-step cooling of cadaveric bone

[0119] Once the cryoprotectant has infiltrated the cadaveric bone (with or without the use of a vacuum), the cadaveric bone is then subjected to an initial cooling period. To this end, the cadaveric bone is placed in a static-80°C freezer that is set at a temperature below about -60°C, e.g., about -70°C to about -80°C, or below about -100°C. Thus, the cadaveric bone is subjected to an initial cooling period. In some embodiments, the cadaveric bone is initially cooled in a static-80°C freezer that is set at a temperature of about -86°C. Data showing the dynamics of the initial cooling period are shown in PCT / US2021 / 042064 at Figure 6 A.

[0120] In some cases, the static freezer is set at a range of temperatures: about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C, or about -100°C. In some cases, the freezer can be set at a range of temperatures: at least about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, or about -95°C. In some cases, the freezer can be set at a range of temperatures: up to about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C or about -100°C.

[0121] The cadaver bone can be initially cooled at a rate of about -0.3°C / min to about -5°C / min. In some embodiments, the cadaver bone is initially cooled at a rate of about -0.4°C / min to about -0.9°C / min. As an example, the initial cooling rate can be about -0.3°C / min, -0.4°C / min, -0.5°C / min, -0.6°C / min, -0.7°C / min, -0.8°C / min, -0.9°C / min, to about -1°C / min. In other examples, the initial cooling rate can be -1°C / min, -2°C / min, -3°C / min, -4°C / min, or about -5°C / min. In these rates, the minus sign ("-") means that the temperature drops by the stated amount.

[0122] The duration of the initial cooling period can vary from a few hours to overnight. The time should be sufficient for the cadaveric bone to reach a temperature below about -50°C, for example, -60°C to -80°C. In some embodiments, the bone reaches the desired temperature in about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or about 12 hours. In some embodiments, the cadaveric bone is initially cooled in a -80 freezer for at least 12 hours or at least overnight.

[0123] Without wishing to be bound by theory, the initial cooling period, in the presence of extracellular ice, increases intracellular solute concentrations to an amount that permits intracellular vitrification on subsequent cooling.

[0124] The cadaveric bone may temporarily attain a temperature of about -5°C to about -15°C, but this occurs as the temperature of the cadaveric bone continues to decrease toward the desired temperature, e.g., a temperature below about -50°C. Even if the cadaveric bone is not maintained in a static freezer set at a temperature of about -5°C to about -15°C for a period of about 1 to about 30 minutes during the initial cooling period, the cadaveric bone will attain a temperature of about -5°C to about -15°C as the bone continues to cool to the desired temperature.

[0125] Once the cadaveric bone has reached the desired temperature, it is subjected to a subsequent cooling period. To this end, the cadaveric bone is placed in liquid nitrogen or liquid nitrogen vapor, for example, at a temperature of about -200° C. Data showing the dynamics of the subsequent cooling period are shown in PCT / US2021 / 042064 at Figure 6 B. In some embodiments, the subsequent cooling period can occur in a suitable static freezer capable of maintaining a temperature equivalent to liquid nitrogen without the use of liquid nitrogen, for example, a cryogenic freezer.

[0126] During the subsequent cooling period, the cadaveric bone is cooled at a rate of about -2°C / min to about -6°C / min. In some embodiments, the cadaveric bone is initially cooled at a rate of about -2°C / min, -2.2°C / min, -2.4°C / min, -2.6°C / min, -2.8°C / min, -3°C / min, -3.2°C / min, -3.4°C / min, -3.6°C / min, -3.8°C / min, -4°C / min, -4.2°C / min, -4.4°C / min, -4.6°C / min, -4.8°C / min, -5°C / min, -5.2°C / min, -5.4°C / min, -5.6°C / min, -5.8°C / min, or about -6°C / min. In these rates, the minus sign ("-") means that the temperature drops by the stated amount.

[0127] Cryopreserved cadaveric bone can be stored indefinitely in liquid nitrogen, liquid nitrogen vapor, or a suitable static freezer. As an example, cryopreserved cadaveric bone can be stored for at least one day, at least one week, at least one month, at least one year, at least five years, or at least 20 years. Cryopreserved cadaveric bone can be stored in liquid nitrogen, liquid nitrogen vapor, or a suitable static freezer for hundreds or thousands of years.

[0128] Without wishing to be bound by theory, the two-step cooling method of cadaveric bone as disclosed herein improves the survival rate of extracted bone marrow cells (hematopoietic stem cells (HSC; CD34+ cells) and / or mesenchymal stromal / stem cells (MSC)) relative to methods that do not use the two-step cooling method. Thus, by using the methods of the present disclosure, a greater number of viable cells (HSC and / or MSC) are obtained relative to standard methods.

[0129] In some cases, the methods of the present disclosure provide from about 1% more viable cells to about 100% more viable cells, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 112%, 113%, 114%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% of the cells are viable.

[0130] In some cases, the methods of the present disclosure provide about 101% more viable cells to about 200% more viable cells, e.g., about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 200%, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2011, 2021, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2012, 2013, 2014, 2015 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144% , 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173 In some embodiments, the present invention relates to a method for producing a cell-free living cell in an amount of at least 100% or about 200% of the cells to be viable. In some embodiments, the present invention relates to a method for producing a cell-free living cell in an amount of at least 100% or about 200% of the cells to be viable.

[0131] In some cases, the methods of the present disclosure provide about 2-fold to about 10-fold more viable cells, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, about 10-fold, or any times therebetween, compared to methods that do not use the two-step cooling methods disclosed herein. As an example, the methods of the present disclosure provide 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold more viable cells than methods that do not use the two-step cooling methods disclosed herein.

[0132] In some cases, the methods of the present disclosure provide about 10 times more viable cells to about 100 times more viable cells, e.g., about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, about 100 times, or any times therebetween, compared to methods that do not use the two-step cooling methods disclosed herein. As an example, the methods of the present disclosure provide 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, or 90 to 100 times more viable cells than methods that do not use the two-step cooling methods disclosed herein.

[0133] In some cases, the methods of the present disclosure provide about 100 times more viable cells to about 1000 times more viable cells, e.g., about 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, about 1000 times, or any times therebetween, compared to methods that do not use the two-step cooling methods disclosed herein. As an example, the methods of the present disclosure provide 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, 600 to 700 times, 700 to 800 times, 800 to 900 times, or 900 to 1000 times more viable cells than methods that do not use the two-step cooling methods disclosed herein.

[0134] In some cases, the methods of the present disclosure provide about 1000 times more viable cells to about 10,000 times more viable cells, e.g., about 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, about 10,000 times, or any times therebetween, compared to methods that do not use the two-step cooling method disclosed herein. As an example, the methods of the present disclosure provide 1000-fold to 200-fold, 2000-fold to 3000-fold, 3000-fold to 4000-fold, 4000-fold to 5000-fold, 5000-fold to 6000-fold, 6000-fold to 7000-fold, 7000-fold to 8000-fold, 8000-fold to 9000-fold, or 9000-fold to 10000-fold more viable cells than a method that does not use the two-step cooling method disclosed herein.

[0135] Method for rapid warm cryopreservation of cadaveric bones

[0136] In some cases, the present disclosure provides a method for rapidly warming cadaveric bone to provide bone marrow or its derivatives. PCT / US2021 / 042064 discloses a method for rapidly warming cryopreserved bone; the contents of which are incorporated by reference in their entirety. These disclosed methods can be used in the methods of the present disclosure.

[0137] In some cases, a method for rapidly warming cadaveric bone comprises the steps of obtaining cryopreserved cadaveric bone; segmenting the cryopreserved cadaveric bone to obtain cryopreserved bone fragments; transferring the cryopreserved bone fragments to a grinding medium having a temperature of about 35°C to about 45°C for a sufficient time to warm the cadaveric bone fragments to a surface temperature of about 20°C.

[0138] In some embodiments, the cryopreserved cadaveric bone is transferred to a grinding medium (as disclosed herein) without having been divided into fragments. Preferably, the cryopreserved cadaveric bone has a temperature of at least below 0°C when transferred to the grinding medium.

[0139] In an alternative embodiment, the method comprises dividing the cryopreserved cadaveric bone to obtain fragments of the cryopreserved bone.Preferably, the cryopreserved cadaveric bone has a temperature below 0°C when divided into fragments.

[0140] In order to simplify the process and to increase the safety of the processing personnel, a custom bone cutting tool as described in US 2019 / 0343112 (which is incorporated herein by reference in its entirety) is used to divide the cryopreserved cadaveric bone into smaller pieces. Another bone cutting tool can be used in combination with or instead of the custom bone cutting tool as described in US 2019 / 0343112.

[0141] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably a hardened steel that can withstand the forces required to cut through frozen bone. During the cleaning process, the tool is steam sterilized, which may be harmful to the steel. Therefore, in one feature of the present disclosure, the surface of the stainless steel element is passivated to prevent oxidation of the steel element during sterilization.

[0142] A manual bone cutting device for segmenting cryopreserved cadaveric bone is capable of generating up to 1000 lbf when less than 50 lbf is applied. The manual bone cutting device includes: a force transmission mechanism, wherein the force transmission mechanism includes a slender force-conducting member pivotally connected to a gear mechanism; and a manually operable handle connected to an end of the slender force-conducting member, wherein the end is opposite to the gear mechanism. The manual bone cutting device includes an upper cutting element and / or a lower cutting element. Its upper cutting element and / or lower cutting element each include one or more cutting blades extending outwardly from a central portion of the upper cutting element and / or the lower cutting element. When the one or more cutting blades segment the cryopreserved cadaveric bone into roughly fan-shaped fragments.

[0143] The manual bone cutting device divides the cryopreserved cadaveric bone into cryopreserved bone fragments. The cryopreserved bone fragments are transferred to a grinding medium having a temperature of about 35°C to 45°C for a sufficient time to warm the cadaveric bone fragments to a surface temperature of about 20°C. Alternatively, a whole cryopreserved bone that has not been divided is transferred to a grinding medium having a temperature of about 35°C to 45°C for a sufficient time to warm the cadaveric bone fragments to a surface temperature of about 20°C. In some embodiments, the surface temperature of the cadaveric bone fragments is above 20°C, e.g., 25°C or higher.

[0144] A suitable volume of grinding media is warmed and maintained at a temperature of about 35°C to about 45°C, for example, by placing the container containing the grinding media on a hot plate or in a water bath. In some examples, 300 ml, 500 ml, or 1 liter of grinding media is used to warm the cadaveric bone. Preferably, the grinding media has a temperature of about 37°C to about 40°C when the cryopreserved bone fragments are transferred to the grinding media.

[0145] The cadaveric bone fragments are warmed at a rate of about 100°C / min to about 500°C / min to a surface temperature of about 20°C. In some embodiments, the warming rate is greater than about 300°C / min, for example, about 300°C / min, 310°C / min, 320°C / min, 330°C / min, 340°C / min, 350°C / min, 360°C / min, 370°C / min, 380°C / min, 390°C / min, 400°C / min, 410°C / min, 420°C / min, 430°C / min, 440°C / min, 450°C / min, 460°C / min, 470°C / min, 480°C / min, 490°C / min, and about 500°C / min. In some embodiments, the warming rate is about 400°C / min to about 500°C / min. In some cases, the cadaveric bone fragments are warmed to a surface temperature of about 20°C in less than 1 minute. In some cases, the cadaveric bone fragments are warmed to a surface temperature of about 20° C. in about 1 minute or more, e.g., about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or about 10 minutes. Data showing the dynamics of rapid warming are shown in PCT / US21 / 42064. Fig.15 middle.

[0146] When whole cadaveric bone is warmed in the grinding media, the warming rate will be slower than when bone fragments are warmed. As an example, cadaveric bone is warmed to a surface temperature of about 20°C at a rate of about 100°C / min to about 250°C / min.

[0147] Without wishing to be bound by theory, the rapid warming rate of the disclosed method prevents ice recrystallization during thawing of the bone fragments (or whole cadaveric bone).

[0148] Without wishing to be bound by theory, the methods disclosed herein for rapid warming of cadaveric bone improve the survival of extracted bone marrow cells (hematopoietic stem cells (HSC; CD34+ cells) and / or mesenchymal stromal / stem cells (MSC)) relative to methods that do not use the rapid warming method. Thus, by using the methods of the present disclosure, a greater number of viable cells (HSC and / or MSC) are obtained relative to standard methods.

[0149] In some cases, the methods of the present disclosure provide from about 1% more viable cells to about 100% more viable cells, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 112%, 113%, , 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% of the cells are viable.

[0150] In some cases, the methods of the present disclosure provide about 101% more viable cells to about 200% more viable cells, e.g., about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 200%, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2011, 2021, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2012, 2013, 2014, 2015, 2016, 2017, 2021 16%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173 In some embodiments, the present invention relates to a method for producing a cell-free living cell in an amount of at least 100% or about 200% of the cells to be viable. In some embodiments, the present invention relates to a method for producing a cell-free living cell in an amount of at least 100% or about 200% of the cells to be viable.

[0151] In some cases, the methods of the present disclosure provide about 2-fold to about 10-fold viable cells, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, about 10-fold, or any times therebetween, compared to methods not using the rapid warming methods disclosed herein. As an example, the methods of the present disclosure provide 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold viable cells compared to methods not using the rapid warming methods disclosed herein.

[0152] In some cases, the methods of the present disclosure provide about 10 times more viable cells to about 100 times more viable cells, e.g., about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, about 100 times, or any times therebetween, compared to methods not using the rapid warming methods disclosed herein. As an example, the methods of the present disclosure provide 10 to 20 times, 20 to 30 times, 30 to 40 times, 40 to 50 times, 50 to 60 times, 60 to 70 times, 70 to 80 times, 80 to 90 times, or 90 to 100 times more viable cells than methods not using the rapid warming methods disclosed herein.

[0153] In some cases, compared to methods not using the rapid warming method disclosed herein, the methods of the present disclosure provide about 100 times the viable cells to about 1000 times the viable cells, for example, about 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, about 1000 times or any times therebetween. As an example, compared to methods not using the rapid warming method disclosed herein, the methods of the present disclosure provide 100 to 200 times, 200 to 300 times, 300 to 400 times, 400 to 500 times, 500 to 600 times, 600 to 700 times, 700 to 800 times, 800 to 900 times or 900 to 1000 times the viable cells.

[0154] In some cases, the methods of the present disclosure provide about 1000 times more viable cells to about 10,000 times more viable cells, e.g., about 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, about 10,000 times, or any times therebetween, compared to methods that do not use the rapid warming methods disclosed herein. As an example, the methods of the present disclosure provide 1000-fold to 200-fold, 2000-fold to 3000-fold, 3000-fold to 4000-fold, 4000-fold to 5000-fold, 5000-fold to 6000-fold, 6000-fold to 7000-fold, 7000-fold to 8000-fold, 8000-fold to 9000-fold, or 9000-fold to 10000-fold more viable cells than a method that does not use the rapid warming method disclosed herein.

[0155] Extraction of bone marrow

[0156] Remove the bone from the bag and PLASMA-LYTE TM Remove and absorb any fluid remaining on the VB using sterile gauze or sponge. In one method, the VB is cut into smaller pieces, such as 1.5 cm, using a saw and / or anvil shears. 2Blocks that are small enough to be broken with a bone grinder. In order to simplify the process and to increase the safety of the processing personnel, a customized bone cutting tool as described in US 2019 / 0343112 (which is incorporated herein by reference in its entirety) is provided to cut the VB into smaller pieces. Another customized bone cutting tool can be used in combination with or instead of the customized bone cutting tool as described in US 2019 / 0343112. Other bone cutting tools are described in US 2020 / 0325451, which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the bone is freshly obtained from a cadaver. Alternatively, the bone has been previously frozen and / or cryopreserved.

[0158] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably a hardened steel that can withstand the forces required to cut through bone. During the cleaning process, the tool is steam sterilized, which may be harmful to the steel. Therefore, in one feature of the present disclosure, the surface of the stainless steel element is passivated to prevent oxidation of the steel element during sterilization.

[0159] The block created by the bone cutting tool is immediately placed in a sterile jar and submerged in 300-500 ml of grinding media. In one aspect of the present system and method, the grinding media is PLASMA-LYTE TM -A is used as a base component with heparin, human serum albumin (HSA) and nuclease (Merck KGAA Corporation). Heparin is used as an anticoagulant. Other anticoagulants may also be used in various amounts. HSA provides a protein source to prevent cell adhesion and adsorption to the surface as well as reactive oxygen scavenging. Note that conventional grinding media use DNA enzymes, but for the present disclosure, or Reagents to replace DNA enzyme TM Reagent (Qiagen Sciences LLC). While DNases are only effective against DNA, modern pharmaceutical biotechnology processing relies on enzymes that can cleave all forms of DNA and RNA and can reduce the viscosity of the solution in which the cells are suspended. Note that IMDM (Iscove's modified Dulbecco's medium) can be substituted for PLASMA-LYTE TM -A, because IMDM is suitable for rapid proliferation of high density cell cultures and is ideal for supporting T and B lymphocytes. It is also noted that Denarase reagent (C-Lecta GmbH) is equivalent to the same amount of Benzonase reagent in this process.

[0160] In some embodiments, the amount of heparin in the grinding medium is about 5 U / ml to about 15 U / ml. In some embodiments, the amount of heparin in the grinding medium is about 5 U / ml, about 6 U / ml, about 7 U / ml, about 8 U / ml, about 9 U / ml, about 10 U / ml, about 11 U / ml, about 12 U / ml, about 13 U / ml, about 14 U / ml, or about 15 U / ml. In some embodiments, the amount of heparin in the grinding medium is from about 5 U / ml to about 6 U / ml, from about 5 U / ml to about 7 U / ml, from about 5 U / ml to about 8 U / ml, from about 5 U / ml to about 9 U / ml, from about 5 U / ml to about 10 U / ml, from about 5 U / ml to about 11 U / ml, from about 5 U / ml to about 12 U / ml, from about 5 U / ml to about 13 U / ml, from about 5 U / ml to about 14 U / ml, from about 5 U / ml to about 15 U / ml, from about 6 U / ml to about 7 U / ml, from about 6 U / ml to about 8 U / ml, from about 6 U / ml to about 9 U / ml, from about 6 U / ml to about 10 U / ml, from about 5 U / ml to about 11 U / ml, from about 5 U / ml to about 12 U / ml, from about 5 U / ml to about 13 U / ml, from about 5 U / ml to about 14 U / ml, from about 5 U / ml to about 15 U / ml, l to about 10U / ml, about 6U / ml to about 11U / ml, about 6U / ml to about 12U / ml, about 6U / ml to about 13U / ml, about 6U / ml to about 14U / ml, about 6U / ml to about 15U / ml, about 7U / ml to about 8U / ml, about 7U / ml to about 9U / ml, about 7U / ml to about 10U / ml, about 7U / ml to about 11U / ml, about 7U / ml to about 12U / ml, about 7U / ml to about 13U / ml, about 7U / ml to about 14U / ml, about 7U / ml to about 15U / ml, about 8U / ml to about 9U / ml, / ml, about 8U / ml to about 10U / ml, about 8U / ml to about 11U / ml, about 8U / ml to about 12U / ml, about 8U / ml to about 13U / ml, about 8U / ml to about 14U / ml, about 8U / ml to about 15U / ml, about 9U / ml to about 10U / ml, about 9U / ml to about 11U / ml, about 9U / ml to about 12U / ml, about 9U / ml to about 13U / ml, about 9U / ml to about 14U / ml, about 9U / ml to about 15U / ml, about 10U / ml to about 11U / ml, about 10U / ml to about 12U / ml ml, about 10 U / ml to about 13 U / ml, about 10 U / ml to about 14 U / ml, about 10 U / ml to about 15 U / ml, about 11 U / ml to about 12 U / ml, about 11 U / ml to about 13 U / ml, about 11 U / ml to about 14 U / ml, about 11 U / ml to about 15 U / ml, about 12 U / ml to about 13 U / ml, about 12 U / ml to about 14 U / ml, about 12 U / ml to about 15 U / ml, about 13 U / ml to about 14 U / ml, about 13 U / ml to about 15 U / ml, or about 14 U / ml to about 15 U / ml.In some embodiments, the amount of heparin in the grinding medium is at least about 5 U / ml, about 6 U / ml, about 7 U / ml, about 8 U / ml, about 9 U / ml, about 10 U / ml, about 11 U / ml, about 12 U / ml, about 13 U / ml, or about 14 U / ml. In some embodiments, the amount of heparin in the grinding medium is at most about 6 U / ml, about 7 U / ml, about 8 U / ml, about 9 U / ml, about 10 U / ml, about 11 U / ml, about 12 U / ml, about 13 U / ml, about 14 U / ml, or about 15 U / ml.

[0161] In various embodiments, heparin is omitted from the grinding media.

[0162] In some embodiments, in the grinding medium In some embodiments, the amount of Benzonase in the grinding medium is about 1U / ml to about 2U / ml, about 1U / ml to about 3U / ml, about 1U / ml to about 4U / ml, about 1U / ml to about 5U / ml, about 1U / ml to about 6U / ml, about 1U / ml to about 7U / ml, about 1U / ml to about 8U / ml, about 1U / ml to about 9U / ml, about 1U / ml to about 10U / ml, about 2U / ml to about 3U / ml, about 1U / ml to about 4U / ml, about 1U / ml to about 5U / ml, about 1U / ml to about 6U / ml, about 1U / ml to about 7U / ml, about 1U / ml to about 8U / ml, about 1U / ml to about 9U / ml, about 1U / ml to about 10U / ml, about 2U / ml to about 3U / ml , about 2 U / ml to about 4 U / ml, about 2 U / ml to about 5 U / ml, about 2 U / ml to about 6 U / ml, about 2 U / ml to about 7 U / ml, about 2 U / ml to about 8 U / ml, about 2 U / ml to about 9 U / ml, about 2 U / ml to about 10 U / ml, about 3 U / ml to about 4 U / ml, about 3 U / ml to about 5 U / ml, about 3 U / ml to about 6 U / ml, about 3 U / ml to about 7 U / ml, about 3 U / ml to about 8 U / ml, ml, about 3 U / ml to about 9 U / ml, about 3 U / ml to about 10 U / ml, about 4 U / ml to about 5 U / ml, about 4 U / ml to about 6 U / ml, about 4 U / ml to about 7 U / ml, about 4 U / ml to about 8 U / ml, about 4 U / ml to about 9 U / ml, about 4 U / ml to about 10 U / ml, about 5 U / ml to about 6 U / ml, about 5 U / ml to about 7 U / ml, about 5 U / ml to about 8 U / ml, about 5 U / ml to about In some embodiments, the amount of Benzonase in the grinding medium is at least about 1 U / ml, about 2 U / ml, about 3 U / ml, about 4 U / ml, about 5 U / ml, about 6 U / ml, about 7 U / ml, about 8 U / ml, or about 9 U / ml. In some embodiments, the amount of Benzonase in the grinding medium is at least about 1 U / ml, about 2 U / ml, about 3 U / ml, about 4 U / ml, about 5 U / ml, about 6 U / ml, about 7 U / ml, about 8 U / ml, or about 9 U / ml. In some embodiments, the amount of Benzonase in the grinding medium is at most about 2 U / ml, about 3 U / ml, about 4 U / ml, about 5 U / ml, about 6 U / ml, about 7 U / ml, about 8 U / ml, about 9 U / ml, or about 10 U / ml.

[0163] In some cases, in the grinding media The amount of was about 3 U / ml and the amount of heparin in the grinding medium was about 10 U / ml.

[0164] In some embodiments, in the grinding medium or In some embodiments, the amount of Benzonase in the grinding medium is about 11 U / ml, about 15 U / ml, about 20 U / ml, about 25 U / ml, about 30 U / ml, about 35 U / ml, about 40 U / ml, about 45 U / ml, about 50 U / ml, or about 55 U / ml. In some embodiments, the amount of Benzonase in the grinding medium is at least about 11 U / ml, about 15 U / ml, about 20 U / ml, about 25 U / ml, about 30 U / ml, about 35 U / ml, about 40 U / ml, about 45 U / ml, or about 50 U / ml. In some embodiments, the amount of Benzonase in the grinding medium is about 11 U / ml to about 15 U / ml, about 11 U / ml to about 20 U / ml, about 11 U / ml to about 25 U / ml, about 11 U / ml to about 30 U / ml, about 11 U / ml to about 35 U / ml, about 11 U / ml to about 40 U / ml, about 11 U / ml to about 45 U / ml, about 11 U / ml to about 50 U / ml, about 11 U / ml to about 55 U / ml, about 15 ... about 15 U / ml to about 25 U / ml, about 15 U / ml to about 30 U / ml, about 15 U / ml to about 35 U / ml, about 15 U / ml to about 40 U / ml, about 15 U / ml to about 45 U / ml, about 15 U / ml to about 50 U / ml, about 15 U / ml to about 55 U / ml, about 20 U / ml to about 25 U / ml, about 20 U / ml to about 30 U / ml, about 20 U / ml to about 35 U / ml, about 20 U / ml to about 40 U / ml, about 20 U / ml to about 45 U / ml, about 20 U / ml to about 50 U / ml, about 20 U / ml to about 55 U / ml, about 25 U / ml to about 30 U / ml, about 25 U / ml to about 35 U / ml, about 25 U / ml to about 40 U / ml, about 25 U / ml to about 45 U / ml, about 25 U / ml to about 50 U / ml, about 25 U / ml to about 55 U / ml, about 30 U / ml to about 35 U / ml, about 30 U / ml to about 40 U / ml, about 30 U / ml to about 45 U / ml, about 30 U / ml to about 50 U / ml, about 25 U / ml to about 55 U / ml, about 30 U / ml to about 35 U / ml, about 30 U / ml to about 40 U / ml, about 30 U / ml to about 45 U / ml, about 30 U / ml l to about 50U / ml, about 30U / ml to about 55U / ml, about 35U / ml to about 40U / ml, about 35U / ml to about 45U / ml, about 35U / ml to about 50U / ml, about 35U / ml to about 55U / ml, about 40U / ml to about 45U / ml, about 40U / ml to about 50U / ml, about 40U / ml to about 55U / ml, about 45U / ml to about 50U / ml, about 45U / ml to about 50U / ml, about 45U / ml to about 55U / ml, or about 50U / ml to about 55U / ml.In some embodiments, the amount of Benzonase in the grinding medium is at most about 15 U / ml, about 20 U / ml, about 25 U / ml, about 30 U / ml, about 35 U / ml, about 40 U / ml, about 45 U / ml, about 50 U / ml, or about 55 U / ml.

[0165] Notice that Reagents (C-Lecta GmbH) were used in the same amount as in this process. Reagents are equivalent.

[0166] Significantly, it has been found that in the grinding media There is a relationship between the amount of heparin and the amount of heparin, such that as the amount of heparin decreases, progressively lower amounts of Benzonase can be used. Without wishing to be bound by theory, heparin may help prevent cell aggregation by chelating with calcium; however, more importantly, heparin chelates with magnesium. Magnesium is an important cofactor for Benzonase. Therefore, in the presence of heparin, the presence and / or relative amount of magnesium in the solution is reduced, and this reduction in the amount of magnesium reduces Benzonase activity. Therefore, in some embodiments, the amount of heparin is reduced, while in some embodiments, heparin is omitted.

[0167] In some embodiments, HSA is present in the grinding media at about 0.5% to about 5%. In some embodiments, HSA is present in the grinding media at about 0.5% to about 1%, about 0.5% to about 1.5%, about 0.5% to about 2%, about 0.5% to about 2.5%, about 0.5% to about 3%, about 0.5% to about 3.5%, about 0.5% to about 4%, about 0.5% to about 4.5%, about 0.5% to about 5%, about 1% to about 1.5%, about 1% to about 2%, about 1% to about 2.5%, about 1% to about 3%, about 1% to about 3.5%, about 1% to about 4%, about 1% to about 4.5%, about 1% to about 5%, about 1.5% to about 2%, about 1.5% to about 2.5%, about 1.5% to about 3%, about 1.5% to about 3.5%, about 1.5% to about 4%. %, about 1.5% to about 4.5%, about 1.5% to about 5%, about 2% to about 2.5%, about 2% to about 3%, about 2% to about 3.5%, about 2% to about 4%, about 2% to about 4.5%, about 2% to about 5%, about 2.5% to about 3%, about 2.5% to about 3.5%, about 2.5% to about 4%, about 2.5% to about 4.5%, about 2.5% to about 5%, about 3% to about 3.5%, about 3% to about 4%, about 3% to about 4.5%, about 3% to about 5%, about 3.5% to about 4%, about 3.5% to about 4.5%, about 3.5% to about 5%, about 4% to about 4.5%, about 4% to about 5%, or about 4.5% to about 5% is present in the grinding media. In some embodiments, HSA is present in the grinding medium at about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In some embodiments, HSA is present in the grinding medium at least about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, or about 4.5%. In some embodiments, HSA is present in the grinding medium at most about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

[0168] After grinding, another jar of 300-500 ml of grinding media is reserved to collect bone fragments, and an additional supply of about 100 ml of grinding media is reserved for rinsing the grinder during the grinding process to prevent bone fragments from adhering to the jar surface of the grinding components. In some embodiments, the additional grinding media may have different amounts of heparin, HSA, and Benzonase compared to the initial grinding media.

[0169] An electric bone grinder or a specially configured bone grinder, such as that of Biorep Technologies Inc, (Miami, FL) can be used in an ISO-7 clean room in an ISO-5 environment. If processing the VB and iliac crest from the same donor, the bone types are kept separate. The bones are kept submerged in the grinding media at all times during and after the grinding process. Once the donor bone block is completely ground, the chamber of the bone grinder is thoroughly rinsed with fresh processing media. The bone fragments are discharged from the grinder into a tank containing the grinding media.

[0170] In some cases, bone marrow and bone grindings were shaken at 150 RPM for 10 minutes.

[0171] The contents of the jar are transferred to a sterile bag. Next, the contents of the sterile bag are filtered to extract the solid components. In one embodiment, the contents of each bag are passed through a series of stainless steel sieves. In this embodiment, a 425 μm or 500 μm sieve is stacked on a 177 μm or 200 μm sieve, which is seated above a catch pan to receive the liquid filtered contents. The sterile bag containing the output from the grinder is rotated and then poured evenly onto the sieve stack or filter group. The filtration process is observed to ensure that excessive aggregation does not occur, which may indicate the presence of soft tissue or other contaminants. The bone fragments retained on the sieve surface are evenly distributed on the sieve and rinsed with 250 ml of fresh processing medium. In one embodiment, the processing medium used for rinsing is the above-mentioned grinding medium or PLASMA-LYTE containing 2.5% HSA. TM The sieved bone marrow product (which in a well-performed process may be approximately 1000 ml) is transferred to sterile packaging for subsequent processing and analysis. The contents of each bag are visually inspected to confirm that the contents do not contain any visible bone fragments or soft tissue.

[0172] In some embodiments, the flushing medium can include various amounts of HSA as described with respect to the grinding medium. In some embodiments, the flushing medium can also include heparin and / or Benzonase.

[0173] In some cases, in the flushing medium The amount of DRUG® was about 3 U / ml and the amount of heparin in the flushing medium was about 10 U / ml.

[0174] In another embodiment, the contents of each bag are passed through a bone marrow filtration unit, such as Figure 1As shown in . In this embodiment, system 150 includes a support 154, which is configured to support a sterile collection bag 152, which contains bone fragments and media from the above-mentioned grinding operation. The support includes a container suspension 155, which is configured to engage the lid 153 of the sterile bag to suspend the container. The bottom of the bag includes a discharge assembly 160, which includes a pre-filter 162 extending into the collection bag body. In a specific embodiment, the pre-filter 162 is an 850μm filter. In some embodiments, the bone marrow first passes through an 800μm pre-filter. Filter 162 is connected to an output tube 164, which is connected to an input line 171 of a first online filter 170 by a container claim 166. In this specific embodiment, the first online filter is a 200μm or 500μm filter. The output line 172 of the first online filter is connected to the input line 176 of the second online filter 175. The second online filter is a 200μm or 500μm filter. Both in-line filters are initially 500 μm for the first pass through the filter system 150. Then, the grindings are rinsed a second time, and the two in-line filters are 200 μm. This double pass filtration results in a cleaner suspension and enhances the removal of fat from the suspension. The second in-line filter 175 has an output line 177 that can be coupled to a sterile bag such as bag 152 for a second filtration pass. During the second pass through the system, the output line 177 of the second in-line filter 175 can be coupled to the container fixture 181 of the transfer packaging container 180. The transfer packaging container can be a 600-2000 ml bag to accommodate the filtered bone marrow product, which can be approximately 1000 ml in a well-performed process.

[0175] The total nucleated count (TNC) from the filtered bone marrow product can be calculated as follows: TNC (×10 3 cells / μL) = cell count (×10 3 cells / μL)×total mass of bone marrow extract (g)×1000)

[0176] Agitation of bone grindings and / or bone grinding filtrate

[0177] In some embodiments, described herein is a method for processing bone marrow or its derivatives, the method comprising mechanically agitating bone grindings and / or bone grinding filtrate during the grinding and filtering part of processing bone marrow. In some cases, bone marrow can be obtained from a deceased donor. In some cases, bone marrow can be obtained from a previously cooled sample (e.g., bone or VB). In some cases, bone marrow can be obtained from a previously cooled but unfrozen sample (e.g., bone or VB). In some cases, bone marrow can be obtained from a thawed sample (e.g., bone or VB). In some cases, bone marrow can be processed to obtain bone marrow cells. In some embodiments, bone marrow cells can be hematopoietic stem cells (HSC). In some embodiments, bone marrow cells can be mesenchymal stem cells (MSC).

[0178] Aspects disclosed in the present disclosure include a method for processing bone marrow or a derivative thereof, wherein the bone marrow or a derivative thereof is derived from a deceased donor, the method comprising: obtaining bone or bone fragments from a deceased donor, optionally processing the bone into bone fragments; mechanically grinding the bone or bone fragments in the presence of a grinding solution to produce a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 revolutions per minute ("RPM") for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises bone marrow or a derivative thereof, and wherein the bone marrow or a derivative thereof comprises at least about 1,500,000 CD34+ cells / ml of bone marrow or a derivative thereof. In some embodiments, the method further comprises contacting the solution with a flushing medium, and repeating placing the bone grindings on a shaker and then removing the solution from the shaker. In some embodiments, the method further comprises repeating the steps of placing the bone grindings on a shaker and then removing the solution from the shaker one or more times. In some embodiments, at least about 1,500,000 CD34+ cells / ml bone marrow or derivative thereof comprises at least 85% viable CD34+ cells. In some embodiments, the method further comprises at least about 1,500,000 CD34+ cells / ml bone marrow or derivative thereof comprising at least 90% viable CD34+ cells.

[0179] Mechanical agitation can include stirring bone grinding chips in a linear manner. In some embodiments, mechanical agitation can include stirring bone grinding chips in a three-dimensional manner. In some cases, mechanical agitation of bone grinding chips can include (by orbital shaking table) orbital shaking, for example, bone grinding chips are placed on a shaking table. In some cases, bone grinding chips can be mechanically stirred by a shaking table at least about 10 revs / min (RPM), 20RPM, 30RPM, 40RPM, 50RPM, 60RPM, 70RPM, 80RPM, 90RPM, 100RPM, 110RPM, 120RPM, 130RPM, 140RPM, 150RPM, 160RPM, 170RPM, 180RPM, 190RPM, 200RPM, 210RPM, 220RPM, 230RPM, 240RPM, 250RPM or a larger speed. In some cases, bone grinding chips can be mechanically stirred by centrifugal (e.g., rotation). In some embodiments, bone grinding chips can be rotated at least 10RPM, 20RPM, 30RPM, 40RPM, 50RPM, 60RPM, 70RPM, 80RPM, 90RPM, 100RPM, 110RPM, 120RPM, 130RPM, 140RPM, 150RPM, 160RPM, 170RPM, 180RPM, 190RPM, 200RPM, 210RPM, 220RPM, 230RPM, 240RPM, 250RPM or more. In some embodiments, bone grinding chips can be rotated at least 300RPM, 400RPM, 500RPM, 600RPM or more. In some embodiments, bone grinding chips can be mechanically stirred by both shaking and rotation. In some embodiments, mechanical agitation of the bone grindings can continue for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or longer.

[0180] In some embodiments, mechanical agitation of the bone grindings increases the yield of bone marrow cells obtained. In some cases, the yield of bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the yield of bone marrow cells obtained without mechanical agitation.

[0181] In some embodiments, mechanical agitation of the bone grindings increases the survival rate of the obtained bone marrow cells. In some cases, the survival rate of the bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times or more compared to the survival rate of the bone marrow cells obtained without mechanical agitation.

[0182] In some embodiments, mechanical agitation of the bone grindings increases the number of bone marrow cells expressing CD34 obtained. In some cases, the number of bone marrow cells expressing CD34 obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the number of bone marrow cells expressing CD34 obtained without mechanical agitation.

[0183] In some embodiments, mechanical agitation of bone grindings increases the number of bone marrow cells expressing CD45 obtained by the methods described herein. In some cases, the number of bone marrow cells expressing CD45 obtained by mechanical agitation of bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the number of bone marrow cells expressing CD45 obtained without mechanical agitation.

[0184] The above-mentioned agitation may occur before the aforementioned filtering step.

[0185] In certain embodiments, the amount of CD34+ cells / ml of bone marrow or its derivatives obtained is at least about 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,050,000, 1,100,000, 00,000, 1,150,000, 1,200,000, 1,250,000, 1,300,000, 1,350,000, 1,400,000, 1,450,000, 1,500,000, 1,550,000, 1,600,000, 1,650,000, 1,700,000, 1,750,000, 1,800,000, 1,850,000, 1,900,000, 1950,000, 2,000,000 or more CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or its derivatives obtained is at least about 1,500,000 CD34+ cells / ml to about 2,000,000 CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or its derivatives obtained is at least about 1,500,000 CD34+ cells / ml to about 1,750,000 CD34+ cells / ml, about 1,500,000 CD34+ cells / ml to about 2,000,000 CD34+ cells / ml, or about 1,750,000 CD34+ cells / ml to about 2,000,000 CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or its derivatives obtained is at least about 1,500,000 CD34+ cells / ml, about 1,750,000 CD34+ cells / ml, or about 2,000,000 CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or its derivatives obtained is at least about 1,500,000 CD34+ cells / ml, or about 1,750,000 CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or its derivatives obtained is at most about 1,750,000 CD34+ cells / ml, or about 2,000,000 CD34+ cells / ml.

[0186] In some embodiments, the survival rate of CD34+ cells is at least about 70% to about 95%. In some embodiments, the survival rate of CD34+ cells is at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the survival rate of CD34+ cells is at least about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. In some embodiments, the survival rate of CD34+ cells is at least about 70%, about 75%, about 80%, about 85% or about 90%. In some embodiments, the viability of CD34+ cells is at most about 75%, about 80%, about 85%, about 90%, or about 95%.

[0187] For quality control, a small amount (e.g., 0.3 mL) of bone marrow is extracted from sterile packaging 152 using a syringe at the injection site 157 and mixed by inversion before withdrawing the sample. The sample can be tested by a blood analyzer, such as a Sysmex blood analyzer, to determine the total nucleated cell (TNC) content of the sample as an indication of the TNC content of the bone marrow that is subsequently processed.

[0188] Fat removal and concentration

[0189] The bone marrow product collected from the filtration is essentially a fat emulsion. The fat content of the suspension obtained from the sieve filtration method disclosed above is greater than the fat content of the suspension obtained from the double-pass filtration system 150. However, in both cases, it is necessary to remove the fat content from the suspension. The suspension obtained from the filtration is recovered into a 250 ml bag, which is airtightly sealed with a pipette. Pairs of sterile bags and taring sticks are placed in a centrifuge with the bag opening facing down and balanced. A volume compensation plate is used to prevent wrinkling of the bag during centrifugation. In one embodiment, the bag is centrifuged at 500 x g for 15 minutes at room temperature to concentrate the cells, preferably to 2-3 x 10 8 After centrifugation, each bag was hung individually on a ring stand. The different layers within the bag were visible, with the fat layer clearly delineated at the top of the supernatant and the bone marrow pellet at the bottom, as shown in Figure 1. Figure 2 As shown in . A new sterile bag is welded to the bag removed from the centrifuge. A bag clamp or clip 190 is placed on the bag, just below the fat layer, as shown in . Figure 3As shown, the bag is clamped or squeezed to close it beneath the fat layer. The sediment is then drained from the centrifuge bag into a new sterile bag, with a bag clip used to prevent the fat layer from passing through. The sediment is agitated during drainage to resuspend all of the sediment. After approximately half of the sediment has been drained into the new bag, the tubing is sealed with a hemostat or tube sealer. The second centrifuge bag is then welded to the new bag containing the sediment, and the contents of this second centrifuge bag are drained into the new bag.

[0190] The result is that the new sterile bag contains bone marrow that has been centrifuged to remove fat. These bags of defatted bone marrow are then centrifuged at 500 x g for 15 minutes at room temperature, with a volume compensating plate used to prevent wrinkling of the bags. Each bag is removed and hung on a ring stand, a waste bag is welded to the bag, and a plasma extractor is used to remove the supernatant into the waste bag, as Figure 4 shown. When the sediment rises or breaks up, the tubing is clamped with a hemostat. The tubing is then sealed and cut to remove the bag containing the sediment from the waste bag, and the waste bag is discarded. A Luer connector is welded to the bag containing the sediment. The sediment from each bag is combined into a bulk bag by using a large syringe. The bag containing the sediment is rinsed into the bulk bag by using a flushing medium. The bulk bag is inverted several times to ensure that all of the sediment is resuspended. A small amount (e.g., 0.5 mL) of processed BM can be removed for testing density and cell count for quality control. Human leukocyte antigen, CCR5δ32 mutation, apolipoprotein (APOE), etc. of the test sample can also be evaluated.

[0191] In some embodiments, centrifuge setting can be increased in one or more steps.In some embodiments, centrifuge rotates at about 400g to about 650g.In some embodiments, centrifuge rotates at about 400g to about 450g, about 400g to about 500g, about 400g to about 550g, about 400g to about 600g, about 400g to about 650g, about 450g to about 500g, about 450g to about 550g, about 450g to about 600g, about 450g to about 650g, about 500g to about 550g, about 500g to about 600g, about 500g to about 650g, about 550g to about 600g, about 550g to about 600g, about 550g to about 650g or about 600g to about 650g. In some embodiments, the centrifuge rotates at about 400g, about 450g, about 500g, about 550g, about 600g or about 650g. In some embodiments, the centrifuge rotates at least about 400g, about 450g, about 500g, about 550g or about 600g. In some embodiments, the centrifuge rotates at most about 450g, about 500g, about 550g, about 600g or about 650g. In some embodiments, the centrifuge rotates for about 10 minutes to about 40 minutes. In some embodiments, the centrifuge spins for about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 40 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 40 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, or about 35 minutes to about 40 minutes. In some embodiments, the centrifuge spins for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, centrifuge rotates at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes or about 35 minutes. In some embodiments, centrifuge rotates at most about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes or about 40 minutes. In some cases, the bag comprising the bone marrow extracted can be concentrated by centrifuging for 30 minutes at 600x g (about 2315rpm). By using a plasma extractor, supernatant is removed from the bone marrow precipitation and enters the waste bag. By using standard biohazard protocols, waste is discarded. Then, precipitation is merged in the bulk bag weighed in advance and resuspended by using flushing medium.In some embodiments, centrifuge is stopped without using brakes. In some embodiments, centrifuge is stopped with brakes. In some embodiments, centrifuge brake is set at about 25% to about 100%. In some embodiments, centrifuge brake is set at about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%. In some embodiments, centrifuge brake is set at about 25%, about 50%, about 75% or about 100%. In some embodiments, centrifuge brake is set at at least about 25%, about 50% or about 75%. In some embodiments, centrifuge brake is set at about 50%, about 75% or about 100% at most.

[0192] In some cases, fat removal can be performed using commercial cell processing devices (e.g. 2991 Cell Processor, TerumoBCT). See the World Wide Web at terumobct.com / 2991. This commercial cell processing device can also concentrate cell products.

[0193] The cell product is aliquoted into one or more second volumes, known as isolation vials.

[0194] Cryopreservation of bone marrow

[0195] The present method provides a system for extracting and storing bone marrow according to the above processing method for future clinical use, e.g. Figure 6 This method can eliminate the failure of existing methods to match bone marrow donors to difficult-to-match groups (such as certain minority groups). Once the bone marrow is cryopreserved and stored, there is no uncertainty about the source of the bone marrow, future recipients do not need to wait, and the bone marrow can be obtained in large quantities and reproducibly.

[0196] The methods of the present disclosure also provide different containers for a given bone marrow product, with a first (larger) volume containing cells to be provided to a subject in need thereof, a second (smaller) volume serving as a surrogate for the first volume, and the cells in the second volume (i.e., the surrogate) being used in an assay to determine the suitability of the first volume for administration to a subject in need thereof.

[0197] It is contemplated that, based on ten vertebrae and / or iliac bones obtained from the donor, three or more bags of marrow can be produced per bone donor through the above process. If three bags of marrow are not obtained at the end of the process for a given donor, the donor can be marked as potentially failing overall quality control. It is contemplated that a predetermined volume of marrow is contained in each bag, for example 70 ml in a 250 ml bag. This predetermined volume is used to calculate the volume of the freezing medium component required for effective cryopreservation of the bone marrow pellet. The freezing medium is a solution of the flushing medium and the cryopreservation composition. The cryoprotectant can be a cell permeable medium, such as dimethyl sulfoxide (DMSO); 1,2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; formamide; ethane diol or butane 2,3 diol; and / or an impermeable medium, such as hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribitol, mannitol or polyvinyl pyrrolidone (PVP). Each bone donor may also provide at least three surrogate vials, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more surrogate vials. The greater the number of cryopreservation bags obtained from the donor, the greater the number of surrogate vials that can be prepared, such that each cryopreservation bag has at least one vial, preferably two, three, four or more vials / cryopreservation bags.

[0198] HAS also provides cryoprotection by colloid osmotic pressure, cell surface protein stabilization and reactive oxygen scavenging. In a preferred embodiment, the cryoprotectant is DMSO. The flushing medium can be an electrolyte medium, such as PlasmaLyte, Isolyte, IMDM or other electrolyte solutions suitable for infusion. The freezing medium can also include a deoxyrase concentration to reduce the oxygen content to below atmospheric concentration, such as less than 3% of atmospheric concentration. The addition of deoxyrase produces a low-pressure composition that can promote cryopreservation.

[0199] In some embodiments, for the methods provided herein, the bone marrow product is cryopreserved in a freezing medium, wherein the freezing medium comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.

[0200] In some embodiments, the freezing medium and / or flushing medium comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%. To about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% HSA. In some embodiments, the freezing medium and / or flushing medium contains about 1% to about 5% HSA. In some embodiments, the freezing medium and / or flushing medium contains about 2.5% HSA.

[0201] In some embodiments, the freezing medium comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%. In some embodiments, the freezing medium comprises about 1% to about 10% DMSO. In some embodiments, the freezing medium comprises about 2.5% DMSO, about 5% DMSO or about 10% DMSO.

[0202] In some embodiments, the electrolyte formulation is Plasmalyte A.

[0203] In various embodiments, the flushing medium and / or the freezing medium lacks heparin.

[0204] In some embodiments, the flushing medium is fresh.

[0205] In some cases, the freezing medium is ≤ 25°C prior to the freezing medium being added to the bone marrow bulk bag.

[0206] Freezing medium can be added to the bone marrow bulk bag at a predetermined rate (10% of the freezing medium volume / minute) based on the following formula:

[0207] Volume of freezing medium to be added / minute = total volume of freezing medium (mL) x 0.1 Preferably, the elapsed time for adding the cryoprotectant to the bone marrow bulk bag does not exceed 9-11 minutes.

[0208] According to the calculated total volume of freezing medium required for the volume of collected bone marrow, freezing medium is prepared by mixing cryoprotectants and flushing medium. The bag containing bone marrow is placed on a shaker for mixing, and the freezing medium is introduced into the bag by a syringe. The freezing medium is introduced at a specific rate during a predetermined time. In one embodiment, the freezing medium is added at a rate of 10% of the medium / minute during a period of 10 minutes. Once the medium has been mixed with the concentrated bone marrow, the test sample is extracted by a syringe. The remaining mixture of freezing medium and bone marrow is injected into a separate cryopreservation bag (and a substitute vial) in a predetermined amount. In one embodiment, 70ml of the bone marrow mixture is introduced into each cryopreservation bag and the air is extracted with a syringe. At the end of the process, 8ml of sample can be taken out for sterility testing. Each cryopreservation bag is sealed to produce four compartments, which are then separated to be stored in a box to be stored in a low-temperature freezer. In another embodiment, the separated compartments are stored in a passive cooling box, such as Figure 5 or the cooling box described in US7,604,930, which are incorporated herein by reference in their entirety. Standard freezer boxes with or without box racks can be used in these embodiments. In some embodiments, the boxes are not stored in a passive cooling box. In some embodiments, the boxes are arranged in a specific configuration within the cryogenic freezer to induce a specific freezing rate. In some embodiments, the arrangement is Fig.14 or Fig.15 As shown, the boxes preferably do not touch the inner walls of the freezer shelves. In addition, preferably, the boxes are not stacked on top of each other.

[0209] Aspects of the present disclosure provide cryopreserved cell products, which are divided into two volumes, a first volume (e.g., a cryopreservation bag) comprising a cell product for transplantation into an object in need thereof, and a second volume acting as a substitute for the first volume. As used herein, a surrogate vial is typically a cell product of a smaller volume, and the surrogate can be thawed and measured as needed, such as cell viability (especially "functional viability" determined by proliferation after thawing). The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using a surrogate, it is not necessary to thaw the first volume for determination, but to thaw when needed, for example, for transplantation into an object in need thereof.

[0210] Without wishing to be bound by theory, for a given cell type, a specific optimal cooling rate is required so that the cell type or cell product survives cryopreservation. This optimal cooling rate balances the damage caused by intracellular ice formation (IIF) with the damage caused by the high solute concentration generated by extracellular ice formation. If the cells are cooled too quickly, damaging IIF is possible; if the cells are cooled too slowly, damaging solute effects are possible. In order for the surrogate vial to accurately represent the first volume, the cells should be frozen at approximately the same rate (i.e., the optimized rate) in the two volumes; this common rate results in equivalent survival and viability of the cells in the two volumes. Importantly, the second volume can be stored in the same long-term storage system as the first volume, and therefore will be exposed to the same conditions during the long-term storage duration, thereby facilitating the ability of the surrogate vial to accurately represent the cryopreservation bag containing cells for transplantation. These ultimately allow the second volume to be tested to at least determine whether the storage of the cryopreservation bag is properly maintained and it is not necessary to manipulate and test the cells of the first volume. More specifically, by assaying the surrogate vial, the cryopreservation bag does not need to be warmed and / or treated prior to its immediate use. This feature is particularly helpful to the outcome and health of the subject in two ways. First, the cells used for transplantation are thawed only when they are ready to be administered to the subject (and preferably at the site of administration), rather than being thawed approximately two weeks prior to use so that assays can be performed to ensure that the cell product is suitable for use; this two-week delay (during which the cells are kept at room temperature, on ice, or at 37°C, or worse, refrozen and returned to cold storage) may adversely affect their survival and utility once transplanted. Second, because the subject may undergo myeloablative conditioning prior to transplantation, the patient may be able to prevent myeloablative conditioning until the cell product has been assayed and determined to be suitable for use, which typically takes about two weeks; because of the surrogate vial, the subject begins myeloablative conditioning once a suitable product has been identified, thereby shortening the length of time the subject remains immunocompromised.

[0211] The present disclosure provides a method for ensuring that the two volumes are cooled at the same rate. At least based on the laws of physics, the smaller volume of a cell product will cool at a faster rate than the larger volume of a cell product. Thus, relative to a larger volume with a slower cooling rate, a smaller volume with a faster cooling rate should have an increased IIF. The method of the present disclosure promotes the equivalent rate of cell cooling between the first (larger) volume and the second (smaller) volume, so that each volume will have a similar amount of IFF, so that a surrogate bottle (smaller volume) will accurately represent the larger first volume. Without wishing to be bound by theory, the method of the present disclosure is based in part on using different types of containers that directly or indirectly hold the first volume or the second volume and / or placing the container in the same freezer (e.g., static temperature freezer), slowing the cooling rate of the second (smaller) volume to the rate that the first (larger) volume undergoes. Thus, the cells in the smaller volume (i.e., the second volume / surrogate vial) experience a similar cooling rate (e.g., about -1°C / minute) as the cells in the larger volume (i.e., the first volume / cryopreservation bag) when placed in the same static freezer (e.g., a -86°C static freezer). Importantly, to slow the cooling rate of the smaller second volume, the surrogate vial is placed directly in an insulated vial container (e.g., In a cryopreservation system, the cells in the surrogate vials are subjected to a freezing rate of about -1°C / minute when the container is placed in a static freezer below -80°C (e.g., a -86°C static freezer); without the use of an insulated vial container, the cells in the surrogate vials may be subjected to a freezing rate of about -10°C / minute, thereby potentially causing damage due to IIF. On the other hand, the larger first volume (cryopreservation bag) does not need to be placed directly in an insulated container, but rather the cells in the cryopreservation bag are preferably subjected to a freezing rate of about -1°C / minute when the bag is placed in a non-insulated (to avoid slowing the cooling rate of the bag) box and moved to a -86°C static freezer. "Directly placed" means that each vial is in close proximity to the insulating material of the insulated container. These manipulations allow the first and second volumes of cells to have approximately equal penetration of intracellular water into the extracellular space of the cells; this penetration increases intracellular solute concentrations and helps avoid the formation of (harmful) intracellular ice crystals and promotes extracellular ice formation (which is less harmful to cells). Once the first step of freezing (in a -86°C static freezer), the cryopreservation bag and surrogate vial are placed in the same long-term storage device (e.g., a liquid nitrogen storage tank) and positioned in approximately similar locations within the long-term storage device.

[0212] Thus, the methods of the present disclosure allow for the production of surrogate samples of cell products that are expected to accurately represent the portion of the cell product to be administered to a subject in need thereof, and provide a cell product that not only therapeutically benefits the subject, but also promotes the subject's outcome and health, in a manner that is not achievable when the cryopreserved cell product is contained solely in a single bag and without a surrogate vial.

[0213] In some cases, the first volume (i.e., cryopreservation bags) and the second volume (i.e., surrogate cryovials) are placed in a -86°C static freezer. The bags are placed in a box, which may lack insulation, and the surrogate vials are placed separately. In a frozen storage system, the box front is then placed in the freezer.

[0214] When the test sample from a specific bone marrow batch has been verified for cell count and sterility, the cryopreservation bags and surrogate bottles of the cryopreserved bone marrow can be further cooled for long-term storage. In one embodiment, the bag and bottle are cooled at a controlled rate to prevent damage to the bone marrow and cells. The optimal cooling scheme for producing the optimal amount of surviving bone marrow and cells includes changing the cooling rate at different stages of the cooling process. In some embodiments, the stage of the cooling process is called "super freezing" (about 17 ° C to nucleation point) and "sub freezing" (about -10 ° C to -40 ° C). Typically, nucleation occurs at about 7 ° C to about 15 ° C.

[0215] Once the first step of freezing (eg, in the same -86°C static freezer), the cryo bags and surrogate vials are placed in the same long-term storage device (eg, liquid nitrogen storage tank) and positioned in substantially similar locations within the long-term storage device.

[0216] Aspects described in the present disclosure include a method for processing bone marrow or a derivative thereof (e.g., a bone marrow-derived cell composition), wherein the bone marrow or a derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally processing the bone into a bone fragment; extracting the bone marrow or a derivative thereof from the bone or bone fragment; and cryopreserving the bone marrow or a derivative thereof, wherein the cryopreservation comprises reducing the temperature of the bone marrow or a derivative thereof at a freezing rate greater than about -1°C / min in a static freezer. In some embodiments, cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -5°C / min until at least at least one cell of the bone marrow or a derivative thereof is nucleated. In some embodiments, cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -4°C / min until at least at least one cell of the bone marrow or a derivative thereof is nucleated. In some embodiments, cryopreservation comprises cooling the bone marrow or a derivative thereof at a superfreezing rate of about -2.5°C / min to about -3.5°C / min until at least at least one cell of the bone marrow or a derivative thereof is nucleated. In some embodiments, cryopreservation includes cooling the bone marrow or its derivatives at a subfreezing rate of about -1°C / min to about -2°C / min. In some embodiments, the superfreezing rate and the subfreezing rate are maintained without using a passive cooling box. In some embodiments, cryopreservation includes arranging one or more aliquots of the bone marrow or its derivatives in a static freezer so that the aliquots do not contact the walls of the static freezer. In some embodiments, the bone marrow or its derivatives comprise a CD34+ cell population. In some embodiments, the CD34+ cell population comprises at least 70% of viable CD34+ cells after the bone marrow or its derivatives are thawed. In some embodiments, the CD34+ cell population comprises at least 80% of viable CD34+ cells after the bone marrow or its derivatives are thawed. In some embodiments, the static freezer is set at about -70°C to -90°C. In some embodiments, the static freezer is set at -86°C. In some embodiments, the static freezer is set at less than -80°C.

[0217] In a specific embodiment, the cryopreservation bag and the surrogate vial are cooled at a rate of -1 to -40°C / minute until the bag has reached a temperature suitable for immersing the bag in liquid nitrogen. Preferably, the bag is cooled at a rate of -1°C to -5°C. Suitable temperatures range from -40 to -100°C. Once this temperature has been reached, the bag is further cooled at a faster rate to a temperature below -130°C for storage. Once the first step of freezing (e.g., in the same -86°C static freezer), the cryopreservation bag and the surrogate vial are placed in the same long-term storage device (e.g., a liquid nitrogen storage tank) and are positioned in a roughly similar position within the long-term storage device.

[0218] In some embodiments, the temperature for freezing bone marrow or bone marrow cells includes the temperature and freezing rate shown in Example 5. In some embodiments, bone marrow or bone marrow cells can be cryopreserved under ultra-freezing rate or ultra-freezing range. In some embodiments, bone marrow or bone marrow cells can be cryopreserved by freezing under ultra-freezing rate and sub-freezing rate. For example, bone marrow or bone marrow cells can be cryopreserved as follows: first freezing under ultra-freezing rate, until reaching a predetermined temperature, freezing bone marrow or bone marrow cells are switched to sub-freezing rate thereafter. In some embodiments, the nucleation temperature of bone marrow or bone marrow cells can be reached during ultra-freezing. In some embodiments, the nucleation temperature of bone marrow or bone marrow cells can be reached during sub-freezing. In some embodiments, the nucleation temperature of bone marrow or bone marrow cells can be reached during ultra-freezing and sub-freezing.

[0219] In some cases, the bone marrow or bone marrow cells can first be cryopreserved under super freezing. For example, when the bone marrow or bone marrow cells have just been processed and are at room temperature, the bone marrow or bone marrow cells can be cryopreserved. In some cases, the super freezing rate is generally higher (e.g., the temperature is reduced at a faster rate) than the subfreezing rate. In some embodiments, the super freezing rate is about -6°C / min to about -0.5°C / min. In some embodiments, the super freezing rate is about -0.5°C / min to about -1°C / min, about -0.5°C / min to about -1.5°C / min, about -0.5°C / min to about -2°C / min, about -0.5°C / min to about -2.5°C / min, about -0.5°C / min to about -3°C / min, about -0.5°C / min to about -3.5°C / min, about -0.5°C / min to about -4°C / min, about -0.5°C / min to about -4.5°C / min, about -0.5°C / min to about - ...3.5°C / min, about -0.5°C / min to about -3.5°C / min, about -0.5°C / min to about -4°C / min, about -0.5°C / min to about -5.5°C / min, about -0.5°C / min to about to about -5.5°C / min, about -0.5°C / min to about -6°C / min, about -1°C / min to about -1.5°C / min, about -1°C / min to about -2°C / min, about -1°C / min to about -2.5°C / min, about -1°C / min to about -3°C / min, about -1°C / min to about -3.5°C / min, about -1°C / min to about -4°C / min, about -1°C / min to about -4.5°C / min, about -1°C / min to about -5°C / min, about -1°C / min to about -5.5 ... about -6°C / min, about -1.5°C / min to about -2°C / min, about -1.5°C / min to about -2.5°C / min, about -1.5°C / min to about -3°C / min, about -1.5°C / min to about -3.5°C / min, about -1.5°C / min to about -4°C / min, about -1.5°C / min to about -4.5°C / min, about -1.5°C / min to about -5°C / min, about -1.5°C / min to about -5.5°C / min, about -1.5°C / min to about -6°C / min, about -2°C / min to about -2.5°C / min min, about -2°C / min to about -3°C / min, about -2°C / min to about -3.5°C / min, about -2°C / min to about -4°C / min, about -2°C / min to about -4.5°C / min, about -2°C / min to about -5°C / min, about -2°C / min to about -5.5°C / min, about -2°C / min to about -6°C / min, about -2.5°C / min to about -3°C / min, about -2.5°C / min to about -3.5°C / min, about -2.5°C / min to about -4 ...5°C / min, about -2.5°C / min to about -5°C / min, about -2.5°C / min to about -5.5°C / min, about -2.5°C / min to about -6°C / min, about -3°C / min to about -3.5°C / min, about -3°C / min to about -4°C / min, about -3°C / min to about -4.5°C / min, about -3°C / min to about -5°C / min, about -3°C / min to about -5.5°C / min, about -3°C / min to about -6°C / min, about -3.5°C / min to about -4°C / min, about -3.5°C / min to about -4.5°C / min, about -3°C / min to about -5°C / min, about -3°C / min to about -5.5°C / min, about -3°C / min to about -6°C / min, about -3.5°C / min to about -4°C / min, about -3.5°C / min to about -4.5°C / min, about -3.5°C / min to about -5°C / min in, about -3.5°C / min to about -5.5°C / min, about -3.5°C / min to about -6°C / min, about -4°C / min to about -4.5°C / min, about -4°C / min to about -5°C / min, about -4°C / min to about -5.5°C / min, about -4°C / min to about -6°C / min, about -4.5°C / min to about -5°C / min, about -4.5°C / min to about -5.5°C / min, about -4.5°C / min to about -6°C / min, about -5°C / min to about -5.5°C / min, about -5°C / min to about -6°C / min, or about -5.5°C / min to about -6°C / min. In some embodiments, the super freezing rate is about -0.5 ° C / min, about -1 ° C / min, about -1.5 ° C / min, about -2 ° C / min, about -2.5 ° C / min, about -3 ° C / min, about -3.5 ° C / min, about -4 ° C / min, about -4.5 ° C / min, about -5 ° C / min, about -5.5 ° C / min or about -6 ° C / min. In some embodiments, the super freezing rate is at least about -0.5 ° C / min, about -1 ° C / min, about -1.5 ° C / min, about -2 ° C / min, about -2.5 ° C / min, about -3 ° C / min, about -3.5 ° C / min, about -4 ° C / min, about -4.5 ° C / min, about -5 ° C / min or about -5.5 ° C / min. In some embodiments, the super freezing rate is at most about -1°C / min, about -1.5°C / min, about -2°C / min, about -2.5°C / min, about -3°C / min, about -3.5°C / min, about -4°C / min, about -4.5°C / min, about -5°C / min, about -5.5°C / min, or about -6°C / min. In some embodiments, the super freezing rate is -3.2°C. In some embodiments, the super freezing rate is from about -2.54°C / min to about -4.09°C / min. .

[0220] In some embodiments, the bone marrow or bone marrow cells can be cryopreserved at a subfreezing rate or subfreezing range. In some embodiments, the subfreezing rate is about -2.5°C / min to about -0.1°C / min. In some embodiments, the subfreezing rate is about -0.1°C / min to about -0.2°C / min, about -0.1°C / min to about -0.4°C / min, about -0.1°C / min to about -0.6°C / min, about -0.1°C / min to about -0.8°C / min, about -0.1°C / min to about -1°C / min, about -0.1°C / min to about -1.2°C / min, about -0.1°C / min to about -1.4°C / min, about -0.1°C / min to about -1.6°C / min, about -0.1°C / min to about -1.8°C / min, about -0.1°C / min to about -2°C / min, From about -0.1°C / min to about -2.5°C / min, from about -0.2°C / min to about -0.4°C / min, from about -0.2°C / min to about -0.6°C / min, from about -0.2°C / min to about -0.8°C / min, from about -0.2°C / min to about -1°C / min, from about -0.2°C / min to about -1.2°C / min, from about -0.2°C / min to about -1.4°C / min, from about -0.2°C / min to about -1.6°C / min, from about -0.2°C / min to about -1.8°C / min, from about -0.2°C / min to about -2. 5°C / min, about -0.4°C / min to about -0.6°C / min, about -0.4°C / min to about -0.8°C / min, about -0.4°C / min to about -1°C / min, about -0.4°C / min to about -1.2°C / min, about -0.4°C / min to about -1.4°C / min, about -0.4°C / min to about -1.6°C / min, about -0.4°C / min to about -1.8°C / min, about -0.4°C / min to about -2°C / min, about -0.4°C / min to about -2.5°C / min, about -0.6°C / min to about -0.8°C / min, about -0.6°C / min to about -1.2°C / min, about -0.4°C / min to about -1.4°C / min, about -0.4°C / min to about -1.6°C / min, about -0.4°C / min to about -1.8°C / min, about -0.4°C / min to about -2°C / min, about -0.4°C / min to about -2.5°C / min, about -0.6°C / min to about -0.8°C / min, about -0.6°C / min to about in to about -1°C / min, about -0.6°C / min to about -1.2°C / min, about -0.6°C / min to about -1.4°C / min, about -0.6°C / min to about -1.6°C / min, about -0.6°C / min to about -1.8°C / min, about -0.6°C / min to about -2°C / min, about -0.6°C / min to about -2.5°C / min, about -0.8°C / min to about -1°C / min, about -0.8°C / min to about -1.2°C / min, about -0.8°C / min to about -1.4°C / min, about -0.8°C / min to about -1.6°C / min, about -0.8°C / min to about -1.8°C / min, about -0.8°C / min to about -2°C / min, about -0.8°C / min to about -2.5°C / min, about -1°C / min to about -1.2°C / min, about -1°C / min to about -1.4°C / min, about -1°C / min to about -1.6°C / min, about -1°C / min to about -1.8°C / min, about -1°C / min to about -2°C / min, about -1°C / min to about -2.5°C / min, about -1.2°C / min to about -1.4°C / min, about -1.2°C / min to about -1.6°C / min, about -1.2°C / min to about -1.8°C / min, about -1.2°C / min to about -1.8°C / min, about -1.2°C / min to about -1.6°C / min / min to about -2°C / min, about -1.2°C / min to about -2.5°C / min, about -1.4°C / min to about -1.6°C / min, about -1.4°C / min to about -1.8°C / min, about -1.4°C / min to about -2°C / min, about -1.4°C / min to about -2.5°C / min, about -1.6°C / min to about -1.8°C / min, about -1.6°C / min to about -2°C / min, about -1.6°C / min to about -2.5°C / min, about -1.8°C / min to about -2°C / min, about -1.8°C / min to about -2.5°C / min, or about -2°C / min to about -2.5°C / min. In some embodiments, the subfreezing rate is about -0.1°C / min, about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, about -2°C / min, or about -2.5°C / min. In some embodiments, the subfreezing rate is at least about -0.1°C / min, about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, or about -2°C / min. In some embodiments, the subfreezing rate is at most about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, about -2°C / min, or about -2.5°C / min. In some embodiments, the subfreezing rate may be -1.36°C / min. In some embodiments, the subfreezing rate includes a range of -1.13°C / min to -1.62°C / min. .

[0221] In some embodiments, the freezing rate for cryopreservation of bone marrow or bone marrow cells described herein includes determining a nucleation temperature. In some embodiments, the nucleation temperature is about -24°C to about -2°C. In some embodiments, the nucleation temperature is about -2°C to about -4°C, about -2°C to about -6°C, about -2°C to about -8°C, about -2°C to about -10°C, about -2°C to about -12°C, about -2°C to about -14°C, about -2°C to about -16°C, about -2°C to about -18°C, about -2°C to about -20°C, about -2°C to about -22°C, about -2°C to about -24°C, about -4°C to about -6°C, about -4°C to about -8°C, about -4°C to about -10°C, about -4°C to about -12°C, about -4°C to about -14°C, about -4°C to about -16°C, about -4°C to about -18°C, about -4°C to about -20°C, about -4°C to about -22°C, about -4°C to about -24°C, about -6°C to about -8°C, about -6°C to about -10°C, about -6°C to about -12°C, about -6°C to about -14°C, about -6°C to about -16°C, about -6°C to about -18°C, about -6°C to about -20°C, about -6°C to about -22°C, about -6°C to about -24°C, about -8°C to about -10°C, about -8°C to about -12°C, about -8°C to about -14°C, about -8°C to about -16°C, about -8°C to about -18°C, about -8°C to about -20°C, about -8°C to about -22°C, about -8°C to about -24°C, about -10°C to about -12°C, about -10°C to about -14°C, about -10°C to about -16°C, about -10°C to about -18°C, about -10°C to about -20°C, about -10°C to about -22°C, about -10°C to about -24°C, about -12°C to about -14°C, about -12°C to about -16°C, about -12°C to about -18°C, about -12°C to about -20°C, about -12°C to about -22°C, about -1 ℃ to about -24 ℃, about -14 ℃ to about -16 ℃, about -14 ℃ to about -18 ℃, about -14 ℃ to about -20 ℃, about -14 ℃ to about -22 ℃, about -14 ℃ to about -24 ℃, about -16 ℃ to about -18 ℃, about -16 ℃ to about -20 ℃, about -16 ℃ to about -22 ℃, about -16 ℃ to about -24 ℃, about -18 ℃ to about -20 ℃, about -18 ℃ to about -22 ℃, about -18 ℃ to about -24 ℃, about -20 ℃ to about -22 ℃, about -20 ℃ to about -24 ℃, or about -22 ℃ to about -24 ℃. In some embodiments, the nucleation temperature is about -2°C, about -4°C, about -6°C, about -8°C, about -10°C, about -12°C, about -14°C, about -16°C, about -18°C, about -20°C, about -22°C, or about -24°C. In some embodiments, the nucleation temperature is at least about -2°C, about -4°C, about -6°C, about -8°C, about -10°C, about -12°C, about -14°C, about -16°C, about -18°C, about -20°C, or about -22°C.In some embodiments, the nucleation temperature is at most about -4°C, about -6°C, about -8°C, about -10°C, about -12°C, about -14°C, about -16°C, about -18°C, about -20°C, about -22°C, or about -24°C. In some embodiments, the nucleation temperature may be about -12.31°C / min. In some embodiments, the nucleation temperature may include a range of about -7.24°C to about -17.52°C.

[0222] In some embodiments, the bone marrow or bone marrow cells to be cryopreserved can be placed in a container or bag, such as a cryopreservation bag. In some cases, the cryopreservation bag can be subsequently placed in a cooling box that lacks insulation for freezing. Alternatively, the cryopreservation bag is not placed in a cooling box. In some cases, the cryopreservation bag can be placed in a box and subsequently placed in a freezing environment (e.g., placed in a freezer such as a -86°C static freezer). In some cases, the cryopreservation bag can be placed in a freezing environment of liquid nitrogen or vapor derived from liquid nitrogen. In some cases, the cryopreservation bag can be placed in different compartments or different levels of a shelf in a freezer or in liquid nitrogen or liquid nitrogen vapor. In some embodiments, the cryopreservation bag containing the bone marrow or bone marrow cells can be placed in a container such as a freezer. Fig.14 or Fig.15 The location shown in .

[0223] The cryopreservation bag is placed in the corresponding compartment 201-203 of the cooling box 200, and the overlapping cover 205 is closed on the compartment to provide a sealed environment for cryopreservation of the contents of the bag. The cooling box is placed in a low temperature freezer so that the cooling box produces a cooling rate of -0.5 to -5C ° / min, typically -1C ° / min, with a nucleation temperature above -20 ° C. The freezing process continues at a specified rate until the temperature of the bone marrow reaches a suitable temperature. The temperature suitable for storing the bag is a temperature <-80 ° C or <-150 ° C.

[0224] In another embodiment, the bag is cooled at a static chamber temperature, as opposed to the controlled rate cryopreservation described above. In the passive cooling method, the cooling box is placed in a -86°C freezer until the bag reaches a stable temperature. In some cases, the freezer can be set at a temperature range of about -100°C to about -60°C. In some cases, the freezer can be set at a temperature range of about -60°C to about -65°C, about -60°C to about -70°C, about -60°C to about -75°C, about -60°C to about -80°C, about -60°C to about -82°C, about -60°C to about -84°C, about -60°C to about -86°C, about -60°C to about -88°C, about -60°C to about -90°C, about -60°C to about -95°C, about -60°C to about -100°C, about -65°C to about -70°C, about -65°C to about -75°C, about -65°C to about -80°C, about -65 ... ℃ to about -84 ℃, about -65 ℃ to about -86 ℃, about -65 ℃ to about -88 ℃, about -65 ℃ to about -90 ℃, about -65 ℃ to about -95 ℃, about -65 ℃ to about -100 ℃, about -70 ℃ to about -75 ℃, about -70 ℃ to about -80 ℃, about -70 ℃ to about -82 ℃, about -70 ℃ to about -84 ℃, about -70 ℃ to about -86 ℃, about -70 ℃ to about -88 ℃, about -70 ℃ to about -90 ℃, about -70 ℃ to about -95 ℃, about -70 ℃ to about -100 ℃, about -75 ℃ to about -80 ℃, about -75 ℃ to about -82 ℃, about -70 ℃ to about -84 ℃, about -70 ℃ to about -86 ℃, about -70 ℃ to about -88 ℃, about -70 ℃ to about -90 ℃, about -70 ℃ to about -95 ℃, about -70 ℃ to about -100 ℃, about -75 ℃ to about -80 ℃, about -75 ℃ to about -82 ℃, about -75 ℃ to about -84 ℃, about -75 ℃ to about -86 ℃, about -75 ℃ to about -88 ℃, about -75 ℃ to about -90 ℃, about -75 ℃ to about -95 ℃, about -75 ℃ to about -100 ℃, about -80 ℃ to about -82 ℃, about -80 ℃ to about -84 ℃, about -80 ℃ to about -86 ℃, about -80 ℃ to about -88 ℃, about -80 ℃ to about -90 ℃, about -80 ℃ to about -95 ℃, about -80 ℃ to about -100 ℃, about -82 ℃ to about -84 ℃, about -82 ℃ to about -86 ℃, about -82 ℃ to about -88 ℃, about -82 ℃ to about -90 ℃, about -80 ℃ to about -95 ℃, about -80 ℃ to about -100 ℃, about -82 ℃ to about -84 ℃, about -82 ℃ to about -86 ℃, about -82 ℃ to about -88 ℃, about -82 ℃ to about -90 ℃, about -82 ℃ To about -95°C, about -82°C to about -100°C, about -84°C to about -86°C, about -84°C to about -88°C, about -84°C to about -90°C, about -84°C to about -95°C, about -84°C to about -100°C, about -86°C to about -88°C, about -86°C to about -90°C, about -86°C to about -95°C, about -86°C to about -100°C, about -88°C to about -90°C, about -88°C to about -95°C, about -88°C to about -100°C, about -90°C to about -95°C, about -88°C to about -100°C, about -90°C to about -95°C, about -90°C to about -100°C, or about -95°C to about -100°C.In some cases, the freezer can be set at a temperature range of about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C, or about -100°C. In some cases, the freezer can be set at a temperature range of at least about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, or about -95°C. In some cases, the freezer can be set at a temperature range of up to about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C, or about -100°C.

[0225] In some cases, cryopreservation bags and surrogate vials were frozen less effectively when placed in a static freezer set at -80° C. Instead, better results were obtained when the static freezer was set to a temperature below -80° C. (e.g., -86° C.).

[0226] It is contemplated that cryopreservation storage can be in many forms. For example, cryopreserved bone marrow can be contained in a bag of 1 ml to 5 ml volume or a vial of 0.1 to 15 ml volume. In a preferred embodiment, a bag with 70 ml of bone marrow is stored in a cooling box in a cryogenic freezer.

[0227] Cryopreserved bone marrow is cryogenically stored for later thawing and extraction of desired cells. Thawed bone marrow can be provided for a wide range of treatments, including treatment of leukemia, brain tumors, breast cancer, Hodgkin's disease, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, blood cancer, ovarian cancer, sarcoma, testicular cancer, other solid organ cancers, rheumatoid arthritis, multiple sclerosis, diabetes, cystic fibrosis (cystic fibrosus), Alzheimer's disease, inherited immunodeficiency, metabolic disorders, bone marrow failure syndrome and HIV. Bone marrow can also be used to induce immune tolerance, to alleviate the potential rejection of implants obtained from organ donors. It has also been shown that bone marrow therapy can be used for casualties caused by radiation and certain biological weapons.

[0228] Another aspect of the present disclosure includes a method for processing a biological sample comprising cells or derivatives thereof, the method comprising: generating a first volume of the biological sample comprising cells or derivatives thereof, wherein the first volume comprises a first concentration of cells or derivatives thereof; generating a second volume of the biological sample comprising cells or derivatives thereof, wherein the second volume is less than the first volume and comprises a second concentration of cells, wherein the second concentration of cells differs from the first concentration of cells by no more than 30%; and cooling the first volume at a first cooling rate and cooling the second volume at a second cooling rate, wherein the first cooling rate is about the same as the second cooling rate; wherein a post-thaw cell proliferation rate of the cells in the first volume differs from a post-thaw cell proliferation rate of the cells in the second volume by no more than 30%. In some embodiments, the first volume is contained in a first container, wherein the second volume is contained in a second container, and wherein the first container and the second container are exposed to a common temperature.

[0229] The vast majority of literature indicates that cells stored below the glass transition temperature of water (-130°C) are indefinitely stable, with an estimated 200-30,000 years based on biophysical properties (see, e.g., Woods et al., "Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use". Cytotherapy 18(6): 697-711. (2016), the contents of which are incorporated by reference in their entirety). The main potential source of damage is thermal cycling due to improper storage. The method of the present disclosure allows detection of improper storage and associated damage to biological samples to be administered to subjects in need. However, preferably, the storage unit is monitored for alarms 24 hours a day and manually checked weekly to ensure that the temperature remains constant.

[0230] A biological sample having at least two volumes allows a subset of the biological sample (the second volume) to be tested without having to manipulate the portion of the biological sample to be administered to the subject (the first sample), with the second volume acting as a surrogate for the first volume. As used herein, a surrogate vial is typically a smaller volume of a cell product, and the surrogate can be thawed and assayed, for example, for cell viability, as needed. The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using a surrogate, the first volume does not need to be thawed for assay, but is thawed when needed, for example, for transplantation into a subject in need. In order for the surrogate vial to accurately represent the first volume, the cells in the two volumes should be frozen at the same rate; this common rate results in equivalent functional viability of the cells in the two volumes. In some cases, the first volume (i.e., a cryopreservation bag) and the second volume (i.e., a surrogate frozen vial) are placed in a -86°C static freezer. The bag is placed in a box, which may lack insulation, and the surrogate vials are placed separately. In a frozen storage system, the box front is then placed in the freezer.

[0231] In some embodiments, the second volume is less than about 0.5% of the first volume to about 50% of the first volume. In some embodiments, the second volume is less than about 50% of the first volume to about 40% of the first volume, about 50% of the first volume to about 30% of the first volume, about 50% of the first volume to about 20% of the first volume, about 50% of the first volume to about 10% of the first volume, about 50% of the first volume to about 5% of the first volume, about 50% of the first volume to about 1% of the first volume, about 50% of the first volume to about 0.5% of the first volume, about 40% of the first volume to about 30% of the first volume, about 40% of the first volume to about 20% of the first volume, about 40% of the first volume to about 10% of the first volume, about 40% of the first volume to about 5% of the first volume, about 40% of the first volume to about 1% of the first volume, about 40% of the first volume to about 0.5% of the first volume, about 30% of the first volume to about 10% of the first volume, about 40% of the first volume to about 5% of the first volume, about 40% of the first volume to about 1% of the first volume, about 40% of the first volume to about 0.5% of the first volume, about 30% of the first volume to about 10% of the first volume

[0045] From about 20% of the first volume, from about 30% of the first volume to about 10% of the first volume, from about 30% of the first volume to about 5% of the first volume, from about 30% of the first volume to about 1% of the first volume, from about 30% of the first volume to about 0.5% of the first volume, from about 20% of the first volume to about 10% of the first volume, from about 20% of the first volume to about 5% of the first volume, from about 20% of the first volume to about 1% of the first volume, from about 20% of the first volume to about 0.5% of the first volume, from about 10% of the first volume to about 5% of the first volume, from about 10% of the first volume to about 1% of the first volume, from about 10% of the first volume to about 0.5% of the first volume, from about 5% of the first volume to about 1% of the first volume, from about 5% of the first volume to about 0.5% of the first volume, or from about 1% of the first volume to about 0.5% of the first volume. In some embodiments, the second volume is less than about 50% of the first volume, about 40% of the first volume, about 30% of the first volume, about 20% of the first volume, about 10% of the first volume, about 5% of the first volume, about 1% of the first volume, or about 0.5% of the first volume. In some embodiments, the second volume is less than at least about 50% of the first volume, about 40% of the first volume, about 30% of the first volume, about 20% of the first volume, about 10% of the first volume, about 5% of the first volume, or about 1% of the first volume. In some embodiments, the second volume is less than at most about 40% of the first volume, about 30% of the first volume, about 20% of the first volume, about 10% of the first volume, about 5% of the first volume, about 1% of the first volume, or about 0.5% of the first volume. In some embodiments, the second volume is less than 50% of the first volume. In some embodiments, the second volume is less than 40% of the first volume. In some embodiments, the second volume is less than 37.5% of the first volume. In some embodiments, the second volume is less than 35% of the first volume. In some embodiments, the second volume is less than 30% of the first volume.In some embodiments, the second volume is less than 20% of the first volume. In some embodiments, the second volume is less than 15% of the first volume. In some embodiments, the second volume is less than 10% of the first volume. In some embodiments, the second volume is less than 5% of the first volume. In some embodiments, the second volume is less than 1% of the first volume.

[0232] In some embodiments, the post-thaw viability (e.g., functional viability) of the cells in the first volume differs by no more than about 0.5% from the post-thaw viability of the cells in the second volume to about 30% from the post-thaw viability of the cells in the second volume. In some embodiments, the post-thaw viability of the cells in the first volume differs by no more than about 30% from the post-thaw viability of the cells in the second volume to about 25% from the post-thaw viability of the cells in the second volume, by about 30% from the post-thaw viability of the cells in the second volume to about 20% from the post-thaw viability of the cells in the second volume, by about 30% from the post-thaw viability of the cells in the second volume to about 15% from the post-thaw viability of the cells in the second volume, by about 30% from the post-thaw viability of the cells in the second volume to about 10% from the post-thaw viability of the cells in the second volume, by about 15% from the post-thaw viability of the cells in the second volume to about 10% from the post-thaw viability of the cells in the second volume, by ... The post-thaw viability of the cells in the second volume differs from about 30% to about 5% from the post-thaw viability of the cells in the second volume, from about 30% to about 1% from the post-thaw viability of the cells in the second volume, from about 30% to about 0.5% from the post-thaw viability of the cells in the second volume, from about 25% to about 20% from the post-thaw viability of the cells in the second volume, from about 25% to about 10% from the post-thaw viability of the cells in the second volume, The post-thaw viability of the cells in the second volume is about 15% different from the post-thaw viability of the cells in the second volume, about 25% different from the post-thaw viability of the cells in the second volume to about 10% different from the post-thaw viability of the cells in the second volume, about 25% different from the post-thaw viability of the cells in the second volume to about 5% different from the post-thaw viability of the cells in the second volume, about 25% different from the post-thaw viability of the cells in the second volume to about 1% different from the post-thaw viability of the cells in the second volume, about 25% different from the post-thaw viability of the cells in the second volume to about 0.5% different from the post-thaw viability of the cells in the second volume, and about 25% different from the post-thaw viability of the cells in the second volume to about 10% different from the post-thaw viability of the cells in the second volume. The post-thaw viability of the cells in the second volume may differ from about 20% to about 15% from the post-thaw viability of the cells in the second volume, from about 20% to about 10% from the post-thaw viability of the cells in the second volume, from about 20% to about 5% from the post-thaw viability of the cells in the second volume, from about 20% to about 1% from the post-thaw viability of the cells in the second volume, from about 20% to about 0.5%, about 15% different from the post-thaw viability of the cells in the second volume to about 10% different from the post-thaw viability of the cells in the second volume, about 15% different from the post-thaw viability of the cells in the second volume to about 5% different from the post-thaw viability of the cells in the second volume, about 15% different from the post-thaw viability of the cells in the second volume to about 1% different from the post-thaw viability of the cells in the second volume, about 15% different from the post-thaw viability of the cells in the second volume to about 0.5% different from the post-thaw viability of the cells in the second volume, about 10% different from the post-thaw viability of the cells in the second volume to about 5% different from the post-thaw viability of the cells in the second volume , a post-thaw viability of the cells in the second volume that differs by about 10% to about 1% from the post-thaw viability of the cells in the second volume, a post-thaw viability of the cells in the second volume that differs by about 10% to about 0.5% from the post-thaw viability of the cells in the second volume, a post-thaw viability of the cells in the second volume that differs by about 5% to about 1% from the post-thaw viability of the cells in the second volume, a post-thaw viability of the cells in the second volume that differs by about 5% to about 0.5% from the post-thaw viability of the cells in the second volume, or a post-thaw viability of the cells in the second volume that differs by about 1% to about 0.5% from the post-thaw viability of the cells in the second volume. In some embodiments, the post-thaw viability of the cells in the first volume differs by no more than about 30% from the post-thaw viability of the cells in the second volume, differs by about 25% from the post-thaw viability of the cells in the second volume, differs by about 20% from the post-thaw viability of the cells in the second volume, differs by about 15% from the post-thaw viability of the cells in the second volume, differs by about 10% from the post-thaw viability of the cells in the second volume, differs by about 5% from the post-thaw viability of the cells in the second volume, differs by about 1% from the post-thaw viability of the cells in the second volume, or differs by about 0.In some embodiments, the post-thaw viability of the cells in the first volume does not differ by more than at least about 30% from the post-thaw viability of the cells in the second volume, by about 25% from the post-thaw viability of the cells in the second volume, by about 20% from the post-thaw viability of the cells in the second volume, by about 15% from the post-thaw viability of the cells in the second volume, by about 10% from the post-thaw viability of the cells in the second volume, by about 5% from the post-thaw viability of the cells in the second volume, or by about 1% from the post-thaw viability of the cells in the second volume. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than about 25%, about 20%, about 15%, about 10%, about 5%, about 10%, or about 0.5%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 30%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 25%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 20%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 15%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 13.6%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 10%. In some embodiments, the post-thaw survival rate of the cells in the first volume differs from the post-thaw survival rate of the cells in the second volume by no more than 5%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 25%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 20%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 15%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 13.6%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 10%. In some embodiments, the post-thaw cell proliferation rate of the cells in the first volume differs from the post-thaw cell proliferation rate of the cells in the second volume by no more than 5%. In some embodiments, the post-thaw viability of the cells is at least 50%. The viability can relate to one or both of functional viability (which measures the ability of the cells to proliferate) and conventional viability (which relates to the number or percentage of viable cells, e.g., as measured by Trypan Blue). .

[0233] In some embodiments, the post-thaw proliferation rate of the cells (which represents the functional survival rate of the cells) is at least 1 CFU-GM / 10 5 In some embodiments, the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 In some embodiments, the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 10 5 cells to about 10 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 20 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 30 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 40 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 50 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 1 CFU-GM / 10 5cells to about 200 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 20 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 30 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 40 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 50 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 30 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 40 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 50 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5cells to about 90 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 40 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 50 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 50 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5cells to about 200 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells to about 60 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 5 cells, about 50 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells to about 70 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells to about 80 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5 cells to about 90 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5cells to about 100 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, about 90 CFU-GM / 10 5 cells to about 100 CFU-GM / 10 5 cells, about 90 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 cells, or about 100 CFU-GM / 10 5 cells to about 200 CFU-GM / 10 5 In some embodiments, the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5 cells, about 90 CFU-GM / 10 5 cells, about 100 CFU-GM / 10 5 cells or about 200 CFU-GM / 10 5 In some embodiments, the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 10 5 cells, about 10CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5 cells, about 90 CFU-GM / 10 5 cells or about 100 CFU-GM / 10 5In some embodiments, the post-thaw proliferation rate of the cells is at most about 10 CFU-GM / 10 5 cells, about 20 CFU-GM / 10 5 cells, about 30 CFU-GM / 10 5 cells, about 40 CFU-GM / 10 5 cells, about 50 CFU-GM / 10 5 cells, about 60 CFU-GM / 10 5 cells, about 70 CFU-GM / 10 5 cells, about 80 CFU-GM / 10 5 cells, about 90 CFU-GM / 10 5 cells, about 100 CFU-GM / 10 5 cells or about 200 CFU-GM / 10 5 Cells. Assays to determine functional viability may require about 10 days to about 2 weeks of culture. Methods of culturing cell products related to the present disclosure are well known in the art.

[0234] In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -0.1 ° C / min to about -5 ° C / min, until at least ice has nucleated in the freezing medium. In some cases, the biological sample or its derivative can be cryopreserved at super freezing. For example, when the biological sample or its derivative has just been processed and at room temperature, the biological sample or its derivative can be cryopreserved. In some cases, compared with the sub-freezing rate, the super freezing rate is generally higher (for example, with a faster cooling rate). In some embodiments, the super freezing rate is about -6 ° C / min to about -0.5 ° C / min. In some embodiments, the super freezing rate is about -0.5°C / min to about -1°C / min, about -0.5°C / min to about -1.5°C / min, about -0.5°C / min to about -2°C / min, about -0.5°C / min to about -2.5°C / min, about -0.5°C / min to about -3°C / min, about -0.5°C / min to about -3.5°C / min, about -0.5°C / min to about -4°C / min, about -0.5°C / min to about -4.5 ... about -5°C / min, about -0.5°C / min to about -5.5°C / min, about -0.5°C / min to about -6°C / min, about -1°C / min to about -1.5°C / min, about -1°C / min to about -2°C / min, about -1°C / min to about -2.5°C / min, about -1°C / min to about -3°C / min, about -1°C / min to about -3.5°C / min, about -1°C / min to about -4°C / min, about -1°C / min to about -4.5°C / min, about -1°C / min to about -5°C / min, about -1°C / min to about -5.5°C / min, about -1°C / min to about -6°C / min, about -1.5°C / min to about -2°C / min, about -1.5°C / min to about -2.5°C / min, about -1.5°C / min to about -3°C / min, about -1.5°C / min to about -3.5°C / min, about -1.5°C / min to about -4°C / min, about -1.5°C / min to about -4.5°C / min, about -1.5°C / min to about -5°C / min, about -1 .5℃ / min to about -5.5℃ / min, about -1.5℃ / min to about -6℃ / min, about -2℃ / min to about -2.5℃ / min, about -2℃ / min to about -3℃ / min, about -2℃ / min to about -3.5℃ / min, about -2℃ / min to about -4℃ / min, about -2℃ / min to about -4.5℃ / min, about -2℃ / min to about -5℃ / min, about -2℃ / min to about -5.5℃ / min, about -2℃ / min to about -6℃ / min, about -2.5°C / min to about -3°C / min, about -2.5°C / min to about -3.5°C / min, about -2.5°C / min to about -4°C / min, about -2.5°C / min to about -4.5°C / min, about -2.5°C / min to about -5°C / min, about -2.5°C / min to about -5.5°C / min, about -2.5°C / min to about -6°C / min, about -3°C / min to about -3.5°C / min, about -3°C / min to about -4°C / min, about -3°C / min to about -4.5°C / min, about -3°C / min to about -5°C / min, about -3°C / min to about -5.5°C / min, about -3°C / min to about -6°C / min, about -3.5°C / min to about -4°C / min, about -3°C / min to about -4.5°C / min, about -3°C / min to about -5°C / min, about -3°C / min to about -5.5°C / min, about -3°C / min to about -6°C / min, about -3.5°C / min to about -4°C / min, about -3 .5°C / min to about -4.5°C / min, about -3.5°C / min to about -5°C / min, about -3.5°C / min to about -5.5°C / min, about -3.5°C / min to about -6°C / min, about -4°C / min to about -4.5°C / min, about -4°C / min to about -5°C / min, about -4°C / min to about -5.5°C / min, about -4°C / min to about -6°C / min, about -4.5°C / min to about -5°C / min, about -4.5°C / min to about -5.5°C / min, about -4.5°C / min to about -6°C / min, about -5°C / min to about -5.5°C / min, about -5°C / min to about -6°C / min, or about -5.5°C / min to about -6°C / min. In some embodiments, the super freezing rate is about -0.5 ° C / min, about -1 ° C / min, about -1.5 ° C / min, about -2 ° C / min, about -2.5 ° C / min, about -3 ° C / min, about -3.5 ° C / min, about -4 ° C / min, about -4.5 ° C / min, about -5 ° C / min, about -5.5 ° C / min or about -6 ° C / min. In some embodiments, the super freezing rate is at least about -0.5 ° C / min, about -1 ° C / min, about -1.5 ° C / min, about -2 ° C / min, about -2.5 ° C / min, about -3 ° C / min, about -3.5 ° C / min, about -4 ° C / min, about -4.5 ° C / min, about -5 ° C / min or about -5.5 ° C / min. In some embodiments, the super freezing rate is at most about -1°C / min, about -1.5°C / min, about -2°C / min, about -2.5°C / min, about -3°C / min, about -3.5°C / min, about -4°C / min, about -4.5°C / min, about -5°C / min, about -5.5°C / min, or about -6°C / min. In some embodiments, the super freezing rate is -3.2°C. In some embodiments, the super freezing rate is from about -2.54°C / min to about -4.09°C / min. In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -2.5°C / min to about -4°C / min, until at least ice has nucleated in the freezing medium. In some embodiments, the first cooling rate and the second cooling rate include a super freezing rate of about -2.5°C / min to about -3.5°C / min, until at least ice has nucleated in the freezing medium. Preferably, the first cooling rate and the second cooling rate are about -1°C to about -5°C. .

[0235] In some embodiments, the first cooling rate and the second cooling rate differ by about 1% to about 500%. The first cooling rate and the second cooling rate can differ by about 1% to about 100%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The first cooling rate and the second cooling rate may differ by about 100% to about 200%, for example, 100%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 19 21%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147% , 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 4%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199% or 200%.The first cooling rate and the second cooling rate may differ by about 200% to about 300%, for example, 200%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%, 215%, 216%, 217%, 218%, 219%, 220%, 221%, 222%, 223%, 224%, 225%, 226%, 227%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247%, 248%, 249%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281 21%, 222%, 223%, 224%, 225%, 226%, 227%, 228%, 229%, 230%, 231%, 232%, 233%, 234%, 235%, 236%, 237%, 238%, 239%, 240%, 241%, 242%, 243%, 244%, 245%, 246%, 247% , 248%, 249%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 4%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, 299% or 300%.The first cooling rate and the second cooling rate may differ by about 400% to about 500%, for example, 300%, 302%, 303%, 304%, 305%, 306%, 307%, 308%, 309%, 310%, 311%, 312%, 313%, 314%, 315%, 316%, 317%, 318%, 319%, 320%, 321%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%, 331%, 332%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%, 344%, 345%, 346%, 347%, 348%, 349%, 350%, 351%, 352%, 353%, 354%, 355%, 356%, 357%, 358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%, 370%, 371%, 372%, 373%, 374%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%, 383%, 384%, 385 21%, 322%, 323%, 324%, 325%, 326%, 327%, 328%, 329%, 330%, 331%, 332%, 333%, 334%, 335%, 336%, 337%, 338%, 339%, 340%, 341%, 342%, 343%, 344%, 345%, 346%, 347% , 348%, 349%, 350%, 351%, 352%, 353%, 354%, 355%, 356%, 357%, 358%, 359%, 360%, 361%, 362%, 363%, 364%, 365%, 366%, 367%, 368%, 369%, 370%, 371%, 372%, 373%, 374%, 4%, 375%, 376%, 377%, 378%, 379%, 380%, 381%, 382%, 383%, 384%, 385%, 386%, 387%, 388%, 389%, 390%, 391%, 392%, 393%, 394%, 395%, 396%, 397%, 398%, 399% or 400%.The first cooling rate and the second cooling rate may differ by about 400% to about 500%, for example, 400%, 402%, 403%, 404%, 405%, 406%, 407%, 408%, 409%, 410%, 411%, 412%, 413%, 414%, 415%, 416%, 417%, 418%, 419%, 420%, 421%, 422%, 423%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%, 435%, 436%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447%, 448%, 449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%, 461%, 462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%, 474%, 475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%, 487%, 488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 49 21%, 422%, 423%, 424%, 425%, 426%, 427%, 428%, 429%, 430%, 431%, 432%, 433%, 434%, 435%, 436%, 437%, 438%, 439%, 440%, 441%, 442%, 443%, 444%, 445%, 446%, 447% , 448%, 449%, 450%, 451%, 452%, 453%, 454%, 455%, 456%, 457%, 458%, 459%, 460%, 461%, 462%, 463%, 464%, 465%, 466%, 467%, 468%, 469%, 470%, 471%, 472%, 473%, 474%, 4%, 475%, 476%, 477%, 478%, 479%, 480%, 481%, 482%, 483%, 484%, 485%, 486%, 487%, 488%, 489%, 490%, 491%, 492%, 493%, 494%, 495%, 496%, 497%, 498%, 499% or 500%.

[0236] In some embodiments, the first cooling rate and the second cooling rate include a sub-freezing rate of about -1°C / min to about -2°C / min. In some embodiments, the sub-freezing rate is about -2.5°C / min to about -0.1°C / min. In some embodiments, the sub-freezing rate is about -0.1°C / min to about -0.2°C / min, about -0.1°C / min to about -0.4°C / min, about -0.1°C / min to about -0.6°C / min, about -0.1°C / min to about -0.8°C / min, about -0.1°C / min to about -1°C / min, about -0.1°C / min to about -1.2°C / min, about -0.1°C / min to about -1.4°C / min, about -0.1°C / min to about -1.6°C / min, about -0.1°C / min to about -1.8°C / min, about -0.1°C / min to about -2°C / min in, about -0.1°C / min to about -2.5°C / min, about -0.2°C / min to about -0.4°C / min, about -0.2°C / min to about -0.6°C / min, about -0.2°C / min to about -0.8°C / min, about -0.2°C / min to about -1°C / min, about -0.2°C / min to about -1.2°C / min, about -0.2°C / min to about -1.4°C / min, about -0.2°C / min to about -1.6°C / min, about -0.2°C / min to about -1.8°C / min, about -0.2°C / min to about -2°C / min, about -0.2°C / min to about -1.8°C / min to about -2.5°C / min, about -0.4°C / min to about -0.6°C / min, about -0.4°C / min to about -0.8°C / min, about -0.4°C / min to about -1°C / min, about -0.4°C / min to about -1.2°C / min, about -0.4°C / min to about -1.4°C / min, about -0.4°C / min to about -1.6°C / min, about -0.4°C / min to about -1.8°C / min, about -0.4°C / min to about -2°C / min, about -0.4°C / min to about -2.5°C / min, about -0.6°C / min to about -0.8°C / min, about -0.6°C / min to about -1°C / min, about -0.6°C / min to about -1.2°C / min, about -0.6°C / min to about -1.4°C / min, about -0.6°C / min to about -1.6°C / min, about -0.6°C / min to about -1.8°C / min, about -0.6°C / min to about -2°C / min, about -0.6°C / min to about -2.5°C / min, about -0.8°C / min to about -1°C / min, about -0.8°C / min to about -1.2°C / min, about -0.8°C / min to about -1.4°C / min, about -0.8°C / min to about -1.6°C / min, about -0.8°C / min to about -1.8°C / min, about -0.8°C / min to about -2°C / min, about -0.8°C / min to about -2.5°C / min, about -1°C / min to about -1.2°C / min, about -1°C / min to about -1.4°C / min, about -1°C / min to about -1.6°C / min, about -1°C / min to about -1.8°C / min, about -1°C / min to about -2°C / min, about -1°C / min to about -2.5°C / min, about -1.2°C / min to about -1.4°C / min, about -1.2°C / min to about -1.6°C / min, about -1.2°C / min to about -1.8°C / min, about -1°C / min to about -2°C / min, about -1°C / min to about -2.5°C / min, about -1.2°C / min to about -1.4°C / min, about -1.2°C / min to about -1.6°C / min, about -1.2°C / min to about -1.8°C / min, From about -1.2°C / min to about -2°C / min, from about -1.2°C / min to about -2.5°C / min, from about -1.4°C / min to about -1.6°C / min, from about -1.4°C / min to about -1.8°C / min, from about -1.4°C / min to about -2°C / min, from about -1.4°C / min to about -2.5°C / min, from about -1.6°C / min to about -1.8°C / min, from about -1.6°C / min to about -2°C / min, from about -1.6°C / min to about -2.5°C / min, from about -1.8°C / min to about -2°C / min, from about -1.8°C / min to about -2.5°C / min, or from about -2°C / min to about -2.5°C / min. In some embodiments, the subfreezing rate is about -0.1°C / min, about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, about -2°C / min, or about -2.5°C / min. In some embodiments, the subfreezing rate is at least about -0.1°C / min, about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, or about -2°C / min. In some embodiments, the subfreezing rate is at most about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min, about -1°C / min, about -1.2°C / min, about -1.4°C / min, about -1.6°C / min, about -1.8°C / min, about -2°C / min, or about -2.5°C / min. In some embodiments, the subfreezing rate may be -1.36°C / min. In some embodiments, the subfreezing rate includes a range of -1.13°C / min to -1.62°C / min. .

[0237] In some embodiments, wherein the super freezing rate, sub-freezing rate and nucleation temperature of a given biological sample are unknown, the cryogenic storage method described herein also includes determining the super freezing rate, sub-freezing rate and nucleation temperature of the biological sample. In some embodiments, super freezing rate, sub-freezing rate and nucleation temperature are derived from the freezing curve of the biological sample. In some embodiments, the freezing curve is modeled by using a computer. In some embodiments, the freezing curve (e.g., Example 5) is determined empirically according to the procedures and methods described herein.

[0238] In some embodiments, the post-thaw survival rate of cells (e.g., the functional survival rate of cells) is at least about 60% to about 95%. In some embodiments, the post-thaw survival rate of cells is at least about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95%. In some embodiments, the post-thaw survival rate of cells is at least about 60%, about 70%, about 80%, about 90% or about 95%. In some embodiments, the post-thaw survival rate of cells is at least about 60%, about 70%, about 80%, about 90% or about 95%. In some embodiments, the post-thaw survival rate of cells is at least about 60%, about 70%, about 80% or about 90%. In some embodiments, the post-thaw survival rate of cells is at most about 70%, about 80%, about 90% or about 95%. In some embodiments, the post-thaw survival rate of cells is at least 60%. In some embodiments, the post-thaw survival rate of the cell is at least 70%. In some embodiments, the post-thaw survival rate of the cell is at least 80%. In some embodiments, the post-thaw survival rate of the cell is at least 90%. The survival rate can be related to one or both of the functional survival rate (which measures the ability of cell proliferation) and the conventional survival rate (which involves the number or percentage of living cells, for example, as measured by trypan blue).

[0239] In some embodiments, (c) occurs in one or more freezers. In some embodiments, the first container and the second container are disposed in a first freezer in one or more freezers. In some embodiments, the first container is contained in a first freezer in one or more freezers, and the second container is contained in a second freezer in one or more freezers. In some embodiments, one or more freezers comprise a static freezer. In some embodiments, the first freezer, the second freezer, or both are static freezers. The method of any of the preceding claims, wherein one or more freezers comprise a controlled rate freezer. In some embodiments, the first freezer, the second freezer, or both are controlled rate freezers. In some embodiments, one or more freezers are set at about -70°C to -90°C. In some embodiments, one or more freezers are set at -80°C. In some embodiments, one or more freezers are set at -86°C. In some cases, one or more freezers may be set at a temperature range of about -100°C to about -60°C.In some cases, the freezer can be set at a temperature range of about -60°C to about -65°C, about -60°C to about -70°C, about -60°C to about -75°C, about -60°C to about -80°C, about -60°C to about -82°C, about -60°C to about -84°C, about -60°C to about -86°C, about -60°C to about -88°C, about -60°C to about -90°C, about -60°C to about -95°C, about -60°C to about -100°C, about -65°C to about -70°C, about -65°C to about -75°C, about -65°C to about -80°C, about -65 ... ℃ to about -84 ℃, about -65 ℃ to about -86 ℃, about -65 ℃ to about -88 ℃, about -65 ℃ to about -90 ℃, about -65 ℃ to about -95 ℃, about -65 ℃ to about -100 ℃, about -70 ℃ to about -75 ℃, about -70 ℃ to about -80 ℃, about -70 ℃ to about -82 ℃, about -70 ℃ to about -84 ℃, about -70 ℃ to about -86 ℃, about -70 ℃ to about -88 ℃, about -70 ℃ to about -90 ℃, about -70 ℃ to about -95 ℃, about -70 ℃ to about -100 ℃, about -75 ℃ to about -80 ℃, about -75 ℃ to about -82 ℃, about -70 ℃ to about -84 ℃, about -70 ℃ to about -86 ℃, about -70 ℃ to about -88 ℃, about -70 ℃ to about -90 ℃, about -70 ℃ to about -95 ℃, about -70 ℃ to about -100 ℃, about -75 ℃ to about -80 ℃, about -75 ℃ to about -82 ℃, about -75 ℃ to about -84 ℃, about -75 ℃ to about -86 ℃, about -75 ℃ to about -88 ℃, about -75 ℃ to about -90 ℃, about -75 ℃ to about -95 ℃, about -75 ℃ to about -100 ℃, about -80 ℃ to about -82 ℃, about -80 ℃ to about -84 ℃, about -80 ℃ to about -86 ℃, about -80 ℃ to about -88 ℃, about -80 ℃ to about -90 ℃, about -80 ℃ to about -95 ℃, about -80 ℃ to about -100 ℃, about -82 ℃ to about -84 ℃, about -82 ℃ to about -86 ℃, about -82 ℃ to about -88 ℃, about -82 ℃ to about -90 ℃, about -80 ℃ to about -95 ℃, about -80 ℃ to about -100 ℃, about -82 ℃ to about -84 ℃, about -82 ℃ to about -86 ℃, about -82 ℃ to about -88 ℃, about -82 ℃ to about -90 ℃, about -82 ℃ To about -95°C, about -82°C to about -100°C, about -84°C to about -86°C, about -84°C to about -88°C, about -84°C to about -90°C, about -84°C to about -95°C, about -84°C to about -100°C, about -86°C to about -88°C, about -86°C to about -90°C, about -86°C to about -95°C, about -86°C to about -100°C, about -88°C to about -90°C, about -88°C to about -95°C, about -88°C to about -100°C, about -90°C to about -95°C, about -88°C to about -100°C, about -90°C to about -95°C, about -90°C to about -100°C, or about -95°C to about -100°C. In some cases, the freezer can be set at a temperature range of about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C, or about -100°C.In some cases, the freezer can be set at a temperature range of at least about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, or about -95°C. In some cases, the freezer can be set at a temperature range of at most about -65°C, about -70°C, about -75°C, about -80°C, about -82°C, about -84°C, about -86°C, about -88°C, about -90°C, about -95°C, or about -100°C.

[0240] In some cases, cryopreservation bags and surrogate vials were frozen less effectively when placed in a static freezer set at -80° C. Instead, better results were obtained when the static freezer was set to a temperature below -80° C. (e.g., -86° C.).

[0241] In some embodiments, the second volume is directly placed in an insulated container so that each vial is adjacent to the insulating material of the insulated container. In some embodiments, the method also includes arranging the first volume in a static freezer so that the first volume is not in contact with the wall of one or more freezers. In some embodiments, the biological sample containing cells or their derivatives in the first volume and the biological sample containing cells or their derivatives in the second volume are subjected to the same cooling rate. In some embodiments, the cell is a stem cell or an immune cell. In some embodiments, the stem cell includes hematopoietic stem cells (HSC), mesenchymal stem cells (MSC) or both. In some embodiments, the biological sample includes whole bone marrow. In some embodiments, the biological sample includes mobilized bone marrow cells, that is, produced by treating the donor with a bone marrow mobilizing agent (e.g., colony stimulating factor (CSF)). In some embodiments, the biological sample includes one or more organs, blood or both. In some embodiments, immune cells include T cells. In some embodiments, blood is cord blood or peripheral blood. In some embodiments, the biological sample includes plasma or serum. In some embodiments, HSC includes CD34+ cells. It is envisioned that the container can be in various forms. For example, the biological sample or its derivative can be contained in a bag of 1 ml to 5 ml volume or a vial of 0.1 to 15 ml volume. In a preferred embodiment, the sample containing less than 15 ml of the biological sample is stored in an insulated container (e.g., a cooling box) in a freezer.

[0242] In some embodiments, a method for cryopreservation of bone marrow or bone marrow cells is described herein. In some embodiments, the method utilizes the system described herein. In some embodiments, the method includes processing bone to obtain bone marrow or its derivatives, thereby obtaining bone marrow cells. In some cases, the bone marrow cells can be any cells that can be separated from the bone marrow. In some embodiments, the bone marrow cells can be hematopoietic stem cells. In some embodiments, the bone marrow cells can be mesenchymal stem cells. In some embodiments, the bone marrow or bone marrow cells are cryopreserved at a freezing rate comprising at least -0.1°C / min, -0.2°C / min, -0.5°C / min, -1°C / min, -1.5°C / min, -2°C / min, -2.5°C / min, -3°C / min, -3.5°C / min, -4°C / min, -4.5°C / min, -5°C / min, -5.5°C / min, -6°C / min, -7°C / min, -7.5°C / min, -8°C / min, -8.5°C / min, -9°C / min, -9.5°C / min, -10°C / min, -11°C / min, -12°C / min, -13°C / min, -14°C / min, -15°C / min, -20°C / min or more. Preferably, the cryopreservation bags and surrogate vials are cooled at a rate of -1°C to -5°C.

[0243] In some embodiments, the freezing rate includes a temperature drop measured by directly contacting the bone marrow or bone marrow cells with a thermometer. In some embodiments, the freezing rate includes a temperature drop measured in a microenvironment or environment immediately adjacent to the bone marrow or bone marrow cells. In some embodiments, the freezing rate includes a temperature drop measured in a freezing device (e.g., a freezing bag, a cryopreservation bag, a freezing tube, a cryotank, a freezing box, a freezer, or a container containing liquid nitrogen).

[0244] In some embodiments, the method for cryopreserving bone marrow or bone marrow cells as described herein increases the yield of bone marrow cells after thawing, compared to bone marrow cells that have not been cryopreserved at a freezing rate as described herein. In some cases, the yield of bone marrow cells cryopreserved at a freezing rate as described herein increases by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times or more, compared to the yield of bone marrow cells that have not been cryopreserved at a freezing rate as described herein. In some embodiments, the method for cryopreserving bone marrow or bone marrow cells as described herein increases the survival rate of bone marrow cells after thawing, compared to bone marrow cells that have not been cryopreserved at a freezing rate as described herein. In some cases, the survival rate of bone marrow cells cryopreserved by the freezing rates described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the survival rate of bone marrow cells that were not cryopreserved by the freezing rates described herein. In some embodiments, the methods of cryopreserving bone marrow or bone marrow cells described herein increase the number of CD34+ bone marrow cells after thawing compared to the number of CD34+ bone marrow cells that were not cryopreserved by the freezing rates described herein. In some cases, the number of CD34+ bone marrow cells cryopreserved by the freezing rates described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the number of CD34+ bone marrow cells that were not cryopreserved by the freezing rates described herein. In some embodiments, the methods of cryopreserving bone marrow or bone marrow cells described herein increase the number of CD45+ bone marrow cells after thawing compared to the number of CD45+ bone marrow cells that were not cryopreserved by the freezing rates described herein. In some cases, the number of CD45+ bone marrow cells cryopreserved by a freezing rate described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the number of CD45+ bone marrow cells that have not been cryopreserved by a freezing rate described herein.

[0245] In some embodiments, compared to known cryopreservation schemes, after thawing a sample frozen using a scheme described herein (e.g., Example 5), the sample comprises an increased amount of viable CD34+ cells. In some embodiments, the percentage of CD34+ cells surviving in the thawed sample is at least about 70% to about 95%. In some embodiments, the percentage of CD34+ cells surviving in the thawed sample is at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the percentage of CD34+ cells that survive in the thawed sample is at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the percentage of CD34+ cells that survive in the thawed sample is at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the percentage of CD34+ cells that survive in the thawed sample is at most about 75%, about 80%, about 85%, about 90%, or about 95%.

[0246] Exemplary methods for obtaining, producing, cryopreserving and / or storing bone marrow products containing hematopoietic stem cells for use in the methods of the present disclosure can be described in PCT / US2020 / 025778 and in Woods et al., “Ischemia considerations for the development of an organ and tissue donor derived bone marrow bank.” J Transl Med 18, 300 (2020); the contents of each of which are incorporated by reference in their entirety.

[0247] Automated system for recovering bone marrow

[0248] The present disclosure contemplates an automated process for recovering bone marrow and even selecting cells from the bone marrow. In one aspect, the automated system 209 includes Figure 7A-7B The first station 210 of the automated process cleans the VBs to remove all soft tissue. As opposed to the manual process that operates on one VB at a time, the automated process is configured to clean the entire set of donor VBs (which may be at least ten vertebral bodies). The VBs are placed on a rack or tray 212 that is configured to support a set of vertebral bodies from a given donor. Figures 8A-8BAs shown, a tray 212 is placed on a transfer track 216 of a housing 215, and the tray is automatically or manually advanced to the interior of the housing. The housing 215 supports a plurality of hydraulic jets 220 that direct high-pressure and high-velocity saline jets onto the VB. In known manual processes, the manual hydraulic jets operate at lower speeds and pressures to direct a stream of detergent onto the VB. In manual processes, detergents are required to remove the soft tissue of the VB. In contrast, the automated cleaning station 210 of the present disclosure uses a saline medium, and the speed and pressure of the water jet are sufficient to remove all soft tissue from the VB. The automated cleaning station of the present disclosure includes jets configured to produce a direct stream or narrow "V" water / saline jet that produces high concentrated impact forces at different distances. In order to achieve good coverage of the VB, the device includes a number of direct jets that are closely spaced in different directions relative to the VB, thereby allowing uniform cleaning to be achieved independent of the position of the VB in the device. Fig. 8A In the illustrative embodiment of the invention, the hydraulic jets are arranged in an upper row 220 and a lower row 221. The "V" shaped jets are arranged at different angles to achieve full coverage of the VB surface. Additionally or alternatively, the hydraulic jets 220, 221 can be configured to swing over the tray of the VB to ensure complete coverage.

[0249] like Figure 8B As shown, a visualization device 225 is arranged at the exit of the cleaning station 210, which is operable to visualize and interpret the VB leaving the cleaning station to determine whether all soft tissues have been removed. If not, the VB is returned to the housing along the track 216 to further perform the hydraulic jetting process. It is envisioned that a controller (not shown) can be set to control the movement of the tray 212 along the track 216, and interpret the signal generated by the visualization device 225. The visualization device can include a camera that obtains the VB image, and the controller can include imaging software that can identify the soft tissue in the acquired image. At the end of the hydraulic jetting cleaning process, dye can be applied to the cleaned VB, wherein the dye is absorbed by the soft tissue, but not by the bone. Therefore, the dye can provide contrast to facilitate the identification of any remaining soft tissue from the bone. The visualization device 225 can be configured to move and shoot VB everywhere, such as by translating along the frame 226, and by translating the frame, so as to observe the VB from all angles.

[0250] Back to Figures 9A-9B, once it is determined that the VB is clear of all soft tissue, the cleaned VB is then fed to an automated grinding station 240 via a conveyor 230 to produce appropriately sized fragments for tumbling and eventual cell extraction. The manual "dicing" process described above can be variable, time consuming, and potentially unsafe for the operator. The automated system includes a grinding station that combines "dicing" the VB (i.e., cutting the VB into small pieces) and grinding the diced VB to reduce the VB to 2-3 mm fragments. The track 216 and tray 212 can be connected to deposit the cleaned VB onto the conveyor 230, which then automatically transfers the VB to an input hopper 242 of the grinding station 240, shown in more detail in Figures 9A-9B .like Fig. 9A As shown, VB is directed through the primary grinding cutter module 244 and then through the funnel 246 to the fine grinding cutter module 248. Fig. 9B As shown, the primary grinding cutter module 242 includes opposing rotating grinders 245, which are separated by a predetermined gap, such as 5-8 mm gap, to grind the incoming VB into coarse size segments. The coarsely ground segments are fed to the fine grinding cutter module 248, in which smaller diameter grinders 249 are provided. The fine grinders 249 are separated by a smaller gap, about 2-3 mm, to produce finely ground VB segments. Fig. 9A As shown, hopper 246 delivers the coarsely ground segments to a second grinder 248, while hopper 250 directs the finely ground VB segments to a collection tray 252 supported on a plate 253. During the grinding operation, a measured volume of processing / resuspension medium containing DNA enzymes can be directed through an upper hopper to the grinding cutter. Such media can be manually introduced during operation of the grinding station 240, or can be automatically implemented through a nozzle incorporated into the hopper 242.

[0251] The finely ground VB segment and process media are collected in the collection tray 252, and the plate 253 can be moved to the screening station 260 ( Figures 8A-8B Once at the screening station 260, the contents of the pan 252 fall into a screening drum unit, which includes two 12" diameter filter screens, a No. 40 screen 262 on top, followed by a finer No. 80 screen 264, such as Fig.10Described. Funnel 266 directs the filtered contents to a collection container 268. The grindings retained by the filter are rinsed in a screening station 260 with a processing / resuspending medium that does not contain DNase. The liquid bone marrow product in the collection container 268 can be analyzed to determine the cell content and then concentrated and packaged in an appropriate volume for cryopreservation, as described below. Alternatively, some or all of the processed bone marrow can be further processed for specialized cell products, such as CD34+ cells, by using automated cell selection methods. Because large volumes of cells can be recovered from a single organ donor using this method, a single donor can produce multiple product types. In addition, since the source is original bone marrow (relative to peripheral blood mobilized by G-CSF), the cell product will withstand cryopreservation processing.

[0252] In one modification, the output from the grinding station 240 or the screening station 260 can be automatically fed to a collection bag for cryogenic processing. In this modification, the lower funnel 250 can be configured to direct the contents to a fluid line connected to a sterile bag. A peristaltic pump can be engaged with the fluid line to pump the output from the grinding station into the sterile bag. A similar device can be engaged with the funnel 266 of the screening station.

[0253] The contents of the collection container 268, which is essentially bone marrow slurry, are manually or automatically transferred to an adjacent drum station 270, which includes a mechanical drum 272 and a large disposable container 274 that can hold the entire contents of 10 processed VBs and associated processing / resuspension media. The drum 272 has paddles for agitating the ground slurry to mechanically release the cells. When the tumbling cycle is complete, the contents of the drum are poured through a screen bin into container 274. The contents of container 274 can be further processed or prepared for cryogenic storage.

[0254] Additional teachings on packaging are disclosed in Woods, EJ and S Thirumala. "Packaging considerations for biopreservation." Transfusion Medicine and Hemotherapy 38: 149-156 (2011); the contents of which are incorporated by reference in their entirety.

[0255] Isolation of CD34+ cells

[0256] In some aspects, described herein is a method for processing (e.g., isolating) CD34+ cells obtained from bone marrow or bone marrow derivatives. In some cases, the bone marrow or bone marrow derivative can be fresh (e.g., never frozen) or thawed from a previously frozen state. In some embodiments, the bone marrow or bone marrow derivative can be ground by the methods and systems described herein. In some embodiments, the ground bone marrow or bone marrow cells can be contacted with a stabilization buffer described herein. In some embodiments, stabilization prevents the formation of aggregates of bone marrow cells. In some cases, bone marrow cells contacted and suspended in a stabilization buffer can be separated by attaching to an antibody, such as a conjugated antibody. For example, bone marrow cells expressing CD34+ can be separated and enriched by contacting the bone marrow cells with a CD34 antibody conjugated to iron, wherein the bone marrow cells expressing CD34 are then separated by a magnetic separation column (e.g., ) capture. Bone marrow cells that do not express CD34 can be washed away. The captured CD34+ bone marrow cells can be collected by eliminating the magnetic field and eluting the targeted CD34+ bone marrow cells. This method does not require the use of a Ficoll gradient to separate bone marrow cells.

[0257] Aspects described in the present disclosure include a method for processing a CD34+ cell population obtained from bone marrow or its derivatives, wherein the bone marrow or its derivatives are derived from a deceased donor, the method comprising: obtaining bone or bone fragments from a deceased donor, optionally processing the bone into bone fragments; extracting bone marrow or its derivatives from the bone or bone fragments; and contacting the bone marrow or its derivatives with a stabilization buffer, wherein the stabilization buffer comprises greater than about 3U / ml of a nuclease; performing a CD34+ cell separation assay to produce a cell composition comprising a CD34+ cell population, wherein the composition comprising the CD34+ cell population comprises at least about 80,000 CD34+ cells / 750μl of bone marrow or its derivatives contacted with the stabilization buffer. In some embodiments, at least about 80,000 CD34+ cells / 750μl of bone marrow or its derivatives contacted with the stabilization buffer comprise at least 70% viable CD34+ cells. In some embodiments, at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with a stabilization buffer comprises at least 80% viable CD34+ cells. In some embodiments, at least about 80,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with a stabilization buffer comprises at least 90% viable CD34+ cells.

[0258] Another aspect of the present disclosure includes a stabilization buffer comprising: at least 5 U / ml of an anticoagulant; and greater than 3 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 5 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 10 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 15 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises greater than about 20 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises about 20 U / ml of a nuclease. In some embodiments, the nuclease is or In some embodiments, the stabilization buffer further comprises an anticoagulant greater than about 10 U / ml. In some embodiments, the stabilization buffer further comprises an anticoagulant of about 10 U / ml. In some embodiments, the anticoagulant is heparin. In some embodiments, the stabilization buffer further comprises human serum albumin (HSA). In some embodiments, the stabilization buffer comprises 0.5% HSA.

[0259] In some embodiments, the stabilization buffer comprises a nuclease. In some embodiments, the nuclease is or In some embodiments, the stabilization buffer comprises about 3U / ml, 4U / ml, 5U / ml, 6U / ml, 7U / ml, 8U / ml, 9U / ml, 10U / ml, 11U / ml, 12U / ml, 13U / ml, 14U / ml, 15U / ml, 16U / ml, 17U / ml, 18U / ml, 19U / ml, 20U / ml, 21U / ml, 22U / ml, 23U / ml, 24U / ml, 25U / ml, 26U / ml, 27U / ml, 28U / ml, 29U / ml, 30U / ml, 50U / ml, 100U / ml, 200U / ml or more U / ml of nuclease. In some embodiments, the stabilization buffer comprises an anticoagulant. In some cases, the anticoagulant is heparin. In some cases, the stabilization buffer comprises about 0.1 U / ml, 0.2 U / ml, 0.3 U / ml, 0.4 U / ml, 0.5 U / ml, 0.6 U / ml, 0.7 U / ml, 0.8 U / ml, 0.9 U / ml, 1.0 U / ml, 2.0 U / ml, 3.0 U / ml, 4.0 U / ml, 5.0 U / ml, 6.0 U / ml, 7.0 U / ml, 8.0 U / ml, 9.0 U / ml, 10 U / ml, 11 U / ml, / ml, 12U / ml, 13U / ml, 14U / ml, 15U / ml, 16U / ml, 17U / ml, 18U / ml, 19U / ml, 20U / ml, 21U / ml, 22U / ml, 23U / ml, 24U / ml, 25U / ml, 26U / ml, 27U / ml, 28U / ml, 29U / ml, 30U / ml, 50U / ml, 100U / ml, 200U / ml or more U / ml of anticoagulant.

[0260] In various embodiments, the stabilization buffer lacks heparin.

[0261] In some embodiments, the stabilizing buffer comprises about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05% HSA, 0.1% HSA, 0.2% HSA, 0.3% HSA, 0.4% HSA, 0.5% HSA, 0.6% HSA, 0.7% HSA, 0.8% HSA, 0.9% HSA, 1.0% HSA, 1.5% HSA, 2% HSA, 2.5% HSA, 5% HSA, 10% HSA, 20% HSA, or more HSA.

[0262] In some embodiments, described herein is a method for processing bone marrow to obtain bone marrow cells. In some embodiments, the method comprises contacting the bone marrow or bone marrow cells with a stabilization buffer described herein.

[0263] Another aspect of the present disclosure includes a method for processing a CD34+ cell population contained in bone marrow or its derivatives, wherein the bone marrow or its derivatives are derived from a deceased donor, the method comprising: obtaining bones or bone fragments from a deceased donor, optionally processing the bones into bone fragments; extracting bone marrow or its derivatives from the bones or bone fragments; and contacting the bone marrow or its derivatives with a stabilization buffer, wherein the stabilization buffer comprises greater than about 3 U / ml of a nuclease; performing a CD34+ cell separation assay to produce a cell composition comprising a CD34+ cell population, wherein the composition comprising the CD34+ cell population comprises at least about 80,000 CD34+ cells / 750 μl of bone marrow or its derivatives contacted with the stabilization buffer.

[0264] In some embodiments, compared with the yield of bone marrow cells processed in the absence of a stabilizing buffer, processing or contacting bone marrow or bone marrow cells with a stabilizing buffer as described herein increases the yield of bone marrow cells obtained from the methods described herein. In some cases, compared with the yield of bone marrow cells processed in the absence of a stabilizing buffer, processing or contacting bone marrow or bone marrow cells with a stabilizing buffer as described herein increases the yield of bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times or more. In some embodiments, compared with the survival rate of bone marrow cells processed in the absence of a stabilizing buffer, processing or contacting bone marrow or bone marrow cells with a stabilizing buffer as described herein increases the survival rate of bone marrow cells obtained from the methods described herein. In some cases, processing or contacting the bone marrow or bone marrow cells with a stabilizing buffer as described herein increases the survival of the bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more compared to the survival of bone marrow cells processed in the absence of the stabilizing buffer.

[0265] In some embodiments, compared with the number of CD34+ bone marrow cells processed in the absence of a stabilizing buffer, the number of CD34+ bone marrow cells or bone marrow cells processed or contacted with a stabilizing buffer as described herein increases. In some cases, compared with the number of CD34+ bone marrow obtained by processing in the absence of a stabilizing buffer, the number of CD34+ bone marrow obtained by processing with a stabilizing buffer increases by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times or more. In some embodiments, compared with the number of CD45+ bone marrow cells processed in the absence of a stabilizing buffer, the number of CD45+ bone marrow cells or bone marrow cells processed or contacted with a stabilizing buffer as described herein increases. In some cases, the number of CD45+ bone marrow obtained by processing with a stabilization buffer is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or more compared to the number of CD45+ bone marrow obtained by processing in the absence of a stabilization buffer.

[0266] In some embodiments, the cell composition described herein comprising CD34+ cells obtained from a bone marrow sample processed with a stabilization buffer has an increased amount of CD34+ cells compared to a cell composition produced from a known CD34+ separation method. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilization buffer described herein is at least about 70,000 CD34+ cells / 750 μl of bone marrow or a derivative thereof contacted with a stabilization buffer described herein. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilization buffer described herein is at least about 70,000 cells / 750 μl to about 100,000 cells / 750 μl.In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilizing buffer described herein is at least about 70,000 cells / 750 μl to about 75,000 cells / 750 μl, about 70,000 cells / 750 μl to about 80,000 cells / 750 μl, about 70,000 cells / 750 μl to about 85,000 cells / 750 μl, about 70,000 cells / 750 μl to about 90,000 cells / 750 μl, about 70,000 cells / 750 μl to about 100,000 cells / 750 μl, about 150,000 cells / 750 μl to about 160,000 cells / 750 μl, about 170,000 cells / 750 μl to about 180,000 cells / 750 μl, about 190,000 cells / 750 μl to about 200,000 cells / 750 μl, about 210,000 cells / 750 μl to about 220,000 cells / 750 μl, about 230,000 cells / 750 μl to about 240,000 cells / 750 μl, about 250,000 cells / 750 μl to about 260,000 cells / 750 μl, about 260,000 cells / 750 μl to about 270,000 cells / 750 750 μl to about 95,000 cells / 750 μl, about 70,000 cells / 750 μl to about 100,000 cells / 750 μl, about 75,000 cells / 750 μl to about 80,000 cells / 750 μl, about 75,000 cells / 750 μl to about 85,000 cells / 750 μl, about 75,000 cells / 750 μl to about 90,000 cells / 750 μl, about 75,000 cells / 750 μl to about 95,000 cells / 750 μl, l, about 75,000 cells / 750 μl to about 100,000 cells / 750 μl, about 80,000 cells / 750 μl to about 85,000 cells / 750 μl, about 80,000 cells / 750 μl to about 90,000 cells / 750 μl, about 80,000 cells / 750 μl to about 95,000 cells / 750 μl, about 80,000 cells / 750 μl to about 100,000 cells / 750 μl, about 85,000 cells / 750 μl to about About 90,000 cells / 750 μl, about 85,000 cells / 750 μl to about 95,000 cells / 750 μl, about 85,000 cells / 750 μl to about 100,000 cells / 750 μl, about 90,000 cells / 750 μl to about 95,000 cells / 750 μl, about 90,000 cells / 750 μl to about 100,000 cells / 750 μl, or about 95,000 cells / 750 μl to about 100,000 cells / 750 μl. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilization buffer described herein is at least about 70,000 cells / 750 μl, about 75,000 cells / 750 μl, about 80,000 cells / 750 μl, about 85,000 cells / 750 μl, about 90,000 cells / 750 μl, about 95,000 cells / 750 μl, or about 100,000 cells / 750 μl.In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilization buffer described herein is at least about 70,000 cells / 750 μl, about 75,000 cells / 750 μl, about 80,000 cells / 750 μl, about 85,000 cells / 750 μl, about 90,000 cells / 750 μl, or about 95,000 cells / 750 μl. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilization buffer described herein is at most about 75,000 cells / 750 μl, about 80,000 cells / 750 μl, about 85,000 cells / 750 μl, about 90,000 cells / 750 μl, about 95,000 cells / 750 μl, or about 100,000 cells / 750 μl.

[0267] In some embodiments, the CD34+ cells obtained from a bone marrow sample processed with a stabilization buffer described herein also exhibit a higher survival rate compared to cell compositions produced from known CD34+ isolation methods.

[0268] In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises a survival percentage of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises a survival percentage of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises a survival percentage of at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95% or about 90% to about 95% survival percentage. In some embodiments, the amount of CD34+ cells separated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises at least about 70%, about 75%, about 80%, about 85%, about 90% or about 95% survival rate percentage. In some embodiments, the amount of CD34+ cells separated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises at least about 70%, about 75%, about 80%, about 85% or about 90% survival rate percentage. In some embodiments, the amount of CD34+ cells separated from a bone marrow sample contacted with a stabilizing buffer as described herein comprises at most about 75%, about 80%, about 85%, about 90% or about 95% survival rate percentage. Survival rate can be related to one or both of functional survival rate (which measures the ability of cells to proliferate) and conventional survival rate (which involves the number or percentage of living cells, for example, as measured by trypan blue).

[0269] In aspects of the present disclosure, a method is provided for selecting CD34-expressing (CD34+) cells from dead donor bone marrow by using a reduced density Ficoll and an immunomagnetic CD34+ cell separation kit. Surprisingly, it has been found that cell separation by using a reduced density Ficoll prior to CD34 selection is beneficial for obtaining high purity and viability CD45 / CD34+ cells from freshly prepared dead donor bone marrow. On the other hand, it has been found that Ficoll at conventional density is optimal for selecting CD45 / CD34+ cells from thawed cryopreserved dead donor bone marrow.

[0270] Vertebral sections obtained from recently deceased donors are processed as described above. Thus, in one embodiment, the bones are cleaned of all soft tissue and then cut into small pieces which are then immersed in 500 ml of grinding media. The grinding media may be PLASMA-LYTE TMA Injection, pH 7.4, Multiple Electrolytes, Injection Type 1 USP (PLASMA-LYTE TM ), which contains 2.5% human serum albumin (HSA), 3U / ml denarase and 10U / ml heparin. The sliced ​​VB was ground using a bone grinder, filtered and washed with a flushing medium (e.g., PLASMA-LYTE TM The whole cell suspension was centrifuged to concentrate the cells to 2-3x10 8 / ml and extract the cell concentrate. Part or all of the resulting BM preparation can be used immediately for CD34 selection, while the remainder can be prepared for cryopreservation. The cryopreservation portion involves adding DMSO and 5% HSA to the BM cells at a final concentration of 10%, and bringing the preparation to -86°C by passive cooling or by controlled cooling at a rate of about -1°C / min, after which the cryopreserved portion is immersed in liquid nitrogen.

[0271] In order to select CD34+ cells, a newly processed BM preparation is used, or the previously cryopreserved part is thawed for use. Ficoll-Paque PLUS is added to the BM preparation to separate the desired CD34+ cell components of bone marrow. It has been found that in order to select cells from the bone marrow of cryopreservation, the conventional density of Ficoll 1.077g / ml produces acceptable results. However, in one aspect of the present disclosure, in order to select cells from the freshly prepared dead donor bone marrow, Ficoll density is reduced from conventional density. Specifically, reducing this density is by mixing Ficoll-Paque PLUS (density 1.077g / mL, GE Company) with Plasma Lyte-A Injection pH 7.4 (Baxter Healthcare 2B2544X) in a specific ratio and obtaining a total density less than 1.077g / ml, particularly 1.063-1.052g / ml. In a specific embodiment, a density of 1.063 g / ml has been found to be optimal for isolating CD34+ cells, taking into account the amount, viability and purity of CD34+ cells.

[0272] In one embodiment, 5 ml of 1.063 g / ml density Ficoll solution is pipetted into a 15-ml centrifuge tube, and the BM solution generated from the VB of the deceased donor is carefully layered on the Ficoll gradient. The tube is centrifuged at room temperature for 30 min at 400 g without interruption. After centrifugation, the buffy coat cells are carefully collected and the cells are washed in a phosphate-buffered saline (PBS) containing 0.5% HSA and 2 mM ethylenediaminetetraacetic acid (EDTA) (MACS buffer, Miltenyi). In a specific embodiment, centrifugation is performed at 400 g for 5 min, the resulting cell pellet is resuspended in 10 ml PBS, and then centrifuged for a second time at 400 g for 5 min.

[0273] Sysmex XP-300 can be used to count the nucleated cells in the separated buffy coat. Cellometer Vision (Nexcellom) or flow cytometer can be used to determine the cell count of purified CD34 cells. 20 microliters of AOPI can be added to 20 microliters of cells, and after mixing, total viable cells can be determined. CD34+ cells can be selected by positive immunoseparation using CliniMACS system (Miltenyi, Bergisch Gladbach, Germany) or EasySep CD34 kit (Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer's scheme. Unexpectedly, according to testing under various Ficoll densities, it has been determined that the lower Ficoll density (i.e., 1.063-1.052gm / ml compared to conventional 1.077gm / ml density) envisioned in the present disclosure results in more optimized cell recovery. Optimization is based on the purity, viability and yield of the selected CD34 cells. The preferred target is >90% purity and >90% viable CD34+ cells. Although lower Ficoll density results in greater purity and fewer dead cells, it was unexpectedly found that by using lower Ficoll density to prepare the buffy coat, most of the CD34+ cells present in the whole bone marrow of the deceased donor before selection were lost. Therefore, the high viability and purity of CD45 / CD34+ cells achieved under conventional Ficoll density gradients also resulted in a large loss of yield (about 60% loss of input CD34+ cells).

[0274] Therefore, according to one aspect of the present disclosure, for freshly prepared, the optimal density of Ficoll for selecting CD45 / CD34+ with >90% purity and viability is 0.77, particularly 1.063-1.052. This Ficoll density provides a higher CD45 / CD34+ cell yield at a purity and cell viability similar to that of conventional Ficoll density method.

[0275] In another aspect of the present disclosure, CD34+ cells can be initially obtained from freshly prepared deceased donor bone marrow using the reduced density Ficoll-Paque described above. BM can be cryogenically frozen and then CD34+ cells can be subsequently obtained using regular density Ficoll-Paque. This method essentially allows for selective recovery of cells from deceased donor bone marrow using a modified Ficoll density prior to freezing or a regular Ficoll density after freezing and thawing.

[0276] Recovery of MSCs from processed bone marrow

[0277] Bone marrow is a well-known source of mesenchymal stromal / stem cells (MSC), which can be collected from the bone marrow obtained using the above method. MSC is a self-renewing multipotent progenitor cell with the multi-lineage potential of differentiating into cell types (such as adipocytes, osteocytes and chondrocytes) of mesodermal origin. In addition, MSC can migrate to the site of inflammation and play effective immunosuppressive and anti-inflammatory effects by the interaction between the lymphocytes related to the innate immune system and the adaptive immune system. MSC can be used to treat osteogenesis imperfecta, cartilage defects, myocardial infarction, Crohn's disease, multiple sclerosis, autoimmune diseases, such as lupus erythematosus, cirrhosis, osteoarthritis and rheumatoid arthritis. The HSC / MSC unit of matching can be used for co-transplantation to treat graft-versus-host disease (GVHD), and is used for hematopoietic stem cell transplantation support.

[0278] In another feature of the systems and methods disclosed herein, a method for recovering mesenchymal stem cells (MSCs) from enzymatically hydrolyzed vertebral body (VB) bone fragments, which are byproducts of VB grinding and elution of the methods described herein, is provided. In this method, a mixture of collagenase and neutral protease is used to obtain the highest possible yield of vertebral bone adherent MSCs (vBA-MSCs). MSCs can be recovered from cryopreserved VB bone fragments that are subsequently processed according to the present disclosure. In a specific aspect, recombinant Clostridium histolyticum collagenase containing two active isoforms is used in an effective amount in the MSC extraction process. The cell mixture released by digesting the VB bone fragments is cultured on tissue-coated plastic in the presence of Mesencult medium to select for proliferative vBA-MSCs. Freshly digested preparations and vBA-MSCs of different passages can be characterized by flow cytometry, fibroblast colony forming unit (CFU-F) potential, population doubling time (PDT), and in vitro tri-lineage (adipogenic, chondrogenic, and osteogenic) differentiation. In some embodiments, mesenchymal stem cells can be recovered or cultured in Alpha-MEM supplemented with human platelet lysate and epidermal growth factor and / or fibroblast growth factor.

[0279] Therefore, the present disclosure contemplates a method for optimizing digestion of vertebral fragments and MSC recovery using a combination of purified collagenase and neutral protease. In a specific embodiment, the collagenase is DE collagenase (Vitacyte), which is composed of purified Clostridium histolyticum collagenase and Paneibacillus polymyxa neutral protease. According to one aspect of the present disclosure, the optimal neutral protease concentration and collagenase concentration (C1 and C2 collagenase) and the optimal ratio of solution volume (ml) to bone fragment weight (mg) are determined.

[0280] In some embodiments, the collagenase may include Clostridium histolyticum, which also includes two active isoforms C1 and C2. In some embodiments, one or more collagenases including isoforms C1 and C2 may be present in the digestion solution in a ratio that includes more collagenase isoform C1 than collagenase isoform C2. In some embodiments, the ratio of collagenase isoform C1 to collagenase isoform C2 may be about 30 to about 70: about 10 to about 29. In some embodiments, the ratio of collagenase isoform C1 to collagenase C2 may be 35:15. In some embodiments, for each concentration, the mass ratio of C1 to C2 may be 70:30, 54:46, 37:63, 82:18, 54:46, and 90:10.

[0281] In some embodiments, neutral protease can be Paenibacillus polymyxa neutral protease. In some embodiments, neutral protease concentration can be about 2U / ml to about 21U / ml. In some embodiments, neutral protease concentration can be about 2U / ml to about 7U / ml, about 2U / ml to about 12U / ml, about 2U / ml to about 17U / ml, about 2U / ml to about 21U / ml, about 7U / ml to about 12U / ml, about 7U / ml to about 17U / ml, about 7U / ml to about 21U / ml, about 12U / ml to about 17U / ml, about 12U / ml to about 21U / ml, or about 17U / ml to about 21U / ml. In some embodiments, neutral protease concentration can be about 2U / ml, about 7U / ml, about 12U / ml, about 17U / ml or about 21U / ml. In some embodiments, the neutral protease concentration can be at least about 2U / ml, about 7U / ml, about 12U / ml, or about 17U / ml. In some embodiments, the neutral protease concentration can be at most about 7U / ml, about 12U / ml, about 17U / ml, or about 21U / ml. In some embodiments, the digestion solution can contain about 19.6U / ml of active neutral protease.

[0282] In some embodiments, the collagenase concentration is about 0.05U / ml to about 1.6U / ml. In some embodiments, the collagenase concentration is about 0.05U / ml to about 0.1U / ml, about 0.05U / ml to about 0.15U / ml, about 0.05U / ml to about 0.2U / ml, about 0.05U / ml to about 0.25U / ml, about 0.05U / ml to about 0.3U / ml, about 0.05U / ml to about 0.35U / ml, about 0.05U / ml to about 0.4U / ml, about 0.05U / ml to about 0.8U / ml, about 0.05U / ml to about 1.2U / ml, about 0.05U / ml to about 1.6U / ml, about 0.1U / ml to about 0.15U / ml, about 0.1U / ml to about 0.2U / ml, about 0.1 U / ml to about 0.25 U / ml, about 0.1 U / ml to about 0.3 U / ml, about 0.1 U / ml to about 0.35 U / ml, about 0.1 U / ml to about 0.4 U / ml, about 0.1 U / ml to about 0.8 U / ml, about 0.1 U / ml to about 1.2 U / ml, about 0.1 U / ml to about 1.6 U / ml, about 0.15 U / ml to about 0.2 U / ml, about 0.15 U / ml to about 0.25 U / ml, about 0.15 U / ml to about 0.3 U / ml, about 0.15 U / ml to about 0.35 U / ml, about 0.15 U / ml to about 0.4 U / ml, about 0.15 U / ml to about 0. 8U / ml, about 0.15U / ml to about 1.2U / ml, about 0.15U / ml to about 1.6U / ml, about 0.2U / ml to about 0.25U / ml, about 0.2U / ml to about 0.3U / ml, about 0.2U / ml to about 0.35U / ml, about 0.2U / ml to about 0.4U / ml, about 0.2U / ml to about 0.8U / ml, about 0.2U / ml to about 1.2U / ml, about 0.2U / ml to about 1.6U / ml, about 0.25U / ml to about 0.3U / ml, about 0.25U / ml to about 0.35U / ml, about 0.25U / ml to about 0.4U / ml, about 0.25U / ml to about 0.8U / ml, about 0.2U / ml to about 1.2U / ml, about 0.2U / ml to about 1.6U / ml, about 0.25U / ml to about 0.3U / ml, about 0.25U / ml to about 0.35U / ml, about 0.25U / ml to about 0.4U / ml, about 0.25U / ml to about 0. .8 U / ml, about 0.25 U / ml to about 1.2 U / ml, about 0.25 U / ml to about 1.6 U / ml, about 0.3 U / ml to about 0.35 U / ml, about 0.3 U / ml to about 0.4 U / ml, about 0.3 U / ml to about 0.8 U / ml, about 0.3 U / ml to about 1.2 U / ml, about 0.3 U / ml to about 1.6 U / ml, about 0.35 U / ml to about 0.4 U / ml, about 0.35 U / ml to about 0.8 U / ml, about 0.35 U / ml to about 1.2 U / ml, about 0.35 U / ml to about 1.6 U / ml, about 0.4 U / ml to about 0.8 U / ml, about 0.4 U / ml to about 1.In some embodiments, the collagenase concentration is about 0.05 U / ml, about 0.1 U / ml, about 0.15 U / ml, about 0.2 U / ml, about 0.25 U / ml, about 0.3 U / ml, about 0.35 U / ml, about 0.4 U / ml, about 0.8 U / ml, about 1.2 U / ml, or about 1.6 U / ml. In some embodiments, the collagenase concentration is about 0.05 U / ml, about 0.1 U / ml, about 0.15 U / ml, about 0.2 U / ml, about 0.25 U / ml, about 0.3 U / ml, about 0.35 U / ml, about 0.4 U / ml, about 0.8 U / ml, about 1.2 U / ml, or about 1.6 U / ml. In some embodiments, the collagenase concentration is at least about 0.05 U / ml, about 0.1 U / ml, about 0.15 U / ml, about 0.2 U / ml, about 0.25 U / ml, about 0.3 U / ml, about 0.35 U / ml, about 0.4 U / ml, about 0.8 U / ml, or about 1.2 U / ml. In some embodiments, the collagenase concentration is at most about 0.1 U / ml, about 0.15 U / ml, about 0.2 U / ml, about 0.25 U / ml, about 0.3 U / ml, about 0.35 U / ml, about 0.4 U / ml, about 0.8 U / ml, about 1.2 U / ml, or about 1.6 U / ml. .

[0283] According to one aspect of the present disclosure, the neutral protease concentration and collagenase concentration (C1 and C2 collagenase) and the ratio of solution volume (ml) to bone chip weight (mg) were determined.

[0284] In some embodiments, the total collagenase concentration (C1 and C2 collagenase) is about 25 μg / ml to about 100 μg / ml. In some embodiments, the total collagenase concentration is about 25 μg / ml to about 32.5 μg / ml, about 25 μg / ml to about 47.5 μg / ml, about 25 μg / ml to about 42.5 μg / ml, about 25 μg / ml to about 50 μg / ml, about 25 μg / ml to about 65 μg / ml, about 25 μg / ml to about 77.5 μg / ml, about 25 μg / ml to about 85 μg / ml, about 25 μg / ml to about 100 μg / ml, about 32.5 μg / ml to about 47.5 μg / ml, about 25 μg / ml to about 50 μg / ml, about 25 μg / ml to about 65 μg / ml, about 25 μg / ml to about 77.5 μg / ml, about 25 μg / ml to about 85 μg / ml, about 25 μg / ml to about 100 μg / ml, about 32.5 μg / ml to about 47.5 μg / ml. 5 μg / ml, about 32.5 μg / ml to about 42.5 μg / ml, about 32.5 μg / ml to about 50 μg / ml, about 32.5 μg / ml to about 65 μg / ml, about 32.5 μg / ml to about 77.5 μg / ml, about 32.5 μg / ml to about 85 μg / ml, about 32.5 μg / ml to about 100 μg / ml, about 47.5 μg / ml to about 42.5 μg / ml, about 47.5 μg / ml to about 50 μg / ml, about 47.5 μg / ml to about about 65 μg / ml, about 47.5 μg / ml to about 77.5 μg / ml, about 47.5 μg / ml to about 85 μg / ml, about 47.5 μg / ml to about 100 μg / ml, about 42.5 μg / ml to about 50 μg / ml, about 42.5 μg / ml to about 65 μg / ml, about 42.5 μg / ml to about 77.5 μg / ml, about 42.5 μg / ml to about 85 μg / ml, about 42.5 μg / ml to about 100 μg / ml, about 50 μg / ml to About 65μg / ml, about 50μg / ml to about 77.5μg / ml, about 50μg / ml to about 85μg / ml, about 50μg / ml to about 100μg / ml, about 65μg / ml to about 77.5μg / ml, about 65μg / ml to about 85μg / ml, about 65μg / ml to about 100μg / ml, about 77.5μg / ml to about 85μg / ml, about 77.5μg / ml to about 100μg / ml, or about 85μg / ml to about 100μg / ml. In some embodiments, the total collagenase concentration is about 25 μg / ml, about 32.5 μg / ml, about 47.5 μg / ml, about 42.5 μg / ml, about 50 μg / ml, about 65 μg / ml, about 77.5 μg / ml, about 85 μg / ml, or about 100 μg / ml. In some embodiments, the total collagenase concentration is at least about 25 μg / ml, about 32.5 μg / ml, about 47.5 μg / ml, about 42.5 μg / ml, about 50 μg / ml, about 65 μg / ml, about 77.5 μg / ml, or about 85 μg / ml.In some embodiments, the total collagenase concentration is at most about 32.5 μg / ml, about 47.5 μg / ml, about 42.5 μg / ml, about 50 μg / ml, about 65 μg / ml, about 77.5 μg / ml, about 85 μg / ml, or about 100 μg / ml.

[0285] In some embodiments, for each concentration, the mass ratios of C1 and C2 are 70:30, 54:46, 37:63, 82:18, and 90:10, respectively.

[0286] The volume to weight ratio of the digestion solution to the captured ground bone is about 1:1 to about 15:1, for example, about 5:1. In some embodiments, the ratio can be 1:1, 2.5:1, 5:1, 7.5:1, 10:1 and 15:1 (volume:weight). In some embodiments, the incubation period is about 1 hour to about 4 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours or about 2.5 hours. In some embodiments, the incubation period is up to about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, or about 4 hours. In some cases, the digestion solution is in contact with the captured ground bone for up to about 4 hours.

[0287] In some cases, it has been found that the optimal volume to weight ratio is 5: 1 at an optimal incubation time of 2.5 hours. The optimal protease produced a neutral protease activity of 19.6U / ml. On the other hand, it was found that the total viable MSC cell count was generally insensitive to the collagenase concentration. It was also found that the yields produced by recombinant collagenase isoforms Cl and C2 were similar to the yields of purified collagenase regardless of the Cl / C2 ratio. Further details of the MSC recovery process of the present disclosure are found in the technical article of Johnstone et al., "Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities" bioRxiv 2020.05.04.076950; doi.org / 10.1101 / 2020.05.04.076950, the entire disclosure of which is incorporated herein by reference.

[0288] According to this procedure, fragments of VB bone (fresh or cryopreserved) are placed in a cryoprotectant solution (containing PLASMA-LYTE TM , 2.5% human serum albumin and 10% dimethyl sulfoxide (DMSO)) and incubated at 4°C for 1 hour. The solution was removed and the bone fragments were cooled to -86°C at a rate of about 1° / min and then immersed in liquid nitrogen. After 24-48 hours in liquid nitrogen, the bone fragments were quickly thawed in a water bath set at 37°C, then washed in saline and digested with the above collagenase / protease solution.

[0289] Prediction of cell survival based on ischemia time

[0290] As described above, the ischemic time of the donor bone affects the survival rate of the cells extracted using the above process. According to the present disclosure, total ischemia is defined as the interval starting at the time of death (the time point when the donor's arterial system is cross-clamped and stops circulation) and ending at the beginning of cell recovery from the bone. For the purpose of statistical modeling, this total interval can be divided into three continuous and mutually exclusive time parts: (a) warm ischemic time (Warm Ischemia Time, WIT)-starting at the time of death and ending when the bone is recovered and packed on ice or when the body is placed in a cooler; (b) body cooling time (Body Cooling Time, BCT)-starting when the body is placed in a cooler and ending when the bone is packed on ice; and (c) cold ischemic time (Cold Ischemia Time, CIT)-starting when the bone is packed on ice and ending when processing begins to extract cells (such as HSPC). Therefore, total ischemic time = (WIT) + (BCT) + (CIT). For the case where no whole body cooling is used, BCT is zero and total ischemic time = (WIT) + (CIT).

[0291] In addition to total ischemic time, a variable corresponding to processing experience can also be incorporated into the survival determination. Learning curves are known to exert a significant influence on the results, so to control for this fact, the variable EXP can be defined as the number of donors processed before the current donor, i.e., for the i-th donor, EXP = i-1. Other variables can include bone type (e.g., vertebral and iliac), donor gender, and donor age.

[0292] In one aspect, the outcome variable is: the proportion of a particular cell population (e.g., CD34+ cells) that survives, per 10 5 The total number of colony forming units (CFU) detected in each nucleated cell, as well as the number of 5 The number of CFU-granulocyte macrophages (CFU-GM) detected in each nucleated cell.

[0293] According to the present disclosure, an ordinary least squares (OLS) beta regression model can be used to predict the outcome variable, a linear regression model is used for CFU and CFU-GM, and a beta regression model is used for the proportion of viable CD34+ cells, or %CD34+, where 0<(%CD34+)<1. The beta regression equation for predicting %CD34+ is:

[0294] The regression models were based on unadjusted models that only considered ischemia-based variables without considering experience, bone type, donor sex, and donor age variables. The fully adjusted models for %CD34+ considered all variables. The results of these models are described in Tables 1-3.

[0295] Table 1. %CD34+ coefficient values

[0296]

[0297] Table 2. CFU coefficient values

[0298]

[0299] Coefficient β 1 We attempted to quantify the effect of the number of donors processed (i.e., experience) on cell number and viability. In the fully adjusted CFU model, the coefficient β 2 Either or both of these coefficients may be modified, or even eliminated, corresponding to experience at a particular facility.

[0300] Table 3. CFU-GM coefficient values

[0301]

[0302] Applying these models to the observed data can be used to determine the effect of ischemic time variables on %CD34+, such as Figures 11A-11C The effects on total CFU are reflected in the table shown. Figures 12A-12C As shown in the table, the effect on the amount of CFU-GM, as Figures 13A-13C The data in these tables can be used to determine whether a particular donor bone can generate enough cells to warrant further processing of the donor bone. In other words, the predictive model can be used to establish ischemic tolerance limits and HSPC quality acceptance criteria. For example, with respect to the %CD34+ outcome variable, a predicted value of more than 80% may be required in order to consider a particular donor bone.

[0303] The above models and Figures 11A-11CThe examples shown in the table show that, despite the inevitable extension of ischemic time when bones must be purchased by geographically dispersed OPOs and shipped long distances to processing centers, acceptable HSPC quality levels are still achievable. Even under such conditions, a favorable combination of warm ischemic time and cold ischemic time can be achieved, so that %CD34+ survival rate can be in the range of 80-90%. These models also show that refrigerating the body before bone recovery (which is a common practice when recovering tissue) is less beneficial in the context of bone marrow recovery. For example, when whole body cooling is used, CD34+ survival rate averages 72.75%, while when body cooling is not used, the average is slightly less than 90%. These models show that best practices can be exempted from body cooling and move the recovered bone into a cold ischemic environment as soon as possible. These models further show that limiting WIT (warm ischemic time) to less than eight (8) hours and limiting CIT (cold ischemic time) to less than 40 hours optimizes the chance of recovering a meaningful amount of viable cells from donor bone.

[0304] The model disclosed herein predicted survival, with a threshold of 80% CD34+ cell survival determined to be acceptable. As reflected in the graph, the relationship between warm ischemia time and cold ischemia time follows a curve from the point where WIT is 10 hours and CIT is 18 hours to the point where WIT is 1 hour and CIT is 27 hours.

[0305] Further details of the methods of predicting cell viability of the present disclosure are found in Woods et al., “Ischemia considerations for the development of an organ and tissue donor derived bonemarrow bank.” J Transl Med 18, 300(2020).doi.org / 10.1186 / s12967-020-02470-1, the entire disclosure of which is incorporated herein by reference.

[0306] Store corpse BM

[0307] Typically, less than half of patients waiting for an allogeneic bone marrow (BM) transplant receive the required transplant. Live donor BM registries, BM cryopreservation and autologous transplantation, and umbilical cord blood storage have provided life-saving solutions for thousands of patients with at least one hematological disease; however, these methods are still subject to severe limitations associated with supply and logistics, and will benefit from the systems and methods of the present disclosure. In addition, although rare, adverse events from live bone marrow donations are also possible (i.e., the risk of death associated with bone marrow donation is 1:10,000), and although peripheral blood stem cell donations are currently more utilized, almost all of these donors will experience bone pain, 1 / 4 of the donors will have significant headaches, nausea or citric acid poisoning, and 1 / 5,000 of the donors will experience spleen rupture or other fatal complications. In addition, it is unclear what the long-term effects of administering stem cell mobilizers to donors during donation. The technical feasibility of cadaveric BM storage has been demonstrated in principle; however, there are still many challenges. These challenges are directly addressed by the present disclosure.

[0308] Storage BM disclosed herein provides a ready-made mechanism for providing BM to patients who have not yet identified a live donor match. In addition, it also provides a more effective method for providing BM to patients with live donor matches, because at least the delay associated with identifying donor matches, locating donor matches, and arranging donations is reduced. Therefore, for many patients with rapidly progressive diseases and poor prognosis, by allowing transplantation on demand and shortening the waiting time of these patients from multiple months to only 1-2 days, storage BM can greatly increase the survival rate after transplantation. Importantly, this method provides a large amount of BM from a single donor, enough to allow hematopoietic stem cells and progenitor cells (HSPC) to be implanted in several patients, and can achieve immediate repeated BM transplantation when needed. In addition, since a single donor provides enough BM for transplantation, it is not necessary to pool BM from multiple donors, or provide subsequent donations for different donors, each of which increases the possibility of adverse reactions due to allogeneic transplantation.

[0309] The methods and systems disclosed herein can provide a large amount of bone marrow (BM) supply on demand for national emergency preparedness. The billions of dollars of Project Bioshield of HHS, BARDA and the U.S. Department of Defense have fully documented the unmet urgent need for BM and stem cell transplantation on demand as a medical countermeasure for nuclear accidents or attacks. The present disclosure also provides the required BM for emerging applications such as immune tolerance induction. For this method, the key is to process and store BM from deceased organ donors and preserve BM during an extended period of time. In addition, for patients who receive organ transplants from deceased donors today, if BM from these donors is stored, it may benefit from this therapy when this therapy becomes available in the future, making this method immediately beneficial to important organ transplant recipients. In other words, compared with the currently utilized method, the system and method described in the present disclosure allows the collection and storage of an increased amount of bone marrow or bone marrow cells. If successful, other promising approaches and treatments being investigated have the potential to greatly enhance the value of cadaveric BM procurement and storage using the proposed approach, making large supplies of stored marrow immediately available to most recipients in need of rapid BM transplantation, particularly to address autoimmune disorders, genetic diseases, multiple sclerosis, and severe forms of type 1 diabetes.

[0310] The present disclosure provides a clinically oriented research protocol and system that is modified to be implemented in an industrial setting in a state-of-the-art cleanroom. One aspect of the disclosed system includes, among other things, cleaning of incoming donor bone, initial fragmentation using custom surgical stainless steel cutters, and grinding of the fragmented bone into bone fragments approximately 3 mm in size. These improvements provide a system in which a skilled tissue processing technician can process donor bone tissue within a 6 hour window to produce large amounts of viable bone marrow.

[0311] In the process described herein, it is an assessment of potential sources of deceased donor bone marrow. When processing donor long bones such as tibiae, it has been found that due to the conversion of red bone marrow into yellow bone marrow with age, red bone marrow is limited to the ends of long bones and varies greatly between different donors. It has also been determined that compared with complete red bone marrow (e.g., bone marrow from vertebral bodies or ilium), mixed yellow bone marrow-red bone marrow is of poor quality and mixed yellow bone marrow-red bone marrow contains fatty infiltration, thereby complicating subsequent processing. In some clinical experiments, the best donor long bones, compared with cells obtained from the ilium of the same donor, only produced 1 / 100 of BM cells / kg. Therefore, it has been determined that long bone processing is preferably performed only in special cases, such as bone marrow involving additional valuable "universal" HLA types or having HIV-resistant δ32 (CCR5-δ32) mutations.

[0312] In contrast, the vertebral bodies and iliac bones represent the largest sustainable reserves of high-quality red marrow. Utilizing one or both sources has optimized the recovery of bone marrow, particularly in the implementation of the industrialized scalable GMP process disclosed herein. Completion of the process disclosed herein results in cryopreservation of the final product configuration, which is a target storage volume of 60-70 ml at 100-150 million total nucleated cells (TNC) / ml in standard blood bags, similar to the product configuration of cryopreserved BM that has been used for autologous transplantation.

[0313] The present disclosure should be considered as illustrative rather than restrictive. It should be understood that only certain embodiments have been presented, and all changes, modifications, and further applications that fall within the spirit of the present disclosure are desired.

[0314] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is intended to be not limited to the specific examples provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be interpreted in a limiting sense. Without departing from the present invention, many variations, changes and substitutions can now be imagined by those skilled in the art. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportion stated herein according to various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted when practicing the present invention. Therefore, it is contemplated that the present invention will also cover any such substitutions, modifications, changes or equivalents. The appended claims are intended to define the scope of the present invention, and thus cover methods and structures and their equivalents within the scope of these claims.

[0315] In order to promote an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the accompanying drawings and described in the following written specification. It should be understood that this is not intended to limit the scope of the present disclosure. It should also be understood that the present disclosure includes any changes and modifications to the illustrated embodiments, and includes further applications of the principles disclosed herein that would normally be conceivable to a person skilled in the art to which the present disclosure belongs.

[0316] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein.

[0317] definition

[0318] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present disclosure, many modifications, changes and substitutions may now be conceived by those skilled in the art. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be adopted when practicing the present disclosure. The appended claims are intended to define the scope of the present disclosure, and therefore cover methods and structures and their equivalents within the scope of these claims.

[0319] The use of absolute or sequential terms, such as "shall," "shall not," "should," "shall not," "must not," "must not," "first," "initial," "next," "subsequently," "before," "after," "last," and "ultimately," are intended not to limit the scope of the present embodiments disclosed herein but rather to be exemplary.

[0320] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, to the extent that the terms "includes", "comprising", "having", "containing" or variations thereof are used in the detailed description and / or claims, these terms are intended to be encompassed in a manner similar to the term "comprising".

[0321] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are conjunctions and disjunctions in operation. For example, the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" each mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0322] As used herein, "or" may mean "and", "or", or "and / or", and may be used both exclusively and inclusively. For example, the term "A or B" may mean "A or B", "A but not B", "B but not A", and "A and B". In some cases, the context may determine the specific meaning.

[0323] Any systems, methods, software, and platforms described herein are modular. Therefore, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of operation.

[0324] When referring to a numerical value or a numerical range, the term "about" means that the numerical value or numerical range mentioned is an approximate value within the experimental variability (or statistical experimental error), and the numerical value or numerical range can vary, for example, from 1% to 15% of the numerical value or numerical range. In an example, the term "about" refers to ±10% of the number or value.

[0325] The term "from" in "from 1 to 10" includes the initial and final values ​​recited. Thus, "from 1 to 10" includes integers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and includes fractions thereof, (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and about 0.9).

[0326] The terms "increase", "enhancement" or "increase" used herein generally refer to an increase in a statistically significant amount relative to a reference level or historical control. Historical controls relate to data obtained from another subject or subject population, and the other subject is not treated according to the method of the present disclosure, and is similar to the subject in various characteristics (e.g., age, sex, health status, comorbidities, hematological cancer types and cancer severity). In some aspects, the term "increase" or "increase" refers to an increase of at least 10% compared to a reference level or historical control, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10-100%. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, ...

Claims

1. A method for processing a biological sample containing cells, the method comprising include: (a) generating an implantable unit of the biological sample comprising cells, wherein the implantable unit comprises a first volume of the cells at a first concentration; (b) generating a surrogate unit of the biological sample comprising cells, wherein the surrogate unit comprises a second volume of the cells at a second concentration, wherein the second volume is less than the first volume, and the second concentration differs from the first concentration by no more than 30%; as well as (c) cooling the cells of the transplantable unit in a first container and the cells of the surrogate unit in a second container at a common optimized rate that balances damage due to intracellular ice formation with damage due to extracellular ice formation, wherein the second container is thermally insulated; and (d) storing said transplantable unit and said replacement unit in the same long-term storage container, said long-term storage container exposing said cells of said transplantable unit and said cells of said replacement unit to similar conditions during the duration of long-term storage, thereby providing equivalent survival and viability for cells from the transplantable unit and cells from the surrogate unit, wherein the post-thaw cell proliferation rate of the cells of the surrogate unit differs by no more than 30% from the post-thaw cell proliferation rate of the cells of the transplantable unit, such that the surrogate unit accurately represents the transplantable unit, Wherein step (c) occurs in a static freezer or a controlled rate freezer.

2. The method of claim 1, wherein the transplantable unit and the replacement unit are located in the same freezer during a period of a first cooling rate and during a period of a second cooling rate. 3 . The method of claim 1 , wherein the second volume is less than 0.5% of the first volume to less than 50% of the first volume.

4. The method of claim 1, wherein the post-thaw survival rate of the cells in the transplantable unit differs from the post-thaw survival rate of the cells in the surrogate unit by no more than 10% to no more than 30%.

5. The method of claim 1, wherein the post-thaw cell proliferation rate of the cells of the transplantable unit differs from the post-thaw proliferation rate of the cells of the surrogate unit by no more than 5% to no more than 25%.

6. The method of claim 1, wherein the post-thaw viability of the cells of the transplantable unit and the cells of the surrogate unit is at least 50% to at least 90%.

7. The method of claim 1, wherein the post-thawing proliferation rate of the cells of the transplantable unit and / or the cells of the surrogate unit is at least 1 CFU-GM / 10 5 cells.

8. The method of claim 1, wherein the first cooling rate and the second cooling rate are super freezing rates of -0.1°C / min to -5°C / min until at least ice has nucleated in the freezing medium.

9. The method of claim 8, wherein the first cooling rate and the second cooling rate are super-freezing rates of -1°C / min to -4°C / min until at least when ice has nucleated in the freezing medium.

10. The method according to claim 8, wherein the first cooling rate and the second cooling rate are super freezing rates of -1°C / min.

11. The method of claim 1, wherein the freezer is set at -82°C to -90°C.

12. The method of claim 11, wherein the transplantable unit and the replacement unit are disposed in the same shelf of the freezer.

13. The method of claim 11, wherein the freezer is set at -86°C.

14. The method of claim 1, wherein the surrogate unit second volume is placed directly in an insulated container.

15. The method according to claim 1, wherein the method further include: The implantable unit and / or the replacement unit are arranged within the static freezer such that the implantable unit and / or the replacement unit do not contact a wall of the freezer.

16. The method of claim 1, wherein the cell-containing biological sample of the transplantable unit and the cell-containing biological sample of the surrogate unit are subjected to the same cooling rate.

17. The method of claim 16, wherein the cell is a stem cell or an immune cell.

18. The method of claim 17, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, or both.

19. The method of claim 1, wherein the biological sample is one or more organs, blood, or both.

20. The method of claim 1 further comprising the step of transferring the transplantable unit and the replacement unit to a long term storage container at less than -86°C such that the replacement unit experiences the same temperature fluctuations as the transplantable unit.

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