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 deceased donors, for preparing the bone marrow for cryopreservation, and for obtaining desired cells from cryopreserved and fresh bone marrow.

Description

SYSTEMS AND METHODS FOR EXTRACTION AND CRYOPRESERVATION OF BONE MARROWCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority7to U.S. Provisional Application No. 63 / 549911, entitled “SYSTEMS AND METHODS FOR EXTRACTION AND CRYOPRESERVATION OF BONE MARROW,’7which was filed February 5. 2024. The entire disclosure of which is expressly incorporated herein by reference.BACKGROUND

[0002] Bone marrow for clinical purposes is currently harvested from HLA matched siblings or optimally matched unrelated donors. Other graft sources are also now utilized including mismatched haploidentical related or unrelated donors and umbilical cord blood (CB). When transplanted into patients with certain diseases, the hematopoietic stem cells (HSCs) in the donor bone marrow engraft in the patient and reconstitute immune and hematopoietic systems. Bone marrow is also a good source for mesenchymal stromal / stem cells (MSCs) which are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell ty pes of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes.

[0003] Currently bone marrow is typically collected through a hole created in the cortical bone with a trocar needle and then using a bone marrow aspiration needle and a syringe to draw the marrow into the syringe. Multiple syringes are usually necessary to extract sufficient marrow from the bone. The syringes are then removed from the sterile field and each syringe is connected to a collection bag containing anticoagulants and the marrow is pushed into the bag. This step is repeated many times, typically in both pelvic bones, and can result in contamination of the aspirate.

[0004] It was recognized that whole bone marrow (BM) can be obtained from deceased donors. However, multiple barriers have prevented mainstream use of cadaveric bone marrow. One significant barrier has been in finding a streamlined process for controlled extraction and preservation of deceased donor bone marrow and the cell yields from that bone marrow. Another concern regarding the use of cadaveric bone relates to the cryopreservation and recovery7of the bone. In particular, the concern relates to the quality7of viable cells, such as HSCs, which can be obtained from cryopreserved donor bone.SUMMARY

[0005] An aspect of the present disclosure comprises a method for processing a biological sample comprising cells or a derivative thereof, the method comprising: generating a first volume of the biological sample comprising cells or a derivative thereof, wherein the first volume comprises a first concentration of cells or a derivative thereof; generating a second volume of the biological sample comprising cells or a derivative thereof, wherein the second volume is less than the first volume and comprises a second concentration of the cells wherein the second concentration of the cells is no more than 30% different than the first concentration of the cells; 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 is no more than 30% different than a post-thaw proliferation rate of the cells in the second volume. 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 a post-thaw viability rate of the cells in the first volume is no more than 30% different than a post-thaw viability rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 25% different than a post-thaw viability rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 20% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 15% different than a post-thaw viability' rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 13.6% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 10% different than a post-thaw viability7rate ofthe cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 25% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 20% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 15% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 13.6% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 10% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 5% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments the post-thaw viability rate of the cells is at least 50%. In some embodiments the post-thaw proliferation rate of the cells is at least 1 CFU-GM / 105cells. In some embodiments the first cooling rate and the second cooling rate comprise a suprafreeze rate from about -0. l°C / min to about -5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about -4°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about -3.5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a sub-freeze rate from about -l°C / min to about -2°C / min. In some embodiments the post-thaw viability rate of the cells is at least 60%. In some embodiments the post-thaw viability rate of the cells is at least 70%. In some embodiments the post-thaw viability7rate of the cells is at least 80%. In some embodiments the post-thaw viability rate 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 disposed in a first freezer of the one or more freezers. In some embodiments the first container is contained in a first freezer of the one or more freezers and the second container is contained in a second freezer of the one or more freezers. In some embodiments the one or more freezers comprise a static freezer. In some embodiments the first freezer, the second freezer, or both is a static freezer. In some embodiments, the one or more freezers comprise a controlled-rate freezer. In someembodiments the first freezer, the second freezer, or both is a controlled-rate freezer. In some embodiments the one or more freezers are set at about -70°C to -90°C. In some embodiments the one or more freezers are set at less than -80°C. In some embodiments the one or more freezers are set at -86°C. In some embodiments the second volume is placed directly in an insulating container, such that each vial is in close proximity to the insulating material of the insulating container. In some embodiments the method further comprises arranging the first volume inside the static freezer such that the first volume does not contact a wall of the one or more freezers. In some embodiments the biological sample comprising cells or a derivative thereof, in the first volume and the biological sample comprising cells or a derivative thereof, in the second volume experience a same cooling rate. In some embodiments the cells are stem cells or immune cells. In some embodiments the stem cells comprise 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 comprise T cells. In some embodiments the blood is cord blood or peripheral blood. In some embodiments the HSCs comprise CD34+ cells. In various embodiments, the method further comprises a step of transferring the first volume and the second volume to a long-term storage container, e g., a long-term storage container is colder than -86°C.

[0006] Another aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, the method comprising: generating a first volume of the bone marrow or a derivative thereof, wherein the first volume comprises a first concentration of the bone marrow or a derivative thereof; generating a second volume of the 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 is no more than 30% different than the first concentration; 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 a population of bone marrow derived cells in the first volume is no more than 30% different than a post-thaw proliferation rate of a population of bone marrow derived cells in the second volume. 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 secondvolume 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, a post-thaw viability rate of a population of bone marrow derived cells in the first volume is no more than 30% different than a post-thaw viability rate of a population of bone marrow derived cells in the second volume. In some embodiments, a post-thaw viability rate of the population of bone marrow derived cells in the first volume is no more than 25% different than a postthaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments, a post-thaw viability rate of the population of bone marrow derived cells in the first volume is no more than 20% different than a post-thaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments a postthaw viabili ty rate of the population of bone marrow derived cells in the first volume is no more than 15% different than a post-thaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments a post-thaw viability rate of the population of bone marrow derived cells in the first volume is no more than 13.6% different than a postthaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments, a post-thaw viability rate of the population of bone marrow derived cells in the first volume is no more than 10% different than a post-thaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments a postthaw viability rate of the population of bone marrow derived cells in the first volume is no more than 5% different than a post-thaw viability rate of the population of bone marrow derived cells in the second volume. In some embodiments, a post-thaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 25% different than a post-thaw proliferation rate of the population of bone marrow derived cells in the second volume. In some embodiments, a post-thaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 20% different than a post-thaw proliferation rate of the population of bone marrow derived cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 15% different than a post-thaw proliferation rate of the population of bone marrow derived cells in the second volume. In some embodiments a postthaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 13.6% different than a post-thaw proliferation rate of the population of bonemarrow derived cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 10% different than a post-thaw proliferation rate of the population of bone marrow derived cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the population of bone marrow derived cells in the first volume is no more than 5% different than a post-thaw proliferation rate of the population of bone marrow derived cells in the second volume. In some embodiments the post-thaw viability rate of a population of bone marrow derived cells is at least 50%. In some embodiments the post-thaw proliferation rate of a population of bone marrow derived cells is at least 1 CFU-GM / 105cells. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -0.TC / min to about - 5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about -4°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about - 2.5°C / min to about -3.5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a sub-freeze rate from about -rC / min to about -2°C / min. In some embodiments the post-thaw viability rate of a population of bone marrow derived cells is at least 60%. In some embodiments the post-thaw7viability rate of a population of bone marrow derived cells is at least 70%. In some embodiments the post-thaw viability rate of a population of bone marrow derived cells is at least 80%. In some embodiments the post-thaw viability rate of a population of bone marrow derived 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 disposed in a first freezer of the one or more freezers. In some embodiments the first container is contained in a first freezer of the one or more freezers and the second container is contained in a second freezer of the one or more freezers. In some embodiments the one or more freezers comprise a static freezer. In some embodiments the first freezer, the second freezer, or both is a static freezer. In some embodiments, the one or more freezers comprise a controlled-rate freezer. In some embodiments the first freezer, the second freezer, or both is a controlled-rate freezer. In some embodiments the one or more freezers are set at about -70°C to -90°C. In some embodiments the one or more freezers are set at less than -80°C. In some embodiments the one or more freezers are set at -86°C. In some embodiments the second volume is placed directly in an insulating container, such that each vial is in close proximity to the insulating material of the insulating container. In some embodiments the method further comprises arranging the firstvolume inside the static freezer such that the first volume does not contact a wall of the one or more freezers. In some embodiments the bone marrow or a derivative thereof, in the first volume and the bone marrow or a derivative thereof, in the second volume experience a same cooling rate. In some embodiments the bone marrow derived cells are stem cells or immune cells. In some embodiments the stem cells comprise hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), or both. In some embodiments the HSCs comprise CD34+ cells. In various embodiments, the method further comprises a step of transferring the first volume and the second volume to a long-term storage container, e.g., a long-term storage container is colder than -86°C.

[0007] Another aspect of the present disclosure comprises a method for processing MSCs, the method comprising: generating a first volume of the MSCs. wherein the first volume comprises a first concentration of the bone marrow or a derivative thereof; generating a second volume of the MSCs, wherein the second volume is less than the first volume and comprises a second concentration wherein the second concentration is no more than 30% different than the first concentration; 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 MSCs in the first volume is no more than 30% different than a post-thaw proliferation rate of the MSCs in the second volume. 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 a post-thaw viability rate of the MSCs in the first volume is no more than 30% different than a post-thaw viability rate of the MSCs in the second volume. In some embodiments a post-thaw viability rate of the MSCs in the first volume is no more than 25% different than a post-thaw viability rate of the MSCs in the second volume. In some embodiments, a post-thaw viability rate of the MSCs in the first volume is no more than20% different than a post-thaw viability rate of the MSCs in the second volume. In some embodiments, a post-thaw viability rate of the MSCs in the first volume is no more than 15% different than a post- thaw viability rate of the MSCs in the second volume. In some embodiments, a post-thaw viability rate of the MSCs in the first volume is no more than 13.6% different than a post- thaw viability rate of the MSCs in the second volume. In some embodiments a post-thaw viability rate of the MSCs in the first volume is no more than 10% different than a post- thaw viability rate of the MSCs in the second volume. In some embodiments, a post-thaw viability rate of the MSCs in the first volume is no more than 5% different than a post-thaw viability rate of the MSCs in the second volume. In some embodiments, a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 25% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments, a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 20% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 15% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 13.6% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 10% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments a post-thaw cell proliferation rate of the MSCs in the first volume is no more than 5% different than a post-thaw proliferation rate of the MSCs in the second volume. In some embodiments the post-thaw viability rate of the MSCs is at least 50%. In some embodiments the post-thaw proliferation rate of the MSCs is at least 1 CFU- GM / 105MSCs. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -0.1°C / min to about -5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about -4°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about - 3.5°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a sub-freeze rate from about -l°C / min to about -2°C / min. In some embodiments the post-thaw viability rate of the MSCs is at least 60%. In some embodiments the post-thaw viability rate of the MSCs is at least 70%. In some embodiments the post-thaw viability’ rate of the MSCs is at least 80%. In some embodimentsthe post-thaw viability rate of the MSCs 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 of the one or more freezers. In some embodiments the first container is contained in a first freezer of the one or more freezers and the second container is contained in a second freezer of the one or more freezers. In some embodiments the one or more freezers comprise a static freezer. In some embodiments the first freezer, the second freezer, or both is a static freezer. In some embodiments the one or more freezers comprise a controlled-rate freezer. In some embodiments the first freezer, the second freezer, or both is a controlled-rate freezer. In some embodiments the one or more freezers are set at about -70°C to -90°C. In some embodiments the one or more freezers are set at less than -80°C. In some embodiments the one or more freezers are set at -86°C. In some embodiments the second volume is placed directly in an insulating container, such that each vial is in close proximity to the insulating material of the insulating container. In some embodiments the method further comprises arranging the first volume inside the static freezer such that the first volume does not contact a 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-MSC) or vertebral bone adherent MSCs (vBA- MSC).

[0008] Another aspect of the present disclosure comprises a method for processing a biological sample comprising cells or a derivative thereof, the method comprising: generating a first volume of the biological sample comprising cells or a derivative thereof, wherein the first volume comprises a first concentration of cells or a derivative thereof; generating a second volume of the biological sample comprising cells or a derivative thereof, wherein the second volume is less than the first volume and comprises a second concentration of the cells wherein the second concentration of the cells is no more than 30% different than the first concentration of the cells; generating a freezing curve specific for the cells; cooling the first volume at a first cooling rate, wherein the first cooling rate is generated from the freezing curve; and cooling the second volume at a second cooling rate, wherein the first cooling rate is generated from the freezing curve; wherein the first cooling rate is about the same as than the second cooling rate and wherein a post-thaw cell proliferation rate of the cells in the first volume is no more than 30% different than a post-thaw proliferation rate of the cells in the second volume. 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 volumeis 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 a post-thaw viability rate of the cells in the first volume is no more than 30% different than a post-thaw viability rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 25% different than a post-thaw viability rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 20% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 15% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 13.6% different than a post-thaw viability7rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 10% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 25% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 20% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 15% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 13.6% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 10% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 5% different than a post-thaw proliferation rate of the cells in the second volume. In someembodiments the post-thaw viability rate of the cells is at least 50%. In some embodiments the post-thaw proliferation rate of the cells is at least 1 CFU-GM / 105cells. In some embodiments the post-thaw viability rate of the cells is at least 60%. In some embodiments the post-thaw viability rate of the cells is at least 70%. In some embodiments the post-thaw viability rate of the cells is at least 80%. In some embodiments the post-thaw viability rate 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 disposed in a first freezer of the one or more freezers. In some embodiments the first container is contained in a first freezer of the one or more freezers and the second container is contained in a second freezer of the one or more freezers. In some embodiments the one or more freezers comprise a static freezer. In some embodiments the first freezer, the second freezer, or both is a static freezer. In some embodiments, the one or more freezers comprise a controlled-rate freezer. In some embodiments the first freezer, the second freezer, or both is a controlled-rate freezer. In some embodiments the one or more freezers are set at about -70°C to -90°C. In some embodiments the one or more freezers are set at less than -80°C. In some embodiments the one or more freezers are set at -86°C. In some embodiments the second volume is placed directly in an insulating container, such that each vial is in close proximity to the insulating material of the insulating container. In some embodiments the method further comprises arranging the first volume inside the static freezer such that the first volume does not contact a wall of the one or more freezers. In some embodiments the biological sample comprising cells or a derivative thereof, in the first volume and the biological sample comprising cells or a derivative thereof, in the second volume experience a same cooling rate. In some embodiments the cells are stem cells or immune cells. In some embodiments the stem cells comprise 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 comprise T cells. In some embodiments the blood is cord blood or peripheral blood. In some embodiments the HSCs comprise CD34+ cells. In various embodiments, the method further comprises a step of transferring the first volume and the second volume to a long-term storage container, e.g.. a long-term storage container is colder than -86°C.

[0009] Another aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow7or the 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 bone fragments; extracting the bone marrow or the derivative thereof from the bone or bone fragment; and cry opreserving the bonemarrow or the derivative thereof, wherein the cry opreserving comprises decreasing temperature of the bone marrow or the derivative thereof at a freeze rate of more than about - rC / min in a static temperature freezer. In some embodiments, the cry opreserving comprises cooling the bone marrow or the derivative thereof at a supra-freeze rate from about -2.5°C / min to about -5°C / min at least until ice has nucleated in a freezing medium. In some embodiments, the cryopreserving comprises cooling the bone marrow or the derivative thereof at a supra- freeze rate from about -2.5°C / min to about -4°C / min at least until ice has nucleated in a freezing medium. In some embodiments, the cryopreserving comprises cooling the bone marrow or the derivative thereof at a supra-freeze rate from about -2.5°C / min to about - 3.5°C / min at least until ice has nucleated in a freezing medium. In some embodiments, the cry opreserving comprises cooling the bone marrow or the derivative thereof at a sub-freeze rate from about -l°C / min to about -2°C / min. In some embodiments, the supra-freeze rate and the sub-freeze rate are maintained without the use of a passive cool box. In some embodiments, the cryopreserving comprises arranging one or more aliquots of the bone marrow or the derivative thereof inside the static freezer such that no aliquot contacts a wall of the static freezer. In various embodiments, within the static freezer, no aliquot of the bone marrow or the derivative thereof is stored directly on top of another aliquot of the bone marrow or the derivative thereof. In some embodiments, the bone marrow or the derivative thereof comprises a population of CD34+ cells. In some embodiments, the population of CD34+ cells comprises at least 70% viable CD34+ cells after the bone marrow or the derivative thereof is thawed. In some embodiments, the population of CD34+ cells comprises at least 80% viable CD34+ cells after the bone marrow or the derivative thereof 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.

[0010] An aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a 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 submerged in the bleach solution; extracting the bone marrow or the derivative thereof from the bone, wherein at least 90% of CD34+cells comprised in the bone marrow or the derivative thereof are viable. In some embodiments, the bone marrow or derivative thereof is contacted with the bleach solution for at least about 25 minutes. In some embodiments, the bleach solution comprises 10% bleach. In some embodiments, the bone is a vertebral body. In some embodiments, the hydrogen peroxide is a 3% hydrogen peroxidesolution. In some embodiments, the method further comprises transferring the bleached bone product from a container comprising the bleach solution to a container containing the hydrogen peroxide solution. In some embodiments, the method further comprises agitating the bleached bone product within the hydrogen peroxide solution. In some embodiments, c) of submerging the bleached bone product in a solution that includes hydrogen peroxide includes submerging the bleached bone product in a container containing the hydrogen peroxide solution, detecting foam or froth associated with the bleached bone product, and repeating i and / or ii until no foam or froth is detected. In some embodiments, the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth in ii. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.

[0011] Yet another aspect of the present disclosure is a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the 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 bone fragments; mechanically grinding the bone or bone fragment in the presence of a grinding solution to generate a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 rotations per minute (“RPM”) for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises the bone marrow or the derivative thereof and wherein the bone marrow or the 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, 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, 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 CD34+ cells / ml, 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, 2,000,000CD34+ cells / ml, 3,000,000 CD34+ cells / ml, 5,000,000 CD34+ cells / ml, or 10,000,000 CD34+ cells / ml or more, of the bone marrow or the derivative thereof.

[0012] In some embodiments, the method further comprises, contacting the solution with a rinse media and repeating c. and then removing the solution from the shaker. In some embodiments, the method further comprises repeating step c. and then removing the solution from the shaker one or more times. In some embodiments, the at least about 1.500,000 CD34+ cells / ml of the bone marrow or the derivative thereof comprises at least 85% viable CD34+ cells. In some embodiments, the at least about 1,500,000 CD34+ cells / ml of the bone marrow or the derivative thereof comprises at least 90% viable CD34+ cells.

[0013] Another aspect of the present disclosure comprises a method for processing a population of CD34+ cells obtained from bone marrow or a derivative thereof, wherein the bone marrow or the 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 bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U / ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80.000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer. In some embodiments, the at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 70% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 80% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 90% viable CD34+ cells. In some embodiments, the stabilization buffer comprises more than about 5 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 10 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 15 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises about 20 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 20 U / ml of a nuclease. In some embodiments, the nucleases are Benzonase® or Denarase®. In some embodiments, the stabilization buffer further comprises more than about 5 U / ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises more thanabout 10 U / ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises about 10 U / ml of an anticoagulant. 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.

[0014] Another aspect of the present disclosure relates to a stabilization buffer that includes at least 5 U / ml of an anticoagulant, and more than 3 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 5 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 10 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 15 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more 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 Benzonase® or Denarase®. In some embodiments, the stabilization buffer further comprises more than about 10 U / ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises about 10 U / ml of an anticoagulant. 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.

[0015] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

[0016] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the invention are set forth with particularity 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 that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "Figure ' and “FIG.” herein), of which:

[0018] FIG. 1 is a view of a filtration system according to one feature of the present disclosure.

[0019] FIG. 2 is a view of a sterile bag containing a bone marrow pellet processed according to the methods of the present disclosure.

[0020] FIG. 3 is a view of the sterile bag of FIG. 2 with a clip engaging the bag to separate the fat from the bone marrow pellet.

[0021] FIG. 4 shows the set-up for isolation of the bone marrow pellet.

[0022] FIG. 5 is a perspective view of a cooling box according to one aspect of the present disclosure.

[0023] FIG. 6 is a flowchart of one method according to the present disclosure.

[0024] FIGS. 7A and 7B are side and perspective views of an automated bone processing system according to one aspect of the present disclosure.

[0025] FIGS. 8A and 8B are perspective views of a bone debriding station of the system shown in FIGS. 7A and 7B.

[0026] FIGS. 9A and 9B are perspective and front views of a bone grinding station of the system shown in FIG. 7A and FIG. 7B.

[0027] FIG. 10 is a perspective view of a sieve station of the system shown in FIGS. 7A and FIG. 7B.

[0028] FIGS. 11A-11C are tables of CD34+ cell viability as a function of warm and cold ischemia times, without and without body cooling.

[0029] FIGS. 12A-12C are tables ofCFU-Total as afunction ofwarm and cold ischemia times, with and without body cooling.

[0030] FIGS. 13A-13C are tables ofCFU-Total as afunction of arm and cold ischemiatimes, with and without body cooling.

[0031] FIG. 14 illustrates HPC, Marrow experimental trials cassette location in shelf one of the -86°C Eppendorf Cryocube Model F740hi.

[0032] FIG. 15 illustrates an exemplary alternative arrangement of the cassette location in shelf one of the -86°C Eppendorf Cryocube Model F740hi.

[0033] FIG. 16 illustrates an example of graph used to determine supra / sub-freeze cooling rates and nucleation temperatures.

[0034] FIGs. 17A-17E illustrate the prevention of formation of aggregates when the bone marrow cells w ere processed from chilled sample with the stabilization buffer. FIG. 17A shows the bone marrow7cells slurry after antibody labeling. The numerical numbering corresponds to the buffer used. Bone marrow cell sample processed with the stabilization buffer (4) exhibited absence of aggregates. FIG. 17B shows lack of aggregate being trapped after filtration in the bone marrow cell sample processed with the stabilization buffer. FIG. 17C and FIG. 17Dillustrate the formation of aggregates of bone marrow cells processed with CliniMACS buffer (FIG. 17C) or absence of aggregates of bone marrow cells processed with the stabilization buffer (FIG. 17D). FIG. 17E shows that the bone marrow cells processed with the stabilization buffer exhibited increased yield of viability and CD34 expression of bone marrow cells. Purity was greater than 60%, and above 60% CD34 cells were recovered. The ratio between CD3 count and CD34 count was 0.5% (e.g. 5 cells expressing CD3 per 100 cells expressing CD34).

[0035] FIG. 18 illustrates an exemplary cleanroom diagram for the Cleanroom C in Example 9.

[0036] FIG. 19 illustrates an exemplary' cleanroom workflow for decontamination of VBs as described in Example 9.

[0037] FIG. 20 illustrates an exemplary flow cytometry plots of human CD34+ in blood at 8 weeks.

[0038] FIG. 21 illustrates gating strategy7used to determine phenoty pes of BM cells isolated from deceased and living donors.

[0039] FIG. 22 illustrates relative and absolute values for CD45+ leukocytes, CD34+ HSPC and CD3+ T cells in living versus deceased donor BM. Bars represent averages + / - standard deviations.

[0040] FIG. 23 illustrates CFU potential comparison between HPC, Marrow and living donor BM.

[0041] FIG. 24 illustrates similar viability and number of CD34+ HSC isolated from organ donor (OD) and living donor (LD) BM. Means from five studies.

[0042] FIG. 25 illustrates levels of human CD45+ cells in bone marrow of irradiated NSG mice 16 weeks after injection of CD34+ cells. Sham control is bone marrow from nonirradiated, untreated mice. Cell surface CD45 expression was determined by flow cytometry. Thick bars represent means of N= 5 (cord blood) or 10 (HPC, Marrow) mice. Standard deviations are shown by thick vertical lines.

[0043] FIG. 26 illustrates the percentage of human CD45+ and CD34+ cells in bone marrow (BM), peripheral blood (PB) and spleens 16 weeks after irradiation and transplantation of NSG mice with CD34+ cells.

[0044] FIG. 27 depicts secondary transplants. Levels of human CD45+ cells in bone marrow of irradiated NSG mice 16 weeks after injection of total bone marrow from mice engrafted with CD34+ cells from the indicated donors. Sham control is bone marrow from non-irradiated, untreated mice. Cell surface CD45 expression was determined by flow cytometry. Thick bars represent means of N= 10 mice. Standard deviations are shown by thick vertical lines.

[0045] FIG. 28 is an overall continuous manufacturing and process control flowchart that produces cryopreserved bone marrow ( HPC. Marrow”) and as described in Example 16.

[0046] FIG. 29 shows engraftment of frozen / thawed CD34+ cells isolated from Fresh (Fresh or freeze-thaw (“FT”) lx) or Cryopreserved (Frozen FT 2x) BM transplanted into NSG mice.

[0047] The novel features of the 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 that sets forth illustrative embodiments.DETAILED DESCRIPTION

[0048] The compositions, systems, and methods disclosed herein provide a needed complement to existing bone marrow and stem cell sources. Specifically, the compositions, systems, and methods disclosed herein provide techniques for the isolation, processing, and use of bone marrow and hematopoietic stem cells (“HSCs”) from human cadavers. Features that are unique to the compositions, systems, and methods described herein result in improvements to the current state of the art. These features result in significantly improved yield of functional bone marrow and HSCs, lessening the overall burden on resources typically required for bone marrow and HSC isolation, processing, and use.

[0049] The compositions, systems, and methods disclosed herein provide a departure from the isolation and processing techniques of bone marrow and HSCs known in the art, particularly those techniques utilized for processing bone marrow and HSCs from living donors. The present disclosure describes various embodiments of the processing techniques utilized to generate the bone marrow and HSC compositions described herein.

[0050] As described below, various bones are removed from deceased donors and prepared for mechanic and enzy matic processing. The bones are then mechanically processed where bone marrow and / or bone marrow derived cells (e.g. HSCs) filtered to produce viable bone marrow (and HSCs contained in or adjacent to the bone marrow) compositions. The bone and cellular compositions are further mechanically / enzymatically processed to produce optimal yields of viable cells. These processing steps provide deviations from the current state of the art that result in improved cellular compositions and methods of processing.

[0051] The bone marrow is then further processed for either immediate use or preservation. In some embodiments, the bone marrow is further processed to generate cellular compositions comprising specific HSC populations (e.g. CD34+ cells). Optimized compositions, systems.and methods are described herein, providing for improved bone marrow and HSC processing techniques and compositions.

[0052] These optimized compositions, systems, and methods described herein present unique solutions to the current problems realized by medical practitioners (e.g. immunology, regenerative medicine). Utilizing the optimized compositions, systems, and methods described herein can result in bone marrow “banks'’ or depots that will have more viable cells than current depots, and the 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, resulting in a larger population of potential subjects that can benefit from the bone marrow depots generated using the compositions, systems, and methods described herein.

[0053] Aspects of the present disclosure provide a cryopreserved cell product that is divided into two volumes, with a first volume (e.g., cryopreservation bag) for containing the cell product for transplant into a subject in need thereof and a second volume that acts as a surrogate for the first volume. As used herein, a surrogate vial is typically a smaller volume of the cell product and the surrogate can be thawed and assayed as needed, e.g.. for cell viability (and especially “function viability ” as determined by post-thaw proliferation). The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using the surrogate it is unnecessary to thaw the first volume for assaying and, instead, it is thawed when needing to be used, e.g.. for transplanting into a subject in need.

[0054] Without wishing to be bound by theory, for a given cell type, a specific, optimum cooling rate is required for that cell type or cell product to survive cryopreservation. This optimum cooling rate balances damage from intracellular ice formation (IIF) with damage from high solute concentration resulting from extracellular ice formation. If cells are cooled too fast, damaging IIF is likely; if cells are cooled too slowly, damaging solute effects are likely. For the surrogate vial to accurately represent the first volume, the cells in both volumes should be frozen at about the same rate, for example, the optimized rate; this common rate results in equivalent survival and viability for cells in the two volumes. Importantly, the second volume may be stored in the same long-term storage system as the first volume, and will therefore be exposed to the same conditions over long-term storage durations; thus, promoting the ability of the surrogate vial to accurate represent the cryopreservation bag that contains cell for transplant. These ultimately allow- the second volume to be tested to determine, at least, if storage of the cryopreservation bag was maintained appropriately and without having to manipulate and test the cells of the first volume. More specifically, by assaying the surrogatevial, the cry opreservation bag does not need to be warmed and / or handled prior to its immediate use. This feature is especially helpful to a subject’s outcome and welfare in two ways. First, the cells for transplanting are thawed only when ready to be administered to the subject (and preferably at the site of administration) rather than being thawed two weeks or so before 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 - could adversely affect their viability and uti 1 i ty once transplanted. Second, since a subject likely undergoes myeloablative conditioning prior to transplant, the patient can forestall myeloablative conditioning until a cell product has been assayed and determined to be suitable for use, which usually takes about two weeks; by having an surrogate vial, a subject begins myeloablative conditioning once a suitable product has been identified, thereby shortening the length of time that the subject remains immune compromised.

[0055] The present disclosure provides methods for ensuring that the two volumes cool at the same rate. Based, at least, on laws of physics, a smaller volume of a cell product will cool at a faster rate than a larger volume of the cell product. And, the smaller volume, with a faster cooling rate, should have increased IIF relative to the larger volume, which has a slower cooling rate. Methods of the present disclosure promote an equivalent rate of cell cooling between the first (larger) volume and the second (smaller) volume such that each volume will have similar amounts of IFF and, as such, the surrogate vial (smaller volume) will accurately represent the larger first volume. Without wishing to be bound by theory, methods of the present disclosure slow the rate of cooling for the second (smaller) volume to the rate experienced by the first (larger) volume based, in part, on use of different types of containers that directly holds or indirectly holds either a first volume or the second volume and / or positioning of containers within the same freezer, e.g., a static temperature freezer. As a result, cells in the smaller volume (for example, the second volume / surrogate vial) and cells in the larger volume (for example, the first vol ume / cry opreservation bag) experience similar rates of cooling (e.g., about -l°C / minute) 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, a surrogate vial is placed directly into an insulated vial container, e.g.. CoolCell® freezing storage system, which when placed in a static freezer that is colder than -80°C, e.g., a -86°C static freezer, the cells in the surrogate vial experience rate of freezing at the rate of about -l°C / minute; without use of the insulated vial container, the cells in the surrogate vial would experience rate of freezing at the rate of about - 10°C / minute, which would likely cause damage from IIF On the other hand, the larger first volume, cry opreservation bag, does not need to be directly placed in an insulated container andinstead, when the bags are placed in cassettes - which are not insulated (to avoid slowing the cooling rate of the bag) - and moved to -86°C static freezer, the cells in the cry opreservation bag preferably experience freezing rate of about -l°C / minute. By '‘directly placed” means that each vial is in close proximity to the insulating material of the insulating container. These manipulation cause cells of the first and the second volumes to have roughly equivalent osmosis of intracellular water into the extracellular space for cells; this osmosis increases the solute concentration intracellularly, and helps avoid formation of (harmful) intracellular ice crystals and promotes extracellular ice formation (which is less harmful to the cell). Once the first step of freezing (in the -86°C static freezer), the cr o preservation bag and the surrogate vial are placed in the same long-term storage device (e g., a liquid nitrogen storage tank) and in a roughly similar position within the long-term storage device.

[0056] Accordingly, methods of the present disclosure allow production of a surrogate sample of the cell product that is expected to accurately represent the portion of the cell product that is to be administered to a subject in need thereof and provides a cell product that is, not only therapeutically beneficial to the subject, but promotes subject’s outcome and welfare in ways that are not achievable when a cryopreserved cell product in merely contained in a single bag and without a surrogate vial.Preparing donor bone

[0057] The vertebral body and the ilium represent the largest consistent reservoirs of high- quality red marrow. Utilizing one or both sources has optimized the recovery of bone marrow, particularly with the implementation of an industrialized, scalable, GMP process disclosed herein. In some embodiments, completion of the process disclosed herein results in cry opreservation of a final product of storing a 60-70 ml volume at a target of 100-150 million total nucleated cells / ml in standard blood bags. In some cases, the methods of manufacturing provide a system in which skilled tissue processing technicians can process sets of donor bones within a six-hour window to yield meaningful quantities of viable marrow.

[0058] In some embodiments, the donor bone is vertebral bodies. However, it is understood that the methods described herein can be used on the ilium, a combination of the vertebral bodies and ilium, or other bones suitable for extraction of bone marrow and cells from the marrow, even donor bones with lower expected yields.

[0059] It is understood that the donor bones can be procured according to fixed protocols for clinical recovery. Bones can be recovered by surgeons or by personnel at a trained organ procurement organization (OPO) using an osteotome and mallet from consented organ and tissue donors. Vertebral segments must be carefully recovered, preferably from the thoracicand lumbar vertebrae. The segments are incised and removed using an osteotome and mallet. As much of the spinal cord as possible is removed. A licensed surgeon may have oversight of these steps to assure effective recovery of VBs and prevention of disease transmission and translocation of bacteria.

[0060] Once recovered, the vertebral segments are swabbed for microbial culture testing and placed in a sterile, labeled bag with saline-soaked sterile pads, sponges, or towels to ensure moisture retention during hypothermic shipment. These are then positioned between wet ice packs in a cooler for shipment. Recovery of VBs must occur with a minimal warm ischemia time (< 8 hours). Shipment and initiation of processing must be completed within a minimal cold ischemia time (< 40 hours). The package is finally shipped to a processing facility.

[0061] VB logs are wrapped and double bagged. Bags are placed in an insulated shipper with bagged wet ice surrounding them. Shippers are sealed and sent to Ossium via medical courier. Upon arrival, packaging is checked for compliance with protocols and vertebral body temperature is measured to ensure compliance with shipping requirements.

[0062] The process for preparing the donor bone can occur soon after the bone is obtained from the deceased donor or can occur after the donor bone has been shipped in a hypothermic environment to a processing facility. Since the donor bone can experience prolonged periods of ischemia during recovery and shipment 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, bone subject to predetermined periods of warm ischemia and / or cold ischemia are suitable for obtaining meaningful quantities of viable bone marrow cells.

[0063] During the processing of the donor bone, the bone is debrided in an ISO-5 (class 100) environment (biosafety cabinet) with an ISO-7 (class 10,000) background (clean room), with special care taken to sterilize the bag containing the donor bone, such as by spraying with 70% isopropanol. In one embodiment, the debridement is conducted manually using scalpels, osteotomes and gouges. In processing vertebrae, typically a spinal segment including multiple vertebral levels will be provided. In a typical case, the spine segment runs from T8 to L5, for ten vertebral bodies. During initial debridement of the spinal segment, when enough soft tissue has been removed to visualize the pedicles, the pedicles are removed using either a tissue processing band saw or a bone saw, such as the Stryker System 6 Saw (Stryker, Kalamazoo, MI), or with the hand tool shown in FIG. 1A to FIG. ID. Special care is taken to avoid breaching the cortical bone which would expose the cancellous bone, to ensure that the hypoxic cancellous bone marrow remains protected throughout the entire debriding process. Theanterior element of the vertebral bodies, which contain the cancellous bone material, remain, while the pedicles and posterior elements are discarded.

[0064] Using a boning knife or tissue processing band saw, the vertebral bodies (VB) are separated at the intervertebral discs. The intervertebral disc and soft tissue remaining on each vertebral body is removed with a scalpel, scissors and / or osteotomes, leaving clean, separated VBs. In the case of donor ilium, the soft tissue can be removed with gouges and a scalpel, with special care taken to ensure that the cortical bone is not breached. Any anatomical pathologies or injuries of the bone are noted and recorded as part of the batch record for the marrow ultimately obtained from the bones. Bones damaged during the recovery' process are discarded.

[0065] In some cases, cadaver bones undergo a “pre-processing” to reduce contaminants carried by the cadaver bone and which risk transferring the contamination to the facility that the bone is processed. In these cases, two technicians perform different aspects of the preprocessing. A first technician opens a package containing harvested cadaver bones, preferably contained in a sealed, inner bag. The second technician, wearing sterile gloves, removes the cadaver bone from the package and places the tissue in a first (rinse) basin. The second technician scrubs all surfaces of cadaver bone vigorously with an about 4% chlorhexidine gluconate solution for about 3 minutes while in or above the rinse basin. The first technician, wearing sterile gloves, pours sterile saline onto the scrubbed cadaver bone, with the runoff being captured in the rinse basin. A sufficient amount of saline is poured onto the cadaver bone to rinse all of its surfaces. The rinsed cadaver bone is then placed on a sterile cloth adjacent to the rinse basin. The saline rinse may be repeated as necessary. Alcohol, e.g., 70% isopropyl alcohol, is poured over the cadaver bone. A sufficient amount of alcohol is poured onto the cadaver bone to contact all of its surfaces. The alcohol runoff is captured in the rinse basin. The cadaver bone is placed in an open container which is sprayed with alcohol and then the open container and bone is transferred to a hood, where further processing of the bone can take place.

[0066] An aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a 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 submerged in the bleach solution; extracting the bone marrow or the derivative thereof from the bone, wherein at least 90% of CD34+ cells comprised in the bone marrow or the derivative thereof are viable. In some embodiments, the bone marrow or derivative thereof is contacted with the bleach solution for at least about 25 minutes. In someembodiments, the bleach solution comprises 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 comprising 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 within the hydrogen peroxide solution. In some embodiments, the submerging the bleached bone product in a solution that includes hydrogen peroxide includes submerging the bleached bone product in a container containing the hydrogen peroxide solution, detecting foam or froth associated with the bleached bone product, and repeating the submerging until no foam or froth is detected. In some embodiments, the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.

[0067] In some embodiments, the bone in the bleach solution is agitated (e.g., shaken).

[0068] The VBs. either pre-processed or not, are placed into a sterile bag and submerged in an about 10% bleach solution (0.5% sodium hypochlorite in sterile water), yielding a concentration of 5,000 ppm free chlorine, for a predetermined period, ty pically from about 5 minutes to about 25 minutes, e.g., 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 a toxic residue, is unaffected by water hardness and is fast acting.

[0069] Bone marrow from each group of VBs processed at different duration of bleach treatment can be tested by' flow cytometry' to assess the viability of the cells isolated from the bone marrow. As exemplified in Table 4, soaking the VBs for more than 10 minutes yields no significant difference in cell viability compared to when the VBs are soaked for up to 25 minutes. However, without wishing to be bound by theory, an increase in bleaching time improves the ultimate product. For example, increasing the soaking of the VBs in bleach for longer period of time allows the bleach to fill the cavity' or crevice of the VBs to further decontaminate or sterilize the VBs.

[0070] The bleach solution may be from about 5% bleach to about 15% bleach, e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or about 15% bleach. In some embodiments,the bleach treatment comprises using 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%. 15%. 20%, 25%, 30%, 35%, 40%, 45%, 50%, or higher percentage of bleach. In some embodiments, the bleach treatment comprises contacting the VBs with 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 VBs are submerged in the bleach solution for at least about 10 minutes to at least about 25 minutes, e.g., about 10 to about 12 minutes, about 1 1 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 viability of the bone marrow cells isolated from the VBs treated with the bleach treatment is not decreased at any duration of bleach treatment described herein compared to bone marrow cells isolated from the VBs without the bleach treatment. In some embodiments, the viabi li ty of the bone marrow cells isolated from the VBs treated with 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 of the bleach treatment is not decreased or is decreased by less than 3% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 2% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 1% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment.

[0071] Interestingly, bleach treatment provides surface sterilization of the bone, but does not penetrate the BM-containing compartment. Therefore, the bleach treatments disclosed herein do not substantially reduce the yield of viable cells obtained from BM.

[0072] In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80% to about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in 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 comprised in the bone marrow or derivative thereof extractedfrom the bone submerged in bleach is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80%, about 85%, or about 90%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at most about 85%. about 90%, or about 95%.

[0073] At the end of the bleaching period, the bones are transferred to another sterile bag and submerged in a 3% hydrogen peroxide (H2O2) solution. In some cases, the H2O2 solution comprises PLASMA-LYTE™ (a multiple electrolyte injection that is a sterile, nonpyrogenic isotonic solution that is a base source of water and electrolyte-balanced crystalloids for the cells, obtained from Baxter Healthcare, Ltd.). In some cases, the H2O2 solution comprises PLASMA-LYTE™ and Human Serum Albumin (HSA) which is a stabilizing reagent and storage agent (it may be diluted in the H2O2 solution to achieve 2.5% HSA). The bag is closed and shaken briefly to ensure that the entire surface of the bone is in contact with the solution. Most living cells include catalase, which is an enzyme that catalyzes the break down of H2O2 into H2O and O2. This breakdown manifests as foam or froth when the H2O2 solution contacts soft tissue but not bone. The foam level can be observed as an indication of the amount of soft tissue remaining on the bone. This observation can be performed manually by a human processor or. in another embodiment, by an automated processor. The automated processor incorporates a visualization device, such as a camera, and object recognition software that can determine foam levels within the bag. The addition of an inert contrast dye can help the human or automated processor detect the foam level. If any foam or froth is observed, the bone is returned for further processing to remove all of the remaining soft tissue from the bone. Once the VBs or ilium has been cleaned of all soft tissue, the bones are transferred to a new sterile bag. The bag is filled with IL of PLASMA-LYTE™, or other suitable sterile, nonpyrogenic isotonic solution. The bag is closed and shaken briefly to ensure that the entire bone is contacted with the PLASMA-LYTE™.

[0074] In some embodiments, the method further comprises a step of agitating the bleached bone product within the hydrogen peroxide solution. In some embodiments, the submerging the bleached bone product in a solution that includes hydrogen peroxide includes submerging the bleached bone product in a container containing the hydrogen peroxide solution, detecting foam or froth associated with the bleached bone product, and repeating the submerging until no foam or froth is detected. In some embodiments, the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth. Insome embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.

[0075] The bleaching step and the hydrogen peroxide steps may be repeated multiple times.

[0076] Without wishing to be bound by theory, it is believed that the H2O2 solution not only helps surface sterilize the bone, but it also helps break down any residual bleach into salt, oxygen, and water.

[0077] After the surface sterilization, the cadaver bone may be rinsed with water, a saline, or with a cryoprotectant solution. Then the surface sterilized cadaver bone may be placed in a closed container comprising a cryoprotectant solution and the pressure is reduced.Cryoprotectant infiltration into cadaver bone

[0078] Cadaver bone can be contacted with a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of a cryoprotectant solution into the cadaver bone. Methods for cryopreserving bone is described below and elsewhere. In some cases, the conditions sufficient to allow infiltration of a cryoprotectant solution involve use of vacuum- assisted infiltration of a cryoprotectant into a cadaver bone as disclosed in PCT / US2021 / 042064, the contents of which are incorporated by reference in its entirety. In other cases, cadaver bone is submerged in a cryoprotectant solution and without use of a vacuum.

[0079] An aspect of the present disclosure is a method for cryopreserving a cadaver bone using vacuum to assist infiltration of a cryoprotectant into the cadaver bone. The method comprises steps of (a) placing a cadaver bone in a closed container comprising a cryoprotectant solution; (b) reducing the pressure in the closed container, and optionally, holding the closed container at reduced pressure, to remove at least a portion of the w ater present in the cadaver bone; (c) raising the pressure in the closed container and holding the closed container at a raised pressure to allow infiltration of the cryoprotectant solution into the cadaver bone; (d) removing the cadaver bone from the closed container; and (e) chilling the cadaver bone to a temperature at least below- 0°C, thereby cryopreserving the cadaver bone.

[0080] Surprisingly, by immersing a cadaver bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cry oprotectant infiltrates the cadaver bone significantly more rapidly that would occur by’ passive diffusion. With respect to PCT / US2021 / 042064, compare FIG. 2 with FIG. 4A and FIG. 4B and FIG. 3 with FIG. 5. Such effective infiltration of cryoprotectant contributes to reduced ice cry stal formation duringfreezing of the cadaver bone and, ultimately, extraction of viable bone marrow cells that have replicative potential.

[0081] 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, repeating the reduced pressure and the raised pressure may increase infiltration of a cryoprotectant into a cadaver bone. See, e.g, FIG. 5 of PCT / US2021 / 042064. In other embodiments, there is sufficient infiltration of cryoprotectant into a cadaver bone after a single cycle of reduced pressure and raised pressure.

[0082] In various embodiments, a cadaver bone (e.g., vertebral body) is bisected, cut into quarters, or more extensively divided prior to vacuum-assisted infiltration of the cryoprotectant.

[0083] The reduced pressure in the closed container may any pressure value from about -400 mmHg to about -800 mmHg. The pressure requirement should be sufficient to remove at least a portion of the water present in the cadaver bone. The reduced pressure in the closed container may have a value of about -400 mmHg, -425 mmHg, -450 mmHg, -475 mmHg, -500 mmHg, -525 mmHg, -550 mmHg, -575 mmHg, -600 mmHg, -625 mmHg, -650 mmHg, -675 mmHg, -700 mmHg, -725 mmHg, -750 mmHg, -775 mmHg, or - 800 mmHg. In some embodiments, the reduced pressure in the closed container is from about -400 mmHg to about -500 mmHg.

[0084] In some embodiments, it takes from about one minute to about 10 minutes for the closed container to reach a desired reduced pressure once the pressure in the closed container begins reducing. As examples, the closed container is may take 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 length of time in between (e.g. , a fraction of a minute, e.g.. about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween) to reach the desired reduced pressure. In some embodiments, the cadaver bone reaches the desired reduced pressure rapidly, e.g., from about one second to about one minute.

[0085] In some embodiments, the cadaver bone is held at the reduced pressure once the reduced pressure has been reached. The cadaver bone may be held for from less than one minute to about 50 minutes. As examples, the closed container is held at reduced pressure for less than one 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, 48minutes, 49 minutes, or about 50 minutes and any length of time in between (e.g. , a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds. 40 seconds, about 50 seconds, and any number of seconds therebetween). In some embodiments, the cadaver bone is not held at reduced pressure for any measurable time and instead, the method progresses to step (c) of raising the pressure in the closed container.

[0086] In step (c), the pressure of the closed container is raised until the pressure is from about 0 mmHg to about 760 mmHg. In other words, the pressure is raised to up to standard atmospheric temperature. The exact raised pressure may be any amount within the specified range, e.g., 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 raised pressure must be high enough to allow infiltration of the cryoprotectant solution into the cadaver bone.

[0087] The closed container may be held at the raised pressure for less than about two hours. As examples, for less than one hour, less than one-half hour, about one-half hour, or less time. In some embodiments, the closed container is held at the raised pressure for ten minutes. The duration that the closed container is held at the raised pressure must be long enough to allow infiltration of the cryoprotectant solution into the cadaver bone. As examples, the closed container is held at the raised pressure 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, and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween).

[0088] The closed container and the cryoprotectant contained therein may be at room temperature. Alternately, the closed container and the cryoprotectant contained therein may be below room temperature, e.g., as low as 4°C. The closed container and the cryoprotectant contained therein may be above room temperature, e.g., as high as 37°C.

[0089] Any suitable cryoprotectant may be used in a cryoprotectant solution. Examples of cryoprotectant include dimethyl sulfoxide (also known as DMSO, C2H6OS, 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 polyvinylpyrrolidone (PVP). In some embodiments, the cryoprotectant is DMSO. The cryoprotectant solution may comprise from about 5% DMSO to about 100% DMSO, e.g, about 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 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%, or about 100% DMSO. The cryoprotectant solution may comprise about 10% DMSO. The cryoprotectant solution may comprise about 20% DMSO. In some embodiments, the cryoprotectant solution may comprise about 40% DMSO or 60% DMSO. In some embodiments, a higher percentage of cry oprotectant is preferred, e.g. , percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.

[0090] The cryoprotectant solution may have water or a saline as base. In some embodiments, the saline is isotonic to human tissues. In embodiments the saline is a 0.9% saline solution. Any commercially available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate. The saline may be 0.9% sodium chloride.

[0091] The cryoprotectant solution may further comprise a protein. As examples, the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate. An example of a commercially available human platelet lysate product is Stemulate™ (from Cook® Regentec).

[0092] In some embodiments, the cryoprotectant solutions comprise about 10% protein, e.g, 10% human platelet lysate or 10% albumin.

[0093] In one example, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0094] In another example, the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0095] In yet another example, the cry oprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0096] In a further example, the cry oprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0097] In an additional example, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.

[0098] In any of the above aspects, the method may comprise a step of increasing the pressure in the closed container comprising a cryoprotectant to above 760 mmHg by introducing a compressed gas (e.g.. nitrogen, xenon, CO2, argon, H2S, or helium), a gas released bysublimination (e.g. , CO2 via dry ice), or a gas provided by evaporation (e.g. , nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone. In embodiments, the gas is CO2.. e.g., compressed CO2. In some embodiments, the gas is nitrogen, e.g.. compressed nitrogen. The time required for gas infiltration into a vertebral body is less when the gas is compressed versus a gas obtained by sublimination.

[0099] Alternately, in any of the above-mentioned aspects, rather than placing a cadaver bone in closed container comprising a cryoprotectant solution, the cadaver bone is placed in a closed container that lacks a cryoprotectant solution. In these alternate aspects, the method comprises a step of increasing the pressure in the closed container (which lacks a cryoprotectant solution) to above 760 mmHg by introducing a compressed gas (e.g., nitrogen, xenon, CO2, argon, H2S, or helium), a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g, nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone. Any method disclosed herein may be adapted by comprising initial steps of placing a cadaver bone in closed container that lacks a cryoprotectant solution and increasing the pressure in the closed container to above 760 mmHg by introducing a compressed gas, a gas released by sublimination, or a gas provided by evaporation; in a later step, a cryoprotectant solution is added to the closed container. In embodiments, the gas is CO2 , e.g., compressed CO2. In some embodiments, the gas is nitrogen, e.g., compressed nitrogen.

[0100] Without wishing to be bound by theory, increasing the pressure in a closed container by introducing a compressed gas (e.g, nitrogen, xenon, CO2, argon. H2S, or helium) , a gas released by sublimination (e g, CO2 via dry ice), or a gas provided by evaporation (e.g, nitrogen via liquid nitrogen), promotes infiltration of the cryoprotectant solution into the cadaver bone.

[0101] In some cases, the closed container comprises solid materials, e.g, metal, plastic, or other polymers. In some cases, the closed container comprises a foam material, e.g. Styrofoam.

[0102] In alternate aspects, a cadaver bone is infiltrated with a cryoprotectant without use of a vacuum. Here, an intact vertebral body, a vertebral body that has been bisected, cut into quarters, or more extensively divided is submerged into a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of the cryoprotectant solution into the cadaver bone.

[0103] The bone or bone fragment is placed, e.g., submerged, in a cryoprotectant solution and incubated for 1 hour at about 4°C. 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 about1.5 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 about2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about1.5 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, about2.5 hours, or about 3 hours.

[0104] Any suitable cryoprotectant may be used in a cryoprotectant solution. Examples of cryoprotectant include dimethyl sulfoxide (also known as DMSO, CzHeOS. 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 polyvinylpyrrolidone (PVP). In some embodiments, the cryoprotectant is DMSO. The cry oprotectant solution may comprise from about 5% DMSO to about 100% DMSO, e.g, 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%, or about 100% DMSO. The cryoprotectant solution may comprise 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, e.g. , percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.

[0105] The cryoprotectant solution may have water or a saline as base. In some embodiments, the saline is isotonic to human tissues. In embodiments the saline is a 0.9% saline solution. Any commercially available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate. The saline may be 0.9% sodium chloride.

[0106] The cryoprotectant solution may further comprise a protein. As examples, the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate. An example of a commercially available human platelet lysate product is Stemulate™ (from Cook® Regentec).

[0107] In some embodiments, the cryoprotectant solutions comprise about 10% protein, e.g, 10% human platelet lysate or 10% albumin.

[0108] In one example, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0109] In another example, the cry oprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0110] In yet another example, the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0111] In a further example, the cry oprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.

[0112] In an additional example, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.Two-Step Chilling of Cadaver Bone

[0113] Once a cadaver bone is infiltrated with cryoprotectant (either with or without use of a vacuum), the cadaver bone then undergoes an initial chilling period. For this, the cadaver bone is placed in a static minus 80 freezer set at a temperature of colder than about - 60°C, e.g., from about - 70°C to about -80°C, or colder than about -100°C. There, the cadaver bone undergoes an initial chilling period. In some embodiments, the cadaver bone is initially chilled in a static minus 80 freezer set at a temperature of about -86°C. Data showing the dynamics of the initial chilling period is shown in PCT / US2021 / 042064 at FIG. 6A.

[0114] In some cases, the static freezer is set at a range of temperature from 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 temperature from 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 temperature from 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.

[0115] The cadaver bone may be initially chilled at a rate of from about -0.3°C / min to about -5°C / min. In some embodiments, the cadaver bone is initially chilled at a rate of from about - 0.4°C / min to about -0.9°C / min. As examples, the initial chilling rate may 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 / mm, -0.9°C / min, to about -l°C / min. In other examples, the initial chilling rate may be about -l°C / min, -2°C / min. -3°C / min, -4°C / min, or about -5°C / min. In these rates, the minus signmeans that the temperature is dropping by the stated amount.

[0116] The duration of the initial chilling period may vary from a few hours to overnight. The time should be sufficient for the cadaver bone to reach a temperature of colder than about - 50°C, e.g., at -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 cadaver bone is initially chilled in the minus 80 freezer for at least 12 hours or at least overnight.

[0117] Without wishing to be bound by theory, it appears that the period of initial chilling in the presence of extracellular ice increases intracellular solute concentrations to an amount that allows intracellular vitrification in the subsequent chilling.

[0118] The cadaver bone may temporarily acquire a temperature of from about - 5°C to about - 15° C, but this occurs as the temperature of the cadaver bone is continuously dropping towards the desired temperature, e.g., colder than about -50°C. Even though during the period of initial chilling, the cadaver bone is not held in a static freezer having its temperature set to from about - 5°C to about - 15°C and for a period of time from about 1 to about 30 minutes, the cadaver bone achieves a temperature of from about - 5°C to about - 15°C (as the bone continues to chill to the desired temperature.

[0119] Once the cadaver bone has reached the desired temperature, the cadaver bone undergoes a subsequent chilling period. For this, the cadaver bone is placed in liquid nitrogen or in liquid nitrogen vapor, e.g., at a temperature of about -200°C. Data showing the dynamics of the subsequent chilling period is shown in PCT / US2021 / 042064 at FIG. 6B. In some embodiments, the subsequent chilling period may occur in a suitable static freezer that is capable of maintaining temperatures equivalent to liquid nitrogen yet without use of liquid nitrogen, e.g., a cryogenic freezer.

[0120] During the subsequently chilling period, the cadaver bone is cooled at a rate of from about -2°C / min to about -6°C / min. In some embodiments, the cadaver bone is initially chilled 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 / mm, -5.4°C / min, -5.6°C / min, -5.8°C / min, or about - 6°C / min. In these rates, the minus signmeans that the temperature is dropping by the stated amount.

[0121] The cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or in a suitable static freezer indefinitely. As examples, the cryopreserved cadaver bonemay be held for at least a day, at least a week, at least a month, at least a year, at least five years, or at least 20 years. The cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or suitable static freezer for hundreds or thousands of years.

[0122] Without wishing to be bound by theory, the two-step chilling of cadaver bone method, as disclosed herein, improves the viability of the extracted bone marrow cells (hematopoietic stem cells (HSCs; CD34+ cells) and / or mesenchymal stromal / stem cells (MSCs)) relative to methods that do not use the two-step chilling method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and / or MSCs) are obtained relative to standard methods.

[0123] 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%, or about 100% more viable cells than from methods that do not use two-step chilling, as disclosed herein.

[0124] In some cases, the methods of the present disclosure provide from about 101% more viable cells to about 200% more viable cells, e.g, about 101%, 102%, 103%, 104%. 105%, 106%, 107%, 108%, 109%, 10%, 1 1 1 %, 1 12%, 1 13%, 1 14%, 1 15%, 1 16%, 117%, 1 18%, 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%,197%, 198%, 199%, or about 200% more viable cells than from methods that do not use two- step chilling, as disclosed herein.

[0125] In some cases, the methods of the present disclosure provide from about 2-fold more viable cells 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 fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, 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 from methods that do not use two-step chilling, as disclosed herein.

[0126] In some cases, the methods of the present disclosure provide from about 10-fold more viable cells to about 100-fold more viable cells, e.g., about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, about 100-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 60-fold, 60-fold to 70-fold, 70-fold to 80-fold, 80-fold to 90-fold, or 90-fold to 100-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.

[0127] In some cases, the methods of the present disclosure provide from about 100-fold more viable cells to about 1000-fold more viable cells, e.g., about 100-fold, 200-fold, 300-fold, 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, about 1000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 100-fold to 200 fold, 200-fold to 300-fold, 300-fold to 400-fold, 400-fold to 500-fold, 500-fold to 600-fold. 600-fold to 700-fold, 700-fold to 800-fold, 800-fold to 900-fold, or 900-fold to 1000-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.

[0128] In some cases, the methods of the present disclosure provide from about 1000-fold more viable cells to about 10000-fold more viable cells, e.g.. about 1000-fold. 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, about 10000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, 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 from methods that do not use two-step chilling, as disclosed herein.Methods for Rapidly Warming a Cryopreserved Cadaver Bone

[0129] In some cases, the present disclosure provides a method for rapidly warming cadaver bone for providing bone marrow or a derivative thereof. PCT / US2021 / 042064 discloses methods for rapidly warming cryopreserved bone: the contents of which are incorporated by reference in its entirety. These disclosed methods may be useful in the methods of the present disclosure.

[0130] In some cases, the method for rapidly warming cadaver bone comprises steps of: obtaining a cryopreserved cadaver bone; dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone; transferring the fragments of the cryopreserved bone into a grinding medium having a temperature of from about 35°C to about 45°C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20°C.

[0131] In some embodiments, a cryopreserved cadaver bone transferred into a grinding medium (as disclosed herein) without having been divided into fragments. Preferably, the cryopreserved cadaver bone has a temperature of at least below 0°C when transferred into a grinding medium.

[0132] In alternate embodiments, the method comprises dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone. Preferably, the cryopreserved cadaver bone has a temperature of below 0°C when dividing into fragments.

[0133] In order to simplify the process and for increased safety to the processing personnel, a custom bone cutting tool as described in US 2019 / 0343112, which is hereby incorporated byreference in its entirety, is used to divide the cryopreserved cadaver bone into smaller pieces. Another bone cutting tool may be used in combination, or in lieu of the custom bone cutting tool as described in US 2019 / 0343112.

[0134] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably hardened steel capable of withstanding the forces required to cut through frozen bone. In the cleaning process, the tool is subjected to steam sterilization, which can be deleterious to the steel. Thus, in one feature of the present disclosure, the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.

[0135] The manual bone-cutting device for dividing the cryopreserved cadaver bone is capable of generating up to 1000 Ibf when less than 50 Ibf is applied. Such a manual bonecutting device comprises: a force transmission mechanism, wherein the force transmission mechanism comprises an elongated force transducing member pivotally coupled to a gear mechanism; and a manually operable handle coupled to an end of the elongated force transducing member, wherein the end is opposite of the gear mechanism. The manual bonecutting device comprises an upper cutting element and / or a lower cutting element. Its upper cutting element and / or lower cutting element each comprises one or more cutting blades that radiate outwards from a central portion of the upper cutting element and / or the lower cutting element. When the one or more cutting blades divide the cryopreserved cadaver bone into fragments that are generally sector shaped.

[0136] The manual bone-cutting device divides the cryopreserved cadaver bone into fragments of the cryopreserved bone. The fragments of the cryopreserved bone are transferred into a grinding medium having a temperature of from about 35°C to 45°C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20°C. Alternately, whole cryopreserved bone, which has not been divided, is transferred into a grinding medium having a temperature of from about 35°C and 45°C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20°C. In some embodiments, the surface temperature of the cadaver bone fragments is higher than 20°C, e.g, 25°C or higher.

[0137] A suitable volume of grinding medium is warmed and held at a temperature of from about 35°C to about 45°C, for example, by placing a container holding the grinding medium on a hot plate or in a water bath. In some examples, 300ml, 500ml or one liter of grinding medium is used to warm the cadaver bone. Preferably, the grinding medium has a temperature of about 37°C to about 40°C when the fragments of the cry opreserved bone are transferred to the grinding medium.

[0138] The cadaver bone fragments are warmed to a surface temperature of about 20°C at a rate of from about 100°C / min to about 500°C / min. Is some embodiments, the warming rate is greater than about 300°C / min, e.g, 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 from about 400°C / min to about 500°C / min. In some instances, the cadaver bone fragments are warmed to a surface temperature of about 20°C in less than one minute. In some cases, the cadaver bone fragments are warmed to a surface temperature of about 20°C in about one 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 the fast warming is shown in PCT / US21 / 42064 at FIG. 15.

[0139] When whole cadaver bone is warmed in the grinding medium, the w arming rate will be slower than when bone fragments are warmed. As examples, the cadaver bone is warmed to a surface temperature of about 20°C at a rate of from about 100°C / min to about 250°C / min.

[0140] Without wishing to be bound by theory, the fast warming rate of the present disclosed methods prevents ice recrystallization during thawing of the bone fragments (or whole cadaver bone).

[0141] Without wishing to be bound by theory’, the rapid warming of cadaver bone method, as disclosed herein, improves the viability’ of the extracted bone marrow cells (hematopoieticstem cells (HSCs; CD34+ cells) and / or mesenchymal stromal / stem cells (MSCs)) relative to methods that do not use the rapid warming method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and / or MSCs) are obtained relative to standard methods.

[0142] 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%, or about 100% more viable cells than from methods that do not use the rapid warming method, as disclosed herein.

[0143] In some cases, the methods of the present disclosure provide from about 101% more viable cells to about 200% more viable cells, e.g., about 101%, 102%, 103%, 104%. 105%, 106%, 107%, 108%, 109%. 10%, 111%. 112%. 113%. 114%, 115%, 116%. 117%. 1 18%. 1 19%, 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%,197%, 198%, 199%, or about 200% more viable cells than from methods that do not use the rapid warming method, as disclosed herein.

[0144] In some cases, the methods of the present disclosure provide from about 2-fold more viable cells 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 fold therebetween more viable cells than from methods that do not use rapid warming, as disclosed herein. As examples, 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 from methods that do not use rapid warming, as disclosed herein.

[0145] In some cases, the methods of the present disclosure provide from about 10-fold more viable cells to about 100-fold more viable cells, e.g., about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, about 100-fold or any fold therebetween more viablecells than from methods that do not use rapid warming, as disclosed herein. As examples, the methods of the present disclosure provide 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40- fold, 40-fold to 50-fold, 50-fold to 60-fold, 60-fold to 70-fold, 70-fold to 80-fold, 80-fold to 90-fold, or 90-fold to 100-fold more viable cells than from methods that do not use rapid warming, as disclosed herein.

[0146] In some cases, the methods of the present disclosure provide from about 100-fold more viable cells to about 1000-fold more viable cells, e.g., about 100-fold. 200-fold. 300-fold. 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, about 1000-fold or any fold therebetween more viable cells than from methods that do not use rapid warming, as disclosed herein. As examples, the methods of the present disclosure provide 100-fold to 200 fold, 200- fold to 300-fold, 300-fold to 400-fold. 400-fold to 500-fold, 500-fold to 600-fold, 600-fold to 700-fold, 700-fold to 800-fold, 800-fold to 900-fold, or 900-fold to 1000-fold more viable cells than from methods that do not use rapid warming, as disclosed herein.

[0147] In some cases, the methods of the present disclosure provide from about 1000-fold more viable cells to about 10000-fold more viable cells, e.g., about 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold. 8000-fold. 9000-fold, about 10000-fold or any fold therebetween more viable cells than from methods that do not use rapid warming, as disclosed herein. As examples, 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 from methods that do not use rapid warming, as disclosed herein.Extracting the Bone Marrow

[0148] The bone is removed from the bag and from the PLASMA-LYTE™, and a sterile gauze or sponge is used to absorb any liquid remaining on the VBs. In one approach, a saw and / or anvil shears are used to cut the VBs are cut into smaller pieces, such as 1.5 cm2pieces, that are small enough for fragmenting with a bone grinder. In order to simplify the process and for increased safety to the processing personnel, a custom bone cutting tool as described in US 2019 / 0343112. which is hereby incorporated by reference in its entirety, is provided is used to cut the VBs into the smaller pieces. Another custom bone cutting tool can be used in combination, or in lieu of the custom bone cutting tool as described in US 2019 / 0343112. The additional bone cutting tool is described in US 2020 / 0325451, which is hereby incorporated by reference in its entirety.

[0149] In some embodiments, the bone is freshly obtained from a cadaver. Alternately, the bone has previously been frozen and / or cryopreserved.

[0150] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably hardened steel capable of withstanding the forces required to cut through bone. In the cleaning process, the tool is subjected to steam sterilization, which can be deleterious to the steel. Thus, in one feature of the present disclosure, the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.

[0151] The pieces produced by the bone cutting tool are immediately placed into a sterile pitcher and submerged in 300-500 ml of a grind media. In one aspect of the present system and method, the grind media uses PLASMA-LYTE™-A as a base with heparin, human serum albumin (HSA), and a nuclease (Merck KGAA Corporation). Heparin is used as an anticoagulant. Other anticoagulants at various quantities can also be used. HSA provides a protein source to prevent cell adherence and adsorption to surfaces, as well as reactive oxygen scavenging. It is noted that conventional grind media utilizes DNase, but for the present disclosure Benzonase® or Denarase® reagent is substituted for DNase™ reagent (Qiagen Sciences LLC). Whereas DNase works only on DNA, modem 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. It is noted that IMDM (Iscove's Modified Dulbecco's Media) can substitute for the PLASMA-LYTE™-A. since IMDM is suitable for rapidly proliferating high-density cell cultures and ideal for supporting T- and B- lymphocytes. It is further noted that Denarase reagent (C-Lecta GmbH) is equivalent to Benzonase reagent in the same quantity in the present process.

[0152] In some embodiments, the amount of heparin in the grind media is about 5 U / ml to about 15 U / ml. In some embodiments, the amount of heparin in the grind media 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 grind media is 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 9 U / ml, about 5 U / ml to about 10 U / ml, about 5 U / ml to about 11 U / ml, about 5 U / ml to about 12 U / ml, about 5 U / ml to about 13 U / ml, about 5 U / ml to about 14 U / ml, about 5 U / ml to about 15 U / ml, about 6 U / ml to about 7 U / ml, about 6 U / ml to about 8 U / ml, about 6 U / ml to about 9 U / ml, about 6 U / ml to about 10 U / ml, about 6 U / ml to about 11 U / ml. about 6 U / ml to about 12 U / ml. about 6 U / ml to about 13 U / ml, about 6 U / ml to about 14 U / ml, about 6 U / ml to about 15 U / ml, about 7 U / ml to about8 U / ml, about 7 U / ml to about 9 U / ml, about 7 U / ml to about 10 U / ml, about 7 U / ml to about 11 U / ml, about 7 U / ml to about 12 U / ml. about 7 U / ml to about 13 U / ml, about 7 U / ml to about 14 U / ml, about 7 U / ml to about 15 U / ml, about 8 U / ml to about 9 U / ml, about 8 U / ml to about 10 U / ml, about 8 U / ml to about 11 U / ml, about 8 U / ml to about 12 U / ml, about 8 U / ml to about 13 U / ml, about 8 U / ml to about 14 U / ml, about 8 U / ml to about 15 U / ml, about 9 U / ml to about 10 U / ml, about 9 U / ml to about 11 U / ml. about 9 U / ml to about 12 U / ml, about 9 U / ml to about 13 U / ml. about 9 U / ml to about 14 U / ml. about 9 U / ml to about 15 U / ml, about 10 U / ml to about 11 U / ml, about 10 U / ml to about 12 U / 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 grind media 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 grind media 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.

[0153] In various embodiments, heparin is omitted from a grind medium.

[0154] In some embodiments, the amount of Benzonase® in the grind media is about 1 U / ml to about 10 U / ml. In some embodiments, the amount of Benzonase in the grind media is 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, about 9 U / ml, or about 10 U / ml. In some embodiments, the amount of Benzonase in the grind media is about 1 U / ml to about 2 U / ml, about 1 U / ml to about 3 U / ml, about 1 U / ml to about 4 U / ml, about 1 U / ml to about 5 U / ml, about 1 U / ml to about 6 U / ml, about 1 U / ml to about 7 U / ml, about 1 U / ml to about 8 U / ml, about 1 U / ml to about 9 U / ml, about 1 U / ml to about 10 U / ml, about 2 U / ml to about 3 U / 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, 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 5U / ml to about 8 U / ml, about 5 U / ml to about 9 U / ml, about 5 U / ml to about 10 U / ml, about 6 U / ml to about 7 U / ml, about 6 U / ml to about 8 U / ml, about 6 U / ml to about 9 U / ml, about 6 U / ml to about 10 U / ml, about 7 U / ml to about 8 U / ml, about 7 U / ml to about 9 U / ml, about 7 U / ml to about 10 U / ml, about 8 U / ml to about 9 U / ml, about 8 U / ml to about 10 U / ml, or about 9 U / ml to about 10 U / ml. In some embodiments, the amount of Benzonase in the grind media 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 grind media 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.

[0155] In some cases, the amount of Benzonase® in a grind medium is about 3 U / ml and the amount of heparin in the grind medium is about 10 U / ml.

[0156] In some embodiments, the amount of Benzonase® or Denarase® in the grind media is about 11 U / ml to about 55 U / ml. In some embodiments, the amount of Benzonase in the grind media 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 grind media 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 grind media 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 U / ml to about 20 U / ml, 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 30 U / ml to about 55 U / ml, about 35 U / ml to about 40 U / ml, about 35 U / ml to about 45 U / ml, about 35 U / ml to about 50 U / ml, about 35 U / ml to about 55 U / ml, about 40 U / ml to about 45 U / ml. about 40 U / ml to about 50 U / ml, about 40 U / ml to about 55 U / ml, about 45 U / ml to about 50 U / ml, about 45 U / ml to about 55 U / ml, orabout 50 U / ml to about 55 U / ml. In some embodiments, the amount of Benzonase in the grind media 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.

[0157] It is noted that Denarase® reagent (C-Lecta GmbH) is equivalent to Benzonase® reagent in the same quantity in the present process.

[0158] Notably, it has been discovered that a relationship exists between the amount of Benzonase® in a grinding medium and the amount of heparin, such that progressively lower amounts of Benzonase can be used as the amounts of heparin is reduced. Without wishing to be bound by theory', it is likely that heparin, through calcium chelation, helps prevent clumping of cells; however, more importantly, heparin chelates magnesium. Magnesium is an important co-factor for Benzonase. Therefore, in the presence of heparin, the presence and / or relative amounts of magnesium in a solution is reduced and this reduction in magnesium amounts reduces Benzonase activity. Thus, in some embodiments, the amount of heparin is lowered and in some embodiments, heparin is omitted.

[0159] In some embodiments, HSA is present in the grind media at about 0.5% to about 5%. In some embodiments, HSA is present in the gnnd 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%. In some embodiments, HSA is present in the grind media 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 grind media 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 grind media 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%.

[0160] Another pitcher of 300-500 ml of grind media is retained for collecting the bone fragments after grinding, and another supply of about 100 ml of the grind media is retained for rinsing through the grinder during the grinding process to prevent bone fragments from sticking to the surface of the pitcher of the grinding components. In some embodiments, the additional grind media may have different quantities of heparin, HSA, and Benzonase as compared to the initial grind media.

[0161] An electric bone grinder or a purpose-built bone grinder, such as the grinder of Biorep Technologies Inc, (Miami, FL) can be used in an ISO-5 environment within an ISO-7 clean room. Bone types are kept separate if both VB and ilium from the same donor are being processed. The bone is kept submerged in grind media at all times during and after the grinding process. Once all of the donor bone pieces are ground, the chamber of the bone grinder is thoroughly rinsed with fresh processing media. The bone fragments are discharged from the grinder into the pitcher containing grind media.

[0162] In some cases, bone marrow and bone grindings from are shaken for 10 minutes at 150 RPM.

[0163] The contents of the pitcher are transferred to sterile bags. Next, the contents of the sterile bags 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 pm or 500 pm sieve is stacked on top of a 177 pm or 200 pm sieve, which is seated over a catch-pan to receive the liquid filter contents. The sterile bags containing the output from the grinder are swirled and then poured evenly over the sieve stack or filtration sets. The filtering process is observed to ensure that excessive clumping is not occurring, which can signal the presence of soft tissue or other contaminants. Bone fragments retained on the surface of the sieves are distributed evenly on the sieves and rinsed with 250 ml of fresh processing medium. In one embodiment, the processing medium used for rinsing is the grind media described above or PLASMA-LYTE™ with 2.5% HSA. The sieved bone marrow product, which can be approximately 1000 ml in a well-performed process, is transferred to sterile packs for subsequent processing and analysis. The contents of each bag are visually inspected to confirm that the contents do not include any visible bone fragments or soft tissue.

[0164] In some embodiments, the rinse media can contain the various amounts of HSA as described for the grind media. In some embodiments, the rinse media can contain, additionally, heparin and / or Benzonase.

[0165] In some cases, the amount of Benzonase® in a rinse medium is about 3 U / ml and the amount of heparin in the rinse medium is about 10 U / ml.

[0166] In another embodiment, the contents of each bag are passed through bone marrow filtration units, as depicted in FIG. 1. In this embodiment, the system 150 includes a stand 154 configured to support a sterile collection bag 152 which contains the bone fragments and media from the grinding operation described above. The stand includes a container hanger 155 configured to engage the cap 153 of the sterile bag to suspend the container. The bottom of the bag includes a discharge assembly 160 that includes a pre-filter 162 projecting into the body of the collection bag. In one specific embodiment the pre-filter 162 is an 850pm filter. In some embodiments, the bone marrow passes first through an 800 pm pre-filter. The filter 162 is connected to an output tube 164 that is connected by a container claim 166 to the input line 171 of a first in-line filter 170. In the specific embodiment, the first in-line filter is a 200pm or a 500pm filter. The output line 172 of the first in-line filter is connected to the input line 176 of a second in-line filter 175. The second in-line filter is a 200pm or a 500pm filter. The two inline filters are initially both 500 pm for a first pass through the filter system 150. A second rinse is then performed on the grindings with the two in-line filters being 200pm. This doublepass filtration results in a cleaner suspension and enhances removal of fat from the suspension. The second in-line filter 175 has an output line 177 that can be engaged to a sterile bag. such as bag 152 for the second filtration pass. On the second pass through the system, the output line 177 of the second in-line filter 175 can be engaged to a container clamp 181 of a transfer pack container 180. The transfer pack 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.

[0167] The Total Nucleated Count (TNC) from a filtered bone marrow product can be calculated:TNC (MO3cells fL) = Cell Count (MO cells / iL) x Total Mass of Bone Marrow Extract (g) x 1000)Agitation of Bone Grindings and / or Bone Grinding Filtrate

[0168] Described herein, in some embodiments, is a method for processing bone marrow or derivative thereof, the method comprises mechanically agitating the bone grindings and / or bone grinding filtrate during the grinding and filtration portion of the processing of the bone marrow. In some instances, the bone marrow can be obtained from a deceased donor. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled but not frozen. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that is thawed. In some cases, the bone marrow can be processed forobtaining bone marrow cells. In some embodiments, the bone marrow cells can be hematopoietic stem cells (HSCs). In some embodiments, the bone marrow cells can be mesenchymal stem cells (MSCs).

[0169] Aspect disclosed in the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the 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 bone fragments; mechanically grinding the bone or bone fragment in the presence of a grinding solution to generate a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 rounds per minute (“RPM”) for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises the bone marrow or the derivative thereof and wherein the bone marrow or the derivative thereof comprises at least about 1,500,000 CD34+ cells / ml of the bone marrow or the derivative thereof. In some embodiments, the method further comprises contacting the solution with a rinse media and repeating the placing of the bone grindings on the shaker and then removing the solution from the shaker. In some embodiments, the method further comprises repeating step placing the bone grinding on the shaker and then removing the solution from the shaker one or more times. In some embodiments, the at least about 1,500,000 CD34+ cells / ml of the bone marrow or the derivative thereof comprises at least 85% viable CD34+ cells. In some embodiments, the method further comprises the at least about 1,500,000 CD34+ cells / ml of the bone marrow or the derivative thereof comprises at least 90% viable CD34+ cells.

[0170] The mechanical agitation can comprise agitating the bone grindings in a linear fashion. In some embodiments, the mechanical agitation can comprise agitating the bone grindings in a three-dimensional fashion. In some cases, the mechanical agitation of the bone grindings can comprise orbital shaking (via an orbital shaker) such as placing the bone grinding on a shaker. In some cases, the bone grindings can be mechanically agitated by the shaker at a rate at least about 10 rounds per minute (RPM), 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM, 120 RPM, 130 RPM. 140 RPM, 150 RPM, 160 RPM, 170 RPM, 180 RPM, 190 RPM, 200 RPM. 210 RPM. 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more. In some cases, the bone grindings can be mechanically agitated by centrifugation (e.g. spinning). In some embodiments, the bone grindings can be spun at least 10 RPM, 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM. 120 RPM, 130 RPM, 140 RPM. 150 RPM, 160 RPM, 170 RPM. 180 RPM, 190 RPM, 200 RPM, 210 RPM, 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more. In someembodiments, the bone grindings can be spun at least 300 RPM, 400 RPM, 500 RPM, 600 RPM, or more. In some embodiments, the bone grindings can be mechanically agitated by both shaking and spinning. In some embodiments, the mechanical agitation of the bone grindings can be 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.

[0171] In some embodiments, the mechanical agitation of the bone grindings increases the yield of the bone marrow cells obtained. In some instances, the yield 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 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to yield of bone marrow cells obtained without the mechanical agitation.

[0172] In some embodiments, the mechanical agitation of the bone grindings increases the viability of the bone marrow cells obtained. In some instances, the viability 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 fold, 3 fold, 4 fold. 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the viability of bone marrow cells obtained without the mechanical agitation.

[0173] In some embodiments, the mechanical agitation of the bone grindings increases the number of CD34 expressing bone marrow cells obtained. In some instances, the number of CD34 expressing 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 number of CD34 expressing bone marrow cells obtained without the mechanical agitation.

[0174] In some embodiments, the mechanical agitation of the bone grindings increases the number of CD45 expressing bone marrow cells obtained by the methods described herein. In some instances, the number of CD45 expressing 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 number of CD45 expressing bone marrow cells obtained without the mechanical agitation.

[0175] The above mentioned agitation can occur before the filtration steps described previously.

[0176] In certain embodiments, the amount of CD34+ cells / ml of the bone marrow or the derivative thereof 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, 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 than 2,000,000 CD34+ cells / ml. In some embodiments, the amount of CD34+ cells / ml of the bone marrow or the derivative thereof 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 the derivative thereof 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 the derivative thereof 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 the derivative thereof obtained is at least 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 the derivative thereof obtained is at least at most about 1,750,000 CD34+ cells / ml, or about 2,000,000 CD34+ cells / ml.

[0177] In some embodiments, the viability of the CD34+ cells is at least about 70% to about 95%. In some embodiments, the viability of the 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 viability’ of the CD34+ cells is at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the viability of the CD34+ cells is at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the viability of the CD34+ cells is at least at most about 75%, about 80%, about 85%, about 90%. or about 95%.

[0178] For quality control, a small quantity of bone marrow, such as 0.3 mL. is extracted from the sterile pack 152 using a syringe at an injection site 157 and conducting inversion mixing before pulling the sample. The sample can be tested by a hematology analyzer, such as a Sysmex Hematology' Analyzer, to determine the total nucleated cell (TNC) content of the sample, as an indicator of the TNC content of the bone marrow being subsequently processed. Fat Removal and Concentration

[0179] The bone marrow product collected from the filtering is essentially a fatty' emulsion. The fat content of the suspension obtained from the sieve filtering approach disclosed above is greater than the fat content of the suspension obtained from the double-pass filtration system 150. However, in both cases, there is a need to remove the fat content from the suspension. The suspension obtained from the filtering is recovered into 250 ml bags which are hermetically sealed with tube welders. Pairs of sterile bags and taring sticks are mounted within a centrifuge with bag ports facing down, and balanced. Volume compensating plates are used to prevent creasing of the bags during centrifugation. In one embodiment, the bags are centrifuged at 500xg for 15 minutes at room temperature to concentrate the cells, preferably to 2-3xl08 / ml. After centrifugation is complete, each bag is individually hung on a ring stand. The distinct layers within the bag are visible, with the fat layer clearly delineated on top of the supernatant with the bone marrow pellet at the bottom, as shown in FIG. 2. 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 FIG. 3, to clamp off or squeeze the bag closed beneath the fat layer. The pellet is then drained from the centrifuge bag into the new sterile bag, with the bag clip preventing passage of the fat layer. The pellet is agitated as it is drained to resuspend all of the pellet. After about half of the pellet has drained into the new bag, the tubing is closed with a hemostat or tube sealer. The second centrifuge bag is then welded to the new bag containing the pellet, and the contents of this second centrifuge bag are drained into the new bag.

[0180] The result is new sterile bags containing the bone marrow centrifuged to remove the fat. These bags of de-fatted bone marrow are then centrifuged at 500xg for 15 minutes at room temperature, with volume compensating plates to prevent creasing of the bags. Each bag is removed and suspended on a ring stand and a waste bag is welded to the bag, and a plasma extractor is used to remove the supernatant into the waste bag, as shown in FIG. 4. The tubing is clamped with a hemostat when the pellet rises or breaks. The tubing is then sealed and severed to remove the pellet — containing bag from the waste bag, which is discarded. A Luer connection is welded to the pellet-containing bag. The pellets from each bag are combined into a bulk bag using a large syringe. The pellet-containing bags are rinsed into the bulk bag using a rinse media. The bulk bag is inverted several times to ensure that all of the pellet is resuspended. A small quantity of the processed BM, such as 0.5 m , can be removed for quality control testing for density7and cell count. The test sample can also be evaluated for human leukocyte antigens, CCR5delta 32 mutation and apolipoprotein (APOE), among other things.

[0181] In some embodiments, the centrifuge settings at one or more steps can be increased. In some embodiments, the centrifuge is spun at about 400 g to about 650 g. In someembodiments, the centrifuge is spun at about 400 g to about 450 g, about 400 g to about 500 g, about 400 g to about 550 g, about 400 g to about 600 g, about 400 g to about 650 g, about 450 g to about 500 g, about 450 g to about 550 g, about 450 g to about 600 g, about 450 g to about 650 g, about 500 g to about 550 g, about 500 g to about 600 g, about 500 g to about 650 g, about 550 g to about 600 g, about 550 g to about 650 g, or about 600 g to about 650 g. In some embodiments, the centrifuge is spun at about 400 g, about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun at least about 400 g. about 450 g, about 500 g, about 550 g, or about 600 g. In some embodiments, the centrifuge is spun at most about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun for about 10 minutes to about 40 minutes. In some embodiments, the centrifuge is spun 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 is spun 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, the centrifuge is spun for at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the centrifuge is spun for at most about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some cases, a bag comprising extracted bone marrow can be concentrated by centrifuging at 600 x g (~2315 rpm) for 30 minutes. The supernatant is removed from the bone marrow pellets using a plasma extractor and into a waste bag. Waste is discarded using standard biohazard protocol. The pellets are then combined into a pre-weighed bulk bag and resuspended using rinse media. In some embodiments, the centrifuge is stopped without the use of a brake. In some embodiments, the centrifuge is stopped with a brake. In some embodiments, the centrifuge brake is set at about 25% to about 100%. In some embodiments, the 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, the centrifugebrake is set at about 25%, about 50%, about 75%, or about 100%. In some embodiments, the centrifuge brake is set at least about 25%, about 50%, or about 75%. In some embodiments, the centrifuge brake is set at most about 50%, about 75%, or about 100%.

[0182] In some cases, fat removal can occur using a commercial cell processing device (e.g., COBE® 2991 cell processor, TerumoBCT). See the World Wide Web (at) terumobct.com / 2991. Such commercial cell processing devices may also concentrate cell products.

[0183] The cell product is aliquoted into one or more second volumes, for example, segregate vials.Cryopreservation of the Bone Marrow

[0184] The present method provides a system for extracting and banking bone marrow for future clinical use according to the processing methods described above, as summarized in the flowchart of FIG. 6. This method can eliminate the failures of the current methods of matching bone marrow donors to groups that are tough to match, such as certain minorities. Once the bone marrow is cryopreserved and banked there is no uncertainty as to the source of the bone marrow, there is no wait for a future recipient and the bone marrow is available in large, repeatable volumes.

[0185] Methods of the present disclosure further provide distinct containers for a given bone marrow product with a first (larger) volume that contains cells to be provided to a subject in need and a second (smaller) volume that acts as a surrogate for the first volume, with cells of the second volume, for example, the surrogate, being used for assays to determine suitability of the first volume for administration to a subject in need.

[0186] It is contemplated that each bone donor can yield three or more bags of bone marrow through the process described above, based on ten vertebrae and / or the ilium obtained from the donor. If at the end of the process for a given donor three bags of bone marrow are not obtained, the donor can be flagged as potentially not passing overall quality control. A predetermined volume of bone marrow in each bag is contemplated, such as 70 ml contained in 250 ml bags. This predetermined volume is used to calculate the volume of freeze media components necessary for efficient cryopreservation of the bone marrow pellet. The freeze media is a solution of a rinse media and a cry opreservation composition. The cryoprotectant can be a cell- permeable media, such as dimethyl sulfoxide (DMSO); 1, 2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; formamide; ethanediol or butane 2, 3 diol; and / or a non-permeable media, such as hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribitol. Mannitol or polyvinylpyrrolidone (PVP). Each bone donor can alsoprovide at least three surrogate vials, e.g., 3, 4, 5, 6. 7, 8, 9, 10, 11, 12 or more surrogate vial. The greater number of cryopreservalion bags obtained from a donor, the greater number of surrogate vials that can be prepared, so that each cry opreservation bag has at least one vial, preferably, two, three, four, or more vials per cry opreservation bag.

[0187] HSA also provides cryoprotection through oncotic pressure, cell surface protein stabilization and reactive oxygen scavenging. In a preferred embodiment, the cryoprotectant is DMSO. The rinse media can be an electrolyte medium, such as PlasmaLyte, Isolyte, IMDM or other electrolyte solutions suitable for infusion. The freeze media can also include concentrations of oxyrase to reduce oxygen content to less than atmospheric, such as to less than 3% of atmospheric concentrations. The addition of oxyrase produces a hypobaric composition that can facilitate cry opreservation.

[0188] In some embodiments, for a method provided herein, a bone marrow product is cryopreserved in a freeze media, wherein said freeze media comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.

[0189] In some embodiments, said freeze media and / or rinse media 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 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, said freeze media and / or rinse media comprises about 1% to about 5% HSA. In some embodiments, said freeze media and / or rinse media comprises about 2.5% HSA.

[0190] In some embodiments, said freeze media 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 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% DMSO. In some embodiments, said freeze media comprises about 1% to about 10% DMSO. In some embodiments, said freeze media comprises about 2.5% DMSO, about 5% DMSO, or about 10% DMSO.

[0191] In some embodiments, said electrolyte formulation is Plasmalyte A.

[0192] In various embodiments, a rinse medium and / or freeze medium lacks heparin.

[0193] In some embodiments, the rinse media is fresh.

[0194] In some cases, the freeze media is <25°C before adding it to the bone marrow bulk bag.

[0195] The freeze media may be added to the bone marrow bulk bag at a predetermined rate (10% of the Freeze Media volume per minute) based on the following formula:Volume of Freeze Media to add per minute = Total Volume of Freeze Media (mL) x 0.1 Preferably, the elapsed time for adding the cryoprotectant to the bone marrow bulk bag does not exceed 9-1 1 minutes.

[0196] The freeze media is prepared by mixing the cryoprotectant and the rinse media according to the calculated total volume of freeze media needed for the volume of bone marrow collected. The bag containing the bone marrow is placed on a rocker for mixing and the freeze media is introduced into the bag by syringe. The freeze media is introduced at a particular rate over a predetermined time. In one embodiment, the freeze media is added at a rate of 10% of the media per minute, for a time of ten minutes. Once the media has been mixed with the concentrated bone marrow, a test sample is extracted by syringe. The remaining mixture of freeze media and bone marrow is injected in predetermined amounts into separate cry opreservation bags (and surrogate vials). In one embodiment, 70 ml of bone marrow mixture is introduced into each cryopreservation bag and air is drawn out with a syringe. At the end of the process, an 8 ml sample can be removed for sterility testing. Each cryopreservation bag is sealed to create four compartments, which are then separated for storage in cassettes to be stored in a cryo-freezer. In another embodiment, the separated compartments are stored in apassive cooling box, such as cooling box 200 shown in FIG. 5 or the cooling boxes described in US 7,604,930. which is hereby incorporated by reference, in its entirety. A standard freezer box with or without a box rack may be used in these embodiments. In some embodiments, the cassettes are not stored in a passive cooling box. In some embodiments, the cassettes are arranged inside a cryo-freezer in a particular configuration to induce specific freezing rates. In some embodiments, the arrangement is the arrangement depicted in FIG. 14 or FIG. 15. As shown, the cassettes are preferably not touching an internal wall of a freezer shelf. Also, it is preferable that cassettes are not stacked on top of each other.

[0197] Aspects of the present disclosure provide a cryopreserved cell product that is divided into two volumes, with a first volume (e.g., cryopreservation bag) for containing the cell product for transplant into a subject in need thereof and a second volume that acts as a surrogate for the first volume. As used herein, a surrogate vial is ty pically a smaller volume of the cell product and the surrogate can be thawed and assayed as needed, e.g., for cell viability (and especially “function viability” as determined by post-thaw proliferation). The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using the surrogate it is unnecessary to thaw the first volume for assaying and, instead, it is thawed when needing to be used, e g., for transplanting into a subject in need.

[0198] Without wishing to be bound by theory, for a given cell type, a specific, optimum cooling rate is required for that cell type or cell product to survive cryopreservation. This optimum cooling rate balances damage from intracellular ice formation (IIF) with damage from high solute concentration resulting from extracellular ice formation. If cells are cooled too fast, damaging IIF is likely; if cells are cooled too slowly, damaging solute effects are likely. For the surrogate vial to accurately represent the first volume, the cells in both volumes should be frozen at about the same rate, for example, the optimized rate; this common rate results in equivalent survival and viability for cells in the two volumes. Importantly, the second volume may be stored in the same long-term storage system as the first volume, and will therefore be exposed to the same conditions over long-term storage durations; thus, promoting the ability of the surrogate vial to accurate represent the cryopreservation bag that contains cell for transplant. These ultimately allow the second volume to be tested to determine, at least, if storage of the cry opreservation bag was maintained appropriately and without having to manipulate and test the cells of the first volume. More specifically, by assaying the surrogate vial, the cry opreservation bag does not need to be warmed and / or handled prior to its immediate use. This feature is especially helpful to a subject’s outcome and welfare in two ways. First, the cells for transplanting are thawed only when ready to be administered to the subject (andpreferably at the site of administration) rather than being thawed two weeks or so before 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 - could adversely affect their viability and utility’ once transplanted. Second, since a subject likely undergoes myeloablative conditioning prior to transplant, the patient can forestall myeloablative conditioning until a cell product has been assayed and determined to be suitable for use, which usually takes about two weeks; by having an surrogate vial, a subject begins myeloablative conditioning once a suitable product has been identified, thereby shortening the length of time that the subject remains immune compromised.

[0199] The present disclosure provides methods for ensuring that the two volumes cool at the same rate. Based, at least, on laws of physics, a smaller volume of a cell product will cool at a faster rate than a larger volume of the cell product. And, the smaller volume, with a faster cooling rate should have increased IIF relative to the larger volume, which has a slower cooling rate. Methods of the present disclosure promote an equivalent rate of cell cooling between the first (larger) volume and the second (smaller) volume such that each volume will have similar amounts of IFF and. as such, the surrogate vial (smaller volume) will accurately represent the larger first volume. Without wishing to be bound by theory, methods of the present disclosure slow the rate of cooling for the second (smaller) volume to the rate experienced by the first (larger) volume based, in part, on use of different types of containers that directly holds or indirectly holds either a first volume or the second volume and / or positioning of containers within the same freezer, e.g., a static temperature freezer. As a result, cells in the smaller volume (for example, the second volume / surrogate vial) and cells in the larger volume (for example, the first volume / cry opreservation bag) experience similar rates of cooling (e.g., about -l°C / minute) 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, a surrogate vial is placed directly into an insulated vial container, e.g., CoolCell® freezing storage system, which when placed in a static freezer that is colder than -80°C, e.g., a -86°C static freezer, the cells in the surrogate vial experience rate of freezing at the rate of about -l°C / minute; without use of the insulated vial container, the cells in the surrogate vial would experience rate of freezing at the rate of about - 10°C / minute, which would likely cause damage from IIF On the other hand, the larger first volume, cry opreservation bag, does not need to be directly placed in an insulated container and instead, when the bags are placed in cassettes - which are not insulated (to avoid slowing the cooling rate of the bag) - and moved to -86°C static freezer, the cells in the cryopreservation bag preferably experience freezing rate of about -l°C / minute. By '‘directly placed” means thateach vial is in close proximity to the insulating material of the insulating container. These manipulation cause cells of the first and the second volumes to have roughly equivalent osmosis of intracellular water into the extracellular space for cells; this osmosis increases the solute concentration intracellularly, and helps avoid formation of (harmful) intracellular ice crystals and promotes extracellular ice formation (which is less harmful to the cell). Once the first step of freezing (in the -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 in a roughly similar position within the long-term storage device.

[0200] Accordingly, methods of the present disclosure allow production of a surrogate sample of the cell product that is expected to accurately represent the portion of the cell product that is to be administered to a subject in need thereof and provides a cell product that is. not only therapeutically beneficial to the subject, but promotes subject’s outcome and welfare in ways that are not achievable when a cryopreserved cell product in merely contained in a single bag and without a surrogate vial.

[0201] In some cases, a first volume (for example, a cryopreservation bag) and a second volume (for example, a surrogate cryovials) are placed in -86°C static freezer. The bags are placed in cassettes, which may lack insulation, while the surrogate vials are placed separately in a CoolCell® freezing storage system and then in front of the box of cassettes into the freezer.

[0202] When the test samples from the particular bone marrow batch have been validated for cell count and sterility, the cryopreservation bags and surrogate vials of cryopreserved bone marrow can be further cooled for long-term storage. In one embodiment, the bags and vials are cooled at a controlled rate to prevent damage to the bone marrow and cells. An optimal cooling scheme to yield an optimal amount of viable bone marrow and cells comprises varying the cool rate at various stages of the cooling process. In some embodiments, the stages of the cooling process are referred to as '‘Supra-Freeze” (about 17°C to the point of nucleation) and ‘'SubFreeze” (from about -10°C to -40°C). Typically, nucleation occurs from about 7°C to about 15°C.

[0203] 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 in a roughly similar position within the long-term storage device.

[0204] Aspects described in the present disclosure comprises a method for processing bone marrow or a derivative thereof (e.g. bone marrow derived cellular compositions), wherein the bone marrow or the derivative thereof is derived from a deceased donor, the methodcomprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; extracting the bone marrow or the derivative thereof from the bone or bone fragment; and cry opreserving the bone marrow or the derivative thereof, wherein the cryopreserving comprises decreasing temperature of the bone marrow or the derivative thereof at a freeze rate of more than about -l°C / min in a static freezer. In some embodiments, the cryopreserving comprises cooling the bone marrow or the derivative thereof at a suprafreeze rate from about -2.5°C / min to about -5°C / min at least until at least one cell of the bone marrow or the derivative thereof is nucleated. In some embodiments, the cry opreserving comprises cooling the bone marrow or the derivative thereof at a supra-freeze rate from about -2.5°C / min to about -4°C / min at least until at least one cell of the bone marrow or the derivative thereof is nucleated. In some embodiments, the cryopreserving comprises cooling the bone marrow or the derivative thereof at a supra-freeze rate from about -2.5°C / min to about - 3.5°C / min at least until at least one cell of the bone marrow or the derivative thereof is nucleated. In some embodiments, the cryopreserving comprises cooling the bone marrow or the derivative thereof at a sub-freeze rate from about -l°C / min to about -2°C / min. In some embodiments, the supra-freeze rate and the sub-freeze rate are maintained without the use of a passive cool box. In some embodiments, the cryopreserving comprises arranging one or more aliquots of the bone marrow or the derivative thereof inside the static freezer such that no aliquot contacts a wall of the static freezer. In some embodiments, the bone marrow or the derivative thereof comprises a population of CD34+ cells. In some embodiments, the population of CD34+ cells comprise at least 70% viable CD34+ cells after the bone marrow or the derivative thereof is thawed. In some embodiments, the population of CD34+ cells comprise at least 80% viable CD34+ cells after the bone marrow or the derivative thereof 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.

[0205] In one specific embodiment, the cry opreservation bags and surrogate vials are cooled at a rate of -1 to -40°C per minute until the bags have reached a temperature suitable for plunging the bags into liquid nitrogen. Preferably, the bags are cooled at a rate of -1°C to -5°C. A suitable temperature is in the range of -40 to -100°C. Once that temperature has been reached, the bags are cooled further at a more rapid rate to a temperature of 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 in a roughly similar position within the long-term storage device.

[0206] In some embodiments, the temperatures for freezing the bone marrow or bone marrow cells comprise the temperatures and freeze rates shown in Example 5. In some embodiments, the bone marrow or bone marrow cells can be cryopreserved at a supra-freeze rate or a suprafreeze range. In some embodiments, the bone marrow or bone marrow cells can be cryopreserved by freezing at both supra-freeze rate and sub-freeze rate. For example, the bone marrow or bone marrow cells can be cryopreserved by freezing at first with supra-freeze rate until a predetermined temperature is reached, which is then followed by switching freezing the bone marrow or bone marrow cells to a sub-freeze rate. In some embodiments, the nucleation temperature of the bone marrow or bone marrow cells can be reached during the supra-freeze. In some embodiments, the nucleation temperature of the bone marrow or bone marrow cells can be reached during the sub-freeze. In some embodiments the nucleation temperature of the bone marrow or bone marrow cells can be reached during the switching between the supra- freeze and the sub-freeze.

[0207] In some instances, the bone marrow or bone marrow cells can be cryopreserved first with supra-freeze. For example, the bone marrow or bone marrow cells can be cryopreserved while the bone marrow or bone marrow cells are just processed and at room temperature. In some instances, the supra-freeze rate is generally higher (e g. decreasing of the temperature at a faster rate) compared to the sub-freeze rate. In some embodiments, the supra-freeze rate is from about -6°C / min to about -0.5°C / min. In some embodiments, the supra-freeze rate is from about -0.5°C / min to about -l°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 -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 -l°C / min to about -1.5°C / min, about - l°C / min to about -2°C / min, about -l°C / min to about -2.5°C / min, about -l°C / min to about - 3°C / min, about -l°C / min to about -3.5°C / min, about -l°C / min to about -4°C / min, about - l°C / min to about -4.5°C / min, about -l°C / min to about -5°C / min, about -l°C / min to about - 5.5°C / min, about -l°C / min to about -6°C / min, about -1.5°C / min to about -2°C / min, about - I.5°C / min to about -2.5°C / min, about -I.5°C / min to about -3°C / min, about -1.5°C / mm 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, 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°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 -3.5°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 supra-freeze rate is about -0.5°C / min, about -l°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 supra-freeze rate is at least about - 0.5°C / min. about -l°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 supra-freeze rate is at most about -l°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 supra-freeze rate is -3.2°C. In some embodiments, the supra-freeze rate is from about -2.54°C / min to about -4.09°C / min.

[0208] In some embodiments, the bone marrow or bone marrow cells can be cryopreserved at a sub-freeze rate or a sub-freeze range. In some embodiments, the sub-freeze rate is from about -2.5°C / min to about -0. l°C / min. In some embodiments, the sub-freeze rate is from 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 -l°C / min, about -0. l°C / min to about -1 2°C / min, about -0. l°C / min to about -1 ,4°C / min, about -0. l°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, about -0. l°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 -l°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 -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 -l°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 -l°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 -l°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 -l°C / min to about -1.2°C / min, about -l°C / min to about -1.4°C / min, about -l°C / min to about -1.6°C / min. about -l°C / min to about -1.8°C / min, about -l°C / min to about -2°C / min, about -l°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 -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 sub-freeze 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 -l°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 sub-freeze 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 -l°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 sub-freeze 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 -l°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 sub-freeze rate can be -1.36°C / min. In some embodiments, the sub-freeze rate comprises a range of -I. I3°C / min to -1.62°C / min.

[0209] In some embodiments, the freeze rate for cry opreserving the bone marrow or the bone marrow cells described herein comprises determining the nucleation temperature. In some embodiments, the nucleation temperature is from about -24°C to about -2°C. In some embodiments, the nucleation temperature is from 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 -12°C to about -24°C, about -14°C to about -16°C, about -14°C to about -18°C, about -14°C to about -20°C, about -14°C to about -22°C, about -14°C to about -24°C, about - 16°C to about -18°C. about -16°C to about -20°C. about -16°C to about -22°C. about -16°C to about -24°C, about -18°C to about -20°C, about -18°C to about -22°C, about -18°C to about - 24°C, about -20°C to about -22°C, about -20°C to about -24°C, or about -22°C to about -24°C. 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 can be about - 12.31 °C / min. In some embodiments, the nucleation temperature can comprise a range of from about -7.24°C to about -17.52°C.

[0210] 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 into a cooling box which lacks insulation for freezing. Alternative, the cry opreservation bag is not placed in a cooling box. In some cases, the cryopreservation bag can be placed in a cassette and the 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 vaporstemmed from liquid nitrogen. In some cases, the cryopreservation bag can be placed in different compartments or different levels of shelves in the freezer or the in the 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 position as depicted in FIG. 14 or FIG. 15.

[0211] A cry opreservation bag is placed within a corresponding compartment 201-203 of the cooling box 200 and the overlapping cover 205 is closed over the compartments to provide a sealed environment for cryo-preservation of the contents of the bags. The cooling box is placed within a cryo freezer such that the cooling box produces a cooling rate of -0.5 to -5 C° / min, and typically -1 C° / min, with nucleation temperatures above -20°C. The freezing process continues at the prescribed rate until the temperature of the bone marrow reaches a suitable temperature. The suitable temperature for storage of the bags is a temperature < -80°C or < - 150°C.

[0212] In another embodiment, the bags are cooled in a static chamber temperature as opposed to the controlled rate cryopreservation described above. In the passive cooling approach, the cooling box is placed in a -86°C freezer until the bags reach a stable temperature. In some cases, the freezer can be set at a range of temperature from about -100°C to about - 60°C. In some cases, the freezer can be set at a range of temperature from 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°C to about -82°C, about -65°C to about -84°C, about -65°C to about -86°C, about -65°C to about -88°C, about -65°C to about -90°C, about -65°C to about -95°C, about - 65°C to about -100°C, about -70°C to about -75°C. about -70°C to about -80°C. about -70°C to about -82°C, about -70°C to about -84°C, about -70°C to about -86°C, about -70°C to about -88°C, about -70°C to about -90°C, about -70°C to about -95°C, about -70°C to about -100°C, about -75°C to about -80°C, about -75°C to about -82°C, about -75°C to about -84°C, about - 75°C to about -86°C, about -75°C to about -88°C. about -75°C to about -90°C, about -75°C to about -95°C. about -75°C to about -100°C, about -80°C to about -82°C, about -80°C to about -84°C, about -80°C to about -86°C, about -80°C to about -88°C, about -80°C to about -90°C, about -80°C to about -95°C, about -80°C to about -100°C, about -82°C to about -84°C, about -82°C to about -86°C, about -82°C to about -88°C, about -82°C to about -90°C, about -82°C to about -95°C. about -82°C to about -I00°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 -90°C to about -100°C, or about -95°C to about -100°C. In some cases, the freezer can be set at a range of temperature from 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 temperature from 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 temperature from 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.

[0213] In some cases, freezing the cryopreservation bags and surrogate vials is less effective when placed in a static freezer set at -80°C. Instead, better results were obtained when the static freezer was set to temperatures less than -80°C, e.g., -86°C.

[0214] It is contemplated that the cryopreservation storage can be in many forms. For instance, the cryopreserved bone marrow can be contained in bags of 1 ml to 5 ml volume or vials of 0.1 to 15 ml volumes. In a preferred embodiment, the bags with 70 ml bone marrow are stored in a cooling box within a cryogenic freezer.

[0215] The cryopreserved bone marrow is cryobanked for later thawing and extraction of desired cells. The thawed bone marrow can be provided for a wide range of treatments including treatment for leukemias, brain tumors, breast cancer, Hodgkin's disease, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, blood cancers, ovarian cancer, sarcoma, testicular cancer, other solid organ cancer, rheumatoid arthritis, multiple sclerosis, diabetes mellitus, cystic fibrosus, Alzheimer's disease, genetic immunodeficiencies, metabolic disorders, marrow failure syndromes, and HIV. Bone marrow can also be used for induction of immunotolerance to reduce the potential rejection of an implant obtained from an organ donor. Bone marrow treatments can also be indicated for casualties caused by radiation and certain biological weapons.

[0216] Another aspect of the present disclosure comprises a method for processing a biological sample comprising cells or a derivative thereof, the method comprising: generating a first volume of the biological sample comprising cells or a derivative thereof, wherein the first volume comprises a first concentration of cells or a derivative thereof; generating a second volume of the biological sample comprising cells or a derivative thereof, wherein the second volume is less than the first volume and comprises a second concentration of the cells whereinthe second concentration of the cells is no more than 30% different than the first concentration of the cells; 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 than the second cooling rate; wherein a post-thaw cell proliferation rate of the cells in the first volume is no more than 30% different than a post-thaw proliferation rate of the cells in the second volume. 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.

[0217] The preponderance of literature indicates cells stored below the glass transition temperature of water (-I30°C) are stable indefinitely, with estimates based on biophysical properties ranging from 200-30,000 years (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 its entirety). The major potential source of damage is through thermocy cling due to inappropriate storage. Methods of the present disclosure allow detection of inappropriate storage and associated damage to the biological sample that is to be administered to a subject in need. Nonetheless, preferably storage units are alarm monitored 24 hours per day and manually checked weekly to ensure temperature is maintained.

[0218] Having at least two volumes of a biological sample allows testing a subset of the biological sample (the second volume) without having to manipulate the portion of the biological sample that is to be administered to a subject (the first sample), with the second volume acting as a surrogate for the first volume. As used herein, the surrogate vial is typically a smaller volume of the cell product and the surrogate can be thawed and assayed as needed, e.g., for cell viability. The assay results for the surrogate vial represent the expected assay results for the first (larger) volume; however, by using the surrogate it is unnecessary to thaw the first volume for assaying and, instead, it is thawed when needing to be used, e.g., for transplanting into a subject in need. For the surrogate vial to accurate represent the first volume, the cells in both volumes should be frozen at the same rate; this common rate results in equivalent functional viability for cells in the two volumes. In some cases, a first volume (for example, a cry opreservation bag) and a second volume (for example, a surrogate cryovials) are placed in -86°C static freezer. The bags are placed in cassettes, which may lack insulation, while the surrogate vials are placed separately in a CoolCell® freezing storage system and then in front of the box of cassettes into the freezer.

[0219] 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 20% of the first volume, about 30% of the first volume to about 10% of the first volume, about 30% of the first volume to about 5% of the first volume, about 30% of the first volume to about 1% of the first volume, about 30% of the first volume to about 0.5% of the first volume, about 20% of the first volume to about 10% of the first volume, about 20% of the first volume to about 5% of the first volume, about 20% of the first volume to about 1% of the first volume, about 20% of the first volume to about 0.5% of the first volume, about 10% of the first volume to about 5% of the first volume, about 10% of the first volume to about 1% of the first volume, about 10% of the first volume to about 0.5% of the first volume, about 5% of the first volume to about 1% of the first volume, about 5% of the first volume to about 0.5% of the first volume, or 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 secondvolume 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.

[0220] In some embodiments a post-thaw viability rate (e.g., the functional viability) of the cells in the first volume is no more than about 0.5% different than a post-thaw viability rate of the cells in the second volume to about 30% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than about 30% different than a post-thaw viability rate of the cells in the second volume to about 25% different than a post-thaw viability rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 20% different than a post-thaw viability rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 15% different than a post-thaw viability rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 10% different than a post-thaw viability rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 5% different than a post-thaw viability' rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 1% different than a post-thaw viability rate of the cells in the second volume, about 30% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume to about 20% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability' rate of the cells in the second volume to about 15% different than a post-thaw viability’ rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume to about 10% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume to about 5% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume to about 1% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volumeto about 15% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volume to about 10% different than a post-thaw viability’ rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volume to about 5% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volume to about 1 % different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume to about 10% different than a post- thaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume to about 5% different than a post-thaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume to about 1% different than a post-thaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability’ rate of the cells in the second volume to about 5% different than a postthaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability rate of the cells in the second volume to about 1% different than a post-thaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, about 5% different than a post-thaw viability rate of the cells in the second volume to about 1% different than a post-thaw viability rate of the cells in the second volume, about 5% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume, or about 1% different than a post-thaw viability rate of the cells in the second volume to about 0.5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than about 30% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability rate of the cells in the second volume, about 5% different than a post- thaw viability rate of the cells in the second volume, about 1% different than a post-thaw viability rate of the cells in the secondvolume, or about 0.5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than at least about 30% different than a post-thaw viability rate of the cells in the second volume, about 25% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a post-thaw viability7rate of the cells in the second volume, about 15% different than a post- thaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability rate of the cells in the second volume, about 5% different than a post-thaw viability rate of the cells in the second volume, or about 1% different than a post-thaw viability7rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than at most about 25% different than a post-thaw viability rate of the cells in the second volume, about 20% different than a postthaw viability rate of the cells in the second volume, about 15% different than a post-thaw viability rate of the cells in the second volume, about 10% different than a post-thaw viability rate of the cells in the second volume, about 5% different than a post-thaw viability7rate of the cells in the second volume, about 1% different than a post-thaw viability rate of the cells in the second volume, or about 0.5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 30% different than a post-thaw viability7rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 25% different than a post-thaw viability rate of the cells in the second volume. In some embodiments, a post-thaw viability rate of the cells in the first volume is no more than 20% different than a post-thaw viability7rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 15% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 13.6% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 10% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw viability rate of the cells in the first volume is no more than 5% different than a post-thaw viability rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 25% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 20% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the firstvolume is no more than 15% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 13.6% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 10% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments a post-thaw cell proliferation rate of the cells in the first volume is no more than 5% different than a post-thaw proliferation rate of the cells in the second volume. In some embodiments the post-thaw viability rate of the cells is at least 50%. Viability may relate to either or both of functional viability which measures the cells’ ability to proliferate and routine viability which relates to the numbers or percentages of live cells, e.g., as measured by Trypan Blue.

[0221] In some embodiments the post-thaw proliferation rate of the cells (which represents the cells’ functional viability) is at least 1 CFU-GM / 105cells. In some embodiments the postthaw proliferation rate of the cells is at least about 1 CFU-GM / 105cells to about 200 CFU- GM / 105cells. In some embodiments the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 105cells to about 10 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 20 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 30 CFU-GM / 105cells, about 1 CFU- GM / 105cells to about 40 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 50 CFU- GM / 105cells, about 1 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 1 CFU-GM / 15cells to about 100 CFU-GM / 105cells, about 1 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 20 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 30 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 40 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 50 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 10 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 30 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 40 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 50 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 20 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 20CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 40 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 50 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 30 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 50 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 40 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 50 CFU-GM / 105cells to about 60 CFU-GM / 105cells, about 50 CFU-GM / 105cells to about 70 CFU-GM / 1055 cells, about 50 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 50 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 50 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 50 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 60 CFU-GM / 105cells to about 70 CFU-GM / 105cells, about 60 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 60 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 60 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 60 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 70 CFU-GM / 105cells to about 80 CFU-GM / 105cells, about 70 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 70 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 70 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 80 CFU-GM / 105cells to about 90 CFU-GM / 105cells, about 80 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 80 CFU-GM / 105cells to about 200 CFU-GM / 105cells, about 90 CFU-GM / 105cells to about 100 CFU-GM / 105cells, about 90 CFU-GM / 105cells to about 200 CFU-GM / 105cells, or about 100 CFU-GM / 105cells to about 200 CFU-GM / 105cells. In some embodiments the post-thaw proliferation rate of the cells is at least about 1 CFU-GM / 105cells, about 10 CFU-GM / 105cells, about 20 CFU-GM / 105cells, about 30 CFU-GM / 105cells, about 40 CFU-GM / 105cells, about 50 CFU-GM / 105cells, about 60 CFU-GM / 105cells, about 70 CFU-GM / 105cells, about 80 CFU-GM / 105cells, about 90 CFU-GM / 105cells, about 100 CFU-GM / 105cells, or about 200 CFU-GM / 105cells. In some embodiments the post-thaw proliferation rate of the cells is at least at least about 1 CFU-GM / 105cells, about 10 CFU-GM / 105cells, about 20 CFU-GM / 105cells, about 30 CFU-GM / 105cells, about 40 CFU-GM / 105cells, about 50 CFU-GM / 105cells, about 60 CFU-GM / 105cells, about 70 CFU-GM / 105cells, about 80 CFU-GM / 105cells, about 90 CFU-GM / 105cells, or about 100 CFU-GM / 105cells. In some embodiments the post-thawproliferation rate of the cells is at least at most about 10 CFU-GM / 105cells, about 20 CFU- GM / 105cells, about 30 CFU-GM / 105cells, about 40 CFU-GM / 105cells, about 50 CFU- GM / 105cells, about 60 CFU-GM / 105cells, about 70 CFU-GM / 105cells, about 80 CFU- GM / 105cells, about 90 CFU-GM / 105cells, about 100 CFU-GM / 105cells, or about 200 CFU- GM / 105cells. Assays to determine functional viability may take about ten days to about two weeks of culturing. Method for culturing cell products relevant to the present disclosure are well-known in the art.

[0222] In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -0. l°C / min to about -5°C / min at least until ice has nucleated in a freezing medium. In some instances, the biological sample or derivative thereof can be cryopreserved first with supra-freeze. For example, the biological sample or derivative thereof can be cryopreserved while the biological sample or derivative thereof are just processed and at room temperature. In some instances, the supra-freeze rate is generally higher (e.g. decreasing of the temperature at a faster rate) compared to the sub-freeze rate. In some embodiments, the supra-freeze rate is from about -6°C / min to about -0.5°C / min. In some embodiments, the supra-freeze rate is from about -0.5°C / min to about -l°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 -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 -l °C / min to about -1.5°C / min, about -l°C / min to about -2°C / min, about -l°C / min to about -2.5°C / min, about -l°C / min to about -3°C / min, about -l°C / min to about -3.5°C / min, about -l°C / min to about -4°C / min, about -l°C / min to about -4.5°C / min, about -l°C / min to about -5°C / min, about -l°C / min to about -5.5°C / min, about -l°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°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, 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°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 -3.5°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 supra-freeze rate is about -0.5°C / min, about -l°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 supra-freeze rate is at least about -0.5°C / min, about -l°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 supra-freeze rate is at most about -l°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 supra-freeze rate was -3.2°C. In some embodiments, the supra-freeze 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 comprise a supra-freeze rate from about -2.5°C / min to about -4°C / min at least until ice has nucleated in a freezing medium. In some embodiments the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -2.5°C / min to about -3.5°C / min at least until ice has nucleated in a freezing medium. Preferably, the first cooling rate and the second cooling rate are from about -1°C to about -5°C.

[0223] In some embodiments first cooling rate and the second cooling rate differ by from about 1% to about 500%. The first cooling rate and the second cooling rate may differ by from about 1% to about 100%, e.g., 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%. or 100%. The first cooling rate and the second cooling rate may differ by from about 100% to about 200%, e.g., 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%, 197%, 198%, 199%, or 200%. The first cooling rate and the second cooling rate may differ by from about 200% to about 300%, e.g., 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%, 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 from about 400% to about 500%, e.g., 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%. 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 from about 400% to about 500%, e.g., 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%, 497%, 498%, 499%, or 500%.

[0224] In some embodiments the first cooling rate and the second cooling rate comprise a sub-freeze rate from about -l°C / min to about -2°C / min. In some embodiments, the sub-freeze rate is from about -2.5°C / min to about -0.1°C / min. In some embodiments, the sub-freeze rateis from 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 -l°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. l°C / min to about -1.6°C / min, about -0. l°C / min to about -1.8°C / min, about -0. l°C / min to about -2°C / min, 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 -l°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 -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 -l°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 -l°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 -l°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 -l°C / min to about -1.2°C / min. about -l°C / min to about -1.4°C / min, about -l°C / min to about - 1.6°C / min, about -l °C / min to about -1.8°C / min, about -l °C / min to about -2°C / min, about - l°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 -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 sub-freeze rate is about -0. l°C / min, about -0.2°C / min, about -0.4°C / min, about -0.6°C / min, about -0.8°C / min. about -l°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 sub-freeze 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 - l°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 sub-freeze 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 -l°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 sub-freeze rate can be -1.36°C / min. In some embodiments, the sub-freeze rate comprises a range of -1.13°C / min to -1.62°C / min.

[0225] In some embodiments, wherein the supra-freezing rate, sub-freezing rate, and nucleation temperature for the given biological sample is not known, the cyrobanking methods described herein further comprise determining the supra-freezing rate, sub-freezing rate, and nucleation temperature for the biological sample. In some embodiments, the supra-freezing rate, sub-freezing rate, and nucleation temperature are derived from a freezing curve for the biological sample. In some embodiments, the freezing curve is modelled using a computer. In some embodiments, the freezing curve is determined empirically by following the procedures and methods described herein (e.g. Example 5).

[0226] In some embodiments the post-thaw viability rate of the cells (e.g., the cells’ functional viability ) is at least about 60% to about 95%. In some embodiments the post-thaw viability7rate of the 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 viability7rate of the cells is at least about 60%, about 70%, about 80%. about 90%, or about 95%. In some embodiments the post-thaw viability rate of the cells is at least at least about 60%, about 70%, about 80%, or about 90%. In some embodiments the post-thaw viability rate of the cells is at least at most about 70%, about 80%, about 90%, or about 95%. In some embodiments the post-thaw viability7rate of the cells is at least 60%. In some embodiments the post-thaw viability rate of the cells is at least 70%. In some embodiments the post-thaw viability rate of the cells is at least 80%. In some embodiments the post-thaw viability rate of the cells is at least 90%. Viability may relate to either or both of functional viability7which measures the cells’ ability to proliferate and routine viability' which relates to the numbers or percentages of live cells, e.g., as measured by Trypan Blue.

[0227] 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 of the one or more freezers. In some embodiments the first container is contained in a first freezer of the one or more freezers and the second container is contained in a second freezer of the one or more freezers. In some embodiments the one or more freezers comprise a static freezer. In some embodiments the first freezer, the second freezer, or both is a static freezer. In some embodiments, the one or more freezers comprise a controlled-rate freezer. In someembodiments the first freezer, the second freezer, or both is a controlled-rate freezer. In some embodiments the one or more freezers are set at about -70°C to -90°C. In some embodiments the one or more freezers are set at -80°C. In some embodiments the one or more freezers are set at -86°C. In some cases, the one or more freezers can be set at a range of temperature from about -100°C to about -60°C. In some cases, the freezer can be set at a range of temperature from 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°C to about -82°C, about -65°C to about -84°C, about -65°C to about -86°C, about -65°C to about -88°C, about -65°C to about -90°C, about - 65°C to about -95°C, about -65°C to about -100°C, about -70°C to about -75°C, about -70°C to about -80°C, about -70°C to about -82°C, about -70°C to about -84°C, about -70°C to about -86°C, about -70°C to about -88°C, about -70°C to about -90°C, about -70°C to about -95°C, about -70°C to about -100°C, about -75°C to about -80°C, about -75°C to about -82°C, about -75°C to about -84°C. about -75°C to about -86°C. about -75°C to about -88°C. about -75°C to about -90°C, about -75°C to about -95°C, about -75°C to about -100°C, about -80°C to about -82°C, about -80°C to about -84°C, about -80°C to about -86°C, about -80°C to about -88°C, about -80°C to about -90°C, about -80°C to about -95°C, about -80°C to about -100°C, about -82°C to about -84°C, about -82°C to about -86°C. about -82°C to about -88°C. about -82°C to about -90°C, about -82°C 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 -90°C to about -100°C, or about -95°C to about -100°C. In some cases, the freezer can be set at a range of temperature from 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 temperature from 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 temperature from 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.

[0228] In some cases, freezing the cryopreservation bags and surrogate vials is less effective when placed in a static freezer set at -80°C. Instead, better results were obtained when the static freezer was set to temperatures less than -80°C, e.g., -86°C.

[0229] In some embodiments the second volume is placed directly in an insulating container, such that each vial is in close proximity to the insulating material of the insulating container. In some embodiments the method further comprises arranging the first volume inside the static freezer such that the first volume does not contact a wall of the one or more freezers. In some embodiments the biological sample comprising cells or a derivative thereof, in the first volume and the biological sample comprising cells or a derivative thereof, in the second volume experience a same cooling rate. In some embodiments the cells are stem cells or immune cells. In some embodiments the stem cells comprise hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), or both. In some embodiments the biological sample comprises whole bone marrow. In some embodiments the biological sample comprises mobilized bone marrow cells, for example, that result from treatment of a donor with a bone marrow mobilizing agent, e.g., a colony stimulating factors (CSFs). In some embodiments the biological sample comprises one or more organs, blood, or both. In some embodiments the immune cells comprise T cells. In some embodiments the blood is cord blood or peripheral blood. In some embodiments the biological sample comprises plasma or blood serum. In some embodiments the HSCs comprise CD34+ cells. It is contemplated that the containers can be in many forms. For instance, the biological sample or derivative thereof can be contained in bags of 1 ml to 5 ml volume or vials of 0.1 to 15 ml volumes. In a preferred embodiment, the samples with less than 15 ml of biological sample are stored in an insulating container (e.g. a cooling box) within a freezer.

[0230] Described herein, in some embodiments, is a method for cryopreserving bone marrow or bone marrow cells. In some embodiments, the method utilizes the systems described herein. In some embodiments, the method comprises processing bone to obtain bone marrow- or derivative thereof to obtain bone marrow^ cells. In some cases, the bone marrow cells can be any cells that can be isolated from 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 to be cryopreserved at a freeze rate comprising at least -0.1°C / min, -0.2°C / min, -0.5°C / min, - l°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. -l l°C / min, -12°C / min, -13°C / min, -14°C / min, -15°C / min, -20°C / min,or higher rate. Preferably, the cryopreservation bags and surrogate vials are cooled at a rate of -l°C to -5°C.

[0231] In some embodiments, the freeze rate comprises the temperature decrease as measured by directly contacting the bone marrow or bone marrow cells with a thermometer. In some embodiments, the freeze rate comprises the temperature decrease as measured in the microenvironment or environment immediately adjacent the bone marrow or bone marrow cells. In some embodiments, the freeze rate comprises the temperature decrease as measured in the freezing apparatus (e.g. freezing bag, cry opreservation bag, cryotube, cryo tank, freezing cassette, freezer, or vessel holding liquid nitrogen).

[0232] In some embodiments, the method of cry opreserving the bone marrow or bone marrow cells described herein increases the yield of the bone marrow cells after thawing compared to bone marrow cells that are not cryopreserved by the freezer rate described herein. In some instances, the yield of the bone marrow cells cryopreserved by the freezer rate described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 50 folds, or more compared to yield of bone marrow cells not cryopreserved by the freezer rate described herein. In some embodiments, the method of cryopreserving the bone marrow or bone marrow cells described herein increases the viability of the bone marrow cells after thawing compared to bone marrow cells that are not cryopreserved by the freezer rate described herein. In some instances, the viability of the bone marrow cells cryopreserved by the freezer rate described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 50 folds, or more compared to viability of bone marrow cells not cryopreserved by the freezer rate described herein. In some embodiments, the method of cry opreserving the bone marrow or bone marrow cells described herein increases the number of CD34+ bone marrow cells after thawing compared to the number of CD34+ bone marrow cells that are not cryopreserved by the freezer rate described herein. In some instances, the number of CD34+ the bone marrow cells cryopreserved by the freezer rate described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 folds, 3 folds. 4 folds. 5 folds, 10 folds. 20 folds, 50 folds, or more compared to the number of CD34+ the bone marrow cells not cryopreserved by the freezer rate described herein. In some embodiments, the method of cryopreserving the bone marrow or bone marrow cells described herein increases the number of CD45+ bone marrow cells after thawing compared to the number of CD45+ bone marrow cells that are not cryopreserved by the freezer rate described herein. In some instances, the number of CD45+ the bone marrow cells cryopreserved by thefreezer rate described herein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%. 100%, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 50 folds, or more compared to the number of CD45+ the bone marrow cells not cryopreserved by the freezer rate described herein.

[0233] In some embodiments, after thawing the samples frozen utilizing the schemes described herein (e.g. Example 5), the samples contain an increased amount of viable CD34+ cells as compared to known cryopreservation protocols. In some embodiments, the percentage of viable CD34+ cells in the thawed sample is at least about 70% to about 95%. In some embodiments, the percentage of viable CD34+ cells 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 viable CD34+ cells 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 viable CD34+ cells in the thawed sample is at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the percentage of viable CD34+ cells in the thawed sample is at least at most about 75%, about 80%, about 85%, about 90%, or about 95%.

[0234] Illustrative methods for obtaining, manufacturing, cryopreserving, and / or storing bone marrow products comprising hematopoietic stem cells used in methods of the present disclosure may 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 its entirety.Automated System for Recovery of Bone Marrow

[0235] The present disclosure contemplates an automated process for recovery of the bone marrow, and even selection of cells from the bone marrow. In one aspect, an automated system 209 includes sequential stations, as depicted in FIGS. 7A-7B. The first station 210 of the automated process debrides the VBs to remove all soft tissue. In contrast to the manual process that operates on one VB at a time, the automated process is configured to debride an entire donor VB set (which can be at least ten vertebral bodies). The VBs are mounted on a rack or tray 212 that is configured to support the vertebral body set from a given donor. The tray 212 is placed on transfer rails 216 of a housing 215, as shown in FIGS. 8A-8B, with the trayadvanced automatically or manually into the interior of the housing. The housing 215 supports a plurality of hydrojets 220 that direct high pressure and high velocity jets of saline onto the VBs. In the known manual process, a manual hydrojet, operating at lower velocities and pressures, directs a stream of detergent onto the VB. In the manual process, the detergent is needed to clean the VBs of the soft tissue. In contrast, the automated cleaning station 210 of the present disclosure uses a saline medium, with the velocity and pressure of the water jets being sufficient to dislodge all soft tissue from the VBs. The automated cleaning station of the present disclosure includes jets configured to produce a direct stream or narrow “V” water / saline jet that generates a high concentrated impact force at varying distances. To achieve good coverage of the VBs, the device includes many direct jets at close spacing at different orientations relative to the VBs, which allows for uniform cleaning independent of the position of the VB in the device. In the illustrated embodiment of FIG. 8A, the hydrojets are provided in an upper 220 and a lower row 221. The “V” jets are aligned at different angles to achieve full coverage of the surfaces of the VBs. In addition, or alternatively, the hydrojets 220, 221 can be configured to oscillate over the tray of VBs to ensure complete coverage.

[0236] A visualization device 225 is arranged at the outlet of the debridement station 210 that is operable to visualize and interpret the VBs exiting the station to determine if all of the soft tissue has been removed, as shown in FIG. 8B. If not, then the VBs are returned along the rails 216 back into the housing for further hydrojet processing. It is contemplated that a controller (not shown) can be provided to control the movement of the tray 212 along the rails 216 and to interpret the signals generated by the visualization device 225. The visualization device can include a camera that obtains an image of the VBs and the controller can include imaging software capable of recognizing the soft tissue in the acquired image. A dye can be applied to the cleaned VBs at the end of the hydrojet debridement process, in which the dye is absorbed by soft tissue but not bone. The dye can thus provide contrast to facilitate differentiation of any remaining soft tissue from the bone. The visualization device 225 can be configured to pan across the VBs, such as by translating along a frame 226 and by translating the frame in order to view the VBs at all angles.

[0237] Returning to FIGS. 9A-9B, once it is determined that the VBs are cleaned of all soft tissue, the debrided VBs are then fed by a conveyor 230 to an automated grinding station 240 to produce appropriately sized pieces for tumbling and final cell extraction. The manual “cubing’" process described above can be variable, time consuming, and potentially not safe for the operator. The automated system includes a grinding station that combines “cubing"’ the VBs (for example, cutting the VBs into small pieces) and grinding the cubed VBs to reducethe VBs to 2-3mm pieces. The rails 216 and tray 212 can be conOd to deposit the debrided VBs onto the conveyor 230 which then automatically transfers the VBs to an input hopper 242 of the grinding station 240, shown in more detail in FIGS. 9A-9B. The VBs are directed through an initial mill cutter module 244, then through a funnel 246 to a fine mill cutter module 248, as show n in FIG. 9A. As shown in FIG. 9B the initial mill cutter module 242 includes opposed rotating grinding mills 245 that are separated by a predetermined gap, such as a 5-8mm gap, so that the incoming VBs are ground into coarse-sized segments. The coarse ground segments are fed to the fine mill cutter module 248 in which smaller diameter grinding mills 249 are provided. The fine grind mills 249 are separated by a smaller gap, on the order of 2-3mm, to produce finely ground VB segments. As shown in FIG. 9A, a funnel 246 conveys the coarse ground segments to the second grinding mill 248, and a funnel 250 directs the finely ground VB segments to a collection pan 252 supported on a plate 253. During the milling operation, a measured volume of processing / resuspension medium with DNase can be directed through the upper hopper, onto grinding cutters. This medium can be manually introduced during the operation of the grinding station 240, or can be automatically implemented through nozzles incorporated into the hopper 242.

[0238] The finely ground VB segments and processing medium are collected in the collection pan 252 and the plate 253 can be moved to a sieve station 260 (FIGS. 8A-8B), whether manually or automatically. Once at the sieve station 260 the contents of the pan 252 are dropped into a sieve cartridge unit which includes two 12?’ diameter filter sieves- a #40 sieve 262 on top followed by a finer #80 sieve 264, as depicted in FIG. 10. A funnel 266 directs the filtered contents to a collection container 268. The grindings retained by the filters are rinsed within the sieve station 260 with processing / resuspension medium that does not include DNase. The liquid bone marrow product in the collection container 268 can be analyzed to determine cell content and then concentrated and packaged in appropriate volumes for cryopreservation, as described below . Alternatively, some or all of the processed bone marrow can be further processed using automated cell selection approaches for specialized cell products such as CD34+ cells. Because large volumes of cells can be recovered from a single organ donor with this approach, one donor could yield multiple product types. Moreover, since the source is primary bone marrow (as opposed to G-CSF mobilized peripheral blood) the cell product will endure cry opreservation processing.

[0239] In one modification, the output from the grinding station 240 or the sieve station 260 can be automatically fed to a collection bag for cryogenic treatment. In this modification, the lower funnel 250 can be configured to direct the contents to a fluid line connected to a sterilebag. A peristaltic pump can engage the fluid line to pump the output from the grinding station to the sterile bag. A similar arrangement can be engaged to the funnel 266 of the sieve station.

[0240] The content of the collection container 268, which is essentially a bone marrow slurry, is conveyed, either manually or automatically, to an adjacent tumbler station 270 that includes a mechanical tumbler 272 and a large disposable vessel 274 that can contain the entire contents of ten processed VBs and associated processing / resuspension medium. The tumbler 272 has a paddle for agitation of the grinding slurry to mechanically liberate cells. When the tumbling cycle is complete, the contents of the tumbler are poured through a sieve magazine into the vessel 274. The contents of the vessel 274 can be processed further or prepared for cryogenic storage.

[0241] Additional teachings regarding packaging are disclosed in Woods. E.J. and S Thirumala. ‘'Packaging considerations for biopreservation.” Transfusion Medicine and Hemotherapy 38: 149-156 (2011); the contents of which is incorporated by reference in its entirety.Isolation of CD34+ cells

[0242] Described herein, in some aspects, is a method for processing (e.g. isolating) CD34+ cells obtained from bone marrow or bone marrow7derivative. In some cases, the bone marrow or bone marrow derivative can be fresh (e.g. never frozen) or thawed from being previously frozen. In some embodiments, the bone marrow or bone marrow derivative can be ground by the methods and systems described herein. In some embodiments, ground bone marrow or bone marrow cells can be contacted with the stabilization buffer described herein. In some embodiments, the stabilization prevents formation of aggregates of the bone marrow7cells. In some instances, the bone marrow7cells contacted and suspended in the stabilization buffer can be isolated by attaching to antibody such as a conjugated antibody. For example, bone marrow cells expressing CD34+ can be isolated and enriched by contacting the bone marrow cells with the CD34 antibody conjugated with iron, where the bone marrow cells expressing CD34 are then trapped a magnetic separation column (e.g. “CliniMACS®”). The bone marrow cells not expressing CD34 can be washed away. The trapped CD34+ bone marrow cells can be harvested by removing the magnetic field and eluting the targeted CD34+ bone marrow cells. Such approach does not require isolating the bone marrow cells with a Ficoll gradient.

[0243] Aspect described in the present disclosure comprises a method for processing a population of CD34+ cells obtained from bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processingthe bone into bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U / ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer. In some embodiments, the at least about 80.000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 70% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 80% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 90% viable CD34+ cells.

[0244] Another aspect of the present disclosure comprises a stabilization buffer that includes at least 5 U / ml of an anticoagulant, and more than 3 U / ml of a nuclease. In some embodiments, stabilization buffer comprises more than about 5 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 10 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 15 U / ml of a nuclease. In some embodiments, the stabilization buffer comprises more 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 Benzonase® or Denarase®. In some embodiments, the stabilization buffer further comprises more than about 10 U / ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises about 10 U / ml of an anticoagulant. 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.

[0245] In some embodiments, the stabilization buffer comprises nuclease. In some embodiments, the nuclease is Benzonase® or Denarase®. In some embodiments, the stabilization buffer comprises nuclease at about 3 U / ml. 4 U / ml, 5 U / ml, 6 U / ml, 7 U / ml, 8 U / ml, 9 U / ml, 10 U / ml, 11 U / ml, 12 U / ml, 13 U / ml, 14 U / ml, 15 U / ml, 16 U / ml, 17 U / ml, 18 U / ml, 19 U / ml, 20 U / ml, 21 U / ml, 22 U / ml, 23 U / ml, 24 U / ml, 25 U / ml, 26 U / ml, 27 U / ml, 28 U / ml, 29 U / ml. 30 U / ml, 50 U / ml, 100 U / ml, 200 U / ml, or more U / ml. In some embodiments, the stabilization buffer comprises an anticoagulant. In some cases, the anticoagulant is Heparin. In some instances, the stabilization buffer comprises anticoagulant at 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, 12 U / ml, 13 U / ml, 14 U / ml, 15 U / ml, 16 U / ml, 17 U / ml, 18 U / ml, 19 U / ml, 20 U / ml, 21 U / ml, 22 U / ml, 23 U / ml, 24 U / ml, 25 U / ml, 26 U / ml, 27 U / ml, 28 U / ml, 29 U / ml, 30 U / ml, 50 U / ml, 100 U / ml, 200 U / ml, or more U / ml.

[0246] In various embodiments, a stabilization buffer lacks heparin.

[0247] In some embodiments, the stabilization 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.

[0248] Described herein, in some embodiments, is a method of processing bone marrow to obtain bone marrow cells. In some embodiments, the method comprises contacting the bone marrow or the bone marrow cells with the stabilization buffer described herein.

[0249] Another aspect of the present disclosure comprises a method for processing a population of CD34+ cells comprised in bone marrow or a derivative thereof, wherein the bone marrow or the 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 bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U / ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer.

[0250] In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield of the bone marrow cells obtained from the methods described herein compared to the yield of the bone marrow cells processed in the absence of the stabilization buffer. In some instances, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield 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 yield of bone marrow cells processed in the absence of the stabilization buffer. In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability of the bone marrow cells obtained from themethods described herein compared to the viability of the bone marrow cells processed in the absence of the stabilization buffer. In some instances, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability 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 viability of bone marrow cells processed in the absence of the stabilization buffer.

[0251] In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD34+ bone marrow cells compared to the number of CD34+ bone marrow cells processed in the absence of the stabilization buffer. In some cases, the number of CD34+ bone marrow obtained from processing with the 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 CD34+ bone marrow obtained from processing in the absence of stabilization buffer. In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD45+ bone marrow cells compare to the number of CD45+ bone marrow cells processed in the absence of the stabilization buffer. In some cases, the number of CD45+ bone marrow obtained from processing with the 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 from processing in the absence of stabilization buffer.

[0252] In some embodiments, cellular compositions comprising CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein have an increased amount of CD34+ cells, as compared to cellular compositions generated from known CD34+ isolation methods. In some embodiments. The amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 CD34+ cells / 750 pl of bone marrow or a derivative thereof contacted with the stabilization buffers described herein. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells / 750 pl to about 100,000 cells / 750 pl. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70.000 cells / 750 pl to about 75,000 cells / 750 pl, about 70,000 cells / 750 pl to about 80,000 cells / 750 pl, about 70,000 cells / 750 pl to about 85,000 cells / 750 pl, about 70,000 cells / 750 pl to about 90,000 cells / 750 pl, about70,000 cells / 750 pl to about 95,000 cells / 750 pl, about 70,000 cells / 750 pl to about 100,000 cells / 750 jil, about 75,000 cells / 750 pl to about 80,000 cells / 750 .1, about 75,000 cells / 750 pl to about 85,000 cells / 750 pl, about 75,000 cells / 750 pl to about 90,000 cells / 750 pl, about 75,000 cells / 750 pl to about 95,000 cells / 750 pl, about 75,000 cells / 750 pl to about 100,000 cells / 750 pl, about 80,000 cells / 750 pl to about 85,000 cells / 750 pl, about 80,000 cells / 750 pl to about 90,000 cells / 750 pl, about 80,000 cells / 750 pl to about 95,000 cells / 750 pl, about 80.000 cells / 750 pl to about 100,000 cells / 750 pl, about 85,000 cells / 750 pl to about 90.000 cells / 750 pl, about 85,000 cells / 750 pl to about 95,000 cells / 750 pl, about 85,000 cells / 750 pl to about 100,000 cells / 750 pl, about 90,000 cells / 750 pl to about 95,000 cells / 750 pl, about 90,000 cells / 750 pl to about 100,000 cells / 750 p.1, or about 95,000 cells / 750 pl to about 100,000 cells / 750 | l. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells / 750 pl, about 75,000 cells / 750 pl, about 80,000 cells / 750 pl, about 85,000 cells / 750 pl, about 90,000 cells / 750 pl, about 95,000 cells / 750 pl, or about 100,000 cells / 750 pl. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at least about 70.000 cells / 750 pl. about 75,000 cells / 750 pl, about 80,000 cells / 750 pl, about 85,000 cells / 750 pl, about 90,000 cells / 750 pl, or about 95,000 cells / 750 pl. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at most about 75,000 cells / 750 pl, about 80,000 cells / 750 pl, about 85.000 cells / 750 pl, about 90,000 cells / 750 pl, about 95,000 cells / 750 pl, or about 100,000 cells / 750 pl.

[0253] In some embodiments, the CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein also exhibit higher viability as compared to cellular compositions generated from known CD34+ isolation methods.

[0254] In some embodiments, the amount of CD34+ cells isolated from the bone marrow^ samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability 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%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at least about 70%, about 75%. about 80%, about 85%, or about 90%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at most about 75%, about 80%, about 85%, about 90%. or about 95%. Viability may relate to either or both of functional viability which measures the cells’ ability to proliferate and routine viability which relates to the numbers or percentages of live cells, e.g., as measured by Trypan Blue.

[0255] In an aspect of the present disclosure, a method is provided for selecting CD34 expressing (CD34+) cells from deceased donor bone marrow using density reduced Ficoll and an immunomagnetic CD34+ cell isolation kit. Surprisingly, it has been found that cell isolation using density’ reduced Ficoll prior to CD34 selection is beneficial to obtain high purity and viability CD45 / CD34+ cells from freshly prepared deceased donor bone marrow. On the other hand. Ficoll at conventional density has been found to be optimal for CD45 / CD34+ cell selection from thawed cryopreserved deceased donor bone marrow.

[0256] Vertebral sections obtained from a recently deceased donor were processed as described above. Thus, in one embodiment, the bone is cleaned of all soft tissue and then cut into small pieces that were immediately submerged into 500 ml of grinding media. The grinding media can be PLASMA-LYTE™ A injection pH 7.4, multiple electrolytes, injection type 1 USP (PLASMA-LYTE™) containing 2.5% human serum albumin (HSA), 3 U / ml denarase, and 10 U / ml heparin. The sectioned VB are ground using a bone grinder, filtered and rinsed with rinse media (such as PLASMA-LYTE™ with 2.5% HSA). The entire cell suspension is centrifuged to concentrate cells to 2-3xl08 / ml and the cell concentration is extracted. A portion or all of the resulting BM preparation can be used immediately for CD34 selection, while the remainder can be prepared for cry opreservation. The cryopreserved portion involves adding a final concentration of 10% DMSO and 5% HSA to the BM cells and bringing the preparation to - 86°C, either by passive cooling or by controlled cooling at a rate of approximately -rc / min. after which the cryopreserved portion is plunged into liquid nitrogen.

[0257] For selection of CD34+ cells, either the newly processed BM preparation is used or a previously cryopreserved portion is thawed for use. Ficoll-Paque PLUS is added to the BM preparation to separate the desired CD34+ cell component of the bone marrow. It has been found for cell selection from cryopreserved bone marrow that the conventional density for the Ficoll of 1.077 g / ml produces acceptable results. However, in one aspect of the present disclosure, for cell selection from freshly prepared deceased donor bone marrow the Ficoll density is reduced from the conventional density. In particular, the density is reduced by mixing Ficoll-Paque PLUS (density 1.077 g / mL, GE Company) with Plasma Lyte-A Injection pH 7.4 (Baxter Healthcare 2B2544X) in specific proportions to obtain an overall density of less than 1.077 g / ml, particularly 1.063 — 1.052 g / ml. In one specific embodiment, the density of 1.063 g / ml was found to be optimal for isolation of CD34+ cells, taking into account quantity, viability and purity’ of the CD34+ cells.

[0258] In one embodiment, 5 ml of the 1.063 g / ml density Ficoll solutions is pipetted into 15- ml centrifuge tubes, and the BM solution generated from VBs of deceased donors is carefully layered over the Ficoll gradient. The tubes are centrifuged for 30 min at 400 g without break at room temperature. After centrifugation, buffy coat cells are harvested carefully, and the cells are washed in phosphate-buffered saline (PBS) containing 0.5% HSA and 2mM Ethylenediaminetetraacetic acid (EDTA) (MACS buffer, Miltenyi). In one specific embodiment, centrifugation is performed for 5 min at 400 g, and the resulting cell pellets are resuspended in 10 ml PBS, followed by a second centrifugation for 5 min at 400 g.

[0259] Nucleated cells in the isolated huffy coat can be counted using a Sysmex XP-300. A Cellometer Vision (Nexcellom) or flow cytometer can be used to determine cell counts 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. The CD34+ cells can be selected by a positive immune separation method using a CliniMACS system (Miltenyi, Bergisch Gladbach, Germany) or an EasySep CD34 kit (Stemcell Technologies, Vancouver, BC, Canada) in accordance with the protocol of the manufacturer. From testing at various Ficoll densities it has been surprisingly determined that the lower Ficoll density contemplated in the present disclosure (for example, 1.063 — 1.052 gm / ml vs. the conventional 1.077 gm / ml density) leads to more optimum cell recovery’. Optimization is based on purity, viability and yield of selected CD34 cells. A target of >90% purity' and >90% viable CD34+ cells is preferred. While lower Ficoll densities resulted in greater purity and fewer dead cells, it was surprisingly found that a greater portion of the CD34+ cells present in the deceased donor whole bone marrow before selection are lost using the lower Ficoll densities to prepare buffy coat. Thus, the high viabilityand purity of CD45 / CD34+ cells achieved at the conventional Ficoll density gradient also leads to a large loss in yield (approximately 60% loss of input CD34+ cells).

[0260] Thus, in accordance with one aspect of the present disclosure, for freshly prepared the optimal density of Ficoll for selection of CD45 / CD34+ cells at >90% purity and viability is less than 1.077 and particularly 1.063- 1.052. This Ficoll density provides a higher yield of CD45 / CD34+ cells with similar purity and cell viability to the conventional Ficoll density approach.

[0261] In another aspect of the present disclosure, the CD34+ cells can be initially acquired from a freshly prepared deceased donor bone marrow using the reduced density Ficoll-Paque described above. The BM can be cryogenically frozen and then the CD34+ cells can be acquired later using conventional density Ficoll-Paque. This approach essentially allows selective recovery of cells from deceased donor bone marrow — either before freezing using the modified Ficoll density7or after freezing and thawing using conventional Ficoll density. Recovery of MSCs from Processed Bone Marrow

[0262] Bone marrow is a well-known source for mesenchymal stromal / stem cells (MSCs) which can be harvested from bone marrow obtained using the methods described above. MSCs are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell types of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes. In addition, MSCs can migrate to sites of inflammation and exert potent immunosuppressive and antiinflammatory effects through interactions between lymphocytes associated with both the innate and adaptive immune system. MSCs can be used in treating osteogenesis imperfect, cartilage defects, myocardial infarction, Crohn's disease, multiple sclerosis, autoimmune disease such as Lupus, liver cirrhosis, osteo arthritis, and rheumatoid arthritis. Matched HSC / MSC units which can be used in co-transplant for treatment of graft vs. host disease (GVHD), and for hematopoietic stem cell transplant support.

[0263] In another feature of the systems and methods disclosed herein, a method is provided for recovering mesenchymal stem cells (MSCs) from enzy matically digested vertebral body (VB) bone fragments that are the byproduct of the VB grinding and elution of the methods described herein. In this method, a mixture of both collagenase and neutral protease is used to obtain the highest possible yields of vertebral bone adherent MSC (vBA-MSC). The MSCs can be recovered from cryopreserved VB bone fragments that are later processed according to the present disclosure. In one specific aspect, recombinant Clostridium histolyticum collagenase, comprised of the two active isoforms, is used in effective amounts in the MSC extraction process. The mixture of cells liberated by digesting VB bone fragment is cultured on tissue-coated plastic in the presence of Mesencult medium to select proliferative vBA-MSC. Freshly digested preparations as well as different passages of vBA-MSC can be characterized by flow cytometry, colony forming unit-fibroblast (CFU-F) potential, population doubling time (PDT) and trilineage (adipogenic, chondrogenic and osteogenic) differentiation in vitro. In some embodiments, the 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.

[0264] The present disclosure thus contemplates a method for optimizing digestion and MSC recovery from vertebral bone fragments using a combination of purified collagenase and neutral protease. In one specific embodiment, the collagenase is DE collagenase (Vitacyte), which is comprised of purified Clostridium histolyticum collagenase and Paneibcicillus polymyxa neutral protease. In accordance with one aspect of the disclosure, optimal neutral protease concentration and collagenase concentrations (Cl and C2 collagenase) and optimal ratio of solution volume (mis) to bone fragment weight (mgs) are determined.

[0265] In some embodiments, a collagenase may include Clostridium histolyticum further comprising two active isoforms. C 1 and C2. In some embodiments, one or more collagenases comprising isoforms Cl and C2 may be present in the digestion solution at a ratio comprising more collagenase isoform Cl than collagenase isoform C2. In some embodiments, the ratio of collagenase isoform Cl to collagenase isoform C2 may be about 30 to about 70: about 10 to about 29. In some embodiments, the ratio of collagenase isoform Cl to collagenase C2 may be 35: 15. In some embodiments, the mass ratio of Cl and C2 for each concentration may be 70:30, 54:46, 37:63, 82: 18, 54:46, and 90: 10.

[0266] In some embodiments, the neutral protease may be Paneibacillus polymyxa neutral protease. In some embodiments, the neutral protease concentration may be about 2 U / ml to about 21 U / ml. In some embodiments, the neutral protease concentration may be about 2 U / ml to about 7 U / ml, about 2 U / ml to about 12 U / ml, about 2 U / ml to about 17 U / ml, about 2 U / ml to about 21 U / ml, about 7 U / ml to about 12 U / ml, about 7 U / ml to about 17 U / ml, about 7 U / ml to about 21 U / ml, about 12 U / ml to about 17 U / ml, about 12 U / ml to about 21 U / ml, or about 17 U / ml to about 21 U / ml. In some embodiments, the neutral protease concentration may be about 2 U / ml, about 7 U / ml, about 12 U / ml, about 17 U / ml, or about 21 U / ml. In some embodiments, the neutral protease concentration may be at least about 2 U / ml, about 7 U / ml, about 12 U / ml, or about 17 U / ml. In some embodiments, the neutral protease concentration may be at most about 7 U / ml, about 12 U / ml, about 17 U / ml, or about 21 U / ml. In someembodiments, the digestion solution may comprise the neutral protease at an activity of about19.6 U / ml.

[0267] In some embodiments, the collagenase concentration is about 0.05 U / ml to about 1.6 U / ml. In some embodiments, the collagenase concentration is about 0.05 U / ml to about 0.1 U / ml, about 0.05 U / ml to about 0.15 U / ml, about 0.05 U / ml to about 0.2 U / ml, about 0.05 U / ml to about 0.25 U / ml, about 0.05 U / ml to about 0.3 U / ml, about 0.05 U / ml to about 0.35 U / ml, about 0.05 U / ml to about 0.4 U / ml. about 0.05 U / ml to about 0.8 U / ml, about 0.05 U / ml to about 1.2 U / ml, about 0.05 U / ml to about 1.6 U / ml, about 0.1 U / ml to about 0.15 U / ml, about 0.1 U / ml to about 0.2 U / 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 0.8 U / ml, about 0.15 U / ml to about 1.2 U / ml, about 0.15 U / ml to about 1.6 U / ml, about 0.2 U / ml to about 0.25 U / ml, about 0.2 U / ml to about 0.3 U / ml, about 0.2 U / ml to about 0.35 U / ml. about 0.2 U / ml to about 0.4 U / ml, about 0.2 U / ml to about 0.8 U / ml. about 0.2 U / ml to about 1.2 U / ml, about 0.2 U / ml to about 1.6 U / ml, about 0.25 U / ml to about 0.3 U / ml, about 0.25 U / ml to about 0.35 U / ml, about 0.25 U / ml to about 0.4 U / ml, about 0.25 U / 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 1.2 U / ml, about 0.4 U / ml to about 1.6 U / ml. about 0.8 U / ml to about 1.2 U / ml. about 0.8 U / ml to about1.6 U / ml, or about 1.2 U / ml to 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.

[0268] In accordance with one aspect of the disclosure, neutral protease concentration and collagenase concentrations (C 1 and C2 collagenase) and ratio of solution volume (mis) to bone fragment weight (mgs) are determined.

[0269] In some embodiments, the total collagenase concentrations (C 1 and C2 collagenase) are about 25 pg / ml to about 100 pg / ml. In some embodiments, the total collagenase concentrations are about 25 pg / ml to about 32.5 pg / ml, about 25 pg / ml to about 47.5 pg / ml, about 25 pg / ml to about 42.5 pg / ml, about 25 pg / ml to about 50 pg / ml, about 25 pg / ml to about 65 pg / ml, about 25 pg / ml to about 77.5 pg / ml, about 25 pg / ml to about 85 pg / ml, about 25 pg / ml to about 100 pg / ml, about 32.5 pg / ml to about 47.5 pg / ml, about 32.5 pg / ml to about42.5 pg / ml, about 32.5 pg / ml to about 50 pg / ml, about 32.5 pg / ml to about 65 pg / ml, about32.5 pg / ml to about 77.5 pg / ml, about 32.5 pg / ml to about 85 pg / ml, about 32.5 pg / ml to about 100 pg / ml, about 47.5 pg / ml to about 42.5 pg / ml, about 47.5 pg / ml to about 50 pg / ml, about47.5 pg / ml to about 65 pg / ml, about 47.5 pg / ml to about 77.5 pg / ml, about 47.5 pg / ml to about 85 pg / ml, about 47.5 pg / ml to about 100 pg / ml, about 42.5 pg / ml to about 50 pg / ml, about42.5 pg / ml to about 65 pg / ml, about 42.5 pg / ml to about 77.5 pg / ml, about 42.5 pg / ml to about 85 pg / ml, about 42.5 pg / ml to about 100 pg / ml, about 50 pg / ml to about 65 pg / ml. about 50 pg / ml to about 77.5 pg / ml, about 50 pg / ml to about 85 pg / ml, about 50 pg / ml to about 100 pg / ml, about 65 pg / ml to about 77.5 pg / ml, about 65 pg / ml to about 85 pg / ml, about 65 pg / ml to about 100 pg / ml, about 77.5 pg / ml to about 85 pg / ml, about 77.5 pg / ml to about 100 pg / ml, or about 85 pg / ml to about 100 pg / ml. In some embodiments, the total collagenase concentrations are about 25 pg / ml, about 32.5 pg / ml, about 47.5 pg / ml, about 42.5 pg / ml, about 50 pg / ml, about 65 pg / ml, about 77.5 pg / ml, about 85 pg / ml, or about 100 pg / ml. In some embodiments, the total collagenase concentrations are at least about 25 pg / ml. about 32.5 pg / ml, about 47.5 pg / ml. about 42.5 pg / ml, about 50 pg / ml, about 65 pg / ml, about 77.5 pg / ml, or about 85 pg / ml. In some embodiments, the total collagenase concentrations are at most about32.5 pg / ml, about 47.5 pg / ml, about 42.5 pg / ml, about 50 pg / ml, about 65 pg / ml, about 77.5 pg / ml, about 85 pg / ml, or about 100 pg / ml.

[0270] In some embodiments, the mass ratio of Cl and C2 for each concentration are 70:30, 54:46, 37:63. 82: 18 and 90: 10. respectively.

[0271] The volume to weight ratio of digestion solution to captured ground bone is about 1 :1 to about 15: 1, e.g., about 5: 1. In some embodiments, the ratio may 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 3hours, 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, about 3 hours, or about 4 hours. In some cases, the digestion solution is contacted with the captured ground bone for up to about 4 hours.

[0272] In some cases, the optimal volume-to-weight ratio has been found to be 5: 1 at an optimal incubation time of 2.5 hours. The optimal protease produced neutral protease activity of 19.6 U / ml. On the other hand, it was found that total viable MSC cell count is generally insensitive to collagenase concentration. It was also found that the yields produced by recombinant collagenase isoforms Cl and C2 are similar to the yields with purified collagenase, regardless of the C1 / C2 ratio. Further details of the MSC recovery' process of the present disclosure are found in the technical article in 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.

[0273] According to the process, fragments of VB bone (either fresh fragments or cryopreserved fragments) are placed in cryoprotectant solution comprised of PLASMA- LYTE™, 2.5% human serum albumin and 10% dimethyl sulfoxide (DMSO) and incubated for 1 hour at 4°C. The solution is removed and the bone fragments cooled at a rate of ~l° / min to - 86°C and then plunged into liquid nitrogen. After 24-48 hours in liquid nitrogen, the bone fragments are thawed rapidly in a water bath set at 37°C and then washed in saline and digested using the collagenase / protease solution described above.Predicting Cell Viability Based on Ischemia Time

[0274] As discussed above, ischemia time of the donor bone impacts the viability of the cells extracted using the processes descnbed above. According to the present disclosure, total ischemia is defined as the interval starting at time of death (the point at which the donor's arterial system was cross-clamped and circulation ceased) and ending with the start of the recovery of cells from the bone. For purposes of statistical modeling, this total interval can be separated into three successive and mutually exclusive time components: (a) Warm Ischemia Time (WIT) - beginning at time of death and ending either when bones are recovered andpacked on ice or when the body is placed in a cooler; (b) Body Cooling Time (BCT) - beginning when the body is placed in the cooler and ending when bones are packed on ice; and (c) Cold Ischemia Time (CIT) - beginning when bones are packed on ice and ending when processing begins for extraction of cells, such as HSPCs. Thus, Total Ischemia Time = (WIT) + (BCT) + (CIT). For cases where whole-body cooling is not used, BCT is zero and Total Ischemia Time = (WIT) + (CIT).

[0275] In addition to Total Ischemia Time, a variable corresponding to processing experience can be incorporated into the viability’ determination. It is known that learning curves exert significant effects on outcomes, so to control for this fact a variable EXP can be defined as the number of donors processed prior to the current donor — for example, for the i* donor, EXP = i — 1. Other variables can include bone type (such as vertebral bodies and ilia), donor sex and donor age.

[0276] In one aspect, the outcome variables are: the proportion of a particular cell population, such as CD34+ cells, that are viable, the total number of colony forming units (CFUs) per 105nucleated cells detected following cell processing, and the number of CFU granulocyte macrophages (CFU-GM) detected per 105nucleated cells.

[0277] According to the present disclosure, an ordinary least squares (OLS) beta regression model can be used to predict the outcome variables, with linear regression models used for CFU and CFU-GM and a beta regression model used for the proportion of viable CD34+ cells, or%CD34+, where 0 < (%CD34+) < 1. The beta regression equation for predicting%CD34+ is:

[0278] The regression models are based on un-adjusted models that only account for the ischemia-based variables and not the experience, bone type, donor sex and donor age variables. A fully adjusted model for%CD34+ that accounts for all of the variables. The results of these models are depicted in Tables 1-3.Table 1.%CD34+ values for the coefficientsTable 2. CFU values for the coefficients

[0279] The coefficient Pi attempts to quantify the effect of the number of donors processed (for example, experience) on cell quantity and viability. In the fully adjusted CFU model, coefficient 2 corresponds to the experience at a particular facility based on the assumption that facilities can have different learning trajectories. Either or both of these coefficients may be modified or even eliminated.Table 3. CFU -GM values for the coefficients

[0280] Applying these models to observed data can be used to determine the effect of ischemia time variables on%CD34+, as reflected in the tables shown in FIGS. 11A-11C, on total CFU, as shown in the tables of FIGS. 12A-12C, and on the amount of CFU-GM, as shown in the tables of FIGS. 13A-13C. The data in these tables can be used to decide whether a particular donor bone can yield sufficient cells to warrant further processing of the donor bone. In other words, the predictive models can be used to establish ischemia tolerance limits and HSPC quality acceptance criteria. For instance, with respect to the%CD34+ outcome variable, predicted values of over 80% may be required in order to consider the particular donor bone.

[0281] The models described above and the examples show n in the tables of FIGS. 11A-11C suggest that acceptable levels of HSPC quality are achievable despite the prolonged ischemia times that are inevitable when bones must be procured by geographically-dispersed OPOs and shipped long distances to processing centers. Even under such conditions, favorable combinations of warm- and cold-ischemia times can be achieved, enabling%CD34+ viabilities in the range of 80-90%. The models also suggest that refrigerating the body prior to bone recovery, a practice that is common in the recovery of tissues, is less beneficial in the contextof bone marrow recovery'. For instance, when whole-body cooling was used, CD34+ viability averaged 72.75%, whereas when body cooling was not used, the average was just under 90%. These models suggest that an optimal practice would be to dispense with body cooling and move recovered bone as quickly as possible to a cold ischemic environment. The models further suggest that limiting WIT (warm ischemia time) to less than eight (8) hours and CIT (cold ischemia time) to less than 40 hours optimizes the opportunity to recover meaningful quantities of viable cells from donor bone.

[0282] The models disclosed herein predict viability in which an 80% CD34+ cell viability threshold is determined to be acceptable. As reflected in the chart, the relationship between warm and cold ischemia times follows a curve from a point at which the WIT is 10 hours and the CIT is 18 hours, to a point at which the WIT is 1 hour and the CIT is 27 hours.

[0283] Further details of the method for 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 bone marrow bank.” J Transl Med 18, 300 (2020). doi.org / 10. 1186 / s 12967-020- 02470-1, the entire disclosure of which is incorporated herein by reference.Banking Cadaveric BM

[0284] Typically, less than one-half of the patients waiting for an allo-bone marrow (BM) transplant receive the need transplant. The living donor BM registry', BM cry opreservation and auto-transplantation, and umbilical cord blood banking have provided lifesaving solutions for thousands of patients with, at least, hematologic diseases; however, these methods still suffer from severe limitations tied to supply and logistics and would benefit from the systems and methods of the present disclosure. Additionally, though rare, adverse events are possible from living bone marrow donation (for example, the risk of death associated with bone marrow donation is 1: 10.000)). and while peripheral blood stem cell donation is currently much more utilized, nearly all of those donors will experience bone pain, 1 in 4 will have significant headache, nausea, or citrate toxicity', and 1 in 5,000 will experience splenic rupture or other fatal complication. Additionally, the long-term effects of stem cell mobilizing agents administered to a donor during donation are not yet known. The technical feasibility of cadaveric BM banking has been demonstrated in principle; however, numerous challenges in this remain. These challenges are directly addressed by this disclosure.

[0285] Banking BM as disclosed herein provides a ready mechanism to provide BM to patients for whom living donor match has not been identified. Additionally, it provides a more efficient method for providing BM to the patient for whom a living donor match exists, in that there is reduced delay associated with, at least, identifying a donor match, locating the donormatch, and arranging for the donation. Accordingly, banking BM can greatly increase posttransplant survival rates for many patients with rapidly progressing diseases and poor prognosis by allowing on-demand transplantation and reducing waiting times for these patients from many months to only 1-2 days. And importantly, this approach provides large quantities of BM from a single donor, sufficient to allow engraftment of hematopoietic stem and progenitor cells (HSPCs) for several patients and enabling immediate repeat BM transplantation when needed. Furthermore, since a single donor provides sufficient BM for transplantation, it is unnecessary to pool BM from multiple donors or provide a subsequent donations for different donors, each of which increases the likelihood of adverse reaction due to allografting.

[0286] The methods and systems disclosed herein enable large supplies of on-demand bone marrow (BM) for national emergency preparedness efforts. The urgent unmet need for on- demand BM and stem cell transplants as a medical countermeasure for nuclear accidents or attacks has been well documented by HHS, BARDA's multi -billion-dollar Project Bioshield, and the United States Dept, of Defense. The present disclosure also provides needed BM for emerging applications such as immune tolerance induction and beyond. A protocol for processing and the banking of BM from deceased organ donors that presen es the BM for extended periods of time is critical to this approach. Additionally, patients who receive deceased donor organ transplants today could benefit from this therapy when it becomes available in the future if BM from these donors is banked - making this method immediately beneficial to vital organ transplant recipients. In other words, the systems and methods described in the present disclosure allows harvesting and banking of increased number of bone marrow or bone marrow cells compared to methods currently utilized. If successful, other promising methods and treatments being researched have the potential to greatly enhance the value of cadaveric BM procurement and banking using the proposed method for making large supplies of banked bone marrow immediately usable for most recipients who need a BM transplant quickly, particularly to address severe forms of autoimmune disorders, genetic diseases, Multiple Sclerosis, and Type 1 Diabetes.

[0287] The present disclosure provides a clinically oriented research protocol and system that is modified to be implemented in an industrial context within state-of-the-art clean rooms. One aspect of the disclosed system involves, among other things, debridement of the incoming donor bone, initial fragmentation using a custom-made surgical stainless-steel cutter, and grinding of the fragmented bone to approximately 3 mm-sized bone fragments. Theserefinements provide a system in which skilled tissue processing technicians can process sets of donor bones within a 6-hour window to yield meaningful quantities of viable marrow.

[0288] In the process described herein is the evaluation of potential sources of deceased donor bone marrow. In processing long bones from a donor, such as the tibia, it has been found that due to conversion of red marrow to yellow7with age, red marrow is limited to the ends of the long bones and varies dramatically from donor to donor. It has also been determined that mixed yellow-red marrow is poor quality, compared to wholly red marrow, such as marrow from the vertebral bodies or the ilium, and mixed yellow-red marrow contains fatty infiltrate that complicates subsequent processing. The best donor long bone in certain clinical experiments yielded only l / lOO* BM cells / kg compared to cells obtained from the ilia of the same donor. It has been determined, then, that long bone processing is preferably only performed in special cases, such as involving extra valuable ‘'universal” HLA types or bone marrow with the HIV resistant delta 32 (CCR5-delta 32) mutation.

[0289] In contrast, the vertebral body and the ilium represent the largest consistent reservoirs of high-quality red marrow. Utilizing one or both sources has optimized the recovery of bone marrow, particularly with the implementation of the industnalized. scalable. GMP process disclosed herein. The completion of the process disclosed herein results in cry opreservation of a final product configuration of storing a 60-70 ml volume at a target of 100-150 million total nucleated cell (TNC) / ml in standard blood bags, similar to the product configuration already used for cryopreserved BM for autologous transplants.

[0290] The present disclosure should be considered as illustrative and not restrictive in character. It is understood that only certain embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the disclosure are desired to be protected.

[0291] While preferred embodiments of the present invention have been shown and described herein, it w ill be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now7occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodimentsof the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0292] For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and described in the following written specification. It is understood that no limitation to the scope of the disclosure is thereby intended. It is further understood that the present disclosure includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles disclosed herein as would normally occur to one skilled in the art to which this disclosure pertains.

[0293] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.DEFINITIONS

[0294] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0295] Use of absolute or sequential terms, for example, “will,’" “will not “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next.” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[0296] 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. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0297] As used herein, the phrases "at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of 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” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0298] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”. “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0299] Any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[0300] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.

[0301] The term “from” as in “from 1 to 10” includes the initial and final number recited. Therefore, “from 1 to 10” includes the whole numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and includes fractions thereof, (e.g, about .1, .2, .3, .4, .5, .6, .7. .8, and about .9).

[0302] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount relative to a reference level or a historical control. A historical control relates to data obtained from another subject or population of subjects who have not received a treatment according to methods of the present disclosure and are similar to the subject in various characteristics (e.g., age, sex. health status, comorbidities, hematologic cancer type, and cancer severity). In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level or a 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 a 100% increase or any increase between 10-100% as compared to a reference level, standard, or historical control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level or a historical control.

[0303] The terms “decreased'’, “decreasing”, or “decrease"’ are used herein generally to mean a decrease in a value relative to a reference level or a historical control. A historical control relates to data obtained from another subject or population of subjects who have not received a treatment according to methods of the present disclosure and are similar to the subject in various characteristics (e.g., age, sex, health status, comorbidities, hematologic cancer type, and cancer severity). In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level or a historical control, for example a decrease by 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 a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level or a historical control. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0304] An “effective amount” or “therapeutically-effective amount” refers to that amount of a bone marrow product and / or HSCs contained in a bone marrow product as described herein which, when administered to a subject (e.g., human), that sufficient to promote treating a disease, e.g.. a hematologic cancer. The amount of a bone marrow product and / or HSCs contained in the bone marrow product that constitutes a “therapeutically-effective amount” will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0305] CD34: Antigen present on immature hematopoietic precursor cells and all hematopoietic colony-forming cells in bone marrow and blood. Certain populations of non- hematopoietic (for example, CD45 negative) cells also express CD34. Of hematopoietic (for example, CD45+ cells), the CD34 antigen expression is highest on early progenitor cells and decreases with the maturation of cells. The CD34 antigen is absent on fully differentiated hematopoietic cells. Normal peripheral blood lymphocytes, monocytes, granulocytes, and platelets do not express the CD34 antigen.

[0306] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.ADDITIONAL EMBODIMENTS

[0307] In another aspect, a method of the present disclosure provides for recovering cells from deceased donor bone marrow that comprises the steps of: obtaining bone from a deceased donor; processing the bone to extract bone marrow cells from the bone; obtaining a reduced density Ficoll solution having a density7of 1.063- 1.052 gm / mL; introducing the reduced density Ficoll solution into a centrifuge tube to form a Ficoll gradient; layering the extracted bone marrow cells over the Ficoll gradient in the centrifuge tube; centrifuging the tubes containing the Ficoll gradient and bone marrow cells; harvesting the buffy coat cells from within the centrifuge tubes; and washing the harvested cells for subsequent use or processing. In some embodiments, the bone is a vertebral body. In some embodiments, the harvested cells are CD34+ cells. In some embodiments, the processing of the bone comprises: cleaning the bone of soft tissue; cutting the bone into pieces and grinding the pieces; filtering and rinsing the ground pieces of bone; and centrifuging a suspension of the filtered and rinsed pieces of bone to concentrate bone marrow cells. In some embodiments, the obtaining of a reduced density Ficoll comprises mixing Ficoll-Paque at a density of 1.077 g / mL with PLASMA- LYTE™ in a proportion to obtain a density of 1.063- 1.052 g / mL. In some embodiments, the centrifuging of the tubes includes centrifuging the tubes for 30 minutes at 400g. In some embodiments, the washing of the harvested cells includes washing the cells in phosphate- buffered saline (PBS) containing 0.5% human serum albumin (HSA) and 2mM Ethylenediaminetetraacetic acid (EDTA).

[0308] Another aspect of the present disclosure comprises a method for obtaining bone marrow cells from deceased donor bone comprising: obtaining a bone from a deceased donor; cleaning the bone of soft tissue; grinding the bone into bone pieces; filtering and rinsing the ground bone to produce a liquid composition; centrifuging the liquid composition of the filtered and rinsed ground bone to concentrate bone marrow cells into a bone marrow cell composition; and extracting the bone marrow cell composition into a sterile container. In some embodiments, the donor bone is one or more vertebral bodies and / or the ilium of the deceased donor. In some embodiments, the donor bone is freshly obtained and not frozen. In some embodiments, the donor bone is thawed after being frozen for transfer to a processing facility. In some embodiments, the cleaning of the bone of soft tissue comprises: removing soft tissue from the bone using a tool; and submerging the bone in one or more solutions adapted to remove soft tissue and soft tissue cells from the bone. In some embodiments, the submerging of the bone in one or more solutions comprising: submerging the bone in a bleach solution; and submerging the bone in a hydrogen peroxide solution. In some embodiments, thesubmerging of the bone in one or more solutions includes; submerging the bone in a container; detecting a level of foam within the container; and repeating at least the step of submerging the bone in a hydrogen peroxide solution until no foam is detected. In some embodiments, an inert contrast dye is added to the hydrogen peroxide solution to enhance the visibility of any foam in the container. In some embodiments, the grinding of the bone comprises: cutting the bone into fragments; and grinding the bone fragments in a bone grinder with a grind media. In some embodiments, the grind media comprises PLASMA-LYTE™ as a base with 10 U / m...

Claims

WHAT IS CLAIMED IS:

1. A method for processing a biological sample comprising cells or a derivative thereof, the method comprising:(a) generating a first volume of said biological sample comprising cells or a derivative thereof, wherein said first volume comprises a first concentration of cells or a derivative thereof;(b) generating a second volume of said biological sample comprising cells or a derivative thereof, wherein said second volume is less than said first volume and comprises a second concentration of said cells wherein said second concentration of said cells is no more than 30% different than said first concentration of said cells; and(c) cooling said first volume at a first cooling rate and cooling said second volume at a second cooling rate, wherein said first cooling rate is about the same as second cooling rate; wherein a post-thaw cell proliferation rate of said cells in said first volume is no more than 30% different than a post-thaw proliferation rate of said cells in said second volume.

2. The method of claim 1, wherein the biological sample comprises one or more organs, blood, or both, optionally wherein the blood is cord blood or peripheral blood.

3. A method for processing a bone marrow or a derivative thereof, the method comprising:(a) generating a first volume of said bone marrow or a derivative thereof, wherein said first volume comprises a first concentration of bone marrow or a derivative thereof;(b) generating a second volume of said bone marrow or a derivative thereof, wherein said second volume is less than said first volume and comprises a second concentration of said bone marrow wherein said second concentration of said bone marrow is no more than 30% different than said first concentration; and(c) cooling said first volume at a first cooling rate and cooling said second volume at a second cooling rate, wherein said first cooling rate is about the same as second cooling rate; wherein a post-thaw cell proliferation rate of a population of bone marrow derived cells in said first volume is no more than 30% different than a postthaw proliferation rate of a population of bone marrow derived cells in said second volume.

4. A method for processing mesenchymal stem cells (MSCs), the method comprising:(a) generating a first volume of the MSCs, wherein the first volume comprises a first concentration of the bone marrow or a derivative thereof;(b) generating a second volume of the MSCs, wherein the second volume is less than the first volume and comprises a second concentration wherein the second concentration is no more than 30% different than the first concentration; and(c) 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 MSCs in the first volume is no more than 30% different than a post-thaw proliferation rate of the MSCs in the second volume.

5. The method of claim 4. wherein the MSCs comprise bone marrow derived MSCs (BM-MSC) and / or vertebral bone adherent MSCs (vBA-MSC).

6. A method for processing a biological sample comprising cells or a derivative thereof, the method comprising:(a) generating a first volume of the biological sample comprising cells or a derivative thereof, wherein the first volume comprises a first concentration of cells or a derivative thereof;(b) generating a second volume of the biological sample comprising cells or a derivative thereof, wherein the second volume is less than the first volume and comprises a second concentration of the cells wherein the second concentration of the cells is no more than 30% different than the first concentration of the cells;(c) generating a freezing curve specific for the cells; cooling the first volume at a first cooling rate, wherein the first cooling rate is generated from the freezing curve; and(d) cooling the second volume at a second cooling rate, wherein the first cooling rate is generated from the freezing curve; wherein the first cooling rate is about the same as than the second cooling rate and wherein a post-thaw cell proliferation rate of the cells in the first volume is no more than 30% different than a post- thaw proliferation rate of the cells in the second volume.

7. The method of claim 6, wherein the biological sample comprises one or more organs, blood, or both, optionally wherein the blood is cord blood or peripheral blood.

8. The method of claim 6, wherein the first volume and the second volume are exposed to a common temperature.

9. The method of claim 6, wherein the second volume is less than 50. 40, 37.5, 35,30, 20, 15, 10, 5. or 1% of the first volume.

10. The method of claim 6, wherein a post-thaw viability rate of the first volume is no more than 30, 25, 20, 15, 13.6, 10, or 5% different than a post-thaw viability rate of the second volume.

11. The method of claim 10, wherein the post-thaw viability rate is at least 50, 60, 70.

80. or 90%.

12. The method of claim 6, wherein the post-thaw cell proliferation rate of the first volume is no more than 25, 20, 15, 13.6, 10, or 5% different than a post-thaw proliferation rate of the second volume.

13. The method of claim 12, wherein the post-thaw proliferation rate of the cells is at least 1 CFU-GM / 105cells.

14. The method of claim 6, wherein the first cooling rate and the second cooling rate comprise a supra-freeze rate from about -O.T’C / min to about -5°C / min, or any integer between -0. 1 and -5°C / min, at least until ice has nucleated in a freezing medium.

15. The method of claim 6, wherein the cooling step for the first volume and / or the second volume occurs in at least one freezer.

16. The method of claim 15, wherein the first volume and the second volume are contained in separate freezers.

17. The method of claim 15, wherein the at least one freezer is a static freezer, and / or a controlled-rate freezer.

18. The method of claim 15, wherein the at least one freezer is set at about -70°C to about -90°C, or any integer between -70°C and -90°C.

19. The method of claim 15, wherein the first volume and / or the second volume are arranged such that the first and / or second volume does not contact a wall of the at least one freezer.

20. The method of claim 6, wherein the first and / or the second volume is placed in an insulating container, such that the first and / or second volume is in close proximity to the insulating material of the insulating container.

21. The method of claim 6, wherein the first and second volume have the same cooling rate.

22. The method of claim 6, wherein the cells in the first and / or the second volume are stem cells or immune cells.

23. The method of claim 22, wherein the cells comprise T cells, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), or any combination thereof.

24. The method of claim 6, wherein the cells comprise CD34+ cells.

25. The method of claim 6, further comprising a step of transferring the first volume and / or the second volume to a long-term storage container, optionally wherein the long-term storage container is colder than -70, -80. -86, or -90°C.

26. A method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising:(a) obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments;(b) extracting the bone marrow or the derivative thereof from the bone or bone fragment; and(c) cryopreserving the bone marrow or the derivative thereof, wherein the cry opreserving comprises decreasing temperature of the bone marrow or the derivative thereof at a freeze rate of more than about -l°C / min in a static temperature freezer.

27. The method of claim 26, wherein the cry opreserving comprises cooling the bone marrow or the derivative thereof at a supra-freeze rate from about -l°C / min to about -5°C / min, or any integer that is between -2 and -5°C / min, at least until ice has nucleated in a freezing medium.

28. The method of claim 26, wherein the cry opreserving comprises cooling the bone marrow or the derivative thereof at a sub-freeze rate from about -l°C / min to about -2°C / min, or any integer that is between -1 and -2°C / min.

29. The method of claim 26, wherein the supra-freeze rate and / or the sub-freeze rate are maintained without the use of a passive cool box.

30. The method of claim 26, wherein the cry opreserving comprises arranging one or more aliquots of the bone marrow or the derivative thereof inside the static temperature freezer such that no aliquot contacts a wall of the static temperature freezer.

31. The method of claim 26. wherein no aliquot of the bone marrow or the derivative thereof is stored directly on top of another aliquot of the bone marrow or the derivative thereof.

32. The method of claim 26, wherein the bone marrow or the derivative thereof comprises a population of CD34+ cells, optionally wherein the bone marrow or the derivative thereof comprises at least 70 or 80% viable CD34+ cells after the bone marrow or the derivative thereof is thawed.

33. A method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising:(a) obtaining a bone from a deceased donor;(b) contacting the bone with a bleach solution for at least about 10 minutes to at least about 25 minutes, wherein the bone is submerged in the bleach solution to form a bleached bone product;(c) extracting the bone marrow or the derivative thereof from the bleached bone product, wherein at least 90% of CD34+cells comprised in the bone marrow or the derivative thereof are viable.

34. The method of claim 33, wherein the bone marrow or derivative thereof is contacted with the bleach solution for at least about 5.

10.

15.

20. or 25 minutes.

35. The method of claim 33, wherein the bleach solution comprises about 1, 5, 10, 15, or 20% bleach, or any integer that is between 1 and 20%.

36. The method of claim 33, wherein the bone is a vertebral body bone.

37. The method of claim 33, wherein the hydrogen peroxide is a 1, 2, 3, 4, or 5% hydrogen peroxide solution, or any integer that is between 1 and 5%.

38. The method of claim 33, further comprising a step of transferring the bleached bone product from a container comprising the bleach solution to a container containing the hydrogen peroxide solution.

39. The method of claim 33, further comprising a step of agitating the bleached bone product within the hydrogen peroxide solution.

40. The method of claim 33, wherein the step of submerging the bleached bone product in a solution comprising hydrogen peroxide further comprises:(a) submerging the bleached bone product in a container containing the hydrogen peroxide solution;(b) detecting foam or froth associated with the bleached bone product; and(c) repeating (a) and / or (b) until no foam or froth is detected.

41. The method of claim 33, further comprising removing soft tissue from the bleached bone product, optionally wherein the soft tissue is removed manually.

42. The method of claim 33, further comprising adding an inert contrast dye to the solution comprising hydrogen peroxide.

43. A method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising:(a) obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments;(b) mechanically grinding the bone or bone fragment in the presence of a grinding solution to generate a plurality of bone grindings;(c) shaking the plurality' of bone grindings on a shaker at about 100 to about 200 rotations per minute C’RPM") for about 1 to about 20 minutes to generate a solution; and(d) removing the solution from the shaker, wherein the solution comprises the bone marrow or the derivative thereof, wherein the bone marrow or the derivative thereof comprises at least about 1.000, 1,500, 3.000, 5,000, 10,000. 15,000. 30.000, 50,000, 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, 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, 2,000,000, 3,000,000,5,000,000, or 10,000,000 CD34+ cells / ml, or any integer that is between 1,000 and 10,000,000 CD34+ cells / mL, of the bone marrow or the derivative thereof.

44. The method of claim 43, wherein the method further comprises contacting the plurality of bone grindings and / or the solution with a rinse media.

45. The method of claim 43, the method further comprising repeating the shaking of the plurality of bone grindings and / or the solution at least once.

46. The method of claim 43, wherein the solution comprises at least about 70, 80, 85, or 90% viable CD34+ cells.

47. A method for processing a population of CD34+ cells obtained from bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising:(a) obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments;(b) extracting the bone marrow or derivative thereof from the bone or bone fragment;(c) contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U / ml of a nuclease; and(d) performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells / 750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer.

48. The method of claim 47, wherein the population of CD34+ cells comprises at least about 70, 80, 85, or 90% viable CD34+ cells.

49. A composition for use as a stabilization buffer, the composition comprising at least about 5 U / ml of an anticoagulant, and at least about 3 U / ml of a nuclease.

50. The composition of claim 49, comprising at least about 5, 10, 15, or 20 U / mL of the anticoagulant and / or the nuclease.

51. The composition of claim 49, wherein the nuclease comprises Benzonase® and / or Denarase®.

52. The composition of claim 49, wherein the anticoagulant comprise heparin.

53. The composition of claim 49, wherein the composition further comprises human serum albumin (HSA), optionally wherein the HSA is present at about 0.1, 0.3, 0.5, 0.7, or 1%, or any integer that is between 0.1 and 1%.