# US 2020/0399604 A1 · System and method for extraction and cryopreservation of bone marrow > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399604 A1 - Document ID: US20200399604A1 - Status: Published application - Kind: A1 - Application number: 17/013,379 - Filed: 2020-09-04 - Published: 2020-12-24 - Earliest claimed: 2019-04-15 - Assignees: Ossium Health, Inc. - Applicants: Ossium Health, Inc. - Inventors: Erik John WOODS; Brian H. JOHNSTONE; Dongsheng GU; Aubrey Marie SHERRY; Kelsey Gwen MUSALL; John R. WOODS; James HARDIN; Alan HOOKS - Web page: https://patentgrep.com/patent/US20200399604A1 ## CPC - C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)} - A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators - A61B 17/1611: {the two jaw elements being integral with respective elongate shaft elements} - A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells - C12M 45/02: {by mechanical forces; Stirring; Trituration; Comminuting (crushing, pulverizing, disintegrating in general B02C)} - C12M 45/05: {by centrifugation (centrifuges in general B04B)} - A61B 17/1606: {of forceps type, i.e. having two jaw elements moving relative to each other} - A61B 2017/00831: {Material properties} - A61B 2017/00969: {used for transplantation} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} ## Abstract Methods 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. ## Claims ### Claim 1 A method for high-yield recovery of stem cells from cadaver bone or cadaver bone fragments, the method comprising: (a) obtaining cadaver bone or cadaver bone fragments; (b) processing the bone or bone fragments to extract bone marrow cells; (c) combining the extracted bone marrow cells with a reduced density Ficoll solution having a density gradient of 1.063-1.052 g/ml; (d) centrifuging the extracted bone marrow cells, thereby separating out buffy coat cells comprising stem cells; and (e) harvesting the buffy coat cells, thereby recovering stem cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the reduced-density Ficoll solution comprises a mixture of PLASMA-LYTE™ and Ficoll-Paque solution at a density of 1.077 g/ml. ### Claim 3 (depends on 1) The method of claim 1, wherein after centrifuging in step (d), the buffy coat cells are located in the Ficoll gradient at a density of about 1.063 g/ml. ### Claim 4 (depends on 1) The method of claim 1 wherein the centrifuging occurs for about 30 minutes at 400 g. ### Claim 5 (depends on 1) The method of claim 1, wherein the centrifuging occurs for about 5 minutes at 400 g. ### Claim 6 (depends on 1) The method of claim 1, wherein the cadaver bone or cadaver bone fragment has not been cryopreserved prior to processing in step (b). ### Claim 7 (depends on 1) The method of claim 1, wherein the purity of recovered stem cells is at least about 90%. ### Claim 8 (depends on 1) The method of claim 1, wherein the viability of recovered stem cells is at least about 90%. ### Claim 9 (depends on 1) The method of claim 1, wherein the processing in step (b) comprises grinding the cadaver bone fragments to obtain ground cadaver bone, wherein grinding uses a bone grinder in the presence of a grinding medium, and, optionally, further comprises dividing cadaver bone into cadaver bone fragments. ### Claim 10 (depends on 9) The method of claim 9, wherein the grinding medium comprises two or more of Benzonase® or Denarase®, HSA, heparin, and PLASMA-LYTE A. ### Claim 11 (depends on 9) The method of claim 9, wherein the grinding medium promotes extraction of bone marrow cells from the ground cadaver bone. ### Claim 12 (depends on 11) The method of claim 11, wherein the ground cadaver bone is captured on a filter or sieve and a filtrate that passes thru the filter or sieve comprises the extracted bone marrow cells. ### Claim 13 (depends on 1) The method of claim 1, further comprising after step (e) a step of washing the harvested buffy coat cells for subsequent use or further processing. ### Claim 14 (depends on 13) The method of claim 13, wherein the washing includes contacting the cells with phosphate-buffered saline (PBS) containing human serum albumin (HSA) and ethylenediaminetetraacetic acid (EDTA) or with PBS alone. ### Claim 15 (depends on 1) The method of claim 1, further comprising a step of performing a separation method that isolates CD34+ cells from the recovered stem cells. ### Claim 16 (depends on 1) The method of claim 1, wherein the cadaver bone or the cadaver bone fragments are derived from a vertebral body, an ileum, or a combination thereof. ### Claim 17 (depends on 1) The method of claim 1, wherein the cadaver bone or cadaver bone fragments are derived from a plurality of cadaver bones from the same donor. ### Claim 18 (depends on 1) The method of claim 1, wherein the recovered stem cells comprise hematopoietic stem cells and/or mesenchymal stem cells. ### Claim 19 (depends on 18) The method of claim 18, wherein the hematopoietic stem cells are CD34+ cells. ### Claim 20 (depends on 1) The method of claim 1, wherein the lower density Ficoll gradient provides a recovered stem cell population having higher purity CD34+ cells with higher viability than a recovered stem cell population obtained from a conventional Ficoll gradient.