System and method for extraction and cryopreservation of bone marrow
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.
1. A method for recovery of transplantable stem cells from cadaver bone or cadaver bone fragments, the method comprising:
(a) obtaining cadaver bone or cadaver bone fragments, optionally, processing the cadaver bone into cadaver bone fragments;
(b) combining the cadaver bone fragments with a grinding medium comprising a nuclease that cleaves both DNA and RNA and one or more of human serum albumin (HSA), heparin, an electrolyte medium, and a growth media, thereby obtaining grinding medium-treated cadaver bone fragments;
(c) processing the grinding medium-treated cadaver bone fragments to extract bone marrow cells; and
(d) collecting the extracted bone marrow cells, thereby recovering the transplantable stem cells.
2. The method of claim 1 , wherein the nuclease is present in the grinding medium at a concentration of about 3 U/mL.
3. The method of claim 1 , wherein the electrolyte medium comprises a sterile, nonpyrogenic, isotonic solution.
4. The method of claim 1 , wherein the electrolyte medium comprises a pH of about 7.4.
5. The method of claim 1 , wherein the grinding medium comprises two or more of HSA, heparin, the electrolyte medium, and the growth media.
6. The method of claim 5 , wherein the grinding medium comprises three or more of HSA, heparin, the electrolyte medium, and the growth media.
7. The method of claim 1 , wherein the growth media is Iscove's Modified Dulbecco's Media (IMDM).
8. The method of claim 1 , wherein the processing in step (c) comprises grinding the cadaver bone fragments to obtain ground cadaver bone.
9. The method of claim 8 , wherein the processing in step (c) further comprises a filtering step thereby producing a filtered product comprising extracted bone marrow cells.
10. The method of claim 9 , wherein the filtering comprises a No. 40 (425 μm) sieve and/or a No. 80 (177 μm) sieve.
11. The method of claim 10 , further comprising combining the filtered product with additional grinding medium having components of step (b), thereby obtaining a diluted bone marrow cell product.
12. The method of claim 11 , further comprising a step of further filtering the diluted bone marrow cell product with one or more filters comprising pore sizes ranging from about 200 μm to about 825 μm and/or selected from filters having pore sizes of 200 μm, 500 μm, or 825 μm.
13. The method of claim of claim 12 , further comprising a step removing fat from an intermediate product.
14. The method of claim 1 , wherein the cadaver bone fragments are present in or divided into 1.5 cm 2 pieces in step (a).
15. 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.
16. The method of claim 15 , wherein the cadaver bone or cadaver bone fragments are derived from a plurality of cadaver bones from the same donor.
17. The method of claim 1 , wherein the extracted stem cells comprise hematopoietic stem cells.
18. The method of claim 1 , wherein the extracted stem cells comprise a Mesenchymal stem cells (MSCs).
19. The method of claim 6 , wherein the grinding medium comprises the electrolyte medium, 10 U/mL heparin, 2.5% HSA, and 3 U/mL of the nuclease.
20. The method of claim 1 , wherein the transplantable stem cells are suitable for transplantation into a human subject.