Compositions and methods for extraction of mesenchymal stem cells
Compositions and methods for the extraction mesenchymal cells from deceased donor bone, and therapeutic applications thereof, along with combination therapeutic applications comprising mesenchymal cells from deceased donors and bone marrow derived from deceased donors are presented.
1 . A method of generating a population of mesenchymal stem cells (MSCs), the method comprising:
a. obtaining a cadaver bone, cadaver bone fragments, or ground cadaver bone, optionally, preparing ground cadaver bone from the cadaver bone or cadaver bone fragments;
b. contacting the ground cadaver bone with a grinding medium comprising at least one of Benzonase®, Iscove's Modified Dulbecco's Media (IMDM), PLASMA-LYTE™ A, or any combination thereof, under conditions sufficient to separate bone marrow from the ground cadaver bone;
c. capturing the ground cadaver bone on a filter or sieve and collecting a filtrate that passes thru the filter or sieve, thereby recovering the bone marrow;
d. contacting the captured ground cadaver bone with a digestion solution comprising a collagenase from Clostridium histolyticum , thereby obtaining fresh MSCs from the captured ground cadaver bone, wherein the digestion solution is present at a ratio of volume to weight of said the captured ground cadaver bone of about 1:1 to about 15:1; and
e. passaging a sample of fresh MSCs at least 4 times, wherein the sample of fresh MSCs comprise a doubling rate of at least about 16 to 36 hours over the at least 4 passages, thereby generating the population of MSCs.
2 . The method of claim 1 , wherein the digestion solution comprises a neutral protease.
3 . The method of claim 1 , wherein the collagenase comprises collagenase isoforms C1 and C2 at a ratio comprising more collagenase isoform C1 than collagenase isoform C2.
4 . The method of claim 3 , wherein the ratio of collagenase isoform C1 to collagenase isoform C2 is about 30:10 to about 70:29.
5 . The method of claim 4 , wherein the ratio of collagenase isoform C1 to collagenase C2 is 35:15.
6 . The method of claim 2 , wherein the digestion solution comprises about 2 to about 20 U/ml of the neutral protease.
7 . The method of claim 6 , wherein the digestion solution comprises the neutral protease at an activity of about 19.6 U/ml.
8 . The method of claim 1 , wherein the digestion solution is present at a ratio of volume to weight of the captured ground cadaver bone is about 5:1.
9 . The method of claim 1 , wherein the digestion solution is contacted with the captured ground cadaver bone for up to about 2.5 hours.
10 . The method of claim 1 , wherein the population of MSCs are passaged at least 10 times.
11 . The method of claim 1 , wherein the population of MSCs comprises more than 1.75% CD45+ cells.
12 . The method of claim 1 , wherein the population of MSCs comprises at least 90% CD105+ cells.
13 . The method of claim 1 , wherein the population of MSCs comprises at least 90% CD166+ cells.
14 . The method of claim 1 , wherein the cadaver bone is a vertebral body.
15 . The method of claim 1 , wherein the doubling rate is less than about 29 hours.
16 . The method of claim 1 , wherein the grinding medium comprises an anticoagulant, wherein the anticoagulant is heparin.
17 . The method of claim 1 , wherein the grinding medium further comprises:
a. a Denarase reagent (C-Lecta GmbH); and/or
b. a DNase reagent.
18 . The method of claim 1 , wherein the grinding medium comprises a protein source and/or a reactive oxygen scavenger, optionally wherein the protein source and/or reactive oxygen scavenger is human serum albumin.
19 . The method of claim 1 , wherein the method further comprises contacting the ground cadaver bone with an isopropyl alcohol and/or a bleach prior to contacting the ground cadaver bone with a grinding medium.
20 . The method of claim 1 , wherein the method further comprises contacting the ground cadaver bone with hydrogen peroxide, then washing the bone at least once in a solution comprising PLASMA-LYTE™ A and/or IMDM, prior to contacting the ground cadaver bone with a grinding medium.
21 . The method of claim 1 , wherein the method further comprises an at least one rinse step after separating the bone marrow from the ground cadaver bone, wherein the rinse step comprising contacting the bone marrow with a rinse media comprising human serum albumin, and at least one of: IMDM, PLASMA-LYTE™ A, Benzonase®, heparin, or any combination thereof.
22 . The method of claim 1 , wherein the method further comprises collecting the filtrate that passes through the filter or sieve in a media comprising human serum albumin, and at least one of: IMDM, PLASMA-LYTE™ A, Benzonase®, heparin, or any combination thereof.
23 . The method of claim 1 , wherein the method further comprises contacting the captured ground cadaver bone with a digestion solution for 1 to 3 hours.