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 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 said captured ground cadaver bone with a digestion solution, thereby obtaining fresh MSCs from the captured ground cadaver bone; and
e. passaging a sample of fresh MSCs at least 4 times, wherein said sample of fresh MSCs comprise a doubling rate of at least about 16 to 36 hours over said at least 4 passages,
thereby generating said population of MSCs.
2 . The method of claim 1 , wherein said digestion solution comprises one or more distinct enzymes.
3 . The method of claim 2 , wherein said one or more distinct enzymes comprise a collagenase.
4 . The method of claim 3 , wherein said one or more distinct enzymes comprise a neutral protease.
5 . The method of claim 3 , wherein said collagenase comprises collagenase isoforms C1 and C2 at a ratio comprising more collagenase isoform C1 than collagenase isoform C2.
6 . The method of claim 5 , wherein said ratio of collagenase isoform C1 to collagenase isoform C2 is about 30:10 to about 70:29.
7 . The method of claim 6 , wherein said ratio of collagenase isoform C1 to collagenase C2 is 35:15.
8 . The method of claim 4 , wherein said digestion solution comprises about 2 to about 20 U/ml of said neutral protease.
9 . The method of claim 8 , wherein said digestion solution comprises said neutral protease at an activity of about 19.6 U/ml.
10 . The method of claim 1 , wherein said digestion solution is present at a ratio of volume to weight of said captured ground cadaver bone of about 1:1 to about 15:1.
11 . The method of claim 10 , wherein the digestion solution is present at a ratio of volume to weight of said captured ground cadaver bone is about 5:1.
12 . The method of claim 1 , wherein said digestion solution is contacted with said captured ground cadaver bone for up to about 2.5 hours.
13 . The method of claim 1 , wherein said population of MSCs comprises a doubling rate of at least about 16 to 36 hours over each of said at least 4 passages.
14 . The method of claim 1 , wherein said population of MSCs are passaged at least 10 times.
15 . The method of claim 1 , wherein said population of MSCs comprises more than 1.75% CD45+ cells.
16 . The method of claim 1 , wherein said population of MSCs comprises at least 90% CD105+ cells.
17 . The method of claim 1 , wherein said population of MSCs comprises at least 90% CD166+ cells.
18 . The method of claim 1 , wherein said cadaver bone is a vertebral body.
19 . The method of claim 1 , wherein the doubling rate is less than about 29 hours.
20 . The method of claim 1 , wherein the doubling rate is about 16 hours.