CELL COMPOSITION AND METHODS OF MAKING THE SAME
Provided herein are improved methods for the formulation of compositions comprising placental stem cells, and improved compositions and cell formulations produced thereby.
1 . A method of making a composition comprising cells, comprising:
(a) contacting said cells with a solution comprising dextran and human serum albumin (HSA) to form a cell-containing solution;
(b) filtering the cell-containing solution;
(c) if said cell-containing solution comprises greater than about 10±3×10 6 cells per milliliter, diluting said cells to no more than about 10±3×10 6 cells per milliliter with a first dilution solution comprising dextran; and
(d) optionally diluting the cell-containing solution with a second dilution solution comprising dextran but not comprising HSA, thereby making a composition comprising cells.
2 . The method of claim 1 , wherein said cells are cryopreserved after step (c) and before step (d).
3 . The method of claim 1 , wherein said dextran in said first dilution solution or said second dilution solution is dextran 40.
4 . The method of claim 1 , wherein said dextran in said first dilution solution and said second dilution solution is dextran 40.
5 . The method of claim 1 , wherein said dextran 40 in said first dilution solution is 5.5% dextran 40.
6 . The method of claim 1 , wherein said HSA in said solution comprising HSA is 10% HSA.
7 . The method of claim 1 , wherein said first dilution solution comprises HSA.
8 . The method of claim 7 , wherein said HSA in said first dilution solution is 10% HSA.
9 . The method of claim 1 , wherein said first dilution solution further comprises a cryoprotectant.
10 . The method of claim 9 , wherein said cryoprotectant is DMSO.
11 . The method of claim 1 , wherein said dextran 40 in said second dilution solution is 10% dextran 40.
12 . The method of claim 1 , wherein said solution in step (a) comprises a cryoprotectant.
13 . The method of claim 1 , wherein said composition comprising cells comprises about 7.5% to about 9% dextran.
14 . The method of claim 1 , wherein said composition comprising cells comprises about 1.0±0.3×10 6 cells per milliliter to about 5.0±1.5×10 6 cells per milliliter.
15 . The method of claim 1 , wherein said cells are placental cells.
16 . The method of claim 15 , wherein said stem cells are isolated human adherent placental cells.
17 . A method of making a composition comprising cells, comprising:
(a) suspending a plurality of isolated human adherent placental cells in a 5.5% dextran 40, 10% HSA solution to form a cell-containing solution;
(b) filtering the cell-containing solution through a 70 μM filter;
(c) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±3×10 6 cells/mL;
(d) cryopreserving the cells;
(e) thawing the cells; and
(f) optionally diluting the cell-containing solution 1:1 to 1:11 with 10% dextran 40 to produce said composition comprising cells.
18 . A method of making a composition comprising cells, comprising:
(a) centrifuging a plurality of isolated human adherent placental cells to collect the cells;
(b) resuspending the cells in 5.5% dextran 40;
(c) centrifuging the cells to collect the cells;
(d) resuspending the cells in a 5.5% dextran 40 solution that comprises 10% HSA to form a cell-containing solution;
(e) filtering the cell-containing solution through a 70 μM to 100 μM filter;
(f) diluting the cell-containing solution in 5.5% dextran 40, 10% HSA, and 5% DMSO to no more than about 10±3×10 6 cells/mL;
(g) cryopreserving the cells;
(h) thawing the cells; and
(i) optionally diluting the cell-containing solution 1:1 to 1:11 with 10% dextran 40 to produce said composition comprising cells.
19 . A method of making a composition comprising isolated human adherent placental cells, comprising:
(a) providing a plurality of isolated human adherent placental cells in a solution comprising 5.5% dextran 40 and 10% human serum albumin (HSA) to form a solution comprising isolated human adherent placental cells;
(b) filtering said solution comprising isolated human adherent placental cells with filter that removes visible cell clumps to produce filtered isolated human adherent placental cells;
(c) diluting said filtered isolated human adherent placental cells with an amount of a solution comprising 5.5% dextran 40, 10% HSA and 5% dimethylsulfoxide (DMSO) sufficient to bring said filtered isolated human adherent placental cells to about 10±3×10 6 cells per milliliter; and
(d) diluting said isolated human adherent placental cells with 10% dextran 40 at a ratio of about 1:1 to about 1:11 isolated human adherent placental cells:dextran 40 to produce said composition.
20 . The method of claim 1 , wherein said filter is a 70 μM filter.
21 . The method of claim 1 , wherein said filter is a 100 μM filter.
22 . The method of claim 1 , wherein said cells are isolated human adherent placental cells.
23 . The method of claim 22 wherein said isolated human adherent placental cells are CD10 + , CD34 − and CD105 + .
24 . The method of claim 23 , wherein said CD10′ − , CD34 − and CD105 + cells are CD200 + .
25 . The method of claim 24 , wherein said CD10 + , CD34 − , CD105 + and CD200 + cells are either CD45 − or CD90 + .
26 . The method of claim 24 , wherein said CD10 + , CD34 − , CD105 + and CD200 + cells are CD45 − and CD90 + .
27 . A composition made by the method of claim 1 .
28 . A method of making a composition comprising isolated human adherent placental cells, comprising contacting said isolated human adherent placental cells with a solution comprising 5.5% dextran and 10% HSA; filtering the isolated human adherent placental cells; and diluting said isolated human adherent placental cells to about 10±3×10 6 cells per milliliter with a solution comprising 10% dextran 40 (w/v).
29 . A composition comprising a plurality of isolated human adherent placental cells in a solution comprising 10% dextran 40, wherein said composition comprises between about 1.0±0.3×10 6 cells per milliliter to about 5.0±1.5×10 6 cells per milliliter, and wherein said composition comprises no macro cell clumps.
30 . The composition of claim 29 , wherein said placental stem cells are thawed cryopreserved cells.
31 . The composition of claim 29 , wherein said composition is contained within a bag.
32 . The composition of claim 29 , wherein said composition comprises no macro cell clumps.
33 . The composition of claim 29 , wherein said composition comprises fewer than about 200 micro cell clumps per 10 6 cells.
34 . The composition of claim 29 , wherein said composition comprises fewer than about 150 micro cell clumps per 10 6 cells.
35 . The composition of claim 29 , wherein said composition comprises fewer than about 100 micro cell clumps per 10 6 cells.
36 . The composition of claim 29 , wherein said isolated human adherent placental cells are CD10 + , CD34 − and CD105 + .
37 . The composition of claim 36 , wherein said CD10 + , CD34 − and CD105 + cells are CD200 + .
38 . The composition of claim 37 , wherein said CD10 + , CD34 − , CD105 + and CD200 + cells are either CD45 − or CD90 + .
39 . The composition of claim 37 , wherein said CD10 + , CD34 − , CD105 + and CD200 + cells are CD45 − and CD90 + .
40 . The composition of claim 29 , wherein said composition is a pharmaceutical composition.
41 . The method of claim 1 , further comprising concentrating the composition comprising cells to about 5×10 6 cells per milliliter to 1×10 8 cells per milliliter.
42 . The method of claim 41 , further comprising subcutaneously administering the composition comprising cells to a subject.