Enhanced MSC preparations
View Patent ↗The present invention provides preparations of MSCs with important therapeutic potential. The MSC cells are non-primary cells with an antigen profile comprising less than about 1.25% CD45+ cells (or less than about 0.75% CD45+), at least about 95% CD105+ cells, and at least about 95% CD166+ cells. Optionally, MSCs of the present preparations are isogenic and can be expanded ex vivo and cryopreserved and thawed, yet maintain a stable and uniform phenotype. Methods are taught here of expanding these MSCs to produce a clinical scale therapeutic preparations and medical uses thereof.
1. A method of making an MSC preparation which inhibits IL2Ra expression by CD3/CD28 activated PBMCs, comprising the steps of:
a. making a plurality of preparations, each preparation made by the steps comprising:
i. obtaining from a single donor at least about 70 ml of non-fetal human bone marrow aspirate containing viable cells;
ii. expanding by passage expansion the number of viable cells to provide a preparation of at least about 1 billion of the viable cells, wherein the passage expansion comprises establishing a primary culture of isolated MSCs and then serially establishing a first non-primary (P1) culture of isolated MSCs from the previous culture;
iii. expanding by passage expansion the P1 culture of isolated MSCs to a second non-primary (P2) culture of MSCs;
iv. preparing and cryopreserving an in-process intermediate MSC preparation from the P2 culture of MSCs; and
v. thawing the cryopreserved in-process intermediate MSC preparation and expanding by passage expansion the in-process intermediate MSC preparation; and
b. selecting from the plurality of preparations at least one preparation that has an antigen profile and an activity profile comprising:
i. less than about 0.75% CD45±cells;
ii. at least about 95% CD105+ cells;
iii. at least about 95% CD166+ cells; and
iv. capable of inhibiting IL2Ra expression by CD3/CD28-activated PBMCs by at least about 30% relative to a control; and
wherein the step of selecting comprises testing the at least one preparation to determine the percent CD45+ cells, the percent CD155+ cells, the percent CD166+ cells, and the percent inhibition of IL2Ra expression by CD3/CD28-activated PBMCs.
2. The method of making an MSC preparation of claim 1 , wherein the step of expanding comprises expanding the number of cells by at least about 20 population doublings.
3. The method of making an MSC preparation of claim 2 , wherein the step of expanding comprises expanding the number of cells by less than about 30 population doublings.
4. The method of making an MSC preparation of claim 2 , wherein the step of expanding comprises producing a preparation of at least about 20 billion cells.
5. The method of making an MSC preparation of claim 1 , further comprising a step of cryogenically preserving the selected preparation, optional′ wherein at least about 70% of the MSCs are viable after thawing.
6. The method of making an MSC preparation of claim 1 , wherein the step of establishing a primary culture of isolated MSCs comprises:
a. isolating an MSC-containing population from the bone marrow aspirate, optionally wherein the MSC-containing population is a population of isolated nucleated bone marrow cells (INBMCs);
b. seeding the MSC-containing population on a substrate at a concentration in a range of about 50 to about 1000 cells per cm 2 or 146±about 100 cells per cm 2 ; and
c. culturing the MSC-containing population for a period of time.
7. The method of making an MSC preparation of claim 1 , wherein the step of serially establishing at least a first non-primary (P1) culture of isolated MSCs from the previous culture comprises:
a. seeding isolated MSCs from the previously established culture on a substrate in a range of about 1,000 to about 10,000 cells per cm 2 or at about 5,900±about 20% cells per cm 2 ; and then
b. culturing the isolated MSCs for a period of time.
8. The method of making an MSC preparation of claim 1 , wherein the step of serially establishing at least a first non-primary (P1) culture of isolated MSCs from the previous culture comprises:
a. splitting the previous culture at a ratio of about 1:4 to about 1:8 into a plurality of cultures;
b. culturing the plurality of cultures for a period of time; and then
c. optionally, pooling the plurality of cultures.
9. A mesenchymal stem cell (MSC) preparation made according to the method of claim 1 .
10. The MSC preparation of claim 9 , wherein the at least about 1 billion human bone marrow-derived MSCs are isogenic.
11. The MSC preparation of claim 10 , wherein the MSC preparation comprises at least about 20 billion human hone marrow-derived MSCs in number.
12. The MSC preparation of claim 9 , wherein the MSCs express at least about 13 pg to about 44 pg TNFRI per million MSCs and wherein the MSCs, when mixed with peripheral blood mononuclear cells (PBMCs) at a ratio of about 5 PBMCs per MSC, are capable of inhibiting IL2Ra expression by CD3/CD28-activated PBMCs cells by at least about 30%, relative to a control.
13. The MSC preparation of claim 12 , wherein the MSCs are isogenic and are at least 4.5×10 9 human bone marrow-derived MSCs in number.
14. The MSC preparation of claim 13 , further comprising a cryopreservative.
15. The MSC preparation of claim 14 , wherein after a freeze-thaw cycle, at least about 70% of the MCSs are viable, as assessed by dye exclusion.
16. The MSC preparation of claim 13 , wherein the MSCs:
a. are capable of at least 1 population doubling; and
b. retain said antigen profile after the population doubling.
17. The MSC preparation of claim 13 , wherein the MSCs:
are capable of at least 1 population doubling; and maintain a differentiation capacity after the population doubling.
18. The MSC preparation of claim 9 , wherein the preparation contains less than 55 μg/ml BSA and less than 42 μg/ml trypsin.
19. The MSC preparation of claim 9 , wherein the preparation is substantially free of:
a. mycoplasma , endotoxin, and fungi; and
viral nucleic acids, wherein viral nucleic acids comprise HTL V1, HTL V2, HBV, CMV, EBV, HHV-6A, HHV-6B, HHV-8, HIV, Parvovirus B-19, HCV, and HPV Type 18 nucleic acids.