Enhanced MSC preparations

Patent metadata

Field	Value
Publication number	US-9828586-B2
Application number	US-201314138265-A
Country	US
Kind code	B2
Filing date	Dec 23, 2013
Priority date	Oct 8, 2010
Publication date	Nov 28, 2017
Grant date	Nov 28, 2017

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Mesoblast Int Sarl
• Mesoblast Int Sárl

Inventors

• Tom Samson
• Ton Christopher
• Danilkovitch Alla

Classifications

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P5/14

  of the thyroid hormones, e.g. T3, T4 · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61P19/02

  for joint disorders, e.g. arthritis, arthrosis · CPC title