# US 10,865,385 B2 · Allogeneic mesenchymal stem cell compositions and methods thereof

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- Number: US 10,865,385 B2
- Document ID: US10865385B2
- Status: Granted
- Kind: B2
- Application number: 14/624,110
- Filed: 2015-02-17
- Published: 2015-06-11
- Granted: 2020-12-15
- Earliest claimed: 2009-11-27
- Assignees: Stempeutics Research Pvt. Ltd.
- Applicants: Stempeutics Research Pvt. Ltd.
- Inventors: Rakhi Pal; Pawan Kumar Gupta; Prasanna Kumar Kemburu; Jyothi Prasanna; Satish Totey; Raviraja Neelavar Seetharam; Umesh Baikunje Golithadka; Anish Sen Majumdar
- Web page: https://patentgrep.com/patent/US10865385B2

## CPC

- C12N 5/0668: {Mesenchymal stem cells from other natural sources}
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 19/00: Drugs for skeletal disorders
- A61P 25/00: Drugs for disorders of the nervous system
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention discloses a method of isolation, pooling and further culturing of Mesenchymal Stem cells (MSC) for clinical application. Present invention also discloses the method of establishing Master Cell bank, followed by Working Cell Bank from which the final therapeutic composition referred to as Investigational Product/Investigational Medicinal Product comprising of allogenic bone marrow-derived MSC is formulated for clinical applications.

## Claims

### Claim 1

A cryopreserved pharmaceutical composition comprising pooled and cultured Mesenchymal Stem Cells (MSCs), Human Serum Albumin (HSA), Multiple Electrolytes Injection, Type I, USP, and Dimethyl Sulphoxide (DMSO), prepared by the method comprising:
a) creating 3 or more master cell banks and cryopreserving the master cell banks, wherein each master cell bank is created from MSCs from an individual donor cultured to passage 1 (P1);
b) thawing the 3 or more master cell banks and pooling MSCs from the 3 or more master cell banks, followed by culturing the pooled MSCs to passage 2 (P2);
c) culturing the pooled MSCs to passage 3 (P3) to create a working cell bank comprising pooled and cultured MSCs, followed by cryopreserving the working cell bank;
d) thawing the working cell bank and further culturing said pooled and cultured MSCs to passage 4 (P4) and subsequently to passage (P5); and
e) cryopreserving the pooled and cultured MSCs after d) to obtain the cryopreserved pharmaceutical composition,
wherein the pooled and cultured MSCs in the cryopreserved pharmaceutical composition have a concentration ranging from about 25 million to 200 million cells, the HSA has a concentration ranging from about 1% to 6%, the Multiple Electrolytes Injection, Type I, USP has a concentration ranging from about 80% to 90%, and the DMSO has a concentration ranging from about 5% to 10%; and wherein the pooled and cultured MSCs after P5 have increased cytokine secretion compared to the pooled and cultured MSCs after P3.

### Claim 2 (depends on 1)

The cryopreserved pharmaceutical composition of claim 1, wherein preparation of the composition comprises:
a) preparing a Working Cell Bank composition by pooling mesenchymal stem cells from multiple master cell bank compositions, wherein each master cell bank composition is obtained from a single donor, said master cell bank composition comprising un-pooled mesenchymal stem cells, Serum and DMSO, said preparation comprising acts of:
i) thawing un-pooled mesenchymal stem cells from the master cell bank compositions, reviving the cells by neutralizing with culture media to obtain a neutralized cell suspension and centrifuging the neutralized cell suspension;
ii) suspending the cell pellet from each master cell bank composition in the culture media and pooling the cells from each of the master cell bank compositions in equal proportions to obtain pooled cells; and
iii) harvesting the pooled cells, centrifuging the harvested cells and freezing in freezing media to obtain the working cell bank composition comprising pooled mesenchymal stem cells from multiple donors, serum ranging from about 85% to 95% and DMSO ranging from about 5% to 15%;
b) obtaining pooled mesenchymal stem cells from the Working Cell Bank composition obtained in a), followed by culturing the cells with culture media to achieve about 80% to 85% confluency;
c) harvesting the confluent cells by aspirating spent culture media, washing the confluent cells with Dulbecco's Phosphate Buffered Saline (DPBS) and treating the cells with 0.25% trypsin;
d) neutralizing the trypsin of the treated cells with the culture media and thereafter centrifuging the cells to obtain a cell pellet and suspending the cell pellet in the complete media;
e) culturing the cells of step (d), in the complete media to achieve about 80% to 85% confluency;
f) harvesting the confluent cells by washing the cells with the DPBS; treating the washed cells with 0.25% trypsin followed by neutralizing the trypsinized cells with the complete media;
g) centrifuging the neutralized cell suspension and washing the centrifuged cells with the DPBS followed by re-centrifuging to obtain a pellet; and
h) adding the Multiple Electrolytes Injection, Type I, USP, the DMSO and the Human Serum Albumin to the pellet and cryopreserving to obtain the cryopreserved pharmaceutical composition.

### Claim 3 (depends on 2)

The cryopreserved pharmaceutical composition of claim 2, wherein the pooled Mesenchymal stem cells of the Working Cell Bank composition ranges from about 1 million cells to 3 million cells.

### Claim 4 (depends on 2)

The cryopreserved pharmaceutical composition of claim 2, wherein the centrifuging is carried out at about 1200 rpm to 1500 rpm, at temperature ranging from about 20° C. to 25° C. for time duration ranging from about 10 minutes to 20 minutes.

### Claim 5 (depends on 2)

The cryopreserved pharmaceutical composition of claim 2, wherein the confluency is obtained by replenishing the culture media after about 7 days to 8 days.

### Claim 6 (depends on 2)

The cryopreserved pharmaceutical composition of claim 2, wherein the culture media comprises Dulbecco's Modified Eagle Medium-Knock Out at a concentration ranging from about 85% to 95%, Serum at a concentration ranging from about 5% to 15%, Glutamine at a concentration ranging from about 0.5% to 2%; Pen-Strep at a concentration ranging from about 0.1% to 1%, and basic Fibroblast growth Factor at a concentration ranging from about 0.5 ng/ml to 5 ng/ml.

### Claim 7 (depends on 1)

A kit comprising the cryopreserved pharmaceutical composition of claim 1, and an instruction manual.

### Claim 8 (depends on 1)

The cryopreserved pharmaceutical composition as claimed in claim 1, wherein the Mesenchymal Stem Cells comprise at least about 80% of cells which are positive for CD166, CD73, CD105 and CD90 and less than about 10% of cells which are positive for CD14, CD19, CD34, CD45, CD133 and HLA-DR.

### Claim 9

A cryopreserved pharmaceutical composition comprising pooled and cultured Mesenchymal Stem Cells (MSCs) prepared by the method comprising:
a) creating 3 or more master cell banks and cryopreserving the master cell banks, wherein each master cell bank is created from MSCs from an individual donor cultured to passage 1 (P1);
b) thawing the 3 or more master cell banks and pooling MSCs from the 3 or more master cell banks, followed by culturing the pooled MSCs to passage 2 (P2);
c) culturing the pooled MSCs to passage 3 (P3) to create a working cell bank comprising pooled and cultured MSCs, followed by cryopreserving the working cell bank;
d) thawing the working cell bank, and further culturing said pooled and cultured MSCs to passage 4 (P4) and subsequently to passage 5 (P5); and
e) cryopreserving the pooled and cultured MSCs after d) to obtain the cryopreserved pharmaceutical composition,
wherein the pooled and cultured MSCs in the cryopreserved pharmaceutical composition have a concentration ranging from about 25 million to 200 million cells; and wherein the pooled and cultured MSCs after P5 have increased cytokine secretion compared to the pooled and cultured MSCs after P3.