# US 11,077,145 B2 · Method for mass producing proteins in mesenchymal stem cells

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- Number: US 11,077,145 B2
- Document ID: US11077145B2
- Status: Granted
- Kind: B2
- Application number: 15/562,382
- Filed: 2016-04-01
- Published: 2018-10-04
- Granted: 2021-08-03
- Earliest claimed: 2015-04-03
- Assignees: Stemmedicare Co., Ltd.
- Applicants: Stemmedicare Co., Ltd.
- Inventors: Jang Ho Lee
- Web page: https://patentgrep.com/patent/US11077145B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 9/0014: {Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00)}
- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- A61P 17/16: Emollients or protectives, e.g. against radiation
- C07K 14/435: from animals; from humans
- C07K 14/47: from mammals
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/49: Platelet-derived growth factor [PDGF]
- C07K 14/50: Fibroblast growth factor [FGF]
- C07K 14/525: Tumour necrosis factor [TNF]
- C07K 14/54: Interleukins [IL]
- C07K 14/5409: {IL-5}
- C07K 14/545: IL-1
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 5/0031: {Serum-free culture media}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to the method for mass production of mesenchymal stem cell-derived proteins including various growth factors and cytokines, a mesenchymal stem cell conditioned medium containing a large amount of protein and produced by the above production method, cosmetic composition and pharmaceutical composition including the above conditioned medium for skin regeneration, anti-wrinkle, alopecia treatment, prevention of hair loss and promotion of hair growth. The method for mass production of the mesenchymal stem cell-derived protein of the present invention can greatly increase the amount of various growth factors and proteins which are known to be not expressed from mesenchymal stem cells or expressed only in very small amounts by the existing culture method, and the mesenchymal stem cell conditioned medium prepared by the above method contains a large amount of various kinds of cytokines and growth factors so that it has the excellent effect on skin regeneration, wrinkle improvement, prevention of hair loss, alopecia treatment and hair growth promotion.

## Claims

### Claim 1

A method for mass producing a mesenchymal stem cell-derived protein, the method comprising:
(A) storing mesenchymal stem cells in a cryopreservation medium solution at a temperature of −80° C.;
(B) inoculating the mesenchymal stem cells at a density of 20,000 cells/cm2;
(C) culturing the mesenchymal stem cells in a serum-free medium;
(D) obtaining a stem cell conditioned medium containing the protein produced by the mesenchymal stem cells after 120 hours of culture; and
(E) extracting the mesenchymal stem cell-derived protein from the obtained stem cell conditioned medium,
wherein a total concentration of the extracted protein is 30 μg/ml to 70 μg/ml, and
wherein said protein is selected from the group consisting of AR, bFGF, BMP-5, BMP-7, GH, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, SCF, TGF α, TGF β1, VEGF R3, VEGF-D, ICAM-1, IL-1a, IL-5, MIP-1a, MIP-b, MIP-d, RANTES, TNF R1, TNF RII, BDNF, BMP-4, b-NGF, EGF R, FGF-4, FGF-7, GDF-15, GDNF, HGF, IGFBP-6, IGF-I, Insulin, MCSF R, NGF R, NT-3, NT-4, OPG, PDGF-AA, PIGF, SCF R, VEGF, G-CSF, IL-2, IL-6, IL-8, IL-11, MCP-1, MCSF, MIG, TIMP-1, TIMP-2, TNFα, and TNFβ, or a combination thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells derived from amniotic fluid, fat, bone marrow, umbilical cord blood, or amniotic membrane.

### Claim 3 (depends on 1)

The method of claim 1, wherein the mesenchymal stem cells are amniotic fluid-derived mesenchymal stem cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the step (D) of obtaining the stem cell condition medium further comprises obtaining the stem cell conditioned medium an additional one to three times at intervals of 114 to 126 hours.