Method for mass producing proteins in mesenchymal stem cells
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.
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/cm 2 ;
(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.
2. 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.
3. The method of claim 1 , wherein the mesenchymal stem cells are amniotic fluid-derived mesenchymal stem cells.
4. 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.