KR-20260064040-A - Method for the Production of Specific Proteins and Exosomes by Culturing Amnion-Derived Stem Cells

Abstract

The present invention relates to a method for producing amniotic membrane-derived stem cells or a culture medium thereof, comprising the step of culturing amniotic membrane-derived stem cells under hypoxic conditions having an oxygen partial pressure of 0.5 to 15%. The amniotic membrane-derived adult stem cells or a culture medium thereof produced according to the above method have significantly increased expression of Osteopontin, IGFBP-3, Thrombospondin1, Chitinase3-like1, EMMPRIN, uPAR, GDF-15, KGF, VEGF, FGF-19, IL-17A, IL-1a, EGF, or MMP-9, and exhibit excellent anti-inflammatory, wrinkle improvement, cell regeneration, wound healing, skin moisturization, hair growth promotion, and hair loss prevention effects, so they can be usefully used as a skin and hair condition improvement agent or a wound healing agent.

Inventors

• 신용우
• 탁성길
• 허재녕
• 권제영

Assignees

• 주식회사 도프

Dates

Publication Date

20260507

Application Date

20241031

Claims (7)

1. A method for producing exosomes comprising the steps of: culturing amniotic-derived stem cells under hypoxic conditions having an oxygen partial pressure of 0.5 to 15%; and recovering exosomes from the culture.
2. A method for preparing amniotic membrane-derived stem cells or a culture medium thereof, comprising the step of culturing amniotic membrane-derived stem cells under hypoxic conditions having an oxygen partial pressure of 0.5 to 15%.
3. A manufacturing method according to claim 2, further comprising the step of confirming that the expression of one or more selected from the group consisting of Osteopontin, IGFBP-3, Thrombospondin1, Chitinase3-like1, EMMPRIN, uPAR, GDF-15, KGF, VEGF, FGF-19, IL-17A, IL-1a, EGF, and MMP-9 is increased.
4. A method of manufacturing according to claim 2, characterized in that the amniotic membrane-derived stem cells are amniotic mesenchymal stem cells derived from human amniotic membranes isolated in vitro or amniotic epithelial cells isolated in vitro.
5. A method for manufacturing according to claim 2, characterized in that the culture is performed for 24 to 48 hours in an alpha-MEM medium containing 10% fetal bovine serum and 10 μg/mL gentamicin.
6. A cosmetic composition comprising amniotic membrane-derived stem cells or their culture medium produced by the manufacturing method of claim 2.
7. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, alopecia, acne, folliculitis, or scars comprising amniotic membrane-derived stem cells or a culture medium thereof prepared by the manufacturing method of claim 2.

Description

Method for the Production of Specific Proteins and Exosomes by Culturing Amnion-Derived Stem Cells The present invention relates to a method for producing exosomes comprising the step of culturing amniotic membrane-derived stem cells and a method for producing amniotic membrane-derived stem cells or a culture medium thereof, and more specifically, to a method for producing exosomes comprising the step of culturing amniotic membrane-derived stem cells under hypoxic conditions having an oxygen partial pressure of 0.5 to 15% and a method for producing amniotic membrane-derived stem cells or a culture medium thereof. Recently, various studies are being conducted regarding the potential of stem cells, such as embryonic and adult stem cells, for cell therapy by differentiating them into diverse cell types. Although embryonic stem cells have garnered attention as cell therapies due to their pluripotency and ability to differentiate into various cells, their practical application is difficult due to ethical concerns. Consequently, research utilizing adult stem cells is actively underway to circumvent these ethical issues. Representative adult stem cells include those derived from bone marrow, fat, and umbilical cord blood, and various studies are being conducted on methods to differentiate these cells into specific cell types. Regarding differentiation induction, the types of cells produced can vary significantly depending on the type of stem cell. For example, it has been reported that preadipocytes differentiate into adipocytes when exposed to hormones that induce differentiation into adipocytes, but do not differentiate into adipocytes under hypoxic conditions even when exposed to such hormones (Lin et al., J Biol Chem. 13;281(41):30678-83, 2006). To develop cell therapies using adult stem cells that are free from ethical issues, it is essential to establish a method to effectively proliferate specific adult stem cells while maintaining their stemness. However, adult stem cells have a problem in that they have a low proliferation rate and age easily, limiting the number of cells that can be obtained from a single tissue. Placenta-derived stem cells, which have recently garnered attention, have advantages such as not requiring invasive procedures compared to other adult stem cells; however, they still have the same problem of limited cell counts from a single placenta, and the reduced duration of cell proliferation even when subculture is performed to increase the number of cells, resulting in a small number of cells ultimately obtained. Against this technical background, the inventors have made diligent efforts to develop a culture method capable of proliferating amniotic membrane-derived adult stem cells at a high proliferation rate while maintaining their stem cell characteristics. As a result, they confirmed that when amniotic membrane-derived adult stem cells are cultured under hypoxic conditions in a serum-free medium, they effectively express various growth factors known to be related to cell growth, wound healing, tissue repair, improvement of skin wrinkles, and improvement of skin elasticity, thereby completing the present invention. The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs. Figure 1 shows the results of stem cell marker analysis of cultured amniotic stem cells. Figure 2 shows the particle number and size measurement results of the amniotic stem cell culture medium. Figure 3 shows the results of the component analysis of amniotic stem cells. Figure 4 shows the results of a comparative analysis of components during amniotic stem cell culture under hypoxia and normoxia conditions. Figure 5 shows the results of a component analysis comparison between the amniotic stem cell culture medium and a competitor's product. Figure 6 shows the results of the evaluation of the cell growth-promoting ability of the amniotic stem cell culture medium. Figure 7 shows the results of the evaluation of the hair follicle cell growth-promoting ability of amniotic stem cell culture medium. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art. The inventors confirmed that when amniotic stem cells (AMSC) are cultured in a serum-free medium under 2% O2 hypoxic conditions, the expression of various factors increases compared to adipose stem cells, placental stem cells, or commercially available stem cell culture media. Accordingly, they confirmed that the stem cells or their culture media produced by the above manufacturing method can be utilized as cosmetic compositions or pharmaceutical