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KR-20260063924-A - High-efficiency Technology for increasing stem cell-derived extracellular vesicles and proteins using a nucleic acid fragments mixture

KR20260063924AKR 20260063924 AKR20260063924 AKR 20260063924AKR-20260063924-A

Abstract

The medium containing the nucleic acid fragment mixture of the present invention and the stem cell culture technology for high-efficiency production of stem cell-derived extracellular vesicles and proteins based thereon can significantly increase the expression levels of proteins such as growth factors of stem cells and the production of extracellular vesicles. Stem cells cultured by the above method or extracellular vesicles obtained therefrom can be usefully applied in the beauty field, such as skin whitening, improvement of skin wrinkles, change of hair color, alleviation or prevention of hair loss, alleviation of acne, UV protection, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or skin texture refinement and elasticity enhancement, due to their high protein content, and in the medical field, such as the management of hair loss, inflammatory diseases, acne and folliculitis, and prevention of scars.

Inventors

  • 신용우
  • 탁성길
  • 허재녕
  • 권제영

Assignees

  • 주식회사 도프

Dates

Publication Date
20260507
Application Date
20241031

Claims (18)

  1. A step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments; and A method for producing extracellular vesicles comprising the step of recovering extracellular vesicles from a culture.
  2. A method for preparing an extracellular vesicle according to claim 1, characterized in that the nucleic acid fragment mixture has an average molecular weight of 100 kDa to 3000 kDa.
  3. A method for preparing an extracellular vesicle according to claim 1, characterized in that the nucleic acid fragment mixture is a mixture of nucleic acid fragments having a molecular weight of 1 to 100,000 kDa.
  4. A method for preparing an extracellular vesicle according to claim 1, wherein the nucleic acid fragment mixture is a DNA fragment mixture.
  5. A method for preparing an extracellular vesicle according to claim 4, characterized in that the DNA fragment mixture is PDRN (polydeoxyribonucleotide) or PN (polynucleotide).
  6. A method for preparing an extracellular vesicle according to claim 1, wherein the nucleic acid fragment mixture is derived from an organism selected from the group consisting of microbiome, animals, plants, and algae.
  7. A method for preparing an extracellular vesicle according to claim 6, characterized in that the nucleic acid fragment mixture is a mixture of DNA fragments derived from fish.
  8. A method for producing extracellular vesicles according to claim 1, characterized in that the culture medium is a serum-free or serum-containing culture medium.
  9. A method for manufacturing an extracellular vesicle according to claim 1, characterized in that the extracellular vesicle is an exosome.
  10. A method for preparing an extracellular vesicle according to claim 9, characterized in that the exosome expresses one or more exosome markers selected from the group consisting of CD9, CD81, CD63, CD82, TSG101, and ALIX.
  11. A method for preparing an extracellular vesicle according to claim 1, characterized in that the concentration of the nucleic acid fragment mixture is 50 μg/ml to 1000 μg/ml.
  12. A culture medium composition for the culture of stem cells or the production of extracellular vesicles comprising a mixture of nucleic acid fragments.
  13. A method for culturing stem cells comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.
  14. Stem cells cultured by the culture method of paragraph 13 or a culture medium thereof.
  15. Extracellular vesicles manufactured by the manufacturing method of claim 1.
  16. A cosmetic composition comprising as an active ingredient any one or more of the stem cells or culture medium thereof of claim 14 and the extracellular vesicles of claim 15.
  17. A cosmetic composition according to claim 16 for skin whitening, improvement of skin wrinkles, change of hair color, alleviation or prevention of hair loss, alleviation of acne, protection against UV rays, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or skin texture refinement and elasticity enhancement.
  18. A pharmaceutical composition for the prevention or treatment of hair loss, inflammatory diseases, neurodegenerative diseases, acne and folliculitis or scars comprising any one or more of the stem cells or culture medium thereof of claim 14 and the extracellular vesicles of claim 15.

Description

High-efficiency Technology for increasing stem cell-derived extracellular vesicles and proteins using a nucleic acid fragments mixture culture method and culture medium The present invention relates to a technology for producing high-efficiency stem cell-derived extracellular vesicles and proteins through stem cell culture using a nucleic acid fragment mixture base. Stem cells are cells capable of differentiating into various types of cells that constitute biological tissues; they are undifferentiated or intermediately differentiated cells obtained from the tissues of embryos, fetuses, and adults, representing a stage prior to final differentiation. Stem cells undergo differentiation into specific cell types in response to differentiation stimuli (environmental factors). Unlike cells that have completed differentiation and ceased cell division, stem cells possess the characteristic of proliferation (expansion) as they can self-renewal by dividing to produce identical cells. Furthermore, they exhibit plasticity in differentiation, as they can differentiate into other cell types under different environments or different differentiation stimuli. In particular, stem cells are reported to produce and secrete biological substances, such as various growth factors and cytokines, at high levels; consequently, their applications are being researched in various fields, including medicine and cosmetics. Recently, extracellular vesicles, particularly exosomes, have garnered attention as a novel modality capable of efficiently delivering various payloads to all types of cells within living organisms. While the precise nature and mechanisms of this action are still under investigation, methods to produce exosomes at high titers have been required to exert desired biological and clinical effects (Cheng and Schorey, 2016, Biotech Bioeng 113(6): 1315-1324; Kalluri, 2016, J Clin Invest 126(4): 1208-1215). Although various methods for the mass production of exosomes have been reported, they generally utilize media supplemented with the blood of cancer patients or animal serum (Chaput and Thery, 2011, Semin Immunopathol (2011) 33:419-440; Tarte K et al., 2010, Blood 115, 1549-1553, et al.). Generally, culture methods used for the culture of stem cells and the production of exosomes utilize basal culture media (e.g., DMEM, alpha-MEM) supplemented with 5 to 20% fetal bovine serum (FBS) and additional growth agents. Animal-derived components, such as fetal bovine serum, are known to cause problems such as transmissible spongiform encephalopathy (TSE). Including a process to remove animal-derived components can significantly reduce the yield of the final product (stem cells or exosomes) (Cell Prolif. 2013 Sep 30;46(6):608-627). Furthermore, trace components within fetal bovine serum are not only very difficult to analyze but can also vary depending on the time or location of production; this variability makes it difficult to trust the consistency of experimental results and acts as a limiting factor in establishing reproducible testing and production processes (Stem Cell Research & Therapy volume 1, Article number: 8 (2010)). Therefore, there is a strong demand for the development of serum-free media that do not contain heterologous serum, such as bovine fetal serum. Meanwhile, polydeoxyribonucleotide (PDRN®) is a mixture of DNA fragments extracted from animals or plants with specific specifications (e.g., defined by molecular weight range or average molecular weight) that is known to have anti-inflammation, cell proliferation, and tissue regeneration effects by stimulating adenosine A2 receptors (Kim, Y.H., et al., 2010). In Europe and Korea, a mixture of DNA fragments obtained from the sperm of fish such as salmon or trout was named PDRN®. PDRN has been used in the medical field, particularly in dermatology, as an injectable composition for tissue regeneration to accelerate the body's natural wound healing process. Recently, the anti-inflammatory effects of PDRN on scar formation were investigated, and it was discovered that PDRN prevents scar formation by exerting anti-inflammatory and collagen synthesis effects through the inhibition of HMGB-1 (Jeong W et al Int J Mol Sci. 2017 Aug 3;18(8):1698.). Polynucleotide (PN) is a mixture of DNA fragments similar to PDRN, but has a longer nucleic acid chain length and a larger average molecular weight than PDRN. These polynucleotides are used as raw materials for medical devices to provide cell adhesion, lubrication, and buffering effects by acting as a physical support. Under the background technology described above, the inventors made diligent efforts to develop a novel serum-free culture process for stem cells. As a result, they confirmed that when stem cells are cultured using a serum-free culture medium containing a mixture of nucleic acid fragments such as PDRN or PN, the amount of protein and the number of exosome particles in the stem cell culture medium increase significantly