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CN-122012388-A - Serum-free culture medium and preparation method and application thereof

CN122012388ACN 122012388 ACN122012388 ACN 122012388ACN-122012388-A

Abstract

The application relates to a serum-free culture medium in the technical field of cell culture, and a preparation method and application thereof. The serum-free culture medium comprises the following components of 5-15ng/mL of dexamethasone, 200-600ng/mL of hydrocortisone, 20-200 mu g/mL of transferrin, 0.2-1mg/mL of human serum albumin, 40-70 mu M of beta-mercaptoethanol, 5-12 mu M of glutathione, 10-65mg/L of ascorbic acid, 1-10ng/mL of EGF, 1-1 1-20ng/mL of IGF, 1-10ng/mL of bFGF and 1-10ng/mL of PDGF-BB, wherein the basal medium is IMDM. The serum-free culture medium is added with fewer culture medium components, so that an excellent culture effect can be obtained, the complexity of industrial production is greatly reduced, the industrial production cost is reduced, and the large-scale clinical requirements can be better met.

Inventors

  • WANG JIANGANG
  • WU CHANGYOU
  • QIAO LEI
  • ZHU HE
  • HE XIAOYAN
  • ZHENG BINBIN
  • WANG YANNA
  • HAN QIAN
  • LIN SHILONG
  • CHEN HUANHUAN

Assignees

  • 康立泰生物医药(青岛)有限公司

Dates

Publication Date
20260512
Application Date
20260320

Claims (10)

  1. 1. A serum-free medium, comprising a basal medium and the following final concentrations of each component: Dexamethasone is 5-15ng/mL, 200-600Ng/mL hydrocortisone, Transferrin 20-200 μg/mL, Human serum albumin 0.2-1mg/mL, Beta-mercaptoethanol 40-70 mu M, Glutathione is 5-12 mu M, 10-65Mg/L of ascorbic acid, EGF 1-10ng/mL, IGF-1 1-20ng/mL, bFGF 1-10ng/mL, PDGF-BB 1-10ng/mL; The basal medium is IMDM.
  2. 2. The serum-free medium of claim 1, wherein the serum-free medium comprises the following final concentrations of the components: 8-10ng/mL of dexamethasone, Hydrocortisone 350-450ng/mL, Transferrin 50-70 μg/mL, Human serum albumin 0.4-0.8mg/mL, Beta-mercaptoethanol 55-65 mu M, Glutathione is 8-11 mu M, Ascorbic acid 55-65mg/L, EGF 1-10ng/mL, IGF-1 2-8ng/mL, bFGF 4-7ng/mL, PDGF-BB 1-3ng/mL; The basal medium is IMDM.
  3. 3. A method of culturing mesenchymal stem cells, characterized in that the method of culturing mesenchymal stem cells is performed using the serum-free medium of any one of claims 1 to 2.
  4. 4. A method of culturing mesenchymal stem cells according to claim 3, wherein culturing comprises primary culturing.
  5. 5. A method of culturing mesenchymal stem cells according to claim 4, wherein the primary culturing conditions comprise culturing at 36-38℃under 4.5% -5.5% (v/v) CO 2 for 10-14 days.
  6. 6. The method according to claim 5, wherein the primary culture is carried out until the confluence of the cells reaches 80% -90%.
  7. 7. The method of culturing mesenchymal stem cells of any one of claims 3 to 6, wherein culturing comprises subculturing.
  8. 8. The method according to claim 7, wherein the conditions for subculturing comprise culturing at 36-38deg.C under 4.5% -5.5% (v/v) CO 2 for 48-96h per generation.
  9. 9. The method of claim 8, wherein the mesenchymal stem cells are subcultured to any one of P3 to P10.
  10. 10. The method of culturing mesenchymal stem cells according to any one of claims 3 to 6 and 8 to 9, wherein the mesenchymal stem cells are derived from umbilical cord, amniotic membrane, placenta or bone marrow.

Description

Serum-free culture medium and preparation method and application thereof Technical Field The application relates to the technical field of cell culture, in particular to a serum-free culture medium and a preparation method and application thereof. Background Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs for short) are adult stem cells which originate from mesoderm and widely exist in various tissues and organs such as bone marrow, adipose tissues, umbilical cords, placenta and the like, and have the core biological characteristics of being capable of differentiating into mesoderm-derived cells such as osteoblasts, chondroblasts, adipocytes and the like under specific induction conditions, being capable of differentiating into nerve cells, cardiac muscle cells and the like, being capable of regulating immunity, inhibiting T lymphocyte proliferation, regulating dendritic cell maturation and reducing immune rejection reaction through secretion of cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) and the like, being capable of releasing bioactive substances including cytokines, chemokines, exosomes and the like, participating in regulation of tissue repair microenvironment and promoting regeneration of damaged tissues. These properties make MSCs a central subject of research in the field of regenerative medicine and cell therapy. In recent years, clinical research and application of mesenchymal stem cells have been significantly progressed, and a plurality of disease fields have been covered. In the field of nervous system diseases, animal experiments and early clinical researches aiming at ischemic cerebral apoplexy and Alzheimer disease show that MSCs can promote nerve cell survival and improve nerve loop connection through paracrine action, and in immune related diseases, MSCs are used for adjuvant therapy of Graft Versus Host Disease (GVHD), systemic lupus erythematosus and the like, and can reduce tissue damage caused by immune overactivation. Up to now, thousands of MSCs related clinical studies have been developed worldwide, and some of them have entered the phase III clinical trial stage, showing broad clinical conversion prospects. Recent researches show that umbilical cord-derived mesenchymal stem cells can be an ideal substitute for bone marrow mesenchymal stem cells and have greater application potential. The umbilical mesenchymal stem cells express the special molecular markers of various embryonic stem cells, and have the characteristics of large differentiation potential, strong proliferation capability, low immunogenicity, convenient material acquisition, no limitation of ethical issues, easy industrial preparation and the like, so the umbilical mesenchymal stem cells are likely to become the pluripotent stem cells with the most clinical application prospect. At present, culture schemes of umbilical cord-derived mesenchymal stem cells are reported as follows: A human mesenchymal stem cell culture medium is described, wherein the culture medium is based on DMEM/F12 and comprises the following components of human serum albumin, human transferrin, human insulin, fibronectin, cholesterol, sodium selenite, hydrocortisone, putrescine, beta-mercaptoethanol, ethanolamine, L-ascorbic acid, bFGF, EGF, PDGF-BB, SCF, nicotinamide and acarditin, and the concentration of each component in the basal medium is 10-20g/L of human serum albumin, 5-20mg/L of human transferrin, 1-10mg/L of human insulin, 1-5mg/L of fibronectin, 5-30 mug/mL of cholesterol, 30-50nmol/L of sodium selenite, 10-50 mug/L of hydrocortisone, 5-30mg/L of putrescine, 5-10× 10 -5 M of beta-mercaptoethanol, 1-3 mug/mL of ethanolamine, 1-50mg/L, bFGF 5-10ng/mL of PDascorbic acid, 5-10 ng/BB, 5-10ng of EGF, 5-10ng/mL of novel and 1-10 mM of acarditin. The culture method of mesenchymal stem cells is also described in literature, wherein a serum-free culture medium is adopted to culture the mesenchymal stem cells, the serum-free culture medium consists of 1vt% of nonessential amino acid, 2mM of glutamine, 50mg/L of hydrocortisone, 10 mug/L of dexamethasone, 2-10g/L of polyvinyl alcohol, 10mg/L of recombinant human insulin, 5.5mg/L of recombinant human transferrin, 20 mug/L of recombinant human epidermal growth factor, 20 mug/L of recombinant human basic fibroblast growth factor, 20 mug/L of recombinant human Wnt-3a protein, 50 mug/L of recombinant human fibronectin, 20 mug/L of recombinant human laminin, 4mg/L of L-glutathione, 50mg/L of L-ascorbic acid, 2.5mg/L of beta-mercaptoethanol, 0.2g/L of ethanolamine, 500mg/L of Pluronic F-68 mg/L of selenium, and 25 mg/L of basic cholesterol. However, the existing mesenchymal stem cell culture medium has the disadvantages of complex formula, multiple raw material types, high protein consumption and high cost. In view of this, the present application has been proposed. Disclosure of Invention Based thereon, one or more embodiments of the present application prov