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Culture medium for culturing human adipose-derived stem cells

A technology for human adipose stem cells and culture medium, applied in the biological field, can solve the problems of complex methods, stem cell contamination, and stem cells are not suitable for direct application.

Inactive Publication Date: 2018-08-17
李倩
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method of cultivating stem cells is that the method is complicated, the number of extracted stem cells is small, the purity is not high, and the subculture proliferation is slow. More importantly, the use of feeder cells and animal serum is easy to cause stem cell contamination, especially the potential animal sources in animal serum. Sexual endotoxins or viruses will pose a great risk to human health, and the stem cells cultured in this way are not suitable for direct clinical application

Method used

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  • Culture medium for culturing human adipose-derived stem cells
  • Culture medium for culturing human adipose-derived stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: the culture medium preparation that does not add polypeptide

[0014] 1) Prepare a predetermined amount of 1000mL, take 900mL high-glucose DMEM cell culture medium (manufacturer: Sigma), add penicillin 100IU / ml, streptomycin 100μg / ml, L-glutamine 2mmol / L, basic fibroblasts grow Factor l0ng / ml, vitamin C 50μg / ml, human serum albumin 13μg / mL, transferrin 6μg / mL, reduced glutathione 10μg / mL, linoleic acid 5μg / mL.

[0015] 2) adjust the pH value: adjust the pH to 7.0 with 5% NaHCO3, and adjust the volume to 1000 ml with DMEM cell culture medium.

[0016] 3) Sterilization by filtration: Use one filter membrane of 0.45um and 0.22um each, the upper layer is 0.45um and the lower layer is 0.22um to ensure the filtering effect.

Embodiment 2

[0017] The extraction of embodiment 2 polypeptide

[0018] Clean the leaves of Pyrethia chinensis in western Sichuan, mince them, squeeze the juice, add papain and trypsin, the amount of enzyme added is 15000IU / g leaves, the enzymatic hydrolysis temperature is 45°C, the pH value is 7.0, the enzymatic hydrolysis time is 2h, and the enzymatic hydrolysis is completed at 92°C Inactivate the enzyme for 13 minutes; filter the insoluble matter after inactivating the enzyme to obtain a solution, add 4% activated carbon to the obtained polypeptide solution for adsorption and decolorization, use dextran G-50 (Sephadex G-50) to separate the polypeptide, and wash with 20mmol / L HCl solution The flow rate was 1.3mL / min, and the eluted products of different time periods were collected respectively, and the solution was adjusted to pH 7.0, centrifuged at 10,000 rpm for 15 minutes, desalted by macroporous resin DA201-C, concentrated in vacuo, and the supernatant was Freeze-dried for later use;...

Embodiment 3

[0019] Embodiment 3 The preparation of the culture medium that contains polypeptide

[0020] 1) Prepare a predetermined amount of 1000mL, take 900mL high-glucose DMEM cell culture medium (manufacturer: Sigma), add penicillin 100IU / ml, streptomycin 100μg / ml, L-glutamine 2mmol / L, basic fibroblasts grow Factor l0ng / ml, vitamin C 50μg / ml, human serum albumin 13μg / mL, transferrin 6μg / mL, reduced glutathione 10μg / mL, linoleic acid 5μg / mL, CXXDC-1 polypeptide 30μg / ml .

[0021] 2) adjust the pH value: use 5% NaHCO 3 Adjust the pH to 7.0, and dilute to 1000ml with DMEM cell culture medium.

[0022] 3) Sterilization by filtration: Use one filter membrane of 0.45um and one filter membrane of 0.22um, the upper layer is 0.45um, and the lower layer is 0.22um. The prepared medium is CXXDC-1 medium.

[0023] In the same manner as above, culture media for CXXDC-2-27 were prepared respectively.

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Abstract

The invention discloses a culture medium for culturing human adipose-derived stem cells. The culture medium consists of DMEM basal culture medium, human serum albumin, transferrin, reduced glutathione, linoleic acid, penicillin, streptomycin, L-glutamine, basic fibroblast growth factor, vitamin C and plant extract polypeptide. The culture medium has culture rate, and the cells maintain good integrity and activity. The culture medium can be used for large-scale cell culture, and is low in cost and convenient to prepare.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a culture medium for human fat stem cells. Background technique [0002] Adipose-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential isolated from adipose tissue in recent years. They have the characteristics of general stem cells such as rapid expansion and not easy to age. Human adipose stem cells, namely human adipose-derived mesenchymal stem cells, are a kind of adult stem cells widely used in the field of tissue engineering and regenerative medicine, which have the same multi-directional differentiation potential as bone marrow mesenchymal stem cells. ADSCs showed fibroblast-like growth, abundant cytoplasm and nucleoli, arranged in parallel or swirl-like arrangement. Cell cycle analysis showed that 69% of cells were in G0 / G1 phase, 24% in S phase, and 8% in G2 / M phase. The cells were subcultured for 2-3 days in the presence of f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C07K7/08C12P21/06C07K1/14C07K1/16
CPCC07K7/08C12N5/0667C12N2500/24C12N2500/30C12N2500/32C12N2500/36C12N2500/38C12N2501/115C12N2501/998C12N2501/999C12P21/06
Inventor 李倩
Owner 李倩
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