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Cell culture substrate, and preparation method and application thereof

A technology of cell culture and culture substrate, which is applied in the field of cell culture substrate and its preparation, can solve the problems of in vitro proliferation of stem cells, differences in directional differentiation ability, and inability to meet the clinical needs of mesenchymal stem cells, and achieve low cost and easy operation , the effect of improving efficiency

Inactive Publication Date: 2014-01-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 2) Traditional in vitro subculture methods cannot meet the clinical needs of autologous mesenchymal stem cells in patients of different ages
At present, autologous mesenchymal stem cells have been gradually applied in clinical treatment, but stem cells of different ages have great differences in their ability to proliferate and differentiate in vitro

Method used

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  • Cell culture substrate, and preparation method and application thereof
  • Cell culture substrate, and preparation method and application thereof
  • Cell culture substrate, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1 Preparation of cell culture substrates from umbilical cord fibroblast secretions

[0047] (1) Preparation of induction culture medium, its components include: commercial minimum essential medium α-MEM 80% by volume, fetal bovine serum 20% by volume, antibiotics 10-200 U / mL, adenine 0.25 mM, preserved Glycine 50μg / mL, dexamethasone 100nM.

[0048] (2) Induce umbilical cord fibroblasts to secrete matrix: inoculate umbilical cord fibroblasts in a commercial cell culture plate at a density of 20,000 cells / cm2, add induction culture medium from step (3), and store at 37oC in 5% CO 2 Cultivate in the incubator for 7-20 days, and change the medium every other day.

[0049] (3) Decellularization treatment: wash with pH 7.4 phosphate buffer for 3 times, then add decellularization treatment solution containing Triton X-100 5% volume ratio and ammonia water 4% volume ratio. CO 2 Let stand in the incubator for 5 minutes, wash with pH7.4 phosphate buffer for 3 times, then add 5% ...

Embodiment 2

[0055] 1 Cell morphology detection of target mesenchymal stem cells expanded by cell culture substrate

[0056] The target mesenchymal stem cells were inoculated on the cell culture substrate obtained in Example 1, and the Olympus IX71 microscope was used to take fluorescent photos to obtain the following: figure 2 Morphological photos of target mesenchymal stem cells. Mesenchymal stem cells cultured on cell culture substrates maintain good fibroblast morphology and high cell density, which can provide a basis for in vitro expansion of mesenchymal stem cells.

[0057] 2 Active oxygen analysis of target mesenchymal stem cells amplified by the cell culture substrate prepared by the present invention and common culture substrate

[0058] The target mesenchymal stem cells were inoculated on the cell culture substrate obtained in Example 1 and a common culture plate, and the cells were collected when the cell density reached about 90%, and the cells were collected using 2′,7...

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Abstract

The invention belongs to the technical field of biological material of tissue engineering. The invention discloses a cell culture substrate, a preparation method thereof, and an application thereof. According to the invention, umbilical cord fibroblast cell self-secretion substrate is adopted; and through decellularization treatment and drying and sterilization, a natural tissue engineering material is obtained. The material comprises collagen type I, collagen type III, fibronectin, laminin, decorin, and the like, and can be used in in-vitro amplification of target mesenchymal stem cells. With the cell culture substrate provided by the invention, mesenchymal stem cell in-vitro amplification efficiency can be substantially improved, special immune phenotype of stem cells can be maintained, and stem cell active oxygen content can be substantially reduced. Also, raw material source is wide, and no ethic and moral problem is caused.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering biomaterials, and in particular relates to a cell culture substrate and a preparation method and application thereof. Background technique [0002] Mesenchymal stem cells have been widely used in regenerative medicine, gene therapy, tissue engineering, cytokine replacement therapy and other fields due to their high self-renewal ability and multi-lineage differentiation potential. [0003] Although mesenchymal stem cell therapy is steadily moving towards clinical practice, there are still many difficulties in the widespread application of mesenchymal stem cell transplantation technology in treatment. First, the number of transplanted mesenchymal stem cells: clinically effective transplantation requires mesenchymal stem cells to reach a certain level, but practice has proved that it is difficult to obtain sufficient tissue-specific mesenchymal stem cells in vitro without losing their stem...

Claims

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Application Information

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IPC IPC(8): C12M3/00C12N5/0775
CPCC12N5/0662C12N2533/90
Inventor 何帆刘小珍谭子芳龚逸鸿
Owner SUN YAT SEN UNIV
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