A kind of cell culture substrate and its preparation method and application

A technology of cell culture and culture substrate, applied in the field of cell culture substrate and its preparation, which can solve the problems of inability to meet the clinical needs of mesenchymal stem cells, in vitro proliferation of stem cells, and differences in directional differentiation ability, with low cost and simple method , the effect of reducing the content of active oxygen

Inactive Publication Date: 2015-12-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of cell culture substrate and its preparation method and application
  • A kind of cell culture substrate and its preparation method and application
  • A kind of cell culture substrate and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1 Preparation of cell culture substrate from umbilical cord fibroblast secretion

[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 10~200U / mL, adenine 0.25mM, proline 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 5% volume ratio of TritonX-100 and 4% volume ratio of ammonia water, in 5% CO at 37oC 2 Let stand in the incubator for 5 minutes, wash with pH7.4 phosphate buffer for 3 times, then ad...

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 OlympusIX71 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′-dic...

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Abstract

The invention belongs to the technical field of tissue engineering biomaterials, and discloses a cell culture substrate, a preparation method and application thereof. The present invention utilizes the umbilical cord fibroblast self-secreted matrix, and after decellularization, drying and sterilization, the natural tissue engineering material obtained contains type I collagen, type III collagen, fibronectin, laminin, decorin, etc. , which can be used for in vitro expansion of target mesenchymal stem cells. The cell culture substrate prepared by the invention can significantly improve the in vitro expansion efficiency of mesenchymal stem cells, maintain the unique immune phenotype of stem cells, significantly reduce the active oxygen content of stem cells, and meanwhile have a wide range of sources of raw materials without causing ethical and moral problems.

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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Patent Type & Authority Patents(China)
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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