A method for large-scale expansion of mesenchymal stem cells

A technology of stem cells and bone marrow mesenchyme, applied in the field of stem cells, can solve the problems of high cost, high consumption, non-biodegradability, etc., and achieve the effects of low cost, avoiding virus infection, and promoting culture.

Inactive Publication Date: 2019-01-18
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of the currently used commercial microcarriers are biodegradable. When cultivating

Method used

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  • A method for large-scale expansion of mesenchymal stem cells
  • A method for large-scale expansion of mesenchymal stem cells
  • A method for large-scale expansion of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0050] Example 1 Preparation of microcarriers

[0051] Weigh 5 g of gelatin, add 50 mL of ultrapure water, and heat at 70° C. to dissolve completely to obtain a gelatin solution.

[0052] First, by volume, mix 5 parts of gelatin solution and 1 part of emulsifier ethyl acetate to form the first oil phase, stir at 900 rpm to form the first emulsion; then, mix 1 part of the first emulsion Add 2 parts of emulsifier olive oil to form a second oil phase. After stirring at 450 rpm for 1 min, use an ice-water bath to quickly cool to below 5°C, then continue stirring at 450 rpm for 30 min to obtain the second emulsion; finally, add the second emulsion Two times the volume of the emulsion with 20°C dehydration and deoiling agent absolute ethanol is stirred and dehydrated to obtain gelatin microspheres.

[0053] The initial product of the gelatin microspheres was washed alternately with dehydrating and deoiling reagent isopropanol and acetone, then washed with absolute ethanol, dried at 80°C, ...

Example Embodiment

[0055] Example 2

[0056] Take Life A1033201 MSC SFM CTS TM Serum-free medium for human mesenchymal stem cells was used as the cell culture medium to prepare a gelatin microcarrier suspension with a microcarrier density of 10-20g / L and a cell density of 4×10. 5 Cells / mL of P3 generation adipose-derived mesenchymal stem cell suspension, then P3 generation adipose-derived mesenchymal stem cells were seeded in a cell culture flask containing the microcarrier suspension, and placed in 5% CO 2 , 37 ℃, 95% humidity in a cell incubator.

[0057] In the cell culture process, first use a slow magnetic stirrer to rotate at 30 rpm for 10 min, and then stand still for 20 min. The cycle repeats for 6 hours, and then stirs the culture at a constant speed at 65 rpm, and replaces the medium in half the next day.

[0058] When the growth confluence of the cells to be attached to the microcarrier reaches 80%~90%, remove the culture medium, wash twice with PBS~EDTA, and then add 0.25% trypsin~EDTA to ...

Example Embodiment

[0059] Example 3

[0060] Take Life A1033201 MSC SFM CTS TM Serum-free medium for human mesenchymal stem cells was used as the cell culture medium to prepare a gelatin microcarrier suspension with a microcarrier density of 10-20g / L and a cell density of 9×10. 5 Cells / mL of P3 generation adipose-derived mesenchymal stem cell suspension, then P3 generation adipose-derived mesenchymal stem cells were seeded in a cell culture flask containing the microcarrier suspension, and placed in 5% CO 2 , 37 ℃, 95% humidity in a cell incubator.

[0061] In the cell culture process, first use a slow magnetic stirrer to rotate at 50 rpm for 2 min, and then stand still for 20 min. The cycle repeats for 12 hours, and then stirs the culture at a constant speed at 65 rpm, with half of the medium exchanged every other day.

[0062] When the growth confluence of the cells to be attached to the microcarrier reaches 80%~90%, remove the culture medium, wash twice with PBS~EDTA, and then add 0.25% trypsin~EDT...

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Abstract

The invention belongs to the technical field of stem cells, in particular to a method for large-scale expansion of mesenchymal stem cells. The method provided by the invention adopts a biodegradable gelatin microcarrier and combines with an intermittent stirring method to carry out suspension culture and jointly promotes the culture of mesenchymal stem cells. At the same time, in the cell cultureprocess, the serum-free medium is used instead of animal serum medium as the cell culture medium, which will not bring any mycoplasma and virus, thus avoiding the risk of virus infection. The method of the invention is simple in operation and low in cost, and can not only promote the culture of mesenchymal stem cells, but also maintain cell dryness and differentiation potential.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a method for expanding mesenchymal stem cells on a large scale. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells with self-replication ability and multi-directional differentiation potential, which can develop into bone, cartilage, fat and other types of cells. Mesenchymal stem cells can be transplanted, and the type of cells they grow into depends on where they are injected. For example, mesenchymal stem cells injected into the heart can form healthy new tissue, among other things. Mesenchymal stem cells are clinically used to solve a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, partial knee meniscectomy injury repair, and autoimmune diseases, and have achieved major breakthroughs, saving the lives of more patients. Therefore, large-scale expansion of mesenchymal stem cells ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N5/0664C12N5/0665C12N5/0667C12N2531/00C12N2533/54
Inventor 葛啸虎陈海佳王一飞戚康艺王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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