Culture method of mesenchymal stem cells

A culture method and mesenchymal stem cell technology, applied in the field of mesenchymal stem cell culture, can solve problems such as unknown specific components, inability to completely exclude heterologous viruses, and inability to grow cells

Active Publication Date: 2021-03-19
YUNNAN KEY LAB OF PRIMATE BIOMEDICINE RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, CN 111424011 A provides a three-dimensional culture method capable of maintaining the morphology of umbilical cord mesenchymal stem cells. However, the serum-free medium used contains serum substitute additives provided by a third party, and this type of serum-free culture The specific composition of the base is unknown, and the purchased serum-free additives often contain animal-derived proteins, which cannot completely eliminate the risk of heterologous viruses
In addition, the cells cultured in this traditional three-dimensional bioreactor are vulnerable to mechanical damage, and the cells cannot sustain long-term growth on it

Method used

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  • Culture method of mesenchymal stem cells
  • Culture method of mesenchymal stem cells
  • Culture method of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] This embodiment provides a 2D culture method of mesenchymal stem cells, the formula of the serum-free medium used is as follows:

[0084] Each 1L medium consists of the following components:

[0085] IMDM 17.662g, L-alanyl-glutamine 2mM, chemical lipid 0.6v / v%, cholesterol 15mg, insulin 12.5mg, transferrin 25mg, recombinant human albumin 2g, hydrocortisone 500μg, dexamethasone 4μg, progesterone 5.66μg, putrescine 9mg, ascorbic acid 100mg, β-mercaptoethanol 75μM, soybean lecithin 10mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.2mg, N-Acetyl-L-Cysteine ​​1mM, reduced glutathione Glycine 2mg, Taurine 5mg, Y-27632 5μM, Ethanolamine 2mg, b-FGF 20μg, EGF 20μg, PDGF-BB 10μg, IGF-1 15μg, TGF-β3 2μg, HGF 10μg, CTGF 2μg and VEGF 15μg; The amount is water for cell culture.

[0086] The configuration method of the serum-free medium is as follows:

[0087] Dissolve IMDM powder in cell culture water, stir well, filter with 0.22um filter membrane, add recombinant human insulin...

Embodiment 2

[0096] This example provides a 2D culture method for mesenchymal stem cells, the difference from Example 1 is that the formula of the serum-free medium used is as follows:

[0097] Each 1L medium consists of the following components:

[0098] IMDM 17.662g, L-alanyl-glutamine 2mM, chemical lipid 0.1v / v%, cholesterol 5mg, insulin 8mg, transferrin 10mg, recombinant human albumin 1g, hydrocortisone 0.1mg, dexamethasone 4μg, progesterone 1μg, putrescine 5mg, ascorbic acid 25mg, β-mercaptoethanol 50μM, soybean lecithin 2mg, zinc sulfate heptahydrate 1.25mg, lipoic acid 0.1mg, N-Acetyl-L-Cysteine ​​0.2mM, reduced glutathione Glycine 1mg, Taurine 2mg, Y-27632 2μM, Ethanolamine 1mg, b-FGF 5μg, EGF 5μg, PDGF-BB 2μg, IGF-1 10μg, TGF-β3 1μg, HGF 2μg, CTGF 1μg and VEGF 5μg; The amount is water for cell culture.

[0099] Its configuration method is the same as embodiment 1.

Embodiment 3

[0101] This example provides a 2D culture method for mesenchymal stem cells, the difference from Example 1 is that the formula of the serum-free medium used is as follows:

[0102] IMDM 17.662g, L-alanyl-glutamine 2mM, chemical lipid 1v / v%, cholesterol 30mg, insulin 25mg, transferrin 30mg, recombinant human albumin 5g, hydrocortisone 2mg, dexamethasone 20μg, Progesterone 10μg, putrescine 15mg, ascorbic acid 200mg, β-mercaptoethanol 100μM, soybean lecithin 20mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.5mg, N-Acetyl-L-Cysteine ​​2mM, reduced glutathione 5mg , taurine 10mg, Y-27632 10μM, ethanolamine 5mg, b-FGF 40μg, EGF 50μg, PDGF-BB 20μg, IGF-1 40μg, TGF-β3 5μg, HGF 10μg, CTGF 5μg and VEGF 20μg; the balance is cell culture use water.

[0103] Its configuration method is the same as embodiment 1.

[0104] The same strain of P1 generation human umbilical cord mesenchymal stem cells was taken for testing, and three groups of repeated experiments were carried out in each o...

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Abstract

The invention relates to the technical field of cell culture, and particularly relates to a culture method of mesenchymal stem cells. In the culture method, a serum-free culture medium optimized by the invention is used for culturing the mesenchymal stem cells. In addition, the invention further provides a better culture method for 3D culture. The cells obtained by the culture method disclosed bythe invention are strong in proliferation capacity, intact in shape and strong in secretion capacity, and are in accord with the international quality control standards of the mesenchymal stem cells.Meanwhile, according to the culture method disclosed by the invention, a large number of mesenchymal stem cells (particularly human umbilical cord mesenchymal stem cells) can be obtained by amplifyinga small number of mesenchymal stem cells under the conditions of small space occupation, less culture medium consumption, simpler and more convenient operation and greatly reduced workload; and a solid technical scheme and a theoretical basis are provided for obtaining a large number of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretions thereof in the clinical field.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) refer to pluripotent stem cells that exist in various tissues of the human body. Stem cells of functional cells. Mesenchymal stem cells have the ability to induce differentiation and repair in vivo, that is, to realize the possibility of on-demand repair and regeneration of damaged tissues. [0003] After entering the body, mesenchymal stem cells return to the heart through the blood circulatory system, and then reach various organs and tissues of the body through the heart with the blood, or reach the damaged site under the influence of recruitment factors. Under the induction of growth factors, mechanical pressure and other conditions, the gene expression of stem cells undergoes a series of changes, and finally differentiates into terminal cells with the same specif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2513/00C12N2500/92C12N2501/15C12N2500/38
Inventor 李天晴冯春
Owner YUNNAN KEY LAB OF PRIMATE BIOMEDICINE RES
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