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Induction composition of human mesenchymal stem cells, induction differentiation culture liquid and in-vitro induction method and application

A technology for stem cells and cell culture, applied in cell culture active agents, biochemical equipment and methods, tissue culture, etc., can solve the problems of complex induction and culture process, limited number of nerve cells, etc., to improve survival, strong activity, low cost effect

Active Publication Date: 2019-04-30
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this prior art can shorten the induction time of neural stem cells, it still needs time to be induced into specific nerve cells, and the number of induced nerve cells is limited, and the induction culture process is complicated, etc.

Method used

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  • Induction composition of human mesenchymal stem cells, induction differentiation culture liquid and in-vitro induction method and application
  • Induction composition of human mesenchymal stem cells, induction differentiation culture liquid and in-vitro induction method and application
  • Induction composition of human mesenchymal stem cells, induction differentiation culture liquid and in-vitro induction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Isolation and culture of umbilical cord mesenchymal stem cells

[0060] According to the anatomical structure of the umbilical cord, under aseptic conditions, cut it longitudinally, remove the blood vessels and the umbilical cord skin, wash the umbilical cord with normal saline until there is no blood and turn white, then cut the tissue into small sections with ophthalmic scissors, and put Walton glue Cut into small pieces. Put a small piece of umbilical cord tissue in a 50mL centrifuge tube, cut it into mud, and plant it in a 75cm 2 In the culture bottle, MSCM-acf serum-free culture solution (manufacturer Sciencell product number SC-7521) is installed in the culture bottle, and the 2 Cultured in a saturated humidity incubator. After 2 hours, add liquid (MSCM-acf serum-free culture medium) to the primary extracted umbilical cord tissue block, and then change the liquid every 3 days, and repeat the operation until cells crawl out from the edge of the tissue block. ...

Embodiment 2

[0085] Example 2 Comparison of Different Methods Inducing Umbilical Cord Mesenchymal Stem Cells to Differentiate into Functional Nerve Cells

[0086] 1. Induction and culture of neuroblast-like cells

[0087] Taking Example 1 as the induction method 1, and using the conventional cytokine induction method as the induction method 2, the induction method 1 and the induction method 2 were compared.

[0088] Induction method 1:

[0089] Take the 5th generation UC-MSCs, 5×10 4 Seed in 6-well plates at 37 °C 5% CO 2 Cultivate in a saturated humidity incubator. When the cell growth density reaches 80%, replace with induction differentiation medium;

[0090] The composition of the induction differentiation medium is: BDNF (brain-derived neurotrophic factor) 25ng / mL, NGF (nerve growth factor) 15ng / mL, N2 cell culture additive volume percentage content 2%, EGF (epidermal growth factor) 30ng / mL mL, FGF (fibroblast growth factor) is 30ng / mL, the volume percentage composition of B27 cel...

Embodiment 3

[0100] Detection of cell viability: 5 batches of human mesenchymal stem cells at passage 5 were randomly selected from the liquid nitrogen bank, induced and differentiated according to the method described in Example 1, and 1 mL of cell suspension was taken before and after induction of differentiation for cell viability detection. The amount of cells determines whether to dilute. Cell viability was calculated by dividing the number of viable cells in the hemocytometer grid by the total number of cells. Dead cells are stained blue, live cells cannot be stained. The results are shown in Table 1.

[0101] The specific operation method is:

[0102] (1) Measure the cell density of the cell suspension using a counting plate.

[0103] (2) Prepare a trypan blue solution with a concentration of 0.4% and a pH value of 7.2-7.4 with an isotonic buffered saline solution (ie, phosphate buffered saline solution).

[0104] (3) Add 0.1 mL of trypan blue solution to 1 mL of cells.

[0105...

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Abstract

The invention provides an induction composition of human mesenchymal stem cells, an induction differentiation culture liquid and an in-vitro induction method and an application, and relates to the field of nerve cell regeneration. The induction composition includes a brain-derived neurotrophic factor, a nerve growth factor, an epidermal cell growth factor, a fibroblast growth factor, a N2 cell culture additive, a B27 cell culture additive, rhodioloside and astragalus polysaccharide. In the induction composition provided by the invention, rhodioloside, astragalus polysaccharide and cytokines are compounded, induced differentiation nerve cells have the advantages of high content, strong activity, short period and low cost, can solve the problem of low differentiation efficiency when human mesenchymal stem cells are induced in vitro, improve the survival status of the induced nerve cells, and have little toxic and side effects on cells. The induction composition can be used for inducing the human mesenchymal stem cells, is easy to operate, enhances the activity of the differentiated nerve cells and shortens the induction differentiation time.

Description

technical field [0001] The invention relates to the technical field of nerve cell regeneration, in particular to an induction composition of human mesenchymal stem cells, a culture medium for inducing differentiation, and an in vitro induction method and application thereof. Background technique [0002] The repair of nervous system injury has always been a difficult problem in clinical prevention and treatment. The development of microsurgical technology and brain stereotaxic technology has provided technical support for the restoration of nerve function, which has greatly promoted the prevention and treatment of nerve injury and improved the effect of prevention and treatment. . Neural stem cell transplantation can promote the recovery of nervous system function after injury, and neural stem cells are mainly induced from embryonic stem cells, or directly isolated and cultured from the central nervous system of developing and adult mammals, but ethics, safety and cell safet...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N5/0775A61K35/30A61K38/38A61P25/00A61P25/16A61P25/28
CPCA61K35/30A61K38/385A61P25/00A61P25/16A61P25/28C12N5/0619C12N5/0622C12N2501/11C12N2501/115C12N2501/13C12N2501/90C12N2501/999C12N2506/1353C12N2506/1384C12N2506/1392A61K2300/00
Inventor 田娜张石林王秀
Owner JILIN TUO HUA BIOTECH
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