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Method for improving secretion of glial cell line-derived neurotrophic factors from human umbilical cord mesenchymal stem cells

A neurotrophic factor and mesenchymal stem cell technology, applied in cell dissociation methods, cell culture active agents, animal cells, etc., can solve the problem of ectopic expression of exogenous gene transfection, unclear conditioned medium composition, and difficulty in controlling the expression level and other issues, to achieve the effect of increasing secretion, safety and easy industrialization, and strong in vitro amplification ability

Active Publication Date: 2020-07-07
千石生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the existing technology is concerned, although the secretion of glial cell-derived neurotrophic factor has increased, there are still many safety problems, such as unclear composition of conditioned medium, ectopic expression of exogenous gene transfection, insertion mutation , loss of gene fragments and difficulty in controlling expression levels, etc., all limit its further clinical application

Method used

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  • Method for improving secretion of glial cell line-derived neurotrophic factors from human umbilical cord mesenchymal stem cells
  • Method for improving secretion of glial cell line-derived neurotrophic factors from human umbilical cord mesenchymal stem cells
  • Method for improving secretion of glial cell line-derived neurotrophic factors from human umbilical cord mesenchymal stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Cultivation of human umbilical cord mesenchymal stem cells

[0034] 1) Take P3~5 human umbilical cord mesenchymal stem cells, when the cells grow to 80%-90% confluence, aspirate the culture supernatant, add 10mL 0.01M phosphate buffer PBS into the T75 culture flask and wash it Abandon, a total of twice;

[0035] 2) Add 1.0 mL of 0.125% trypsin-0.01% EDTA digestion solution to the bottom of the T75 culture flask. When the cells are round and floated under an inverted microscope, add 1.0 mL of fetal bovine serum to stop trypsin digestion;

[0036] 3) Add 20mL 0.01M phosphate buffered saline PBS and repeatedly pipet and rinse, at 1000 rpm at room temperature for 5 minutes; after discarding the supernatant, add 10mL DMEM / F-12 medium (ThermoFisher Gibco TM Cat:12500096, prepared according to its instructions) resuspend the cells in a T75 culture flask according to 1~2×10 4 / cm 2 Inoculate human umbilical cord mesenchymal stem cells;

[0037] 4) The culture system is DMEM / F-12 ...

Embodiment 2

[0056] 1. Cultivation of human umbilical cord mesenchymal stem cells

[0057] 1) Take P3~5 generation human umbilical cord mesenchymal stem cells, when the cells grow to 80%-90% confluence, aspirate the culture supernatant, and add 10~15mL 0.01M phosphate buffer PBS to T75 culture flask to wash After sucking and discarding, a total of two times;

[0058] 2) Add 1.0 mL of 0.125% trypsin-0.01% EDTA digestion solution to the bottom of the T75 culture flask. When the cells are round and floated under an inverted microscope, add 1.0 mL of fetal bovine serum to stop trypsin digestion;

[0059] 3) Add 20mL 0.01M phosphate buffered saline PBS and repeatedly pipet and rinse, at 1000 rpm at room temperature for 5 minutes; after discarding the supernatant, add 10mL DMEM / F-12 medium to resuspend the cells, in a T75 culture flask According to 1~2×10 4 / cm 2 Inoculate human umbilical cord mesenchymal stem cells;

[0060] 4) The culture system is DMEM / F-12 basal medium supplemented with 10% fetal ...

Embodiment 3

[0079] 1. Cultivation of human umbilical cord mesenchymal stem cells

[0080] 1) Take P3~5 generation human umbilical cord mesenchymal stem cells, when the cells grow to 80%-90% confluence, aspirate the culture supernatant, and add 10~15mL 0.01M phosphate buffer PBS to T75 culture flask to wash After sucking and discarding, a total of twice;

[0081] 2) Add 1.0 mL of 0.125% trypsin-0.01% EDTA digestion solution to the bottom of the T75 culture flask. When the cells are round and floated under an inverted microscope, add 1.0 mL of fetal bovine serum to stop trypsin digestion;

[0082] 3) Add 20mL 0.01M phosphate buffered saline PBS and repeatedly pipet and rinse, at 1000 rpm at room temperature for 5 minutes; after discarding the supernatant, add 10mL DMEM / F-12 medium to resuspend the cells, in a T75 culture flask According to 1~2×10 4 / cm 2 Inoculate human umbilical cord mesenchymal stem cells;

[0083] 4) The culture system is DMEM / F-12 basal medium supplemented with 10% fetal bovi...

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Abstract

The invention provides a method for improving secretion of glial cell line-derived neurotrophic factors from human umbilical cord mesenchymal stem cells. The method comprises the step of cultivating human umbilical cord mesenchymal stem cells in a neurobasal medium which is supplemented with cytokine bFGF and FGF8. The method is capable of obviously increasing the secretion quantity of the glial cell line-derived neurotrophic factors of the mesenchymal stem cells, and has safety and easy industrialization. The ability of secretion of the glial cell line-derived neurotrophic factors from the human umbilical cord mesenchymal stem cells is improved through a cytokine induction method instead of a method of genetic modification, co-culture and conditioned medium cultivation, risks and uncertainties caused by other methods are avoided, and the method is more suitable for industrialization and clinical application. When the method is compared with stimulation or induction techniques which are reported in existing literature, the secretion quantity of the glial cell line-derived neurotrophic factors is increased significantly compared with that of a previous culture method after the mesenchymal stem cells are cultivated through the method for 6 days.

Description

Technical field [0001] The invention provides a method for culturing mesenchymal stem cells, in particular a method for enhancing the secretion of glial cell-derived neurotrophic factors from human umbilical cord mesenchymal stem cells. Background technique [0002] Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor isolated and purified from the rat B49 glial cell line by Lin et al in 1993. It belongs to the TGF-β superfamily . It exists in the entire central nervous system during embryonic development, and is mainly distributed in the cortex, hippocampus, striatum, substantia nigra, thalamus, cerebellum, corpus callosum, etc. in mature brain tissue. Glial cell-derived neurotrophic factor is a multi-potency neurotrophic factor. It has a strong neurotrophic effect not only on motor neurons but also on sensory neurons, especially neurons that are relatively insensitive to other neurotrophic factors. It can also specifically protect dopaminergic neurons a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12P21/02C07K14/475
CPCC12N5/0668C12N5/0663C12N5/0667C12N5/0665C12N5/0664C07K14/4756C12N2501/115C12N2501/119C12N2509/00
Inventor 谢姜王斌董方
Owner 千石生物科技(上海)有限公司
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