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Cell culture fluid inducing umbilical cord mesenchymal stem cells into neural stem cells, and using method thereof

A cell culture, stem cell technology, applied in the direction of cell culture active agent, animal cells, culture process, etc., to achieve the effect of high proportion, shortened time and obvious characteristics

Active Publication Date: 2017-11-24
深圳市沃英达生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention develops a method for efficiently inducing UC-MSCs into NSCs in a short period of time, which can effectively make up for the technical defect of the prior art that the induction time is too long

Method used

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  • Cell culture fluid inducing umbilical cord mesenchymal stem cells into neural stem cells, and using method thereof
  • Cell culture fluid inducing umbilical cord mesenchymal stem cells into neural stem cells, and using method thereof
  • Cell culture fluid inducing umbilical cord mesenchymal stem cells into neural stem cells, and using method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Take fresh healthy umbilical cord and umbilical cord blood, collect the umbilical cord blood and centrifuge, take the upper serum and inactivate it for later use. After rinsing with PBS, remove the umbilical vessels with scissors and tweezers, peel off the Wahrenheit jelly tissue inside, and cut the resulting tissue to a size of 1mm3, add α-MEM culture medium and place at 37°C, 5% CO 2 Cultivate in an incubator, and the culture medium contains 10% inactivated umbilical cord serum, 100U / ml streptomycin, and 100U / ml penicillin. After 5-8 days of umbilical cord tissue culture, it can be seen that some cells crawled out from around the tissue block, showing a small spindle shape. After one week, the cells began to proliferate rapidly and formed cell colonies of various sizes. After the cells were full, use 0.25 % Trypsinization for passage. Such as figure 1 As shown, the human umbilical cord mesenchymal stem cells grow in the form of fibroblast-like adherence, and the cel...

Embodiment 2

[0032] Take P3 UC-MSCs in the logarithmic growth phase, digest with trypsin, and adjust the cell density to 1x10 6 cells / ml, transfer the cells to 9 flow tubes, 1ml in each tube, centrifuge at 600g for 3min, discard the supernatant, add 1ml PBS to wash 2 times. After resuspending the cells in 200 μl PBS, add 5 μL each of CD19-FITC, CD34-FITC, CD45-PE, CD73-PE, CD90-PE, CD105-PE and HLA-DR-PE mouse anti-human monoclonal antibodies, and respectively Add 5 μL of anti-mouse IgG1-FITC and anti-mouse IgG1-PE to the other 2 tubes as isotype controls. After incubating at 4°C in the dark for 30 min, wash with PBS three times, discard the supernatant, resuspend the cells in PBS, and detect the cell phenotype by flow cytometry.

[0033] The result is as figure 2 As shown, the UC-MSCs obtained by isolation and culture fully meet the identification criteria, that is, high expression of CD73, CD90, and CD105, low expression or no expression of CD19, CD34, CD45, and HLA-DR.

Embodiment 3

[0035] Take P3 UC-MSCs in the logarithmic growth phase, digest with trypsin, and adjust the cell density to 1x10 5 cells / ml, inoculated in 6-well plates, 2ml per well, replaced with fresh culture medium after 24 hours, and randomly divided into 7 groups of A, B, C, D, E, F, G, and added inducing factors according to Table 1. . The concentrations of EGF and bFGF were added according to the reported concentration of 20 ng / ml, and the concentrations of AS were 0 μg / ml, 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, respectively. Each group was placed in a 3% hypoxic environment every day to induce culture for 1 hour, and then transferred to normal 37°C, 5% CO 2 , and a cell culture incubator under saturated humidity conditions for 10 days of intermittent induction of hypoxia. Cell growth and morphological changes were observed once a day under an inverted phase-contrast microscope. In each group, 10 non-overlapping fields of view were randomly selected, the total num...

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Abstract

The invention relates to a cell culture fluid used for inducing umbilical cord mesenchymal stem cells into neural stem cells, and a using method thereof. In the method, UC-MSC is efficiently induced into NSC within a short time by utilizing an intermittent hypoxia induction method through induced culture fluid containing basic fibroblast growth factors, epidermal growth factors and astragaloside, the technical defect in the prior art of over-long induction time can be effectively overcome, and large-scale clinical promotion is facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell culture solution for inducing umbilical cord mesenchymal stem cells into neural stem cells and a method for using the same. Background technique [0002] Neural stem cells (Neural Stem Cells, NSCs) are a group of cells that can self-renew and have multiple differentiation potentials. astrocytes. [0003] The view for a long time in the past held that neurogenesis in the central nervous system of adult mammals was very limited and that neuronal numbers gradually decreased with age. However, with the continuous development of stem cell technology, scientists have confirmed that neurogenesis still exists in the central nervous system of adult mammals. The currently identified sites include the dentate gyrus of the hippocampus and the subependymal area. Endogenous NSCs in these parts have the ability to proliferate, migrate and differentiate into neurons and glial cells, and ca...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/02C12N2500/30C12N2501/11C12N2501/115C12N2506/1369
Inventor 林词雄王旭李陶林洁璇朱刚
Owner 深圳市沃英达生命科学有限公司
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