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A kind of primary culture medium of umbilical cord mesenchymal stem cells and primary culture method thereof

A technology of primary culture medium and stromal cells, applied in the field of umbilical cord mesenchymal stem cell primary culture medium and its primary culture, can solve the problems of unclearness and time-consuming primary culture, achieve good effect and shorten the primary culture The effect of incubation time, avoidance of the introduction of xenogeneic substances

Active Publication Date: 2020-08-21
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing conventional culture technology uses exogenous serum (such as fetal bovine serum) to promote the proliferation of umbilical cord mesenchymal stem cells. Another part is still unclear, and the composition and content of serum often vary with the sex, age, physiological conditions and nutritional conditions of the blood-supplying animals

Method used

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  • A kind of primary culture medium of umbilical cord mesenchymal stem cells and primary culture method thereof
  • A kind of primary culture medium of umbilical cord mesenchymal stem cells and primary culture method thereof
  • A kind of primary culture medium of umbilical cord mesenchymal stem cells and primary culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, mixture proportioning

[0056] Mixture working concentration:

[0057] (1) The working concentration of tranexamic acid is 500mg / mL and 10000mg / mL;

[0058] (2) The working concentration of G-CSF is 10ng / mL, 20ng / mL;

[0059] (3) The working concentration of EGF is 10ng / mL and 20ng / mL.

[0060] Prepare two media:

[0061] Experimental group: named group A, which is DMEM / F12+ mixture;

[0062] Control group: named group B, DMEM / F12+10%FBS.

[0063] The specific groups are as follows:

[0064] Table 1 Concentration ratio of each component in medium of group A

[0065]

Embodiment 2

[0066] Embodiment 2, observe and compare primary cell climbing out situation

[0067] (1) peel off the umbilical cord adventitia, 1 vein and 2 arteries to obtain Wharton's jelly;

[0068] (2) Shred Wharton's jelly to 1~2mm 3 , 3ml / 15cm dish was inoculated with Wharton's jelly;

[0069] (3) Cultivate Wharton's jelly with two types of medium in Example 1; culture condition is: 37 ℃, 5% CO 2 Cultured in an incubator, the medium was changed every three days.

[0070] (4) Observe every 3 days, and compare the situation of crawling out of the cells.

[0071] The situation of climbing out of cells is shown in the table below:

[0072] Table 2 Climbing out of cells in each group

[0073] number of days 3d 6d 9d 12d A1 0 CFU 1CFU 3CFU 5CFU A2 0 CFU 1CFU 5CFU 7CFU A3 1CFU 3CFU 6CFU 8CFU A4 1CFU 2CFU 5CFU 9CFU A5 1CFU 3CFU 7CFU 10CFU A6 3CFU 6CFU 15CFU 16CFU A7 1CFU 4CFU 12 CFU 13 CFU ...

Embodiment 3

[0075] Embodiment 3, AB group differentiation ability comparison

[0076] Take the 3rd generation cells of formula A8 and B, press 2.5×10 4 The cells / mL concentration were inoculated in a 24-well plate containing DMEM / F12+20% FBS medium, and when the cells were 80% confluent, they were replaced with 1nmol / L dexamethasone, 10mg / L insulin, 0.5mmol / L 3- The culture solution of isobutyl-1-methylxanthine and 100 μmol / L indomethacin was changed twice a week, fixed with paraformaldehyde after 3 weeks, and stained with Oil Red O. For staining results, see figure 1 . Clearly stained lipid droplets can be seen, which proves that the umbilical cord mesenchymal stem cells cultured in group A8 of the present invention have adipogenic differentiation ability.

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Abstract

The invention especially relates a primary culture medium and primary culture method for umbilical cord mesenchymal stem cells, belonging to the technical field of stem cell culture. The primary culture medium comprises tranexamic acid with a concentration of 500-15000 mg / L, G-CSF with a concentration of 10-50 ng / L, EGF with a concentration of 5-30 ng / mL and a basal culture medium, being the balance. According to the invention, tranexamic acid, G-CSF and EGF are into the basal culture medium, so primary culture time can be shortened, and the primary culture medium has better effect than conventional culture mediums and culture mediums with a single factor; and at the same time, the introduction of heterologous substances can be avoided and higher clinical safety is obtained.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a primary culture medium for umbilical cord mesenchymal stem cells and a primary culture method thereof. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with high self-renewal ability and multi-lineage differentiation potential, which widely exist in bone marrow, fat, amniotic fluid, placenta, umbilical cord blood and umbilical cord tissue. Umbilical cord mesenchymal stem cells (UC-MSCs) are a kind of stem cells present in umbilical cord Wharton's jelly (Wharton's jelly) and perivascular tissue. [0003] Umbilical cord mesenchymal stem cells have advantages over MSCs derived from embryos, bone marrow, adipose and other tissues. First, the source of the umbilical cord is not subject to ethical disputes, and the cost is low; second, hUC-MSCs do not cause teratoma, and have tumor suppressor properties, because hUC-MSCs also have low ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2501/11C12N2501/22C12N2501/999
Inventor 陈海佳葛啸虎王一飞黄幸王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD