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A serum-free step-by-step culture method of huc-msc and huc-msc obtained by said method

A culture method, serum-free technology, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problem that stem cell adherence, maintenance of proliferation stability and other characteristics cannot achieve expected results, and there is no transmission of xenogeneic pathogens. , the complex composition of non-human serum, etc., to achieve good proliferation ability and multi-directional differentiation potential, eliminate the risk of spreading xenogeneic pathogens, and shorten the effect of primary culture time

Active Publication Date: 2019-06-21
郭镭 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing hUC-MSC culture methods mostly use the addition of FBS and penicillin to the basal medium, but the complex components of non-human serum make hUC-MSC easy to differentiate during the long-term culture process, and there is no risk of spreading heterogeneous pathogens. Danger
[0005] In addition, although researchers have developed various types of serum substitutes, the current commercially available serum substitutes and complete medium for hUC-MSC culture are still unsatisfactory, especially for the adherence of stem cells. , proliferation, and maintenance of cell stability after long-term culture were unable to achieve the expected results

Method used

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  • A serum-free step-by-step culture method of huc-msc and huc-msc obtained by said method
  • A serum-free step-by-step culture method of huc-msc and huc-msc obtained by said method
  • A serum-free step-by-step culture method of huc-msc and huc-msc obtained by said method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Culture of hUC-MSC in conventional medium with serum

[0058] Test medium: 89 parts by volume of α-MEM, 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 U / ml streptomycin, 0.1 parts by volume of β-mercaptoethanol, 10 ng / ml of b-FGF , 1 part by volume of non-essential amino acid aqueous solution (11140, Gibco Company).

[0059] In the biosafety cabinet, the hUC-MSCs of the third generation isolated from the umbilical cord Huatong glue tissue of natural delivery newborns were collected and divided into 2×10 4 cells / cm 2 Density inoculation in T75 cell culture flask, add 15mL test medium, transfer into CO 2 The concentration is 5% in a 37°C constant temperature incubator. Observe the cell adhesion 2 hours after inoculation, a large number of hUC-MSC cells adhere to the wall, and tentacles protrude; observe after 48 hours, hUC-MSC reaches 90% confluence; the cell tentacles are stretched and bright.

[0060] For cell morphology see figure 1 . However...

Embodiment 2

[0061] Example 2 Culture hUC-MSC in conventional commercially available serum-free medium

[0062] Referring to the method in Example 1, inoculate with the same cell source and the same density, and add 15 mL of commercially available serum-free medium (product of Saiye Company, product number HUXUC-90061) to culture the cells. 2 hours after inoculation, it was observed that the cells were attached to the wall, the cells were bright, mostly round, and the antennae were stretched; 24 hours after the inoculation, the cells were observed, hUC-MSCs were bright under the microscope, the antennae were extended, and the expansion was not obvious; after 48 hours of inoculation Finally, the confluence rate of the cells reached about 50%. Observing the cells 72 hours after inoculation, the hUC-MSC cells were bright and reached more than 90% confluence. The cells were collected by trypsinization and frozen.

[0063] Optionally, after the cells reach 100% confluency, the cells begin to...

Embodiment 3

[0064] Example 3 Screening of medium composition

[0065] (1) Screening of TME medium composition

[0066] Test medium: 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3 or 0.5 parts by volume of β-mercaptoethanol, 1 part by volume of non-essential amino acid aqueous solution (11140, Gibco), 99 parts by volume of a-MEM .

[0067] Referring to the method in Example 1, inoculate with the same cell source and the same density, and add 15 mL of test medium to culture the cells. Observe the cell adhesion.

[0068] Results: In the two concentration groups containing 0.01 and 0.02 parts by volume of β-mercaptoethanol in the medium, the speed of cell attachment was slow. After 4 hours of inoculation, some cells still did not adhere to the wall. After about 8 hours, The cells basically adhered to the wall; in the four concentration groups containing 0.05, 0.1, 0.15 and 0.2 volume parts of β-mercaptoethanol in the medium, the cells were completely adhered to the wall after 4 hours of inoculati...

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Abstract

The invention discloses a serum-free culture scheme for human umbilical cord mesenchymal stem cells (hUC-MSC). According to the scheme, the hUC-MSC are cultured by adopting a stepped method, wherein the hUC-MSC are cultured with a TME culture medium for 3-4 hours to promote adherence of the hUC-MSC, and then the hUC-MSC are cultured in a TMD culture medium for rapid amplification. According to the TME-TMD serum-free stepped culture method, the problems that in a serum-free culture method for the hUC-MSC, the cell adherence capacity is poor, multiplication is slow and differentiation is prone to occur are better solved, and a base is laid for a long-term stable serum-free culture technique for the hUC-MSC.

Description

technical field [0001] The invention relates to the research field of stem cells, in particular to a novel and efficient serum-free culture method of hUC-MSC. Background technique [0002] Mesenchymal stem cells are ubiquitous in various tissues and organs of the human body, have multi-directional differentiation potential, stimulate tissue regeneration, regulate immunity and other functions, and have broad application prospects in the field of cell therapy. [0003] Bone marrow mesenchymal stem cells have been widely used clinically, and current research shows that umbilical cord-derived mesenchymal stem cells can not only become an ideal substitute for bone marrow mesenchymal stem cells, but also have greater application potential. Among them, human umbilical cord mesenchymal stem cells (human Umbilical Cord mesenchymal stem cells, hUC-MSCs) derived from human umbilical cord express a variety of unique markers of embryonic stem cells, and have high differentiation potentia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0606
Inventor 郭镭
Owner 郭镭
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