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A kit for culturing huc-msc step by step and huc-msc obtained by using the kit

A kit and culture medium technology, applied in the field of serum-free step-by-step culture kits, can solve the problems of stem cell adhesion, maintenance of proliferation stability and other characteristics that cannot achieve the expected results, non-existence of transmission of heterogeneous pathogens, and complex non-human serum components To achieve good proliferation ability and multi-directional differentiation potential, eliminate the risk of spreading heterogeneous pathogens, and shorten the 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 kit for culturing huc-msc step by step and huc-msc obtained by using the kit
  • A kit for culturing huc-msc step by step and huc-msc obtained by using the kit
  • A kit for culturing huc-msc step by step and huc-msc obtained by using the kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Culture of hUC-MSC in conventional medium with serum

[0059] 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).

[0060] 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.

[0061] For cell morphology see figure 1 . However...

Embodiment 2

[0062] Example 2 Culture hUC-MSC in conventional commercially available serum-free medium

[0063] 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.

[0064] Optionally, after the cells reach 100% confluency, the cells begin to...

Embodiment 3

[0065] Example 3 Screening of medium composition

[0066] (1) Screening of TME medium composition

[0067] 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 .

[0068] 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.

[0069] 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, a fractional step method is adopted to culture the hUC-MSC: first, a TME culture medium is used for culture for 3-4 hours so as to promote attachment of the hUC-MSC, and then the medium is replaced by a TMD culture medium for rapid amplification. By the adoption of the TME and TMD serum-free fractional step culture method, the problems of poor cell attachment capacity, slow amplification, easy differentiation and the like of a serum-free hUC-MSC culture method are well solved, and a foundation is laid for a long-term stable serum-free hUC-MSC culture technology.

Description

technical field [0001] The invention relates to the research field of stem cells, in particular to a novel and highly efficient serum-free step-by-step culture kit for 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 differ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/32C12N2500/44C12N2500/90C12N2501/115
Inventor 郭镭
Owner 郭镭
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