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Kit for cell culture

A kit, stem cell technology, applied in tissue culture, animal cells, vertebrate cells, etc., can solve the problem of stem cell adherence, maintenance of proliferation stability, etc. Unsatisfactory problems, to achieve the effect of maintaining proliferation ability and multi-directional differentiation potential, good proliferation ability and multi-directional differentiation potential, and eliminating the risk of spreading heterogeneous pathogens

Inactive Publication Date: 2016-03-23
郭镭 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing hUC-MSCs culture methods mostly use the addition of FBS and penicillin to the basal medium, but the composition of non-human serum is complex, which makes it easy for hUC-MSCs to differentiate during long-term culture, and there is a risk of spreading heterogeneous pathogens
[0005] In addition, although researchers have developed various types of serum substitutes, the currently commercially available serum substitutes and complete medium for hUC-MSCs 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Screening of medium composition in the kit of the present invention

[0054] (1) Content screening of β-mercaptoethanol

[0055] 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, 10ng / ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech company), 1 Parts by volume of non-essential amino acid aqueous solution (11140, Gibco Company), 5 parts by volume of culture supernatant concentrate, 94 parts by volume of a-MEM.

[0056] 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 seeding in T75 cell culture flask, add 12-15ml test medium, observe cell growth.

[0057] 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 inoculatio...

Embodiment 2

[0066] Example 2 Preparation and application of serum-free medium in the kit of the present invention

[0067] Serum-free medium preparation:

[0068] Recipe: 0.1 parts by volume of β-mercaptoethanol, 1 part by volume of non-essential amino acid aqueous solution (11140, Gibco company), 5 parts by volume of the prepared culture supernatant concentrate, 94 parts by volume of a-MEM / DMEM-F12 and Recombinant human basic fibroblast growth factor at a final concentration of 10 ng / ml.

[0069] Take β-mercaptoethanol, non-essential amino acid aqueous solution, and a-MEM / DMEM-F12 to prepare a premix, and mix the culture supernatant concentrate and recombinant human basic fibroblast growth factor with the premix.

[0070] Cell culture: In a biosafety cabinet, hUC-MSCs of the third passage isolated from the umbilical cord Huatong glue tissue of natural delivery newborns were collected and divided into 2×10 4 cells / cm 2 Density inoculate in T175 cell culture flask, add 15mL serum-free...

Embodiment 3

[0071] Example 3 Preparation and application of serum-free medium in the kit of the present invention

[0072] Serum-free medium preparation:

[0073] Recipe: 0.05 parts by volume of β-mercaptoethanol, 2 parts by volume of non-essential amino acid aqueous solution, 4 parts by volume of culture supernatant concentrate, 90 parts by volume of DMEM-F12 and a final concentration of 15 ng / ml recombinant human basic synthetic Fiber growth factor.

[0074] According to the method in Example 2, serum-free medium was prepared.

[0075]Cell culture refers to the method of Example 2, observe that the cells are in good condition after 24 hours of inoculation, and the confluence reaches 40%, and continue to cultivate. After 48 hours, the cells are confluent in a spindle-shaped vortex, reaching 80%, and continue to cultivate without rolling up.

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Abstract

The invention discloses a novel kit for serum-free stem cell culture. The kit comprises a serum-free culture medium, wherein the serum-free culture medium contains 0.05-0.2 part of beta-mercaptoethanol, 0.5-2 parts of a non-essential amino acid aqueous solution, 4-6 parts of human mesenchymal stem cell culture supernatant concentrate, 90-95 parts of a-MEM / DMEM-F12 and human recombinant basic fibroblast growth factors with the final concentration of 5-15 ng / ml by volume. The culture supernatant concentrate is prepared by adopting the following steps of collecting the umbilical cord mesenchymal stem cell culture supernatant, centrifugally removing cells, cellular debris, impurities and the like, performing filtration by using a micro-filtration membrane and performing ultra-filtration concentration. By utilizing the kit for stem cell culture, the multi-directional differentiation potential and stronger proliferation ability of the cells are still kept under the condition of long-term culture.

Description

technical field [0001] The invention relates to the research field of stem cells, in particular to a novel, high-efficiency serum-free medium and its preparation method and application. 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 (humanUmbilicalCordmesenchymalstemcells, hUC-MSCs) derived from human umbilical cord express a variety of unique markers of embryonic stem cells, with high differ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/00
Inventor 郭镭
Owner 郭镭
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