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Serum-free medium and preparing method and application thereof

A technology of serum-free medium and culture supernatant, applied to bone/connective tissue cells, vertebrate cells, animal cells, etc. Complex serum components, unsatisfactory culture effects, etc., 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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  • Serum-free medium and preparing method and application thereof
  • Serum-free medium and preparing method and application thereof
  • Serum-free medium and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Screening of medium composition

[0057] (1) Content screening of β-mercaptoethanol

[0058] 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.

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

[0060] 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 t...

Embodiment 2

[0069] Example 2 Preparation and application of serum-free medium

[0070] Serum-free medium preparation:

[0071] 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.

[0072] 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.

[0073] 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 medium of the present invention, tr...

Embodiment 3

[0074] Example 3 Preparation and application of serum-free medium

[0075] Serum-free medium preparation:

[0076] 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.

[0077] According to the method in Example 2, serum-free medium was prepared.

[0078]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 serum-free medium. The serum-free medium comprises, by volume, 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 cultural concentrated supernatant and 90-95 parts of a-MEM / DMEM-F12 and recombination human basic fibroblast growth factors with the final concentration of 5-15 ng / ml. A preparing method of the cultural concentrated supernatant includes the following steps that umbilical cord mesenchymal stem cell cultural supernatant is collected; cells, cell debris, impurities and the like are centrifugally removed; filtering is carried out through a micro-filtration membrane; ultrafiltration and concentration are carried out. When cultured through the medium, stem cells still keep the multi-directional differentiation potential and the high multiplication capacity under the long-time culturing condition.

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): C12N5/0775
CPCC12N5/0663C12N5/0667C12N5/0668
Inventor 郭镭
Owner 郭镭
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