Mesenchymal stem cell serum-free culture medium

A technology of serum-free medium and basal medium, applied in the direction of cell culture active agent, culture process, tissue culture, etc., can solve the problem of not supporting primary cell culture, complex components, and inability to effectively maintain the differentiation potential of mesenchymal stem cells and other problems, to achieve the effect of clear ingredients and simple addition of ingredients

Active Publication Date: 2017-06-30
北京赛斯达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defects of the prior art, the object of the present invention is to provide a serum-free medium for mesenchymal stem cells, which overcomes the inability to effectively ma

Method used

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  • Mesenchymal stem cell serum-free culture medium
  • Mesenchymal stem cell serum-free culture medium
  • Mesenchymal stem cell serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of mesenchymal stem cell serum-free medium

[0049] This example provides a serum-free medium for mesenchymal stem cells, using DMEM / F12 as the basal medium, and adding the following components to the basal medium:

[0050]

[0051]

[0052] See the table below for the cell culture reagents and cytokine manufacturers and article numbers used in this example:

[0053]

[0054]

[0055] Adjust the parameters of the medium as follows:

[0056] pH: 7.2-7.4

[0057] Osmotic pressure: 260-320mosm

[0058] Bacteria, fungi test: negative

[0059] Chlamydia, mycoplasma detection: negative

[0060] Endotoxin: 0-0.5EU / mL

[0061] The complete medium prepared above was sterilized by filtration with a 0.22um filter, and stored at -20°C in the dark.

Embodiment 2

[0062] Embodiment 2: preparation of human umbilical cord mesenchymal stem cells

[0063] Separate primary cultures with the following three groups of media:

[0064] (1) Group 1: the serum-free medium for mesenchymal stem cells according to Example 1 of the present invention;

[0065] (2) Group 2: traditional serum-containing medium (DMEM / F12+10% FBS);

[0066] (3) Group 3: serum-free medium for human mesenchymal stem cells (produced by LONZA, product number 00190632).

[0067] Experimental steps:

[0068] (1) Under aseptic conditions, the umbilical cords of healthy newborns (including the blood test results of the puerpera's physical examination) were collected, both ends were ligated with silk thread, and soaked in a preservation bottle containing 2% double antibody (penicillin and streptomycin mixture).

[0069] (2) Take out the umbilical cord from the biological safety cabinet, disinfect the surface of the umbilical cord with 75% medical alcohol for 30-40 seconds, cut o...

Embodiment 3

[0074] Embodiment 3: Three groups of cultured umbilical cord mesenchymal stem cell proliferation ability contrast

[0075] Experimental procedure: take the three groups of medium in Example 2 to culture well-growing P3 human umbilical cord mesenchymal stem cells, enzymatically hydrolyze to make a single cell suspension, and adjust the concentration to 2.4x 10 4 cells / mL, add to 12-well plate, add corresponding three groups of media respectively, 1 mL per well, place at 37°C, 5% CO 2 Culture in an incubator. From the second day, take three wells of each group every other day to calculate the cell volume, calculate the average value, and measure continuously for 8 days. Take the culture time as the horizontal axis and the total number of cell proliferation as the vertical axis to draw the cell growth curve . Such as image 3 shown.

[0076] from image 3 It can be seen that when using the medium of Example 1 of the present invention to culture mesenchymal stem cells, the cel...

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Abstract

The invention discloses a mesenchymal stem cell serum-free culture medium. The mesenchymal stem cell serum-free culture medium comprises a basic culture medium and added ingredients added in the basic culture medium, and the added ingredients comprise L-glutamine, nonessential amino acid, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, a trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-beta1 and PDGF-BB. By means of the culture medium, the problems that in the prior art, the serum-free culture medium is poor in cell adherence, components are relatively complex, and primary cell culture is not supported are solved.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a serum-free medium for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells derived from mesoderm, which have the potential of self-renewal, multilineage differentiation and low immunogenicity. In vitro, mesenchymal stem cells can be induced to differentiate into epithelial, bone, cartilage, fat, nerve, heart and other tissue cells, which can promote blood vessel formation, protect nerves and cell replacement therapy. Mesenchymal stem cells also have potential clinical application prospects in clinical hematopoietic support, promotion of stem cell implantation, and immune regulation. [0003] Although there are abundant sources of mesenchymal stem cells, such as fat, umbilical cord blood, umbilical cord, placenta and other tissues, mesenchymal stem cells are adult stem cells, and the original concentration in th...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2501/115C12N2501/135C12N2501/11C12N2500/90C12N2500/32C12N2500/38C12N2500/46C12N2500/25C12N2501/998C12N2501/734C12N2501/15C12N2501/39C12N5/0668C12N2500/30
Inventor 穆小生李晋李刚毅盖丽云
Owner 北京赛斯达生物技术有限公司
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