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Culture method of adipose tissue-derived stromal cells

A culture method and stem cell technology, applied in the direction of cell culture active agent, tissue culture, animal cells, etc., can solve the problems of poor mobility, limited expansion ability, etc., to solve the limited expansion ability, improve the expansion ability, high The effect of migratory ability

Inactive Publication Date: 2017-03-22
海南博鳌一龄迈迪精准医学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the embodiments of the present invention is to overcome the above-mentioned deficiencies of the prior art, and provide a method for culturing adipose-derived mesenchymal stem cells, so as to solve the technical problems such as limited expansion ability and poor migration of existing adipose-derived mesenchymal stem cells.

Method used

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  • Culture method of adipose tissue-derived stromal cells
  • Culture method of adipose tissue-derived stromal cells
  • Culture method of adipose tissue-derived stromal cells

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Embodiment 1

[0036] This embodiment provides a method for culturing adipose-derived mesenchymal stem cells, comprising the following steps:

[0037] 1. Preparation of recombinant human IL-3 (rhIL-3)

[0038] 1.1 Construction of pET-30a-rhIL-3 prokaryotic expression strain

[0039] The base codon of the human full-length IL-3 gene from the GenBank database was optimized to make it easy to express in Escherichia coli. The optimized base sequence is as follows:

[0040] 5'-ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC CTG GTC CGC CCCGGA CTC CAA GCT CCC ATG ACC CAG ACA ACG CCC TTG AAG ACA AGC TGG GTT AAC TGCTCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTGGAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGAAGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAC GCA TCA GCAATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCCACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTGACG TTC TAT C...

Embodiment 2

[0065] This embodiment provides a method for culturing adipose-derived mesenchymal stem cells, comprising the following steps:

[0066] 1. For the preparation of recombinant human IL-3 (rhIL-3), refer to step 1 of Example 1;

[0067] 2. Isolation and culture of primary adipose-derived mesenchymal stem cells

[0068] 2.1 Day 0:

[0069] Cell culture flask: Take 1 T75 culture flask without coating treatment.

[0070] Preparation of 1# complete medium: high sugar DMEM+2% Ultroser G+10 μmol / L SCF+10ug / ml IL-3+1% penicillin and streptomycin. Wherein, IL-3 is recombinant human IL-3 (rh IL-3) obtained in step 1.

[0071] Step 2.2 to step 2.8 refer to step 2.2 to step 2.8 of Example 1; the cells obtained from culture are labeled as S / I-ADSC.

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Abstract

The invention provides a culture method of adipose tissue-derived stromal cells. The culture method of the adipose tissue-derived stromal cells comprises the following culture step: inoculating acquired primary-generation adipose tissue-derived stromal cells into a stem cell culture medium, wherein the stem cell culture medium contains stem cell factors and interleukin-3 factors, and the content ratio of the stem cell factors to the interleukin-3 factors is (5 to 20 [mu]mol / L) to (5 to 20 [mu]mol / L). The culture method can improve the multiplication ability of the adipose tissue-derived stromal cells and achieve relatively high transfer ability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for culturing adipose-derived mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) were first discovered in the bone marrow and have been confirmed to exist in various tissues and organs of the body. They are a group of pluripotent stem cells with multi-directional differentiation potential. Compared with MSCs from other sources, adipose derived stromal cells (ADSCs) have great advantages in isolation and culture: [0003] (1) It is convenient to obtain materials. Adipose tissue reserves are abundant, and it is less painful to obtain samples. At present, 200ml of fat can be obtained in one liposuction operation, and about 1×10 6 stem cells, which is 40 times that of bone marrow; (2) The separation is simple. ADSCs can be obtained from peripheral fat by mechanical shearing, collagenase digestion and simple...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2501/125C12N2501/2303C12N2501/998
Inventor 李玮覃绍君陈瑞华
Owner 海南博鳌一龄迈迪精准医学研究院有限公司
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