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Method for Rapid Culture and Differentiation of Adipose Stem Cells

An adipose stem cell and cell technology, applied in the field of stem cells, can solve the problems of endotoxin, cell activity needs to be strengthened, and fat stem cells cannot be obtained.

Active Publication Date: 2020-07-28
北京佑仁生物科技集团有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of this method: ①The operation time is too long and the cost is high, especially when it is necessary to separate a large number of samples
③The purity of the enzyme is not high, and it may contain endotoxin, pigment, foreign antigen, etc.
④The required sample size is relatively large. When the sample size is insufficient, a sufficient amount of adipose-derived stem cells cannot be obtained for experimental research
Similarly, the number of cell passages prepared by this method also needs to be improved, and the cell activity also needs to be strengthened

Method used

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  • Method for Rapid Culture and Differentiation of Adipose Stem Cells
  • Method for Rapid Culture and Differentiation of Adipose Stem Cells
  • Method for Rapid Culture and Differentiation of Adipose Stem Cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Separation of Adipose Stem Cells

[0021] The subcutaneous adipose tissue of healthy volunteers was taken, washed with PBS buffer 2 to 3 times, and the adipose tissue was cut into pieces of 0.5 mm under sterile conditions. 2The tissue block was digested by adding 0.075% collagenase type Ⅰ and 0.1% trypsin at 2 times the volume at 37°C for 1 hour with shaking at 20 r / min to carry out digestion and separation. Filter through a 70 μm cell sieve, centrifuge at 1500 r / min for 10 min at 4°C, and discard the supernatant. The erythrocyte lysate was treated for 5 minutes, PBS was added, centrifuged at 1500 r / min for 10 minutes at 4°C, and the supernatant was discarded. Inoculate in a culture flask at a density of 4000 cells / cm2, and the culture medium is DMEM containing 10% fetal bovine serum and 0.05% cell activity stimulating peptide (SEQ ID NO: 1). After 48 hours, the medium was changed for the first time to remove non-adherent cells and residual red blood cells. ...

Embodiment 2

[0022] The proliferation speed experiment of embodiment 2 cells

[0023] Using the MTT method, the P3, P4, and P5 generation cells in the logarithmic growth phase were inoculated in a 96-well plate at 4000 cells / well, divided into 4 groups, and each group had three replicate wells, and cultured in a 37°C, 5% CO2 incubator. Add MTT solution (5mg / mL) every 24h, continue to incubate for 4h, terminate the culture, and discard the culture supernatant. Add 150 μL DMSO to each well and shake for 10 minutes to fully melt the crystals. Colorimetry: select 490nm wavelength, measure the light absorption value of each hole on the enzyme-linked immunosorbent monitor, record the result, take the time as the abscissa, and the light absorption value as the ordinate to draw the cell growth curve, and use the culture medium without polypeptide as a contrast, from figure 2 It can be seen that the adipose stem cell culture method prepared by the present invention has a relatively fast cell cul...

Embodiment 3

[0024] Example 3 Identification of Inducible Differentiation Ability of Adipose Stem Cells

[0025] DMEM containing 10 nM dexamethasone (Sigma, St Louis, MO, USA) and 15 mg / L hydroxyapatite was used to induce osteogenic differentiation of human adipose-derived stem cells at passage 16 for 24 h. The next day the cells were replaced with osteogenic culture medium: cell culture medium DMEM+10% fetal bovine serum, 10mM 3-phosphate glyceraldehyde, 60mM ascorbic acid, 1OnM dexamethasone, 0.05% cell activity stimulating peptide SEQ ID NO: 1 and 15mg / L hydroxyapatite. The osteogenic culture medium was replaced every two days, and cultured for 8-14 days. The inducibility without peptide was used as positive control, and DMEM without inducer and fetal bovine serum were used as negative control. Analysis of qRT-PCR results Through the detection of induced ADSCs gene expression, the influence of ADSCs osteogenesis-related gene expression was analyzed. The results are shown in Table 1 ...

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Abstract

The invention provides a method for separating and cultivating adipose-derived stem cells. Cell activity excitation peptide is adopted for improving cell reproduction activity, the cell reproduction speed is increased, particularly, in the later inducted differentiation process, the differentiation cell growth speed can be well increased, the differentiation success rate is increased, the differentiation effect is improved, and the good application prospect is achieved.

Description

technical field [0001] The application belongs to the field of stem cells, and in particular relates to a rapid culture method for adipose stem cells. Background technique [0002] Adipose stem cells (adipose tissue-derived stem cells, ADSCs) are also called adipose-derived mesenchymal stem cells or processed fat-absorbing (PLA) cells. In 2001, it was isolated for the first time from adipose tissue removed by human liposuction by Zuk et al. ADSCs are a kind of adult mesenchymal stem cells with self-renewal, long-lasting vitality and multi-lineage differentiation potential, with stable growth and proliferation ability. Compared with embryonic stem cells and bone marrow mesenchymal stem cells, ADSCs are easy to obtain and have no ethical issues. ADSCs can differentiate into mesoderm cells such as adipocytes, osteoblasts, chondrocytes, cardiomyocytes, and even nerve cells in different induction differentiation media. In addition, ADSCs have a wide range of secretory function...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/077A61K35/32A61P19/08
CPCA61K35/32A61P19/08C12N5/0654C12N5/0667C12N2500/05C12N2500/38C12N2501/39C12N2501/998C12N2501/999C12N2506/1384C12N2509/00
Inventor 李红臣寇佳琳张永强吴梦妍朱刘明王青
Owner 北京佑仁生物科技集团有限公司
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