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Isolated culture and identification method of porcine adipose-derived stem cells

A technology of adipose stem cells and identification methods, which is applied in the field of isolation, culture and identification of porcine adipose stem cells, which can solve problems such as the aging limitation of adipose stem cells, the failure to induce chondrocytes, and the change of surface antigens, and achieve good multidirectional differentiation performance , Improving the fecundity of high-quality livestock, and the effect of simple operation

Inactive Publication Date: 2016-05-25
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, porcine adipose stem cells are prone to aging after continuous culture in vitro, with changes in karyotype and surface antigens, and their multidirectional differentiation ability is limited to osteoblasts or osteoblasts and adipocytes (Zhang et al. .(2014).PLoSONE9(1):e85089.doi:10.1371 / journal.pone.0085089; Huang etal.StemCellResearch&Therapy (2015)6:77), there is no report of successful induction of chondrocytes
Aging of adipose stem cells limits its research and application in adipose tissue engineering and other tissue engineering

Method used

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  • Isolated culture and identification method of porcine adipose-derived stem cells
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  • Isolated culture and identification method of porcine adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1 Isolation and culture of porcine adipose stem cells

[0105] A fat tissue block from the groin of a dead Guangxi Bama miniature boar within 24 hours of birth was selected. After being sterilized with 75% alcohol, the tissue was washed three times with high-resistant PBS, and then transferred to a sterile centrifuge tube containing culture medium (specification 50mL) back to the laboratory. In a sterile ultra-clean bench, the adipose tissue was fully shredded into small pieces of about 1mm×1mm×1mm in size and transferred to a 50mL sterile centrifuge tube. The fat volume was about 5mL / tube, and 20 μg / mL LiberaseTL, which was 1 times the fat volume, was added to each tube. , mix well, and then put it into a constant temperature water bath shaker (100rpm / min) at 37°C for digestion for 2 hours, then take out the centrifuge tube and add 20mL low-sugar DMEM containing 10% FBS to stop the digestion, mix the digestion mixture gently, and use 100μm cells Sieve to remov...

Embodiment 2

[0106] Example 2 Biological Characteristic Identification of Porcine Adipose Stem Cells

[0107] Collect the P3, P5, P10 and P15 generations of Guangxi Bama miniature pig adipose stem cells with good growth conditions in Example 1, carry out cell counting and inoculate to a 96-well plate with a density of 2000 cells per well, and set blank wells (no cells) And control wells (medium without drugs, with cells), set 3 replicate wells for each group. Operate in accordance with the CCK-8 assay instructions. Continuously measure for one week, the time is the abscissa, and the reading of the microplate reader (the average number of 5 wells) is the ordinate to draw the growth curve, the growth curve is as attached figure 1 shown.

[0108] It can be seen from the growth curve that the passaged porcine adipose stem cells proliferate rapidly, but with the increase of the number of passages, the growth rate decreases.

[0109] Select Guangxi Bama mini-pig adipose stem cells with good g...

Embodiment 3

[0115] Example 3 Flow Cytometry Detection of Surface Antigens of Bama Miniature Pig Adipose Stem Cells in Guangxi

[0116] Take Guangxi Bama mini-pig adipose stem cells with different generations and good growth conditions in Example 1. When the cell abundance reaches 80%-90%, first use LiberaseTL (20 μg / mL) in CO 2 The incubator pre-digested the adipose stem cells for 2 minutes, then added 0.25% trypsin + 0.04% EDTA to continue the digestion for 2-3 minutes, then took out the culture bottle and shook the bottom vigorously to free the cells from the adherent state. Add culture medium to stop the digestion, collect the cells, and discard the supernatant by centrifugation. After washing with PBS twice, centrifuge to discard the supernatant, then add an appropriate amount of PBS to mix the cells, then divide the adipose-derived stem cells into 12 tubes, and add antibodies CD29-AF647, CD44-FITC, CD31-FITC, CD45-FITC, HLA-DA-FITC, CD90-FITC, CD105-PE, IgG1-AF647, IgG1-FITC, IgG2a-...

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Abstract

The invention discloses an isolated culture and identification method of porcine adipose-derived stem cells. The method comprises the following steps: 1) isolated culture and purification of adipose-derived stem cells; and 2) subculturing of adipose-derived stem cells. The adipose-derived stem cells obtained by the method disclosed by the invention have good genetic stability, and the genetic stability is still kept after the adipose-derived stem cells are continuously cultured in vitro to the 15th generation; with good dryness, the adipose-derived stem cells still have the surface antigen characteristics of mesenchymal stem cells after cultured in vitro to the 18th generation; and with good multi-directional differentiation property, the adipose-derived stem cells have the ability of differentiating into osteoblasts, chondroblasts and lipoblasts.

Description

technical field [0001] The invention relates to the field of animal cell culture, in particular to a method for separating, culturing and identifying pig adipose stem cells. Background technique [0002] After the research on embryonic stem cells encountered ethical and legal bottlenecks, adult stem cells have increasingly become a research hotspot. Mesenchymal stem cells (mesenchymal stem cells, MSCs) are a type of adult stem cells that exist in tissues such as umbilical cord blood, bone marrow, muscle, skin and fat. For fat, bone, cartilage, muscle, nerve, liver, myocardium, endothelial and other tissue cells. Adipose-derived stem cells (Adipose-derived stem cells) are a type of adult mesenchymal stem cells isolated from adipose tissue in recent years, which have the potential of self-renewal and differentiation. Adipose-derived stem cells are the most ideal source for existing stem cell bioengineering research due to their safety, easy acquisition, rapid expansion and m...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12Q1/02C12Q1/68G01N15/14
CPCC12N5/0667C12Q1/6888C12Q2600/158G01N15/14G01N33/5005
Inventor 杨素芳韩杰李海洋邓彦飞石德顺朱鹏杨海燕韦精卫李湘萍黄奔
Owner GUANGXI UNIV
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