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Isolated culture method of porcine fat stem cells

A technology for separating and culturing and stem cells, applied in the field of life science, can solve the problems of long time to digest adipose tissue, affect the effect of enzymolysis, cytotoxicity, etc. Effect

Inactive Publication Date: 2015-06-03
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adipose tissue contains a lot of blood and other impurities that affect the effect of enzymatic hydrolysis, so collagenase takes too long to digest adipose tissue
In addition, the current medium for the isolation and culture of adipose stem cells is usually DMEM / F12 complete medium containing fetal bovine serum, and serum may have toxic effects on cells and serum-derived contamination

Method used

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  • Isolated culture method of porcine fat stem cells
  • Isolated culture method of porcine fat stem cells
  • Isolated culture method of porcine fat stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043](1) After the subcutaneous adipose tissue was extracted by liposuction under aseptic operation, the adipose tissue was fully mixed with 1 volume of PBS buffer solution containing high concentrations of penicillin and streptomycin, and the adipose tissue was allowed to stand for 5 minutes. Layered with PBS. Use a 25ml pipette to absorb the mixture of PBS and blood in the lower layer, and then add PBS to repeat washing once.

[0044] In step 1, the PBS buffer is phosphate buffered saline.

[0045] (2) Cut up a small amount of large-volume adipose tissue, remove the connective tissue, transfer the adipose tissue into 50ml centrifuge tubes, add 1 times the volume of fat, mix the above PBS, and centrifuge at 700g for 3 minutes.

[0046] Use a pipette with a 10ml range (it is more convenient to use a pipette with a 10ml range for a 50ml centrifuge tube to absorb liquid, and a pipette with a 25ml range is too large in diameter, which will destroy the layering effect after sepa...

Embodiment 2

[0057] Example 2 Flow cytometric identification of porcine adipose stem cell P1 generation cells

[0058] (1) After the subcutaneous adipose tissue was extracted by liposuction under aseptic operation, the adipose tissue was fully mixed with 2 times the volume of PBS buffer solution containing high concentrations of penicillin and streptomycin, and the adipose tissue was allowed to stand for 10 minutes. Layered with PBS. Use a 25ml pipette to absorb the mixture of PBS and blood in the lower layer, and then add PBS to repeat washing once.

[0059] In step 1, the PBS buffer is phosphate buffered saline.

[0060] (2) Cut up a small amount of large-volume adipose tissue, remove the connective tissue, transfer the adipose tissue into 50ml centrifuge tubes, add 2 times the volume of fat in the above PBS, mix well, and centrifuge at 500g for 5 minutes.

[0061] Use a pipette with a 10ml range (it is more convenient to use a pipette with a 10ml range for a 50ml centrifuge tube to ab...

Embodiment 3

[0072] (1) After the subcutaneous adipose tissue was extracted by liposuction under aseptic operation, the adipose tissue was fully mixed with 1.5 times the volume of PBS buffer solution containing high concentrations of penicillin and streptomycin, and the adipose tissue was allowed to stand for 8 minutes. Layered with PBS. Use a 25ml pipette to absorb the mixture of PBS and blood in the lower layer, and then add PBS to repeat washing once.

[0073] In step 1, the PBS buffer is phosphate buffered saline.

[0074] (2) Cut up a small amount of large-volume adipose tissue, remove the connective tissue, and transfer the adipose tissue into 50ml centrifuge tubes, add 1.5 times the volume of fat in the above PBS, mix well, and centrifuge at 600g for 4 minutes.

[0075] Use a pipette with a 10ml range (it is more convenient to use a pipette with a 10ml range for a 50ml centrifuge tube to absorb liquid, and a pipette with a 25ml range is too large in diameter, which will destroy the...

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Abstract

The invention discloses an isolated culture method of porcine fat stem cells, which comprises the following steps: extracting porcine subcutaneous fat tissues, washing the fat tissues with a double-antibody-containing PBS (phosphate buffer solution), adding a I-type collagenase digestive juice into the fat tissues for digestion to obtain porcine fat stem cells, and culturing in an EGF-containing serum-free culture medium. The porcine fat stem cell isolated culture method can reduce the influence of blood and other impurities in the fat tissue source on the enzymolysis fat and shorten the fat enzymolysis time of the I-type collagenase; the serum-free culture medium is utilized to culture the primary cells; and proper growth factors are added into the culture medium, so that the porcine fat stem cells can be better subjected to isolated culture and maintain high proliferation and growth capacity, thereby establishing a porcine fat stem primary isolated culture method conforming to the Standard Specification.

Description

technical field [0001] The invention belongs to the field of life sciences, and in particular relates to a method for separating and culturing pig adipose stem cells. Background technique [0002] Adipose-Derived Stem Cells (ADSCs) are mesenchymal stem cells derived from adipose tissue with continuous self-renewal and multidirectional differentiation potential. Adipose stem cells are fibroblast-like. Adipose stem cells express low HLA-ABC but not HLA-DR, which shows that AMSCs have low immunogenicity and will not cause immune rejection after allogeneic or xenogeneic transplantation Under certain conditions, it can differentiate into adipocytes, chondrocytes, muscle cells and osteoblasts. The basic morphology, proliferation cycle, immunogenicity and multi-directional differentiation potential of adipose stem cells are similar to those of bone marrow mesenchymal stem cells, which are more researched at present. resemblance. [0003] ADSCs are derived from adipose tissue, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎冯德龙马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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