Culture and identification method for very small porcine embryonic-like stem cells

An embryonic-like stem cell and identification method technology, applied in the fields of stem cell engineering, tissue engineering and biological pacing, stem cell culture and identification, can solve the lack of separation of VSELs from large artiodactyls, the difficulty of culture and expansion of very small embryonic-like stem cells, and the purity No advanced problems, to achieve the effect of practical method, strong repeatability and high cell yield

Inactive Publication Date: 2016-03-30
NORTHERN JIANGSU PEOPLES HOSPITAL
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Problems solved by technology

[0004] Therefore, in view of the current outstanding problems such as difficulty in culture and expansion of very small embryonic-like stem cells, low efficiency, easy differentiation, low purity, difficulty in identification, and the lack of reports on the isolation, culture, and identification of VSELs in large artiodactyls so far

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  • Culture and identification method for very small porcine embryonic-like stem cells
  • Culture and identification method for very small porcine embryonic-like stem cells
  • Culture and identification method for very small porcine embryonic-like stem cells

Examples

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Embodiment 1

[0043] 1. In vitro culture method of porcine very small embryonic-like stem cells

[0044] 1. Isolation of allogenic suckling pig myoblasts

[0045] (1) After anesthetizing the piglets, separate the skeletal muscle samples of the newborn piglets under aseptic conditions, wash them with PBS, and remove the surface connective tissue as much as possible;

[0046] (2) Place the skeletal muscle tissue sample in a centrifuge tube and cut it into 1mm 3 Add 1g / L collagenase II, place in a constant temperature water bath at 37°C for digestion, after 20-40 minutes, centrifuge at 1200rpm for 6 minutes, and discard the supernatant;

[0047] (3) Add 0.25% trypsin digestion solution, gently pipette for 3-5 minutes, it can be seen that the digestion solution becomes cloudy, at this time, take the supernatant in another clean test tube, add an equal volume of 10-15% fetal bovine serum DMEM terminates the digestion, and the remaining tissue can be digested once again;

[0048] (4) Gently pi...

Embodiment 2

[0062] 1. In vitro culture method of porcine very small embryonic-like stem cells

[0063] The steps are the same as above, and the description will not be repeated.

[0064] 2. Method for identification of porcine VSELs by indirect immunofluorescence

[0065] (1) Aspirate the isolated or cultured VSELs culture solution from the culture well plate, add 4% paraformaldehyde to fix the cells for 30 minutes, rinse with PBS for 3 times; treat with 0.2% TritonX-100 permeabilization membrane for 20 minutes, rinse with PBS for 3 times ;PBS containing 10% FBS blocked for 2h;

[0066] (2) Add primary antibodies Sox-2, Oct-4, and Nanog to different wells, and freeze overnight at 4°C;

[0067](3) After overnight cell incubation, add the corresponding secondary antibody respectively; add 0.5% Tween20 in PBS solution (PBS-T) to wash 3 times, each time for 5min, add the secondary antibody dropwise, and incubate at 37°C for 2h (protected from light) ); Rinse with PBS-T 3 times, 5min each t...

Embodiment 3

[0072] 1. In vitro culture method of porcine very small embryonic-like stem cells

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Abstract

The invention relates to the technical field of stem cell culture and identification, and in particular relates to a culture and identification method for very small porcine embryonic-like stem cells of cloven-hoofed animals. The method comprises the following technical contents: (1) separating same roaster myoblasts and preparing a myoblast feeder layer; (2) inoculating the separated very small embryonic-like stem cells of porcine bone marrow into the myoblast feeder layer, and carrying out culture with an RPMI 1640 culture medium, adding cytokines to carry out culture and amplification on the very small embryonic-like stem cells; and (3) identifying the very small embryonic-like stem cells by morphology, immunofluorescence and genetic level series. The efficient culture method and accurate identification steps for the very small embryonic-like stem cells provided by the invention have great application prospects; and an important technical support is provided for further research on the very small embryonic-like stem cells.

Description

technical field [0001] The invention relates to the fields of stem cell engineering, tissue engineering and biological pacing, in particular to the technical field of stem cell culture and identification. technical background [0002] Very small embryonic-like stem cells (VSELs) were successfully isolated and named from mouse bone marrow mononuclear cells by Professor M. Ratajczak of the University of Louisville in Kentucky in 2006. The cell phenotype is Sca-1 (+) lin (-) CD45 (-), 2-4 μm in diameter. Further research by the team found that it also exists in human bone marrow, umbilical cord blood, peripheral blood, brain, myocardium, kidney, pancreas and other tissues, and the cell phenotype is SSEA-4+ / Oct-4+ / CD133+ / CXCR4+ / Lin- / CD45- , with a diameter of 4-6 μm. The biological characteristics of human and mouse VSELs mainly include: larger than platelets, smaller red blood cells, larger nuclei, containing euchromatin, expressing primitive pluripotent stem cell markers Oct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/077C12Q1/68
CPCC12N5/0607C12N5/0658C12N2501/115C12N2501/20C12N2502/03C12N2502/1335C12N2509/00C12Q1/6851C12Q2531/113C12Q2565/125
Inventor 顾翔李碧春顾健孙加斌金凯蒋一秀罗雪朱业孙磊胡茂志石青青朱睿王竟悟顾艺荀苗书航
Owner NORTHERN JIANGSU PEOPLES HOSPITAL
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