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A method for isolating porcine spermatogonial stem cells

A technology of spermatogonial stem cells and separation methods, which is applied in the direction of germ cells, animal cells, vertebrate cells, etc., can solve the problem of large loss of spermatogonial stem cells, and achieve the effects of simplifying serum components, reducing loss, and increasing yield

Inactive Publication Date: 2015-11-04
GUANGXI UNIV
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AI Technical Summary

Problems solved by technology

[0013] The culture medium used in the above scheme needs to add many types of serum, and the loss of spermatogonial stem cells is relatively large during the separation process of spermatogonial stem cells

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0025] (1) Solution preparation

[0026] 1. DMEM / F12 (Gbico Cat no: 12400-016): Prepare according to the instructions, adjust the pH value to 7.2-7.4.

[0027] 2. Culture medium: DMEM / F12 (Gbico Cat no: 12400-016), prepared according to the instructions, adding FBS, the amount of FBS added is 10% DMEM / F12 volume, 55ng / ml sodium pyruvate, temporarily adding penicillin and streptomycin Add 100u and 100μg of penicillin and streptomycin per ml of DMEM / F12, respectively.

[0028] 3. Formula of PBS without calcium and magnesium ions:

[0029] water

1000ml

NaCl

8.0145g

KCl

0.20115g

[0030] Na 2 HPO 4

1.420g

K H 2 PO 4

0.272g

[0031] Autoclaved, ready to use.

[0032] 4. Preparation of Collagenase IV:

[0033] Preparation of ten times stock solution: 20mg / ml, preparation method:

[0034] Dissolve collagenase in DMEM / F12 at a concentration of 20mg / ml, filter through a 0.22μm filter, dispense 0.5ml...

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Abstract

The invention relates to a method for separating pig spermatogonia stem cells. The method comprises steps of preparing solutions and separating spermatogonia stem cells, wherein in the step of preparing solutions, the solutions to be prepared comprises nutrient solution, digestive enzyme solution, buffer solution, collagen solution, gelatin solution and laminin solution, the step of separating spermatogonia stem cells comprises substeps of cutting pig testicular tissue into pieces and adding enzyme for digestion, filtering by a cell strainer, carrying out enrichment culture and difference adherent culture of the spermatogonia stem cells, finally collecting the spermatogonia stem cells, wherein the difference adherent culture comprises collagen coating adherent culture and laminin coating adherent culture. The method is characterized in that the nutrient solution comprises DMEM / F12, FBS (fetal bovine serum), sodium pyruvate, penicillin and streptomycin; according to the collagen coating adherent culture, after being cultured for required time, the cell is blown and beaten and then continuously cultured. In the collagen coating adherent culture, the spermatogonia stem cell is slightly blown and beaten and is not attached to the wall of the collagen, so that loss of the spermatogonia stem cell is reduced, and the yield of the spermatogonia stem cell is improved.

Description

technical field [0001] The invention relates to a method for separating porcine spermatogonial stem cells, belonging to stem cell bioengineering. Background technique [0002] At present, the separation methods of spermatogonial stem cells include immunomagnetic bead separation, flow cytometry sorting, differential attachment sorting, discontinuous density gradient centrifugation, and differential attachment combined with discontinuous density gradient centrifugation. [0003] Isolation of spermatogonial stem cells is a complex process. When using immunomagnetic bead separation and flow cytometry to separate spermatogonial stem cells, since there are no specific cell surface markers, methods that rely on biomarkers to separate spermatogonial stem cells such as immunomagnetic bead separation and flow cytometry The purity of spermatogonial stem cells obtained by cytometer sorting is not high or many spermatogonial stem cells are missed; technically speaking, because different...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076
Inventor 赵会敏卢克焕卢盛晟陆阳清杨小淦杨欢李敏霞张飒
Owner GUANGXI UNIV
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