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Method for separating and culturing mouse primitive spermatogonia

A spermatogonial stem cell, separation and culture technology, applied in the field of cell separation and culture, can solve the problems of cell state influence, cell state change, low cell efficiency, etc., and achieve the effects of reduced stimulation, reduced invention cost, and simplified traditional separation steps.

Inactive Publication Date: 2010-09-01
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0020] Although MACS and FACS have higher purity in separating and enriching SSCs, the cost of expensive instruments makes the cost of the invention too high, which makes it difficult for conventional invention laboratories to study SSCs in depth; and SSCs need to be processed before passing through the instrument. The label that can be recognized by the instrument, this chemical process will have a certain impact on the cell state, and improper operation may even cause differentiation or apoptosis; the efficiency of the instrument to sort cells is not high, and it takes a long time, which can lead to cell viability Decrease, cell state changes, these are not suitable for the needs of conventional invention laboratories to sort and enrich SSCs in large quantities for scientific research

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  • Method for separating and culturing mouse primitive spermatogonia
  • Method for separating and culturing mouse primitive spermatogonia
  • Method for separating and culturing mouse primitive spermatogonia

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Embodiment Construction

[0050] (1), materials and reagents:

[0051] Source of spermatogonial stem cells: 7-day-old male ICR mice.

[0052] Source of feeder cells: mouse embryonic fibroblasts: 12.5-day pregnant ICR female fetuses; seminiferous tubule-derived cells: testicular tissue of 7-day-old male ICR mice.

[0053] Reagents: DPBS, 1mg / ml neutral protease, 0.01g / L gelatin, 10ug / ml mitomycin C, 0.25% trypsin, L-glutamine, sodium pyruvate, 20ng / ml GDNF (glial cell source neurotrophic factor),

[0054] Solutions: α-MEM (Gibco), DMEM (Gibco), fetal bovine serum (FBS, Hyclone,), non-essential amino acids (NEAA, Sigma,)

[0055] Medium:

[0056] Mouse spermatogonial stem cell culture medium: α-MEM containing 10% FBS by volume or DMEM containing 10% FBS by volume;

[0057] 10% FBS α-MEM and 10% FBS DMEM are added: 1% non-essential amino acid by volume, 1% L-glutamine by volume, 1% sodium pyruvate by volume, 20ng / ml GDNF ;

[0058] Feeder layer culture medium: DMEM containing 10% FBS by volume.

[...

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Abstract

The invention discloses a method for separating and culturing mouse primitive spermatogonia. In the method, the mouse primitive spermatogonia are separated by utilizing the characteristics of neutral protease, such as mild digestibility performance, high efficiency and small stimulation to cells; the step of enrichment and subculture of the mouse primitive spermatogonia are organically combined; and adherent mouse primitive spermatogonium colony is digested by utilizing the neutral protease, so a better enrichment effect is achieved. The method for separating and culturing the mouse primitive spermatogonia is characterized by simple separating and culturing operation, short period, good effect and low cost.

Description

technical field [0001] The invention relates to the field of cell separation and culture, in particular to a method for the separation and culture of mouse spermatogonial stem cells. Background technique [0002] Spermatogonial stem cells (SSCs) are male reproductive stem cells that grow in the epithelium of the seminiferous tubule near the basement membrane. Cells and eventually develop into sperm, to ensure the normal reproductive capacity of male animals. SSCs are the only stem cells in male animals that can transmit genetic information to offspring, and have strong plasticity, so they are important research materials in reproductive medicine, stem cell engineering, developmental biology, animal transgenics, etc., and have great scientific research value . The successful isolation and culture of SSCs is the basis for in-depth research on them. However, due to their extremely small content in animal testes, it is difficult to separate and purify them, which has caused gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
Inventor 宋锐曹鸿国陶勇章孝荣张运海李运生殷慧群刘亚方富贵刘旭光任春环张子军丁建平
Owner ANHUI AGRICULTURAL UNIVERSITY
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