In-vitro culture and passage method of buffalo spermatogonial stem cell-like cells

A technology for culturing spermatogonial stem cells and stem cells, which is applied in the field of in vitro culture and passage of buffalo spermatogonial stem cells, can solve the problems of in vitro culture and passage methods of buffalo spermatogonial stem cells, etc. The effect of reducing the culture time and saving the cost of cell culture

Inactive Publication Date: 2019-03-01
卢克焕 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no in vitro culture and passage method for buffalo spermatogonial stem cells

Method used

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  • In-vitro culture and passage method of buffalo spermatogonial stem cell-like cells
  • In-vitro culture and passage method of buffalo spermatogonial stem cell-like cells
  • In-vitro culture and passage method of buffalo spermatogonial stem cell-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The in vitro culture and passage method of buffalo spermatogonial stem cell-like cells of the present invention includes the following steps:

[0044] (1) Use collagenase digestion solution with a collagenase concentration of 10 mg / ml and DNase digestion solution with a DNase concentration of 0.5 mg / ml to digest the chopped testicular tissue of a 3-month-old buffalo for 50 minutes. Centrifuge, discard the supernatant, and get the precipitate; the addition amount of collagenase digestion solution is 0.5ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 100μl / g buffalo testis tissue;

[0045] (2) Add the trypsin cell digestion solution with a mass fraction of 0.1% and DNase with a DNase concentration of 2 mg / ml to the precipitation in step (1) to digest the precipitate for 10 minutes to obtain a digestion solution; The addition amount is 1ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 800μl / g buffalo testis tissue;

[...

Embodiment 2

[0053] The in vitro culture and passage method of buffalo spermatogonial stem cell-like cells of the present invention includes the following steps:

[0054] (1) Use collagenase digestion solution with a collagenase concentration of 13 mg / ml and DNase digestion solution with a DNase concentration of 0.7 mg / ml to digest the chopped testicular tissue of a 4-month-old buffalo after removing the albuginea and epididymis for 60 minutes. Centrifuge, discard the supernatant, and get the precipitate; the addition amount of collagenase digestion solution is 0.8ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 200μl / g buffalo testis tissue;

[0055] (2) Add 0.2% trypsin cell digestion solution with a mass fraction of 0.2% and DNase with a DNase concentration of 1.7 mg / ml in the precipitation of step (1) to digest the precipitate for 14 minutes to obtain a digestion solution; trypsin cell digestion solution The addition amount of 3ml / g buffalo testis tissue; the a...

Embodiment 3

[0062] The in vitro culture and passage method of buffalo spermatogonial stem cell-like cells of the present invention includes the following steps:

[0063] (1) Use collagenase digestion solution with a collagenase concentration of 15 mg / ml and DNase digestion solution with a DNase concentration of 0.9 mg / ml to digest the chopped 5-month-old buffalo testis tissue for 70 minutes after removing the albuginea and epididymis. Centrifuge and discard the supernatant to obtain the precipitate; the addition amount of collagenase digestion solution is 0.9ml / g buffalo testis tissue; the addition amount of DNase digestion solution is 300μl / g buffalo testis tissue;

[0064] (2) Add 0.25% mass fraction of trypsin cell digestion solution and DNase concentration of 1.5mg / ml DNase to the precipitation of step (1) to digest the precipitate for 16 minutes to obtain digestion solution; trypsin cell digestion solution The addition amount of DNase is 2.5ml / g buffalo testis tissue; the addition amount ...

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Abstract

The invention discloses an in-vitro culture and passage method of buffalo spermatogonial stem cell-like cells. The in-vitro culture and passage method comprises the following steps: putting primary buffalo spermatogonial stem cell-like cells into a culture dish containing buffalo supporting cells; culturing for 60 to 120 h to finish in-vitro culture; adding a PBS (Phosphate Buffer Solution) into the buffalo spermatogonial stem cell-like cells obtained by the in-vitro culture; collecting superficial cells; adding a pancreatin cell digestive juice into the superficial cells and digesting for 10to 60 s; then adding an IMDM (Iscove's Modified Dulbecco's Medium) culture solution to stop enzymic digestion reaction; collecting superficial cells and transferring the superficial cells which are collected at the second time into a culture dish paved with gelatin; culturing for 1.5 to 4 h; taking supernatant cells and transferring into a feeder layer containing the buffalo supporting cells to finish passage culture. The in-vitro culture and passage method provided by the invention is simple and convenient to operate, safe and reliable and high in repeatability, and has great significance ontreatment of infertility of mammals and protection of endangered species resources.

Description

Technical field [0001] The invention relates to the fields of genetic engineering, preservation of rare species and cell engineering, and more specifically to a method for in vitro culture and passage of buffalo spermatogonial stem cell-like cells. Background technique [0002] Spermatogonial stem cells (SSCs) are the precursor cells that form sperm. In male mammals, the proliferation and differentiation of spermatogonial stem cells provide a steady flow of power for the occurrence of sperm, while also ensuring that the genetic material is in the parent and child. Effective transfer between generations. Since the successful development of spermatogonial stem cell transplantation technology in 1994, the research of spermatogonial stem cells has become a hot spot, and in recent years, a method for in vitro culture of spermatogonial stem cells has been successfully established. In 2011, a serum-free and feeder-free culture system for mice was successfully carried out in vitro, brin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/33C12N2500/44C12N2501/115C12N2501/13C12N2501/999C12N2509/00
Inventor 卢克焕李婷婷陆阳清杨小淦梁兴伟
Owner 卢克焕
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