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Preparation method of buffalo testis single-cell suspension solution

A single cell and suspension technology, applied in the field of stem cells and tissues, can solve the problems of difficult to meet the demand and low production performance, and achieve the effects of easy operation, reduced enzyme damage, and good cell separation effect

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

As a unique species in South China, buffalo is mainly used for traditional labor. Its production performance such as milk production and meat production is low, and it is difficult to meet the current demand for animal husbandry products.

Method used

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  • Preparation method of buffalo testis single-cell suspension solution
  • Preparation method of buffalo testis single-cell suspension solution
  • Preparation method of buffalo testis single-cell suspension solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A preparation method for buffalo testis single-cell suspension, comprising the following steps:

[0029] (1) Pretreatment of raw materials: take 3-month-old buffalo testis and soak them in 75% alcohol for 5 minutes to sterilize, then cut off the buffy film in an ultra-clean bench, wash it three times with PBS containing double antibodies, and place it in culture after cleaning. Cut into small pieces in a dish, then transfer to an EP tube and cut into pieces;

[0030] (2) Digestion: transfer the shredded testis tissue in step (1) to a petri dish, add 1% collagenase by mass ratio and 0.001% DNase I enzyme by mass ratio to digest for 5min, place at 37°C, 5 After digestion in the % carbon dioxide incubator until the testicular tissue is basically digested into a single fine tubule, transfer to a centrifuge tube for centrifugation, and discard the supernatant to obtain the lower turbid solution;

[0031] (3) First-level resuspension: the lower turbid liquid obtained in step...

Embodiment 2

[0035] A preparation method for buffalo testis single-cell suspension, comprising the following steps:

[0036] (1) Raw material pretreatment: 7-month-old buffalo testes were soaked and disinfected in 75% alcohol for 15 minutes, then the white film was cut off in an ultra-clean bench, washed three times with PBS containing double antibodies, and then placed in culture Cut into small pieces in a dish, then transfer to an EP tube and cut into pieces;

[0037](2) Digestion: Transfer the shredded testis tissue in step (1) to a petri dish, add 3% collagenase by mass ratio and 0.005% DNase I by mass ratio to digest for 5-15 minutes, and place at 37°C , Digest in a 5% carbon dioxide incubator until the testicular tissue is basically digested into a single fine tubule, transfer to a centrifuge tube for centrifugation, and discard the supernatant to obtain the lower turbid solution;

[0038] (3) First-level resuspension: the lower turbid liquid obtained in step (2) was resuspended wit...

Embodiment 3

[0042] A preparation method for buffalo testis single-cell suspension, comprising the following steps:

[0043] (1) Pretreatment of raw materials: take 5-month-old buffalo testis and soak them in 75% alcohol for 5-15 minutes, then cut off the buffy film in an ultra-clean bench, wash them three times with PBS containing double antibodies, and put them in place after cleaning. Cut into small pieces in a Petri dish, then transfer to an EP tube and cut into pieces;

[0044] (2) Digestion: Transfer the shredded testis tissue in step (1) to a petri dish, add collagenase with a mass ratio of 1-3% and DNase I with a mass ratio of 0.003% to digest for 10 minutes, and place at 37°C , Digest in a 5% carbon dioxide incubator until the testicular tissue is basically digested into a single fine tubule, transfer to a centrifuge tube for centrifugation, and discard the supernatant to obtain the lower turbid solution;

[0045] (3) First-level resuspension: the lower turbid liquid obtained in ...

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Abstract

The invention discloses a preparation method of a buffalo testis single-cell suspension solution. The preparation method comprises the following steps: (1) pre-treating raw materials: taking a buffalotestis and disinfecting, then shearing to remove tunica albuginea; washing and cutting into small blocks; (2) digesting: transferring the sheared testis into a culture dish; adding a primary digestive juice for digesting until single seminiferous tubules are obtained; centrifuging and removing supernatant; (3) carrying out primary re-suspension: re-suspending precipitates by utilizing a PBS (Phosphate Buffer Solution); centrifuging and removing supernatant; after repeating for two times, obtaining a bottom-layer re-suspension solution; (4) carrying out cell separation: after carrying out secondary digestion, placing the solution on a microscope and observing; blowing through a pipette to form single cells; after neutralizing, collecting the cells into a centrifugal tube and centrifuging;removing supernatant to obtain a primary cell suspension solution; (5) carrying out secondary re-suspension: re-suspending by utilizing a culture solution and filtering through a cell net to obtain the buffalo testis single-cell suspension solution. The invention provides the preparation method of a buffalo spermatogonial stem cell suspension solution, which has high separation speed and good effect; the method is simple and feasible to operate and is easy to realize.

Description

technical field [0001] The invention relates to the technical field of stem cells and tissues, in particular to a method for preparing buffalo testicular single-cell suspension. Background technique [0002] Spermatogonial stem cells (SSCs) are precursor cells that form sperm. In male mammals, the proliferation and differentiation of spermatogonial stem cells provide a steady stream of power for spermatogenesis, and at the same time ensure the genetic material in the parent-child Efficient transfer between generations ([1] Kanatsu-Shinohara et al., 2013). Since the successful development of spermatogonial stem cell transplantation technology in 1994 ([2] Brinster et al., 1994), the research of spermatogonial stem cells has become a hot spot, and in recent years, the in vitro culture method of spermatogonial stem cells has been successfully established ([3] ]Shinohara et al., 2003). In 2011, Shinohara et al. successfully developed a serum-free and feeder-free culture system...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/00
Inventor 卢克焕李婷婷陆阳清杨小淦梁兴伟
Owner 卢克焕
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