A method for establishing a sustainable cell line of tree shrew spermatogonial stem cells

A technology of spermatogonial stem cells and cell lines, applied in the field of stem cell research, can solve the problems that affect the in-depth study of stem cell culture systems, the establishment of tree shrew spermatogonial stem cells has no achievements, pollution, etc., and achieve long-term in vitro culture and sorting effects Good, high sorting efficiency

Active Publication Date: 2020-01-14
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor also tried to use mouse MEF as a feeder layer, but has been unsuccessful
Moreover, the interference of unknown components on the identification of stem cells and the contamination of heterologous cells will affect the in-depth study of stem cell cult...

Method used

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  • A method for establishing a sustainable cell line of tree shrew spermatogonial stem cells
  • A method for establishing a sustainable cell line of tree shrew spermatogonial stem cells
  • A method for establishing a sustainable cell line of tree shrew spermatogonial stem cells

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1 (digestion and separation of tree shrew spermatogonial stem cells)

[0067] (1) Tree shrew testis digestion:

[0068] ① Wash the testes of 6-month-old tree shrews with PBS, remove the buffy film, and cut them into pieces (aseptic operation).

[0069] ② Prepare a digestion solution containing 1 mg / mL hyaluronidase, 3 mg / mL collagenase, and 0.2 mg / mL DNaseI, digest the tissue fragments in a 37°C water bath for 20 minutes, and thoroughly mix the tissue and the digestion solution with a pipette every 5 minutes during the period.

[0070] ③ Wash twice with PBS to remove collagenase, centrifuge at 1500rmp for 5min, and discard the supernatant.

[0071] ④ Place the tissue fragments and digested cells in 0.25% trypsin digestion solution and digest the tissue fragments in a 37°C water bath for about 10 minutes. Mix once every 2 minutes with a pipette gun. When most of the cells in the digestive solution are in a single-cell state, the serum stops digestion.

[00...

Embodiment 2

[0093] Embodiment 2 (feeder layer cell culture and processing of tree shrew spermatogonial stem cells)

[0094] The invention provides a method for making feeder cells specially used for culturing tree shrew spermatogonial stem cells in vitro, comprising the following steps:

[0095] (1) Digest into single cells according to the above tree shrew testis digestion method.

[0096] (2) Cells were sorted according to the above tree shrew spermatogonial stem cell sorting method, and Thy1-negative cells were collected by magnetic bead sorting.

[0097] (3) Use DMEM medium as the base medium, add 10% NCS to culture the cells, remove the non-adherent cells after culturing for one day, and use the remaining cells as the feeder layer cells.

[0098] The processing method of the above feeder layer cells comprises the following steps:

[0099] (1) Petri dishes are pre-coated with gelatin;

[0100] (2) Digest the cells obtained by the method for making the feeder layer with 0.025% tryps...

Embodiment 3

[0105] Embodiment 3 (the preparation of the special culture medium of tree shrew spermatogonial stem cells)

[0106] Prepare 10mL of special medium for tree shrew spermatogonial stem cells, add 5μL GDNF (100ug / ml), 1.2mL FBS, 15μL EGF (10ug / mL), 0.05μL bFGF (20ug / ml), 12μL LIF (1*10 6 U / mL), 8μL Wnt3a (10ug / ml), 8.5mL StemPro34 basal culture medium, stemPro34supplement 250μL, mix evenly.

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Abstract

The invention discloses a method for establishing a continuously passable tree shrew spermatogonia stem cell system. A special culture medium comprises main components of bFGF, LIF, EGF, GDNF, wnt3a and FBS. A separation and culture method comprises the following steps: marking an antibody on the tree shrew testicular tissue which is digested into single cells; screening by using flow cytometry or magnetic beads so as to obtain a Thy1+ cell population; transferring the screened tree shrew testicular Thy1+ cells into a culture dish for culturing; and when spermatogonia stem cell colonies of cells are generated in the culture dish, transferring into a new culture dish which is treated with mitomycin and is coated by a special culture layer. The method is high in cell separation efficiency, definite in components of a special culture medium, and free of pollution of heterogenous cells. The culture method is simple and efficient, and has the advantages that the efficiency of obtaining target cells is high when tree shrew spermatogonia stem cells are separated, and a continuously passable cell system is high in establishment success rate, good in cell proliferation passage property and safe and stable.

Description

technical field [0001] The invention relates to a method for establishing a sustainable passaging tree shrew spermatogonial stem cell line, in particular to the screening of antibodies for specifically sorting the spermatogonial stem cells in the tree shrew testis and the establishment of a sustainable passaging tree shrew spermatogonial stem cell line Dedicated culture conditions and establishment of cell lines. Belongs to the field of stem cell research. Background technique [0002] The tree shrew (Tupaia belangeri) has many advantages in biomedical research compared with rodents and nonhuman primates. It is very close to primates in terms of genetic, evolutionary and physiological and biochemical characteristics. Tree shrews are more practical than non-human primates (monkeys) because of their small size, short growth and development cycle, high fecundity, and easy breeding. However, tree shrews, as experimental animals for biomedical research, have not achieved their...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/32C12N2500/44C12N2500/84C12N2500/90C12N2501/11C12N2501/115C12N2501/13C12N2501/2306C12N2501/415
Inventor 郑萍李朝晖班文赞
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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