Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells

A technology of spermatogonial stem cells and Bama miniature pigs, applied in the field of stem cell bioengineering, can solve the problems of inducing sperm cells and achieve the effect of expanding the scope of scientific research

Inactive Publication Date: 2014-05-28
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing technologies are aimed at mice, cattle and humans, and no sperm cells have been induced from pig spermatogonial stem cells

Method used

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  • Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells
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  • Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells

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Experimental program
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Embodiment 1

[0023] (1) Configure culture medium:

[0024] STO culture medium: composed of basal medium DMEM / F12, FBS, β-mercaptoethanol, sodium pyruvate, penicillin, and streptomycin, the amount of FBS added is 7% of the volume of DMEM / F12, and 0.1 mmol β is added to each milliliter of DMEM / F12 - mercaptoethanol, 55ng sodium pyruvate, 100U penicillin, 100 μg streptomycin;

[0025] Sperm stem cell culture medium: the medium is composed of DMEM / F12, sodium pyruvate, BSA, B27 supplement, GDNF, bFGF, GFRα1, β-mercaptoethanol, penicillin, and streptomycin, and BSA is added in 0.3% DMEM / F12 mass , 55ng sodium pyruvate, 20μl 50-fold B27 supplement, 20ngGDNF, 10ngbFGF, 100ngGFRα1, 0.1mmol β-mercaptoethanol, 100U penicillin, 100μg streptomycin were added per ml of DMEM / F12.

[0026] (2) Production of STO feeder layer

[0027] Will be 3.0-4.0×10 6 STO cells were inoculated into a 100mm culture dish, and 10ml of STO culture medium was added, at 37°C, CO 2Cultured under the condition of 5% volume...

Embodiment 2

[0034] (1) Configure culture medium:

[0035] STO culture medium: composed of basal medium DMEM / F12, FBS, β-mercaptoethanol, sodium pyruvate, penicillin, and streptomycin, the amount of FBS added is 7% of the volume of DMEM / F12, and 0.1 mmol β is added to each milliliter of DMEM / F12 - mercaptoethanol, 55ng sodium pyruvate, 100U penicillin, 100 μg streptomycin;

[0036] Sperm stem cell culture medium: the medium is composed of DMEM / F12, sodium pyruvate, BSA, B27 supplement, GDNF, bFGF, GFRα1, β-mercaptoethanol, penicillin, and streptomycin, and BSA is added in 0.3% DMEM / F12 mass , 55ng sodium pyruvate, 20μl 50-fold B27 supplement, 20ngGDNF, 10ngbFGF, 100ngGFRα1, 0.1mmol β-mercaptoethanol, 100U penicillin, 100μg streptomycin were added per ml of DMEM / F12.

[0037] (2) Production of STO feeder layer

[0038] Will be 3.0-4.0×10 6 STO cells were inoculated into a 100mm culture dish, and 10ml of STO culture medium was added, at 37°C, CO 2 Cultured under the condition of 5% volum...

Embodiment 3

[0045] (1) Configure culture medium:

[0046] STO culture medium: composed of basal medium DMEM / F12, FBS, β-mercaptoethanol, sodium pyruvate, penicillin, and streptomycin, the amount of FBS added is 7% of the volume of DMEM / F12, and 0.1 mmol β is added to each milliliter of DMEM / F12 - mercaptoethanol, 55ng sodium pyruvate, 100U penicillin, 100 μg streptomycin;

[0047] Sperm stem cell culture medium: the medium is composed of DMEM / F12, sodium pyruvate, BSA, B27 supplement, GDNF, bFGF, GFRα1, β-mercaptoethanol, penicillin, and streptomycin, and BSA is added in 0.3% DMEM / F12 mass , 55ng sodium pyruvate, 20μl 50-fold B27 supplement, 20ngGDNF, 10ngbFGF, 100ngGFRα1, 0.1mmol β-mercaptoethanol, 100U penicillin, 100μg streptomycin were added per ml of DMEM / F12.

[0048] (2) Production of STO feeder layer

[0049] Will be 3.0-4.0×10 6 STO cells were inoculated into a 100mm culture dish, and 10ml of STO culture medium was added, at 37°C, CO 2 Cultured under the condition of 5% volum...

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Abstract

The invention relates to a method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells, and belongs to stem cell bioengineering. The invention discloses the method of inducing the spermatids with the Ba-ma mini pig spermatogonia stem cells. The method comprises the steps of preparing a culture medium, fabricating an STO feed layer, inducing the spermatids with the spermatogonia stem cells, and identifying the spermatids, wherein the preparation of the culture medium comprises the step of preparing an STO culture solution and a spermatogonia stem cell culture solution; the STO culture solution comprises a basal culture medium DMEM / F12 (Dulbecco Modified Eagle Medium / F12), FBS (Fetal Bovine Serum), beta-mercaptoethanol, sodium pyruvate, penicillin and streptomycin; and the spermatogonia stem cell culture solution comprises the basal culture medium DMEM / F12, BSA (Bovine Serum Albumin), sodium pyruvate, a B27 additive, GDNF (Glial Cell Line-Derived Neurotrophic Factor), bFGF (Basic Fibroblast Growth Factor), GFR alpha 1, beta-mercaptoethanol, penicillin and streptomycin. An application object of the method is a pig. Different from the application object of the existing technical scheme, the method extends a scientific research scope.

Description

technical field [0001] The invention relates to a method for inducing sperm cells from Bama minipig spermatogonial stem cells, which belongs to stem cell bioengineering. Background technique [0002] In 2002, Li-Xin Feng et al. used stem cell factor (stem cell factor) to induce mouse spermatogonial stem cells to produce sperm cells (Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, et al. Generation and in vitro differentiation of a spermatogonial cell line.Science.2002Jul19;297(5580):392-5); in 2003, Fariborz Izadyar et al cultured bovine type A spermatogonia in vitro for a long time to induce sperm cells (Izadyar F, Den Ouden K, Creemers LB, Posthuma G, Parvinen M, De Rooij DG. Proliferation and differentiation of bovine type A spermatogonia during long-term culture. Biol Reprod. 2003 Jan;68(1):272-81.); 2006, Jae Ho Lee et al. cultured rat testicular cells in a three-dimensional culture medium made of collagen, and the tissue could undergo spermatogenesis (Lee JH,...

Claims

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Application Information

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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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