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Preparation method of in-vitro human testicular spermatogenesis model

A technology of spermatogenesis and testis, applied in the field of biomedicine, can solve problems such as unreached

Inactive Publication Date: 2019-07-19
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite multiple attempts, humans have yet to achieve "gold standard" meiosis in vitro

Method used

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  • Preparation method of in-vitro human testicular spermatogenesis model
  • Preparation method of in-vitro human testicular spermatogenesis model
  • Preparation method of in-vitro human testicular spermatogenesis model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 The spermatogonial stem cells still proliferated in the testes of 12-week-embryo cultured for 100 days

[0023] (1) Tissue culture method: prepare 0.25% agar blocks with low melting point agar and sterilized water, cut the agar blocks into 8mm×8mm squares, and replace them with complete testis medium for at least 8 hours; Remove the mesonephros from the human embryonic testes, cut them into 3mm tissue pieces, place them on the prepared 0.25% agar block soaked in the culture set in advance, add the culture solution to two-thirds of the agar block, and 34°C, 5% CO 2 to cultivate;

[0024] (2) Establishment of culture system: During the cultivation process of this experiment, in order to find out the most suitable medium for testicular organogenesis in vitro, three culture schemes were adopted in the experiment. The specific medium components are as follows:

[0025]Complete testis culture medium: 90% (V / V) αMEM, 10% (V / V) KSR, BMP 4 / 7 (20 ng / mL, R&D Systems), ...

Embodiment 2

[0030] Example 2 The spermatocytes appeared in the 12-week-old testis cultured for 10 days

[0031] (1) Tissue culture method: prepare 0.25% agar blocks with low melting point agar and sterilized water, cut the agar blocks into 8mm×8mm squares, and replace them with complete testis medium for at least 8 hours; Remove the mesonephros from the human embryonic testes, cut them into 3mm tissue pieces, place them on the prepared 0.25% agar block soaked in the culture set in advance, add the culture solution to two-thirds of the agar block, and 34°C, 5% CO 2 to cultivate;

[0032] (2) Using complete testis medium to culture 12-week human testis for 10 days, stain the tissue sections from Day 0 before culture and Day 10 after culture, the cell types that play a key role in spermatogenesis, including germ cells, spermatogonia, and spermatozoa Cell markers (DDX4, PLZF, SYCP3, PRM1, SOX9, CYP17A1) of sperm cells, Sertoli cells, and mesenchymal cells were fluorescently stained ( imag...

Embodiment 3

[0033] Example 3 Round spermatozoa appeared in the 12-week-embryo testes after 30 days of culture

[0034] (1) Tissue culture method: prepare 0.25% agar blocks with low melting point agar and sterilized water, cut the agar blocks into 8mm×8mm squares, and replace them with complete testis medium for at least 8 hours; Remove the mesonephros from the human embryonic testes, cut them into 3mm tissue pieces, place them on the prepared 0.25% agar block soaked in the culture set in advance, add the culture solution to two-thirds of the agar block, and 34°C, 5% CO 2 to cultivate;

[0035] (2) The 12-week human testis was cultured for 30 days with complete testis medium, and the tissue sections at 30 days during the culture process were stained, and markers for germ cells, spermatogonia, spermatocytes, sperm cells, Sertoli cells, and mesenchymal cells ( DDX4, PLZF, SYCP3, PRM1, SOX9, CYP17A1) for fluorescent staining ( Figure 4 ), the results showed that there were positive signal...

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Abstract

The invention relates to a preparation method of an in-vitro human testicular spermatogenesis model. According to the method, the human testicular tissues can be separated to small piece and placed ona 0.25% to 1.5% agar block which is replaced with a complete testicular medium at least 8 hours in advance, the complete testicular medium is added to 1 / 3 to 2 / 3 of a height of the agar block, and culture is carried out for 10 to 100 days at 32 DEG C to 37 DEG C in a 5% carbon dioxide atmosphere. The model provides a platform for in-vitro studies of human testicular spermatogenesis and supports research aimed at understanding the complexity of testicular organogenesis, and provides a research platform for the development of much-needed male infertility treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for constructing the complete seminiferous epithelium of human testis in vitro to complete meiosis and obtain sperm cells. Background technique [0002] Spermatogenesis refers to a continuous and complex biological process that develops from spermatogonia into mature sperm, including three stages: self-renewal of spermatogonia, meiosis of spermatocytes, and deformation of sperm cells. Testicular spermatogenesis is prone to genetic mutations, risks of endocrine disturbances, and exposure to environmental endocrine disruptors. Existing data show that the incidence of childhood tumors is 1.0 to 2.5 cases per 1,000 children, and about one-third of men face the risk of infertility after treatment. Human testicular spermatogenesis in vitro establishes genetic, epigenetic and environmental factors for the study of germ cells and testicular somatic cell formation and dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2501/13C12N2501/115C12N2501/11C12N2501/15C12N2501/125C12N2501/155C12N2501/30C12N2501/31
Inventor 沙家豪周琪郭雪江袁艳李来花
Owner NANJING MEDICAL UNIV
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