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Complete testis culture medium and application thereof

A technology of medium and medium components, which is applied in the field of biomedicine and can solve problems such as unreached

Inactive Publication Date: 2022-05-17
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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  • Complete testis culture medium and application thereof
  • Complete testis culture medium and application thereof
  • Complete testis culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 The spermatogonial stem cells still proliferated in the 12-week-embryo testis cultured for 100 days

[0024] (1) Tissue culture method: prepare 0.25% agar blocks with low-melting point agar and sterilized water, cut the agar blocks into squares of 8mm×8mm size, and place them in complete testis medium for at least 8 hours; Remove the mesonephros from the human embryonic testis, cut it into 3mm-sized tissue pieces, place them on the 0.25% agar block soaked in the culture set prepared in advance, add the culture solution to two-thirds of the agar block, and 34°C, 5% CO 2 to cultivate;

[0025] (2) Establishment of the culture system: In the cultivation process of this experiment, in order to explore the most suitable culture medium for testicular organogenesis in vitro, three culture schemes were adopted in the experiment. The specific culture medium components are as follows:

[0026]Complete testis culture medium: 90% (V / V) αMEM, 10% (V / V) KSR, BMP 4 / 7 (20ng...

Embodiment 2

[0031] Example 2 The testes of the 12-week embryo were cultured for 10 days and spermatocytes appeared

[0032] (1) Tissue culture method: prepare 0.25% agar blocks with low-melting point agar and sterilized water, cut the agar blocks into squares of 8mm×8mm size, and place them in complete testis culture medium for at least 8 hours; Remove the mesonephros from the testes of human embryos, cut them into 3mm-sized 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;

[0033] (2) Use the complete testis medium to culture the 12-week human testis for 10 days, stain the tissue sections of the 0 Day before culture and the 10 Day of culture, the cell types that play a key role in spermatogenesis, including germ cells, spermatogonia, spermatozoa, and sperm Cell markers (DDX4, PLZF, SYCP3, PRM1, SOX9, CYP17A1) of cells, supporting cells, and mesenchym...

Embodiment 3

[0034] Example 3 Round spermatozoa appeared in the testis of 12 weeks of embryo culture for 30 days

[0035] (1) Tissue culture method: prepare 0.25% agar blocks with low-melting point agar and sterilized water, cut the agar blocks into squares of 8mm×8mm size, and place them in complete testis culture medium for at least 8 hours; Remove the mesonephros from the testes of human embryos, cut them into 3mm-sized 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;

[0036] (2) Use complete testis medium to culture 12-week human testis for 30 days, stain the tissue sections of 30 Days in the culture process, and marker (DDX4 , PLZF, SYCP3, PRM1, SOX9, CYP17A1) for fluorescent staining ( Figure 4 ), the results showed that there were positive signals of PRM1 in the seminiferous tubules after 30 days of tissue culture, and the nuclei of these pos...

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Abstract

The invention discloses a complete testis culture medium and application thereof. The method comprises the following steps: separating human testis tissues into small blocks, placing the small blocks on 0.25-1.5% of agar blocks which are subjected to complete testis culture medium replacement for at least 8 hours in advance, adding a complete testis culture medium to reach 1 / 3-2 / 3 of the height of the agar blocks, and culturing for 10-100 days in a 5% carbon dioxide environment at 32-37 DEG C. The model provides a platform for in-vitro research on human testicular spermatogenesis, and provides a research platform for supporting research aiming at understanding testicular organ generation complexity and developing urgently needed male infertility treatment.

Description

[0001] This application is the application number 201910184175.5, the application date is March 12, 2019, and the divisional application of the invention title "a method for preparing an in vitro human testicular spermatogenesis model". technical field [0002] 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 [0003] 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 ...

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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