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Method for in vitro culture of testis tissues and induction to generate spermatid

A technology for in vitro culture of sperm cells, applied in the field of cell engineering, can solve the problems of low haploid, no long sperm, etc., and achieve the effect of sufficient nutrition

Inactive Publication Date: 2015-10-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the probability of obtaining haploid by 1.5-3.5-day testicular tissue culture is low, and there is no report about obtaining elongated spermatozoa from 10.5-14.5 days after birth (meiosis has started and no round spermatozoa)

Method used

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  • Method for in vitro culture of testis tissues and induction to generate spermatid
  • Method for in vitro culture of testis tissues and induction to generate spermatid
  • Method for in vitro culture of testis tissues and induction to generate spermatid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 newborn testicular tissue culture round spermatozoa

[0036] 1. Preparation of culture gel blocks

[0037] (1) Preparation of culture medium: the composition and content of the culture medium are: 90% MEMα (invitrogen product number: 11120-022) + 10% KSR (invitrogen product number: 10828).

[0038] (2) Preparation of agarose gel: take 33ml of ultrapure water, add 0.396g of agarose, autoclave at 121°C for 20 minutes to make 1.2% agarose gel, and pour it into a 10cm cell culture plate;

[0039] (3) Preparation of cultured gel blocks: After the agarose gel is solidified, cut the gel blocks into 10×10×5mm gel blocks, place the cut gel blocks in a 6-well plate, put 3 gel blocks in each well, Then add 4.5ml prepared medium to each well to soak the gel block in the medium. After soaking overnight, suck off the old medium and add 1.9ml fresh medium to make the liquid level of the medium reach the height of the gel block. In 4 / 5 places, tissue culture can be perfo...

Embodiment 2

[0047] Example 2 newborn testis tissue culture elongated sperm

[0048] 1. Preparation of culture gel blocks

[0049] Method is with embodiment 1.

[0050] 2. Cultivate the testis tissue of newborn 10.5 days, 12.5 days, 13.5 days and 14.5 days, take the testes of mice, remove the outer layer of white membrane, and divide into 1mm 3 sized tissue samples. Place the tissue samples on the aforementioned gel blocks respectively, put 3 pieces of testicular tissue in each gel block, place the 6-well plate at 34°C, 5% CO 2 Cultures were carried out in a cell culture incubator, and the medium was changed every 4.5 days.

[0051] Taking the testicular tissue samples of 12.5 days old as an example, HE staining was carried out on the tissue samples cultured for 27 days to 40 days respectively, and the observation results (such as Figure 7 Shown), A, B, and C in the figure are the results after 27 days, 31 days, and 40 days of newborn 12.5-day mouse testis tissue culture respectively....

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Abstract

The invention relates to the field of cell engineering, and concretely provides a method for in vitro culture of testis tissues and induction to generate spermatid. The method comprises the following steps: 1, preparing culture gel pieces: placing 1.2% agarose gel pieces in a 6-orifice plate, adding a medium, immersing overnight, extracting the used medium, adding the fresh medium, and using the agarose gel pieces as culture gel pieces, wherein the medium comprises 90% of MEM alpha and 10% of KSR; 2, taking 5.5-days and 10.5-12.5-days mouse testes, removing external layer albuginea, and cutting to form block tissue samples; 3, placing the tissues samples on the culture gel pieces, putting the 6-orifice plate in a cell culture box, and culturing; and 4, culturing the 5.5-days mouse testis tissues for 22-25d to obtain round sperms, and culturing the 10.5-12.5-days mouse testis tissues for about 27d to obtain elongated sperms.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a method for culturing testicular tissue in vitro to induce sperm cells. Background technique [0002] The function of the testis is mainly maintained by the constantly proliferating spermatogonial stem cells. The spermatogonial stem cells are located on the basement membrane of the seminiferous tubules. On the one hand, they can self-proliferate to form more spermatogonial stem cells. On the other hand, they can pass Several mitotic divisions followed by meiosis to form spermatocytes and eventually spermatids. The renewal of spermatogonial stem cells can produce 2 spermatogonial stem cells or split to produce 2 pairs of so-called Apr-type spermatogonia connected by cytoplasmic bridges, and Apr further divides to form Aal-type (the Aaligned spermatogonia) of 4, 8, and 16 cells ) spermatogonia. Aal becomes the first generation of differentiated A1 type spermatogonia. Type A1 the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
Inventor 李向东姚建飞
Owner CHINA AGRI UNIV
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