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In-vitro culture method for cryopreserved bovine spermatogenic cells

A spermatogenic cell and in vitro culture technology, applied in the field of cryopreserved bovine spermatogenic cell in vitro culture, can solve problems such as inability to guarantee transportation, inability to cell culture, inability to guarantee spermatogenic cells, etc. Simple operation, the effect of reducing the number of samples and costs

Pending Publication Date: 2022-01-21
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the long distance from the sampling site, it cannot be guaranteed to be transported to the laboratory for processing in a short time; conventional tissue freezing only puts tissue samples into liquid nitrogen tanks for storage, and recovery cannot be guaranteed without adding cryoprotectants After that, spermatogenic cells can be obtained, and subsequent cell culture cannot be carried out

Method used

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  • In-vitro culture method for cryopreserved bovine spermatogenic cells
  • In-vitro culture method for cryopreserved bovine spermatogenic cells
  • In-vitro culture method for cryopreserved bovine spermatogenic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Fresh bovine testicular tissue was collected in sterile 0.9% NaCl solution and transported to the laboratory on melting ice;

[0059] Place three sterile Petri dishes with a diameter of 100 mm on ice and fill them with 5 mL of DMEM / F12, and use sterile forceps to transfer fresh testicular tissue into the first Petri dish filled with DMEM / F12;

[0060] Transfer fresh bovine testis tissue to a second Petri dish containing DMEM / F12;

[0061] Use sterile scissors or a scalpel to divide fresh bovine testicular tissue into 4cm 3 Remove the residual buffy coat, and keep a piece of fresh bovine testicular tissue as a fresh control;

[0062] Transfer the cut fresh bovine testicular tissue fragments to the last Petri dish containing DMEM / F12;

[0063] Place the cryovials at 4°C, fill 15 cryovials with 800 μL of cryoprotectant, fill each cryovial with four cut pieces of fresh bovine testicular tissue, and mark the cryovials;

[0064] Incubate cryovials at 4°C for 15 minutes;

...

Embodiment 2

[0082] Fresh bovine testicular tissue was collected in sterile 0.9% NaCl solution and transported to the laboratory on melting ice;

[0083] Place three sterile Petri dishes with a diameter of 100 mm on ice and fill them with 5 mL of DMEM / F12, and use sterile forceps to transfer fresh bovine testicular tissue to the first Petri dish filled with DMEM / F12;

[0084] Transfer fresh bovine testis tissue to a second Petri dish containing DMEM / F12;

[0085] Divide fresh bovine testicular tissue into 4 cm sections with sterile scissors or a scalpel 3 Remove the residual buffy coat, and keep a piece of fresh bovine testicular tissue as a fresh control;

[0086] Transfer the cut fresh bovine testicular tissue fragments to the last Petri dish containing DMEM / F12;

[0087] Place the cryovials at 4°C, fill 15 cryovials with 800 μL, 50% cryoprotectant respectively, fill each cryovial with four cut pieces of fresh bovine testicular tissue, and place them at room temperature for 10 minutes;...

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Abstract

The invention discloses an in-vitro culture method for cryopreserved bovine spermatogenic cells, which comprises the following steps: performing programmed freezing or vitrification freezing on fresh bovine testicular tissues; resuscitating bovine testis tissues, and then preparing a single-cell suspension; preparing feeder layer cells by using the prepared single-cell suspension; and co-culturing supporting cells and spermatogenic cells by using the prepared feeder layer cells. Compared with the prior art, the method has the advantages that sufficient samples can be effectively collected at a time and can be stored and transported under the low-temperature condition, and the spermatogenic cells with high motility rate can be obtained by performing enzyme digestion on the resuscitated samples. The spermatogenic cells subjected to resuscitation can be subjected to long-term culture and passage in vitro, and a foundation is laid for subsequent cell experiments. The method has the characteristics of simplicity in operation and no limitation of time and distance, and can reduce sampling times and cost.

Description

technical field [0001] The invention relates to the technical field of cryopreservation and thawing culture and proliferation of spermatogenic cells, in particular to a method for culturing cryopreserved bovine spermatogenic cells in vitro. Background technique [0002] At present, the in vitro culture of bovine spermatogenic cells mainly adopts fresh testicular tissue, which is sampled from slaughterhouses or farms, placed on ice, and transported to the laboratory in a short time, and single-cell suspension is obtained by enzymatic digestion method for subsequent cell separation and culture experiments. In the long-term cryopreservation of tissues and cells, vitrification and programmed freezing are widely used, but their effects vary due to species differences. [0003] However, due to the long distance from the sampling site, it cannot be transported to the laboratory for processing in a short time; conventional tissue freezing only puts tissue samples into liquid nitrog...

Claims

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Application Information

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IPC IPC(8): C12N5/076C12N5/071A01N1/02
CPCC12N5/061A01N1/021A01N1/0252C12N2502/246C12N2509/00C12N2509/10
Inventor 蔡欣易川平赵旺生钟金城汪虹英陈雪梅罗辉柴志欣
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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