3D suspension culture method for in-vitro amplification of spermatogonial stem cells

A spermatogonial stem cell and suspension culture technology, applied in the biological field, can solve the problem of relying on the role of feeder cells, and achieve the effect of maintaining undifferentiated activity, promoting cell proliferation, and maintaining adhesion.

Inactive Publication Date: 2021-02-19
广州市奥宇科技有限公司
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  • Abstract
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Problems solved by technology

At present, there are relatively few studies on the three-dimensional culture of mouse SSCs in vitro, and most of the reports on the three-dimensional culture of mouse SSCs still rely on the role of feeder cells

Method used

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  • 3D suspension culture method for in-vitro amplification of spermatogonial stem cells
  • 3D suspension culture method for in-vitro amplification of spermatogonial stem cells

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Embodiment 1

[0031] 1. Culture medium preparation

[0032] (1) Preparation of RPMI-1640 medium containing 4% v / v Ultroser G: add 4% v / v Ultroser G to RPMI-1640 medium, stir and mix, filter and sterilize to obtain the product.

[0033] (2) Preparation of improved RPMI-1640 medium: add 50 μM β-mercaptoethanol, 1% v / v Ultroser G, 1 mg / mL GP4G, 50 ng / mL GFRα1, 20 ng / mL rrGDNF, 10 ng / mL to RPMI-1640 medium / mL bFGF factor, 50U / mL penicillin and 50μg / mL streptomycin, stir to dissolve, and filter to sterilize.

[0034] 2. Preparation of porous polylactic acid microspheres modified by RGD polypeptide

[0035] (1) Weigh 2g of polylactic acid and dissolve it in 20mL of dichloromethane, stir to dissolve, then add a gelatin aqueous solution with a mass fraction of 5%, the mass ratio of gelatin to polylactic acid is 1:40, and place it in a high-speed shear emulsifier at 10000rpm Mix for 3 minutes to obtain an emulsion; add the emulsion to 50 mL of polyvinyl alcohol solution with a mass fraction of 0....

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Abstract

The invention provides a 3D suspension culture method for in-vitro amplification of spermatogonial stem cells. The method comprises the following steps: inoculating a spermatogonial stem cell suspension on RGD polypeptide modified porous polylactic acid microspheres in a rotary biological reaction container, adding an improved RPMI-1640 culture medium, carrying out three-dimensional static cultureat 30-34 DEG C, starting a rotary system after the spermatogonial stem cells adhere to the RGD polypeptide modified porous polylactic acid microspheres, and carrying out three-dimensional dynamic suspension culture at 30-34 DEG C. By using the method disclosed by the invention, in-vitro large-scale amplification of the mouse SSCs can be realized on the premise of not adding feeder layer cells, and good stem cell proliferation activity and undifferentiated activity are obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a 3D suspension culture method for expanding spermatogonial stem cells in vitro. Background technique [0002] Spermatogonial stem cells (SSCs) are a type of adult stem cells that exist in the inner wall of the seminiferous tubules in the testes of male animals and maintain a balance of proliferation and differentiation. During in vitro culture, SSCs can spontaneously reorganize and transform Become pluripotent stem cells with biological functions similar to embryonic stem cells. It is a focus of adult stem cell research. [0003] The Kanatsu-Shinohara laboratory successfully established the in vitro culture system of mouse spermatogonial stem cells in 2003. After more than 10 years of development, the isolation and culture technology of mouse SSCs has become more and more mature, and mouse SSCs have become the main cell material and model for reproductive stem cell rese...

Claims

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Application Information

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IPC IPC(8): C12N5/076C12N5/02C08J9/40C08L67/04
CPCC08J9/40C08J2367/04C12N5/061C12N2500/40C12N2500/44C12N2501/115C12N2501/13C12N2501/998C12N2513/00C12N2533/40
Inventor 黄秀珍
Owner 广州市奥宇科技有限公司
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