A three-dimensional dynamic culture method for in vitro expansion of spermatogonial stem cells using fact Ⅲ microcarriers

A technology of spermatogonial stem cells and culture methods, applied in the field of three-dimensional dynamic culture, can solve the problems of time-consuming and cumbersome operation, and achieve the effect of simple operation, good repeatability and high success rate of culture

Active Publication Date: 2020-07-31
INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Combining three-dimensional dynamic culture and microcarrier culture technology, this culture method avoids the shortcomings of traditional two-dimensional planar conventional methods, such as multiple digestion passages, cumbersome operations and long time-consuming, etc.

Method used

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  • A three-dimensional dynamic culture method for in vitro expansion of spermatogonial stem cells using fact Ⅲ microcarriers
  • A three-dimensional dynamic culture method for in vitro expansion of spermatogonial stem cells using fact Ⅲ microcarriers
  • A three-dimensional dynamic culture method for in vitro expansion of spermatogonial stem cells using fact Ⅲ microcarriers

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Embodiment 1

[0033] 1. Preparation of materials

[0034] (1) The formula of SSCs medium (DMEM complete medium) is: 1% v / v essential amino acid solution (Gibco company, product number 11130051), 1% v / v Non-essential amino acid solution of v (Gibco, Cat. No. 11140050), 1% v / v vitamin solution (Gibco, Cat. No. 11120052), 55 µM β-mercaptoethanol solution, 2 mM L-glutamine, 2 mM sodium pyruvate , 5 wt% FCS, 1% v / v double antibody solution (Gibco, Cat. No. 15140-122), 20 ng / mL rmbFGF factor, 20 ng / mL rrGDNF factor and 10 3 U / mLIF factor. Preparation: Mix the components of the above medium, dissolve, filter and sterilize to obtain the product.

[0035] The SSCs culture medium of the present invention is an improved culture solution, adding growth factors to the basic culture solution can significantly improve the SSCs proliferation in vitro.

[0036] (2) Preparation of the Reverse Cell Culture System (RCCS):

[0037] The composition of the Zhuanbi's cell culture system (RCCS) used in the pres...

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Abstract

The invention provides a three-dimensional dynamic culture method for expanding spermatogonial stem cells in vitro by using FACT III microcarriers. In this method, a three-dimensional dynamic culture environment was constructed using a Zhuanbi bioreactor (RCCS), and FACTⅢ microcarriers were selected as scaffold materials to establish a three-dimensional dynamic culture system that could support the rapid proliferation of mouse testicular SSCs in vitro and maintain their undifferentiated characteristics. Under this three-dimensional dynamic culture system, after one week of culture in RCCS, SSCs showed an excellent proliferation effect, especially the SSCs obtained after 17-22 days of in vitro culture proliferated rapidly, and the proliferating SSCs cells still retained their biological characteristics in an undifferentiated state. Features and functions. The method of the invention has the obvious advantages of simple operation, low culture cost, good repeatability, high culture success rate, no need for subculture, avoiding feeder layer cell contamination, etc., and provides a reliable SSC in vitro culture technical solution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a three-dimensional dynamic culture method for in vitro expansion of spermatogonial stem cells using FACT III microcarriers. Background technique [0002] Spermatogonial stem cells (SSCs) are male germline stem cells located at the basement membrane of the seminiferous tubules of the testis. They are adult stem cells with the potential to maintain self-renewal and directed sperm differentiation. They can also undergo spontaneous dedifferentiation transformation during in vitro culture. Become pluripotent stem cells and become ideal seed cells for research on stem cell mechanism and clinical application of regenerative medicine. [0003] The number of adult stem cells in humans and animals is very small, and SSCs account for 0.03% of all germ cells in the adult mouse testis. To apply adult stem cells to clinical medicine, the urgent problem to be solved is how to expand th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2531/00C12N2533/32C12N2533/52C12N2509/00
Inventor 张秀娟陈金平白银山张守全王承民
Owner INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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