Long-term culture method for spermatogonial stem cells in vitro without feeder layer

A feeder-free technology for spermatogonial stem cells, applied in the field of long-term cultivation of spermatogonial stem cells in vitro without a feeder layer, to achieve good spermatogonial stem cell characteristics and good cell viability

Active Publication Date: 2018-11-13
NORTHWEST A & F UNIV
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Problems solved by technology

The existing long-term culture system of spermatogonial stem cells cannot fully realize the long-term culture of porcine spermatogonial stem cells in vitro, so we need to optimize the in vitro culture system of pSSCs to lay a foundation for exploring their biological characteristics

Method used

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  • Long-term culture method for spermatogonial stem cells in vitro without feeder layer
  • Long-term culture method for spermatogonial stem cells in vitro without feeder layer
  • Long-term culture method for spermatogonial stem cells in vitro without feeder layer

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Embodiment

[0054] The following is a detailed description of the isolation and culture identification of porcine spermatogonial stem cells, as well as tests such as cell morphological characteristics, marker genes, and immunofluorescence identification. The description is an explanation of the present invention rather than a limitation.

[0055] 1. Isolation and purification of porcine spermatogonial stem cells

[0056] The testes of 20-day-old Guanzhong black pigs were collected from farmers in Yangling District. After two consecutive differential attachments, the positive rate of spermatogonial stem cells was found to be over 80% ( figure 1 ).

[0057] 2. Screening of different extracellular matrices:

[0058] Mainly including the screening of four substrates, the poly-lysine-coated culture dish was at least 4 hours, after which the culture plate was washed twice with PBS and dried naturally. Petri dishes were coated with gelatin (0.1%) for 1 hour and then washed once with PBS before...

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Abstract

The invention relates to a long-term culture method for spermatogonial stem cells in vitro without a feeder layer. According to the method, the spermatogonial stem cells of a pig can be cultured in vitro to three months by screening different extracellular matrices and applying chemical small molecule substances to long-term culture of the spermatogonial stem cells of the pig. A 2D natural artificial synthetic polypeptide matrix coating is utilized as an extracellular matrix, a typical grape beaded spermatogonial stem cell cluster is observed during the culture process, different chemical small molecule combinations are screened, the spermatogonial stem cells of the pig are cultured in vitro for three months by adding Chir99021, Repsox and a CD lipid concentrate with good spermatogonial stem cell characteristics maintained.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a long-term culture method for spermatogonial stem cells without a feeder layer in vitro. Background technique [0002] China is one of the countries with the largest number of pigs in the world. Because pigs are closely related to humans and their physiological and biochemical indicators are similar to humans, they are often used to study the reproductive development mechanism of humans. [0003] Spermatogonial stem cells (SSC) are the only adult stem cells in mammals that can transmit genetic information to the next generation. In early studies, SSCs have always been considered unipotent, but in fact SSCs can autonomously reprogram to form ES-like pluripotent stem cells during culture, although the efficiency of spontaneous reprogramming is relatively low, therefore, spermatogonial stem cells have also been Considered an ideal source of pluripotent cells t...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/32C12N2500/44C12N2501/11C12N2501/115C12N2501/13C12N2501/235C12N2533/32C12N2533/52C12N2533/54
Inventor 华进联张莹周哲沈巧燕祝振硕李娜
Owner NORTHWEST A & F UNIV
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