Spermatogonial stem cell culture system without adding recombinant growth factors

A spermatogonial stem cell and growth factor technology, applied in the field of spermatogonial stem cell culture, can solve the problems of high cost of culture, no support for long-term culture in vitro, easy cell differentiation, etc., and achieve clear medium composition, complete regeneration potential, and good repeatability. Effect

Inactive Publication Date: 2017-03-15
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These recombinant growth factors are expensive and need to be continuously added, and the culture cost is very expensive (although only GDNF is currently recognized as an essential growth factor), the proliferation of spermatogonial stem cells is slow, and the average doubling period is about one week, making the culture operation very difficult. complicated
In addition, it has been reported in the past that serum is needed to support the cultivation of spermatogonial stem cells. The components of serum are extremely complex, and the effects of each component on cell growth are not clear, and the cultured cells are easy to differentiate, which does not support long-term culture in vitro.

Method used

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  • Spermatogonial stem cell culture system without adding recombinant growth factors
  • Spermatogonial stem cell culture system without adding recombinant growth factors
  • Spermatogonial stem cell culture system without adding recombinant growth factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Spermatogonia stem cell (SSC) culture medium 500mL formula: basal medium (MEMα), bovine serum albumin (BSA) 0.6%, transferrin (Tf) 100μg / mL, unsaturated fatty acid (FFA) 15.2μeq / L , sodium selenite (Na 2 SeO 3 )6×10 - 8 M, 2-mercaptoethanol (2-ME) 100μM, insulin (InsuLin) 25μg / mL, 4-hydroxyethylpiperazineethanesulfonic acid (HEPESBUFFER) 10mM, putrescine (Putrescine) 120μM, penicillin (Penicillin) 50units / mL (Gibco15140122 (article number)), streptomycin (Streptomycin) 50μg / mL (Gibco15140122 (article number)), L-glutamine (L-glutamine) 2mM, 0.22μm filter filtration; Every milliliter described unsaturated fatty acid (FFA 100meq / L) includes: Linolenic acid 5.6mM, Oleic acid 13.4mM, Palmitoleic acid 2.8mM, Linoleic acid 35.6mM, Hexadecanoic acid (Palmitic acid) 31mM, octadecanoic acid (Stearicacid) 11.6mM, absolute ethanol as solvent.

[0023] 2. 500mL formula of culture medium used for STO cell line culture: basic high pond medium (DMEM): 455mL, serum (FBS): 35mL...

Embodiment 2

[0045] Compared with Example 1, this example only uses common STO cells as trophoblast cells.

[0046] 1. Spermatogonia stem cell (SSC) serum-free medium 500mL formula: basal medium (MEMα), bovine serum albumin (BSA) 0.6%, transferrin (Tf) 100μg / mL, unsaturated fatty acid (FFA) 15.2μeq / L, sodium selenite (Na 2 SeO 3 )6×10 -8 M, 2-mercaptoethanol (2-ME) 100μM, insulin (InsuLin) 25μg / mL, 4-hydroxyethylpiperazineethanesulfonic acid (HEPESBUFFER) 10mM, putrescine (Putrescine) 120μM, penicillin (Penicillin) 50units / mL (Gibco15140122 (article number)), streptomycin (Streptomycin) 50μg / mL (Gibco15140122 (article number)), L-glutamine (L-glutamine) 2mM, 0.22μm filter filtration; Every milliliter described unsaturated fatty acid (FFA 100meq / L) includes: Linolenic acid 5.6mM, Oleic acid 13.4mM, Palmitoleic acid 2.8mM, Linoleic acid 35.6mM, Hexadecanoic acid (Palmitic acid) 31mM, octadecanoic acid (Stearicacid) 11.6mM, absolute ethanol as solvent.

[0047] 2. 500mL formula of cult...

Embodiment 3

[0053] Compared with Example 1, this example only adds 1% serum (FBS) by volume to the spermatogonial stem cell culture medium.

[0054] 1. Spermatogonia stem cell (SSC) serum-containing culture medium 500mL formula: basal medium MEMα, bovine serum albumin 0.6%, transferrin 100 μg / mL, unsaturated fatty acid 15.2 μeq / L, sodium selenite 6×10 -8 M, 2-mercaptoethanol 100μM, insulin 25μg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 10mM, putrescine 120μM, penicillin 50units / mL, streptomycin 50μg / mL, L-glutamine 2mM,, FBS (Fetal bovine serum): 5mL, filtered through a 0.22μm filter; the unsaturated fatty acids per milliliter include: linolenic acid 5.6mM, octadecenoic acid 13.4mM, palm oleic acid 2.8mM, linoleic acid 35.6mM, hexadecane Acid 31mM, octadecanoic acid 11.6mM, absolute ethanol as solvent.

[0055] 2. 500mL formula of culture medium used for STO cell line culture: basic high pond medium (DMEM): 455mL, serum (FBS): 35mL, penicillin-streptomycin (P&S): 5mL, L-glutamine (...

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Abstract

The invention relates to a spermatogonial stem cell culture system without adding recombinant growth factors and application thereof. The spermatogonial stem cell culture system comprises a well-defined spermatogonial stem cell culture medium and a trophoblast cell containing glial-derived neurotrophic factors (GDNF). By utilizing a method provided by the invention, a spermatogonial stem cell can be cultured in vivo for a long term; a transplantation experiment verifies that the characteristics of the stem cell are no different from those of a spermatogonial stem cell cultured by a traditional culture method. Compared with the traditional culture method, the method provided by the invention has the benefits that any expensive recombinant growth factor and fetal bovine serum are not required to be added, and the culture medium is very clear in component and good in repeatability. The spermatogonial stem cell cultured by applying the method provided by the invention has a complete regeneration potential and an ability of infinite proliferation. The method provided by the invention is simple and convenient to culture, economic and efficient.

Description

technical field [0001] The invention belongs to the technical field of spermatogonial stem cell culture, and in particular relates to a spermatogonial stem cell culture system without adding growth factors and its application, in particular to a STO trophoblast cell expressing recombinant growth factor GDNF efficiently and spermatogonial stem cells with clear components Medium. Background technique [0002] Spermatogonial stem cells are reproductive stem cells located on the basement membrane of the seminiferous tubules in the testis of male mammals. It accounts for only 0.02% to 0.03% of the germ cells in the whole testis, but it is the only reproductive stem cell that can carry out self-renewal and differentiation, and maintain spermatogenesis throughout the life of male animals. The isolation and culture of spermatogonial stem cells plays a very important role in the study of the proliferation and in vitro differentiation of spermatogonial stem cells and the mechanism of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/10C12N15/86
CPCC12N5/061C12N15/86C12N2500/25C12N2500/30C12N2500/32C12N2500/36C12N2500/44C12N2500/46C12N2501/13C12N2501/998C12N2502/99C12N2800/107
Inventor 吴鑫贾媛媛薛园媛
Owner NANJING MEDICAL UNIV
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