In vitro culture method for avian stem spermatogonium

A technology of spermatogonial stem cells and in vitro culture, applied in the direction of germ cells, tissue culture, animal cells, etc., can solve the problems of long-term culture of chicken embryo spermatogonial stem cells, few line establishments, and no exploration of in vitro culture and passage conditions, etc., to achieve Reproducible, easy-to-operate results

Inactive Publication Date: 2007-03-28
YANGZHOU UNIV
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Problems solved by technology

[0004] Studies have shown that on the basis of a feeder layer, DMEM as a basal medium and adding some additives and some growth factors have good effects on the in vitro survival, division and proliferation, and clone formation of mammalian spermatogonial stem cells, but for chicken embryo spermatogonial stem cells. There are few research reports on the long-term culture and line establishment of original stem cells, and the optimal in vitro culture and passage conditions have not yet been explored

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  • In vitro culture method for avian stem spermatogonium

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Embodiment

[0017] Step 1: Isolation of Chicken Embryo SSCs

[0018] The chicks within one week of the shell were taken out, the chicken testes were aseptically obtained, dipped in PBS three times, and the albuginea was peeled off under a dissecting microscope. Add appropriate amount of PBS and pipette vigorously. First digest with collagenase (1 mg / mL) 10 times the volume of the tissue block, at 37 °C, 5% CO 2 Under the condition of high concentration, it acts for 5-8 minutes to remove the interstitial cells and some vascular components of the seminiferous tubule, and then digest it with a mixture of hyaluronidase (1.5mg / mL) and trypsin (0.25%) for 3-5 minutes. Minutes, the SSCs and supporting cells on the seminiferous epithelium were obtained, the digestion was terminated with 10% FCS in the digestion solution, and the undigested tissue components were filtered out with a 350-mesh filter. The filtrate was centrifuged at 1000r / min for 8 minutes, the supernatant was discarded, the cells...

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Abstract

The invention discloses to the method used to culture bird stem spermatogonium in vitro. It includes the following steps: inoculating the bird stem spermatogonium to CEF feeder layer; adding 10% calf blood serum, 2% chicken blood serum, 2mmol/L L-glutamine, 1mmol/L sodium pyruvate, 5.5*10-15ng/ml beta-mercaptoethanol, 10 mul/ml non-essential amino acid, 15-20ng/ml hSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11, 10ng/ml IGF, 100U/ml gentamicin sulphate culture solution into DMEM high glucose culture medium. This culturing method can process vitro operation for chicken stem spermatogonium, and lay study foundation for chicken embryo stem spermatogonium further series building.

Description

technical field [0001] The invention relates to the fields of transgene, animal cloning, preservation of rare animals, tissue and cell engineering and the like. Background technique [0002] Spermatogonia stem cells (SSCs) are the precursor cells of spermatogenesis and a kind of pluripotent stem cells with self-renewal and multilineage differentiation potential. With the continuous development and maturity of in vitro culture technology, cryopreservation technology and transplantation technology of spermatogonial stem cells, the application of spermatogonial stem cells becomes possible. Moreover, spermatogonial stem cells are of great significance in human medicine, establishment of transgenic animal models and protection of endangered animals. The research on spermatogonial stem cells has gradually become a hot spot in the field of stem cell research. Due to the unique reproductive physiological structure and anatomical characteristics of poultry, there are very few studi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/076
Inventor 李碧春魏彩霞陈国宏孙思宇吴信生赵文明徐琪吴洪周冠月孙国波戴建明孙鹏翔葛剑辉
Owner YANGZHOU UNIV
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