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Passage culture type sheep testis cell and passage domesticating culture method, and special culture system and application thereof

A technique for cell passage and domestication culture, applied in the field of cell and tissue culture, can solve the problems of easy contamination, limited number of passages, poor cell morphology, etc., and achieves the effect of being suitable for mass production and increasing the number of passages

Active Publication Date: 2017-08-18
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, sheep testicular cells (LT) are all primary cells derived from lamb testes, which are dissected, shredded, digested, and cultured. The cells grow slowly and stop growing after a certain number of generations (generally within 10 generations). The number of times is limited. With the increase of the number of passages, the viability of the cells decreases, and the shape of the cells becomes worse and worse, and finally the passage cannot be carried out; and the source of lamb testes is limited, the collection workload is heavy, it is easy to pollute, and it is time-consuming and troublesome; the cell production process Complicated and time-consuming, not conducive to mass production

Method used

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  • Passage culture type sheep testis cell and passage domesticating culture method, and special culture system and application thereof
  • Passage culture type sheep testis cell and passage domesticating culture method, and special culture system and application thereof
  • Passage culture type sheep testis cell and passage domesticating culture method, and special culture system and application thereof

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Embodiment 1

[0043] Embodiment 1, subculture domestication culture (acquisition) method and preservation of sheep testis passage cell LT-1 (CGMCC No.12988)

[0044] The culture system used to subculture and domesticate sheep testis passage cells (LT-1) includes cell culture medium I, cell culture medium II and cell culture medium III, and the cell culture medium I contains 10% (V / V) newborn bovine Serum (purchased from Jinyuankang Company, batch number 20151208) and 1%-3% (W / V) L-glutamine (purchased from Gibco Company, batch number 21051024) MEM culture medium (purchased from Yixing Saier Biotechnology Co., Ltd., batch number is 150130), the cell culture solution II is DMEM culture solution containing 10% (V / V) newborn bovine serum, and the cell culture solution III is DMEM containing 5% (V / V) fetal bovine serum ( DMEM culture fluid (purchased from gibco company, batch number 10099-141) and 5% (V / V) newborn bovine serum (purchased from gibco company, batch number 12800082);

[0045] L-gl...

Embodiment 2

[0056] Example 2, the use of sheep testis passage cell LT-1 (CGMCC No.12988) for the proliferation and culture of sheep oral ulcer virus

[0057] The sheep testis passage cell LT-1 (CGMCC No.12988) is used for the proliferation and culture of the sheep oral ulcer virus, and the specific method comprises the following steps:

[0058] 1) Cultivate sheep testis passage cells LT-1 (CGMCC No.12988) with cell culture medium III (DMEM medium containing 5% (V / V) fetal bovine serum and 5% (V / V) newborn bovine serum), The culture temperature is 37°C. After the monolayer of cells is overgrown, the supernatant of the culture solution is discarded, and a mouth ulcer virus (derived from Jin Yubao Ling Biological Pharmaceutical Co., Ltd.), after 1 hour of adsorption, add maintenance solution Ⅰ (DMEM culture solution containing 2% (V / V) fetal bovine serum), the amount of maintenance solution Ⅰ is 10mL (8-10mL is acceptable), Culture in a constant temperature incubator at a temperature of 37°...

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Abstract

The invention discloses a passage culture type sheep testis cell and a passage domesticating culture method, and a special culture system and application thereof, and provides a passage culture type sheep testis cell LT-1 (CGMCC No.12988) and a method for obtaining the cell by domesticating culture. The method has the advantages that optimized cell culture liquid is used for performing passage domesticating culture on a sheep testis primary cell (LT), the passage times of the sheep testis primary cell can be increased, and the method is suitable for batched production; the sheep orf virus is subject to multiplication culture by the cell; proofed by results, the sheep orf virus can be continuously subject to passage culture for three generations, the obvious lesion is produced, the content of virus reaches 107.5TCID50 / mL, and the method can be applied to the multiplication culture and vaccine preparation of the sheep orf virus.

Description

technical field [0001] The invention belongs to a cell culture method and a culture system in the field of cell and tissue culture, in particular to a method for subculture and domestication of sheep testis primary cells (LT) to obtain subcultured sheep testis cells (LT-1) and Its special cultivation system and application. Background technique [0002] At present, sheep testicular cells (LT) are all primary cells derived from lamb testes, which are dissected, shredded, digested, and cultured. The cells grow slowly and stop growing after a certain number of generations (generally within 10 generations). The number of times is limited. With the increase of the number of passages, the viability of the cells decreases, and the shape of the cells becomes worse and worse, and finally the passage can not be carried out; and the source of lamb testes is limited, the collection workload is large, easy to pollute, and time-consuming; the cell production process Complicated and time-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12N7/02C12R1/91
CPCC12N5/0683C12N7/00C12N2710/24251
Inventor 路荣刘国英范秀丽张燕红高艳华
Owner JINYUBAOLING BIO PHARMA CO LTD
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