Sheep testis immortalized cell domestication method suitable for full suspension culture

Abstract

The invention discloses a sheep testis immortalized cell domestication method suitable for full suspension culture. The method comprises the following steps: (1) carrying out water bath resuscitationon undomesticated sheep testis cells, and centrifuging; (2) resuspending the cells with a DMEM culture solution containing 5% of newborn bovine serum, inoculating the cells into a cell culture bottle,and culturing; (3) subculturing, and digesting; (4) carrying out cyclotron suspension culture; and (5) transferring into a suspension culture bottle, culturing until the cell mass disappears, and ensuring that the cell density can be stable during repeated culture so as to obtain the sheep testis immortalized cells adapting to full suspension. Under the condition of suspension culture, the passage cells domesticated and cultured by the method are spherical and transparent, and are in a single or small-ball suspension state; the suspension domesticated cells subjected to 10 generations can bestably cultured to 2-3*10<6> cells / ml within 96 hours; and the virus culture adaptability results show that the virus titer reaches the peak value and is similar to the virus production level of adherent culture cells after the domesticated suspension cells are inoculated with orf virus for 72 hours.

Description

technical field [0001] The invention relates to a domestication method for sheep testis immortalized cells suitable for full suspension culture, and belongs to the field of suspension culture of immortalized sheep testis cell lines. Background technique [0002] Compared with the traditional spinner bottle culture technology, the cell suspension culture technology has the advantages of simple culture, safe operation, batch-to-batch uniformity, automation, and large-scale preparation, which can reduce labor, cost, site and production cycle in the actual production process. [0003] Since most of the cells used for vaccine virus production are adherent growth, the key to restricting the application of cell suspension culture technology is to adapt to the cell line of full suspension culture. Carrier suspension culture of cells is similar to adherent culture. It needs spinner bottles to provide seed cells, uses high-cost microcarriers, undergoes cumbersome steps such as trypsin...

AI Technical Summary

Problems solved by technology

Carrier suspension culture of cells is similar to adherent culture. It needs spinner bottles to provide seed cells, uses high-cost microcarriers, undergoes cumbersome steps such as trypsin digestion, and requires medium with high serum content, which is not conducive to industrial production.

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