Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line

Abstract

The invention discloses a method for preparing an MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and the MDCK cell line. The method includes steps of 1), discarding culture media for cultivating MDCK cells, cleaning cell layers by the aid of pancreatin solution, then discarding the pancreatin solution, adding pancreatin solution into the MDCK cells again, digesting the MDCK cells, stopping digesting the MDCK cells after the cells are rounded, centrifuging cell suspension and then discarding supernatant to obtain cell clusters; 2), re-suspending the cell clusters obtained at the step 1) by the aid of serum-free culture media to obtain cell re-suspension; 3), arranging the cell re-suspension obtained at the step 2) in a shaking table, cultivating the cell re-suspension in culture tanks, diluting the cell re-suspension by the aid of the serum-free culture media and carrying out passage on the cell re-suspension to obtain the MDCK cell line. The method and the MDCK cell line have the advantages that the MDCK cell line is high in cell density and activity and uniform in size, individually grows in a dispersion manner, is plump in form and is suitable for serum-free suspension culture by the aid of bioreactors.

Description

technical field [0001] The invention relates to the field of cell line culture and domestication, in particular to a preparation method of an MDCK cell line adapted to serum-free full suspension culture and the MDCK cell line obtained by the preparation method. Background technique [0002] Canine kidney epithelial cells (Madin-Darby canine kidney cells, MDCK) are regarded as one of the most suitable cell lines for the production of influenza A and B influenza virus vaccines, and can be used to replace chicken embryos to culture influenza viruses. At present, a microcarrier adherent culture production process has been developed that utilizes MDCK cell lines instead of chicken embryos to cultivate influenza virus. Although this process has a certain production scale that can be achieved, it still has certain defects: 1. Microcarriers are difficult to multiply 2. The culture density of adherent cells is limited by the adherent area, resulting in a decrease in virus production;...

AI Technical Summary

Problems solved by technology

At present, a microcarrier adherent culture production process has been developed that utilizes MDCK cell lines instead of chicken embryos to cultivate influenza virus. Although this process has a certain production scale that can be achieved, it still has certain defects: 1. Microcarriers are difficult to multiply 2. The culture density of adherent cells is limited by the adherent area, resulting in a decrease in virus production; 3. Serum is usually added to help cell attachment and growth in adherent culture, and medium replacement is required , the process is complicated, and it will introduce mycoplasma, chlamydia or animal protein pollution, which poses a potential threat to the safety of vaccine products

However, the MDCK suspension cells domesticated by the above methods have been adapted to the serum-free culture and then domesticated to the suspension culture process. Most of them have defects such as long domestication time, cumbersome steps, cell clumping, or uneven cell size, and cannot reach high density. growth requirements

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