Study On Serum-free Culture Domestication For Rabbit Myeloma And Hybridoma Cells

Monoclonal antibody(mAb)has become a key tool for the detection of specific substances in the field of biology and medicine.Rabbit mAb has a higher affinity than mAb and is widely used in the scientific research and disease diagnosis.Hybridoma cell culture in vitro is an important way to prepare mAb.Traditional culture medium needs the addition of serum,which would cause the difference between batches and the instability of culture system,and there is some risk of pollution.In this thesis,the engineering cell lines prepared for mAb,including rabbit myeloma cell lines and hybridoma cell lines,were domesticated in serum-free medium to remove the serum in the medium.Firstly,four kinds of serum-free medium were selected to domesticate the rabbit myeloma cell line(240E-W2).Direct and gradual reduction modes were compared and evaluated to explore the effects of different acclimation methods and medium.It was found that 1640AB medium and CD Hybridoma Medium in a 1:1 volume ratio could efficiently reduce the serum content in the gradual mode,and the content of serum was decreased from 9%to 1%.The cell density reached 4.0-5.0 × 105 cells/ml after 72 h culture.Secondly,rabbit myeloma cell line(240E-W2)was tested in serum-free suspension culture.The initial inoculation density increased from 0.9 × 105 cells/ml to 1.2 × 105 cells/ml,and the content of serum was successfully decreased from 1%to 0.After 72 h culture cell density could reach 4.0-6.0 × 105 cells/ml and cell status was good.The cell line in serum-free medium was subcloned several times and the most stable myeloma cell line(240E-W2-SFM)was selected and maintained.Moreover,the appropriate serum-free cryoprotectant was selected,and the results of frozen recovery test indicated that there was no negative impact to the cell's physiological activity in serum-free cryopreservation.Finally,rabbit myeloma cell lines in serum-free medium(240E-W2-SFM)were fused with rabbit spleen cells to construct hybridoma cell lines.The maintenance effects with normal 9%serum medium and serum-free medium were compared.The positive rate of ELISA,WB test,the positive clone's loss rate and so on were analyzed.The results showed that the hybridoma cell lines could adapt to the serum-free culture with 1:1 volume ratio of 1640AB and CD Hybridoma Medium.The proportion of positive clones was good and the ability of target protein secreting were stable,which demonstrated good adaptability and application prospect.In this thesis,aiming to the removal of serum components in the engineering cell culture medium for rabbit mAb,the culture medium and domestication of cell lines and were optimized.The results indicated the feasibility of cell culture with serum-free medium for rabbit mAb preparation,which showed good potential for further applications.