The Suspension And Serum-free Culture For Trichoplusia Ni Ovarian Cell (Hi-five)

Insect cell baculovirus expression vector system (BEVS) is the fourth largest genetic expression vector system after the E. coli, yeast and animal cell. In comparison to other expression vector system, BEVS has more efficient of cloning exogenous gene and higher standard of expressing recombinant protein. This makes it the research hotspot in the biopharmacy industry. As BEVS are widely used in recombinant protein drug research, the study for in vitro large-scale culture of insect cells and the corresponding insect cell serum-free media is very important. The Insect cell lines, Sf-9 cell line and BTI-TN-5B1-4 (Hi-five) cell line are widely used, and a shows a high production and research value through the high expression of exogenous gene. The majority of the insect cell media are imported products, which are expensive and result in high cost of experiments, this has limited the development of large-scale culture of insect cells in vitro, and hindered the promotion and popularization of producing recombinant protein drug by using BEVS. After domestication of suspension and serum-free culture on Hi-five cells, we obtained a serum-free suspension cell line and a serum-free media for the cell line, and we developed the trial production of the media. So the insect cell serum-free culture method was established, which will be helpful for the production of the recombinant protein drugs by BEVS.As the starting point we use commercialized insect cell media to domesticate the Hi-five adherent cell which come from the China Center for Type Culture Collection (CCTCC). A serum-free Hi-five suspension cell bank, was established cell biology, microbial contamination (fungi, bacteria, mycoplasma and virus exogenous factor, etc.) and oncogenicity were detected. The results showed that:suspension Hi-five cells cultured in triangular flask with HyQ SFX-Insect, the vitality of recovery was up to 79.3%, the maximum proliferation concentration was 3.2×106 cells·mL-1 with the population doubling time (PDT) of 27.4 h for batch culture. Maximum proliferation concentration was 4.0×06 cells·mL-1 with the population doubling time (PDT) of 23.2 h in exponential growth phase with media changed. The cells were not contaminated by bacteria, fungi, mycoplasma, endogenous virus and exogenous virus, and had no tumorigenic ability. Then a Hi-five suspension cell line was establishedBy analysing the Grace's, IPL-41 and TC-100 media, and a litterature study, we designed a basal medium for Hi-five suspension cells. We designed single factor and orthogonal experiments. Glucose, yeast extract, hydrolyzed milk protein, lipid complexes and PF-68 were added to the basal medium, while adjusting the composition. By studying cell growth and metabolism, we screened out and designed a serum-free media witch was suitable for Hi-five suspension cell growth and named H-SFM-2.Comparing with the Hi-five cells cultured by HyQ SFX-Insect, we got the results of the Hi-five cells cultured by H-SFM-2:the maximum proliferation concentration was 4.2×106 cells·mL-1 with population doubling time of 25.4 h in HyQ SFX-Insect, and the maximum proliferation concentration was 4.4×106 cells·mL-1 with the population doubling time of 24.4 h in H-SFM-2. After continuously cultured for 20 passages in H-SFM-2, the average maximum proliferation concentration of the Hi-five suspension cells was 4.7×106 cells·mL-1, the average cell viability was 88.8%, and the average doubling time was 27.5 h. Cultured in 5L Sartorius bioreactor with fed-batch culture, the maximum proliferation concentration of the Hi-five suspension cells was 3.5×106 cells·mL-1 with the population doubling time of 26.9 h. The cell number reached 1.4×1010 cells. In a conclusion, the Hi-five suspension cell line we domesticated and the H-SFM-2 serum-free media we designed present a positive development prospect.