| Taxol(Paclitaxel)is an effective anticancer drug which is mainly extracted from the tissues such as bark of Taxus genus and chemically synthesized.However,these methods have disadvantages such as shortage of resources and low yield of Taxol.At present,it is widely accepted that the large-scale culture of Taxus tissue and cell are the most promising approaches to solve the serious shortage of Taxol.In this paper,the scale-up culture of Taxus cuspidata suspension cells was studied in order to provide a basis for the large-scale production of Taxol using Taxus cuspidata suspension cells.In this work,callus tissues were initiated from young stems of Taxus cuspidata,which were subcultured and then used to establish suspension cell lines.Also,the effect of vitamin content in B5 medium on Taxol production by suspension cells was studied.To solve the problem of variation in the process of the scale-up culture of Taxus cuspidata suspension cells,the cryopreservation of Taxus cuspidata suspension cells by vitrification was studied.Then,the characteristic parameters of scale-up culture of Taxus cuspidata suspension cells and the rheological characteristics of fermentation broth in the airlift bioreactor were studied.Finally,the CFD numerical simulation of two-phase flow in 2L airlift bioreactor was carried out,and the effect of aeration rate on gas holdup and velocity of liquid circulation was investigated.The conclusions are as follows.1.Stable Taxus cuspidata suspension cell lines were established using the callus tissues which were induced from young stems of Taxus cuspidata.Based on the optimization of vitamin content in B5 culture medium,it was concluded that the optimal vitamin concentration for Taxol production of Taxus cuspidata suspension cells was vitamin B1 of 21.58mg/L,vitamin B6 of 3.09mg/L,inositol of 201.29mg/L and nicotinic acid of 1.77mg/L.The maximum Taxol yield was 2.767mg/L.2.The optimal scheme of cryopreserving Taxus cuspidata suspension cells by vitrification was as follows.Taxus cuspidata suspension cells were cultured for 12 d,after that the suspension cells were pre-cultured for 1.69 d in the B5 medium containing 0.7mol/L sucrose and 5% DMSO(w/v).Afterwards,the cells were pre-treated for 32.25 min using 60% PVS4,and then they were dehydrated for 41.55 min using 100% PVS4 at 0°C.Then the cells were separated,mixed with fresh 100% PVS4 at 0°C and preserved in liquid nitrogen.After 48 h,the cells were thawed in 60 s at 37°C,and then the cells were washed with 1.2mol/L sucrose.The survival rate was 76.62%.3.The cell growth and Taxol synthesis in the conical flask of 250 m L and 500 m L,2L airlift bioreactor was similar.But the biomass and Taxol yield in the conical flask were higher than that in airlift bioreactor of 15%-25% and 20%,respectively.In the three culture scales,the change regularities of p H value and conductivity were similar,the cell growth can be monitored online by p H value and conductivity.Airlift bioreactor was suitable for suspension culture of Taxus cuspidata cells,and the optimal aeration rate was 0.2vvm.In addition,it was found that culture liquid of Taxus cuspidata suspension cells was pseudoplastic fluid and the rheology constitutive equation of each growth stage was established.It provided a theoretical basis for the control of the bioreactor.4.Using FLUENT software to simulate the 2L airlift bioreactor with different aeration rates,the distribution of gas holdup and velocity of liquid circulation were obtained by visual output.The results showed that the gas holdup and circulating liquid velocity increased with the increase of aeration rate.By simulation analysis,we can know how the aeration rate affected gas holdup and circulating liquid velocity in various stages of cell culture.Based on the above results,cell growth was regulated by controlling the bioreactor. |
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