Establishment of cryopreservation system of Taxus wallichiana var.mairei suspension cells
[Objective]Paclitaxel has a broad-spectrum anticancer effect,but its content in Taxus is extremely low,which was unable to meet the growing clinical needs.At present,Taxus suspension cell culture technology is considered to be one of the effective ways to solve the scarcity of paclitaxel.However,cells are prone to genetic variation after continuous passage,resulting in poor cell growth and decreased ability to synthesize paclitaxel.To solve this problem,this study proposes an optimized method for cryopreservation of Taxus wallichiana var.mairei suspension cells.[Method]The effects of preculture temperature,preculture time,pretreatment agent,dehydration time,cryoprotectant and thawing temperature on cell survival were investigated by single factor experiment with Taxus wallichiana var.mairei suspension cells as materials.On this basis,the response surface optimization of cryopreservation conditions was conducted with cell survival rate as the optimization objective.[Result]According to the single factor test,three significant influencing factors were selected,which were sucrose concentration,dehydration time and thawing temperature.By response surface optimization,the optimal preservation conditions of cells were obtained.Taxus wallichiana var.mairei suspension cells were first precultured in B5 medium for 12 days at a preculture temperature of 5℃,then pretreated with 6.6％sucrose for 30 min at room temperature,removed the pretreatment agent,added 20％ethylene glycol + 35％glycerol + 10％dimethyl sulfoxide + 10％sorbitol as cryoprotectant,dehydrated at 0℃ for 31 min,iced at 0℃ for 30 min,placed at-20℃ for 2 h,and finally placed in a refrigerator at-80℃ for 14 days.The frozen suspension cells were rapidly thawed in a water bath at 41℃ for 60 s,and then washed with B5 medium containing 1.2 mol/L sucrose to remove the protective agent.Finally,the suspension cells were resuscitated on B5 medium.The survival rate of Taxus wallichiana var.mairei suspension cells after the above cryopreservation treatment could reach 68.19％.[Conclusion]In this study,the cryopreservation system of Taxus wallichiana var.mairei suspension cells was established and optimized,and a certain cryopreservation effect was achieved,which provided a technical reference for the long-term maintenance of Taxus wallichiana var.mairei and other medicinal plant cells.
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