PDMS-玻璃微流控芯片上附着态HepG2细胞低温保存条件研究

Research on Cryopreservation Conditions of Attached HepG2 Cells on PDMS-glass Microfluidic Chip

微流控芯片上细胞样本的低温保存能够显著缩短芯片上细胞实验周期、降低实验成本,有助于芯片上细胞实验的广泛应用。本文对PDMS-玻璃微流控芯片上附着态HepG2细胞的低温保存条件进行了研究,包括降温速率、低温保护剂、低温存储时间及存储温度,确定了芯片上附着态HepG2细胞的低温保存方案。结果表明:采用不同的冷却环境、冷却介质,芯片微通道内能够实现0.36~35.12℃/min的降温速率;芯片上附着态HepG2细胞低温保存存在最优降温速率0.5~1.5℃/min;-20℃或液氮表面上方40 cm处冷却环境与-80℃冷却环境组合冻存能够显著提高芯片上冻存7 d的HepG2细胞存活率;体积浓度为20%的DMSO与0.1 mol/L海藻糖联合能够显著提高芯片上冻存HepG2细胞活性。经过上述冻存条件优化后,芯片上附着态HepG2细胞冻存7 d活性超过90%。

Efficient cryopreservation of cell samples on microfluidic chips can significantly shorten the cycle of cell experiments on chips,reduce the cost of experiments,which will contribute to the wide application of cell experiments on chips.In this paper,the cryopreservation of the attached HepG2 cells on PDMS-glass microfluidic chip was examined.Furthermore,the cooling rate,cryoprotectant,storage time and temperature,and the cryopreservation protocol of the attached HepG2 cells on the chip was determined.The results indicated that a cooling rate of 0.36-35.12℃/min can be realized in the chip microchannel by using different cooling environments and cooling mediums.There is an optimal cooling rate of 0.5-1.5℃/min in cryopreservation of HepG2 cells.Furthermore,at-20℃or 40 cm above the surface of liquid nitrogen cooling environment combined with-80℃cooling environment can significantly increase the viability of 7 d frozen HepG2 cells on chip.Additionally,DMSO with a volume concentration of 20%combined with 0.1 mol/L trehalose can significantly enhance the activity of cryopreserved HepG2 cells on chip.After optimization of the aforementioned cryopreservation conditions,the attached HepG2 cells on chip were successfully stored for 7 d with a viability of more than 90%.