For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO–K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).

中文翻译：

---

微流控转染系统和温度强烈影响瞬时转染的效率

在瞬时转染 (TTF) 过程中，通常使用复合物将 DNA 转运到细胞中。目的基因的多聚复合物摄取和随后的瞬时表达对于成功转染非常重要。在这项研究中，我们研究了一种 3D 打印微流体系统，旨在促进直接 TTF 悬浮 CHO-K1 细胞。结果表明，该系统比手动方法取得了明显更好的结果。此外，根据膜相变探讨了转染后孵育时间 (t) 和温度 (T) 对聚合复合物摄取的影响。注意获得尽可能高的转染效率（TFE）、活力（V）和活细胞浓度（VCC）。我们的结果表明，以 VCC 和 TFE 的乘积测量的转染输出在T = 22 °C下t = 1 小时时是最佳的。此外，转染后在T = 22 °C 下孵育，并在T = 40 °C 下短时间增加T，观察到进一步增加了输出。最后，我们发现在T = 19 °C 左右，TFE 急剧增加。这是 CHO-K1 细胞的膜相变T，因此这些结果表明膜顺序与渗透性（进而与 TFE）之间存在相关性。