Indee Labs, Berkeley, CA, USA.
Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia.
Sci Rep. 2021 Jun 3;11(1):11818. doi: 10.1038/s41598-021-91307-y.
Microfluidic vortex shedding (µVS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of µVS intracellular delivery remains undefined and µVS-Cas9 genome editing requires further studies. Herein, we evaluated a series of µVS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a µVS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (eµVS). µVS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eµVS with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5 × 10 cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10 to 10 in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of µVS and eµVS for genome editing human primary T cells with Cas9 RNPs.
微流控涡旋脱落（µVS）可以快速将 mRNA 递送至 T 细胞，具有高产率和对细胞状态的最小干扰。µVS 细胞内递送的机制基础仍未定义，µVS-Cas9 基因组编辑需要进一步研究。在此，我们评估了一系列包含分流板的 µVS 装置以减弱涡旋脱落，并了解计算力和频率对效率和活力的贡献。然后，我们选择了一种 µVS 设计，通过递送 Cas9 核糖核蛋白（RNP）并用和不用短暂暴露于电场（eµVS）来敲除原代人 T 细胞中的内源性 T 细胞受体表达。µVS 单独处理与电穿孔相比，产生了等效产量的基因组编辑 T 细胞，且细胞质量得到改善。用 eµVS 可将编辑效率提高 1.8 倍，而对细胞活力的影响可以忽略不计。在此，我们证明了在不到 5 秒的时间内，在 500µl 的 cGMP OptiMEM 中有效处理 5×10 个细胞，单个装置的处理能力在 1 到 30 秒之间可以处理 10 到 10 个细胞。总之，这些结果证明了 µVS 和 eµVS 在使用 Cas9 RNP 对人类原代 T 细胞进行基因组编辑方面的快速和强大的应用。
