Department of Bioengineering, University of Louisville, Louisville, KY, USA.
Department of Biology, University of Louisville, Louisville, KY, USA.
Ultrasound Med Biol. 2024 Nov;50(11):1646-1660. doi: 10.1016/j.ultrasmedbio.2024.06.010. Epub 2024 Aug 5.
Cell-based therapies have shown significant promise for treating many diseases, including cancer. Current cell therapy manufacturing processes primarily utilize viral transduction to insert genomic material into cells, which has limitations, including variable transduction efficiency and extended processing times. Non-viral transfection techniques are also limited by high variability or reduced molecular delivery efficiency. Novel 3D-printed acoustofluidic devices are in development to address these challenges by delivering biomolecules into cells within seconds via sonoporation.
In this study, we assessed biological parameters that influence the ultrasound-mediated delivery of fluorescent molecules (i.e., calcein and 150 kDa FITC-Dextran) to human T cells using flow cytometry and confocal imaging.
Low cell plating densities (100,000 cells/mL) enhanced molecular delivery compared to higher cell plating densities (p < 0.001), even though cells were resuspended at equal concentrations for acoustofluidic processing. Additionally, cells in the S phase of the cell cycle had enhanced intracellular delivery compared to cells in the G2/M phase (p < 0.001) and G0/G1 phase (p < 0.01), while also maintaining higher viability compared to G0/G1 phase (p < 0.001). Furthermore, the calcium chelator (EGTA) decreased overall molecular delivery levels. Confocal imaging indicated that the actin cytoskeleton had important implications on plasma membrane recovery dynamics after sonoporation. In addition, confocal imaging indicates that acoustofluidic treatment can permeabilize the nuclear membrane, which could enable rapid intranuclear delivery of nucleic acids.
The results of this study demonstrate that a 3D-printed acoustofluidic device can enhance molecular delivery to human T cells, which may enable improved techniques for non-viral processing of cell therapies.
细胞疗法在治疗多种疾病方面显示出巨大的潜力，包括癌症。目前的细胞治疗制造工艺主要利用病毒转导将基因组物质插入细胞中，但存在一些局限性，包括转导效率的可变性和延长的处理时间。非病毒转染技术也受到高变异性或降低的分子传递效率的限制。新型 3D 打印声流控装置正在开发中，以通过声孔作用在几秒钟内将生物分子递送到细胞内，从而解决这些挑战。
在这项研究中，我们使用流式细胞术和共聚焦成像评估了影响人 T 细胞中荧光分子（即钙黄绿素和 150 kDa FITC-葡聚糖）超声介导传递的生物学参数。
与较高的细胞接种密度（p < 0.001）相比，较低的细胞接种密度（100,000 个细胞/mL）增强了分子传递，尽管细胞在声流处理时以相等的浓度重新悬浮。此外，与 G2/M 期（p < 0.001）和 G0/G1 期（p < 0.01）相比，细胞周期 S 期的细胞具有增强的细胞内传递，同时与 G0/G1 期相比也保持了更高的活力（p < 0.001）。此外，钙螯合剂（EGTA）降低了整体分子传递水平。共聚焦成像表明，肌动蛋白细胞骨架对声孔作用后质膜恢复动力学具有重要影响。此外，共聚焦成像表明声流处理可以使核膜穿孔，这可能使非病毒细胞治疗的核内快速传递成为可能。
这项研究的结果表明，3D 打印声流控装置可以增强人 T 细胞的分子传递，这可能为非病毒细胞治疗的处理技术提供改进。
