Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, USA.
Comparative Medicine Institute, North Carolina State University, USA.
Biomater Sci. 2023 Mar 28;11(7):2372-2382. doi: 10.1039/d2bm01753a.
Developing the next generation of cellular therapies will depend on fast, versatile, and efficient cellular reprogramming. Novel biomaterials will play a central role in this process by providing scaffolding and bioactive signals that shape cell fate and function. Previously, our lab reported that dry macroporous alginate scaffolds mediate retroviral transduction of primary T cells with efficiencies that rival the gold-standard clinical spinoculation procedures, which involve centrifugation on Retronectin-coated plates. This scaffold transduction required the scaffolds to be both macroporous and dry. Transduction by dry, macroporous scaffolds, termed "Drydux transduction," provides a fast and inexpensive method for transducing cells for cellular therapy, including for the production of CAR T cells. In this study, we investigate the mechanism of action by which Drydux transduction works through exploring the impact of pore size, stiffness, viral concentration, and absorption speed on transduction efficiency. We report that Drydux scaffolds with macropores ranging from 50-230 μm and with Young's moduli ranging from 25-620 kPa all effectively transduce primary T cells, suggesting that these parameters are not central to the mechanism of action, but also demonstrating that Drydux scaffolds can be tuned without losing functionality. Increasing viral concentrations led to significantly higher transduction efficiencies, demonstrating that increased cell-virus interaction is necessary for optimal transduction. Finally, we discovered that the rate with which the cell-virus solution is absorbed into the scaffold is closely correlated to viral transduction efficiency, with faster absorption producing significantly higher transduction. A computational model of liquid flow through porous media validates this finding by showing that increased fluid flow substantially increases collisions between virus particles and cells in a porous scaffold. Taken together, we conclude that the rate of liquid flow through the scaffolds, rather than pore size or stiffness, serves as a central regulator for efficient Drydux transduction.
开发下一代细胞疗法将依赖于快速、多功能和高效的细胞重编程。新型生物材料将在这个过程中发挥核心作用，提供支架和生物活性信号，从而塑造细胞命运和功能。此前，我们实验室报道称，干燥的大孔海藻酸盐支架介导原代 T 细胞的逆转录病毒转导效率可与金标准的临床旋转接种程序相媲美，后者涉及在 Retronectin 涂层板上进行离心。这种支架转导需要支架既具有大孔又干燥。干燥大孔支架的转导，称为“Drydux 转导”，为细胞治疗提供了一种快速且廉价的转导细胞的方法，包括生产 CAR T 细胞。在这项研究中，我们通过探索孔径大小、刚度、病毒浓度和吸收速度对转导效率的影响，研究了 Drydux 转导作用的机制。我们报告称，孔径范围为 50-230μm，杨氏模量范围为 25-620kPa 的 Drydux 支架均能有效地转导原代 T 细胞，这表明这些参数不是作用机制的核心，但也表明可以在不失去功能的情况下调整 Drydux 支架。增加病毒浓度会导致转导效率显著提高，表明增加细胞-病毒相互作用对于最佳转导是必要的。最后，我们发现细胞-病毒溶液被吸收到支架中的速度与病毒转导效率密切相关，吸收速度越快，转导效率越高。多孔介质中液体流动的计算模型通过表明增加流体流动会大大增加多孔支架中病毒颗粒和细胞之间的碰撞，验证了这一发现。综上所述，我们得出结论，支架中液体的流动速度，而不是孔径或刚度，是高效 Drydux 转导的核心调节因素。
