Absorption rate governs cell transduction in dry macroporous scaffolds

Abstract

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.

Figure 1. Fabrication of dry macroporous alginate (Drydux) scaffolds.

An alginate solution is cross-linked with a calcium solution and the resulting gel is frozen overnight followed by lyophilization for 72 h to create dry macroporous scaffolds. Activated T cells and viral particles are mixed and seeded on top of the scaffold and scaffolds are incubated at 37 °C, 5% CO₂. EDTA is used to dissolve the scaffolds and isolate the transduced T cells.

Figure 2. Impact of porosity and stiffness on Drydux transduction efficiency varying calcium and alginate concentrations.

(A) Photographs of scaffolds with corresponding SEM images and average pore sizes. (B) Quantification of retrovirus transduction efficiency against primary human T cells for each calcium-alginate combination with significance shown between differing calcium concentrations; * p < 0.0001 with all other p-values indicated on plot; concentrations used were ~5000 cells/μL and ~10000 viruses/μL; n = 3 scaffolds per group; two-way ANOVA with Tukey correction used to determine significance. See Supplemental Figure 3 for significance between differing alginate concentrations. (C) Quantification of scaffold pore size using a minimum of 10 pores per scaffold. (D) Spearman correlation between scaffold pore size and transduction efficiency. (E) Quantification of Young’s modulus of each scaffold; n = 3 scaffolds per group. (F) Spearman correlation between scaffold stiffness and transduction efficiency. Data are represented as the mean ± SEM. Statistical analysis was not completed for (C) or (E).

Figure 3. Impact of porosity and stiffness on Drydux transduction efficiency varying freezing temperature and alginate concentration.

(A) Photographs of scaffolds with corresponding SEM images and average pore sizes. (B) Quantification of retroviral transduction efficiency against primary human T cells for each alginate-temperature combination with significance shown between differing temperatures; * p < 0.0001 with all other p-values indicated on plot; concentrations used were ~5000 cells/μL and ~10000 viruses/μL; n = 3 scaffolds per group; two-way ANOVA with Tukey correction used to determine significance. See Supplemental Figure 4 for significance between differing alginate concentrations. (C) Quantification of scaffold pore size using a minimum of 10 pores per scaffold. (D) Spearman correlation between scaffold pore size and transduction efficiency. (E) Quantification of Young’s modulus of each scaffold; n = 3 scaffolds per group. (F) Spearman correlation between scaffold stiffness and transduction efficiency. Data are represented as the mean ± SEM. Statistical analysis was not completed for (C) or (E).

Figure 4. Impact of seed volume on Drydux transduction.

(A) Live-images of scaffold absorbing 20 μL of cell-virus solution. (B) Images of scaffolds 24 hours after absorbing different volumes of cell-virus solution. (C) Quantification of transduction efficiency for each seed volume. (D) Kinetics of absorption for each seed volume. (E) Spearman correlation between absorption rate and transduction efficiency. (F) Calculated volumetric flux of different seed volumes. (G) Spearman correlation between volumetric flux and transduction efficiency. Data are represented as the mean ± SEM; concentrations used were ~2000 cells/μL and ~4000 viruses/μL; n = 3 scaffolds per group; one-way ANOVA was used to determine significance.

Figure 5. Computational model of flow through scaffold pore.

(A) Schematic showing activated T cells and virus seeded together onto dry macroporous scaffold. (B) Particle positions at a statistical equilibrium state for uniform unbounded flow (top) and flow inside the scaffold pore at a volumetric flux of 30 μL/min/cm² (bottom). (C) The flow velocity distribution at the midplane of the scaffold model showing the flow acceleration and deceleration in response to the changes in the model geometry. (D) Quantification of the number of collisions per 1 μL per minute for no flow, unbounded flow, and scaffold pore flow at different volumetric fluxes.