Professor Georgia Institute of Technology, United States
Introduction:: Personalized cell therapies are a breakthrough treatment to combat the hardest-to-treat diseases, such as cancer, autoimmune disorders, diabetes, and sickle cell disease1. While cell therapies are a commercial reality, access remains limited due to the highly expensive and variable genetic payload delivery process, which is typically performed through viral transduction, chemical membrane permeabilization, or electroporation2. Microfluidic mechanoporation approaches may be a solution since they can be less expensive than viral transduction and avoid the toxicity associated with reactive ion species generated in electroporation or lipofection.
Prior mechanoporation studies found a novel cell response to rapid compressions at timescales less than a millisecond that results in a temporary change of volume3, which can be used to deliver cargo such as nanoparticles and gene modifying molecules into cells to solve some of the biggest challenges in cell engineering4,5 (Fig 1). The volume exchange cell transfection (VECT) approach has been used for a number of applications, but there is no fundamental understanding of how cell mechanics affect the volume exchange and cargo uptake process.
In this research, a microfluidic approach has been developed to characterize cell mechanical responses to rapid compressions and shear forces. As cells flow through the ridge-constrictions in the microfluidic channel, cell deformation and relaxation responses are captured using a high-speed video camera and analyzed at sub-millisecond timescales (Fig. 2). The effect of force intensity and duration on cell mechanical response are evaluated by characterizing the behavior of cells of different diameters subjected to different cell-solution flowrates and constriction sizes.
Materials and Methods:: Device Fabrication: A SU-8 silicon wafer mold was produced using standard photolithography. Polydimethylsiloxane (PDMS) and crosslinking agent were mixed in a 10:1 ratio and poured onto the SU-8 mold to form the microfluidic channel features. The PDMS was then degassed and cured at 80°C for an hour, and inlets and outlets were created using biopsy punches. The PDMS was bonded to a clean glass slide using a plasma bonder (PDC-32G Harrick) and then subjected to 80°C oven for 15-minutes.
Cell Culture: Jurkat cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine. The cells were incubated at 37 °C with 5% CO2.
Microfluidic Experimental Setup: Cells suspended in culture media at a concentration of ~2×106 cells/mL were infused into the microfluidic device at controlled flowrates using a pressure controller (OB1 MK3+, Elveflow). The cell mechanical response was captured using a high-speed video camera (PhantomV9, Vision Research Inc.).
Cell Response Analysis: High-speed videos were imported into ImageJ and separated into image frames. Cell velocity is calculated by measuring the distance travelled through the ridge-constriction and dividing the distance by the travel time given by video framerate. In each frame, the cell area and perimeter were measured to calculate cell circularity, 4π(area/perimeter2), to generate a circularity vs. time plot. The slopes of the circularity vs. time plot are further analyzed to extract parameters such as deformation and relaxation rate (Δcircularity/time).
Results, Conclusions, and Discussions:: To understand the effect of force intensity on cell mechanical response, cells of different diameters travelling through a 9 µm constriction at 20 µL/min flowrate were analyzed. As the cell size increases, the cells experience a higher strain, with greater change in cell circularity (Fig. 3A). The cell velocity under the ridge is similar across diameters (Fig. 3B). As cell size increases, cells experience greater strain (and higher intensity of force), causing deformation and longer recovery time (Fig. 3C).
To understand the effect of force duration on cell mechanical response, ~12 µm diameter cells were flowed through a 9 µm constriction at flowrates of 10, 15 and 20 µL/min. At various flowrates, cells experienced similar strain and change in cell circularity (Fig. 4A). As expected, the cell velocity under the ridge increased with flowrate (Fig 4B). Constricted cells with higher velocity experienced a faster deformation and recovery response (Fig. 4C).
To study the combined effect of force intensity and duration on cell mechanical response, ~12 µm-sized cells were infused through microfluidic devices with 5 and 9 µm constrictions at 20 µL/min flowrate. Cells that flowed through the narrower constriction experienced greater strain, corresponding to the greater change in circularity (Fig. 5A). The decrease in constriction size resulted in higher cell velocity through the constriction (Fig. 5B). The combined effects of higher force intensity (greater strain) and shorter force duration (higher cell velocity) resulted in a minimal difference in cell recovery response between 5 and 9 µm constrictions (Fig 5C).
In conclusion, higher strain will decrease the rate of cell deformation and recovery while higher cell velocity will do the opposite. In future studies, additional variables such as cell stiffness, cell viscosity and device dimensions will be explored to further advance understanding of cell mechanical response at high deformation. With knowledge of cell mechanical response at fast timescales, mechanoporation technology can be optimized to improve payload delivery efficiency and reduce cost in cell therapy manufacturing.
Acknowledgements (Optional): : Funding Source: National Institute of Health
References (Optional): : [1] Aijaz, A. et al. (2018) Nat Biomed Eng. 2(6):362-376. [2] Duckert B. et al. (2021) J Control Release. 330:963-975. [3] Liu, A. (2020) Small. 16(2):e1903857. [4] Kiru, L. (2022) Proc Natl Acad Sci U S A. 119(6):e2102363119. [5] Sciolino, N. (2022) Commun Biol. 5(1):451.