Electroporation outcomes in human Jurkat cells are affected by exposure to simulated microgravity.

Electroporation (EP) is commonly used to permeabilize cell membranes to deliver nucleic acids and other biomolecules. EP-based methodologies have the potential to support in-space biomanufacturing; however, it is currently unclear whether cellular adaptations to microgravity influence EP outcomes. To investigate this, human T-lymphocyte (Jurkat) cells were exposed to simulated microgravity (SµG) in a rotating wall vessel (RWV) bioreactor prior to EP to deliver exogenous fluorescent molecules or plasmid DNA. We observed that two hours of SµG exposure led to a 20% decrease in fluorophore uptake and a 31% decrease in transfection efficiency compared to standard gravity controls for several voltages. To explore underlying mechanisms, the effects of SµG on actin organization were assessed using a phalloidin stain, which revealed increased cell area and cortical actin thickness following SµG. Actin targeting drugs jasplakinolide and latrunculin B were used to promote or destabilize F-actin, respectively, and modulated cell area and cortical actin thickness. Promoting F-actin also led to decreased transfection efficiency, mimicking the effect of SµG, while destabilizing F-actin during SµG showed a small increase in transfection efficiency, slightly reversing the impact of SµG. Our results demonstrate that adaptations to microgravity exposure can influence transfection efficiency via cytoskeletal reorganization.