Acoustic Poration and Dynamic Healing of Mammalian Cell Membranes during Inkjet Printing

We have investigated the effect of piezoelectric actuating voltage on cell behavior after drop on demand inkjet printing using mouse 3T3 cells as a model cell line. Cell viability after printing was assessed using a live/dead assay, Alamar Blue as an assay for cell proliferation, and propidium iodide (PI) and Texas Red labeled dextran molecular probes to assess cell membrane integrity. No significant difference was found for the cell death rate compared between an unprinted control population and after printing at 80, 90, and 100 V, respectively. However, cell proliferation was lower than that of the control population at all time points postprinting. Cell membrane integrity was quantified using PI and dextran probes of mean molecular weight of 3, 10, 40, and 70 kDa. Total membrane damage (assessed by PI) increased with increasing piezoelectric actuator driving voltage, and this was always greater than the unprinted control cells. The uptake of the labeled dextran only occurs after inkjet printing and was never observed with the control cells. The largest dextran molecular probe of 70 kDa was only taken up by cells after printing using the lower printing voltages of 80 and 90 V and was absent after printing at 100 V. At the two lower printing voltages, the membrane damage is recovered, and no dextran molecule penetrated the cells 2 h after printing. However, printing at 100 V leads to an increased uptake of 3 and 10 kDa dextran molecules, the retention of membrane porosity, and continued uptake of these 3 and 10 kDa dextran for greater than 2 h postprinting. We hypothesize that the change in membrane porosity with increasing actuation voltage can be explained by distinct nucleation and growth stages for pore formation in response to printing stress.