Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation |
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Authors: | Kohli Vikram Acker Jason P Elezzabi Abdulhakem Y |
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Institution: | Ultrafast Photonics and Nano-Optics Laboratory, Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada. |
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Abstract: | Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein we report on the uniqueness of using high-intensity femtosecond laser pulses for reversibly permeabilizing mammalian cells for biopreservation applications. When mammalian cells were suspended in a impermeable hyperosmotic cryoprotectant sucrose solution, femtosecond laser pulses were used to transiently permeabilize cells for cytoplasmic solute uptake. The kinetics of cells exposed to 0.2, 0.3, 0.4, and 0.5 M sucrose, following permeabilization, were measured using video microscopy, and post-permeabilization survival was determined by a dual fluorescence membrane integrity assay. Using appropriate laser parameters, we observed the highest cell survival for 0.2 M sucrose solution (>90%), with a progressive decline in cell survival towards higher concentrations. Using diffusion equations describing the transport of solutes, the intracellular osmolarity at the inner surface of the membrane (x = 10 nm) and to a diffusive length of x = 10 microm was estimated, and a high loading efficiency (>98% for x = 10 nm and >70% for x = 10 microm) was calculated for cells suspended in 0.2 M sucrose. This is the first report of using femtosecond laser pulses for permeabilizing cells in the presence of cryoprotectants for biopreservation applications. |
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Keywords: | femotsecond ultrashort laser pulses permeabilization cryoprotectant sugars biopreservation cryobiology cryopreservation |
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