title: Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation.
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.
authors.affiliation: Ultrafast Photonics and Nano-Optics Laboratory, Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Vikram
authors.initials: V
authors.lastname: Kohli
authors.forename: Jason P
authors.initials: JP
authors.lastname: Acker
authors.forename: Abdulhakem Y
authors.initials: AY
authors.lastname: Elezzabi
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004285:Dogs; D007834:Lasers; D009995:Osmosis / Q000187:drug effects / Q000528:radiation effects; D013395:Sucrose / Q000494:pharmacology*; D013549:Sweetening Agents / Q000494:pharmacology*
chemical_list: D003451:Cryoprotective Agents; D013549:Sweetening Agents; D013395:Sucrose