title: Highly efficient adenoviral transduction of pancreatic islets using a microfluidic device.
abstract: Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell-cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College St., Toronto, Ontario M5S 3G9, Canada. jon.rocheleau@utoronto.ca.
authors.forename: Pamuditha N
authors.initials: PN
authors.lastname: Silva
authors.forename: Zaid
authors.initials: Z
authors.lastname: Atto
authors.forename: Romario
authors.initials: R
authors.lastname: Regeenes
authors.forename: Uilki
authors.initials: U
authors.lastname: Tufa
authors.forename: Yih Yang
authors.initials: YY
authors.lastname: Chen
authors.forename: Warren C W
authors.initials: WC
authors.lastname: Chan
authors.forename: Allen
authors.initials: A
authors.lastname: Volchuk
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Kilkenny
authors.forename: Jonathan V
authors.initials: JV
authors.lastname: Rocheleau
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D065096:Calcium Chelating Agents / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004229:Dithiothreitol / Q000633:toxicity; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D004867:Equipment Design; D005240:Feasibility Studies; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D056656:Lab-On-A-Chip Devices; D008297:Male; D053768:Metal Nanoparticles / Q000737:chemistry; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D000075082:Proof of Concept Study; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000633:toxicity; D046509:Tissue Culture Techniques; D014771:Virion / Q000502:physiology*
chemical_list: D065096:Calcium Chelating Agents; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D049452:Green Fluorescent Proteins; D006046:Gold; D004229:Dithiothreitol