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.

Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation

“…In contrast, the truncated receptor isoform (⌬C Ven ) showed similar anisotropy to the tandemdimer control. A similar trend was confirmed in the beta-cells of human islets, suggesting that the receptor aggregation state is intrinsic to the receptor and occurs across species (17) (Fig. S2).…”

“…Islets were loaded into a microfluidic device designed for HEAT-on-a-chip (i.e. highly efficient adenoviral transduction) (17). Briefly, transduction medium (RPMI 1640 supplemented with 11 mM glucose, 10 mM HEPES, 10% FBS, and 5 units/ml P/S, 2 mM EDTA) supplemented with 2.61 ϫ 10 7 infectious units/ml of adenovirus was flowed through isletloaded devices at 200 l/h for 75 min.…”

Clarifying intact 3D tissues on a microfluidic chip for high-throughput structural analysis

“…1A. This "spiraling" microfluidic chip was designed to ensure that each spheroid was subjected to equivalent shear stress, flow rates, and concentrations of fluids (24). Once loaded, a monomer solution of 2-8% (wt/vol) acrylamide, 4% (wt/vol) formaldehyde, and 2.5% (wt/vol) of the thermal radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (Va-044) is infused at 800 μL/h through the spheroids in the microfluidic chip for 20 min (Fig.…”

A Microfluidic Device to Enhance Viral Transduction Efficiency During Manufacture of Engineered Cellular Therapies

“…By contrast, the use of microfluidics has the potential to effectively drive the colocalization of virus and target cells without the risk of cell damage or the need for extensive product washing 23–26 . Chuck and Palsson demonstrated high rates of viral transduction (total percentage of cells transduced) achieved in relatively short coincubation times when virus-laden media was flowed past target cells trapped against a cell-impermeable membrane 23 .…”