“Chip-on-a-Transwell” Devices for User-Friendly Control of the Microenvironment of Cultured Cells

Abstract

We present microfabricated poly-(dimethylsiloxane) templates assembled atop Transwell membranes ("Chip-on-a-Transwell") for focal delivery of soluble reagents to cells. This modified Transwell operates based on area-selective diffusion of soluble species to the basal side of cells. The device was capable of addressing small groups of cells, with a maximum spatial resolution of 20 μm, while sustaining focal signal for up to 48 hours. Furthermore, we developed a fluorosilane-based cell micropatterning method that complements the Transwell system, creating well-defined arrays of muscle cell cultures and enabling automated microscopy and image analysis protocols. The micropatterning method is based on plasma-etching hydrophilic cell-adhesive tracks against a hydrophobic fluorosilane cell-repellent background. We applied the platform as a model of the neuromuscular junction (NMJ), in which we effectively substituted motor neurons with apertures delivering agrin stimuli to subcellular regions of micropatterned myotubes. With the Transwell system, we demonstrated that focal agrin application to subcellular sections of myotubes induced localized acetylcholine receptor microclustering, mimicking the in vivo NMJ. We also demonstrated spatially selective transfection of a monolayer of cells using Lipofectamine carrying the plasmid DNA for pMax GFP, which is ubiquitously expressed in transfected cells. In summary, we present a user-friendly cell culture tool that provides spatiotemporal control of the fluidic and substrate microenvironments with potential applications to developmental biology and tissue engineering.

In Vitro Cell Migration through Three-Dimensional Interfaces of Varying Depths, Widths, and Curvatures on Micropatterned Polymer Surfaces

“…Creating cell-free regions in confluent monolayers in conventional wound healing assays is not consistent with high-throughput testing. Micropatterning allows the creation of high-throughput platforms widely used to establish in vitro models. − Traditional wound healing assays could thus be improved by the use of micropatterning methods by allowing groups of tests to be conducted in parallel.…”