A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy.

In our brains, different neurons make appropriate connections; however, there remain few in vitro models of such circuits. We use an open microfluidic approach to build and study neuronal circuits in vitro in ways that fit easily into existing bio-medical workflows. Dumbbell-shaped circuits are built in minutes in standard Petri dishes; the aqueous phase is confined by fluid walls - interfaces between cell-growth medium and an immiscible fluorocarbon, FC40. Conditions are established that ensure post-mitotic neurons derived from human induced pluripotent stem cells (iPSCs) plated in one chamber of a dumbbell remain where deposited. After seeding cortical neurons on one side, axons grow through the connecting conduit to ramify amongst striatal neurons on the other - an arrangement mimicking unidirectional cortico-striatal connectivity. We also develop a moderate-throughput non-contact axotomy assay. Cortical axons in conduits are severed by a media jet; then, brain-derived neurotrophic factor and striatal neurons in distal chambers promote axon regeneration. As additional conduits and chambers are easily added, this opens up the possibility of mimicking complex neuronal networks, and screening drugs for their effects on connectivity.

Stable diffusion gradients in microfluidic conduits bounded by fluid walls.

Assays mimicking in vitro the concentration gradients triggering biological responses like those involved in fighting infections and blood clotting are essential for biomedical research. Microfluidic assays prove especially attractive as they allow precise control of gradient shape allied to a reduction in scale. Conventional microfluidic devices are fabricated using solid plastics that prevent direct access to responding cells. Fluid-walled microfluidics allows the manufacture of circuits on standard Petri dishes in seconds, coupled to simple operating methods; cell-culture medium sitting in a standard dish is confined to circuits by fluid walls made of an immiscible fluorocarbon. We develop and experimentally validate an analytical model of diffusion between two or more aqueous streams flowing at different rates into a fluid-walled conduit with the cross-section of a circular segment. Unlike solid walls, fluid walls morph during flows as pressures fall, with wall shape changing down the conduit. The model is validated experimentally for Fourier numbers < 0.1 using fluorescein diffusing between laminar streams. It enables a priori prediction of concentration gradients throughout a conduit, so allowing rapid circuit design as well as providing bio-scientists with an accurate way of predicting local concentrations of bioactive molecules around responsive and non-responsive cells.

A platform for modular assembly and feeding of micro-organoids on standard Petri dishes.

Organoids grow in vitro to reproduce structures and functions of corresponding organs in vivo. As diffusion delivers nutrients over only ∼200 µm, refreshing flows through organoids are required to avoid necrosis at their cores; achieving this is a central challenge in the field. Our general aim is to develop a platform for culturing micro-organoids fed by appropriate flows that is accessible to bioscientists. As organs develop from layers of several cell types, our strategy is to seed different cells in thin modules (i.e. extra-cellular matrices in stronger scaffolds) in standard Petri dishes, stack modules in the required order, and overlay an immiscible fluorocarbon (FC40) to prevent evaporation. As FC40 is denser than medium, one might expect medium to float on FC40, but interfacial forces can be stronger than buoyancy ones; then, stacks remain attached to the bottom of dishes. After manually pipetting medium into the base of stacks, refreshing upward flows occur automatically (without the need for external pumps), driven mainly by differences in hydrostatic pressure. Proof-of-concept experiments show that such flows support clonal growth of human embryonic kidney cells at expected rates, even though cells may lie hundreds of microns away from surrounding fluid walls of the two immiscible liquids.

Photo and Soft Lithography for Organ-on-Chip Applications.

Organs-on-Chip devices are generally fabricated by means of photo- and soft lithographic techniques. Photolithography is a process that involves the transfer of a pattern onto a substrate by a selective exposure to light. In particular, in this chapter two different photolithography methods will be described: liquid and dry photolithography. In liquid photolithography, a silicon wafer is spin-coated with liquid photoresist and exposed to UV light in order to be patterned. In dry photolithography, the silicon wafer is laminated with resist dry film before being patterned through UV light. In both cases, the UV light can be collimated on top of the wafer either through photomasks or by direct laser exposure. The obtained patterned wafer is then used as a mold for the soft lithographic process (i.e., replica molding) to produce polymer-based microdevices.

Microfluidics on Standard Petri Dishes for Bioscientists.

Few microfluidic devices are used in biomedical labs, despite the obvious potential; reasons given include the devices are rarely made with cell-friendly materials, and liquids are inaccessibly buried behind solid confining walls. An open microfluidic approach is reviewed in which aqueous circuits with almost any imaginable 2D shape are fabricated in minutes on standard polystyrene Petri dishes by reshaping two liquids (cell-culture media plus an immiscible and bioinert fluorocarbon, FC40). Then, the aqueous phase becomes confined by fluid FC40 walls firmly pinned to the dish by interfacial forces. Such walls can be pierced at any point with pipets and liquids added or removed through them, while flows can be driven actively using external pumps or passively by exploiting local differences in Laplace pressure. As walls are robust, permeable to O2 plus CO2 , and transparent, cells are grown in incubators and monitored microscopically as usual. It is hoped that this simple, accessible, and affordable fluid-shaping technology provides bioscientists with an easy entrée into microfluidics.

Predicting flows through microfluidic circuits with fluid walls.

The aqueous phase in traditional microfluidics is usually confined by solid walls; flows through such systems are often predicted accurately. As solid walls limit access, open systems are being developed in which the aqueous phase is partly bounded by fluid walls (interfaces with air or immiscible liquids). Such fluid walls morph during flow due to pressure gradients, so predicting flow fields remains challenging. We recently developed a version of open microfluidics suitable for live-cell biology in which the aqueous phase is confined by an interface with an immiscible and bioinert fluorocarbon (FC40). Here, we find that common medium additives (fetal bovine serum, serum replacement) induce elastic no-slip boundaries at this interface and develop a semi-analytical model to predict flow fields. We experimentally validate the model's accuracy for single conduits and fractal vascular trees and demonstrate how flow fields and shear stresses can be controlled to suit individual applications in cell biology.

Rapid Prototyping of Soft Lithography Masters for Microfluidic Devices Using Dry Film Photoresist in a Non-Cleanroom Setting.

Fabrication of microfluidic devices by soft lithography is by far the most popular approach due to simplicity and low cost. In this approach PDMS (polydimethylsiloxane) is cast on a photoresist master to generate replicas that are then sealed against glass slides using oxygen plasma. In this work, we demonstrated fabrication of soft photolithography masters using lamination of ADEX dry film as an alternative to the now classic SU-8 resist masters formed by spin coating. Advantages of using ADEX dry film include the easily-achievable uniform thickness without edge bead; simplicity of the process with significant time savings due to non-sticky nature of the film; and fewer health concerns due to less toxic developing solution and antimony-free composition. As we demonstrate, the process can be performed in a low-cost improvised fabrication room in ambient light, in place of a conventional yellow-light cleanroom environment. We believe this approach holds the promise of delivering state-of-the-art microfluidic techniques to the broad field of biomedical and pharmaceutical research.