There is an unmet demand for microfluidics in biomedicine. We describe contactless fabrication of microfluidic circuits on standard Petri dishes using just a dispensing needle, syringe pump, 3-way traverse, cell-culture media, and an immiscible fluorocarbon (FC40). A submerged micro-jet of FC40 is projected through FC40 and media on to the bottom of a dish, where it washes media away to leave liquid fluorocarbon walls pinned to the substrate by interfacial forces. Such fluid walls can be built into almost any imaginable 2D circuit in minutes, which we exploit to clone cells using limiting dilution in a way that beats the Poisson limit, sub-culture adherent cells, and feed arrays of cells continuously for a week. This general method should have wide application in biomedicine.
One sentence summaryIn the everyday world, we cannot build complex structures out of liquids as they collapse into puddles; in the microworld we can. Figure 1A illustrates the approach. The bottom of a standard tissue-culture dish is covered with a film of cell-growth media, and an FC40 overlay added to prevent evaporation. A dispensing needle filled with FC40, connected to a syringe pump, and held by a 3-way traverse is now lowered below the surface of the fluorocarbon; starting the pump jets FC40 on to the dish to push media aside. As FC40 has a low equilibrium contact angle (CA) on polystyrene (< 10°), it wets it better than media (equilibrium CA ~50°; Walsh et al., 2017), so it adheres to the bottom. Moving the micro-jet sideways then creates a line of FC40 on the dish, and drawing more lines creates a grid with 256 chambers in <2 min ( Fig. 1B; Movie 1). Each chamber is isolated from others by liquid walls of FC40 pinned to polystyrene. Interfacial forces dictate chamber geometry -a spherical cap sitting on a square footprint (height ~75 µm; volume ~100 nl). Up to ~900 nl more media can be pipetted into chambers as fluid walls morph above unchanging footprints. Chambers are then used like wells in microplates: liquids are added/removed to/from them by pipetting through FC40 instead of air. The maximum and minimum volumes that can be held in chambers without altering footprints are determined by advancing and receding contact angles; addition of too much media inevitably merges adjacent chambers. Even so, chambers accept a manyfold wider range of volume than equally-spaced wells in a microplate, whilst containing ~1,000 th the volume (Soitu et al., 2018). Consequently, if chambers contain cells, the volume ratio of intra-to extra-cellular fluid more closely resembles that in vivo. Importantly, this method is contactless: the nozzle touches neither dish nor media. Moreover, one pipet tip can add/reagents to/from many chambers without detectable cross-contamination (shown -for example -by seeding bacteria in every other chamber, adding media to all through one tip, and finding that bacteria grow only in inoculated chambers as others remain sterile; Soitu et al., 2018). In other words, a tip is washed effectively by passage through FC40 bet...