Self-Regulated Selective Surface Coating Enables Confinement of Adherent Cells in Closed Microfluidic Arrays

Phenotypical screening assays involving adherent cell culture are essential for research in cell biology and toxicology as well as drug discovery. Such assays often involve hundreds of culture chambers and monitor cell responses for hours. Microfluidic arrays have been widely used for cell assays; however, creating dense arrays of adherent cells is challenging because adherent cells may migrate over time. To confine cells, existing techniques rely on actuators such as valves or surface micropatterning; yet, both present important drawbacks. The actuator-based systems often require complex fabrication and equipment for operation. Noncontact surface patterning techniques for closed microfluidic channels typically require expensive reagents (e.g., photosensitive ones) and precise alignment, whereas contact techniques to pattern open surfaces make sealing the device after patterning difficult and are susceptible to contamination. Here, we present an inexpensive, selective surface-coating method for micropatterning proteins to confine adherent cells in closed microfluidic devices. Using capillary valves, pressure-driven flow, and sequential reagent delivery, we can spatially and temporally expose the channels to cell adhesion molecules and blocking agents separately. We show a large window of operating pressures, up to 4 psi, ensuring robust selective patterning. In the patterned devices, we also demonstrate successful cell culture for over 20 h, with cells remaining spatially confined. This technique offers a cost-effective and scalable solution for creating microarrays of adherent cells without complex equipment or expensive reagents. Its simplicity and ability to pattern in closed microchannels may also benefit applications beyond cell-based assays such as biomolecule detection and other surface-based processes.