High-throughput Screening of Erratic Cell Volume Regulation Using a Hydrogel-based Single-cell Microwell Array

Here, we report that a single-cell microwell array based on photocrosslinked hydrogel can be used to screen cells exhibiting a defective regulatory volume decrease (RVD) in high-throughput. The RVD is a regulatory function of cells that maintains cell volume homeostasis in a hypotonic medium. Single Madin-Darby canine kidney (MDCK) cells grown in the microwells were loaded with a volume-sensitive fluorescence dye. Changes in the volume of discrete single cells were traced for 20 min in a hypotonic solution using a wide-field fluorescence microscopy. The volume changes of more than 100 single cells were analyzed simultaneously using time-lapse fluorescence micrographs. Cells showing erratic RVD could be easily screened from the image analysis. Nearly 40% of the MDCK single cells exhibited weak, or no, RVD. Since other previously reported methods could not detect as many changes in the volume of discrete singles cells as the method used in this report, we anticipate that our reported method will provide an efficient way of elucidating the RVD mechanisms of cells that have not yet been completely understood.