Using open-volume microfluidics to develop gap closure assay in a confluent monolayer of cells

Abstract

Understanding the basic mechanisms of wound healing in living organisms is important to enhance healing as well as to avoid infections and scarring. Today, cell-free areas (gaps) in a confluent monolayer of cells in vitro, are commonly formed to mimic wounds in vivo. Migration and proliferation of cells are essential mechanisms for gap closure, in vitro. In many studies, measuring cell migration is of interest, thus suppressing cell proliferation is desired. Standard gap closure assays provide simple ways of monitoring cell migration in vitro. Commonly, these assays focus on formation of gaps with simple geometries, such as lines circles, squares and triangles. Other methods for gap formation have therefore been developed, however these methods have limited flexibility of forming gaps with arbitrary size and geometry. This study aims to use open-volume microfluidics to develop an in vitro gap-closure assay, allowing for time-dependent gap closure to be monitored and analysed for gaps with different initial geometry. Gaps of different initial geometries and sizes were successfully formed in a confluent monolayer of HaCaT cells. Gap closure of square, circle and crescent moon geometries (300x300 μm) was monitored under conditions where cells were allowed to proliferate and under conditions of no proliferation. Additionally, gap closure of larger square gaps (1000x1000 μm) was monitored under conditions of proliferation. This study developed an assay serving as a foundation for future studies of HaCaT gap closure in vitro. Furthermore, the study provides insights about printing fibronectin with the Biopixlar technology, important for modulating cellular migration and adhesion during gap closure. These conclusions, further add to the current research field of gap closure assays and provides principal knowledge needed for future studies aiming towards formation of gaps with more complex geometries.