Characteristics of collective cell migration in human keratinocytes at subatmospheric pressure (694.5)

Abstract

A negative pressure incubator incorporating the electric cell‐substrate impedance sensing (ECIS) technique has been built to quantify monolayer keratinocytes movement in serum free media at ambient pressure (AP) and at the subatmospheric pressure of 635 mmHg (SP). Monolayer cell motion at the two pressures was continuously recorded by ECIS in the frequency range of 22.5‐64 kHz. Cells in both pressures had similar viability. Greater migration distance was found in cells at SP. We underwent fluorescent staining and protein analysis for E‐cadherin, phosphorylated ‐catenin (p‐catenin), and acetylated ‐tubulin in cells at the both pressures serially at 0, 3, 6, 9, and 12 hours after wounding the monolayer cells. A significant decrease of cell‐substratum conductance (AP: SP= ~5 cm0.5: ~3 cm0.5) and increase of membrane capacitance (AP: SP= ~4F/cm2: ~8F/cm2) were observed early in the monolayer cell movement at SP. Increased cell‐substratum distance and membrane ruffling observed in scanning electron microscopic (EM) images may be respectively responsible for these measurements. Long and stable microtubules may also contribute to the cell deformation. In cells treated with SP, a progressive drop of intercellular resistance from 1.2 to 0.8 cm2 and a significant change were observed 6 hours after wound closure. The gradual loss of cell junctions associated with decreased adherent junction proteins was identified in serial fluorescent stains and transmission EM images in cells at SP. Characteristics of collective cell migration at SP include early release of cell‐substratum adhesion, quick formation of membrane ruffling, and an ensued decrease of cell junction protein expressions.Grant Funding Source: Chang Gung Medical Research Programs