Methodology for investigating the duration of intracellular calcium expression in response to mechanical stimulation in a single cell

Abstract

Graphical abstractDisplay Omitted Highlights? A methodology for studying a mechanotransduction using microcell chip was presented. ? Micro contact printing technique was used for cell patterning. ? A micro cell chip was fabricated using a replica molding process with PDMS. ? The intracellular calcium response of a single cell was measured and discussed. This report describes an effective methodology for investigating the duration of intracellular calcium expression in response to mechanical stimulation in a single cell using a microfluidic platform. A micro cell chip was constructed using the sequential processes of micro contact printing for surface patterning with fibronectin on the cell culture substrate, plasma bonding of the cell substrate and the fluidic channel, and cell seeding. All structures such as the micro stamp for the micro contact printing, the cell substrate, and the fluidic channel were fabricated with polydimethylsiloxane (PDMS). Micro stamps having a cell-matrix with various cell densities were fabricated to determine the proper conditions for cell seeding on the substrate. We found that a distance of 65µm between cell sites was preferable for the patterning of MG-63 cells (human osteoblast-like bone cell) using our micro stamp. While the pressure-driven shear stress in the microchannel was applied using a computer-controlled pneumatic system, the intracellular calcium response of a single cell was measured using a laser-scanning microscope. Based on the temporal response of fluorescence intensity under the steady fluid flow, the average period of multiple calcium peaks in the cell was approximately 128?4 s at 1Pa and 164?20 s at 2Pa.This work provides a useful method for fabricating a micro cell chip with a patterned surface for cell culture and for measuring the duration of the intracellular calcium response in a single cell.