Microfluidic chip for monitoring Ca²⁺transport through a confluent layer of intestinal cells

Abstract

The absorption of dietary calcium through the intestinal barrier is essential for maintaining health in general and especially of the bone system. We propose a microfluidic model that studies free calcium (Ca 2+ ) transport through a confluent monolayer of Caco-2 cells. The latter were cultured on a porous membrane that was positioned in between a top and bottom microfluidic chamber. Fresh cell culture medium was continuously supplied into the device at a flow rate of 5 nL s À1 and the culture progress of the cell monolayer was continuously monitored using integrated Transepithelial Electrical Resistance (TEER) electrodes. The electrical measurements showed that the Caco-2 monolayer formed a dense and tight barrier in 5 days. The transported free Ca 2+ from the top microfluidic chamber to the basolateral side of the cell monolayer was measured using the calcium-sensitive dye fura-2. This is a ratiometric dye which exhibits an excitation spectrum shift from 340 nm to 380 nm, when it binds to Ca 2+ with an emission peak at 510 nm. Therefore, the concentration of free Ca 2+ is proportional to the ratio of fluorescence emissions obtained by exciting at 340 nm and 380 nm. The barrier function of the cell monolayer was evaluated by a measured rate of Ca 2+ transport through the monolayer that was 5 times lower than that through the bare porous membrane. The continuous perfusion of cell nutrients and the resultant mechanical shear on the cell surface due to the fluid flow are two key factors that would narrow the gap between the in vivo and in vitro conditions. These conditions significantly enhance the Caco-2 cell culture model for studying nutrients bioavailability.

Intestine-on-a-Chip Microfluidic Model for Efficient in Vitro Screening of Oral Chemotherapeutic Uptake

“…After functionalization of the apical side of the PC membrane with Matrigel, Caco-2 cells were injected inside the apical section of the IOAC device and left to attach for 4 h. At this time point, the apical chamber was then perfused with DMEM cell culture media supplemented with FBS at a flow rate calculated to induce an averaged fluid shear stress of 0.02 dyn cm –2 . In agreement with previous studies, − dense Caco-2 cell monolayers were obtained typically after 5 days of culture in these dynamic culture conditions. The cell monolayers on the PC membrane were prepared for imaging with confocal microscopy which confirmed that the IOAC model allows for the formation of cell monolayers that possess undulating 3D morphology as shown by F-actin mapping of the apical surface of cell monolayer.…”

Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures

“…(2) Model C has one pair of current carrying and pick-up electrodes at the surfaces of both chambers but in opposite sides. Despite the fact that the electrodes are planar and placed on the chamber surface, results are similar to those expected for models where wire electrodes are located at specific positions of the chamber [6,41].…”

Impedance Spectroscopy as a Tool for Monitoring Performance in 3D Models of Epithelial Tissues

“…When integrated into microfluidic organ-on-a-chip devices, impedance measuring electrodes allow long-term monitoring of the cultured cells in a controlled environment (Douville et al, 2010;Booth and Kim, 2012;Griep et al, 2013;Huang et al, 2014;Walter et al, 2016). The size constraint, which is common with respect to microfluidic platforms, dictates the placement of electrodes in close proximity to the cells.…”