Qasem Ramadan

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

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 (Ca2+) 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−1 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 Ca2+ 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 Ca2+ with an emission peak at 510 nm. Therefore, the concentration of free Ca2+ 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 Ca2+ 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.

Integrated Microfluidic Chip for Cell Culture and Stimulation and Magnetic Bead-Based Biomarker Detection

A microfluidic chip has been designed and fabricated for culture and stimulation of cells, and for subsequent cell biomarker monitoring using an on-chip performed magnetic bead-based immunoassay. As a proof of concept, we demonstrate monocytic cell (U937) culture, subsequent stimulation of the cells with lipopolysaccharide (LPS), and fluorescent detection of the cytokine interleukin-6 (IL6) released by the cells using magnetic beads functionalized with fluorescent antibodies (Abs) against IL6. The chip comprises two compartments which are separated by a vertical pillar-based filter. The immune cells are cultured in the first compartment and then differentiated to macrophages, while the second compartment is used for the immunoassay that is performed on magnetic beads, the surface of which is conjugated to an IL6-specific antibody. The magnetic beads are kept in place in the detection zone by soft magnetic tips that are part of an electromagnet circuit. The filter with a pore size of 2 m allows the released cytokines to diffuse from the cell culture / cell stimulation chamber downstream to the chamber containing the magnetic beads, but prevents an exchange of cells and magnetic beads. We found a significant increase of the fluorescence obtained from streptavidin-phycoerythrin (PE)-IL6 conjugates formed on the surface of the magnetic beads, after treating the macrophages with LPS.