A microfluidic cell culture device with integrated microelectrodes for barrier studies.
We present an eight cell culture microfluidic device fabricated using thiol ene 'click' chemistry with embedded microelectrodes for evaluating barrier properties of human intestinal epithelial cells. The capability of the microelectrodes for trans epithelial electrical resistance (TEER) measurements was demonstrated by using confluent human colorectal epithelial cells (Caco 2) and rat fibroblast (CT 26) cells cultured in the microfluidic device. INTRODUCTION Trans epithelial electrical resistance (TEER) is one of the widely used and conceivably the most straightforward technique for understanding the integrity of an epithelial or endothelial cell layer (1, 2) This paper reports on a straight forward method of embedding microelectrodes in a multi layer thiolene microfluidic chip for measuring TEER across cell layers. The reported microelectrodes embedded in the microfluidic device were fabricated using two different metals and are in direct contact with the reagents within the microchannels. The capability of the microelectrodes for TEER measurements was demonstrated by using confluent human colorectal epithelial cells (Caco 2) and rat fibroblast (CT 26) cells cultured in the microfluidic device. Capability of the microelectrodes in sensing dynamic changes of the barrier integrity was demonstrated by challenging the Caco 2 monolayers with a membrane enhancer that temporary disrupted the cell barrier property. Immunofluorescence staining towards the tight junctional protein, ZO 1, and nucleus were also conducted to observe the morphology of the cells. Such a microfluidic cell culture platform, open new opportunities for real time monitoring of the cell barrier properties. METHODS AND MATERIALS EMBEDDING THE MICROELECTRODES ONTO THE MICROCHIP The microfluidic device was fabricated using thiol ene 'click' chemistry. The design and fabrication of the microfluidic chip have been reported earlier (3) Once the top and bottom fluidic layers were cured by UV exposure and demolded from the PDMS molds, microelectrode grooves were micromilled to the bottom layer (Fig. 1b) Briefly, the top and bottom fluidic layers were fabricated by mixing pentaerythritol tetrakis (3mercaptopropionate) (tetra thiols moieties) with tri allyl tri azine (tri allyl moieties) in stoichiometric ratios. The mixture was poured onto the PDMS molds, exposed to UV for 40s and de molded. These layers were used as the upper and lower fluidic layers of the microfluidic chip. Grooves for the microelectrodes (present only in the lower fluidic layer of the microchip) were micro milled into the cured thiol ene piece (Fig. 2a) As for the porous membrane, the surface of the membrane was modified by coating a mixture of tri allyl tri azine (tri allyl moieties) and trimethylopropane tris (2 mercaptopropionate) (tri thiol moieties) Before bonding the fluidic layers and modified Teflon membrane, holes for the inlets, outlets and electrode ports were drilled into the partially cured thiol ene layers (Fig. 1b) The different pieces of cured thiol ene layers were aligned and placed on each other. A slight pressure was applied onto the pieces to ensure good contact between the pieces. The entire microchip was further exposed to UV radiation for another 1min on each side of the microchip. The bottom electrodes were fabricated using an indium alloy, InBiSn, which has a melting temperature of 62 degC. Therefore, to fabricate the bottom electrodes, the sealed microchip was first placed onto a hotplate set at 80 degC and pieces of the InBiSn metal, with length of 5 mm, were inserted into the electrode ports (connecting to the lower fluidic layer) on the thiol ene microchip. Heat from the hotplate and the microchip resulted in InBiSn metal melting. Slight pressure was applied manually to push the melted metal into the electrode grooves. Once the electrodes were formed, electric wires of diameter 0.4 mm were inserted to the liquid metal. These wires would act as the connecting wires to the multimeter. Once the microchip was cooled down in room temperature, the InBiSn electrode solidified, retaining the shape (Fig. 1e) The size of electrode for measuring TEER, was about about 0.6 0.8 mm in diameter and thickness of 0.3 mm (Fig. 1e) The top electrodes were fabricated by fitting two pieces of platinum (Pt) wires (diameter of 0.5 mm, length of 5 mm) into the electrode ports drilled through the top fluidic layer. Two connecting Cu wires were soldered to the Pt wires (Fig. 1c) To fix the electrodes in position, UV epoxy (NOA81; Norland, USA) was applied at the junctions between connecting wires and thiol ene chip. The entire chip was exposed to UV light for 30s. Figure 1. Design and development overview of microelectrodes embedded in the multi layer and multi chamber thiol ene microchip for Caco 2 cell culture: (a) Schematic drawing of the 3 layers present in the thiolene microchip. (b) Schematic drawing of the process to embed the microelectrodes on the microchip. (c) Top view of the completed microchip with embedded electrodes and connecting wires. (d) Underside of thiol ene microfluidic chip with the InBiSn electrode embedded in the microchamber. (e) Expanded view of the InBiSn electrode. (Scale bar 2mm) RESULTS AND DISCUSSION To investigate the working feasibility of the microelectrodes for TEER measurements, two different cell lines were experimented i) Caco 2 cells (human colorectal epithelial cells) which are known to form tight junctions and ii) rat fibroblast (CT 26) cells, which are incapable of forming tight junctions. The TEER measured for Caco 2 cells was 861 Ocm and for CT 26, the TEER measured was 56 Ocm (Fig 2) Further demonstrations of the capability of the microelectrodes in sensing dynamic changes of the barrier integrity of the cell monolayers were presented with the introduction of two different concentrations of membrane enhancer (tetradecyl b D Maltoside (TDM) to the cell monolayers. Challenging the cells cultured in the microfluidic device with 100 mM and 400 mM of TDM, resulted in percentage decrease in TEER values of 60.4 and 20.9 respectively, of initial values (Fig. 3A) To further analyse the morphology of the barrier integrity of the Caco 2 cells, immunostaining of the tight junctional protein, ZO 1, and nucleus of the Caco 2 cells were conducted (Fig 3B D) (d) (e) (c) (a) (b)