Microfluidics based techniques for electrophysiological studies of cells
Doctoral thesis, 2014
This thesis work investigates the application of microfluidics to perform electrophysiological studies on cells, including investigations of the effect of cholesterol on the dynamic ion permeability of TRPV1 ion channels, and the application of a microfluidic device, the multifunctional pipette, in electrophysiological studies on brain slices. In the first part of this thesis, Chinese hamster ovary (CHO) cells overexpressing the TRPV1 ion channel were used in a dynamic ion permeability study, where the activation properties of the TRPV1 ion channel were investigated using the patch clamp technique after depletion of membrane cholesterol. The dynaflow system, an open-volume multichannel microfluidic system, and the multifunctional pipette, a freestanding microfluidic device utilizing hydrodynamically confined flow for spatially confined solution exchange, were used to deliver chemical stimuli exclusively to the patched cell. The result showed that the depletion of membrane cholesterol impaired the dynamic permeability of large cations in TRPV1 in low calcium solutions. The second project focused on the application of the multifunctional pipette in neuropharmacological studies of the brain slices. We developed an experimental setup, performed feasibility studies, characterized the device performance and compared it with common superfusion techniques, using extra- and intracellular electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats. The multifunctional pipette was used in these experiments for highly localized delivery of the competitive AMPA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) to selected locations on the slices. By applying multifunctional pipette, we achieved a multifold gain in solution exchange time and more efficient drug delivery compared to whole slice perfusion. The amount of drugs required in the microfluidics-supported experiments was by several orders of magnitude smaller. The multifunctional pipette enabled selective perfusion of a single dendritic layer in the CA1 region of hippocampus with CNQX, without affecting other layers in this region.