Novel in situ normal streaming potential device for characterizing electrostatic properties of confluent cells

Abstract

The characteristics of transport across confluent cell monolayers may often be attributed to its electrostatic properties. While tangential streaming potential is often used to quantify these electrostatic properties, this method is not effective for transport normal to the apical cell surface where the charge properties along the basolateral sides may be important (i.e., confluent cells with leaky tight junctions). In addition, even when cells have a uniform charge distribution, the shear stress generated by the conventional tangential flow device may dislodge cells from their confluent state. Here we introduce a novel streaming potential measurement device to characterize the normal electrostatic properties of confluent cells. The streaming potential device encompasses a 24 mm cell-seeded Transwell(®) with two AgCl electrodes on either side of the cell-seeded Transwell. Phosphate buffered saline is pressurized transversal to the Transwell and the resultant pressure gradient induces a potential difference. Confluent monolayers of HEK and EA926 cells are used as examples. The corresponding zeta potential of the cell-membrane configuration is calculated using the Helmholtz-Smoluchowski equation and the zeta potential of the confluent cell layer is deconvolved from the overall measurements. For these test models, the zeta potential is consistent with that determined using a commercial dispersed-cell device. This novel streaming potential device provides a simple, easy, and cost-effective methodology to determine the normal zeta potential of confluent cells cultured on Transwell systems while keeping the cells intact. Furthermore, its versatility allows periodic measurements of properties of the same cell culture during transient studies.

FIG. 1.

Illustration of a cell with leaky junction. The figure shows a (a) tangential flow and (b) normal flow configurations of measuring streaming potential.

FIG. 2.

Schematic of the normal streaming potential device. Ag/AgCl electrodes are used to measure the streaming potential with a voltmeter (110 True RMS Multimeter, Fluke, Inc.). PBS (1.87 mS/cm²) was used as the electrolyte solution and the pressure at the inlet was monitored using a pressure gauge. Polycarbonate Transwells seeded with confluent EA926 cells or HEK cells or with no cells are hosted in the chamber.

FIG. 3.

Representative graphs relating the measured potential (ΔΨ) with variation in pressure (ΔP) for polycarbonate membranes with pore size of 3.0 μm.

FIG. 4.

Representative graphs comparing the measured zeta potential (ζ) for three membrane cases: virgin polycarbonate membranes, HEK confluent cell-seeded membrane, and EA926 confluent cell-seeded membrane and the deconvolved confluent cells. Using Eq. (9), the zeta potential (ζ_c) of the HEK cells and EA926 cells were deconvolved from the cell-membrane configurations.

FIG. 5.

Comparing the zeta potential of EA926 and HEK cells deduced from the streaming potential measured using our streaming potential device and that measured using commercially available Malvern Zetasizer.