Cost-Effective Bioimpedance Spectroscopy System for Monitoring Syncytialization In Vitro: Experimental and Numerical Validation of BeWo Cell Fusion

Abstract

The placenta plays a critical role in nutrient and oxygen exchange during pregnancy, yet the effects of medicinal drugs on this selective barrier remain poorly understood. To overcome this, this study presents a cost-effective bioimpedance spectroscopy (BIS) system to assess tight junction integrity and monolayer formation in BeWo b30 cells, a widely used model of the multinucleated maternal-fetal exchange surface of the placental barrier. Cells were cultured on collagen-coated porous membranes and treated with forskolin to induce controlled syncytialization. Electrical impedance was measured using an entry level impedance analyzer, while immunofluorescence staining was used to confirm monolayer formation and syncytialization. The measurements and staining confirmed the formation of a confluent monolayer on day 4. In fact, the electrical resistance tripled for treated samples indicating a more electrically restrictive barrier. This resistance remained constant for treated samples reflecting the intact barrier's integrity over the next 3 days. The measurements show that, on day 4, the electrical capacitance of the cells decreased for the treated samples as opposed to the untreated samples. This reflects that the surface area of the BeWo b30 cells decreased when the samples were treated with forskolin. Finally, a COMSOL model was developed to explore the effects of electrode positioning, depth, and distance on TEER measurements, explaining discrepancies in the literature. In fact, there was a substantial 97% and 39.4% difference in the obtained TEER values. This study demonstrates the AD2 device's feasibility for monitoring placental barrier integrity and emphasizes the need for standardized setups for comparable results. The system can hence be used to analyze drug effects and nutrient transfer across the placental barrier.

Keywords: COMSOL (Version 6.2) multiphysics; bioimpedance spectroscopy; electrical capacitance; syncytiotrophoblast; transepithelial/transendothelial electrical resistance (TEER).

Figure 1

Experimental design: (a) electrode setup, (b) electrical model, (c) theoretical fitting of raw data. (d) experimental fitting of raw data based on electrical model.

Figure 2

Representative fluorescent images of untreated (a) and forskolin-treated (b) BeWo cells after 3 and 5 days of culture. Scale bar: 55 μm and (c) quantification of cell fusion index. *** and **** indicate p ≤ 0.001 and p ≤ 0.0001, respectively.

Figure 3

Impedance spectroscopy measurements and respective statistical analyses: (a) TEER, (b) electrical capacitance, (c) TEER rate of change with respect to day 2, and (d) electrical capacitance rate of change with respect to day 2. * indicates p ≤ 0.05.

Figure 4

COMSOL multiphysics model results: (a) the potential distribution across the transwell setup; (b) the TEER values from the experiments, compared with the simulation results; and (c) the correlation plot between the simulation output and the experimental results. On the one hand, a comparison to the unfused cells will result in a decrease in capacitance. On the other hand, due to the multiple layer formation for the control group, the overall membrane surface area will increase. This phenomenon will result in an increase of the capacitance * indicates p ≤ 0.05.

Figure 5

Error graph and fit of varying electrodes positions: (a) The depth of electrode in the basolateral chamber relative to the support. (b) The distance of the electrode in the apical chamber, relative to the second electrode.