2.4. hCMEC/D3 In Vitro Model

The hCMEC/D3 cell line (Merck Millipore, Darmstadt, Germany) [27] was used for the in vitro experiments. Monolayers of cells were seeded on semipermeable plates coated with rat-tail type I collagen (Discovery Labware, Bedford, MA, USA) at a density of 20,000 cells/well. Medium EndoGro MV Supplement Kit (Merck Millipore) was used as a culturing medium, and cells were incubated at 37 °C, 5% CO₂. Once cells reached 100% of confluence, and a TEER value larger than 20 Ωcm², they were used for experiments. In addition, six hours prior to experiments the culture medium was replaced by a medium without growth supplements. Cultured hCMEC/D3 cells were treated (1%, v/v, 12 h) with thawed plasma either from women with preeclampsia, women with normal pregnancies or non-pregnant women. An epithelial Volt/Ohm meter (EVOM2, World Precision Instruments, Sarasota, FL, USA) with two electrodes in each compartment was used to measure the TEER. Measurements were performed both before adding plasma (baseline), and after incubation with plasma. Delta-TEER values were calculated by subtracting basal TEER values from TEER values after exposure to plasma.

Confirmatory experiments of TEER and cell permeability to high-molecular weight fluorescent dye (Fluorescein-5-isothiocyanate FITC-dextran 70 kDa) were performed as previously reported [30] using randomly selected plasmas from women with preeclampsia (n = 12), and women with normal pregnancies (n = 13).

The hCMEC/D3 cell line was used in passages 5 to 10. Individual plasmas were used in duplicate experimental replicates. None of the plasmas were excluded at the final analysis. More detailed experimental conditions are described in a previous publication [30].