**4. Materials and Methods**

### *4.1. Blood and Tissue Collection*

Preeclampsia is defined as elevation of blood pressure of 140/90 mmHg or more, and proteinuria of more than 0.3 g in a 24 h urine collection or random urine dipstick test of more than 2+ according to the Society of Obstetric Medicine of Australia and New Zealand guidelines [54]. All blood and placental tissues were collected from pregnan<sup>t</sup> women, as detailed below. Written and informed consent was obtained from all individual participants included in the study with the approval of the Monash Health Human Research Ethics Committee (HREC no. 13357B, dated 6 August 2015). Venous blood was collected from women with a singleton healthy pregnancy and from women with established preeclampsia at 24 to 34 weeks of gestation. Women who had received intravenous magnesium sulphate, or had pre-existing or secondary hypertension, diabetes, or a multiple pregnancy, were excluded. None of the women with preeclampsia were in labor at the time of blood sampling. The control (healthy) women were matched for gestation (≤34 weeks). Sera were separated and pooled into two groups: healthy term pregnancy serum and preeclampsia serum. For all in vitro experiments, 20% pooled sera from preeclampsia pregnancies were used for treatment of endothelial cells and were compared with that of the normotensive sera treated cells. There were significant di fferences in the systolic and diastolic blood pressure and proteinuria between normotensive and preeclamptic patients (*p* = 0.007), as previously presented [25].

#### *4.2. Placental Explant Cultures Ex Vivo*

Placental villous explants (*n* = 10) were collected from term uncomplicated pregnancies at elective caesarean section within 20 min of delivery of the placenta. Briefly, placental villous tissue was excised by removing maternal decidua. Villous explants (approximately 50–70 mg wet weight) were then thoroughly washed with cold Hank's balanced salt solution (HBSS, 1:10, Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA) and placed in 24-well plates in M199 supplemented with 1% antibiotics/antimycotics (penicillin G, streptomycin sulphate, and amphotericin B) and 1% of L-glutamine (all from Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA). Incubation details are provided below.
