Perfusion Experiments

The perfusion model used in the current study has been described by Hitzerd et al.¹⁰ Perfusion experiments were conducted in healthy, term placentas. Maternal and fetal perfusion media consisted of Krebs-Henseleit buffer at 37 °C, supplemented with heparin (final concentration; 2500 IU/L) and aerated with 95% O₂ to 5% CO₂. The fetal circulation (closed circuit; flow rate, 6 mL/minute) was established by cannulating the chorionic artery and corresponding vein of an intact cotyledon. Maternal circulation (closed circuit; flow rate 12 mL/minute) was created by placing 4 blunt cannulas in the intervillous space. At t=0, at a concentration of 10×C_max, esomeprazole (50 µmol/L) or omeprazole (30 µmol/L), or no drug as a control were added to the maternal circulation. Samples of the maternal and fetal circulations were taken after 6 minutes and every 30 minutes until the end of the experiment (180 minutes) to determine the fetal-to-maternal (F/M) ratio and sFlt-1 concentration and were immediately stored at −80 °C. To establish good overlap between maternal and fetal circulations, antipyrine (100 mg/L) was added to the maternal buffer, while FITC-dextran (40 kDa, 36 mg/L) was added to the fetal circulation as a marker of integrity of the capillary bed. In order for an experiment to be successful, the F/M ratio of antipyrine had to be >0.75 and the maternal-to-fetal ratio of FITC-dextran <0.03 at t=180.