Phenol red free matrigel substrate

EVT explant cultures were established from first trimester human placentas using the method described previously.16 Briefly, placental villi from 5‐8w gestation placentas with pre‐existing anchoring EVT cell columns were microdissected from the placenta and placed on Millicell‐CM culture dish inserts (pore size 0.4 μm; Millipore Corp, Maryland), pre‐coated with undiluted phenol red‐free Matrigel substrate (0.2 mL; Becton Dickinson, Mississauga, Ontario, Canada). Inserts were cultured under a humidified environment at 3% O₂/5% CO₂ and 37°C overnight prior to addition of serum‐free DMEM‐Ham's F‐12 media (200 μL; Invitrogen, Canada) supplemented with 1% Normocin (Invivogen), pH 7.4 (Explant Media SFM). EVT outgrowths were established over 48 hours‐culture at 3% ambient oxygen and randomized to treatment group. For ABCB1 knockdown experimental treatments, SFM was supplemented with the NC‐1 or Dsi2 siABCB1 transfection reagents (40 nmol/L) and added to the placental EVT explants followed by 48 hours of culture. EVT outgrowth morphology was visually assessed every 24 hours and photographs captured using the DMIL LED inverted microscope (Leica, Concord, ON, Canada) and micropublisher camera 5.0 RTV (Q Imaging).

The explant culture was performed as described previously. In brief, tips of first trimester placental villi (7 weeks) were collected from women and stored in 4 °C sterilized normal saline. After that, villi were dissected into small tissue sections (2–3 mm) and explanted in Millicell-CM culture dish inserts (0.4 mm pore size, Millipore, Carrigtwohill, Co. Cork) precoated with phenol red-free matrigel substrate (Becton Dickinson, Bedford, MA). The dissected tissue pieces were put on top of each drop and allowed anchorage, then were supplemented with 10% FBS and DMEM/F12 medium. Villi successfully anchored on Matrigel matrix and initiated outgrowth and were treated with FUT4 cDNA, siRNA or vehicle alone. EVT sprouting and migration from the distal end of the villous tips were recorded daily for up to 2 days. The extent of migration was taken with an inverted microscope (Olympus). Three separate experiments were repeated.