*4.2. Isolation and In Vitro Culture of VCT*

Chorionic villi were obtained by manual dissection of placental tissues from term placentas as previously described about one hour after delivery [11,55]. Villous tissue was dissected free of membranes, rinsed and minced in Hank's Balanced Salt Solution (HBSS) 1X. The villous sample was then subjected to sequential enzymatic digestion in HBSS 1X containing 0.2% trypsin (*w/v*), 25 IU/mL DNAse I, 0.1 mM MgSO4, 0.1 mM CaCl<sup>2</sup> and 4% milk (*v/v*). Cell dissociation was monitored by light microscopy. The first three digests were discarded to eliminate residual ST fragments and erythrocytes. Cell suspensions resulting from the following four or five sequential digestions were pooled. Cells were then purified on a discontinuous Percoll gradient (5% to 70% in 14 steps) and their viability was determined in *v/v* solution with trypan blue.

Isolated cells were seeded in DMEM containing 10% Fetal Calf Serum (FCS), 1% Penicillin-Streptomycin and 1% L-Glutamin (complete DMEM) at 1000 cells/mm<sup>2</sup> during 15 h at 37 ◦C with 5% CO2. After plating, cells were incubated with RTV, LPV or DMSO control during 6 h, 24 h or 48 h (time necessary to allow fusion process). RTV concentrations used ranged from 5 to 20 µM. LPV chosen concentration was 10 µM (=6.288 µg/mL or 6288 ng/mL) corresponding to the mean LPV blood concentration measured in mothers treated for HIV infection. RTV (SML-0491, Sigma-Aldrich® Inc, St-Louis, MO, USA) or LPV (SML-1222, Sigma-Aldrich® Inc, St-Louis, MO, USA) were dissolved in DMSO. Consequently, control DMSO was equivalent to the percentage of DMSO necessary to dissolve LPV.

Cell culture media were collected at different times of culture, supernatants were kept frozen at −80 ◦C as part of the Equipex 10-PhC/SC-11/243 project "Perinatcollection" until use. Cells were either fixed or snap-frozen for RNA/Protein extraction and stored at −80 ◦C until use.
