Pet transwell inserts

HUVECs (8 × 105/well) were seeded on 2 μm PET Transwell inserts (Falcon, USA) and grown to confluence with EGM-2. The confluence, integrity, and uniformity of the endothelial cell monolayer were examined microscopically. Before performing the experiment, cells were starved for 24 h with rosiglitazone and inhibitors. To determine the permeability through the cell monolayer, Evans Blue (EB) bound to 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) was loaded onto the upper chamber. EB could leak through intercellular spaces without junction or endothelial cell fenestration into the lower chamber. After 1 h, the amount of EB in the lower chamber was determined by spectrophotometer (620 nm). Experiments were performed in triplicate and repeated multiple times.

Cells were differentiated according to the protocol described by Hazim et al. (20 (link)). Briefly, cells were cultivated in T75 flask for two weeks with MEM-nicotinamide medium: 1% N1 (Invitrogen), 1% SVFi, 1% P/S, 0.25mg/mL Taurine (Sigma-Aldrich), Hydrocortisone 20ng/mL (Sigma-Aldrich), Triiodo-thyronine 0.013 ng/mL (Sigma-Aldrich) and 10mM nicotinamide (Sigma-Aldrich). After 3 times 10min TrypLE (Gibco) treatment, the remaining cells were placed on 0.4µM PET transwell inserts (Falcon, 353095) in 24-well-plates, coated with natural mouse laminin (Invitrogen). Cell medium was changed 3 times per week during 8 weeks before use. Differentiation was verified using RT-PCR analysis of markers indicating primary like cell phenotype. Cells on transwells were harvested every week during 8 weeks using Nucleozol (Macherey-Nagel, MN). RNA was extracted with TriZol reagent, according to the manufacturer’s recommendations. Then, 20ng of RNA was reverse transcribed to cDNA using Qscript reagent (VWR). Real-time PCR specific for the BEST1, RPE65, OCLN, MERTK, ZO-1, ITGB5 and housekeeping gene GAPDH mRNA (Table 1) was performed on CFX light cycler and SSoAdvanced SybrGreen (Biorad).