L ascorbic acid 2 phosphate aa2p

HCECs were cultured as indicated above, and then at passages two to four, specific drugs were tested when added to the growth media. L-ascorbic acid 2-phosphate (AA-2P) (Sigma-Aldrich Corp.), versus L-ascorbic acid (AA) at a concentration of 20 μg/mL were tested on HCECs plated at 40,000/well in triplicate and cultured for 2 days to examine survival and function by trans-endothelial electrical resistance assay (TEER; see below). Y27632 ROCK inhibitor (Tocris Bioscience, Minneapolis, MN, USA), SB154352 TGF-β inhibitor (Cayman Chemicals, Ann Arbor, MI, USA), and human recombinant Rspondin-1 (StemRD, Burlingame, CA, USA) were tested on HCECs plated at 5000 cells/well for 12 hours before adding drugs at increasing concentrations for 72 hours. Cells treated with dimethyl sulfoxide (DMSO) or H₂O only were used as controls. To measure survival, a 5 mg/mL stock solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich Corp.) in water was prepared, and added to the culture wells with media in a 1:100 dilution. After 15 minutes at 37°C in 5% CO₂, cells were imaged, and cell counts were compared to control wells. Morphology was assessed by dividing the longest axis by the shortest axis of the cell (length/width ratio), using a grid to image unbiased locations within the culture wells.