Detachkit

HUVECs were purchased from PromoCell (Sickingenstraße, Heidelberg, Germany) and cultured in endothelial cell growth medium (PromoCell) containing 2% (v/v) Fetal Coat Serum (FCS), 0.4% (v/v) endothelial growth supplement: 0.1 ng/mL human Epidermal Growth Factor (EGF), 1.0 μg/mL hydrocortisone, 1 ng/mL human basic fibroblast growth factor (bFGF), 90 µg/mL heparin, and 1 % (v/v) penicillin/streptomycin (DUTSCHER SAS, Brumath, France) in a fully humidified atmosphere at 37 °C and 5% CO₂. Confluent cells were detached with the PromoCell detach kit (Sickingenstraße, Heidelberg, Germany) containing 30 mM Hepes, Trypsin/EDTA Solution (0.04%/0.03%), and Trypsin Neutralizing Solution. FCS was reduced to 1% 24 h before the experiment. All experiments were performed on subconfluent endothelial monolayer cells (80%) after the third passage.

Human umbilical vein endothelial cells pooled from donors (PromoCell, Heidelberg, Germany) were cultured in T75 tissue culture flasks in complete EGM-2 medium (PromoCell) at 37°C in a humidified atmosphere in 5% CO₂ in air. HUVECs were grown to ~80% confluence, after which the culture was trypsinized using the PromoCell Detach Kit (PromoCell). The cells investigated by tubule formation assay (TFA) were used from the third, fourth, or fifth passage only, with HUVEC passaged to ~80% confluence within 24 h of the TFA, as described (14 (link)).

Human-induced pluripotent stem cells (iPSCs) were obtained from the Human Induced Pluripotent Stem Cells Initiative and were cultured in a feeder-free system on six-well plates coated with recombinant human truncated vitronectin (Thermo Fisher Scientific, Waltham, MA) in Essential-8 medium (E8; Thermo Fisher Scientific) at 37° C, 5% CO₂. Cells were routinely passaged by dissociation with Versene (Thermo Fisher Scientific) at a 1:6 split ratio. Human ARPE19 cell line (P25) was cultured in T75 flasks in Dulbecco's modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM; F12; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) at 37°C, 5% CO₂. Mouse NIH3T3 fibroblast cell line (ECACC) was cultured in T75 flasks in high glucose DMEM (Sigma-Aldrich) supplemented with 10% FBS at 37° C, 5% CO₂. ARPE19 and NIH3T3 cells were routinely passaged with trypsin-EDTA solution (Sigma-Aldrich) and split at a 1:3–1:10 ratio. Human dermal microvascular epithelial cell (PromoCell, Heidelberg, Germany) and normal human dermal fibroblasts (NHDFs; PromoCell) were cultured in gelatin-coated T75 culture flasks in Endothelial Cell Growth Medium V2 (PromoCell) or Fibroblast Growth Medium (PromoCell), respectively, at 37° C, 5% CO₂. HDMECs and NHDFs were passaged using the PromoCell DetachKit and split at a 1:3–1:5 ratio.