Endothelial cell growth medium mv kit

ECFC were isolated as described previously.^{41 (link)} In brief, 8–16 mL of venous cord blood was collected in lithium heparin-coated tubes (Greiner Bio-One, Kremsmünster, Austria) immediately after delivery of the placenta. Mononuclear cells (MNC) were separated via density gradient centrifugation using Lymphoprep™ Density Gradient Medium (Axis Shild, Alere Technologies AS, Oslo, Norway). The emerged buffy coat was washed and resuspended in culture medium (Endothelial Cell Growth Medium MV Kit (PromoCell, Heidelberg, Germany) supplemented with 0.1% Gentamycin (ThermoFisher Scientific, Waltham, MA)). 2 × 10⁷ cells were seeded per well of six-well culture plates (ThermoFisher Scientific) pre-coated with rat tail collagen type 1 (Corning, Corning, NY) and cultured at 37 °C, 21% O₂, 5% CO₂ in a humidified incubator. After overnight incubation, the culture medium was changed and then twice a week.

Neonatal ECFCs were isolated, characterised and cultured as described previously (Weiss et al., 2022 (link)) and frozen in culture medium supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 10% dimethyl sulfoxide (SERVA, Heidelberg, Germany) before performing experiments. Endothelial Cell Growth Medium MV Kit (PromoCell, Heidelberg, Germany) with 0.1% gentamycin (Thermo Fisher Scientific) was used as culture medium. ECFCs were cultured at 37°C, 21% O₂, 5% CO₂ in a humidified incubator during experiments and used in passage 5–8.

Preparation of the immortalized mouse skin-derived lymphatic endothelial cell line (iLEC) was previously reported^{27 (link)}. A collagen-coated solution was prepared by adding 1.5 mL of Type I-C (Nitta Gelatin, Osaka, Japan) to 43.5 mL of sterile water (pH 3.0) and passing the mixture through a 0.22 μm filter (Sartorius, Göttingen, Germany). A cell-culture dish was incubated with the collagen-coated solution for 30 min at 37 °C under a 5% CO₂ atmosphere; the solution was discarded and the cell-culture dish was air-dried for 30 min in a sterile environment. Cells were cultured on the collagen-coated dishes in an Endothelial Cell Growth Medium MV Kit (PromoCell, Heidelberg, Germany), which was supplemented with 100 U/mL penicillin / 100 μg/mL streptomycin (Nacalai tesque) and 1 µg/mL Blasticidin S (FUJIFILM Wako Pure Chemical, Osaka, Japan). Cells were maintained at 33 °C under 5% CO₂ conditions to activate the temperature-sensitive SV40T antigen and were sub-cultured from 1/3 to 1/5 every 2–3 days following detachment using Accutase (Funakoshi, Tokyo, Japan). The cells with less than 50 passages were used in the experiments. The numbers of cell seeding for the experiments were as follows: 2.4 × 10⁵ cells/well in 6-well plates, 6 × 10⁴ cells/well in 24-well plates, and 2 × 10⁴ cells/well in 96-well plates.