Endothelial basal medium mv2

ECFCs were isolated from peripheral blood from controls and patients, as previously described^{14 (link)} and the successful yield of ECFC colonies was from individuals was 80%. ECFCs were cultured in Endothelial Basal Medium MV2 (PromoCell) and used between passages 2–5 (n = 3–12) with and without the addition of 1 µM glycomimetic C3^{15 (link)}.

Human adult dermal lymphatic endothelial cells (LEC) were from Promocell (Germany) (CC-12217), and complete Endothelial Cell Growth Medium MV2 containing 5% FCS plus growth factors and supplements (prepared by combining Basal Media with Supplement Pack supplied in the kit) was from Promocell (Germany) (CC-22121). Cells were grown in tissue culture dishes and plates coated with human fibronectin [5 μg/mL, Sigma-Aldrich (United States) #F2006] and used at passage numbers 4–7. Complete, growth factor-free and serum-reduced growth factor-free media solutions were endothelial basal medium MV2 [PromoCell (Germany)] with the addition of 5% fetal calf serum (FCS) and supplement pack, 5% FCS only and 2% FCS only [PromoCell (Germany)], respectively. Cells were incubated at 37°C in a humidified atmosphere of 5% CO_2.

The cells were plated to 90% confluence in 48 well plates. To avoid the use of feeder cells, HLE cells were first expanded as described in the sections above and then separated from the 3T3 cells by using differential trypsinisation. Subsequently, they were plated in a serum-free and feeder-free corneal epithelial culture media CNT-57 (CellnTech, Bern, Switzerland). The HLF cells were placed in their usual culture media (DMEM supplemented with 10% FBS and 1% pen-strep). Prior to irradiation, the culture media was replaced with PBS. The cultures were irradiated at 20mJ/cm² by using a Vilber Lourmat (Eberhardzell, Germany) Bio-Sun UV irradiator set at 265 nm. The PBS was replaced with MV2 basal endothelial medium (Promocell Heidelberg, Germany) supplemented with 2% FBS (basal medium, BM). The produced conditioned media were collected after 24 h, centrifuged at 1500 G to clear from dead cells and debris, aliquoted and stored at −80 °C for a maximum of 2 months before use for analysis.