Figure 1.

Endothelial cell (EC) culture characteristics. (A) Schematic representation of the numbers of EC per 1,000,000 cells in a single lung perfusate sample. Cell numbers were quantified via flow cytometry. (B) Overview of primary lung EC cultures. ECs from different donor perfusate samples were cultured in EBM-2 EC-specific medium. Five different perfusate samples were seeded in gelatin-coated culture flasks, and cell numbers were assessed on different culture days, when cell density was high enough to further passage the cell cultures. Large differences were observed in cell growth rates between individual cell cultures. (C) Light microscopy images of different primary EC cultures. Typical EC morphology could be observed, as well as clustering of EC at higher cell densities. Passage 1, 100×, passage 2, 400×, passage 3, 100×, passage 4, 100×. (D) EC characterization via flow cytometry. Cell cultures were detached using accutase cell detachment solution. ECs were gated as large cells based on forward/sideward scatter. Gated cells were from the non-lymphoid lineage (CD45−) and expressed both CD31 and CD13, characteristic for ECs. Furthermore, intracellular staining revealed these cells to be vWF positive. Density plots were created using FlowJo analysis software.