Type of culture medium determines properties of cultivated retinal endothelial cells: induction of substantial phenotypic conversion by standard DMEM.

Contradictory behavior of microvascular retinal endothelial cells (REC) - a reliable in vitro model to study retinal diseases - have recently been reported which might result from cultivating the cells in standard DMEM not optimized for this cell type. Therefore, we studied DMEM's effects on phenotype and behavior of immortalized bovine REC. Cells were cultivated in endothelial cell growth medium (ECGM) until a confluent monolayer was reached and then further kept for 1-4 days in ECGM, DMEM, or mixes thereof all supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 90 μg/ml heparin, and 100 nM hydrocortisone. Within hours of cultivation in DMEM, the cell index - measured to assess the cell layer's barrier function - dropped to ~5% of the initial value and only slowly recovered, not only accompanied by stronger expression of HSP70 mRNA and secretion of interleukin-6, but also by lower expressions of tight junction proteins claudin-5, claudin-1 or of the marker of cell type conversion caveolin-1. Altered subcellular localizations of EC-typic claudin-5, vascular endothelial cadherin and von Willebrand factor were also observed. Taken together, all experiments with (retinal) EC cultivated in common DMEM need to be interpreted very cautiously and should at least include phenotypic validation.