Tissue & Cell 1998 30 (5) 525-530 © 1998 Harcourt Brace & Co. Ltd Effect of culture conditions on endo- thelial cell growth and responsiveness Ingrid A. M. Relou', Cora A. Damen 1, Daisy W. J. van der Schaft 2, Gerard Groenewegen, Arjan W. Griffioen 2 Abstract. The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosJs induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions. Keywords: Endothelial cell, extracellular matrix, adhesion molecules, angiogenesis, in vitro Introduction The extracellular matrix of endothelial cells (EC) is a dense network composed of macromolecules such as collagen, fibronectin, vitronectin, hyaluronan, laminin and glycosaln- inoglycans. The interaction of EC with these components by specific receptors plays an important role in the growth and ~Department of Internal Medicine and Medical Oncology, Laboratory for Angiogenesis Research, University Hospital Utrecht, Heidelberglaan 100, PO Box 85500, 3508 GA Utrecht, The Netherlands. 2Departmentof Internal Medicine, Tumor Angiogenesis Laboratory University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Received 6 January 1998 Accepted 9 April 1998 Correspondence to: Dr A.W. Griffioen. Fax: +31 43 3875006; E-mail: a.griffioen @intmed.unimaas.nl differentiation of EC and vascular development, e.g. during tumor progression (Liotta, 1986; Dejana et al., 1987; Ingber, 1993; Ingber et al., 1995). EC growth and differentiation is also determined by specific growth factors. Outgrowth of tumors is dependent on the formation of new vasculature and therefore malignant cells produce high levels of angiogenic factors (Folkman, 1990; Folkman & Shing, 1992). We found that these angio- genesis factors down regulate endothelial adhesion molecule expression thereby preventing extravasation and infiltration of leukocytes (Griffioen et al., 1996a, 1996b). To investigate the role of the extracellular matrix in these phenomena we studied the spontaneous and growth factor-induced prolifera- tion of EC in vitro on different types of matrix protein-coated plastic and in the presence of different culture media. In addition, the induction of apoptosis by serum deprivation 525