Abstract A prerequisite for successful tissue engi- neering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-cul- turing endothelial cells (ECs) with tissue-specific ‘by- stander cells’ in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier- based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311–322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were ad- mixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and forma- tion of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-de- rived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully select- ing the experimental parameters of a given in vitro angiogenesis model. Keywords Angiogenesis Æ Co-culture Æ Collagen gel Æ Endothelial cells Æ Fibrin gel Æ Fibroblast cells Æ Microcarrier Æ Serum concentration Æ Smooth muscle cells Abbreviations 3-D three-dimensional AMD average migration distance BAEC bovine aortic endothelial cell bFGF basic fibroblast growth factor (also FGF2) DMEM Dulbecco’s modified Eagle’s medium EC endothelial cell ECM extracellular matrix ECGS endothelial cell growth supplement FB bovine adipose fibroblast cell FBS fetal bovine serum HAEC human aortic endothelial cell HMEC-1 transformed human microvascular endothelial cell HMVEC primary human microvascular endothelial cell F. Dietrich Æ P. I. Lelkes (&) School of Biomedical Engineering, Science, and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA e-mail: pilelkes@drexel.edu Present Address: F. Dietrich Interdisciplinary Center for Clinical Research (IZKF), University of Leipzig, 04103 Leipzig, Germany Angiogenesis DOI 10.1007/s10456-006-9037-x 123 ORIGINAL PAPER Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels Franziska Dietrich Æ Peter I. Lelkes Received: 19 March 2006 / Accepted: 14 June 2006 Ó Springer Science+Business Media B.V. 2006