The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 m were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for Ͼ8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs. endothelium; angiogenesis; human umbilical vein endothelial cell; multicellular spheroid; coculture; tubular structures ANGIOGENESIS IS A COMPLEX morphogenetic process initiated primarily by sprouting of endothelial capillaries from existing blood vessels to form an endothelial plexus. After being remodeled, the vasculature matures by recruitment of perivascular cells and smooth muscle cells. Vascular maturation attenuates the rate of vascular sprouting and prevents vascular collapse and regression (18,24), which is partly reflected by contact inhibition of endothelial cell (EC) proliferation in coculture systems with pericytes or smooth muscle cells (7,12,29,47). In adults, the angiogenic process is essential during wound healing, tissue repair, and remodeling and for female reproductive cycles, among many normal and pathological conditions. Mechanistic studies designed to gain a better understanding of, and an ability to manipulate, the angiogenic process in health and disease have intensified during the past 30 years, having been stimulated by Folkman's (2,16,19,25,27) initial hypotheses regarding the angiogenic switch during tumor cell growth and the development of the concept of antiangiogenic ther...

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