Angiogenesis the growth of new capillaries from pre-existing blood vessels, requiring growth factor driven recruitment, migration, proliferation, and differentiation of endothelial cells (ECs). The study of angiogenesis has many clinical applications in numerous fields, including peripheral and coronary vascular disease, oncology, hematology, wound healing, dermatology, and ophthalmology. In 1980, bovine capillary ECs were found to spontaneously form tubes when cultured in gelatin in vitro. Since then, many in vitro models have been used to recapitulate the basic steps of the in vivo process. Such in vitro models of angiogenesis offer many possibilities: the clinical testing of potential drug therapies; the modeling of pathological conditions; the study of the processes of endothelial cell differentiation, lumen formation, and vascular inoculation; and the investigation of the molecular mechanisms associated with angiogenesis. Endothelial cell differentiation, the lumen or tube formation, can be studied in vitro both in two dimensions and in three dimensions (3D); however, to study the other cellular mechanisms involved, a more complex 3D model that includes cell and extracellular environment interactions is required. Such 3D models involve seeding ECs either on or in gels. Studies involving 3D angiogenesis models have investigated the role of some key design factors on the migration, proliferation, and differentiation of ECs, including endothelial cell type, type of matrix, the concentration and the biochemical conditions of the matrix polymerization that can affect the density and the mechanical properties of the substrate, the culture media composition, and the bioavailability of angiogenic factors. The effect of the key design variables on concentration gradients of oxygen, nutrients, and angiogenic factors within the 3D models has not been fully explored. We have designed a 3D angiogenesis model that can be used to investigate such concentration gradients on the migration, proliferation, and differentiation of ECs. Such a model is comprised of ECs grown on a bovine type I collagen gel formed within a TranswellŽ membrane plate. This system allows access to compartments both above and below the gel that can be used for the delivery of factors or for sampling. Holding all other variables constant, we saw a significant increase in both the kinetics and the amount of migration, proliferation, and differentiation of ECs within the collagen gel for the samples in the TranswellŽ membrane plate compared to samples in a typical solid-bottom well plate, which only allows access to a compartment above the gel. We also tested the effect of adding growth factors to either the top or the bottom compartment of the TranswellŽ membrane plate on both the kinetics and the amount of migration, proliferation, and differentiation of ECs. The results show that both the kinetics and the amount of migration, proliferation, and differentiation of ECs within the collagen was significantly greater for the samples with the growth factors added to the bottom chamber only, showing a clear influence of the concentration gradient on the cellular response. We are continuing to use this system to show that how angiogenic factors are delivered to a 3D system, by taking into account concentration gradients, are just as important as to what angiogenic factors are being delivered.