The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiological relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over of signaling gradients. This paper reviews most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. We observe that the focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications.

Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion. However, many of these cues are missing in current in vitro models of angiogenic sprouting. We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization. The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix. Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix. Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells. A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation. Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel. This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded. Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight. This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels. The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro.

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.