Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

Programmed Topographic Substrates for Studying Roughness Gradient-Dependent Cell Migration Using Two-Photon Polymerization.

The mediation of the extracellular matrix is one of the major environmental cues to direct cell migration, such as stiffness-dependent durotaxis and adhesiveness-dependent haptotaxis. In this study, we explore another possible contact guidance: roughness dependent topotaxis. Different from previously reported studies on topotaxis that use standard photolithography to create micron or submicron structures that have identical height and different spatial densities, we develop a new method to programmatically fabricate substrates with different patterns of surface roughness using two-photon polymerization. Surface roughness ranging from 0.29 to 1.11 µm can be created by controlling the voxel distance between adjacently cured ellipsoid voxels. Patterned Ormocomp® masters are transferred to polypropylene films using the nanoimprinting method for cell migration study. Our experimental results suggest that MG63 cells can sense the spatial distribution of their underlying extracellar roughness and modulate their migration velocity and direction. Three characteristic behaviors were identified. First, cells have a higher migration velocity on substrates with higher roughness. Second, cells preferred to migrate from regions of higher roughness to lower roughness, and their migration velocity also decreased with descending roughness. Third, the migration velocity remained unchanged on the lower roughness range on a graded substrate with a steeper roughness. The last cell migration characteristic suggests the steepness of the roughness gradient can be another environmental cue in addition to surface roughness. Finally, the combination of two-photon polymerization and nanoimprint methods could become a new fabrication methodology to create better 3D intricate structures for exploring topotactic cell migrations.