Hydrogel array patterning using 3D-printed microfluidic inserts to control cell-cell and cell-ECM interactions.

By shaping biochemical gradients and extracellular matrix cues within the local microenvironment, cellular spatial organization plays a critical role in regulating tissue development, homeostasis, and disease progression. Microfluidic platforms are highly suitable for the study of these cell-cell and cell-matrix interactions as they precisely control cell arrangement and gradients compared to conventional experimental systems. Cells are often embedded within hydrogels to improve physiological relevance by enabling matrix-mediated signaling. However, many designs restrict the number and arrangement of hydrogels or generate gradients in only one dimension, limiting their ability to recapitulate complex tissue architectures. To address this need, we introduce a 3D printed microfluidic insert compatible with microplates that allows patterning of up to ten unique hydrogel arrays in two dimensions and generation of parallel or orthogonal concentration gradients. We first develop a physics-based computational model of hydrogel filling to define design parameters that ensure robust hydrogel patterning. We then establish perpendicular concentration gradients on timescales relevant to biological experiments. Furthermore, we demonstrate high cell viability in our 3D-printed devices and control of fibroblast migration across multiple patterned hydrogels. Finally, we monitor the recruitment of primary human monocyte towards cell-free and fibroblast-seeded 3D collagen matrices. Our microfluidic insert platform is compatible with high-throughput automation workflows and allows for interrogation of spatially variant signals that regulate cell migration and cell-cell signaling in physiologically-relevant 3D microenvironments.