Harnessing Thermogel Actuation for Driving Directional Stromal Cell Communication and Migration into Columnar Arrays

Abstract

During developmental processes, cells frequently condense along a preferred axis, creating columnar arrangements—a pivotal step in shaping elongating tissue structures and facilitating gradual building of tissue complexity. Despite advances in biofabrication technology that has allowed researchers to recreate these axial arrangements in vitro in 3D culture, maintaining these patterns for periods of cultivation beyond 7 days has proven challenging, given cells' tendency to exhibit random migratory patterns. In this study, we introduce EXPECT (EXtrusion Patterned Embedded ConstruCTs), a thermosensitive hydrogel based on poly(N-isopropylacrylamide) designed with specific rheological properties enabling the creation of embedded, macroscopic, cell-laden channels within the hydrogel using 3D printing. EXPECT, coupled with mild temperature changes at regular intervals, suppressed the random migratory tendencies of mesenchymal stromal cells (MSCs), guiding the cells to laterally intercalate and aggregate longitudinally. This resulted in the formation of continuous stacked arrangements of MSCs sustained over 36 days of culture. Additionally, EXPECT led to the elongation of initially spaced MSC spheroids toward each other, culminating in their fusion into narrowed, columnar assemblies. Our study presents a versatile and readily applicable approach for orchestrating and maintaining cell communication and movements along a preferred axis outside the developmental niche. By addressing a key limitation in current in vitro 3D culture systems and inducing cell movements reminiscent of both convergent extension and directed chemotaxis, we present a novel tool for studying various facets of development, disease, and repair.