(349i) Award Submission: Dynamic Microstructures for Controlling Spatial Organization of Biological Entities and Materials Within Defined Geometries

Introduction: The architectural complexity and intricate interactions between multiple cell types in their native extracellular microenvironment start in the early developmental stages and continue through the life cycle of a living system. These complex interactions play a role in regulating tissue morphogenesis, organ functions, cellular decisions, and cancer.¹ Recreating these microenvironments could improve our understanding of the early developmental stages and allow us to fabricate disease models for drug discovery and tissues for regenerative medicine. Replicating native biological complexities has remained a challenge. Previous methods, such as the use of static microstructures and patterning techniques,^2,3 have not allowed for targeted spatial control. Herein, we introduce dynamic microstructures to control spatial organization of living materials and biomaterials within defined microenvironments.⁴

Materials and Methods: Prepolymer solutions of poly(N-isopropylacrylamide) (PNIPAAm), a thermoresponsive polymer, were used to generate dynamic microstructures.^4,5 Dynamic microstructures with different patterns, such as microgrooves, circular microwells and square microwells, were fabricated using soft lithographic techniques. The dynamic microstructures responded to temperature by changing their patterned areas. Precursors of an agarose mixture containing different cell types were sequentially molded within dynamic microstructures, which allowed for controlled spatial arrangements of different cell types in different compartments of microgels. In addition, fluorescent beads of different colors were used to show proof of concept for spatially immobilizing chemical compounds within different compartments of a microscale hydrogel. Human hepatoblastoma (HepG2) cells, NIH-3T3 fibroblasts, and Human Umbilical Vein Endothelial cells (HUVECs) were used in the study.⁴

Results: The dynamic microstructures were subjected to different temperatures to test their responsiveness. Their patterned areas gradually increased with temperature increase. The change in patterned areas allowed for patterning biomaterials (containing either biological entities or chemical compounds) at different temperatures. The first layers of microgels were patterned at room temperature and the second layers were fabricated at physiological temperature. Multicompartment striped microgels containing fibroblasts in one compartment and HUVECs in another compartment were fabricated as a model for tissues having striped geometries. Cylindrical and cubic multi-layered hydrogels encapsulated HepG2s in the first layer and HUVECs in the second layer, served as models for tissue engineering and drug discovery. Different fluorescent beads were successfully immobilized into different compartments of the multi-layered microgels, suggesting potential applications for drug delivery or for delivery of soluble factors to microscale complex tissues. With the presented method, controlled spatial distribution of multiple cells and microbeads within a geometrically controlled biological material was achieved.⁴

Conclusion: We have introduced dynamic microstructures for a wide range of applications in life sciences. The shape changing properties of these microstructures could be used to generate complex biological architectures. One could substitute different cells and gels with the ones used in this study to create different microscale complex tissue constructs for regenerative medicine or to study cancer dynamics. Dynamic microstructures may also be useful for studying developmental biology by recapitulating the intricate extracellular organizations of embryonic stages. Thus, the presented methods with dynamic microstructures could be potentially useful for applications in tissue engineering, cancer biology, developmental biology, and drug discovery.⁴

References: (1) C. M. Nelson et al., In An. Rev. of Cell and Dev. Bio. 2006; 22, 287  (2) E. E. Hui et al, PNAS 2007, 104, 5722. (3) Y.-s. Torisawa et al., Int. Bio. 2009, 1, 649. (4) H. Tekin et al., JACS 2011, 133, 12944. (5) H. Tekin et al., Lab Chip 2010, 10, 2411.