(176j) 3D Migration of Fibroblast-Macrophage Co-Cultures in Mechanically-Gradient Collagen Hydrogels

3D Migration of Fibroblast-Macrophage Co-cultures in Mechanically-Gradient Collagen Hydrogels

Rosalyn Hatlen¹, Padmavathy Rajagopalan¹

¹Department of Chemical Engineering, Virginia Tech, Blacksburg, VA 24061

Fibroblasts and macrophages are two of the most common cells found in wounds, diseased tissue and during inflammation. We have recently reported how the interconnectivity of the extracellular matrix (ECM) affects the migration of these two cells. When the ECM is densely interconnected, fibroblasts formed tunnels, which were subsequently used by macrophages to move through the 3D environment. When collagen fibers were loosely interconnected, fibroblasts aligned them to form tracks to promote macrophage migration. Our focus in the current study is to investigate how co-cultures migrate through mechanically gradient hydrogel materials. We focus on how cellular properties change in the interfacial regions.

We have assembled mechanically gradient type I collagen hydrogels by serially stacking gels assembled with different input collagen concentrations. We have assembled gradient gels with input collagen concentrations of 1.1 mg/ml and 2.2 mg/ml. In order to visually identify each part of the gradient, collagen was either covalently conjugated to a fluorescent dye or fluorescent beads were added to the mixture prior to gelation. We have previously shown that collagen gels with an input concentration of 1.1 mg/ml exhibit loosely connected fibers and exhibit a Young’s modulus of approximately 0.2 kPa. We are currently investigating the fiber connectivity in gels assembled with an input collagen concentration of 2.2 mg/ml.

We have monitored the migration and proliferation of fibroblasts through the gradient gels over a 7-day period. Our results indicate that cellular proliferation was greater when the cells were exposed to the softer gel first. For example, cell proliferation was approximately 4-fold when cells were encapsulated in the stiffer gel in comparison to an approximate 6-fold proliferation when they were cultured within a softer gel. There were differences between the different gradient gels in migration distance over a 7-day period. Our early results, indicate that in the presence of mitomycin-C, 3D migration speeds were lower.

Based on these trends, our ongoing and future studies are focused on investigating how migratory patterns in gradient gels change when macrophages are included. In addition, we will focus on the interactions between cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Obtaining an understanding on how CAFs and TAMs interact will provide information on how they invade and migrate through the changing physical environment that is found in tumor microenvironments.