Type I collagen gels for assessing the combined effects of ligand concentration-dependent elasticity and fibril density on cells

• Effects of concentration-dependent elasticity ( C - E ) and fibril density were separated. • Adhesion areas of cells decreased with increasing fibril density at constant C - E . • Adhesion areas of cells increased with increasing C - E in absence of Col-I fibrils. • Adhesion areas remained unchanged when both C - E and fibril density increased. • Combined compositional, mechanical, and topographical cues affect cells. Type I collagen (Col-I) is the most abundant fibrous protein in the extracellular matrix (ECM) and its densification is involved in the progression of various diseases and aging. Elevated Col-I concentrations increase ECM fibril density and stiffening; therefore, the effects of such changes in the microenvironment of cells require examination. To separately examine the effect of ligand concentration-dependent elasticity ( C - E ) and fibril density of Col-I, we developed three types of Col-I gels by introducing reagent-free radiation-induced crosslinking while adjusting the distance between the Col-I monomers or fibrils. The adhesion area of HeLa human cervical cancer epithelial cells, human induced pluripotent stem (hiPS) cells, and 3T3-Swiss albino mouse embryonic fibroblasts, tended to decrease with increasing fibril density when C - E was constant. In the absence of fibrils, the adhesion area of HeLa and 3T3-Swiss cells increased with increasing C - E , whereas hiPS cells clustered regardless of C - E . When C - E and the fibril density increased simultaneously, the adhesion area of HeLa and 3T3-Swiss cells remained unchanged due to the two opposing effects. Our results highlight the importance of evaluating the combined compositional, mechanical, and topographical effects of Col-I on cells. The developed gels will contribute to elucidating the role of Col-I in biological functions.