Cells can sense physical properties of surrounding 3-dimensional (3D) culture substrata; however, the physiological influences of such sensing are not fully understood. Here, we studied the physiological characteristics and activities of the macroscopic structure of a routinely used 3D culture substrata, the RADA16 self-assembling peptide scaffold. We found that RADA16 exhibited three distinct assembly patterns depending on its concentration, and one of these assemblies, formed with 0.01% (w/v) RADA16, was capable of inducing differentiation of human myelocytic leukemia HL-60 cells into monocytes/macrophages. This activity was largely reduced by destroying the 3D structure of the assembly, suggesting that the assembly intrinsically retained the ability to induce HL-60 differentiation. When cultured in the RADA16 scaffold, HL-60 cells accumulated intracellular cholesterol about 10 times more than normally cultured cells. Both the RADA16 culture and cholesterol loading brought about similar gene expression profiles. These results showed that HL-60 cells can sense the physical properties of the RADA16 scaffold through a mechanism that may involve intracellular pathways of cholesterol synthesis and/or transport. (C) 2013 Elsevier Inc. All rights reserved.