Atomic force microscopy nano-characterization of 3D collagen gels with tunable stiffness

As extracellular matrix (ECM) nano-characteristics play a crucial role in cell behavior, including cancer development and metastasis, several ECM in vitro models have been used in order to study cells behavior under different biochemical and mechanical conditions. Among the ECM constituents, collagen (especially collagen type I) has been extensively used as an essential component of ECM models, since it is one of the most abundant ECM protein. Use of three-dimensional (3D) collagen gels provides the advantage of allowing the cells to grow in a 3D environment that bears strong similarities to their natural, in vivo setting. Thus, the ability to form collagen gels with tunable stiffness and well defined naturally occurring nano-characteristics is crucial for these studies. Atomic Force Microscopy (AFM) is a unique tool that is ideal for the complete characterization of such models, in terms of morphology and mechanical properties without destroying the collagen fiber structure. In this protocol, the development and the AFM nano-scale characterization of 3D collagen type I gels is presented. The protocol includes: • The formation of 3D collagen type I gels with tunable stiffness; • The preparation of histological sections from collagen gels; • The AFM-based morphological and mechanical nano-characterization of the gels.