Fabrication of Adhesive Substrate for Incorporating Hydrogels to Investigate the Influence of Stiffness on Cancer Cell Behavior.

Stiffness control of cell culture platforms provides researchers in cell biology with the ability to study different experimental models in conditions of mimicking physiological or pathological microenvironments. Nevertheless, the signal transduction pathways and drug sensibility of cancer cells have been poorly characterized widely using biomimetic platforms because the limited experience of cancer cell biology groups about handling substrates with specific mechanical properties. The protein cross-linking and stiffening control are crucial checkpoints that could strongly affect cell adhesion and spreading, misrepresenting the data acquired, and also generating inaccurate cellular models. Here, we introduce a simple method to adhere to polyacrylamide (PAA) hydrogels on glass coverslips without any special treatment for mechanics studies in cancer cell biology. By using a commercial photosensitive glue, Loctite 3525, it is possible to polymerize PAA hydrogels directly on glass surfaces. Furthermore, we describe a cross-linking reaction method to attach proteins to PAA as an alternative method to Sulfo-SANPAH cross-linking, which is sometimes difficult to implement and reproduce. In this chapter, we describe a reliable procedure to fabricate ECM protein-cross-linked PAA hydrogels for mechanotransduction studies on cancer cells.

Method for the Direct Fabrication of Polyacrylamide Hydrogels with Controlled Stiffness in Polystyrene Multiwell Plates for Mechanobiology Assays.

Polyacrylamide (PAA) hydrogels are now widely used in mechanobiology because the well-defined available protocols allow a robust and reproducible control of substrate stiffness within a physiological range. However, several assays require hydrogels inside traditional plastic substrates and the current methods remain relatively tedious. Here, we present a simple and direct fabrication technique that successfully attaches PAA hydrogels inside polystyrene multiwell plates and Petri dishes of different sizes. It permits a control of the Young's modulus of the gels, within the desired range for mechanobiology. Some critical steps, that had to be overcome to guarantee protein conjugation and cell attachment, are detailed, as they differ from the standardized preparation on glass substrates. To validate our process, we demonstrated that HepG2 and 3T3L1 cell lines as well as primary hepatocytes seeded on PAA gels of different stiffnesses in plastics showed a mechanical response identical to the cells cultured on traditional gels.