A Large-format Polyacrylamide Gel with Controllable Matrix Mechanics for Mammalian Cell Culture and Conditioned Media...

Tissue culture plastic has been used for routine cell culture and in vitro experiments for over 50 years. However, cells are mechanically responsive and behave differently on hard surfaces than they do on softer substrates. Polyacrylamide gels have become a popular hydrogel of choice for controlling surface stiffness and ligand density for cell adhesion. Many synthesis methods use coverslips and small gel surface areas for cell culture, which are amenable to microscopy-based experiments. However, none of the currently published methods can be scaled up to increase the surface area to accommodate conditioned media production, high volume analyte collection, or cell line expansion. To overcome this size limitation, we developed a protocol for synthesizing polyacrylamide in glass dishes using commercially available materials. This enables routine cell culture on soft surfaces and facilitates experiments that require large amounts of analyte, especially studies involving extracellular vesicles and secreted factors.