A comprehensive protocol for PDMS fabrication for use in cell culture.

Cells exhibit remarkable sensitivity to the mechanical properties of their surrounding matrix, particularly stiffness changes, a phenomenon known as cellular mechanotransduction. In vivo, tissues exhibit a wide range of stiffness, from kilopascals (kPa) to megapascals (MPa), which can alter with aging and disease. Traditional cell culture methods employ plastic substrates with stiffness in the gigapascal range, which does not accurately mimic the physiological conditions of most biological tissues. Therefore, employing substrates that can be engineered to span a wide range of stiffnesses, closely resembling the native tissue environment, is crucial for obtaining results that more accurately reflect cellular responses in vivo. Polydimethylsiloxane (PDMS) substrates are widely used in cell culture for their ability to simulate tissue stiffness, but their optimization presents several challenges. Fabrication requires precise control over mixing, weighing, and curing to ensure reproducible mechanical properties. Inconsistent preparation can lead to improperly cured PDMS substrates, compromising experimental outcomes. Additionally, PDMS's inherent hydrophobicity poses challenges for cell attachment, necessitating surface modifications to enhance adhesion. Moreover, the risk of contamination during the sterilization process necessitates stringent protocols to maintain cell culture integrity. These challenges are further compounded by substrate autofluorescence which can cause difficulties when imaging cells. The aim of this study is to develop a standardized method for fabricating PDMS substrates with tuneable stiffness, ranging from kPa to MPa, suitable for diverse cell types using standard laboratory equipment. This method aims to minimize the complexity and equipment required for PDMS fabrication, ensuring reproducibility and ease of use. Achieving consistent and contaminant-free PDMS substrates will facilitate a broader adoption of these substrates in mechanobiology research and improve the relevance of in vitro models to in vivo conditions. Ultimately, contributing to a more comprehensive understanding of cellular responses to mechanical cues in health and disease.