Transparent silk hydrogels as a versatile platform for cell culture and imaging

Recombinant spider silk proteins (spidroins) are emerging as a promising feedstock for biomaterial production due to their inherent ability to form hydrogels at 37 °C. However, their broader application as a robust cell culture platform has been hindered by slow gelation kinetics, CO 2 -induced turbidity, unknown long-term stability, and the use of Tris-HCl buffers that are suboptimal for most mammalian cells. In this study, we aimed to accelerate gelation kinetics of mini-spidroin-based hydrogels, reduce their turbidity, and improve gel stability under physiological conditions. Systematic evaluation of protein pre-treatments and buffer compositions identified parameters governing conformational behavior, gelation dynamics, and structural stability. Multimodal characterization, including turbidity measurements, circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, mechanical assessment, transmission electron microscopy, Thioflavin T assays, and in vitro studies, enabled the formulation of a cytocompatible buffer system optimized for mini-spidroin hydrogels. The formulation improves transparency and accelerates gelation, while maintaining experimental simplicity, thereby advancing the utility of mini-spidroin hydrogels as cell culture platforms.