3D Cell Culture-Based Hybrid Bioanalytical Platform for Optical Imaging Utilizing Silk Fibroin Sponges.

We present an innovative bioanalytical hybrid platform designed for the preclinical evaluation of cellular characteristics. The system combines a three-dimensional (3D) cell culture grown on an artificial extracellular matrix with a chromatography-inspired array configuration. Sponges, made from the structural protein silk fibroin, serve both as a biomimetic extracellular matrix and as a stationary phase. Silk fibroin sponges were produced in-house using a multistep process involving removal of inherent sericin proteins from raw silk fibers, followed by dissolution and dialysis to purify the fibroin solution, dissolution in organic solvent, and subsequent salt-bed casting to generate silk-based sponges with controlled porosity/pore sizes of 500-800 µm. Genetically modified breast cancer cell lines 4T1-iRFP720 and 4T1-wt (non-fluorescent control) were cultured within silk scaffolds using a continuous media flow via a pump, and their cellular growth and characteristics were analyzed non-invasively using optical imaging techniques (in vivo optical imaging instrument). By merging key advantages of chromatographic systems (automatization, reproducibility) with the biological relevance of advanced 3D cell cultures, the platform enables in vitro modeling of tissue-like architecture and morphology while facilitating the monitoring of dynamic cellular behavior. In parallel, the application of medical imaging technology enables real-time and prolonged monitoring of cellular migration and growth, among other factors. This approach offers substantial potential for investigating cellular behaviors at a macroscopic scale in a laminar-like flow system. By improving the physiological relevance of in vitro models, this method may help bridge the translational gap to in vivo studies and is consistent with the reduce, replace, refine (3R) framework for animal experimentation.