Quantifying Biomass and Visualizing Cell Coverage on Fibrous Scaffolds for Cultivated Meat Production

Abstract Cultivated meat represents a transformative solution to environmental and ethical concerns of traditional meat industries, replicating livestock meat's texture and sensory attributes in vitro with a focus on cost, safety, and nutritional quality. Central to this process are biomaterial scaffolds that support tissue development from isolated animal cells grown in or on these matrices. Understanding scaffold interactions with cells, including scaffold degradation and biomass production, is crucial for process design and for scaling‐up goals. In this article, we outline comprehensive methods to quantify scaffold‐cell interactions for such scenarios, focusing on biomaterial scaffold degradation and changes in cell biomass [measured by cell weight, extracellular matrix (ECM) deposition, and cell coverage] during cell culture. We introduce two methodologies for assessing cell coverage: fixation and staining for detailed imaging analysis, and non‐invasive, real‐time evaluation across scaffolds. Here we focus on fiber‐based scaffolds, while the assessments can be extrapolated to 2‐dimensional (2D; films), and in part to 3‐dimensional (3D; sponge) systems. Utilizing the C2C12 mouse myoblast cell line as a gold standard, the protocols deliver precise, step‐by‐step instructions for preparing fiber scaffolds (using silk proteins here), seeding cells, and monitoring key parameters for cultivated meat production, providing a framework for advancing cellular agriculture techniques. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Fabrication and preparation of silk fiber scaffolds for cell seeding Support Protocol 1 : Cultivation of C2C12 cells and seeding onto fibrous scaffolds Basic Protocol 2 : Quantification of decellularized yarn scaffold degradation during cell culture Basic Protocol 3 : Quantification of biomass variation and ECM deposition on yarn scaffolds during C2C12 cell culture Basic Protocol 4 : Visualization of cell‐laden yarn scaffolds and determination of cell coverage ratio using confocal microscopy Support Protocol 2 : Real‐time imaging of cell‐laden yarn scaffolds using Celigo system Support Protocol 3 : Applying green CellTracker fluorescent probes to C2C12 cells seeded on yarn scaffolds