DECELLULARIZED CABBAGE LEAVES AS A SCAFFOLD FOR ADIPOGENICDIFFERENTIATION: A PRELIMINARY STEP TOWARDS EDIBLE FAT STRUCTURES FORCULTURED MEAT PRODUCTION

World population will reach 9.1 billion people by 2050, leading to an increased demand for alternative protein sources(1). Cellular agriculture has advanced considerably over the last decade, aiming to produce edible cellular structures that mimic conventional meat(2). Challenges remain, including recreating the composition of meat, which consists of different tissues. Among these, fat plays a crucial role in meat sensory attributes. In vivo ,intramuscular fat develops within muscle tissue, but the co-culture of cells in vitro remains challenging as adipogenesis inhibits myogenesis (3). To address this issue, researchers are investigating a sequential differentiation approach, where muscle and fat cells are differentiated separately before being combined. Scaffolds and hydrogels are necessary to reach this goal, and decellularized vegetables could offer an edible valid option(3). This study aims to investigate the potential application of decellularized cabbage leaves as a scaffold for culturing and differentiating adipogenic cells, with the final goal of generating cultured adipose structures as an ingredient for in vitro meat. The main hypothesis is that the surface architecture of the leaf, characterized by concave depressions, may promote cell adhesion, aggregation, and adipogenic differentiation. An effective protocol for vegetable decellularization was optimized and preliminary studies with 3T3-L1preadipocytes were performed to validate the model for future application with primary cells. Indirect cytotoxicity (EN ISO 10993-12) and direct cytocompatibility assays were performed. For the indirect cytotoxicity test, decellularized leaves were incubated in culture medium for 1, 3, and 7 days. The resulting eluates were applied to seeded cells for 24h; cell viability was finally assessed using the Alamar Blue assay. For direct cytocompatibility assays, cells were seeded onto the decellularized scaffold, cultured for 16 days, and then differentiated into adipocytes for two weeks. Cell metabolic activity was determined at days 1, 3, and 7 by the Alamar Blue test. Qualitative Live and Dead assay (LD) was performed to evaluate cell viability at day 7 and the end of differentiation. Finally, adipogenic differentiation was tested by Oil Red O staining (OROs). As for indirect cytotoxicity assay, cell viability remained above the lower limit of 70%, with no significant differences between time points. Regarding direct cytocompatibility, no differences in cellular metabolic activity was found between days 1, 3, and 7, suggesting that the cell population remained stable over 7 days. LD assay at day 7 showed viable cells, especially in the concave depressions of the leaf. At the end of differentiation, LD assay confirmed cell viability, OROs demonstrated adipogenic differentiation, highlighting lipid droplets content in the cells. In conclusion, decellularized cabbage leaves were non-cytotoxic and provided a suitable scaffold for adipogenic differentiation. The next phase of the research will shift from murine cell line to primary mammalian cells to develop edible adipose structures for potential applications in cultured meat production.