A novel approach to prepare acid-free chitosan hydrogels as scaffold for fat cultured food ingredients

Introduction Cultured ingredients represent a promising alternative to traditional meat products. However, replicating fat component remains the major challenge. Chitosan hydrogels are widely used in food industry and as scaffold for fat tissue mimicry, as able to prevent lipid oxidation. Chitosan main limitation is represented by the residuals of organic acids commonly used for wet processing which compromise cell viability, organoleptic properties, and product safety. This work investigates a novel approach to fabricate cell-loaded acid-free chitosan hydrogels for cultured fat ingredients, by exploiting water carbonation reversibility, based on Sakai et al. method (1). Materials and Methods Acid-free chitosan hydrogels were prepared by means of an initial dissolution of chitosan in aqueous organic acid, subsequent neutralization, and the resulting gel dissolution in water by introducing carbon dioxide (CO₂). Water carbonation reversibility, and CO₂ evaporation facilitated the removal of acidic residues. Hydrogels stability and rheological properties were studied. 3T3-L1 preadipocytes have been seeded into the hydrogels enriched with DMEM powder by standard mixing (with syringes and three-way Luer Stopcock), as well as by using a novel approach based on embedding pre-differentiated adipocyte pellets: cell viability and lipid droplets production have been evaluated. Results and Discussion The method based on Sakai et al. proved to be effective for the preparation of homogeneous acid-free chitosan hydrogels (CO₂-chitosan). When exposed to a cell culture environment, these hydrogels remained stable for up to 9 days, exhibiting a weight loss of approximately 25% at early time points, in agreement with literature data (2). The rheological properties of CO₂-chitosan hydrogels were analyzed and compared to standard chitosan ones. As shown in Figure 1, CO₂-chitosan hydrogels exhibited significantly lower viscosity and storage modulus values, confirming that CO₂ increases interactions with water and improves extrudability. This effect is attributed to the protonation of chitosan chains due to carbonic acid formation. Hydrogels showed shear thinning behavior, making them suitable for the standard cell seeding protocol employed in this study (Figure 1a). Furthermore, these findings suggest the potential application of CO₂-chitosan hydrogels as a (bio)ink for 3D-(bio)printing. This aspect was further investigated through three-step oscillating test (Figure 1b). It was demonstrated that CO₂-chitosan hydrogels restore their initial rheological properties within minutes after printing. Additionally, as CO₂ evaporates post-printing, the hydrogels reach rheological properties comparable to conventional chitosan hydrogels ones, reinforcing their suitability for biofabrication (Figure 1b). The use of acid-free chitosan hydrogels, with such viscoelastic properties, enables a more homogeneous distribution of shear stress, reducing potential damage to cells (3). Indirect cytotoxicity tests revealed cell viability exceeding 70%, confirming the absence of acidic residues in the hydrogel. 3T3-L1 preadipocytes were homogeneously seeded within the hydrogels, and metabolic activity, assessed via the Alamar Blue assay, increased over time (Figure 2a), indicating good cell viability. This was further corroborated by qualitative Live/Dead staining, which demonstrated a high percentage of viable cells within the hydrogel (93 ± 8%) (Figure 2b). A second, innovative seeding approach was explored, involving the encapsulation of pre-differentiated adipocyte pellets within acid-free hydrogels. This strategy aims to overcome current limitations in the production of functional adipose tissue for cultured food ingredients, in terms of cell proliferation and differentiation capacity (4). Viability assays using Alamar Blue showed an increase in metabolic activity and cell viability over the time. To confirm the maintenance of the adipocyte phenotype, Oil Red O staining was performed, and the results indicated sustained lipid droplet formation (Figure 2c-d). These findings demonstrated that the pellet-based seeding approach supports cell aggregation and lipid accumulation within acid-free chitosan hydrogels, highlighting its potential applications in cultured food ingredients field. Conclusions This study presents an efficient method for producing cultured fat ingredients, using acid-free chitosan hydrogels, overcoming current limitations and improving biofabrication properties. A novel cell seeding approach with pre-differentiated adipocyte pellets enhances cell viability and adipogenic phenotype maintenance. Future research will focus on utilizing animal-derived stem cells and incorporating additives into chitosan to enhance cell adhesion and consumer-relevant properties. Acknowledgement: "Food for Future: 3D plant‐derived structures to produce adipose tissue as innovative food ingredient for cultured meat", PRIN 2022, 2022APBX8X.