Replicating the fat component of meat is a critical challenge in the cultured food industry. Chitosan hydrogels are widely used in food industry and as scaffold for fat tissue mimicry, with some limitations in terms of the residuals of organic acids commonly used for wet processing compromising 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. Acid-free chitosan hydrogels were prepared by means of an initial dissolution of chitosan in aqueous organic acid, subsequent neutralization, and re-dissolved in water by introducing carbon dioxide (CO₂). Upon CO₂ evaporation, acid-free hydrogels (CO₂-chitosan) were obtained, effectively eliminating residual acids and resulting in homogeneous materials. The obtained hydrogels remained stable in cell culture conditions for over 9 days, with a moderate weight loss (~35%). Rheological characterization revealed reduced viscosity and storage modulus compared to conventional hydrogels, indicating improved water interaction and extrudability due to carbonic acid-induced protonation. Hydrogels showed shear thinning behavior, making them suitable for the standard cell seeding protocol, and a potential (bio)ink. A three-step oscillation test confirmed that post-printing CO₂ evaporation restored the mechanical properties to levels comparable with conventional chitosan hydrogels, potentially minimizing shear-induced cell damage (Fig. 1a-b). Biological characterization revealed cell viability exceeding 70% in indirect cytotoxicity test, confirming the absence of acidic residues in the hydrogel. 3T3-L1 preadipocytes have been homogeneously seeded within the hydrogels by standard mixing with syringes, and showed good cell viability. A second innovative approach involving the encapsulation of pre-differentiated adipocytes pellets, was explored to overcome current limitation in functional adipose tissue production: metabolic activity, assessed via the Alamar Blue assay, increased over time, indicating good cell viability (Fig 1c). To confirm the maintenance of the adipocyte phenotype, Oil Red O staining was performed, and a semiquantitative test indicated sustained lipid droplet formation increasing over time (Fig. 1d). This study presents an efficient method for producing cultured fat ingredients, using acid-free chitosan hydrogels, overcoming current limitations and improving biofabrication properties. Future research will focus on the integration of animal-derived stem cells and incorporation of additives into chitosan to improve cell adhesion and consumer-relevant attributes. Authors acknowledge “Food for future: 3D plant-derived structures to produce adipose tissue as innovative food ingredients for cultured meat” PRIN 2022, 2022APBX8X.
PREPARATION AND CHARACTERIZATION OF ACID-FREE CHITOSAN HYDROGELS AS SCAFFOLD FOR FAT CULTURED INGREDIENTS
Beatrice Sottini;Andrea Fiorati;Luigi De Nardo;Lina Altomare
2025-01-01
Abstract
Replicating the fat component of meat is a critical challenge in the cultured food industry. Chitosan hydrogels are widely used in food industry and as scaffold for fat tissue mimicry, with some limitations in terms of the residuals of organic acids commonly used for wet processing compromising 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. Acid-free chitosan hydrogels were prepared by means of an initial dissolution of chitosan in aqueous organic acid, subsequent neutralization, and re-dissolved in water by introducing carbon dioxide (CO₂). Upon CO₂ evaporation, acid-free hydrogels (CO₂-chitosan) were obtained, effectively eliminating residual acids and resulting in homogeneous materials. The obtained hydrogels remained stable in cell culture conditions for over 9 days, with a moderate weight loss (~35%). Rheological characterization revealed reduced viscosity and storage modulus compared to conventional hydrogels, indicating improved water interaction and extrudability due to carbonic acid-induced protonation. Hydrogels showed shear thinning behavior, making them suitable for the standard cell seeding protocol, and a potential (bio)ink. A three-step oscillation test confirmed that post-printing CO₂ evaporation restored the mechanical properties to levels comparable with conventional chitosan hydrogels, potentially minimizing shear-induced cell damage (Fig. 1a-b). Biological characterization revealed cell viability exceeding 70% in indirect cytotoxicity test, confirming the absence of acidic residues in the hydrogel. 3T3-L1 preadipocytes have been homogeneously seeded within the hydrogels by standard mixing with syringes, and showed good cell viability. A second innovative approach involving the encapsulation of pre-differentiated adipocytes pellets, was explored to overcome current limitation in functional adipose tissue production: metabolic activity, assessed via the Alamar Blue assay, increased over time, indicating good cell viability (Fig 1c). To confirm the maintenance of the adipocyte phenotype, Oil Red O staining was performed, and a semiquantitative test indicated sustained lipid droplet formation increasing over time (Fig. 1d). This study presents an efficient method for producing cultured fat ingredients, using acid-free chitosan hydrogels, overcoming current limitations and improving biofabrication properties. Future research will focus on the integration of animal-derived stem cells and incorporation of additives into chitosan to improve cell adhesion and consumer-relevant attributes. Authors acknowledge “Food for future: 3D plant-derived structures to produce adipose tissue as innovative food ingredients for cultured meat” PRIN 2022, 2022APBX8X.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
