Abstract
Cellular agriculture (CellAg) is a rapidly growing field of research focused on the production of agricultural products through cellular culture. To produce cultivated meats that mimic traditional meat, CellAg applies tissue engineering techniques in which muscle cells are cultured on a scaffold. Research in CellAg has expanded to the development of cultivated fish, however the variation in skeletal muscle structure and growth across fish species presents a challenge for the development of biomimetic products. Another major challenge in CellAg is sourcing edible and cost-effective scaffolds that support muscle cell cultivation. Here, a biomimetic approach was applied to the cultivation of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon), two species of commercial importance in New Zealand. Specifically, this research focused on the development of scaffolds that mimic the composition and natural structure of Australasian snapper and Chinook salmon muscle. It was hypothesised that scaffolds for cultivated Australasian snapper and Chinook salmon would have different requirements due to species-specific differences in muscle structure.
Firstly, the structure and growth of early to late juvenile Australasian snapper and Chinook salmon muscle were characterised, revealing distinct muscle growth patterns. A shift from hyperplasia to hypertrophy was observed from early to late juvenile stages in Australasian snapper, while Chinook salmon displayed substantial contributions of both hyperplasia and hypertrophy across juvenile stages. Furthermore, species-specific differences in muscle cellular composition were observed. These findings highlighted the disparities in skeletal muscle growth and composition across fish species. Collagen was prevalent in the extracellular matrix (ECM) of both Australasian snapper and Chinook salmon muscle, with intramuscular accumulation observed from early to late juveniles in both species. The abundance of ECM, and specifically collagen, in the myosepta indicated that biomimetic scaffolds for both species should be structured as thin sheets or films.
These observations led to the extraction of native collagen from Australasian snapper and Chinook salmon skin, waste products of the fishing industry, for use as biomaterials for scaffold production. Australasian snapper and Chinook salmon collagen type I exhibited different biophysical characteristics, with self-assembled hydrogels forming from the former but not the later. Hydrogel stiffness and microfibre bundling were shown to increase with Australasian snapper collagen and sodium chloride concentrations, respectively. Ultraviolet (UV) irradiation and dehydrothermal (DHT) treatments crosslinked collagen within the hydrogel. While hydrogel stiffness and ductility increased with DHT crosslinking, UV crosslinking showed no effect. Microfibril merging was observed with both methods of crosslinking. Irrespective of crosslinking treatment, hydrogels demonstrated initial mass loss in fish cell culture conditions, followed by a period of stability. These findings suggest that self-assembled hydrogels, made from Australasian snapper collagen, are viable candidates for fish muscle cell cultivation. However, further optimisation of hydrogel stiffness would be required to match that of native fish muscle. As fish muscle cells were unavailable, further investigations required the use of mammalian muscle cells though under mammalian cell culture conditions, all hydrogels exhibited 100% mass loss by 72 hours. Thus, an alternative scaffolding approach was investigated.
Denatured whole chain (DWC) collagens extracted from Australasian snapper and Chinook salmon skin were used successfully to produce nanofibrous scaffolds via electrospinning. Food-safe crosslinking techniques were applied, where fructose, glucose, or citric acid were incorporated into collagen electrospinning solutions and scaffolds were heated post-electrospinning. Alternatively, electrospun collagen scaffolds containing no additional agents were treated with DHT or UV. Crosslinking was confirmed following DHT, fructose, glucose and citric acid but not UV treatments. Nanofibrous morphology was preserved following UV, DHT, fructose and glucose crosslinking but not citric acid. The DHT, fructose and glucose crosslinked scaffolds maintained stability under both fish and mammalian cell culture conditions. Further, DHT crosslinked scaffolds were cytocompatible and facilitated the differentiation of mouse C2C12 myoblasts into myotubes. Thus, crosslinked DWC scaffolds, made from Australasian snapper and Chinook Salmon collagen have potential utility for fish muscle cell cultivation.
Overall, this research revealed distinct growth patterns and cellular composition of Australasian snapper and Chinook salmon skeletal muscle. Collagens isolated from these species also displayed distinct properties as biomaterials that impacted their use in scaffold fabrication. These findings highlighted the importance of considering species specificity for the cultivation of fish muscle. Furthermore, biomimetic scaffolds mimicking fish myosepta were successfully produced and shown to support muscle cell differentiation, indicating utility in cultivating Australasian snapper and Chinook salmon muscle for CellAg.