In the past decade, due to sustainability issues associated with traditional meat production, including greenhouse gas emissions, resource consumption, animal welfare and food safety, cell cultured meats are becoming an alternative technology to partially replace the traditional livestock industry in meat production. The composition and structure of tissue-like cell cultured meat are similar to real muscle tissue, mainly comprising mostly adipocytes, and aligned muscle cells. Several meat tissues of livestock such as cow¹ and pig² have been successfully constructed by using 3D bioprinting technology. Seafood is favorite food for many people because of its taste and rich of various proteins, omega-3 fatty acids, and micronutrients. The increase of population, coupled with environmental stress and climate-change, has led to the overexploitation of marine food resources in recent decades, which has had enormous impacts on ecosystem³. Therefore, many aspects, such as scientific assessment and policy management, as well as innovative technologies (for instance: cell cultured fish meat), are urgent for the sustainability of seafood production. Cell cultured fish meats will be not only to release the pressure of traditional fishing and aquaculture industries for world population, but also to cope with the malnutrition by controlling the synthetic technologies for high-quality proteins and micronutrients. The production of tissue-like cultured fish fillets is still a great challenge, because only few studies have focused on the textures of fish muscle tissues and supporting materials of scaffold for fish cell adhesion, proliferation, differentiation and fusion from 2D to 3D culture.

Figure 1. Proliferation and differentiation of large yellow croaker PSCs and PADSCs.

a, Diagrams showing that epaxial muscles and coelom walls of large yellow croaker were used to isolate the primary culture of PSCs and PADSCs respectively. Scale bars: 80 µm. b, Representative images of bright-field and immunofluorescence staining of PSCs during myogenic differentiation at day 0, 1, 6. c, Representative images of bright-field and oil red O staining of PADSCs during adipogenic differentiation at day 0, 1, 6. Scale bars: 40 µm.

In this work, we isolated the piscine satellite cells (PSCs) and piscine adipose-derived stem cells (PADSCs) from marine economic fish of large yellow croaker, and induced two types of cells to differentiate respectively into myotubes and adipocytes in differentiation mediums (Figure 1). Based on the transcriptomic analysis, the myogenic differentiation efficiency was enormously improved by two inhibitors respectively for Tgf-β and Notch signals. Comparing various compositions, we found that gelatins provided good cytocompatibility for both PSCs and PADSCs. The PSC and PADSC adhesions on fish gelatins (FG)-based gel were enforced by calcium ion at appropriate stiffness. The cell viability in the scaffolds was significantly increased by the combination of a p53-inhibitor and a Yap-activator. Similar to the 2D culture, the myogenic differentiation efficiency of 3D culture was also greatly improved by the two chemicals (inhibitors respectively for Tgf-β and Notch signals).

Our 3D scaffold was constructed in high throughput based on FG-gelatin gel with a proper gel point (~27℃), that is the key for bioprinting and maintaining fish cell viability. In a previous report, the beef steak-like cultured meat was manually assembled one by one using linear tendon-gels¹. Inspired by this, we designed a mass customization of FG-gelatin scaffold to simulate native fish muscle tissue (Figure 2). The digital analysis on the textural properties of large yellow croaker muscle tissue showed a regularity of muscle fiber and fat distributions (with a ratio of ~6:4). We extracted the main information from the epixial muscle of large yellow croaker to simplify printable model of 3D scaffold. After proliferation and differentiation culture, the aligned muscle fibers differentiated from PSCs were observed along with the scaffold. Finally, the adipocytes (differentiated PADSCs) were filled into the muscle scaffolds to form cell cultured fish fillets (Figure 3). Analysis on the composition and texture of our cultured fish fillets showed that the numbers of myotubes and adipocytes, the ratio of myofibrils and fat, hardness, gumminess, resilience, springiness, water content, etc., were similar to those of native fish muscle tissue.

Figure 2. Muscle tissue formation under guidance of biomimetic model of scaffold.

a, Schematic diagram of construction of biomimetic 3D-printed model based on native large yellow croaker muscle tissue. b, Micro-CT images of native fish epaxial muscle. Blue: Intermuscular filler tissues; Orange: Muscle tissues; Muscle scaffold model was designed from simplified native muscle texture. Scale bar: 1 mm. c, Calcein-AM fluorescence images of PSCs in scaffolds at day 3 and 10 days of proliferation. Scale bar, 100 μm. d, Representative fluorescence image of myobubes in 3D culture at days 7 of differentiation. Scale bar, 100 μm. e, Growth curve of PSCs in optimized gelatin-based scaffold.

In summary, we have successfully developed a pipeline for the production of tissue-like cultured fish fillets. This will be an advanced protocol for different fish and even other economic animal species. It demonstrates that biomimetic scaffolds based on real tissue structure have a great potential in the production of tissue-like cultured meats. For large scale bioprinting, it is difficult to lift spray flow rate and size of extrusion-based 3D printers, due to the limitation of material properties. Simultaneous extrusion of multiple-nozzles will be better choice for large scale tissue culture meat production in the next step.

Figure 3. Characterization of tissue-like cultured fish fillets.

a, Representative images of cultured and native tissue fillets of large yellow croaker. Scale bar, 5 mm. b, Average numbers of muscle cells and adipocytes in cultured fish fillets and native fish fillets with a size about 0.96 cm³. c, Muscle and fat ratios in cell cultured fish fillets and native fish fillets. d, Representative images of muscle fibers of native fish fillets and cultured fish fillets. Red: hematoxylin-eosin (HE) staining; SEM: scanning electron microscopy; Scale bar, 100 μm. e, Texture properties of empty scaffold, cultured fish fillet and native fish fillet. f, Water distribution of transverse relaxation time (T₂) spectra. “a.u.” denoted arbitrary unit.

References

1. Kang, D. H., et al. Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting. Nat. Commun. 12, 5059 (2021).
2. Zhu, H., et al. Production of cultured meat from pig muscle stem cells. Biomaterials 287, 121650 (2022).
3. Roheim, C. A., Bush, S. R., Asche, F., Sanchirico, J. N., Uchida, H. Evolution and future of the sustainable seafood market. Nat. Sustain. 1, 392-398 (2018).