Reprocessing of seafood waste: a challenge to develop aquatic clean meat from fish cells

Obtaining cell material from fish fins

Cells were obtained from the fin of a filamentous filefish Stephanolepis cirrhifer (Kawahagi in Japanese name) (Fig. 1). The cell was about 20-50 μm in size. Subculture was then performed and stable fibroblast-like cells were obtained at the fifth passage (Fig. 2a, Movie 1). We named the fibroblast-like cells ‘deSc’ (endifferentiated Stephanolepis Cirrhifer). The deSc cells were cultured up to 350 passages without CO₂, and they were immortalized cells. To verify that no chromosomal mutation had occurred in the deSc cells, Q-banding staining analysis was performed and compared to wild-type S. Cirrhiferthose of Murofushi et al.²⁶. Thirty-three chromosomes (2n = 30 + X₁x₂Y) were found in 96% of deSc cells, which was identical to wild type S. Cirrhifer (Supplementary Fig. 1). Four percent of the deSc cells showed 66 chromosomes; They could have been in the midst of cell division with replicated DNA.

Fig. 1: Migratory cells from the fin tissue explants of S. Cirrhifer.

**Fig. 2: deSc cells differentiated into different cell types.**

Differentiation potential of fish fibroblast-like cells

To investigate the optimal culture conditions for the deSc cells, we examined several culture media, serum and extracellular matrix (ECM) and discovered very interesting properties (Table 1). The cells changed their morphology differently depending on the combinations of culture media, serum and ECM (Table 1, Fig. 2b, Movie 2). Of the various results, we focused on the neural-like cells that differentiated their morphology under fewer culture factors, i.e. no serum (L-15 medium only) in an uncoated flask (Fig. 2c, Movie 3).

Table 1 Differentiation of deSc cells under different combinations of culture medium, serum, flask and additive.

The basal state of ES cells in the absence of neural differentiation inhibitors such as serum and transcription factor assumed a neural fate²⁴. The deSc cells cultured under the same conditions also differentiated into neural-like cells within 24 h. This result led us to speculate that the deSc cells would have the potential for neural differentiation, just as if the cells had acquired pluripotency through the dedifferentiation process. To demonstrate this hypothesis, we first attempted to induce neural differentiation directly using KBM Neural Stem Cell Medium (Kohjin Bio Co., Ltd.) and Neural Induction Supplement (Thermo Fisher Scientific). The results showed that neurofilaments were formed with a maximum length of 465 μm and an average strain rate of 45.71 μm/h (Fig. 2d, Movie 4, Table 1). Neural immunofluorescence indicated that the fin cells were practically differentiated into neural cells (Fig. 3). These results showed that the deSc cells have the property of a practicable direct differentiation only with culture medium components. We have succeeded in inducing neuronal differentiation, the basal state of stem cells, both through the presence/absence of serum and through direct differentiation. Next, we examined the differentiation of deSc cells under stimulation with different sera.

**Fig. 3: Immunofluorescence of neural deSc cells.**

Cell differentiation with different sera

In human iPSCs, culturing with various mammalian sera is reported to affect cell proliferation, differentiation, gene expression, and stability of transcriptomes^27.28. Next, we examined cell differentiation with serum shock. First, we evaluated the salmon serum SeaGrow, which was in the same taxonomic group as the deSc cells. Granule-like particles appeared intracellularly in the deSc cells five hours after stimulation with SeaGrow. Another three hours later, the cell morphology became round and larger, which were adipocyte-like cells with 0.5-2.0 μm white droplets (Fig. 4a, Movie 5). In order to examine the white droplets in detail, the cells were stained with Oil Red O or BODIPY and additionally analyzed by gas chromatography. The results showed that the white droplets were fat droplets (Supplementary Fig. 2). Therefore, the use of SeaGrow in the culture media led to the differentiation of the deSc cells into adipocytes.

**Fig. 4: Differentiation of deSc cells into (a) adipocytes, (b) spheroids and (c) CoCoon.**

Next, we examined several mammalian sera other than FBS in cell culture. In one rabbit and one sheep serum, the deSc cells either did not survive or did not show stable and uniform states of differentiation (data not shown).

Three-dimensional culture aimed at cell shaping and design

Organisms are made up of various tissues, such as bone, cartilage, muscle, and skin, built on living scaffolds. By this time, the process of transforming differentiated cells into a tissue body was of significant importance. A three-dimensional spheroid has been described as an important in vitro model in terms of function and organization similar to biological tissue²⁹. The deSc cells formed a spheroid with 3D culture, which was also observed in mammalian cells (Fig. 4b, Movie 6). The spheroids, between 20 and 300 μm in size, were formed by uptake of the surrounding cells. Stimulating the cells with horse serum resulted in colonies, which were cell aggregates, attached to the culture flask (Fig. 4c, Movie 7). We named these colonies “CoCoon”. The CoCoon migrated in the culture flask at an average speed of 38.46 μm/h and moved by merging with the surrounding CoCoon essentially every 3.5 h (Movie 7). The colonies were 20–1000 μm in diameter and the largest could be visualized macroscopically. In addition, our observations confirmed that the colonies in culture were stable for at least three weeks, as was seen in spheroid culture (data not shown). Our results suggested that the fish cells could be cultured in 3D both adherently and in suspension.

This 3D cultivation and the various cell differentiations, such as spheroids, cocoon and skeletal muscle-like cells as well as adipocytes, were reversible processes except for neural differentiation. In other words, the differentiated deSc cells reversed their morphology to fibroblast-like cells when the basic culture conditions were restored: L-15 medium with 10% FBS in a collagen I-coated flask (data not shown). From these results, the processes of differentiation and 3D culturing turned out to be easy since the triggers were the culture media, sera and ECM. Furthermore, by exploiting the simplicity of cell aggregation and processing, we set ourselves the challenge of preparing a cultured meat from normal deSc cells. The deSc cell layer was obtained by further culturing the normal deSc cells after they reached their confluent state at a 25-75 cm stage² Culture vessels with collagen I-coated surface to promote cell adhesion and proliferation.

deSc cells could be cultured and stacked in multiple layers like a sheet (Fig. 5a). We also succeeded in creating an adipocyte cell layer under the adipocyte differentiation culturing method (Fig. 5b). Therefore, it was suggested that the differentiation function observed in single deSc cells was not lost even after the cells formed a sheet structure. Then, for aquatic clean meat, the multilayered deSc cells shrank when the edges of the flask were gently poked with a spatula to detach the cells (Movie 8). The deSc cell layer was shaped like a fish meat sashimi, which at the prototype stage produced aquatic clean meat approximately 70 mm long, 30 mm wide and 2 mm thick (Fig. 5c). A simple sensory test conducted on the artificial sashimi revealed the following characteristics: 1) the color was white, 2) there was no odor (no “fishy” odor normally caused by bacteria), 3) there was no flavor, 4) the texture was smooth, and 5) the firmness was soft. The shape and size of the aquatic clean flesh were flexible. As it is still very different from real sashimi, it needs to be further improved. However, we managed to accumulate tiny 20 μm cells to produce edible sashimi on a laboratory scale, suggesting that fish cells have the potential to support food sustainability (Fig. 6).