Obtaining cellular materials from fish fins
The cells were obtained from the fin of a thread-sail filefish Stephanolepis cirrhiferous (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’ (ofdifferentiated Stephanolepis vsirrigate). deSc cells had been cultured for up to 350 passages without CO2, and they were immortalized cells. To verify that a chromosomal mutation had not occurred in deSc cells, Q band staining analysis was performed and compared to wild type S. cirrhiferouswhich had been reported by Murofushi et al.26. Thirty-three chromosomes (2n = 30 + X1X2Y) were found in 96% of Sc cells, which was identical to wild type S. cirrhiferous (Supplementary Fig. 1). Four percent of deSc cells showed 66 chromosomes; they could have been in the middle of cell division with replicated DNA.
Differentiation power of cells resembling fish fibroblasts
To explore optimal culture conditions for deSc cells, we examined several culture media, serum, and extracellular matrix (ECM), and found some very interesting features (Table 1). Cells changed morphology variably depending on the combinations of culture media, serum, and ECM (Table 1, Figure 2b, Movie 2). Among the various results, we focused on neuronal-like cells, which differentiated their morphology under fewer culture factors, i.e. serum-free (L-15 medium only) in an uncoated flask (Fig. 2c, Movie 3).
The basal state of ES cells in the absence of neuronal differentiation inhibitors such as serum and transcription factor adopted a neuronal fate24. deSc cells cultured under the same conditions also differentiated into neuronal-like cells within 24 h. This result led us to speculate that the deSc cells had the potential for neural differentiation, even if the cells had acquired pluripotency through the process of dedifferentiation. To demonstrate this hypothesis, we first attempted to induce neural differentiation directly with KBM Neural Stem Cell Medium (Kohjin Bio Co., Ltd.) and Neural Induction Supplement (Thermo Fisher Scientific). The results showed that neurofilaments formed with a maximum length of 465 μm and an average elongation rate of 45.71 μm/h (Fig. 2d, Movie 4, Table 1). Neural immunofluorescence suggested that the fin cells were practically differentiated into neural cells (Fig. 3). These results demonstrated that deSc cells possess the characteristic of direct differentiation practicable only with the components of the culture medium. We succeeded in inducing neural differentiation, which is the basal state of stem cells, both by the presence/absence of serum and by direct differentiation. We then studied the differentiation of Sc cells under stimulation with different sera.
Cell differentiation with different sera
In human iPS cells, culture with different mammalian sera would affect cell proliferation, differentiation, gene expression and transcriptome stability27.28. We then examined cell differentiation with serum shock. First, we evaluated SeaGrow salmon serum, which belonged to the same taxonomic group as the cells of Sc. Granule-like particles appeared intracellularly in Sc cells five hours after stimulation with SeaGrow. Three hours later, the cell morphology became round and larger in size, which were adipocyte-like cells with 0.5–2.0 μm white droplets (Fig. 4a, Movie 5). To examine the white droplets in detail, the cells were stained with Oil Red O and BODIPY, respectively, and also analyzed by gas chromatography. The results showed that the white droplets were fat droplets (Supplementary Fig. 2). Therefore, the use of SeaGrow in culture media resulted in the differentiation of Sc cells into adipocytes.
We then examined several non-FBS mammalian sera in cell culture. With rabbit and sheep serum, deSc cells did not survive or exhibit stable and uniform states of differentiation (data not shown).
Three-dimensional culture aimed at cell casting and design
Organisms are made up of various tissues such as bone, cartilage, muscle, and skin, which are established on living scaffolds. At this stage, the process of transforming differentiated cells into a tissue body was very important. A three-dimensional spheroid has been reported as an important in vitro model in terms of functions and organization similar to those of biological tissues29. deSc cells formed a spheroid with 3D culture, which was also observed in mammalian cells (Fig. 4b, Movie 6). Between 20 and 300 μm in size, spheroids were formed by absorbing surrounding cells. Stimulation of cells with horse serum yielded colonies that were aggregates of cells adherent to the culture flask (Fig. 4c, Movie 7). We named these colonies ‘CoCoon’. The CoCoon migrated through the culture flask at an average speed of 38.46 μm/h and moved largely every 3.5 h merging with the surrounding CoCoon (Movie 7). The colonies varied in size from 20 to 1000 μm in diameter, and it was possible to macroscopically visualize the larger ones. Additionally, our observations confirmed that colonies were stable in culture for at least three weeks, as was also observed in spheroid culture (data not shown). Our results suggest that fish cells could be cultured in 3D both in adhesion and in suspension.
This 3D culture and the different 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 baseline culture conditions were restored: L-15 medium with 10% FBS in a collagen I coated flask (data not shown ). From these results, differentiation processes and 3D culture were found to be straightforward as the triggers were culture media, sera and ECM. Additionally, using the ease of cell aggregation and processing, we then challenged to create cultured meat from normal deSc cells. The deSc cell sheet was obtained by continuing to culture the normal deSc cells after they reached their confluent state in a space of 25-75 cm.2 culture utensils with surface coated with collagen I to promote cell adhesion and proliferation.
The deSc cells could be cultured and stacked in multiple layers like a sheet (Fig. 5a). We also succeeded in creating a sheet of adipocyte cells according to the adipocyte differentiation culture method (Fig. 5b). Therefore, it was suggested that the differentiation function that was observed in single SC cells was not lost even after the cells formed a sheet structure. Then, the cells of multi-layered sc for aquatic clean meat shrank when the edges of the flask were gently pressed down with a spatula to detach the cells (Movie 8). The deSc cell sheet was shaped like fish meat sashimi, producing aquatic clean meat about 70 mm long, 30 mm wide and 2 mm thick at the prototype stage (Fig. 5c). A simple sensory test performed with the artificial sashimi found the following characteristics: 1) the color was white, 2) there was no smell (no fishy smell, which is usually caused by bacteria), 3) there was no taste, 4) the texture was smooth, and 5) the firmness was mild. The shape and size of the aquatic clean meat was flexible. Since it was still very different from real sashimi, it will still need to be improved. However, we managed to accumulate tiny 20 μm cells to produce edible sashimi at the laboratory level, suggesting that fish cells have the potential to support food sustainability (Fig. 6).