Cell line development from food-relevant aquatic species

Current challenge

While developing cell lines and establishing mechanisms for increasing access to existing lines is a challenge for cultivated meat generally, aquatic species present additional obstacles. While just three species account for the majority of terrestrial animal meat consumption, there are hundreds of aquatic animal meats consumed – with the top 10 seafood species consumed in the U.S. accounting for only 77% of consumption. The number and diversity of aquatic species that can be used for cultivated seafood is substantially higher than land animal species for cultivated meat. Aquatic animal cell biology has also received limited research funding and attention compared to terrestrial vertebrates. Cell culture techniques for aquatic animal cells still need to be developed and fully optimized, and our understanding of the basic biology of these cells needs to be improved. 

There are few food-relevant aquatic cell lines publicly available

While numerous continuous fish cell lines have been reported over the years, reports of the types of lines most relevant for cultivated seafood are few and far between (Bols et al. 2023) because biomedical research has focused on different species (e.g., zebrafish) and aquaculture research has focused on food-irrelevant cell types. Many of the reported lines that might be useful for cultivated seafood are unavailable from public repositories. Recently, myogenic (Saad et al. 2023, mackerel; Li et al. 2021, goldfish) and myofibroblastic lines (Chong et al. 2022, Australasian snapper) have been described, as well as a fibroblast-like line (Tsuruwaka et al. 2022, filefish) with the capacity to differentiate to meat-relevant cell types. Development of induced pluripotent stem cells (iPSCs) from fish has—thus far—been limited to zebrafish (Xu et al. 2021), and footprint-free reprogramming methods (Rao and Malik 2012) have not been developed. Even less knowledge and fewer cell lines exist when it comes to aquatic invertebrates. For example, the isolation of primary muscle cells from lobster was only recently reported for the first time in the academic literature (Jang et al. 2022).

Aquatic cell biology understanding and tool availability are limited

The lack of cell lines and cell culture methodology for aquatic species stems from a limited understanding of which cell types exist in fish and aquatic invertebrates, the boundaries between types, what cues trigger their development, and which fine-scale cell type differences are relevant in a food context. For example, it is unclear whether an equivalent population to mammalian fibro-adipogenic progenitors (Dohmen et al. 2022) exists in aquatic animals, and while there is some limited evidence that cells from teleost fish may readily transdifferentiate to take on characteristics of other cell types (Saad et al. 2022, Tsuruwaka et al. 2022), it is unclear whether this is true across aquatic species more broadly. In addition, there is no toolset for researchers to use to advance these discoveries. Critical tools like genome annotations, PCR primers, gene delivery systems, and antibodies for fish tissues are under-developed. Therefore, developing appropriate cell lines from aquatic species is more complicated than it should be, and researchers working on downstream questions in cultivated seafood — such as media optimization — lack the necessary tools.

Proposed solution

Furthering basic understanding of aquatic cell biology

Exploratory academic research to grow the currently limited understanding of fish and aquatic invertebrate cell cultures will greatly aid in defining the unique parameters of cultivated seafood cell lines that may require dedicated and specific attention, while also highlighting areas of commonality with other cultivated meat cell lines. Research should be conducted to improve understanding of which cell types exist, which marker genes are reliable, which differentiation cues are effective in aquatic cell lines, and which culture environments are conducive for proliferation and differentiation. While building out robustness of the research toolkit to enable the study of food-relevant aquatic species is critical, there are some interim solutions that can be used to close the gap — such as using tools like EggNogMapper for incomplete genome annotated species and alternative identity methods like lectin staining (Zayas et al. 2011) when antibodies are not available. 

Efforts in bioprospecting to increase the diversity of biological specimens considered in aquatic cell line development could be particularly valuable in identifying the unique parameters of cultivated seafood cell lines; an increased number and variety of aquatic cell lines would facilitate parameter grouping while also establishing a foundation of cells that may be useful for the variety of needs required to develop cell lines from food-relevant aquatic species. For example, these efforts may yield insights as to how the temperature-size rule, which generally states that fish in warmer water grow faster (Lindmark et al. 2022), translates to growth rates of cells in culture for cultivated seafood utility. 

Incentives for knowledge and product sharing for companies in adjacent domains, such as those responsible for breeding lines of high-productivity seafood (Nuetzel et al. 2023), would also be advantageous in understanding and developing high-productivity cell lines. Freshwater and marine aquaculture strives to improve food security, help rebuild protected species and habitats, and grow sustainably. The large overlap in mission with cultivated seafood presents opportunities for values-driven collaboration.

Developing isolation protocols for aquatic species

Development of isolation protocols is a critical prerequisite to robustifying cell line development and particularly important for increasing availability of cell lines from the highly understudied aquatic invertebrates. Fungal and bacterial contamination has been problematic for initiation and stabilization of primary cell cultures for aquatic species, particularly with crustaceans (Fan et al. 2002). Isolation protocols for aquatic species should consider the vast and diverse commensal relationships with other organisms that can drive these contaminations. 

Higher throughput isolation methodologies may help meet the need for a large number of cell lines to respond to the need for cultivated seafoods from a large number of aquatic species. Advances in high-throughput and automated cell line development from adjacent industries could be particularly valuable in maximizing the return on effort in isolation and selection (Tejwani et al. 2021).

Focusing on cell line repositories

Continued development of central cell line repositories, such as those started by ATCC, Extracellular, and Kerafast, will be integral in improving access to the cell lines developed as the outcome from the exploratory research efforts. This is highlighted in a recent cell line survey report as generally important for cultivated meat, but particularly needed for aquatic species. Policy and incentives to encourage industry companies to share their ready-to-use cell lines more broadly with academic R&D would accelerate this process.

A continuous mackerel muscle cell line, MACK1, was recently reported (Saad et al. 2023), representing a breakthrough in food-relevant aquatic species. The MACK1 cell line is available for purchase on Kerafast within the same calendar year as being publicly reported. This represents an important behavior in the cultivated seafood research community and should be further encouraged. The speed from public disclosure to marketplace availability was possible because of the (expedited push from the academic lab and) decentralized nature of the repository, which allows individual research labs to list offerings on the repository.

Academic R&D-based repositories, such as the Phaff Yeast Culture Collection, the Plant Cell Culture Library, and the Indian National Repository of Fish Cell Lines, serve as models that can also be translated into the cultivated seafood space to fuel academic R&D and industry growth. These repositories are often structured as not-for-profit and both centralized and standardized in their offerings. This standardization can drive accessibility by limiting the range of reagents used in the preservation culture medium that researchers need to replicate during adaptation and providing consistency in growth rate reporting that enables like-to-like comparisons between lines. 

Anticipated impact

Advances in cell line development enable more effective and efficient downstream research. An understanding of the relevant cells and their behaviors establishes a basis for building a production platform around them. There are still open questions around production fundamentals for cultivated meat from aquatic species (Rubio et al. 2019), including formulating animal-free culture medium and understanding optimal culture conditions and bioreactor configurations.

Bioprospecting efforts to establish a broad range of cell lines for research and commercialization purposes will have the greatest impact if conducted soon. There are enormous declines in fish species populations with, for example, a third of all freshwater species threatened with extinction and genetic diversity of exploited fish species declining (Petit-Marty et al. 2022). These declines drive the urgency of bioprospecting for cultivated seafood for two primary reasons: 1) this would help to identify a pathway to commercial value for these species that may help drive conservation efforts, and 2) the genetic diversity and associated commercial value should be captured as an asset for cultivated seafood before it is lost.

Integration efforts between cultivated meat and cultivated seafood research and industry communities to identify synergistic areas between cultivated meat and cultivated seafood (e.g., culture media development) would enable cost-benefit analysis for defining a path to commercial success that relies initially on minimal cultivated seafood-specific research requirements. This could result in a mutually beneficial outcome and help to bolster perceptions of the cultivated seafood commercial outcome and fuel additional academic and industry R&D.