Introduction
The global collapse of some fisheries and ongoing challenges in aquaculture highlight the urgent need for alternative sources of marine animal proteins. Cellular aquaculture - the production of these proteins from cells rather than whole animals - offers a sustainable and ethical solution to mitigate seafood shortages by reducing natural resource use and environmental impact. However, key challenges remain, particularly the development of robust marine animal cell lines, the formulation of animal-free culture media, and the reduction/upcycling of waste effluents through circular cultivation systems.
Results
To address the scarcity of marine animal cell lines, we established and characterized lines from the meagre Argyrosomus regius, a commercially valuable species with limited aquaculture production. Cells were obtained from explant cultures of vertebra (VAR3), branchial arch (BAAR1) and jaw (JAR5) tissues using established protocols and propagated in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (FBS). Cultures were maintained for over 100 passages. Growth of spontaneously immortalized lines (>60 passages) was optimized by reducing serum concentrations and adjusting incubation temperatures. Growth curves, population doubling times (PDT), biomass yield, and differentiation potential into myogenic and adipogenic lineages were evaluated, and preliminary results indicate reduced FBS requirements without compromising growth, enhanced proliferation at 33°C, with observed PDTs of 11.1 ± 0.5 h for VAR3, 25.2 ± 0.5 h for BAAR1, and 19.9 ± 0.7 h for JAR5, and successful lipid accumulation throughout adipogenic differentiation (Figure 1).
To enhance the sustainability of cellular aquaculture, we developed a circular bioprocess that integrates microalgae into the cell culture system. This approach addresses two major limitations: the dependence on animal-derived components and the generation of nutrient-rich waste. First, we evaluated the use of a microalgae-derived serum (MS) as a functional substitute for fetal bovine serum (FBS) in culture media. Results demonstrated that fish (MS4 and MS5; Figure 2A) and mammalian (MS3; Figure 2B) cell lines maintained comparable proliferation rates with up to 75% and 99.6% replacement of FBS by MS, respectively. These findings highlight the potential of microalgal-derived nutrients to effectively supply essential growth-supporting factors
Then, we explored the potential of spent cell culture medium as a substrate for microalgae cultivation. Microalgae cultured in spent medium exhibited significantly enhanced growth, achieving a final dry weight of 2.468 ± 0.413 mg/mL by day 7, compared to 0.915 ± 0.436 mg/mL in control medium. Moreover, microalgae grown in spent medium displayed a significantly higher nitrogen content (6.03 ± 2.73%) than those in control medium (1.82 ± 0.25%). Cultivation in spent medium also resulted in substantial reductions in residual nutrients - including ammonia, nitrites/nitrates, and phosphates -indicating efficient nutrient uptake and bioremediation (Table 1). These findings underscore the capacity of microalgae to valorize nutrient-rich effluents and contribute to the sustainable recycling of cell culture medium waste.
Conclusion
Our results demonstrate the feasibility of combining marine animal cell culture with microalgal systems to foster a circular, zero-waste model for cellular aquaculture. Together, the establishment of robust meagre cell lines and the integration of microalgae-based bioprocessing lay the foundation for a scalable, ethical, and resource-efficient platform for the production of cultivated seafood.
Funding
This work was funded by (1) the Good Food institute | EIT Food and the Cultivated Meat Innovation Challenge through µCELLAQUA project (grant no. 32702593), (2) the European Commission and Horizon Europe programme through FEASTS project (grant no. 101136749; call HORIZON-CL6-2023-FARM2FORK-01), (3) the Algarve2030, Portugal2030, and the European Union through INOVACEL project (grant no. 17904) and (4) National funds of the Portuguese Foundation for Science and Technology (FCT) through projects UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020. JR and BC received support from the FCT through the Scientific Employment Stimulus Institutional Call (grant no. CEECINST/00114/2021/CP1780/CT0001) and the PhD Fellowship programme (grant no. 2022.12615.BDANA), respectively.