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Cell-Based Fish: A Novel Approach to Seafood Production and an Opportunity for Cellular Agriculture

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Abstract and Figures

Cellular agriculture is defined as the production of agricultural products from cell cultures rather than from whole plants or animals. With growing interest in cellular agriculture as a means to address the public health, environmental, and animal welfare challenges of animal agriculture, the concept of producing seafood from fish cell- and tissue-cultures is emerging as a means to address similar challenges with industrial aquaculture systems and marine capture. Cell-based seafood - as opposed to animal-based seafood - can combine developments in biomedical engineering with modern aquaculture techniques. Biomedical engineering developments such as closed-system bioreactor production of land animal cells create a basis for large scale production of marine animal cells. Aquaculture techniques such as genetic modification and closed system aquaculture have achieved marked gains in production that can pave the way for innovations in cell-based seafood production. Here, we present the current state of innovation relevant to the development of cell-based seafood across multiple species as well as specific opportunities and challenges that exist for advancing this science. The authors find that the physiological properties of fish cell- and tissue- culture may be uniquely suited to cultivation in vitro. These physiological properties, including hypoxia tolerance, high buffering capacity, and low-temperature growth conditions, make marine cell culture an attractive opportunity for scale production of cell-based seafood; perhaps even more so than mammalian and avian cell cultures for cell-based meats. This, coupled with the unique capabilities of crustacean tissue-friendly scaffolding such as chitosan, a common seafood waste product and mushroom derivative, presents great promise for cell-based seafood production via bioreactor cultivation. To become fully realized, cell-based seafood research will require more understanding of fish muscle culture and cultivation; more investigation into serum-free media formulations optimized for fish cell culture; and bioreactor designs tuned to the needs of fish cells for large scale production.
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Cell-based fish: a novel approach to seafood
production and an opportunity for cellular
Natalie Rubio 1,2, Isha Datar 2David Stachura 3and Kate Krueger 2
1Department of Biomedical Engineering, Tufts University, Medford , MA, USA
2New Harvest, Brooklyn, NY, USA
3Department of Biological Sciences, Chico State University, Chico, CA, USA
Kate Krueger
Cellular agriculture is defined as the production of agricultural products from cell cultures
rather than from whole plants or animals. With growing interest in cellular agriculture as
a means to address the public health, environmental, and animal welfare challenges of
animal agriculture, the concept of producing seafood from fish cell- and tissue-cultures is
emerging as a means to address similar challenges with industrial aquaculture systems
and marine capture.
Cell-based seafood - as opposed to animal-based seafood - can combine developments
in biomedical engineering with modern aquaculture techniques. Biomedical engineering
developments such as closed-system bioreactor production of land animal cells create
a basis for large scale production of marine animal cells. Aquaculture techniques such
as genetic modification and closed system aquaculture have achieved marked gains in
production that can pave the way for innovations in cell-based seafood production.
Here, we present the current state of innovation relevant to the development of cell-
based seafood across multiple species as well as specific opportunities and challenges
that exist for advancing this science. The authors find that the physiological properties
of fish cell- and tissue- culture may be uniquely suited to cultivation in vitro. These
physiological properties, including hypoxia tolerance, high buffering capacity, and low-
temperature growth conditions, make marine cell culture an attractive opportunity for
scale production of cell-based seafood; perhaps even more so than mammalian and
avian cell cultures for cell-based meats. This, coupled with the unique capabilities of cru-
stacean tissue-friendly scaffolding such as chitosan, a common seafood waste product
and mushroom derivative, presents great promise for cell-based seafood production via
bioreactor cultivation. To become fully realized, cell-based seafood research will require
more understanding of fish muscle culture and cultivation; more investigation into serum-
free media formulations optimized for fish cell culture; and bioreactor designs tuned to
the needs of fish cells for large scale production.
Keywords: cellular agriculture, cell-based seafood, fish tissue culture, bioreactor, serum-free media, ocean conservation, marine cell
culture, aquaculture
The concept of producing meat from cell cultures rather than from slaughtered animal flesh has been
proposed as a means to provide nutritional muscle tissue while addressing public health, animal welfare, and
environmental issues associated with industrial animal agriculture (Datar and Betti 2010). The conversation
around this concept has centered on the growth of mammalian or avian cells and tissues to replace meat,
but cellular agriculture could easily be extended to piscine, molluscan, and crustacean cells and tissues to
replace seafoods.
Aquaculture has recently surpassed marine capture as the main source of seafood for human consumption
(Food, F.A.O. 2018). Global landings for marine capture have remained constant at around 90 million
tonnes per year since 1994, while global aquaculture of fish and shellfish has nearly doubled in the same
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© 2018 by the author(s). Distributed under a Creative Commons CC BY license.
Rubio et al. Cell-based fish: an opportunity for cellular agriculture
Figure 1.
Global seafood production by marine capture and aquaculture by year from 2011 to 2016.
Aquaculture production has increased while marine capture has remained fairly constant. Here, fish,
crustaceans, molluscs and other aquatic animals are included, and aquatic mammals, crocodiles, caimans,
seaweeds and other aquatic plants are excluded (Data adapted from the FAO. 2018. The State of World
Fisheries and Aquaculture 2018 - Meeting the sustainable development goals. Rome. Licence: CC BY-NC-
SA 3.0 IGO)
time period (Food, F.A.O. 2018), indicating that aquaculture is not relieving the demand for wild-caught
seafood (Naylor et al., 2000).
While aquaculture is often seen as mitigating overfishing, many farmed fish depend on feed originating
from marine capture. Because several marine captured fish are used feed a single carnivorous farmed fish,
some have argued that aquaculture is decreasing the global fish supply, rather than increasing it (Naylor
and Burke, 2005).
1.1 Trends in seafood production
While aquaculture is a very old technology – pictorial engravings suggest that Egyptians aquacultured
fish as early as 2500 BC (Anon - FAO 1987)- it was only in the 1970s that aquaculture production became
relevant beyond the subsistence and small scale (Asche et al, 2008). Similar to the evolution of agricultural
land animal farming, aquaculture has moved from extensive systems, that depend on natural environments
with minimal human intervention, to semi-intensive and intensive systems, where producers actively control
growing conditions through feeding, breeding, disease control, waste removal, etc., resulting in much
higher production rates (Asche et al., 2008)
With the intensification of aquaculture comes several challenges similar to the intensification of land
animal husbandry. Intensification leads to greater production of nitrogen and phosphorus waste materials
from uneaten feed and metabolic waste products (Yeo et al, 2004), thus creating higher risk of negative
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Rubio et al. Cell-based fish: an opportunity for cellular agriculture
environmental impacts (Piedrahita, 2003), such as the damage of waterways and promotion of algal blooms
(Paerl et al., 2014). Intensification also increases the risk of pathogen spread from farmed fish to native
species (Naylor et al. 2000). Furthermore, an absence of research on the welfare of most aquacultured
species has made it difficult to develop welfare standards for farmed aquatic animals, and thus the ethical
implications and appropriate welfare regulations of aquaculture remain an open question (Browman et al,
While it is acknowledged that fully-controlled aquaculture environments are more efficient production
systems than natural conditions (Tacon and Metian, 2008), there have, to date, been no mentions in the
scientific literature about fully-controlled environments for producing seafood from cell cultures rather
than from marine animal husbandry.
1.2 Engineering biology for seafood production
Genetic modification has resulted in spectacular production increases, feed conversion rates, and reduced
development times for fish in closed systems (Muir 2004). Genetically-modified rohu develop over four
times faster than their traditionally bred counterparts (Venugopal et al. 2004), while genetically-modified
mud loaches are able to grow 35 times faster and substantially larger (Nam et al. 2001). Like the mud
loach, genetically-modified tilapia containing the OPAFPcsGH gene can grow over 320 percent larger
than similarly reared non-transgenic fish (Rahman et al. 1998). The OPAFPcsGH gene, which encodes
for regulatory elements from ocean pout followed by growth hormone, forms the “all fish” chimera gene
sequence used in Aquabounty salmon (Hew and Fletcher 1996). Aquabounty salmon, while approved
for manufacture and sale in Canada, develop twice as fast as their wild type counterparts and sport an
impressive feed conversion rate ten percent higher than farmed salmon (Bisson 2015). These fish are
formidable food sources and an ecosystem threat – and thus are produced only in closed, in-land systems
as triploid, and therefore infertile, females (Reichhardt 2000). Despite these safety measures, significant
regulatory hurdles coupled with legislative pressure have prevented the fish from entering the US market
(Waltz 2017). Regardless, the fish are a prime example of the power of engineering biology to improve
closed-system seafood production. These genome-level advances for closed aquaculture systems may lay
important groundwork for cell-based fish production research and development.
2.1 The progression of cellular agriculture
In the past five years, research focused on cell-based meats has accelerated from the tasting of the
cell-cultured hamburger in 2013 (Zaraska 2013) and an early raft of publications, including life cycle
assessments and basic research (Tuomisto and Teixeira de Mattos 2011; Post 2012; Post 2014), to a growing
start-up community, updated life cycle assessments, and publications focused on refining the technologies
required to accelerate cell-based meat production research (Tuomisto, Ellis, and Haastrup 2014; Krieger et
al. 2018). To date, research decisions in cell-based meat production such as selection of cell species and
cell type have been largely driven by market size and environmental impact (Rodr
ıguez Fern
andez 2017),
rather than suitability of cells species and types for large scale bioreactor cultivation. This is the result of a
general lack of basic research on the cell cultures of commonly-consumed animals.
Cellular agriculture has recently become a topic of interest for regulators in the United States. Given
the regulatory jurisdiction, broadly speaking, of the Food and Drug Administration over products of
biotechnology and the United States Department of Agriculture over livestock, much interest has been
given to the regulation necessary for potentially jurisdiction-blurring cell-based meat products (Stephens et
al. 2018). Despite relative regulatory clarity over seafood, which is regulated entirely by the FDA (except
for animals in the order Siluriformes) (Kobbeman 2004), cell-based seafood production, and its potential
to address a growing demand for seafood while avoiding the challenges of industrial aquaculture, has
remained relatively uninvestigated.
2.2 Basic methodology for cell-based seafood production
Like cell-based meat production, the production of cell-based seafood relies on advanced technological
developments in cell line optimization, media formulation, and bioreactor design (Datar and Betti 2010).
Like any closed cell- or tissue- culture system, a cell-based seafood production system would comprise of
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Rubio et al. Cell-based fish: an opportunity for cellular agriculture
the following integrated elements: an appropriate cell type from the tissue of interest; a growth media to
provide nutrients to proliferating and differentiating cells, and a bioreactor which would provide the closed
environment in which growth would take place. For three-dimensional tissues, a biocompatible scaffold
would offer structure for cell growth and maturation.
Bioreactors are complex closed-system environments for producing biomass that require the constant
monitoring, maintenance, and optimization of several parameters. Marine cell cultures may be more
forgiving compared to mammalian cell cultures, given the unique characteristics of native fish muscle
tissue. This has significant implications for energy usage and thus cost considerations for mass production
of cell-based seafood. It also presents a new set of research opportunities for cellular agriculture and
large-scale tissue engineering as a whole.
Disciplines such as bioengineering, cell biology, and genetics have made great gains in the understanding of
cell- and tissue-culture the past twenty years – however these advances have been mostly within mammalian
systems. Piscine and other marine animal tissues in vitro have not yet been adequately investigated.
Native fish muscle physiology, by contrast, is relatively well-researched because of the implications for
aquaculture. Some experimentation and assaying has been carried out on harvested native muscle tissues
from marine animals, and these studies provide interesting insights into the potential capabilities of fish
muscle in culture.
Unlike mammalian muscle tissues, fish muscle in vivo consists of three muscle types - these muscles
differ between species depending on fish type, location, and feeding strategy. In teleost and elasmobranch
fish, there are three muscle types: red, white, and pink. Red muscle is highly vascularized with capillaries
and comprised of slow twitch fibers with a high density of mitochondria and a rich supply of capillaries
(Johnston 2001). This muscle is for slow, sustainable swimming speeds, and relies on aerobic metabolic
pathways. White muscles are fast twitch, tightly packed with myofibrils, and primarily undergo anaerobic
metabolic pathways. White muscle is used for burst-swimming and fast starts. Pink muscle shares some of
the characteristics of white and red muscle (Johnston 2001).
The variety of fish muscle cell types, which vary in physiology and metabolic pathway, may offer a range
of options when designing closed cultivation systems for cell-based seafood production. Native fish muscle
also has a number of characteristics related to metabolism, described below, that, if mirrored in ex vivo
tissue culture, renders it uniquely suited for cell-based production.
3.1 Oxygen requirements
3.1.1 Of mammalian cell culture
Low oxygen concentrations are detrimental to mammalian cell growth and bioproduct yield (Wang et
al. 1994), and thus dissolved oxygen concentration and air saturation are important variables that must
be monitored and maintained in bioreactor systems. Air saturation levels of 40 percent to 60 percent are
often required for mammalian cell culture at scale (Furukawa and Ohsuye 1998; Link et al. 2004; Zhu et al.
2005). Air saturation and oxygen levels become even more critical in three-dimensional, thick vascularized
tissues where oxygen diffusion limits the development of tissues past 1.8 mm (Griffith et al. 2005).
3.1.2 Of fish
As aquatic animals, fish are well adapted to tolerate low oxygen environments. Fish species are frequently
subjected to low oxygen levels that result in hypoxic conditions (Vaquer-Sunyer and Duarte 2008). Intere-
stingly, many coral reef fish are tolerant of hypoxic conditions as low as 2.8 to 0.5 percent air saturation for
the goby Gobiodon histrio and 1.6 to 0.7 percent air saturation for the blenny Atrosalarias fuscus before
showing signs of distress (Nilsson and Ostlund-Nilsson 2004). Using a microarray and bioinformatics-
based approach, Zhang and colleagues were able to identify over two hundred hypoxia-responsive genes
in the Japanese medaka brain, gill, and liver ranging from cell metabolism to RNA processing to protein
degradation through the ubiquitin system (Zhang et al. 2009). Hif1-alpha is a transcription factor that is
present under hypoxic conditions – when molecular oxygen is present, the protein becomes hydroxylated
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and subsequently degraded by the ubiquitin system (Ivan et al. 2001). This highly tuned system, while
discovered in mammals, plays a notable role in the hypoxia response of fish. Analysis of icefish mRNA
revealed that the functional domains of Hif1-alpha are highly conserved indicating the continued selective
pressure exerted by hypoxia (Rix, Grove, and O’Brien 2017). These and other hypoxia response gene rela-
tionships and predictions have been characterized in greater detail in the hypoxic fish database, HRGFish,
available online, which provides a variety of prediction tools and validation methods to further evaluate the
conservation of hypoxia genes in diverse fish species (Rashid et al. 2017)
In the absence of research on the oxygen requirements of fish cell culture in bioreactor systems, the
authors speculate that the multitude of genetic adaptations of fish to staggering levels of hypoxia suggest
that fish tissues may be uniquely suited to the oxygen-limited environments found in tissue culture and
bioreactor conditions. Further, the presence of hypoxia response genes that are conserved in mammals
suggest potential targets to increase mammalian cells’ ability to tolerate low oxygen environments.
3.2 pH considerations
3.2.1 Of mammalian cells
Extracellular pH directly correlates with intracellular pH in mammalian cell culture (Lagadic-Gossmann,
Huc, and Lecureur 2004), and lactic acid, which is formed by several cells in aerobic conditions (Brooks,
2009), accumulates over extended culture in bioreactor systems (Zhao and Ma 2005), contributing to
acidification of the bioreactor environment. Because of the well-documented relationship between intracel-
lular acidification and cell death (Lagadic-Gossmann, Huc, and Lecureur 2004), and because reduction of
extracellular pH from 6.8 to 6.3 can render cells quiescent (Taylor and Hodson 1984), pH of bioreactor
systems require fine tuning to optimize for cell growth.
Shifting pH can be used as an optimization tool, as changes in pH can affect protein production and
glycosylation (Borys, Linzer, and Papoutsakis 1993), however altering pH to increase protein production
and process performance reduces cell growth and metabolism (Trummer et al., 2006).
For mammalian cells cultures, the pH of a bioreactor system must be actively maintained to counter the
build-up of acidic metabolites, and carefully optimized, for either cell proliferation or protein production,
depending on the goals of the culture system.
3.2.2 Of fish
A key physiological characteristic of in vivo muscle across species is intracellular buffering capacity
(Burton 1978). Buffering capacity is the ability of a cell to maintain a neutral pH in the presence of
metabolic end products such as lactic acid (Rogatzki et al. 2015). It is measured in units of Slykes, the
micromoles of base required to change the pH of homogenized tissue by one unit per gram of wet weight
(Slyke 1922).
While land mammals such as pigs have buffering capacities of 49.7 Slykes, and cows of 51.9 Slykes,
sea mammals such as the fur seal have buffering capacities of 79.2 Slykes, and spotted dolphins of 84.1
Slykes (Castellini and Somero 1981). White muscle in warm-bodied fishes have particularly high buffering
capacity. Capacity was measured at 102 Slykes in the black skipjack, and 107 Slykes in the albacore –
double that of many land mammals (Castellini and Somero 1981). Further, muscle lactate dehydrogenase
(LDH), the enzyme responsible for breaking down lactic acid in muscle tissues, has been shown to exhibit
up to 2-fold higher activity in warm-bodied fish such as the albacore as compared to land animals such as
the cow (Castellini and Somero 1981).
The higher intracellular buffering capacity and the higher muscle lactate dehydrogenase activity unique
to warm bodied white fish muscle in vivo suggest that these muscle tissues may be more resilient than
mammalian cells in vitro tissue culture, with an ability to grow optimally within a wider pH range. More
research is required to determine if buffering capacity of native muscle tissue correlates to the buffering
capacity of tissue cultured in vitro.
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3.3 Temperature requirements
3.3.1 Of mammalian cells
Mammalian cells in culture are often maintained at 37 C, human body temperature. This temperature is
frequently used in mammalian bioreactor process design, however, temperature is a key variable that is
often altered to optimize culture growth. Jenkins and Hovey obtained high viability by alternating culture
temperatures between 39 C and 34 C (Jenkins and Hovey 1993), while others found that cultures grown
under a specific regime at 30 C can live longer and produce more protein (Moore et al. 1997).
3.3.2 Of fish
Fish vary widely in their adaptation strategies to frigid arctic and Antarctic environments, speciating at a
higher rate near the earth’s poles than near the equator (Rabosky et al. 2018). Some of these adaptations
have resulted in the development of antifreeze defense systems that are best optimized for temperatures
of 0- 10 C (Abele and Puntarulo 2004) and increased production of antioxidants, such as marine derived
tocopherol, which is found in greater quantities in salmon from cold waters than fish from equatorial waters
(Yamamoto et al. 2001). Other adaptations include unique oxygen carrying and metabolism capabilities
(Verde, Giordano, and di Prisco 2008). In some cases, these adaptations manifest as lack of hemoglobin
altogether, as is the case in the demersal icefish, Chaenocephalus aceratus – these fish compensate for their
lack of hemoglobin with increased numbers of mitochondria (Johnston 1987). Indeed, some have noted that
adaptations in arctic species exhibit muscle design and mitochondrial density similar to high-performance
adaptations in more equatorial species (Portner 2002).
Fish cell culture conditions typically mirror those of their typical habitats, with culture temperatures
varying from 15 C to 30 C. Some lines can vary their rate of metabolism within a 5-degree temperature
span, with the cells metabolizing more quickly at higher temperatures (Courtenay and Williams 2004).
Because fish cells can be cultured at cooler temperatures, thus reducing 1) the energy required to maintain
a constant temperature of a culture system, and therefore 2) the costs associated with producing cell-based
seafood at scale, cell-based seafood may offer cost benefits at scale compared to cell-based meats.
3.4 Fish cell lines
Until the last decade fish cell lines have been used for limited applications - testing viral load, water
toxicology, and generating vaccines for farmed fish. The American Type Culture Collection (ATCC), the
premier online cell repository in the United States, contains over 4,000 cell lines, and maintains strict
quality control on its collections, however, it does not contain any fish muscle cell lines (Hay, Caputo, and
Macy 1992).
Few cell line databases include fish cell lines, however, two are of note: FICELdb and Cellosaurus.
FICELdb is an unmaintained fish cell database that refers to many legacy lines dating from 1962 to 1999.
Cellosaurus is a cell line compendium created in 2012 and published in 2018, housed within ExPASy, a
database maintained Swiss Institute for Bioinformatics. It consists of cell line repositories from around
the world, categorizing over 100,000 known cell lines (Artimo et al. 2012). Interestingly, only 558 are
immortalized fish cell lines. Only nine are lines of fish cells were isolated from fish muscle, none of which
are myoblastic, all of which have spontaneously immortalized (Bairoch 2018). The nine fish cell lines
are displayed in the table below. Two of the nine fish cells lines originate from salt water species, both of
which are epithelial in nature: a white sturgeon line spontaneously immortalized in 2003, and a bluefin
trevally line from 2006. The cell line originating from barramundi, a euryhaline fish, is fibroblastic. The
remaining six fish cell lines originate from fresh water species. Zebrafish Brachydanio rerio and Goldfish
Carassius auratus are commonly-known model organisms and pets; the remaining cell lines originate from
commonly cultivated species in Southeast Asia; the snakehead murrel, a common food fish in Thailand;
the Sahul Indian catla, a farmed fish that is often grown with other carp species, and the helicopter catfish,
a species farmed commercially in Malaysia.
While these nine cell lines have been useful for culturing a number of fish pathogens, none have yet been
used for bioengineering or tissue engineering applications, and none are myoblastic lines. Derivation and
characterization of myoblastic cell lines from various fish would be an important resource for researchers
advancing cell-based seafood production. The availability of fish cell lines that are well-characterized to the
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level of a biological reagent could be a boon for cell-based seafood research; similar to what the availability
of C2C12 cells have done for mammalian muscle tissue engineering.
Cell Line Publication Morphology Water
White sturgeon Wang et al. 2003 Epithelial Salt
Bluefin Trevally Zhao and Lu 2006 Epithelial Salt
Barramundi Lai et al. 2008 Fibroblastic Euryhaline
Zebrafish Kumar et al. 2016 Fibroblastic Fresh
Goldfish Roug´
ee et al. 2007 Epithelial Fresh
Snakehead Murrel Zhao et al. 2003 Fibroblastic Fresh
Indian Catla Ishaq Ahmed et al. 2008 Epithelial Fresh
Indian Catla Ishaq Ahmed et al. 2009 Fibroblastic Fresh
Helicopter catfish unpublished Unknown Fresh
Table 1.
Immortalized fresh water fish muscle-isolated cell lines with relevant characteristics and
publications from Cellosaurus.
3.5 Cell isolation methods
Because of the lack of fish cell lines, much fish tissue culture work is conducted on primary tissue cultures
isolated from fish.
Fish cell isolation methods are similar in many respects to mammalian cell isolations. In general, the fish
is bathed in ethanol, anesthetized, and a tissue sample is removed with a biopsy. The tissue sample is then
either explanted or enzymatically-digested with collagenase or trypsin. Explanted tissues are allowed to
attach to the culture plate, and cells migrate from the tissue to the culture surface. Enzymatically-digested
tissues are in aqueous solutions, where digestion by trypsin or collagenase releases cells into the liquid
medium. Cells are then rinsed with buffer to remove contaminants and are often filtered to liberate the cells
from residual debris. Enzymatic digestion is often preferred for muscle cells.
While these techniques work well for isolating tissue from adult fish, recent research has begun investiga-
ting the isolation of cells from embryonic teleosts. While embryonic cells from many wild species are hard
to isolate, it is not impossible, especially from farmed strains and wild fish that are artificially fertilized at
fish hatcheries. Additionally, many species of fish have high telomerase activity, so primary embryonic
cells do not senesce as quickly as in mammalian systems (Anchelin et al., 2011). Importantly, while fish
do not have embryonic stem cells per se, they do have embryonic progenitors that may be coaxed down
different differentiation pathways (Ciarlo et al., 2017;Ma et al., 2001; Chandramallika et al., 1997).
As more research is done on fish muscle cell types, it will also be possible to isolate populations of interest
from either adult or embryonic cell preparations. Magnetic beads coupled to muscle-progenitor-specific
antibodies will allow rapid isolation of these cells. Additionally, fluorescence-activated cell sorting (FACS)
is an effective technique for cell isolation. The main challenge is identifying cell-surface proteins that mark
these progenitors and muscle cells; the conservation with extracellular proteins with mammals is very low
(Liongue and Ward, 2007), so most mammalian antibodies do not cross-react.
3.6 Culture conditions
3.6.1 Extracellular environments and scaffolds
Future work determining the correct extracellular matrix (ECM) that these fish cells survive and proliferate
on is of utmost importance; most mammalian cell lines are simply plated on tissue-culture plates coated
with vacuum gas plasma, which makes the polystyrene surface more hydrophilic. Fish cells may require
scaffolds of different ECM proteins such as elastins, collagens, fibronectin, and laminin to adhere and grow
efficiently, and may also require fish glycoaminoglycans. Optimizing these ECM proteins would also likely
be critical to efficient fish culture in vitro, especially since fish protein glycosylation patterns are different
from mammals.
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3.7 Growth media
Parameters for fish cell culture growth media include salt concentration, buffer, temperature, and pH.
Past growth media formulas suggest a trial and error approach that may not be optimal for all species or
purposes. Previous publications used growth media that include mammalian formulations such as Eagle’s
Medium, Modified Eagle’s Medium (MEM), Medium 199 (M199) and Leibowitz’s 15 (L-15) medium
(Fernandez et al. 1993). These media are fully defined and easily replicated, including inorganic salts,
amino acids, and basic nutrients for culture growth. Additional salts are added for some marine species
such as the grunt, but not all (Clem, Moewus, and Michael Sigel 1961). Fetal bovine serum (FBS) and fetal
calf serum (FCS) are common additives. Use of bicarbonate in media formulations aids cell growth and
buffering capabilities as well (Fernandez et al. 1993). This ability to control the buffering capacity of the
media is important; some fish cells require growth at 5 percent carbon dioxide, but others are fine in anoxic
or standard oxygen tension. These conditions vary between species (and even between tissues from the
same fish) but can be controlled for the purposes of cell-based seafood production. Addition of growth
factors, such as fibroblast growth factor (FGF2) has proven helpful to the growth of some muscle cell
cultures. Bain and Schuller reported successful culture of epithelial tuna cells in L-15 media with 10-20
percent FBS (Bain et al. 2013). Attempts to lower the FBS content resulted in reduced cell proliferation,
but the addition of vitamin E and fatty acids improved proliferation. (Scholefield and Schuller 2014).
Clearly, removing FBS or FCS from the media for cell-based seafood productions would be advantageous
due to the high cost of these products, their animal sources, and their potential for carrying mammalian
viruses and prions. Investigations into the molecular components of fish serum would likely be useful to
identify and then generate in vitro the required components to keep fish cells alive and dividing in culture.
Furthermore, fats are nutritionally-relevant in seafood, and more research can be done to understand how
media composition can affect the nutritional quality of a cell-based seafood.
4.1 Cell lines
In recent decades, crustacean cell culture research has been explored for use in the aquaculture and
pharmaceutical industries (Zhao et al. 2003), for studying diseases affecting farmed seafood and identifying
biologics with human clinical relevance. Several studies have attempted to establish cell lines from
crustacean tissues to improve isolation and maintenance methodology, however, in recent decades there
has been a decrease in effort to develop long-term cultures and instead research has proceeded with the use
of short-term cultures obtained from repeated primary cell isolations (Rinkevich 2005). Short-term cultures
are sufficient for bench-scale research but not sufficient for cell-based seafood applications with long-term
goals of mass production and commercialization.
To advance crustacean tissue culture for food purposes, it is vital to attempt new strategies for developing
immortalized or long-term cell lines of relevant lineages such as muscle and fat. Previous crustacean
tissue culture studies have instead focused for ovary-derived epithelial and fibroblast cells. The studies
that examined muscle, eye stalk, and hepatopancreas cells in crustaceans found they have poorer survival
and slower growth compared to ovary-derived epithelial and fibroblast cells (George and Dhar 2010). The
most frequently studied crustacean genus is Penaeus which includes shrimp and prawn species. Studies
of this species have derived cells from ovary, hepatopancreas, nerve, lymphoid and hematopoietic tissue
(Rinkevich 2005).
Much more work is to be done to derive and characterize crustacean cells to the level of a biological
reagent so that they may be used for cell-based seafood research and development. It is possible that
establishment of such cell lines could also be relevant to the aquaculture and pharmaceutical sectors.
4.2 Cell isolation methods
Cell isolation procedures for crustaceans and vertebrates share several similarities. For example, Tong
and Miao found that cells isolated from younger “pre-molting” prawns have a higher success rate for
survival and growth (Tong and Miao 1996). Cell cultivation studies often attempt isolations from both
explanted and enzyme-digested tissues. For the explant method, which is best suited for loose tissues, the
target tissue is removed from the animal, rinsed in custom buffer solution, dissected into small sections and
allowed to attach to a culture surface. If successful, cells within the tissue will proliferate, migrate, and
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adhere to the substrate. For the enzyme digestion method, digestion enzymes (e.g., trypsin, collagenase)
are incubated with dissected explants to degrade the tissue into a single cell suspension. The slurry is often
filtered to remove undigested sections and the enzymes are inhibited after a certain amount of incubation
time to prevent cell damage. For cell isolations from most shrimp tissues, the explant method tends to have
more success than enzyme-digested method in terms of cell proliferation, although enzyme digestion is
preferred for hepatopancreas tissues (Ma, Zeng, and Lu 2017).
4.3 Culture conditions
Parameters such as temperature, pH and osmolality differ between vertebrate and invertebrate cell culture.
The incubation temperature for invertebrates is lower than for vertebrates and typically falls within the
range of 25-30
C. The pH of shrimp and prawn cell cultures is typically within the range of 7.0-7.6
(Ma, Zeng, and Lu 2017). Osmolality can range between 472-760 mmol/kg depending on the hemolymph
osmolality of the origin animal (Chen et al. 1986; Rinkevich 2005). Carbon dioxide exchange is generally
not necessary for invertebrate culture because the basal media tends to be buffered by phosphates rather
than sodium bicarbonate. While cells have been observed to reach confluency and survive for extended
periods of time with media changes once-per-week, the cultures often degenerate after a few rounds of
subculture (Chen et al. 1986; Ma, Zeng, and Lu 2017). This may be due to high sensitivity of the cells to
the enzymes such as trypsin which are employed during passaging.
Like fish cells, crustacean cells can be cultured at lower temperatures, suggesting an energy saving
opportunity for large scale bioreactor culture compared to mammalian cells. Similarly, crustacean cell
culture does not require carbon dioxide exchange, obviating the need for a common mammalian cell culture
bioreactor parameter.
4.4 Growth media
The basal medium for invertebrate cell culture commonly consists of Grace’s medium or L-15 medium,
though formulations such as M199 have at times exhibited superior effects (Ma, Zeng, and Lu 2017). The
standard for mammalian cell culture is basal media supplemented with animal serum from domestic species
like cows, horses or chickens. Fetal bovine serum is also important for shrimp cell culture at a concentration
of ten to twenty percent (Ma, Zeng, and Lu 2017). However, this strategy has not been sufficient to
maintain crustacean cells in culture long-term, so researchers have attempted to supplement media with
marine-relevant factors such as lipid solutions or hemolymph extracted from various sea creatures (Chen et
al. 1986; Rinkevich 2005). Common supplements include shrimp or crab muscle extract and hemolymph
from Penaeus species. These additions likely provide growth factors not supplied by mammalian serum.
Some growth factors have been identified to improve shrimp lymphoid and ovarian cultures, like epidermal
growth factor and transforming growth factor beta (Ma, Zeng, and Lu 2017).
Much more work is needed in the development of a sustainable, animal-free media for the growth of
crustacean cells. It is likely that animal-based media are used merely because a lack of well-researched
5.1 Buffering capacity
Observed buffering capacity varies widely in mollusks (Eberlee and Storey 1984) with some species
exhibiting a particularly high buffering capacity. The channeled whelk, Busycotypus canaliculatus, has
been shown to have an aerobic buffering capacity in the hepatopancreas of 79.4 plus or minus 17.2 Slykes,
and a remarkable buffering capacity of almost 120 Slykes following 24 hours of anoxic stress (Hetrick
et al. 1981). While heart muscle tissues appear to have lower buffering capacity, anoxic stress increases
their buffering capacity to over 60 Slykes (Eberlee and Storey 1984) – still higher than that of many land
The high buffering capacity of bivalves suggests an ability of bivalve cell culture to tolerate a wider pH
range than mammalian cell cultures.
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Rubio et al. Cell-based fish: an opportunity for cellular agriculture
5.2 Cell culture
Bivalve culture has proven even more elusive than fish or crustacean culture. While many have attempted
to continuously culture a bivalve cell line, very few have been able to achieve proliferation (Chen and
Wang 1999). Researchers at the University of Maryland were unable to successfully achieve mitosis in any
of their long-term cultures of clam and oyster lines attempted, in spite of 138 unique attempts in different
media formulations (Hetrick et al. 1981). Odinstova and colleagues observed low, though extant, mitotic
activity in scallop culture over a period of four months (Odintsova and Khomenko 1991). Similarly, mitosis
in the surf clam, Spisula soldissima was observed, though media contained fetal calf serum and whole egg
extract (Cecil 1969). Despite challenges with proliferation in culture, many primary cell cultures have been
generated (Chen and Wang 1999).
An exception to the aforementioned lack of proliferative cultures are cell cultures from abalone species
cultivated for pearl production and hemocyte (blood cell) lines that can be used for toxicological studies.
Mantle of Haliotis varia in Medium 199 containing additional salts, lactalbumin, kanamycin, and sodium
bicarbonate, and fetal bovine serum, has been successfully cultured for 370 days (Suja and Dharmaraj
2005). The authors more recently were able to culture the mantle of Haliotis varia in serum free media
containing a whole-body extract (Suja, Sukumaran, and Dharmaraj 2007).
Even fewer bivalve muscle cells have been isolated. Clam primary cardiomyocytes that could be grown for
up to a month have been isolated(Hanana et al. 2011) and primary culture of clam heart tissue originating
from Meretrix luxoria was successfully cultured for over five months (Chen and Wang 1999).
Bivalve cell culture is understudied, with demonstrated challenges in establishing long term, proliferative,
food-relevant cell lines.
Cultivating three-dimensional tissues relies on the presence of a scaffold – a biocompatible material capable
of supporting cell growth and differentiation. While there are several scaffolding materials employed
in tissue engineering today, we discuss chitosan, a food-relevant material that is derived from chitin; a
primary component of insect and crustacean exoskeletons and one of the most prevalent biopolymers on
earth alongside cellulose. Within exoskeletons, chitin exists as nanofibril structures, providing mechanical
strength to the cuticle. Chitosan can also be derived from non-animal sources like fungi, algae and
yeast. As collagen and other extracellular matrix proteins prove to be successful scaffold biomaterials
for mammalian tissue engineering, it is reasonable (once cell lines are sufficiently available) to employ
chitosan scaffolding techniques as a first step towards three-dimensional culture of seafood-relevant cell
types. Due to the mechanical characteristics of chitosan, it is a popular biomaterial for scaffold construction
for many applications of tissue engineering. Chitosan powder can be isolated from crustacean, mushroom
or microbial sources and dissolved into an aqueous solution which can be cast into a variety of formats
such as membranes, hydrogels and sponges (Croisier and J
ome 2013). Fungal chitosan is a preferred
source as it is (1) non-allergenic, (2) more customizable in terms of molecular weight and (3) approved for
human consumption as a food additive or nutrition supplement (Pochanavanich and Suntornsuk 2002; Tao
Wu et al. 2004; Nitschke et al. 2011).
6.1 Scaffold fabrication
Chitosan can be dissolved in acidic solution such as 1-2 percent acetic acid in distilled water. Agitation
and heat both promote dissolution. As the concentration of dissolved chitosan increases, the solution
becomes increasingly viscous. With time and patience, solutions of 12 percent chitosan can be achieved
if desired (Jana, Cooper, and Zhang 2013). Many scaffolds that support various cell cultures use lower
chitosan concentrations. Solutions with concentrations as low as 0.5 percent chitosan are able to produce
thin membranes with remarkable mechanical strength. To create films for cell culture, chitosan solution
is cast on glass substrates and allowed to dry. The films are subsequently rinsed with buffer to neutralize
remaining acetyl groups, washed with water or PBS and sterilized to prepare for seeding. Chitosan can
also be manipulated to produce physically associated or cross-linked hydrogels (Drury and Mooney 2003),
sponges with tunable mechanical properties and pore size distributions (Jana, Cooper, and Zhang 2013)
and dry or wet spun fibers (Croisier and J´
ome 2013).
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Rubio et al. Cell-based fish: an opportunity for cellular agriculture
Given the progression of aquaculture towards more intensive, more controlled, more efficient systems, the
authors propose cell-based seafood production as a means to potentially avert the challenges of industrial
aquaculture and displace marine capture.
This review presents opportunities for cell-based seafood production research by surveying the literature
on marine cell culture, native marine muscle tissue, and marine animal considerations which may be
directly relevant or adjacent to the development of a cell-based seafood production system. Because of the
minimal availability of literature on marine cell cultures, the authors speculated on the characteristics of
marine cell culture based on the unique properties of native muscle tissue in fish.
Overall, it appears that fish muscle tissue may be innately well-suited for bioreactor cultivation relative to
mammalian muscle tissues, given the ability of fish tissues to 1) endure hypoxic conditions, reducing the
need for active oxygenation in oxygen-limited bioreactor environments; 2) tolerate pH, potentially creating
a wider range of pH in which cell growth is optimized; and 3) grow at lower temperatures, potentially
reducing the heat transfer needs of bioreactor cultivation at scale.
A review of the literature on crustacean cell culture indicates that there is very little research that is
directly relevant for cell-based food production; most research is adjacent, either from species that are
not directly food-relevant, or cell-types that are not directly food-relevant. A review of the literature on
bivalves is even more sparse.
Approaches to cell-based seafood production can range from large scale cell cultivation, resulting in
a large mass of seafood-relevant cells that may have applications in processed seafoods like surimi; to
three-dimensional tissue cultivation, resulting in structured products more akin to fillets. Chitosan is a
promising, marine-relevant biomaterial for three-dimensional tissue culture applications. It is customizable,
edible, and widely available; making it a suitable material to explore for cell-based seafood production, as
well as other tissue engineering and cellular agriculture applications.
There could be several advantages to producing seafood from cell cultures rather than whole marine
animals. The production of cell-based seafood has the distinct potential to alter many of the fundamental
parameters considered immutable in food cultivation, including production of inedible excess tissues such
as bone, skin, shells, and scales. It is also possible that cell-based seafood could be produced from cell
cultures on the order of weeks to months; by comparison, a genetically-modified Aquabounty salmon
grows to market size in 18 months, roughly half the time of a normal salmon (Waltz, 2017).
Producing seafood from marine cell cultures is a novel seafood production method and an intriguing
opportunity for cellular agriculture.
A survey of relevant literature reveals that marine cell and tissue culture is an enormously neglected field
of research. Very few cell lines from marine species have been derived and characterized, and none are
directly relevant to seafood production. Despite the several properties of marine muscle tissue that make it
promising for bioreactor cultivation, the actual behavior of marine cells in large scale culture environments
remains speculative. There are several research gaps that exist for marine cell culture, alongside several
opportunities that make these research gaps worth addressing.
With growing interest in cellular agriculture as a means to produce meat, milk, eggs, and other animal
proteins from cell cultures, and with the rapid intensification of aquaculture systems, the time is right to
investigate the production of seafood without marine animals.
Kate Krueger oversaw the preparation and writing of the manuscript and themes, contributing sections on
bivalve cell culture, and oxygen and pH considerations. Natalie Rubio contributed material on crustacean
cell culture, and David Stachura contributed to fish tissue isolation, embryonic cells, and muscle cell type
sections. Isha Datar contributed to the organization, writing and editing of the manuscript.
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Rubio et al. Cell-based fish: an opportunity for cellular agriculture
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
New Harvest is a 501(c)(3) research institute that supports basic research in cellular agriculture The
research for this review was performed using funding to New Harvest from an anonymous donor.
The authors would like to acknowledge Bradley Silverman for his insights on process engineering conside-
rations, Arif Malik for his insights on the fish musculature section of this review, Andrew Stout for his
insights on the Warburg effect, and Mike Selden for his introductions and support.
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... Their cell line could be useful for providing basic insights into growth, reproduction, and health, creating opportunities for manipulation and thus the cell lines could be used as sources of biochemical products in place of the whole organism [1]. Cell-based aquaculture systems using cell cultures could be a game-changing practice to produce seafood and other aqua food across multiple species for meeting the demand of the burgeoning world population [2]. A cell-based aqua food production system utilizing cells in place of whole fish could also lead to greater preservation of the aquatic environments. ...
... Tissue engineering blend with modern aquaculture techniques can be explored to utilize marine cell culture as an attractive opportunity for the production of in vitro fish meat. Fish muscle cell culture can be used for in vitro fish meat production by exploiting their salient physiological properties like tolerance to a hypoxic-conditions, high buffering capacity, and lower temperature [2]. Fish muscle cell cultures are more adaptable to in vitro conditions than mammalian ones and hence in vitro meat production will be more feasible with fish muscle cell cultures. ...
... A better understanding of the myogenesis involved in the muscle cell and tissue culture would be essential to trap the benefits of muscle cell culture in promoting cellular aquaculture. In vitro models like C2C12 cell lines have been utilized in understanding molecular mechanisms underlying muscle growth and differentiation in mammals [2]. Such studies are in the infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines except for a few fish muscle cell lines [75][76][77]77]. ...
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Introduction Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research. Conclusion The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research.
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Background Skeletal muscle wound healing is dependent on complex interactions between fibroblasts, myofibroblasts, myogenic cells, and cytokines, such as TGF-β1. This study sought to clarify the impact of TGF-β1 signaling on skeletal muscle cells and discern between the individual contributions of fibroblasts and myofibroblasts to myogenesis when in co-culture with myogenic cells. 3D tissue-engineered models were compared to equivalent 2D culture conditions to assess the efficacy of each culture model to predictively recapitulate the in vivo muscle environment. Methods TGF-β1 treatment and mono-/co-cultures containing human dermal fibroblasts or myofibroblasts and C2C12 mouse myoblasts were assessed in 2D and 3D environments. Three culture systems were compared: cell monolayers grown on 2D dishes and 3D tissues prepared via a self-assembly method or collagen 1-based hydrogel biofabrication. qPCR identified gene expression changes during fibroblast to myofibroblast and myoblast differentiation between culture conditions. Changes to cell phenotype and tissue morphology were characterized via immunostaining for myosin heavy chain, procollagen, and α-smooth muscle actin. Tissue elastic moduli were measured with parallel plate compression and atomic force microscopy systems, and a slack test was employed to quantify differences in tissue architecture and integrity. Results TGF-β1 treatment improved myogenesis in 3D mono- and co-cultures containing muscle cells, but not in 2D. The 3D TGF-β1-treated co-culture containing myoblasts and myofibroblasts expressed the highest levels of myogenin and collagen 1, demonstrating a greater capacity to drive myogenesis than fibroblasts or TGF-β1-treatment in monocultures containing only myoblasts. These constructs possessed the greatest tissue stability, integrity, and muscle fiber organization, as demonstrated by their rapid and sustained shortening velocity during slack tests, and the highest Young’s modulus of 6.55 kPA, approximate half the stiffness of in situ muscle. Both self-assembled and hydrogel-based tissues yielded the most multinucleated, elongated, and aligned muscle fiber histology. In contrast, the equivalent 2D co-culture model treated with TGF-β1 completely lacked myotube formation through suppression of myogenin gene expression. Discussion These results show skeletal muscle regeneration can be promoted by treating myogenic cells with TGF-β1, and myofibroblasts are superior enhancers of myogenesis than fibroblasts. Critically, both TGF-β1 treatment and co-culturing skeletal muscle cells with myofibroblasts can serve as myogenesis accelerators across multiple tissue engineering platforms. Equivalent 2D culture systems cannot replicate these affects, however, highlighting a need to continually improve in vitro models for skeletal muscle development, discovery of therapeutics for muscle regeneration, and research and development of in vitro meat products.
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Far more species of organisms are found in the tropics than in temperate and polar regions, but the evolutionary and ecological causes of this pattern remain controversial1,2. Tropical marine fish communities are much more diverse than cold-water fish communities found at higher latitudes3,4, and several explanations for this latitudinal diversity gradient propose that warm reef environments serve as evolutionary ‘hotspots’ for species formation5,6,7,8. Here we test the relationship between latitude, species richness and speciation rate across marine fishes. We assembled a time-calibrated phylogeny of all ray-finned fishes (31,526 tips, of which 11,638 had genetic data) and used this framework to describe the spatial dynamics of speciation in the marine realm. We show that the fastest rates of speciation occur in species-poor regions outside the tropics, and that high-latitude fish lineages form new species at much faster rates than their tropical counterparts. High rates of speciation occur in geographical regions that are characterized by low surface temperatures and high endemism. Our results reject a broad class of mechanisms under which the tropics serve as an evolutionary cradle for marine fish diversity and raise new questions about why the coldest oceans on Earth are present-day hotspots of species formation.
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The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server ( and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences.
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Background Cultured meat forms part of the emerging field of cellular agriculture. Still an early stage field it seeks to deliver products traditionally made through livestock rearing in novel forms that require no, or significantly reduced, animal involvement. Key examples include cultured meat, milk, egg white and leather. Here, we focus upon cultured meat and its technical, socio-political and regulatory challenges and opportunities. Scope and approach The paper reports the thinking of an interdisciplinary team, all of whom have been active in the field for a number of years. It draws heavily upon the published literature, as well as our own professional experience. This includes ongoing laboratory work to produce cultured meat and over seventy interviews with experts in the area conducted in the social science work. Key findings and conclusions Cultured meat is a promising, but early stage, technology with key technical challenges including cell source, culture media, mimicking the in-vivo myogenesis environment, animal-derived and synthetic materials, and bioprocessing for commercial-scale production. Analysis of the social context has too readily been reduced to ethics and consumer acceptance, and whilst these are key issues, the importance of the political and institutional forms a cultured meat industry might take must also be recognised, and how ambiguities shape any emergent regulatory system.
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The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by crestin:EGFP expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases crestin expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation.
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The long evolution of the Antarctic perciform suborder of Notothenioidei in the icy, oxygen-rich waters of the Southern Ocean may have reduced selective pressure to maintain a hypoxic response. To test this hypothesis, cDNA of the key transcriptional regulator of hypoxic genes, hypoxia-inducible factor-1α (HIF-1α), was sequenced in heart ventricles of the red-blooded notothenioid, Notothenia coriiceps, and the hemoglobinless icefish, Chaenocephalus aceratus. HIF-1α cDNA is 4500 base pairs (bp) long and encodes 755 amino acids in N. coriiceps, and in C. aceratus, HIF-1α is 3576 bp long and encodes 779 amino acids. All functional domains of HIF-1α are highly conserved compared to other teleosts, but HIF-1α contains a polyglutamine/glutamic acid (polyQ/E) insert nine amino acids long in N. coriiceps and 34 amino acids long in C. aceratus. Sequencing of this region in four additional species, representing three families of notothenioids, revealed that the length of the polyQ/E insert varies with phylogeny. Icefishes, the crown family of notothenioids, contain the longest polyQ/E inserts, ranging between 16 and 34 amino acids long, whereas the basal, cold-temperate notothenioid, Eleginops maclovinus, contains a polyQ/E insert only 4 amino acids long. PolyQ/E inserts may affect dimerization of HIF-1α and HIF-1β, HIF-1 translocation into the nucleus and/or DNA binding.
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The present study attempts to establish cell culture systems for the oyster, Crassostrea gigas Röding and the hard clam, Meretrix lusoria Thunberg. Treatment with collagenase was better than trypsin at dissociating mollusc tissue fragments for in vitro culture. Heart tissue of oyster and hard clam proved to be the most promising target tissue for the establishment of cell lines in vitro. Primary cultures of clam heart were established and successfully maintained for more than 5 months. Collagenase at a concentration of 100 microg/ml may enhance the growth of oyster and hard clam heart cell cultures in vitro.
We revisit the evidence attributing sentience-pain-suffering to aquatic animals. The objective is to inform readers of the current state of affairs, to direct attention to where research is needed, and to identify "wicked" questions that are difficult to resolve unequivocally. By separating the ethical from the scientific debate, applying organized skepticism to the latter, and taking a pragmatic approach that does not depend on resolving the "wicked" questions, we hope to focus and strengthen research on aquatic animal welfare. A second but closely-related objective is to briefly summarize the research used to support the regulations governing the welfare of aquatic animals, particularly its limitations. If you interact with aquatic animals, these regulations already affect you. If the regulatory environment continues on its current trajec-tory (adding more aquatic animal taxa to those already regulated), activity in some sectors could be severely restricted, even banned. There are surely some lively debates and tough choices ahead. In the end, extending legal protection to aquatic animals is a societal choice, but that choice should not be ascribed to strong support from a body of research that does not yet exist, and may never exist, and the consequences of making that decision must be carefully weighed.
US firm AquaBounty Technologies says that its transgenic fish has hit the market after a 25-year wait.