https://orcid.org/0009-0002-3864-9596
Abstract
Cultivated crustacean meat (CCM) is a means to create highly valued shrimp, lobster, and crab products directly from stem cells, thus removing the need to farm or fish live animals. Conventional crustacean enterprises face increasing pressures in managing overfishing, pollution, and the warming climate, so CCM may provide a way to ensure sufficient supply as global demand for these products grows. To support the development of CCM, this review briefly details crustacean cell culture work to date, before addressing what is presently known about crustacean muscle development, particularly the molecular mechanisms involved, and how this might relate to recent work on cultivated meat production in vertebrate species. Recognizing the current lack of cell lines available to establish CCM cultures, we also consider primary stem cell sources that can be obtained non-lethally including tissues from limbs which are readily released and regrown, and putative stem cells in circulating hemolymph. Molecular approaches to inducing myogenic differentiation and immortalization of putative stem cells are also reviewed. Finally, we assess the current status of tools available to CCM researchers, particularly antibodies, and propose avenues to address existing shortfalls in order to see the field progress.
Introduction
By producing meat through stem cell cultivation rather than animal husbandry, cultivated meat (CM) has emerged as a potentially more sustainable and ethical alternative to conventional production. Since its emergence in the mid-2010s, the field has focused largely on meat from terrestrial species such as beef, pork, and chicken, and to some extent fish, with comparatively little work on marine invertebrates such as crustaceans (Cohen et al. Citation2022). This is despite the fact that global demand for highly valued products such as shrimp, crab, lobster and crayfish is growing, and there are negative aspects to crustacean fishing and farming that may also warrant alternative solutions (FAO Citation2020).
Globally, extensive wild capture of crustaceans is responsible for overfishing and ocean bycatch, and often entails concerns around provenance, microplastics and heavy metal contamination (Baechler et al. Citation2020; Baki et al. Citation2018; Fatema et al. Citation2022; Lira et al. Citation2022; Roda et al. Citation2019). Crustacean aquaculture has emerged over the last several decades as an alternative to wild capture, both to mitigate some of the impacts of fishing and to ensure an adequate global supply of seafood (FAO Citation2020). However, although many modern crustacean aquaculture enterprises place a priority on long-term sustainability, the broader global industry continues to face challenges in this respect. Crustacean farming in many coastal regions is linked to effluent and chemical pollution, mangrove destruction and increased greenhouse gas emissions from land-use change (Abdullah, Barua, and Hossain Citation2019; Ahmed et al. Citation2017; Barraza-Guardado et al. Citation2013; Boone Kauffman et al. Citation2017), and its commercial viability is being increasingly impacted by disease outbreaks and the warming climate (Ahmed et al. Citation2017; Macusi et al. Citation2022). Furthermore, as crustacean animals are now being recognized as sentient and capable of suffering (Albalat et al. Citation2022; Elwood, Barr, and Patterson Citation2009; Passantino, Elwood, and Coluccio Citation2021), current fishing and farming methods are also likely to rate poorly for animal welfare outcomes.
If growing demand is to be met, there is a clear case for investigating the development of cultivated crustacean meat (CCM) as a more sustainable and ethical alternative to conventional production. Some aspects of CCM production may even be advantageous compared to CM from terrestrial species. Being marine or aquatic, crustacean cells require culturing at lower temperatures (25–30 °C), they have a broader pH tolerance (6.8-7.6), and do not require CO2, all of which could translate to lower manufacturing energy consumption (Ma, Zeng, and Lu Citation2017; Rubio, Datar, et al. Citation2019). Additionally, because most crustacean species display continual growth throughout life, have impressive regeneration abilities and high telomerase expression, they may have potential for greater rates of cellular proliferation or even immortalization (acquiring indefinite proliferation ability) (Klapper et al. Citation1998; Specht et al. Citation2021).
Cultivated meat and seafood production requires close control of the ex vivo proliferation of stem cells and their differentiation into meat-relevant tissues. A major research focus is on selecting the best starting stem cells, with the main candidates being the pluripotent types such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the multipotent types such as mesenchymal stem cells or fibro-adipogenic progenitors, or unipotent versions such as adult muscle stem cells (AMSCs) like satellite cells and myoblasts (Post et al. Citation2020; Reiss, Robertson, and Suzuki Citation2021). For fat and connective tissues, unipotent types are preadipocytes and fibroblasts respectively. Starting cells can be obtained from immortalized cell lines or primary tissues, and each cell type is evaluated in terms of its accessibility and proclivity to proliferate or differentiate into meat-relevant tissues such as muscle or fat (Post et al. Citation2020; Reiss, Robertson, and Suzuki Citation2021). Bomkamp et al. (Citation2023) provide a comprehensive review of the developmental interrelationships and advantages and disadvantages of the different cell types considered for CM production in seafood, most of which are of mesodermal origin.
Robust and well characterized immortal cell lines are an obvious choice for CM development. They are able to replicate indefinitely, their requirements are well understood, and once established, they are readily available in unlimited quantities without reliance on source animals (Specht et al. Citation2018). However, they can be quite challenging to create initially and there are currently very few publicly available for CM production.
Although comparatively variable in nature and reliant on continual animal biopsies, primary tissues are also a valid source of CM starter cells, especially due to the current lack of appropriate cell lines. Obtaining AMSCs such as satellite cells from small muscle tissue biopsies to produce meat at the lab bench scale is already a well-established practice and has been employed regularly since the first publicly presented “cultured beef” products in 2013 (Melzener et al. Citation2021; Post et al. Citation2020). summarizes the advantages and disadvantages of each cell source and type.
Figure 1. Advantages and disadvantages of different stem cell sources and types used in cultivated meat development. Stem cell sources are evaluated in terms of ease to establish, immortality and independence from animals. Stem cell types are evaluated in terms of differentiation potential (plasticity), proliferative ability and ease to differentiate. Red down arrows indicate a poor evaluation and blue up arrows indicate a comparatively good evaluation. Orange down arrows indicate a moderate or limited evaluation. Adapted from Bomkamp et al. (Citation2023) and Reiss, Robertson, and Suzuki (Citation2021).
Currently, there are no publicly available cell lines for any crustacean cell type, despite multiple attempts over the last several decades. The desire to develop crustacean cell lines or long-term primary cultures has largely been driven by the urgent need for crustacean pathogen research in aquaculture (Claydon and Owens Citation2008). Now, with the added incentive of CCM development, the need for a crustacean cell line is even more exigent. Until this is achieved, CCM researchers must rely on primary tissue sources to establish meat-relevant cell cultures.
Meat is made up primarily of skeletal muscle (∼90%) with the remainder consisting mostly of fat, and, to a lesser degree, connective, vascular and nerve tissues (Listrat et al. Citation2016). Because intramuscular fat is particularly important for the flavor and mouthfeel of meat, considerable research is directed to cultivating adipocytes in vitro for terrestrial species (Dohmen et al. Citation2022; Jeong et al. Citation2022; Louis et al. Citation2023; Song, Liu, Zheng, et al. Citation2022; Song, Liu, Li, et al. Citation2022; Yuen et al. Citation2023; Zagury et al. Citation2022), and fish (Bomkamp et al. Citation2023; Saad et al. Citation2023). However, the fat content of crustacean muscle tissue does not appear to be generated from intramuscular adipocytes, but from the fatty acids contained in membrane phospholipids (Garofalaki, Miniadis-Meimaroglou, and Sinanoglou Citation2006; Lu et al. Citation2020; Shu-Chien et al. Citation2017). These are delivered to muscle tissues through the hemolymph from the hepatopancreas where they are synthesized or processed from dietary sources (O’Connor and Gilbert Citation1968; Sun et al. Citation2020; Viña-Trillos and Urzúa Citation2021; Vogt Citation2021). This potentially implies that sufficient CCM fat content could be achieved via the supplementation of muscle cell cultures with key fatty acids rather than attempting to recapitulate adipogenesis in vitro (Bomkamp et al. Citation2023). For this reason and in the interests of simplicity, adipogenic differentiation will not be addressed in this review.
The overall intention of this review is to highlight some areas for consideration in CCM development. Section 1 provides an overview of recent crustacean cell culture or immortalization attempts with particular focus on stem cells of embryonic or mesodermal origin. Section 2 considers what is currently known about crustacean muscle development comparative to model species, with particular reference to molecular mechanisms. Section 3 assesses myogenic differentiation and proliferation strategies in current CM research as a background for potential approaches to crustacean cells. Section 4 reviews potential crustacean stem cell sources in terms of accessibility and non-lethality, and propensity for proliferation and myogenic differentiation. Sections 5 and 6 examine molecular tools used in crustacean research and how these might be used to induce myogenesis or immortalization for cell line development. Finally, Section 7 addresses the current status of appropriate tools that may be useful in CCM research. The review finishes by summarizing recommendations for future work.
1. Recent crustacean cell culture and immortalization attempts
We have been able to identify only two publications that detail methods to culture primary crustacean cells specifically for CCM development: one patent covering ESCs, iPSCs and adult muscle and fat stem cells (Sriram and Ling Citation2020); and one experimental paper detailing attempts to isolate and culture satellite cells from adult tail muscle and whole juveniles (Jang, Scheffold, and Bruheim Citation2022). A review of general crustacean cell culture and immortalization attempts reveals comparatively few of these deal with meat-relevant tissues such as muscle, however several do cover hematopoietic or lymphoid cells, which share a mesodermal origin with muscle (). The potential implications of this relationship are discussed further in Section 4.3. Distinction between crustacean hematopoietic and lymphoid tissues is not clear as the latter has only been found in penaeid shrimp and both appear to have circulatory and immune-related functions, leading some to postulate that they are in fact one and the same tissue (Owens and Rusaini Citation2010; Smith, Accorsi, and Malagoli Citation2016; Söderhäll and Söderhäll Citation2022).
Table 1. Crustacean cell culture, transfection/transduction and immortalization attempts reported in the literature with reference to their relevance to cultivated crustacean meat (CCM) research and development.
While these and other crustacean cell culture attempts have been invaluable to crustacean research, a cell line has not been forthcoming which suggests critical aspects specific to this unique taxonomic group are yet to be identified. Most cell culture tools, reagents and standard protocols have been developed for species that differ greatly from crustaceans. Besides specific growth molecules, general metabolic requirements relating to osmolality, pH, gas exchange, and temperature need to be thoroughly understood.
2. Making crustacean muscle: an overview of canonical myogenesis and how this may relate to crustacean muscle development
Compared to vertebrates and even other invertebrates such as insects, there is little known about crustacean myogenesis or the culture of cells relevant to meat production. However, of what is known, there does appear to be some similarity between canonical vertebrate models, insects such as the fruit fly model organism (Drosophila melanogaster) and crustaceans.
2.1. Canonical vertebrate muscle development
In the developing vertebrate embryo, initial myogenesis begins with the differentiation of naïve mesodermal stem cells into myogenic progenitors known as myoblasts, that then replicate before undergoing terminal differentiation to become a population of myocytes (Chal and Pourquié Citation2017). The myocytes fuze together to form preliminary multinucleated myofibers and eventually primary myofibers that produce myofibrillar proteins such as slow myosin heavy chain (MHC) and myosin light chain (MLC) (Chal and Pourquié Citation2017). This occurs via the controlled orchestration between various pathways such as Notch and Wnt, BMP and TGF-β which drive growth and myogenic transcription factors through the early and later stages of myogenesis, particularly the pre-myogenic marker Pax3/7 and the main myogenic regulatory factors (MRFs) including MyoD, Myf5, Mrf4 and MyoG (Chal and Pourquié Citation2017; Kodaka, Rabu, and Asakura Citation2017).
Further muscle growth consists mostly of hypertrophy (i.e., the accumulation of more myofibrillar proteins such myosin, α-actin and tropomyosin) (Sartori, Romanello, and Sandri Citation2021), and the maintenance of a stable pool of Pax3/7+ mononucleated progenitor AMSCs known as satellite cells (Chal and Pourquié Citation2017; Kodaka, Rabu, and Asakura Citation2017). In vertebrate adult skeletal muscle, the pool of Pax3/7+ satellite cells maintain muscle integrity in response to general wear and tear and to replace tissue lost or damaged in injury.
2.2. Crustacean muscle development
2.2.1. Developmental and adult myogenesis
Observations of early crustacean muscle development concur with what has been seen in D. melanogaster where, mononucleated “founder cells” migrate to precise locations in the body and set the scaffold and patterning for subsequent muscle formation (Harzsch and Kreissl Citation2010; Jirikowski et al. Citation2010; Kreissl, Uber, and Harzsch Citation2008). Once in place they fuze with other undifferentiated cells to form bi-nucleated or multinucleated muscle progenitors before undergoing hypertrophic growth to become immature, MHC-producing myofibers (Harzsch and Kreissl Citation2010; Jirikowski et al. Citation2010; Mykles and Medler Citation2015). In crustacean larvae, undifferentiated “myoblasts” have been observed alongside fully differentiated and multinucleated myofibers, suggesting that early myogenesis is staggered (Mykles and Medler Citation2015). These remnant “myoblasts” may be reserved as a pool of myogenic stem cells for future growth, as occurs in vertebrates (Chal and Pourquié Citation2017) and recently observed in the fruit fly (Boukhatmi and Bray Citation2018; Chaturvedi et al. Citation2017).
In most decapod crustaceans muscle growth continues throughout life within the constraints of regular molting, which is required to make room for the additional tissue (Mykles and Medler Citation2015). Like the mammalian model, adult crustacean muscle consists of striated and multinucleated fibers with a comparable nuclei to fiber ratio, as well as a population of mononuclear “myoblasts” that may contribute nuclei to growing muscle fibers, just like vertebrate satellite cells (Mykles and Medler Citation2015; Novotová and Uhrík Citation1992). Continued growth of muscle fibers in most animals is largely the result of hypertrophic protein synthesis (Chal and Pourquié Citation2017; Mykles and Medler Citation2015; Rai and Nongthomba Citation2013), and in many crustaceans this applies more to the expansion of existing, rather than formation of new myofibers (Mykles and Medler Citation2015). Individual crustacean myofibers are also lengthened throughout life via the addition or extension of sarcomeres, a mechanism thought to better enable the plasticity required during molting (Mykles and Medler Citation2015). There are clear similarities between crustaceans and known animal models here, although it remains to be seen how plasticity of in vivo crustacean muscle growth might impact efforts to recapitulate it in vitro.
2.2.2. Myogenesis in limb regeneration
Due to a process known as autotomy, crustaceans can eject their limbs at a preformed breakage plane with almost no blood loss or tissue damage, and then regrow them to their original state () (Almazán et al. Citation2022; Hopkins and Das Citation2015). After the limbs are ejected, they are regrown via epimorphic regeneration which is a process replicative of embryogenesis (Das Citation2015; Read and Govind Citation1998). During this process, undifferentiated (naïve) or dedifferentiated stem cells migrate to the autotomy site and proliferate to form a blastema underneath the breakage plane; these proliferate further, then differentiate and develop into the tissues that make up the mature limb, which is primarily muscle (internally) and cuticle proteins (externally) (Das Citation2015; Hopkins and Das Citation2015).
Figure 2. Graphical depiction and photographs of claw autotomy and six stages claw re-generation in relation to molt cycle in the Australian redclaw crayfish cherax quadricarinatus. Autotomy breakage plane is shown between the basio-ischium (B) and coxa (C) segments. The red line indicates the molt event that occurs between stage 4 and stage 5. Allocation of stage was based on observed morphology (shape, segmentation, and pigment).
After autotomy, dedifferentiated epidermal cells in the limb bud of the fiddler crab Uca pugilator multiply to create a cuticular outer layer. This invaginates after four days to form the beginning of segments, which in turn fill with immature myofibers one to two days after they develop (Hopkins, Chung, and Durica Citation1999; Hopkins and Das Citation2015). It is not known whether the precursors to these early myofibers migrate from nearby muscle tissue or some other source, or transdifferentiate from local epidermal or other cells (Hopkins and Das Citation2015). However, it seems they move into position and differentiate in a seemingly similar process to the “founder” cell model in embryonic development. There appears to be a close link between the severed pedal nerve (after autotomy) and the migration or location of the myogenic precursors, suggesting nerve tissue delivers signals critical to myogenic development and organization (Hopkins, Chung, and Durica Citation1999). What these signals may be remains unknown.
Once the precursors have differentiated into early myofibers and are in place within the growing segments, they undergo hypertrophic growth as in normal development, albeit not continuously, but in distinct growth phases that coincide with the animal’s molt cycle (Hopkins Citation2001; Hopkins, Chung, and Durica Citation1999; Hopkins and Das Citation2015). The muscle fibers are attached at each end to the regenerating cuticle and once molt has taken place, this new and flexible cuticle fills with hemolymph and expands, stretching the young muscle fibers in the process (Hopkins Citation1993; Mykles and Medler Citation2015). This stretching is thought to then stimulate continued hypertrophy until the new limb is filled completely with muscle tissue (Medler et al. Citation2007).
As limb regeneration encompasses crustacean muscle development from an embryonic-like state to fully mature tissue, it may offer numerous lessons for growing muscle in vitro including the important influence of the molt cycle and even mechanical stress.
2.3. Molecular factors influencing crustacean muscle development
As with the morphological aspects, it is possible that various molecular aspects of crustacean muscle development can be related to that of insects or vertebrates (Mykles and Medler Citation2015). highlights a number of genes known to be involved in proliferation and myogenic differentiation in various model and crustacean species. An important caveat to note, however, is that while direct comparisons can be telling, some molecular studies have reported surprising discrepancies in crustacean models. These include an opposite role for the vertebrate muscle growth inhibitor myostatin, and the absence of a clear homolog for insulin growth factor (IGF) in crustaceans.
Table 2. Genes relevant to stem cell proliferation and myogenic differentiation for cultivated meat production in model vertebrate species, with some also noted in insects and crustaceans.
2.3.1. Myogenic factors
In D. melanogaster, instead of the canonical MRFs, the main molecular actors driving myogenesis appear to be Myocyte Enhancer 2 (Mef2), Nautilus and Twist, where Mef2 is important for fusion, Nautilus is closest to MyoD in sequence but has a restricted myogenic function, and Twist is the most likely equivalent of the MRF suite (particularly MyoD) in terms of embryonic myogenesis (Baylies, Bate, and Gomez Citation1998; Michelson et al. Citation1990; Taylor Citation2006). Twist has a key role very early in fruit fly development with its high expression in the initial anterior hemisphere of the mesoderm required for both somatic and cardiac muscle determination (Bothe and Baylies Citation2016). Nautilus on the other hand, has no apparent role during early myogenesis in the fruit fly but rather initiates the myogenic program in adult fibroblasts, while Mef2 is downstream of and works synergistically with Twist, which is similar to its relationship with the MRFs in vertebrates (Taylor Citation2006).
Although references to a crustacean MyoD, or its D. melanogaster ortholog Nautilus, have not been located (Bomkamp et al. Citation2023), crustacean versions of Twist and Mef2 appear to display the same functions and interplay as they do in the D. melanogaster model. In Parhyale hawaiensis Twist is expressed earlier than (although not as early as in the fly) and is seemingly required for Mef2, which again, is required for initial muscle differentiation (Price and Patel Citation2008). Interestingly, in contrast to these models, Mef2 expression in various penaeid shrimp is upregulated well before Twist, although as in the previous models, it too was necessary for subsequent muscle specification and differentiation (Wei et al. Citation2016).
The importance of MyoD-family transcription factors to myogenic progression appears to be well conserved across taxa including invertebrates. Transfection with D. melanogaster Nautilus has been found, along with chicken MyoD, to rescue myogenesis in Caenorhabditis elegans CeMyoD-null cells (Zhang et al. Citation1999). The same study also showed transfection with the fruit fly Nautilus, together with E-protein ortholog Daughterless, was able to initiate myogenesis in mouse fibroblasts. Further investigation into a potential crustacean Myod/Nautilus ortholog is warranted.
2.3.2. Ecdysteroids
The effect of molt hormones on muscle growth has also been investigated in insects and crustaceans. The main molt-activating hormone ecdysone drives molt in all arthropods and because this is intrinsically linked to growth (Hopkins Citation2001) it likely has a specific role in cell proliferation and myogenesis.
In vivo crustacean studies show interesting effects of ecdysteroids where a functioning ecdysteroid signaling pathway coupled with low hemolymph titers appear to be necessary for proliferation of limb bud blastemal cells, while high circulating titers of this hormone have an inhibitory effect (Das and Durica Citation2013). High ecdysteroid hemolymph titers are, however, required for pre-molt muscle protein synthesis and remodeling in non-regenerating muscle (Covi et al. Citation2010; Mykles and Medler Citation2015).
Surprisingly, in vivo RNAi silencing of the ecdysone receptor in the shrimp Litopenaeus vannamei led to increased expression of actin and MHC implying a negative effect on muscle growth (Qian et al. Citation2014).
In vitro insect studies have demonstrated a relationship between ecdysone and myogenesis. In D. melanogaster a functioning ecdysone pathway was found to be necessary for myogenic differentiation, apparently through its activation of Mef2 via Twist, and that spikes in the hormone consistently coincided with increased Mef2 expression (Lovato, Benjamin, and Cripps Citation2005). Concurring with this, terminal differentiation of cultured myoblasts has been achieved with ecdysone media supplementation in several in vitro insect studies (Rubio et al. Citation2020).
It is unclear if the same myogenic effect is possible for crustacean cells. In vivo experiments have demonstrated that high ecdysteroid hemolymph titers (both through injection and molt induction) appear to stimulate muscle protein synthesis, seemingly through increasing translation (rather than transcription) and possibly through myostatin inhibition (Mykles and Medler Citation2015). A recent attempt to culture lobster AMSCs for CCM showed that ecdysone also appears to have a positive effect on their proliferation in vitro, although to a lesser degree than supplementation with the crustacean juvenile hormone methyl farnesoate (Jang, Scheffold, and Bruheim Citation2022).
Ecdysteroids are clearly linked to muscle development in crustaceans, however there are obvious complexities that need to be unraveled, particularly in terms of driving muscle cell proliferation and differentiation in vitro. This should be flagged as a key focus area for further CCM research.
2.3.3. Growth factors
Myostatin (MSTN), a member of the transforming growth factor-β (TGF-β) family, is understood to inhibit myogenesis in vertebrates. Investigations of MSTN in numerous crustacean species have revealed a similar structure to the human ortholog, as well as similar inhibitor interactions (MacLea et al. Citation2010) and it is clearly important to crustacean growth, being intrinsically linked to molting and ecdysteroids (Mykles Citation2021). However, like ecdysteroids, its precise role in crustacean myogenesis remains unclear (Mykles and Medler Citation2015). Studies on the land crab Gecarcinus lateralis showed that high levels of ecdysone generally correlate with low MSTN and high myofibrillar protein synthesis in pre-molt claw muscle, although the molecular pathways involved appeared to be different across different muscle types and stages of the molt cycle (Covi et al. Citation2010; MacLea et al. Citation2012).
A number of studies have attempted to determine whether MSTN is a negative regulator of myogenesis as it is in vertebrates, either by comparing growth phenotypes to MSNT mRNA expression or by conducting RNAi silencing experiments (). These studies have looked at both MSTN and its putative receptor ActivinR2. Although there were interesting indications that MSTN could be a positive myogenic regulator in crustaceans (opposite to vertebrates), overall, the results of these studies point to a negative role. This clearly warrants investigation into MSTN inhibition in CCM cell culture experiments, potentially through delivery of antagonist ligands in cell culture media.
Table 3. Studies investigating a negative or positive role of myostatin (MSTN) or its receptor ActivinR2 in crustacean muscle growth.
Some in vitro and in vivo crustacean studies have identified differing effects of recombinant mammalian growth factors on myogenesis, with many being dependent on dosing (Claydon Citation2009; Jayesh, Philip, and Bright Singh Citation2015). Of particular interest is insulin-like growth factor (IGF) which, delivered via cell culture medium, increased protein synthesis in crayfish muscle explants and increased growth in individuals when delivered via feed (Chaulet et al. Citation2012; Jayesh, Philip, and Bright Singh Citation2015). Despite the fact that no crustacean IGF orthologs have yet been identified, this suggests that related pathways are active (Chandler et al. Citation2017). The closely related sister group to the IGFs, the insulin-like peptides (ILPs), of which there are at least eight known in the fruit fly, may also help facilitate growth (Semaniuk et al. Citation2020). Putative homologs of some of these have been identified in several decapod species and are known to share certain similarities with the IGFs; they both have IGF-like domains and both bind to insulin-like growth factor-binding proteins (IGFBPs) (Chandler et al. Citation2017; Rosen et al. Citation2013). Furthermore, at least one crustacean ILP, the insulin-like androgenic gland hormone (IAG), which is primarily a male-differentiating hormone, has also been associated with increased growth (Rosen et al. Citation2013; Ventura et al. Citation2009).
Epidermal growth factor (EGF) has been directly linked to growth in the freshwater prawn Macrobrachium rosenbergii where temporary silencing of the receptor (EGFR) reduced growth (Sharabi et al. Citation2013). Orthologs of fibroblast growth factor (FGF)-2 and FGF-4 have been found to be involved in very early limb regeneration in the fiddler crab, as apparent targets of retinoic acid which promotes proliferation and inhibits differentiation of blastemal cells (Hopkins Citation2001). Overall, the exact effects and pathways related to growth factors in crustacean muscle growth are not known, and because experiments with recombinant versions have produced varying results, there is a case, particularly in the context of growing cells in vitro, for investigating more species- and tissue-specific factors (Ma, Zeng, and Lu Citation2017).
3. Current approaches to culturing stem cells for cultivated meat
To attain the vast quantities needed for CM production, stem cells must proliferate sufficiently and differentiate into appropriate cell types. Although CM chicken products approved for sale in the United States have been created with fibroblasts (Fasano Citation2022; Overbey Citation2023), efficient differentiation of stem cells into muscle fibers is required if CM is to emulate conventional meat (Lee et al. Citation2023). Approaches to these activities vary depending on the starting stem cell type. While pluripotent stem cells (PSCs) have natural proliferative abilities, they may require greater focus on strategies for efficient myogenic differentiation, whereas naturally myogenic AMSCs need more strategies to encourage and maintain proliferation (Reiss, Robertson, and Suzuki Citation2021). Multipotent stem cells, depending on their relationship to the muscle lineage, require combinations of both strategies.
3.1. Driving myogenic differentiation of pluripotent stem cells
There are two broad approaches to driving the myogenic differentiation of PSCs in vitro: 1) directed differentiation involves the precisely timed delivery of growth factors and other molecules (inhibitors and activators) to simulate the canonical pathways via specific media regimens; and 2) direct reprogramming involves more direct molecular intervention usually with transfection of particular transcription factors such as MyoD, which is considered a “master regulator” of myogenesis () (Chal and Pourquié Citation2017; Genovese et al. Citation2017; Jiwlawat et al. Citation2018; Kodaka, Rabu, and Asakura Citation2017; Post et al. Citation2020). Kodaka, Rabu, and Asakura (Citation2017) and Chal and Pourquié (Citation2017) provide comprehensive reviews of in vitro attempts to drive vertebrate PSCs through the stages of embryonic myogenesis with both approaches.
Figure 3. Directed differentiation vs direct reprogramming for in vitro myogenesis. Myogenesis from a multipotent cell to mature myofibers involves progressive expression of particular myogenic markers (encircled). In directed differentiation (via media), this is enabled with the timed delivery of particular small molecule activators and growth factors (red down arrows). For direct reprograming, myogenesis is enabled with transfection of a master myogenesis regulator MyoD (blue star). Adapted from Chal and Pourquié (Citation2017).
At the time of writing, we know of no published attempts to drive the myogenic differentiation of ESCs toward mature muscle for CM production. We note at least one attempt using direct reprogramming of iPSCs with MyoD (Genovese et al. Citation2017) and another involving direct reprogramming of multipotent fibroblasts (Jeong et al. Citation2022). The direct reprogramming approach can theoretically be applied to all PSC types and is considered by some to be more efficient than the media regime approach (Rao et al. Citation2018).
Three CM companies (Magic Valley, Higher Steaks and Steakholder Foods) use iPSCs to make muscle or fat tissue (Perelmutter Citation2022; Redrup Citation2022; Watson Citation2021), however it is not clear which differentiation techniques they are using. Considerations for direct reprogramming of crustacean cells are covered in Section 5 below.
3.2. Induced pluripotent stem cell considerations
Since their discovery in 2006, iPSCs have been readily created with simple overexpression of four genes known as the “Yamanaka Factors” (Oct3/4[POU5F1], KLF4, Sox2 and c-Myc) (Rosselló et al. Citation2013; Takahashi and Yamanaka Citation2006). They have been used extensively for in vitro myogenesis induction (Chal and Pourquié Citation2017; Kodaka, Rabu, and Asakura Citation2017), and a number of reviewers acknowledge their benefits for CM development (Ben-Arye and Levenberg Citation2019; Kadim et al. Citation2015; Post et al. Citation2020; Verma, Lee, and Salamone Citation2022). Although the use of iPSCs in CM production may entail genetic modification (GM) regulatory hurdles in some places (Post et al. Citation2020), they are considered easier to obtain and maintain in culture than ESCs and they encompass fewer ethical concerns due to their generation from relatively non-lethal tissues (Reiss, Robertson, and Suzuki Citation2021).
Some commentators suggest iPSCs may have the benefit of greater differentiation potential, through epigenetic or other means, if the original cell type is of the same lineage (Scesa, Adami, and Bottai Citation2021; Watanabe et al. Citation2011). For example, several studies have demonstrated successful creation of iPSCs from AMSCs (Lang et al. Citation2009; Polo et al. Citation2010; Watanabe et al. Citation2011), and at least one has shown that these display myogenically-biased gene expression, possibly leading to enhanced myogenic differentiation potential (Polo et al. Citation2010).
To our knowledge, attempts to produce iPSCs from crustacean cells have not been reported so it is unclear if conventional overexpression of “Yamanaka factors” will be applicable. However, since their initial creation, iPSCs have proven to be relatively simple to generate from many different cell types and species, so it may be feasible (Raab et al. Citation2014; Rosselló et al. Citation2013). Rosselló et al. (Citation2013) demonstrated that all four human factors were able to induce pluripotency in numerous species to varying degrees, and that although the fruit fly D. melanogaster (the most divergent from humans), showed the least responsiveness, significant expression of endogenous Oct3/4 and cMyc was achieved indicating induction of some pluripotency (Rosselló et al. Citation2013).
3.3. Adult muscle stem cells in cultivated meat research
Adult muscle stem cells are a popular choice for CM as they readily differentiate into mature muscle cells and can be accessed easily through muscle biopsies and cell dissociation protocols (Guan et al. Citation2021; Post et al. Citation2020; Reiss, Robertson, and Suzuki Citation2021; Zhu et al. Citation2022). Thus, the vast majority of recently published literature on growing meat in vitro involves AMSCs (Choi et al. Citation2021; Lei et al. Citation2022; Li et al. Citation2022; Liu et al. Citation2022; Messmer et al. Citation2022; Okamoto et al. Citation2022; Park, Choi, et al. Citation2022; Park, Choi, et al. Citation2022; Park et al. Citation2023; Stout and Kaplan Citation2022; Stout, Arnett, et al. Citation2023; Stout et al. Citation2022; Stout et al. Citation2020; Stout, Rittenberg, et al. Citation2023; Takahashi et al. Citation2022; Zhang et al. Citation2023; Zheng et al. Citation2022; Zheng et al. Citation2023; Zhu et al. Citation2022). Although these studies largely cover bovine or porcine satellite cells, and to a lesser extent those from chicken or fish, they can serve as initial guides for crustacean AMSC culture. The species differences will likely necessitate use of crustacean-specific supplements or culture conditions for optimal outcomes, however recent attempts to culture lobster tail muscle AMSCs using similar protocols, demonstrate these as a valid place to begin (Jang, Scheffold, and Bruheim Citation2022).
Insect AMSCs are taxonomically closer to crustacean AMSCs than those of vertebrates, and these have also been investigated for CM research. With a D. melanogaster adult muscle progenitor-like cell line (Kerafast #EF4006), Rubio, Fish, et al. (Citation2019) demonstrated some success in promoting proliferation and differentiation using serum-free media and various hormone supplements (20-HE and Methoprene). This and other insect muscle cell culture attempts detailing the effects of hormones (Baryshyan et al. Citation2012; Rubio, Fish, et al. Citation2019), or various small molecules (Shukla, Singh, and Gawri Citation2011) may be valuable to CCM researchers.
Although AMSCs generally have limited proliferative capacity, they may replicate enough in vitro to produce sufficient quantities of muscle tissue for commercial CM production (Melzener et al. Citation2021; Post et al. Citation2020). However, they are generally known to lose their myogenic potential well before they cease proliferating and this could be problematic if it occurs before sufficient numbers are reached (Stout, Arnett, et al. Citation2023; Zhu et al. Citation2022). An alternative method of maintaining both proliferative and myogenic potential indefinitely is to establish an immortalized AMSC line (see Section 6).
4. Prospective primary tissue sources for cultivated crustacean meat
Like vertebrates, crustaceans boast a repertoire of various pluripotent, multipotent and unipotent stem cells (Vogt Citation2022), all of which have differing potential in terms of their ability to proliferate and differentiate into muscle. Despite numerous attempts to culture different types (), a cell line has remained elusive, so CM research must continue to rely on primary sources. In many vertebrate models, primary cells can be taken non-lethally via small tissue biopsies, providing a more practical and humane source of tissue. However, due to their hard exoskeleton, non-lethal tissue biopsies are difficult to obtain from crustaceans. While the numbers of any animals sacrificed for CM cell sourcing will be insignificant compared to those currently required for meat production, limiting this as much as possible is still warranted. We note three potential sources of crustacean stem cells that are non-lethal: a) muscle tissue within fully-grown crustacean limbs such as claws or walking legs; b) tissues within actively regenerating limbs; and c) hematopoietic stem cells (HSCs) circulating within the hemolymph ().
Figure 4. Three potential and non-lethal stem cell sources for cultivated crustacean meat development. (A) Muscle tissue within fully-grown claws or other limbs; (B) tissues within actively regenerating limbs (likely housing pluri- or multipotent stem cells and adult muscle stem cells); (C) circulating hemolymph potentially containing putative (and multipotent) hematopoietic stem cells.
4.1. Fully-grown decapod limb tissue
Due to the process of autotomy, crustaceans can eject their limbs (claws and walking legs) with almost no blood loss or tissue damage, and then regrow them to their original state (Almazán et al. Citation2022) (). This can occur multiple times throughout life, potentially as frequently as every molt. Because they contain fully developed muscle tissue, the ejected limbs may be a very accessible and non-lethal source of AMSCs. Autotomy can be induced by applying some pressure to the limb with forceps (O’Brien Citation1999). Ensuring all care is taken in the autotomy process to limit any stress or discomfort for the animal, this may be a source of primary cells that is ethically on par with small muscle biopsies for larger vertebrates.
Given the similarities between vertebrate and crustacean muscle tissue, particularly regarding the presence of “myoblasts” or putative satellite cells in limb muscle (Konstantinides and Averof Citation2014; White et al. Citation2005), initial efforts to isolate and culture cells from the limb muscle tissue could borrow from the documented attempts with vertebrate or insect AMSCs noted above (section 3.3). Ejected limbs could also be a non-lethal source of programmable cells from which to generate iPSCs. As mentioned, iPSCs reprogrammed from mature AMSCs, like those from limb tissues, may entail certain epigenetic features that could enhance their subsequent myogenic differentiation.
4.2. Regenerating decapod limb tissue
After the initial autotomy, regenerating crustacean limbs can be re-autotomized and regenerated again (before or after the next molt) (Holland and Skinner Citation1976). This provides another non-lethal, but possibly more fruitful, source of stem cells for CCM development. Because the process of epimorphic regeneration begins with undifferentiated blastemal cells and results in mature muscle, regenerating tissues could represent a wide range of stem cell potency. Studies on the fiddler crab suggest that while mitotic activity is predominant in the earliest stages of regeneration, it is hypertrophy (synthesis and elongation of muscle fibers) that is mainly occurring at the immediate pre-molt stage () (Hopkins Citation2001; Hopkins, Chung, and Durica Citation1999; Hopkins and Das Citation2015). Examination of claw regeneration in the crayfish Cherax destructor also found that growth of undifferentiated cells occurs before initial segmentation is visible (equivalent to Stage 3 in ), and myogenic growth occurs afterwards (White et al. Citation2005).
Table 4. Five growth stages of uca pugilator limb regeneration in relation to molt cycle, observed histological activity and description of external morphology (Hopkins Citation2001; Hopkins, Chung, and Durica Citation1999; Hopkins and Das Citation2015).
Together these studies suggest more “potent” (undifferentiated) stem cells may be prevalent earlier in the process, with more myogenic cells predominating later. Further histological and gene expression analyses across the regeneration process may shed light on when key factors are more active. These findings could be augmented with single cell transcriptomics and metabolomics, further assisting researchers to better isolate their cell of choice. Whichever type is preferred, because both are likely to be valuable for CCM development, the regenerating limb presents as a convenient tissue source. Furthermore, the relatively small size of the limb buds and their ability to grow rapidly prior to molting suggest there could be a higher concentration of stem cells than in fully grown limb tissue.
4.3. Hematopoietic stem cells in hemolymph
Accessing stem cells via hemolymph extraction may also be a non-lethal and viable source of CCM stem cells. HSCs have been the focus of numerous crustacean cell culture attempts (), largely because of their specific relevance to pathogen research. After forming and proliferating in the hematopoietic organ (HPT) they enter the hemolymph as mostly, but often not entirely, differentiated hemocytes belonging to one of three immune cell lineages (hyaline, semi-granulated and granulated) (Söderhäll Citation2016; Söderhäll and Söderhäll Citation2022). There is evidence to suggest however, that HSCs in a wide range of animals, including crustaceans, are plastic and can differentiate into other cells besides hemocytes, something that may link with their ability to traverse the body through the circulatory system (Catacchio et al. Citation2013; Ogawa, LaRue, and Mehrotra Citation2013). Such findings have been applied to hepatocytes (Lagasse et al. Citation2000; Theise et al. Citation2000), neural cells (Beltz et al. Citation2011; Eglitis and Mezey Citation1997), epithelial cells of multiple tissues (Krause et al. Citation2001), fibroblasts (Shirai et al. Citation2009) and skeletal muscle cells (Asakura et al. Citation2002; Doyonnas et al. Citation2004; Ferrari et al. Citation1998; Gussoni et al. Citation1999; LaBarge and Blau Citation2002; Uhrík, Rýdlová, and Zacharová Citation1989).
Potentially explaining these observations, it has been suggested that HSC plasticity is triggered by whatever microenvironment they find themselves in, where cell surface receptors receive signals that activate niche transcription pathways, thereby leading the cells down alternative differentiation pathways (Avots et al. Citation2002; Catacchio et al. Citation2013). However, in terms of whether HSCs are capable of true plasticity, there has been some suggestion that “evidence” of transdifferentiation could be the result of other phenomena such as paracrine effects or cell recruitment (Lee and Hong Citation2020), or adoption of new markers and functions via fusion with existing cells (Avots et al. Citation2002; Camargo et al. Citation2003; Wagers and Weissman Citation2004).
Although wide-ranging plasticity of HSCs remains a topic of debate, there is strong evidence for a relationship with myogenic tissues. HSC-derived cells appear to be relatively important in muscle regeneration after injury with the proportion of these cells being much higher in post-injury tissue than in normal physiological conditions (Doyonnas et al. Citation2004; Dunn et al. Citation2018; LaBarge and Blau Citation2002; Polesskaya, Seale, and Rudnicki Citation2003). Single HSCs can give rise to both hematopoietic and myogenic progeny (Corbel et al. Citation2003) and many muscle-resident cells give rise to hematopoietic cell lineages (Asakura et al. Citation2002; Jackson, Mi, and Goodell Citation1999) both of which support a close connection between hematopoietic and myogenic lineages. This relationship might be considered logical given the shared mesodermal origins of these two tissue types (Kanji, Pompili, and Das Citation2011). There is a strong case for the involvement of plastic HSCs in crustacean limb regeneration with histological studies suggesting that hemocytes may directly transdifferentiate into muscle precursors or “satellite” cells (Babu Citation1987; Novotová and Uhrík Citation1992; Read and Govind Citation1998; Uhrík, Rýdlová, and Zacharová Citation1989).
Because there appears to be at least some evidence for the potential of HSCs to differentiate into muscle, and there is a clear relationship between the hematopoietic and myogenic lineages, we hypothesize that HSCs constitute a possible stem cell source for CCM development. Although the canonical view of hemocyte formation sees most mitosis and subsequent differentiation occurring before release from the HPT, there is evidence that, under certain circumstances, a portion of cells entering the circulation could be undifferentiated and proliferative “pro-hemocytes,” closer to HSCs in function (Braasch, Ellender, and Middlebrooks Citation1999; Ellender, Najafabadi, and Middlebrooks Citation1992; Roulston and Smith Citation2011; Sequeira, Tavares, and Arala-Chaves Citation1996; Zhao et al. Citation2022). Others suggest that hyaline cells and pro-hemocytes may be one and the same (Söderhäll Citation2016).
The number of these circulatory mitotic cells is increased up to 5-fold after health challenges such as infection or blood loss (Roulston and Smith Citation2011; Smith, Accorsi, and Malagoli Citation2016). A long-held view is that these mitotic cells are rapidly formed in and released from the HPT in urgent response (Söderhäll Citation2016; Söderhäll et al. Citation2003). However, a very recent study has identified a putative cell cycle regulatory mechanism in circulating hemocytes that appears to enable their rapid proliferation in the face of a health challenge, potentially providing an additional, yet faster and more efficient response mechanism (Zhao et al. Citation2022). The researchers demonstrated accelerated proliferation of cultured hemocytes using RNAi mediated knockdown of the putative negative regulator Csn5, thus presenting a prospective tool to control hemocyte replication in vitro.
In an effort to identify hemocyte lineage markers in crayfish, Wu et al. (Citation2008) found that PCNA could be utilized as a marker to distinguish proliferating HSCs from more differentiated hemocytes (Wu et al. Citation2008). In support of this, a study on the freshwater prawn M. rosenbergii found that PCNA expressing cells were present in both the hematopoietic organ and in hemolymph, the considerably lower proportion in the latter could be reflective of some HSCs entering the circulation (Thansa et al. Citation2021). Roulston and Smith (Citation2011) reported proliferating and undifferentiated hemocytes (HSC-like) through 5-bromo-2-deoxyurdine (BrdU) cell labeling, and then separated them from more differentiated and non-proliferative hemocytes with a two-step Percoll separation procedure (Roulston and Smith Citation2011). If replicated successfully, this could be a way for CCM researchers to non-lethally isolate pluri-, or multipotent stem cells, followed by attempts to maintain proliferation and drive myogenic differentiation either through media regimes or direct reprogramming.
5. Potential for direct reprogramming via transfection/transduction of crustacean cells
Establishing a viable proliferative culture of plastic crustacean cells (akin to PSCs) provides the opportunity to experiment with direct reprogramming for myogenic differentiation. One CM research group has already achieved success with this approach by transfecting porcine iPSCs with human MyoD (Genovese et al. Citation2017). details numerous demonstrations of successful transfection/transduction of crustacean cells including ESCs, lymphoid cells, HSCs, and hemocytes. Gene transfer methods include lenti-, retro- and baculoviral transduction and plasmid transfection with electroporation, lipofection, cationic polymers and lipopolyplexes, sometimes in conjunction with viral or custom-made nuclear localization signals (Arenal et al. Citation2004; Nguyen, Ventura, and Elizur Citation2019).
Although direct reprogramming toward myogenesis has generally been attempted with ESCs or iPSCs (Chal and Pourquié Citation2017), HSCs may also be suitable for this approach in CCM development. This is based on past evidence of their successful transfection and their possible plasticity and/or propensity toward muscle lineage transdifferentiation. In summary, this potential differentiation capacity, together with tools to enhance their in vitro proliferation (i.e., Csn5), suggests that crustacean HSCs successfully isolated from circulating hemolymph could be another non-lethal source of CCM-suitable cells. However, as the target outcome is a food product, the direct reprogramming approach may entail implications around GM regulation like iPSCs, although this may be a less of an issue if transfections are transient.
6. Considerations for immortalizing crustacean cells
Immortalized cell lines offer benefits over primary cell sources in terms of consistency, production yield, and independence from animals, however, they can be difficult to establish and may entail a number of aspects relating to market acceptance.
6.1. Potential methods for immortalizing crustacean cells
To become immortalized, the cells’ DNA must be altered leading to permanent deactivation of cell cycle arrest mechanisms, that can occur either spontaneously or via direct genetic manipulation (Soice and Johnston Citation2021). Spontaneous immortalization involves selecting for cells that have escaped senescence after undergoing random mutations over many population doublings. While this has already been achieved with chicken fibroblasts for CM production (Pasitka et al. Citation2022), it can be difficult depending on the target species and cell type, and may involve additional changes beyond indefinite activation of the cell cycle (Soice and Johnston Citation2021). In contrast, direct manipulation involves very specific changes to cell cycle machinery, largely through overexpression of telomerase reverse transcriptase (TERT) to limit telomere shortening, or knocking out/inhibiting cell cycle arresters such as retinoblastoma (Rb), p15, p16 or p53 (Soice and Johnston Citation2021; Stout, Arnett, et al. Citation2023).
Some genes known to inhibit Rb and p53 include E6, E7, 12SE1A, HPV and SV40-T, all of which have been successfully transfected into crustacean cells for this purpose (Claydon Citation2009; Hu et al. Citation2008; Puthumana et al. Citation2015; Tapay et al. Citation1995). Although immortalization has not been achieved, some reports claim a positive effect on proliferation after transfection with SV40-T (Hu et al. Citation2008; Tapay et al. Citation1995). Investigators of cell immortalization in other animals have observed that success is often only achieved with combinatorial approaches involving both inhibition of cell cycle arresters and TERT overexpression (Fujii et al. Citation2006; Gong et al. Citation2007; Liu et al. Citation2013; Soice and Johnston Citation2021). The benefits of a multi-pronged approach are recognized by CM researchers; Upside Foods have lodged a patent detailing the combination of TERT overexpression and CRISPR knockout of p15 and p16 Genovese, Desmet, and Schulze (Citation2022), and Stout, Arnett, et al. (Citation2023) engineered overexpression of combined TERT and CDK4 (an Rb inhibitor) in bovine AMSCs, resulting in an immortalized line for CM production.
To our knowledge overexpression of TERT in crustacean cells has not been attempted. This is despite crustacean longevity being linked to high telomerase activity in many crustacean tissues (Klapper et al. Citation1998) and TERT overexpression often being recognized as a potential solution to crustacean cell line development (Claydon and Owens Citation2008; Hu et al. Citation2008; Jayesh, Seena, and Bright Singh Citation2012; Puthumana et al. Citation2015). However, while one group reported telomerase activity in cultured shrimp lymphoid cells (Lang et al. Citation2004), another group could not verify this (Jayesh et al. Citation2016), which may suggest TERT overexpression might not be as effective in vitro as hoped.
Nonetheless, the creation of a crustacean cell line is still imperative, both for crustacean research in general and for CCM development in particular, so continued attempts are vital. As the multi-pronged approach has rescued failed immortalization attempts in non-crustacean cells, it is feasible that the same approach is necessary for crustacean cell lines. Moving forward from the already proven ability to transfect and express oncogenes in crustacean cells, more concentrated investigations into telomerase activity, overexpression of TERT or cell cycle promoters, and direct knockdown of cell cycle inhibitors are warranted.
6.2. Potential benefits and limitations of immortalized cell lines
In addition to the technical challenges of establishing an immortalized crustacean cell line for CM production, there are other aspects for prospective developers to consider. Soice and Johnston (Citation2021) provide a comprehensive overview of these, covering the numerous technical possibilities for enhancing immortalized cells for CM development, as well as limitations including government regulation and consumer acceptance considerations. To briefly summarize their review, alongside the benefits already mentioned, immortalized cell lines can be genetically manipulated to improve production efficiency, taste, texture, nutrition and safety of final food products in myriad ways, but they may face more stringent regulation or consumer acceptance hurdles than primary cells in some jurisdictions.
Alongside iPSC generation and direct reprogramming already discussed, direct manipulation for cell immortalization often involves new genomic techniques (NGTs) that include gene editing, mutagenesis or cis-/intragenics, rather than traditional transgenic GM technology. In Europe, NGTs are regulated as strictly as traditional GM products, whereas in the United States and other places, they are not (Campden BRI Chipping Campden Ltd Citation2021; Tachikawa and Matsuo Citation2023). Strict regulation often coincides with more general negative sentiment around GM technology (Campden BRI Chipping Campden Ltd Citation2021) and therefore cell lines produced this way may be less successful in these places.
Cell lines developed with spontaneous techniques have their own limitations; associations with cancer have been made due to the shared processes of senescence evasion through random mutations (Soice and Johnston Citation2021; Stout, Arnett, et al. Citation2023). While there are clear similarities, there are numerous mechanisms researchers can employ to ensure cell lines do not become tumorigenic (Pasitka et al. Citation2022). Related to this is genetic drift which could see other unintended changes occurring in continuously replicating cell populations, potentially affecting desired meat-relevant phenotypes such as texture or taste. However, again, there are techniques that can control for this such as carefully planned cell banking (Soice and Johnston Citation2021). Furthermore, in terms of food safety there is presently no evidence that consumption of immortalized cells, nor genetically modified DNA for that matter, is unsafe (FAO and WHO Citation2023; Nawaz et al. Citation2019).
If the productivity benefits of immortalized cell lines are to be realized for CCM (and CM) development, more education may be required to build public and government support. Interestingly, global regulation of NGTs is converging toward a more lenient middle ground (Tachikawa and Matsuo Citation2023), including that of the European Union which is currently reviewing NGT regulation to become more in line with other jurisdictions and its own sustainability goals (Zimny Citation2022).
7. Available tools for growing crustacean muscle in vitro
A final aspect to consider in CCM development is that alongside the lack of established crustacean cell lines and protocols for long-term proliferative (meat-relevant) cultures, there is a paucity of crustacean-validated tools and reagents for cell isolation, characterization, purification, or manipulation. Online searches reveal that those currently available are primarily validated for model mammalian species (human/mouse etc.), with some also being validated for avian and fish species, and to a lesser extent, non-crustacean invertebrates such as D. melanogaster. Going forward, greater understanding of what works and what does not, can only be achieved with continued attempts. To be effective however, these attempts must be well designed and openly reported, and undertaken in the context of deeper investigations into crustacean-specific or species-specific molecular characteristics.
Fortunately, more genomic and transcriptomic data for various crustacean species are being added to public databases regularly and these, together with in-house sequencing experiments, can be used to identify critical orthologs. Specifically, this includes genes involved in proliferation and differentiation pathways (e.g., receptor-ligand pairs, signal transducers, transcription regulators etc.) and those that can be used for characterization and purification (cell markers). Thorough homology investigations can allow researchers to evaluate the utility of existing tools such as antibodies or growth factors or enable the development of novel, crustacean-specific, alternatives. Alongside general databases such as the National Center for Biotechnology Information, which are being increasingly populated with crustacean genomic and transcriptomic data, there are several crustacean-specific databases that researchers can use to find specific homologs for further investigation: CrusTome (Pérez-Moreno et al. Citation2023), CrusTF (Qin et al. Citation2017) and CrustyBase (Hyde et al. Citation2020).
7.1. Antibodies
Characterization and isolation of specific cell types is critical in CM research, and this is commonly achieved with antibodies in applications such as immunofluorescence, Western Blots, ELISA assays, and flow cytometry. Antibodies used in recent CM research largely include those targeting the satellite cell marker Pax3/7, also the MRFs, mature muscle proteins such as MHC, desmin and actin, and muscle cell surface markers such as CD31, CD45, CD56 and CD29 (Choi et al. Citation2021; Kim et al. Citation2022; Li et al. Citation2022; Saad et al. Citation2023; Stout et al. Citation2022; Takahashi et al. Citation2022; Yamanaka et al. Citation2023; Zheng et al. Citation2023; Zhu et al. Citation2022). Experiments looking to make meat from porcine iPSCs utilized Pou5F1 (Oct3/4) and KLF4 antibodies to confirm pluripotency before the cells were programmed with a mammalian MyoD transfection vector (Genovese et al. Citation2017). Though not specifically reported for CM development, embryonic cell surface markers SSEA-3, SSEA-4, and Nanog are also commonly used to identify PSCs (Vazin and Freed Citation2010).
The vast majority of antibodies utilized by CM researchers have been developed for human proteins, so closer taxonomic distances mean they are much better suited to mammalian, or even avian and fish cells, than those from invertebrates like crustaceans. Searches for crustacean-validated antibodies on public and company databases return very few products and while a search with relevant key words (crustacean, shrimp, crab, lobster, prawn, crayfish) on the public Antibody Registry website (https://www.antibodyregistry.org/), does return several, these mainly target heat shock proteins and neurotransmitters which are not immediately relevant for CM development. A number of crustacean researchers have reported some success using existing (human-validated) antibodies such as Pax7 (Jang, Scheffold, and Bruheim Citation2022), FGF-2 and 4 (Hopkins, Chung, and Durica Citation1999), and PCNA (Pudgerd et al. Citation2019), however further validation of these is certainly required.
Greater success might be achieved with antibodies developed for, or at least validated in, model insects such as D. melanogaster, however there are considerably fewer of these available and depending on the level of conservancy, taxonomic distance may still preclude antibody binding. Discovering favorable homology between target crustacean proteins and the immunogen or epitope sequences of commercial antibodies may assist CCM researchers in antibody selection, however these sequences are often proprietary and rarely available.
Alternatives to commercially available antibodies are custom made options. Some of these generated for crustaceans in the past include those targeting Pax3 (Davis, D’Alessio, and Patel Citation2005), MHC (Kreissl, Uber, and Harzsch Citation2008), hormone receptors (Chang, Keller, and Chang Citation1998; Durica et al. Citation2002), various hemocyte proteins (Claydon Citation2009; Lin et al. Citation2007; Rodriguez et al. Citation1995; Sung, Wu, and Song Citation1999; van de Braak et al. Citation2000; Winotaphan et al. Citation2005), and a glial cell protein (Almazán et al. Citation2022), however most of these are not publicly accessible. Creating and validating new crustacean antibodies is therefore vital to the CCM research field. Alongside traditional antibody development methods, involving animal inoculation and sacrifice, are newer animal-free techniques, which may be more appealing to CM researchers. These include technologies such as phage display screening of established natural (e.g., from B-cells) or fully synthetic (computer randomized) libraries for novel antibody generation, (Shim Citation2015), along with recombinant production methods in microbial, mammalian or plant cell expression systems (DeLuca, Mick, and DeLuca Citation2022; Diamos et al. Citation2019).
Our own recent investigations into these animal-free techniques revealed that although many offer faster service and purportedly more precise products, some were up to 10 times the cost of traditional methods. However, as organizations such as the National Center for the Replacement, Refinement and Reduction of Animals in Research push for more adoption of animal-free tools in the life sciences, costs will hopefully come down.
For CCM production, whichever cell type is targeted (PSCs or AMSCs), the further development of customized tools for public use, strategies for making them, or reports on the suitability of existing tools are all urgently needed. Besides crustacean-validated tools, also needed are more reports on general protocols and media formulations optimized for CCM relevant cell culture, together with validation of key species-specific genes and molecular pathways. For this latter endeavor, investigating molecular mechanisms within limb regeneration from the early, potentially pluripotent stage to the later myogenic stage could be particularly telling as both cell types are important to CM development.
8. Conclusions and outlook
Despite numerous attempts, crustacean cell lines and robust protocols to ensure their sufficient proliferation and differentiation in culture, remain elusive. These are not only vital for the advancement of cultivated crustacean meat (CCM) but will be extremely important to all crustacean researchers. To highlight potential focus areas for prospective researchers, this review seeks to consolidate recent findings in the realms of crustacean cell culture, crustacean muscle development (including what little is known about the molecular mechanisms involved), and common methods to drive in vitro myogenesis.
Well characterized and immortalized cell lines are an optimum choice for CCM development for reasons of reproducibility and non-reliance on animals, however, there are very few available that are relevant to CM, and there are no cell lines at all for any crustacean cell type or species. While the use of primary cells has been very successful for some terrestrial species, hard crustacean exoskeletons often mean primary crustacean cells cannot be obtained without euthanasia. To address this, we highlight three potential non-lethal primary stem cell sources that could be utilized in CCM experiments during the current absence of cell lines.
Novel molecular techniques are also highlighted. Transfection of crustacean cells has shown some, albeit limited, success in the past. This is therefore a promising avenue of investigation, particularly in terms of guiding myogenic differentiation. Transfection and/or silencing of key genes with RNAi could also be explored in the creation of immortalized cell lines.
Aside from the absence of crustacean cell lines, CCM development is hindered by a lack of crustacean-validated tools, particularly antibodies for cell characterization and isolation. More open research around the development of new crustacean-specific tools or the tested success of currently available tools will be extremely useful. In fact, more open research reporting on all aspects of CCM work is vital, including optimized media formulations and protocols as well as more in-depth molecular investigations.
As global demand for delicious shrimp, crab, and lobster products continues to rise, finding alternative, more sustainable sources of these foods is vital. Further progression of open research into CCM development will go a long way toward to providing these alternatives, thus alleviating many of the issues facing their production today.
Disclosure statementThe authors report there are no competing interests to declare.
Acknowledgments
We would like to thank Dr Claire Bomkamp of the Good Food Institute for her kind assistance in reviewing the draft of this manuscript and providing her expertise in all things cultivated seafood.
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Funding
References
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