domain: sdv.bc.ic sde sdv

text:
Isabelle Domart-Coulon

Simon Blanchoud

From Primary Cell and Tissue Cultures to Aquatic Invertebrate Cell Lines: An Updated Overview

Keywords:

The stem cells discipline represents one of the most dynamic areas in biology and biomedicine. The vast majority of research on stem cells is being conducted in vertebrate models. Currently, over 98% of all cell lines are of mammalian origin, which represent only 0.4% of the extant identified metazoan evolution. In particular, aquatic invertebrates as a whole show the largest biodiversity and the widest phylogenetic radiation on Earth but have not yet significantly contributed to cell lines. Yet, with over 500 publications since the 1960s, the current lack of cell lines does not result from a lack of attempts at cultivating these cells but rather from fragmented research efforts in highly taxonomically diverse model species, a paucity in reports of negative results and persistent knowledge gaps in their in vitro metabolic requirements. To promote the establishment of aquatic invertebrate cell lines, there is thus a need for comprehensive knowledge mapping across taxa to identify adequate, possibly cell type-specific, protocols. Here, we review strategies for preparing an optimal inoculum, for optimizing culture conditions and for cell lineage authentication to monitor the quality of cell cultures. Finally, we conclude with our view on promising research perspectives towards establishing aquatic invertebrate cell lines.

Introduction

Currently, the origins of in vitro cell lines are highly biased towards humans. Around 75% of the total number of established cell lines are from Hominidae origin (96,862/128,799) and over 97% are of mammalian origin (126,033/128,799) [START_REF] Bairoch | The Cellosaurus, a Cell-Line Knowledge Resource[END_REF]) (Figure 1). However, mammals represent only 0.4% (1.3% when excluding the Insecta taxon) of the extant identified metazoan evolution [START_REF] Zhang | Animal Biodiversity: An Update of Classification and Diversity in 2013[END_REF][START_REF] Wilson | Class Mammalia Linnaeus[END_REF][START_REF] Chapman | Numbers of Living Species in Australia and the World[END_REF]) (Figure 1). In addition to the scientific interest relative to their sheer diversity, non-mammalian cells have multiple potential applications, including as a source for bio-active molecules or as assays for eco-toxicological tests (e.g., [START_REF] Ribeiro | Cryopreservation: Extending the Viability of Biological Material from Sea Urchin (Echinometra lucunter) in Ecotoxicity Tests[END_REF][START_REF] Rosner | Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts[END_REF]). Yet, with over 500 publications on aquatic invertebrate cell culture alone (Figure 1), the current limited number of invertebrate cell lines does not result from a lack of attempts at cultivating these cells but most likely from inappropriate techniques to cultivate these cells (reviewed in [START_REF] Rinkevich | Marine Invertebrate Cell Cultures: New Millennium Trends[END_REF][START_REF] Yoshino | Molluscan Cells in Culture: Primary Cell Cultures and Cell Lines[END_REF][START_REF] Cai | Marine Invertebrate Cell Culture: A Decade of Development[END_REF]. As exemplified in insects, a breakthrough in culturing conditions [START_REF] Grace | Establishment of Four Strains of Cells from Insect Tissues Grown in Vitro[END_REF]) initiated the emergence of a huge variety of cell lines [START_REF] Bairoch | The Cellosaurus, a Cell-Line Knowledge Resource[END_REF]) (895 cell lines from 104 genera in around 50 years). There is thus a need for a sustained research effort in non-insect invertebrate cell culture to identify adequate culturing conditions and promote the establishment of cell lines. In particular, aquatic invertebrates as a whole show the largest biodiversity and the widest phylogenetic radiation on Earth but have currently contributed to only six cell lines (Figure 1). Figure 1. Comparison of the diversity between metazoan taxa. Depicted is the phylogenetic relation between metazoan taxa and their characteristics with respect to evolutionary radiation, regenerative capacity, lifespan and in vitro cell culture (see Appendix A Table A1 for exact values). Dashed lines represent branchings for which speciation timings have not yet been determined. Colored taxa highlight those whose publication timelines are detailed in Figure 2. Regeneration capacity of the taxa is depicted as follows: + tissue regeneration, ++ appendage regeneration, +++ whole-body regeneration, ++++ from cell aggregates. Longevity is given in years from the maximum reported characteristic of the taxon in the AnAge database [START_REF] Magalhães | An Analysis of the Relationship Between Metabolism, Developmental Schedules, and Longevity Using Phylogenetic Independent Contrasts[END_REF]. Phylogenetic tree based on [START_REF] Halanych | The New View of Animal Phylogeny[END_REF], species numbers on [START_REF] Zhang | Animal Biodiversity: An Update of Classification and Diversity in 2013[END_REF], regeneration potential on [START_REF] Bely | Evolution of Animal Regeneration: Re-Emergence of a Field[END_REF], publications on manually curated online searches (Appendix B Table A2) and cell line numbers on [START_REF] Bairoch | The Cellosaurus, a Cell-Line Knowledge Resource[END_REF]. Source: Graphic by authors.

Figure 2.

Five decades of research on isolation and primary culture of cells from aquatic invertebrates. The figure shows the number of publications for the phyla cnidaria (66 in total), ctenophora (1 in total), and tunicata (44 in total). Publications are grouped by classes of the species used for cell isolation, color-coded as indicated.

Publications were manually curated from online searches, and detailed references are available in the Appendix B Table A2. Source: Graphic by authors.

Cell lines have been established through two main strategies [START_REF] Cai | Marine Invertebrate Cell Culture: A Decade of Development[END_REF][START_REF] Rinkevich | Cell Cultures from Marine Invertebrates: New Insights for Capturing Endless Stemness[END_REF]): either by the isolation of proliferating and self-renewing cells, typically from an embryonic [START_REF] Hansen | Initiating a Cell Line from Embryos of the Snail Biomphalaria Glabrata[END_REF] or cancerous origin [START_REF] Scherer | Studies on the Propagation in Vitro of Poliomyelitis Viruses: Iv. Viral Multiplication in a Stable Strain of Human Malignant Epithelial Cells (Strain Hela) Derived from an Epidermoid Carcinoma of the Cervix[END_REF], or by immortalizing proliferating cells, typically through mutagenesis [START_REF] Earle | Production of Malignancy in Vitro. IV. The Mouse Fibroblast Cultures and Changes Seen in the Living Cells[END_REF] or transfection [START_REF] Russell | Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5[END_REF]. Both strategies thus require, at least transiently, a proliferating primary cell culture. The long-term culture (up to 22 months) of cells from various aquatic invertebrate phyla has been achieved by using a variety of culturing environments [START_REF] Rinkevich | In Vitro Culture of Blood Cells from the Colonial Protochordate Botryllus schlosseri[END_REF][START_REF] Daugavet | Culture of Mussel (Mytiuls edulis L.) Mantle Cells[END_REF][START_REF] Chen | Establishment of Cell Lines Derived from Oyster, Crassostrea Gigas Thunberg and Hard Clam, Meretrix Lusoria Röding[END_REF][START_REF] Kingsley | Scleroblast Cultures from the Gorgonian Leptogorgia Virgulata (Lamarck) (Coelenterata: Gorgonacea)[END_REF]. However, most of these in vitro primary cultures show an apparently ubiquitous cellular quiescence within three days that leads to an absence of proliferation within 1-4 weeks of primary culture [START_REF] Rinkevich | Cell Cultures from Marine Invertebrates: New Insights for Capturing Endless Stemness[END_REF][START_REF] Cai | Marine Invertebrate Cell Culture: A Decade of Development[END_REF]). Yet, transient proliferation events, limited to a subset of acclimated cells, are persistently recorded across most marine invertebrate taxa ~2-4 weeks after the establishment of primary cultures at high seeding density from larval or regenerating adult tissue. For instance, DNA synthesis and mitosis have been observed both in primary cultures of explanted ectodermal tissue monolayers of regenerating Nematostella vectensis [START_REF] Rabinowitz | In Vitro Cultures of Ectodermal Monolayers from the Model Sea Anemone Nematostella vectensis[END_REF], as well as in dissociated cell culture from regenerating tentacles of Anemonia viridis [START_REF] Ventura | Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level[END_REF], and dividing cells have been reported in primary culture of regenerating tissues of Apostichopus japonicus [START_REF] Odintsova | Regenerating Holothurian Tissues as a Source of Cells for Long-Term Cell Cultures[END_REF]. The only established mollusc cell line, Bge, was initiated from the long-term culture of embryonic tissue of the freshwater snail Biomphalaria glabrata [START_REF] Hansen | Initiating a Cell Line from Embryos of the Snail Biomphalaria Glabrata[END_REF]. Taken together, these results suggest that a key to setting efficient primary cultures are to use tissue with high proliferation capacity, potentially due to the presence of stem-like cells. Conveniently, aquatic invertebrates display a variety of asexual reproduction, aging and regeneration phenomena (Figure 1) that indicate high cellular plasticity, cellular proliferation and a likely involvement of stem-like cells [START_REF] Bely | Evolution of Animal Regeneration: Re-Emergence of a Field[END_REF][START_REF] Slack | Animal Regeneration: Ancestral Character or Evolutionary Novelty?[END_REF][START_REF] Bodnar | Marine Invertebrates as Models for Aging Research[END_REF][START_REF] Tomczyk | Hydra, a Powerful Model for Aging Studies[END_REF][START_REF] Rinkevich | A pan-metazoan concept for adult stem cells: The wobbling Penrose landscape[END_REF]). However, established guidelines for the isolation and identification of stem-like cells are currently only available for very few species [START_REF] Hayashi | Isolation of Planarian X-Ray-Sensitive Stem Cells by Fluorescence-Activated Cell Sorting[END_REF][START_REF] Sun | Purification and in Vitro Cultivation of Archaeocytes (Stem Cells) of the Marine Sponge Hymeniacidon Perleve (Demospongiae)[END_REF][START_REF] Hemmrich | Molecular Signatures of the Three Stem Cell Lineages in Hydra and the Emergence of Stem Cell Function at the Base of Multicellularity[END_REF][START_REF] Kassmer | Integrin-Alpha-6+ Candidate Stem Cells Are Responsible for Whole Body Regeneration in the Invertebrate Chordate Botrylloides Diegensis[END_REF]. The recent improvements in next-generation sequencing techniques, and in single cell transcriptomics in particular, are enabling researchers to characterize stem-like cells in an increasing number of taxa [START_REF] Hayashi | Single-Cell Gene Profiling of Planarian Stem Cells Using Fluorescent Activated Cell Sorting and Its 'Index Sorting' Function for Stem Cell Research[END_REF][START_REF] Siebert | Stem Cell Differentiation Trajectories in Hydra Resolved at Single-Cell Resolution[END_REF][START_REF] Rinkevich | A pan-metazoan concept for adult stem cells: The wobbling Penrose landscape[END_REF]), a first important step for their isolation and in vitro culture.

There is an ample body of work that provides numerous quantitative assessments of culturing conditions (e.g., [START_REF] Toullec | Crustacean Primary Cell Culture: A Technical Approach[END_REF][START_REF] Khalesi | Cell Cultures from the Symbiotic Soft Coral Sinularia Flexibilis[END_REF][START_REF] Dessai | Primary Culture of Mantle Cells of Bivalve Mollusc, Paphia Malabarica[END_REF][START_REF] Maselli | A Novel Approach to Primary Cell Culture for Neurons[END_REF], without highlighting one ideal consensus. Given that aquatic invertebrates are phylogenetically very distant, the development of a ubiquitous culturing environment appears rather unlikely. Nevertheless, each phylum could benefit from the advances in primary cell culture made in other phyla. However, a significant fraction of the relevant research data remains unpublished in conventional peer-reviewed journals, being only accessible as chapters in master's or doctoral dissertations, conference proceedings and specialized books. Consequently, in the last five decades, the publication of research efforts has been uneven across phyla, and temporally fragmented, as illustrated for the cnidaria and tunicata phyla (Figure 2).

Here, we review three major drawbacks and limitations of this field of research and their most promising work-around [START_REF] Rinkevich | Marine Invertebrate Cell Cultures: New Millennium Trends[END_REF][START_REF] Cai | Marine Invertebrate Cell Culture: A Decade of Development[END_REF][START_REF] Rinkevich | Cell Cultures from Marine Invertebrates: New Insights for Capturing Endless Stemness[END_REF][START_REF] Yoshino | Molluscan Cells in Culture: Primary Cell Cultures and Cell Lines[END_REF]): (1) seeding the cell culture with a population enriched in proliferating and potentially stem-like cells; (2) devising marine invertebrate specific in vitro culturing environment, including management of oxidative stress and cell adhesion requirements; (3) preventing culture contamination with other cell types and microbes. This review is intended to be accessible both to the non-experts and newcomers to the field of primary cell culture, while providing an updated and curated list of references on the primary cell culture of aquatic invertebrates compiled for the experienced reader.

Given the huge scope of this review (>360,000 species, >60 years of research, >510 publications), we set out to illustrate previous work on aquatic invertebrate cell culture with three summarizing tables (Tables 123), filled with a selection of representative publications in each taxon and focusing on stem cell cultures whenever these have been described. This review is, by nature, not exhaustive and omits, by necessity, many publications, which thus limits generalizations. We conclude this review by providing perspectives on how to solve this limitation, mainly through dramatically extending the present effort in the data mining and metacoding of published work to build an exhaustive knowledge database on aquatic invertebrate cell culture. We also highlight abiotic factors that should be further investigated. We hope that the provided perspectives will help researchers to develop robust and reproducible approaches for culturing dividing aquatic invertebrate cells, a first step towards the possible establishment of cell lines.

Isolating Stem-like Cell Types Suitable for In Vitro Culture

Inoculum type is highly diverse, depending on targeted aquatic invertebrate taxon, tissue and life cycle stage. At the same time, selecting the proper inoculum for a given in vitro culture is certainly the most important decision towards establishing a suitable cell culture.

Selecting Suitable Sources of Cells

Based on the number of publications on primary cultures and the number of reported cell lines across aquatic invertebrate taxa (Figure 1), most research has focused on cell culture establishment from mollusks (mostly bivalves, and comparatively fewer gastropods and cephalopods), followed by porifera (mostly demosponges), cnidaria (historically hydrozoans and currently anthozoans, Figure 2), crustacea (pennaeid shrimps, crabs and crayfish), echinoderms and tunicates. Episodic attempts have also been made for one or two species representatives of ctenophores (Mnemiopsis), annelids (Lumbricidae; Nereidae), nematodes (Caenorhabditis), chelicerates (Limulus) and cephalochordates (Amphioxus). These differences in research efforts illustrate differences in the attractivity of specific taxa for cell culture, which stem from three complementary considerations, detailed below, that every researcher has to take into account when selecting the origin of the cells to be cultured in vitro.

The first pertinent consideration is whether to work on tissue isolated from established experimental models maintained in controlled aquarium or laboratory facilities. These animals, in contrast to wild animal sampling, provide both access to early life stages, as well as increased reproducibility for cell culturing experiments. Their use also meets the biodiversity protection regulations and traceability requirements of the Nagoya protocol. More and more clonal lineages of genotyped animals are becoming available across taxa, and establishing cell cultures from this traceable material is an additional source of reproducibility that reduces complexity and facilitates comparisons between intervention protocols. Ultimately, these biological models will help the optimization of the culturing conditions, for guidelines specific to a few model species. In this context, two attractive taxa for fundamental research are cnidaria (e.g., Hydra vulgaris, Hydractinia echinata, Exaiptasia pallida) and platyhelminthes (e.g., Schmidtea mediterranea) with well-established strains of animals. Mollusks or crustaceans of commercial importance, and with a complete life cycle obtained in captivity and traceable across generations, also represent important taxa to develop stem cell cultures.

A second important consideration when selecting a model species for cell culturing experiments is the wealth of genomic, transcriptomic and metabolomic information available for that species. In addition to allowing the identification of cell-type-specific markers, post-genomic information gained on metabolic pathways and cellular adhesion systems can be used to formulate working hypotheses on taxon-specific in vitro cellular requirements of media components and substrates. Similarly, knowledge acquired on in vivo tissue homeostasis, dynamics of cell proliferation, and somatic stem cell niches [START_REF] Martinez | Articulating the "stem cell niche" paradigm through the lens of non-model aquatic invertebrates[END_REF]) could help in selecting a seed tissue of high proliferative potential. In this respect, the continuous decrease in sequencing costs, as well as in other omics techniques, is allowing more and more research groups working with marine aquatic invertebrates to characterize their favorite species, suggesting that omics information will fast become available for almost all taxa.

The third point to consider is the desired approach for obtaining immortalized cells. Similar to mammalian cell culture, potential sources for immortalized cell lines are artificially reprogrammed cells and spontaneously tumor-like tissue. However, immortalization methodologies are currently limited in aquatic invertebrate cells by low yields and poor stability, as observed in sponges [START_REF] Pomponi | Sponge Hybridomas: Applications and Implications[END_REF][START_REF] Revilla-I-Domingo | Establishment of Transgenesis in the Demosponge Suberites domuncula[END_REF], bivalve mollusks [START_REF] Hetrick | Attempts to Develop a Marine Molluscan Cell Line[END_REF][START_REF] Boulo | Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures[END_REF], and crustaceans [START_REF] Claydon | Attempts at Immortalization of Crustacean Primary Cell Cultures Using Human Cancer Genes[END_REF][START_REF] Xu | Development of a Primary Culture System for Haematopoietic Tissue Cells from Cherax Quadricarinatus and an Exploration of Transfection Methods[END_REF]). As suggested by [START_REF] Odintsova | Naturally Occurring and Artificially Induced Tumor-like Formations in Marine Invertebrates: A Search for Permanent Cell Lines[END_REF], natural tumor-like tissue, characterized by increased (hyperplasia) or altered (neoplasia) cell proliferation patterns, is thus a promising inoculum to initiate primary cultures. However, tumor-like lesions in wild or captive aquatic invertebrate taxa have low registered frequencies [START_REF] Peters | Appendix 1: Invertebrate Neoplasms[END_REF][START_REF] Tascedda | Tumors in Invertebrates[END_REF], and there has been repeated unexpected failures at maintaining the hyperproliferation of successfully isolated cancerous cells in vitro. For instance, in transmissible soft-shell clam (Mya arenaria) leukemia, cancerous hemocytes rapidly undergo in vitro apoptosis, triggered by the release of mortalin-based cytoplasmic sequestration of p53 [START_REF] Walker | Mortalin-Based Cytoplasmic Sequestration of P53 in a Nonmammalian Cancer Model[END_REF]. Other attempts at primary culture initiation from artificially induced tumors of carcinogen-exposed bivalves (Crassostrea virginica) also failed to maintain persistent in vitro cell division [START_REF] Hetrick | Attempts to Develop a Marine Molluscan Cell Line[END_REF].

Consequently, the use of stem-like cells for seeding in vitro cultures appears key to setting dividing primary cultures. Marine invertebrates display a wide variety of intriguing cellular phenomenon, such as asexual reproduction, striking regenerative capacity, reduced aging and dormant stages, which upon arousal restore fully functional individuals (Figure 1). These mechanisms indicate high cellular plasticity, proliferation and a likely involvement of stem-like cells. Although the potency of these cells remains largely uncharacterized in most species, and the orthology between these stem-like cells remains to be assessed [START_REF] Rinkevich | A pan-metazoan concept for adult stem cells: The wobbling Penrose landscape[END_REF], they represent a promising source of proliferating and self-renewable cell types. However, the identification, isolation and characterization of aquatic invertebrate stem cells remains a major, typically species-specific, technical challenge. With few species having established protocols for the isolation of identified stem-like cells [START_REF] Hayashi | Isolation of Planarian X-Ray-Sensitive Stem Cells by Fluorescence-Activated Cell Sorting[END_REF][START_REF] Sun | Purification and in Vitro Cultivation of Archaeocytes (Stem Cells) of the Marine Sponge Hymeniacidon Perleve (Demospongiae)[END_REF][START_REF] Hemmrich | Molecular Signatures of the Three Stem Cell Lineages in Hydra and the Emergence of Stem Cell Function at the Base of Multicellularity[END_REF][START_REF] Kassmer | Integrin-Alpha-6+ Candidate Stem Cells Are Responsible for Whole Body Regeneration in the Invertebrate Chordate Botrylloides Diegensis[END_REF][START_REF] Reyes-Bermudez | Transcription Profiling of Cultured Acropora Digitifera Adult Cells Reveals the Existence of Ancestral Genome Regulatory Modules Underlying Pluripotency and Cell Differentiation in Cnidaria[END_REF], their generalization and transfer by taking advantage of the vast diversity of specific approaches explored in other taxa (Table 1) appear particularly promising.

Selecting a Suitable Type of Inoculum

Aquatic invertebrates have indirect development cycles, with widespread asexual propagation strategies, including colonial budding and the generation of dormant stages, as well as high regenerative abilities, including whole-body regeneration. These developmental properties are suggestive of the presence of proliferative cells, including potential stem-like cells, which are of particular interest to establish proliferating cell cultures. Hence, they provide the following range of theoretically ideal inoculum material: embryonic/larval tissue, regenerating tissue, asexually propagating tissue and dormant stages.

Dissociated tissue from whole embryo/larva consistently yields primary cell cultures dividing over 2-3 weeks, allowing a few rounds of successive subcultures. For example, when applied to cnidarian models, whole dissociated Acropora planula larvae yielded subsets of dividing coral cells that could undergo several successive subcultures (see [START_REF] Reyes-Bermudez | In Vitro Culture of Cells Derived from Larvae of the Staghorn Coral Acropora Millepora[END_REF] for A. millepora, and [START_REF] Kawamura | Establishing Sustainable Cell Lines of a Coral, Acropora Tenuis[END_REF] for A. tenuis).

Dissociated somatic adult tissue sampled from regenerating tissue has also been observed to yield dividing cell cultures that could be subcultured for several weeks. Among other examples, cultures based on regenerating tentacle tips of the sea anemone Anemonia viridis (Barnay-Verdier et al. 2013) could be subcultured for 2-4 weeks, and primary cultures from regenerating intestinal tissue of the holothurian Apostichopus japonicus displayed limited but active in vitro proliferation at ~2 weeks after evisceration [START_REF] Odintsova | Regenerating Holothurian Tissues as a Source of Cells for Long-Term Cell Cultures[END_REF].

The dissociation of asexually growing tissue similarly gave rise to cell cultures with observable proliferative activity for a few weeks. For instance, using fast-growing branch tip fragments of the Acropora millepora coral (Reyes-Bermudez et al. 2021), cells could be subcultured for 2-4 weeks, and delayed senescence was reported in primary cultures from extracted buds of tunicates [START_REF] Rabinowitz | In Vitro Delayed Senescence of Extirpated Buds from Zooids of the Colonial Tunicate Botryllus schlosseri[END_REF].

As implied in the "live slow, grow old" adage, cold adapted hibernating freshwater sponge species from lake Baikal yielded primary cell reaggregate (termed primmorph) cultures with record (max 8 months) longevity [START_REF] Chernogor | Long-Term Cultivation of Primmorphs from Freshwater Baikal Sponges Lubomirskia Baikalensis[END_REF]. Sponge gemmules also represent dormant hibernation/aestivation stages rich in multipotent stem cells [START_REF] Simpson | The Cell Biology of Sponges[END_REF]) that, upon hatching, regenerate a functional adult. Activated gemmules could thus constitute a promising inoculum for primary cultures. Similarly, in the colonial tunicate Botrylloides leachii, arousal from a cold-induced dormancy [START_REF] Burighel | Hibernation of the Colonial Ascidian Botrylloides Leachi (Savigny): Histological Observations[END_REF]) leads to the restoration of multiple adults by proliferating piwi + /pl10+ cells, two markers suggestive of stem-like properties [START_REF] Hyams | Insights into the Unique Torpor of Botrylloides Leachi, a Colonial Urochordate[END_REF]. In both cases, investigating the mechanisms regulating arousal from dormancy may yield cues to stimulate the tissues established in vitro to switch from quiescence to active cell cycling.

In conclusion, despite their initial abundance in cells with proliferative stemness-like properties, the shared in vitro fate of all four above-cited inoculum categories is terminal cell cycle arrest and the gradual accumulation of senescent, necrotic cells in primary culture and subsequent subcultures.

Selecting Suitable Cell Isolation Techniques

Inoculum type is highly diverse, depending on targeted aquatic invertebrate taxon, tissue and life cycle stage. Although no cell culture has yet been observed to sustain its proliferative activity for long, short-term functional primary cultures are routinely established from terminally differentiated cell types of aquatic invertebrates. Differentiated cells being arrested in G 0 can survive in vitro for a limited time with intact function, and hence are best used within hours to ~3 days of isolation. Nevertheless, comparing their tissue-isolation protocols offers opportunities to survey tissue sampling and dissociation methods (Table 1), as well as the cellular interactions and defense mechanisms that may support their in vitro viability, even for short periods of time. Emblematic examples of short-term invertebrate primary culture from quiescent cells include neuron-like cells and circulating hemocytes.

Giant neuronal cells from gastropod mollusks, such as the sensory and motor neurons from the sea hare Aplysia californica, are used to study growth cone motility and synapse plasticity [START_REF] Kaczmarek | The Morphology and Coupling of Aplysia Bag Cells within the Abdominal Ganglion and in Cell Culture[END_REF][START_REF] Lee | Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones[END_REF][START_REF] Zhao | Preparation of Aplysia Sensory-Motor Neuronal Cell Cultures[END_REF][START_REF] Ren | A Single Tyrosine Phosphorylation Site in Cortactin Is Important for Filopodia Formation in Neuronal Growth Cones[END_REF][START_REF] Suter | Live Cell Imaging of Neuronal Growth Cone Motility and Guidance in Vitro[END_REF]. Cultured neurons from the pond snail Lymnea stagnalis are also routinely used for studies on synapse formation, neuronal aging and memory [START_REF] Magoski | A Neuronal Network from the Mollusc Lymnaea Stagnalis[END_REF][START_REF] Prinz | Electrical Synapses by Guided Growth of Cultured Neurons from the Snail Lymnaea Stagnalis[END_REF][START_REF] Walcourt | A Comparison of the Electrophysiological Characteristics of Identified Neurons of the Feeding System of Lymnaea Stagnalis (L.) in Situ and in Culture[END_REF]. The in vitro establishment of nerve cells from jellyfish bell tissue [START_REF] Przysiezniak | Primary Culture of Identified Neurones from a Cnidarian[END_REF][START_REF] Schmid | Transdifferentiation in Medusae[END_REF] or from the solitary tunicate Ciona intestinalis [START_REF] Zanetti | Primary Cultures of Nervous System Cells from the Larva of the Ascidian Ciona Intestinalis[END_REF]) have also been reported. These neuronal cell types are usually micro-dissected from their ganglion, enzyme digested with protease, immobilized on positively charged polylysine-coated coverslips and then used for short-term electrophysiology assays, providing non-conventional in vitro models in neuroscience.

Circulating cells sampled from internal fluids, are another major category of cultured aquatic invertebrate cells. When seeded at high density (>10 6 cells/mL), cultured adherent hemocytes can form partly complete confluent monolayers, with clusters forming in suspension above the monolayer that may then be detached and transferred to new culture dishes. Such cultures have been routinely established since the late 1960s from a wide range of species, including mollusks, crustaceans, tunicates and echinoderms, typically for in vitro cell/microbe interactions and immunopathology assays. Such cultures display short-term conserved functionality, as shown by phagocytosis or immunomodulatory assays. Proliferation may be induced by stimulation with bacterial antigens, as shown for the bivalve Mytilus galloprovincialis [START_REF] Cao | Primary Cultures of Hemocytes from Mytilus Galloprovincialis Lmk.: Expression of IL-2Rα Subunit[END_REF], the tunicate Styela [START_REF] Raftos | Proliferation of Lymphocyte-like Cells from the Solitary Tunicate, Styela clava, in Response to Allogeneic Stimuli[END_REF] and the earthworm Lombricus [START_REF] Bilej | A New Approach to in Vitro Studies of Antigenic Response in Earthworms[END_REF]). These differentiated cell types are drawn directly from internal cavities, lacunae and sinuses using a syringe. To counter their spontaneous self-aggregation (clotting) behavior, hemocytes are collected in syringes half-filled with species-specific anti-clotting saline solution, such as artificial seawater without calcium or magnesium, artificial seawater with a calcium chelator, or Na-citrate based "Alsever" saline solutions. Indeed, hemocytes secrete their own set of taxon-specific lectins (e.g., [START_REF] Matsumoto | A Novel C-Type Lectin Regulating Cell Growth, Cell Adhesion and Cell Differentiation of the Multipotent Epithelium in Budding Tunicates[END_REF]) and extracellular-matrix components (ECM) (e.g., a fibronectin-like ECM in bivalve hemocytes [START_REF] Dyachuk | Identification of β Integrin-like-and Fibronectin-like Proteins in the Bivalve Mollusk Mytilus Trossulus. Development[END_REF]) that support rapid adherence, within hours of sampling, to glass or poly-lysine-coated coverslips.

Aside from the two isolation techniques described above, the quantitative evaluation of various approaches for cell extraction in different species suggests that, for the rapid obtention of single-dissociated cells from soft tissues for RNAseq cell phenotyping, and thus to obtain cells as close as possible to their wild-type state, mechanical isolation is the most efficient method [START_REF] Khalesi | Cell Cultures from the Symbiotic Soft Coral Sinularia Flexibilis[END_REF][START_REF] Dessai | Primary Culture of Mantle Cells of Bivalve Mollusc, Paphia Malabarica[END_REF][START_REF] Daugavet | Culture of Mussel (Mytiuls edulis L.) Mantle Cells[END_REF][START_REF] Maselli | A Novel Approach to Primary Cell Culture for Neurons[END_REF]. For example, cnidarian larval tissue or demosponge and calcisponge adult tissue fragments are dissociated within minutes via shearing in calcium-free seawater and passage through a 40-70 µm nylon mesh. However, species with tough cuticles (e.g., Lombricidae), important extracellular matrices (e.g., Styelidae) or abundant surface mucus (e.g., Dugesiidae) necessitate treatments with specific enzymes to liberate the cells. For instance, in the stony coral Pocillopora damicornis, chemical treatment with a divalent cation chelator followed by a mix of glycosidases and collagenase was reported to help dissolve the mucus and improve the yield of released cells [START_REF] Downs | In Vitro Cell-Toxicity Screening as an Alternative Animal Model for Coral Toxicology: Effects of Heat Stress, Sulfide, Rotenone, Cyanide, and Cuprous Oxide on Cell Viability and Mitochondrial Function[END_REF]. Proteolytic treatments (trypsine, dispase and other protease mixes) are routinely used to dissociate cells from solid tissues dissected from mollusks and crustaceans. Interestingly, protease treatment may induce cellular reprogramming, as shown by the collagenase-induced transdifferentiation of in vitro explanted striated muscle of jellyfish [START_REF] Alder | Cell Cycles and in Vitro Transdifferentiation and Regeneration of Isolated, Striated Muscle of Jellyfish[END_REF][START_REF] Schmid | Isolated, Mononucleated, Striated Muscle Can Undergo Pluripotent Transdifferentiation and Form a Complex Regenerate[END_REF][START_REF] Schmid | Transdifferentiation of Isolated Striated Muscle of Jellyfish in vitro: The Initiation Process[END_REF].

Selecting Suitable Cell-Type Enrichment Strategies

Cells of interest are typically mixed with other cell types after the dissociation of the inoculum. The enrichment of specific cell types, typically proliferative or multipotent ones, relies on the prior development of taxon-specific and custom-designed cell separation methods. For instance, in Stylophora pistillata, stem cells were not identified in the cell atlas established from both larval and adult tissues, following either enzymatic or mechanical dissociation methods [START_REF] Levy | A Stony Coral Cell Atlas Illuminates the Molecular and Cellular Basis of Coral Symbiosis, Calcification, and Immunity[END_REF], which severely limits the development of stem cell-enriched primary cultures.

Sorting methods for enriching inoculum suspensions in proliferative or multipotent cell types are thus required. Initial methods were based on differential sedimentation on density gradients, including sucrose, Percoll, or mixtures of Ficoll and polyethylene glycol. To further discriminate between morphologically similar cell types, and thus target specific cell types, Fluorescence Activated Cell Sorting (FACS) methods have recently been developed and have become highly prominent. For instance, FACS has been used to separate vital-stained coral cells [START_REF] Rosental | Coral Cell Separation and Isolation by Fluorescence-Activated Cell Sorting (FACS)[END_REF], and to isolate cell-type subpopulations for their single-cell gene expression characterization in hydrozoan [START_REF] Siebert | Stem Cell Differentiation Trajectories in Hydra Resolved at Single-Cell Resolution[END_REF], as well as in anthozoan species [START_REF] Levy | A Stony Coral Cell Atlas Illuminates the Molecular and Cellular Basis of Coral Symbiosis, Calcification, and Immunity[END_REF][START_REF] Sebé-Pedrós | Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq[END_REF]. In these diblastic animals, which lack a circulatory system, FACS is necessary to enrich dissociated tissue suspensions in hexacorallian putative immune cells, the amoebocytes recovered from the inter-epithelial mesogleal layer typical of cnidarian, for short-term functional phagocytosis characterization [START_REF] Snyder | Functional Characterization of Hexacorallia Phagocytic Cells[END_REF]. In triploblastic animals, FACS has also been refined to sort tunicate cell subpopulations to study the hematopoietic system [START_REF] Rosental | Complex Mammalian-like Haematopoietic System Found in a Colonial Chordate[END_REF]. Echinoderm coelomocyte subpopulations have been further separated by FACS into distinct cell types, such as the red pigment autofluorescent spherulocytes [START_REF] Hira | Autofluorescence Mediated Red Spherulocyte Sorting Provides Insights into the Source of Spinochromes in Sea Urchins[END_REF].

Consequently, there is a need for stem cell markers suitable for non-invasive stemness tracing in live cells to enable their enrichment. One promising perspective comes from the few aquatic invertebrate experimental models that can be genetically manipulated for which transgenic reporters of stemness properties can be engineered (e.g., in Hydra [START_REF] Juliano | PIWI Proteins and PIWI-Interacting RNAs Function in Hydra Somatic Stem Cells[END_REF]). Another direction of interest is the usage of fluorescent markers conjugated with antibodies specifically labeling stem cells. However, the identification of such markers remains extremely rare for aquatic invertebrates, with the recent notable exception of the colonial tunicate Botrylloides diegensis for which integrin-alpha-6 was shown to specifically label pluripotent cells [START_REF] Kassmer | Integrin-Alpha-6+ Candidate Stem Cells Are Responsible for Whole Body Regeneration in the Invertebrate Chordate Botrylloides Diegensis[END_REF]. Whether this specific marker can be used in other species of aquatic invertebrates to label stem cells will be important to assess.

Selecting Cleansing Techniques to Minimize Contamination

There is a wide consensus across the scientific community that the highest obstacle to continuous marine/freshwater invertebrate cell culture propagation is overgrowth by aquatic microbial contaminants [START_REF] Rinkevich | Marine Invertebrate Cell Cultures: New Millennium Trends[END_REF]. This problem is critical in marine invertebrate primary cell cultures for two main reasons. First, it is because the tissues sampled to initiate the primary cultures come typically from areas directly or semi-directly exposed to environmental microbes, such as the thin epithelial structures at the interface with water (e.g., in porifera and cnidaria), tissues irrigated by a semi-open circulatory system (e.g., in mollusks, echinoderms and tunicates) or digestive and other internal tissues hosting their own microbiota (e.g., gills and hepatopancreas of mollusks). Second, commercial antibiotics/antimycotics/antiparasitic drugs have been designed against microbes isolated from terrestrial animals and mostly from humans and are thus largely ineffective against the mostly underexplored diversity of environmental aquatic microbes. To control the contamination of cell cultures by these aquatic microbes, three main strategies can be attempted.

First, microbial load can be reduced before cell isolation. The inoculum can be sampled from starved animals depurated in oxygenated sterile-filtered seawater to limit environmental microbial contaminants (e.g., for abalone mantle cell culture [START_REF] Suja | Effect of Depuration of Animals and Use of Antimicrobial Agents on Proliferation of Cells and Microbial Contamination in In-Vitro Mantle Explant Culture of the Abalone Haliotis Varia Linnaeus[END_REF]). Microdissecting internal tissues that naturally protect from seawater by epithelial envelopes (e.g., molluscan heart tissue), and thus from aquatic microbes, would also reduce the initial microbial load of the inoculum. Collecting cells that possess natural antiseptic defenses, such as innate immune hemocytes (e.g., from mollusks, crustaceans, tunicates or echinoderms), would also have a positive impact on reducing the contamination of the culture. Alternative strategies include using short-term ubiquitous surface sterilization methods on the surface-exposed tissue, such as dipping for up to 1 hour in 10-70% ethanol (e.g., for molluscan abalone mantle, see [START_REF] Suja | Effect of Depuration of Animals and Use of Antimicrobial Agents on Proliferation of Cells and Microbial Contamination in In-Vitro Mantle Explant Culture of the Abalone Haliotis Varia Linnaeus[END_REF], and for oyster tissue, see [START_REF] Stephens | Decontamination of the American Oyster Tissues for Cell and Organ Culture[END_REF]) or a few seconds in KmnO 4 (e.g., in sea anemone tissue (Doumenc personal communication)) and treating the dissected tissue for up to days in sterile-filtered seawater enriched with a mixture of concentrated large-spectra commercial antibiotics/antimycotics/antiprotist compounds (e.g., molluscan mantle, gill or hepatopancreas tissue).

Second, if specific invertebrate cell types need to be recovered from contaminated primary cultures, the cell-type enrichment strategies established for preparing a suitable inoculum (see Section 2.4) could be reused. For instance, this approach successfully retrieved accessory nidamental gland cells pelleted from native bacteria through a 2% sucrose layer (Figure 3). In addition, the selective rinsing of adherent invertebrate cell types could help to remove cellular debris, toxins and suspended microbes. Third, contamination can be controlled during the primary culture itself. The main strategy for this step to reduce the unwanted mixotrophic growth of contaminants is to use a nutrient-poor basal medium formula, hence limiting the provision of carbon and nitrogen sources that typically exceeds the in vitro energy requirements of the target cells. The culture medium can also be supplemented with antibiotics/antimycotics/antiprotist drugs and changed frequently until the culture appears clean. Proliferating cultures should be closely monitored, and the primary cell cultures containing visible ciliates, bacteria, or clusters of cells with characteristic chytrid-like rhizoid morphology should be discarded.

However, these methods may rescue a subset of the targeted cell-type populations from contaminant overgrowth but carry a high cost in terms of time-consumption and cell yield reduction, for overall limited efficiency.

Defining Optimal Culture Conditions

While obtaining a high-quality inoculum is essential for establishing healthy cell cultures, the culturing conditions used are equally crucial. Indeed, even highly proliferative tissue will undergo terminal cell cycle arrest, typically within weeks after inoculation. Moreover, a breakthrough in the culturing conditions used for the Bge cell line was at the origin of a large expansion in cell lines.

Selecting Suitable Culture Media Composition

Media formulation should strive to provide adequate levels of carbon and nitrogen sources to meet the nutritional needs of each isolated aquatic invertebrate cell-type population. However, the metabolism of stem cells and their nutrient requirements are poorly documented across aquatic invertebrate taxa. Consequently, a large variety of culture media have been tested for their in vitro culture (Table 2). Based on the hypothesis of the conservation of major metabolic pathways across animal phyla, a widespread approach is to use commercial basal formulas originally designed for vertebrate cells, typically MEM, DMEM or Leibovitz L-15, supplemented with salts to adjust to the targeted osmolarity of the specimen's original environment and generally diluted to 10-50% [START_REF] Maramorosch | Invertebrate Cell Culture: Novel Directions and Biotechnology Applications[END_REF][START_REF] Mothersill | Basic Methods and Media[END_REF]). An even simpler option is to provide a minimal medium composed of seawater with pyruvate as a carbon source, and glutamic acid as a nitrogen source. This approach has been used with sponge primmorph spontaneously aggregated from dissociated cells. These media have, however, persistently failed to sustain the in vitro division of cells of aquatic invertebrates. Another much more complex option is to entirely custom design the media's formula based on an extensive biochemical characterization of internal tissue or fluid composition from the targeted animal species (e.g., molluscan hemolymph). However, these taxon-specific media have not yet demonstrated sufficient benefits to justify their development cost.

A more integrated and personalized approach is to adapt the media formulations to meet the needs of the targeted invertebrate cell subpopulations. To check nutrient consumption in vitro, individual uptake experiments of targeted organic carbon (glucose, lipids, etc.) or nitrogen (amino-acid) substrates (see [START_REF] Apte | Transport of Amino Acids into Freshly Isolated Cells from a Sea Anemone[END_REF] for amino-acid transport into sea anemone cells, and Heude-Berthelin et al. (2003) for glucose uptake and glycogen metabolism in oyster cells) may now be updated to metabolomics-based global approaches. Indeed, the search for changes in the metabolite profiles of media sampled at various timepoints in cultured mammalian CHO-CK1 cell lines has helped identify factors that sustain growth and affect in vitro behavior [START_REF] Mohmad-Saberi | Metabolomics Profiling of Extracellular Metabolites in CHO-K1 Cells Cultured in Different Types of Growth Media[END_REF]. A recent breakthrough was reached using this approach to develop an amino-acid-enriched sponge cell culture medium that sustains cell division in primary cultures [START_REF] Conkling | Breakthrough in Marine Invertebrate Cell Culture: Sponge Cells Divide Rapidly in Improved Nutrient Medium[END_REF]. The team used a genetic algorithm to identify suitable amino acid components to supplement a commercial basal formula (M199) for improving the in vitro metabolic activity of Dysidea avara sponge cells [START_REF] Munroe | Genetic Algorithm as an Optimization Tool for the Development of Sponge Cell Culture Media[END_REF].

A striking feature of successful insect culture media that support proliferating primary cultures and cell lines is the addition of lipid-rich supplements, with a trophic role and potential protection against oxidative stress. Lipid addition has been shown to transiently increase metabolic activity (mitochondrial MTT reduction) in cultured oyster heart cells [START_REF] Domart-Coulon | Identification of Media Supplements That Improve the Viability of Primarily Cell Cultures of Crassostrea Gigas Oysters[END_REF]). The lipid-rich "Grace" commercial formula was shown in cnidarian primary cultures to increase octocoral cell numbers [START_REF] Khalesi | Cell Cultures from the Symbiotic Soft Coral Sinularia Flexibilis[END_REF], and is used to obtain a subset of dividing cells and a few rounds of subcultures from cultured sea anemone tentacle [START_REF] Barnay-Verdier | Establishment of Primary Cell Culture from the Temperate Symbiotic Cnidarian, Anemonia Viridis[END_REF]. However, a more global picture of the impact of lipids on culture media for aquatic invertebrate cells is currently lacking.

Medium renewal strategy should be aimed at striking a balance between a conditioned medium supply of undefined trophic factors and cytokines and the removal of senescent cells, debris and toxins from the aging primary cultures. Manipulating inoculum cell densities is an efficient way to facilitate confluence and thus maintain cell-to-cell contacts necessary for the secretion of cytokines that, although currently undefined, are certainly necessary for sustaining cell survival. Old-time tissue explantation methods that rely on the slow outward migration of mixed cell types from a dissected tissue fragment adherent to a culture dish yield the successive outgrowth of distinct morphotypes characterized at minima by their in vitro shape and behavior. These cells can broadly be classified by the following three categories: fibroblast-like, epithelial-like and amoeboid-like cell types [START_REF] Vago | Invertebrate Tissue Culture[END_REF], and can be selected for their ability to survive in vitro on residual native extra-cellular-matrix components. Insect cell lines have emerged from such long-term maintained explant cultures of lepidopteran imaginal discs [START_REF] Echalier | Drosophila Cells in Culture[END_REF]). More recently, the explantation of ectodermal monolayers of regenerating starlet sea anemone yielded mitotically active, mixed cell types, primary cultures [START_REF] Rabinowitz | In Vitro Cultures of Ectodermal Monolayers from the Model Sea Anemone Nematostella vectensis[END_REF].

In addition, culture medium can be complemented with a number of factors to promote cell proliferation: C-type lectins have been shown to have cytostatic effects on the hemocytes of the tunicate Polyandrocarpa misakiensis [START_REF] Matsumoto | A Novel C-Type Lectin Regulating Cell Growth, Cell Adhesion and Cell Differentiation of the Multipotent Epithelium in Budding Tunicates[END_REF]; lectins from another tunicate, Didemnum ternatanum, promote the adhesion of a range of marine invertebrate cells [START_REF] Odintsova | Adhesive and Growth Properties of Lectin from the Ascidian Didemnum Ternatanum on Cultivated Marine Invertebrate Cells[END_REF]; insulin and insulin growth factor, as well as other vertebrate growth factors, were shown to have a positive impact on the transient proliferation of molluscan bivalve cells [START_REF] Domart-Coulon | Identification of Media Supplements That Improve the Viability of Primarily Cell Cultures of Crassostrea Gigas Oysters[END_REF][START_REF] Giard | Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: An in vitro study[END_REF]; and retinoic acid-related molecules are known to be involved in the dedifferentiation process of multipotent cells as reported for tunicate hemocyte cultures (Polyandrocarpa misakiensis) [START_REF] Kawamura | Establishment of cell lines from multipotent epithelial sheet in the budding tunicate, Polyandrocarpa misakiensis[END_REF].

The Oxidative Stress Problem

Very few and exclusively freshwater taxa among the large diversity of aquatic invertebrates have given rise to cell lines, including the snail Biomphalaria (Gastropoda). Salinity is thus a major difference between the primary culture systems that have given rise to cell lines and the unsuccessful attempts based on aquatic invertebrate species. One possible cause for this difference is that higher salinity correlates with lower dissolved oxygen. Consequently, dissolved oxygen levels in the cell cultures might be an important yet overlooked physico-chemical parameter of culture conditions. To date, primary cultures of aquatic invertebrate cells are indeed mostly conducted under standard atmospheric conditions (i.e., ~20% O 2 ), with the cells covered by a thin layer of culture medium where dissolved oxygen is equilibrated by diffusion with the surrounding air. Except for a few cases of full-strength Modified Eagle Medium (or derivatives, osmotically adjusted by salt addition), which requires a bicarbonate/5% CO 2 buffer system, the gaseous atmosphere of most cell cultures is thus composed of air (Table 2). The widely used, amino-acid rich, Leibovitz L-15-based media do not require a 5% CO 2 atmosphere. Seawater/freshwater diluted commercial or custom-made media rely on the addition of Hepes (~20 mM) for pH buffering at 7.4-7.6, depending on species (Tris-HCl is used for sponge cells grown at pH ~8.0). Hence, under typical laboratory conditions (air and 15-25 • C), in vitro aquatic invertebrate cells are exposed to ~20% O 2 , which is largely more than in their natural aquatic environment, and could likely expose them to in vitro oxidative stress.

To circumvent this potential problem, the first step will be to monitor invertebrate intracellular oxidative stress, for instance, via a fluorescent general oxidative stress indicator, such as CM-H 2 DCFDA, which has been used on the spheroid tissue of Fungia coral exposed to short-term acute thermal stress [START_REF] Gardner | Reactive Oxygen Species (ROS) and Dimethylated Sulphur Compounds in Coral Explants under Acute Thermal Stress[END_REF]. Upon the confirmation of oxidative stress, the second step could be medium supplementation with exogenous antioxidants (for example, ascorbic acid [START_REF] Helman | Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro[END_REF], catalase enzyme [START_REF] Domart-Coulon | Identification of Media Supplements That Improve the Viability of Primarily Cell Cultures of Crassostrea Gigas Oysters[END_REF])) or native pigments with high antioxidant properties (e.g., sea urchin spinochrome [START_REF] Ageenko | Pigment cell diferentiation in sea urchin blastula-derived primary cell cultures[END_REF]) and shrimp astaxanthin [START_REF] Lee | Initiation of Cell Cultures from Pandalid Shrimp and Effects of Astaxanthin[END_REF]). Both approaches have reproducibly led to the increased maintenance of the primary cell cultures. An alternative when establishing cultures of tissues containing photosynthetic endosymbionts (e.g., Cyanobacteria-containing sponges, and Symbiodiniaceae-containing sea anemones, corals and octocorals) is to maintain the cultures in the dark to inhibit the photosynthetic processes that generate oxygen and thus increase oxidative stress (Table 2).

Understanding Adhesion and Cell-to-Cell Contact Requirements of Aquatic Invertebrate Stem Cells

To optimize the proliferation of culture cells, transferring genomic knowledge obtained in each aquatic invertebrate taxon on cell-to-cell and cell-to-ECM adhesion systems will be particularly useful for selecting suitable ECM-coatings of culture dishes.

Shifting from classical 2D monolayer culture to 3D "spheroid" culture systems offers opportunities to facilitate the maintenance of cell-to-cell interactions and of native secretions within the cell cluster. Cells from the earliest branching aquatic metazoans, such as poriferans and cnidarians, display spontaneous aggregation properties after tissue dissociation into single cells, sometimes leading to whole-body regeneration (see [START_REF] Simpson | The Cell Biology of Sponges[END_REF] for sponge, [START_REF] Gierer | Regeneration of hydra from reaggregated cells[END_REF] for hydra [START_REF] Vizel | A novel method for coral explant culture and micropropagation[END_REF] for coral). This re-aggregation property is being harnessed for spheroid formation (coral "tissue balls", sponge primmorphs) and their establishment for primary culture (Figure 4A-C). Hemocytes drawn from mollusks, crustaceans, tunicates or echinoderms also self-aggregate into clusters, through sequential migrations of adherent cells on the culture substrate followed by the putative secretion of self-recognition lectins (Figure 4D-F). A recent breakthrough using 3D cultures of sponge cells in ultra-low-gel agarose hydrogel microdroplets has been reported to support cell-ECM interactions and to facilitate the survival of differentiated Geodia neptuni demosponge cells [START_REF] Urban-Gedamke | 3-D Culture of Marine Sponge Cells for Production of Bioactive Compounds[END_REF].

Similarly, improving the in vitro microenvironment of isolated stem cells could potentially sustain their proliferation. Adapting the cellular microenvironment to mimic stem cell niches of a target organism should be pursued in each model taxon, as such information becomes available. In addition, primary cultures that gave rise to cell lines (e.g., insect imaginal disc cells) can provide mechanistic insights into the cellular microenvironment needed to maintain stem cell self-renewal. As shown in mammalian systems, multidirectional signaling by co-culturing stromal "feeder" cells with the target cells (e.g., neurons from the gastropod Aplysia californica [START_REF] Montgomery | Arterial Cells and CNS Sheath Cells from Aplysia Californica Produce Factors That Enhance Neurite Outgrowth in Co-Cultured Neurons[END_REF]; stem-like cells on a monolayer of confluent cephalopod hemocytes (Figure 4E)) might help to generate a microenvironment suitable for stem cell maintenance, proliferation and differentiation, typically by providing cell adhesion molecules, growth factors, hormones and other secreted proteins (see [START_REF] Girard | Neurogenic Heterotopic Ossifications Recapitulate Hematopoietic Stem Cell Niche Development Within an Adult Osteogenic Muscle Environment[END_REF] for hematopoietic stem cell niche, and [START_REF] Ootani | Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche[END_REF] for intestinal stem cell niche). Furthermore, supplementing the culture media with specific growth factors (e.g., Wnt fusion proteins for ISC [START_REF] Ootani | Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche[END_REF])) can lead to the expansion of stem cells with sustained proliferation and multilineage differentiation. As in vivo information on the regulation of aquatic invertebrate stemness becomes available, transferring such information will be particularly important to design optimized cell culture media. To further mimic the in vivo microenvironment of the isolated cells, and their interactions with their environment in particular, new "physiomimetic" approaches should be developed using, for instance, versatile hydrogels to concentrate cells in a 3D microenvironment (see [START_REF] Otero | Oxygen Biosensors and Control in 3D Physiomimetic Experimental Models[END_REF] for a review on such experimental approaches for vertebrate cell systems). The ongoing development of commercial hydrogels (synthetic or derived from jellyfish, i.e., "Jellagel") provides new 3D substrates to test on aquatic invertebrate cells. To determine whether these cells behave in vitro similarly to in vivo, live-cell or live-tissue observations based on the micropropagation of tissue in microfluidic devices should be further established [START_REF] Januszyk | Evaluating the Effect of Cell Culture on Gene Expression in Primary Tissue Samples Using Microfluidic-Based Single Cell Transcriptional Analysis[END_REF].

Controlling the Purity and Quality of Cultured Cells

Upon isolation from their initial tissue microenvironment for establishment in culture dishes, aquatic invertebrate cells change morphology are notoriously difficult to identify by their in vitro shape and behavior [START_REF] Rinkevich | Cell Cultures from Marine Invertebrates: New Insights for Capturing Endless Stemness[END_REF][START_REF] Rinkevich | Marine Invertebrate Cell Cultures: New Millennium Trends[END_REF][START_REF] Cai | Marine Invertebrate Cell Culture: A Decade of Development[END_REF]. Moreover, cultured cells are morphologically highly plastic, changing shape, granularity and sometimes pigmentation with culture age and substratum composition (i.e., with or without surface coating with positive charges or ECM compounds). For cell lineage authentication, checking phenotype and genetic identity is imperative, not only upon culture initiation but also throughout the primary culture and derived subcultures, at least at the time of use for functional assays and before/after cryopreservation.

Proliferation

The monitoring of in vitro cell proliferation is traditionally based on monitoring cell densities (via subsampling a fraction of the culture followed by cell numeration on Malassez-or Neubauer-type hemocytometers, or time-lapse image analysis of microscopy fields of view) and attentive changes in the total protein content or DNA content extracted from cell pellets or monolayers. These methods overestimate live cell densities as they integrate dying cells to the viable cells. Another widely used method relies on the miniaturized high-throughput colorimetric quantification of mitochondrial oxidative phosphorylation (MTT or XTT reduction assays) by the cultured cells. First adapted for screening medium nutritional factors and physico-chemical parameters for molluscan cells (bivalve oyster Crassostrea gigas [START_REF] Domart-Coulon | Identification of Media Supplements That Improve the Viability of Primarily Cell Cultures of Crassostrea Gigas Oysters[END_REF], it has also been adapted to sponge cell mitochondrial activity evaluation in primary culture [START_REF] Zhang | Application of a MTT Assay for Screening Nutritional Factors in Growth Media of Primary Sponge Cell Culture[END_REF]) and to the monitoring of coral larval cell density in primary cultures [START_REF] Kawamura | Establishing Sustainable Cell Lines of a Coral, Acropora Tenuis[END_REF]). However, this type of MTT test detects not only oxidative phosphorylations of the animal cells but also that of bacterial associates in primary culture (e.g., of cephalopod holobiont) tissue [START_REF] Pichon | Cephalopod Bacterial Associations: Characterization and Isolation of the Symbiotic Complex in the Accessory Nidamental Glands[END_REF]). The fluorescence monitoring of cellular esterase activity is also a common method to quantify viable cells in cultures. However, their use for aquatic invertebrate taxa can be limited by the widespread co-occurrence of autofluorescent cell types with fluorescence spectra overlapping those of the enzyme substrates.

By quantifying the proportions of cells in each phase of the cell cycle, flow cytometry allows us to check the proliferative status of the collected tissue sample before culture establishment, and to monitor cell cycling in the derived primary cultures and potential sub-cultures. Applied, for example, in the early 2010s to primary cell cultures from five demosponge species, this flow-cytometry-based approach revealed rapid changes in the cell cycle distribution of a mixed-cell-type suspension over time in primary culture (over a short-term 2-10-day timescale) [START_REF] Schippers | Cell Cycle Analysis of Primary Sponge Cell Cultures[END_REF]. The rapid accumulation of cells with low DNA content together with a drop in the proportion of quiescent (G 1 /G 0 ) and cells (S & G 2 /M) could be visualized, supported by the parallel detection of activated (caspase3) apoptosis pathways. This evidence supported the hypothesis of the rapid senescence of cultured sponge cells, with the accumulation of cellular debris (demonstrated by widely scattered cell size distribution), despite stable or slowly declining cell counts, by only minus ~20% over the 10-day culture period. This observation calls to cautious interpretations of stable or slightly growing cell densities, counted from image analyses of microscopic fields or enumerated on Malassez-type slides, as round empty cell bodies cannot be unambiguously discriminated from living cells based on morphology only, even when using vital stains assays (e.g., neutral red or trypan-blue). Another point of caution when using this approach is the ploidy of the studied samples, and in particular, the presence of mixoploid cell populations that could bias their cell-cycle profile [START_REF] Ermakov | Study of Planarian Stem Cell Proliferation by Means of Flow Cytometry[END_REF].

Phenotyping

Autofluorescent markers (e.g., Green Fluorescent Protein-rich intracellular granules of cnidarian cells,) or chromophore/pigments of specific cell types (e.g., red "echinochrome" pigments of echinoderm coelomocytes) can be used to sort cell types among a mixed cell suspension. However, care should be taken to minimize irradiance energy during fluorescence microscopy examination as it may damage the living cells by DNA photodamage or lipid peroxidation, and thus limit their subsequent in vitro survival. Enzyme activity assays (e.g., phenoloxidase of mollusk and crustacean immune cells), biochemical phenotyping and phagocytosis assays have also been used to characterize the in vitro functionality of hemocytes from molluscan hemolymph, tunicate hemolymph and echinoderm coelomic fluid.

Immunophenotyping requires the prior development and validation of polyclonal or monoclonal antibodies against epitopes of cell-type-specific proteins or membrane preparations. Although labor-intensive and time consuming, this strategy provides the advantage of the unambiguous localization of immuno-positive phenotypes in initial tissue and in primary tissue or mixed cell culture. For instance, low abundant small round coral skeletogenic (calicoblast) cell types were labeled with a polyclonal antibody raised against the biomineral organic matrix [START_REF] Puverel | Antibodies against the Organic Matrix in Scleractinians: A New Tool to Study Coral Biomineralization[END_REF] and antibodies were raised against the Botrylloides piwi sequence to label a specific population of hemocytes [START_REF] Rinkevich | Piwi Positive Cells That Line the Vasculature Epithelium, Underlie Whole Body Regeneration in a Basal Chordate[END_REF]). This antibody-based approach has been successfully used to trace self-sorting processes during cell-to-cell aggregation from mixed-cell-type dissociated tissue suspensions [START_REF] Schmid | Cell-Substrate Interactions in Cnidaria[END_REF]) and for cell fusion experiments [START_REF] Pomponi | Sponge Hybridomas: Applications and Implications[END_REF]). This has an interesting yet still overlooked potential for cell-type enrichment via the antibody panning of immuno-positive cell types (Auzoux-Bordenave and Domart-Coulon 2010).

Novel phenotyping methods have recently been developed from cutting-edge single-cell RNA sequencing methods, which are applied to cultured cells. However, these techniques currently have the three drawbacks: (1) the prior definition of cell-type-specific markers is needed, through the data-mining of single-cell RNAseq libraries obtained from dissociated tissue suspensions, which is still available for a limited number of species of established model organisms (e.g., for the starlet sea anemone [START_REF] Sebé-Pedrós | Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq[END_REF], for planarians [START_REF] Hayashi | Single-Cell Gene Profiling of Planarian Stem Cells Using Fluorescent Activated Cell Sorting and Its 'Index Sorting' Function for Stem Cell Research[END_REF] or for the scleractinian coral [START_REF] Levy | A Stony Coral Cell Atlas Illuminates the Molecular and Cellular Basis of Coral Symbiosis, Calcification, and Immunity[END_REF])); (2) molecular markers should be specific to metabolic pathways restricted to the targeted invertebrate taxon and exclude pathways that are also active in potential contaminating protists/microeucaryotes; (3) assessing the polyclonality (mixture of cell types) versus clonality (single cell line) of the culture requires the quantification of the percentage of reads obtained for each claimed phenotypic marker, relative to the total number of reads.

Genotyping

Despite the proper isolation and cleansing of cells, cultures can easily be overgrown by undesired cells. An undetermined fraction of these aquatic microbes survives the tissue aseptization treatments prior to dissociation or explantation and co-occurs along with metazoan cells in the mixed-cell-type suspensions obtained from soft tissue dissociation or hemolymph syringe-drawings. This large diversity of aquatic microbes is hard to monitor as it requires taxa-specific specialist microbiology knowledge and molecular tools for accurate identification. It is especially difficult to recognize their morphological traits in a mixed-cell-type primary culture that combines the morphological and behavioral plasticities of both the microbe and microbial life stage, and the targeted invertebrate cell types.

To address this problem, genetic markers specific to a species (e.g., Axinella corrugata demosponge, [START_REF] Lopez | Characterization of Genetic Markers for in Vitro Cell Line Identification of the Marine Sponge Axinella Corrugata[END_REF])) or to a genus (e.g., Acropora scleractinian coral [START_REF] Shinzato | Development of Novel, Cross-Species Microsatellite Markers for Acropora Corals Using next-Generation Sequencing Technology[END_REF])) have been developed and validated for identifying cells from the targeted taxon in the initial tissue and over time in primary cultures and subcultures. Marker development is based on molecular genetics methods, such as DNA fingerprinting, amplified fragment length polymorphism (AFLP), single-locus DNA sequence analyses and microsatellites markers designed by next-generation sequencing population genetics methods.

As microbes tend to proliferate more actively than the cells of primary interest of the in vitro culture, it is crucial to check the potential microbial nature of long-term cultured candidate aquatic invertebrate stem cells.

Microbial Contaminants Authentication

Detecting genetic markers specific to the invertebrate taxa of interest does not exclude the potential co-occurrence of microbial contaminants. In fact, because molecular detections are highly sensitive, the detected invertebrate cells could even represent a very small fraction of the cell culture. Thus, it is highly recommended to also systematically molecular probes for microbial taxa to detect potential culture contaminants (Table 3).

Culture contamination is a major obstacle to the development of aquatic invertebrate in vitro models. Indeed, it is widely acknowledged that microbes persistently take over the cultured aquatic invertebrate cell types [START_REF] Rinkevich | Marine Invertebrate Cell Cultures: New Millennium Trends[END_REF], putatively as a result of antagonistic interactions (predation and competition for nutrients) or metabolic plasticity and better adaptation to the in vitro growth conditions. Culture media are commercially designed for vertebrates (e.g., DMEM, Leibovitz L-15) or insects (e.g., Grace Insect Medium) and partly diluted in seawater (or freshwater) or formula custom-prepared to mimic the microenvironment of the sampled tissue, and they are nutrient-rich. Although they may not adequately meet the largely unknown growth requirements of the cultured invertebrate cell types, they provide abundant organic carbon and nitrogen sources that facilitate the overgrowth of opportunistic resident microbial associates. Indeed, epibiotic or endobiotic microbiota (especially unicellular microeukaryotes that are hard to discriminate from animal cells) have repeatedly been shown to take advantage of the medium-derived nutritional resources to fuel their fast heterotrophic growth; see, for example, the consumption of mono and disaccharides, glycerol, glutamate and glycine by the opportunistic unicellular Alveolate Chromera velia [START_REF] Foster | Increased Growth and Pigment Content of Chromera Velia in Mixotrophic Culture[END_REF]. Predatory opisthokonts, ubiquitous in aquatic environments, have a highly plastic morphology, with in vitro growth alternating between a unicellular 'spindle-shape' stage and aggregative or clonal (partly fused) multicellular stages, and they are known to feed on metazoan tissue or derived cells (Tikhonenkov et al. 2020a).

Such eukaryotic microbes, collectively defined as protists, may feed on cellular debris from senescent or dead host/aquatic invertebrate cells, taking over the initial host cell population in long-term primary cultures or their successive subcultures.

Perspectives

While marine invertebrates as a whole show the largest biodiversity and the widest phylogenetic radiation on Earth, they have contributed very little to the in vitro cell lines discipline. The culture of marine invertebrate stem cells and/or their progenitors could thus create new perspectives for fundamental research as well as for biomedical applications. To reach this objective, we thus recommend two main actions.

First, a systematic map of knowledge, built in the form of a database of publications with metacoded information on taxon population, intervention strategies (e.g., cell isolation methods, culture media and physico-chemical conditions) and outcomes (e.g., cell viability, proliferation and differentiation) would be an important tool for increasing the visibility of protocols and know-how in the fragmented scientific community. Furthermore, it would help to incorporate typically unpublished results, including negative results, scientific status that is rarely highlighted in the refereed literature [START_REF] Grasela | Efforts to develop a cultured sponge cell line: Revisiting an intractable problem[END_REF]. Such database would help to build a comprehensive knowledge map to identify optimized culturing conditions for each aquatic invertebrate taxon and cell type, adapted to the expected timescale of utilization. The multiple usage of primary cell and tissue cultures from aquatic invertebrates ranges from short-term use (within hours to <7 days for physiology and cytotoxicity testing) to the long-term (bi-weekly to monthly) selection of subpopulations of dividing cells for serial sub-culturing attempts. Each type of inoculum implies distinct culture media and condition strategies to balance cellular yield, functional stability and proliferation potential. The curated list of 511 relevant publications compiled in Appendix B Table A2 provides a start to this database, allowing us to assess by taxon the extent of research efforts to initiate or develop cell cultures. It should be maintained and completed by the scientific community, for more exhaustive listing and optimized visibility.

Second, best practices would be to develop and adopt robust cell-type authentication protocols applicable to insect or vertebrate cell lines and primary cultures [START_REF] Lynn | Novel Techniques to Establish New Insect Cell Lines[END_REF][START_REF] Dominici | Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society for Cellular Therapy Position Statement[END_REF], and to systematically deposit live or cryopreserved vouchers of "cell lines" in cell repositories. This could lead to the identification of more general stem cell markers for aquatic invertebrates, which would be crucial for obtaining a robust inoculum for in vitro cell cultures. Most recurrent past claims of successfully established aquatic invertebrate cell lines have turned out in fine to be cultures overgrown by microeukaryote contaminants, with examples in each taxon (porifera, colonial cnidarians, crustaceans and others).

Overall, these two actions taken together could help to standardize aquatic invertebrate cell culture to facilitates comparisons between intervention protocols and thus help to optimize the standardized protocols. Given that aquatic invertebrates are phylogenetically very distant, the development of a ubiquitous culturing environment appears rather unlikely. Nevertheless, each phylum could benefit from the scientific and technological advances in primary cell culture made in other phyla.

In particular, the assessment of whether the list of three identification criteria, defined for vertebrate stem cells, are conserved in aquatic invertebrate stem cells, would be of particular interest. The first criterion is whether the stem cells adhere to plastic, and more generally if a specific culture method, such as 3D Matrigel, could lead to decisive improvements (Urban-Gedamke et al. 2021). The second criterion explores the expression of specific surface markers that would allow the robust isolation and enrichment of stem cells/progenitors, as has been attempted by using a single marker in a colonial tunicate [START_REF] Kassmer | Integrin-Alpha-6+ Candidate Stem Cells Are Responsible for Whole Body Regeneration in the Invertebrate Chordate Botrylloides Diegensis[END_REF]. The third criterion aims to define protocols for assessing stem cells' potency differentiation potential, typically by using predefined induction cocktails combined with markers for differentiated cell types, both of which require a precise characterization of gene expression profiles specific to each cell-type for every species of interest [START_REF] Sebé-Pedrós | Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq[END_REF]).

An alternative to identifying suitable stem cells is to immortalize cells of interest in a reproducible manner. One suggested approach is to manipulate adult stem cells of aquatic invertebrates similarly to the approach implemented in mammalian induced pluripotent stem (iPS) cells [START_REF] Rinkevich | Cell Cultures from Marine Invertebrates: New Insights for Capturing Endless Stemness[END_REF]. The second route, probably the most promising and reliable approach, is to control the process of tumorigenesis in aquatic invertebrates, as already suggested [START_REF] Odintsova | Naturally Occurring and Artificially Induced Tumor-like Formations in Marine Invertebrates: A Search for Permanent Cell Lines[END_REF]. Research on this topic is currently very scarce [START_REF] Gardner | Chemically Induced Histopathology in Aquatic Invertebrates[END_REF][START_REF] Robert | Comparative study of tumorigenesis and tumor immunity in invertebrates and nonmammalian vertebrates[END_REF] primarily due to the facts that tumorigenesis in aquatic invertebrates is not as commonly observed as in vertebrates [START_REF] Vogt | How to minimize formation and growth of tumours: Potential benefits of decapod crustaceans for cancer research[END_REF][START_REF] Tascedda | Tumors in Invertebrates[END_REF], that tumor-like lesions in aquatic invertebrates possess a low mitotic index [START_REF] Odintsova | Naturally Occurring and Artificially Induced Tumor-like Formations in Marine Invertebrates: A Search for Permanent Cell Lines[END_REF]) and that the definitions of tumors and tumor cells in aquatic invertebrates are less familiar to pathologists [START_REF] Tascedda | Tumors in Invertebrates[END_REF]). Yet, the tool of tumorigenesis may constitute a very important route for future research, and a potential approach is to use the trait of the vertebrates' cancer cells [START_REF] Vincent | Cancer: A de-repression of a default survival program common to all cells? A life-history perspective on the nature of cancer[END_REF]) as a guiding list for tumors in aquatic invertebrates. A third concept proposes the use of regeneration processes as the source of tumor development [START_REF] Oviedo | Regeneration: The origin of cancer or a possible cure?[END_REF], which is particularly interesting given the broad involvement of aquatic invertebrates' stem cells in regeneration processes, including whole-body regeneration [START_REF] Rinkevich | A pan-metazoan concept for adult stem cells: The wobbling Penrose landscape[END_REF]. For each one of these three approaches, the development of suitable tools for the controlled editing of genetic material of cells, typically through viral transfection, could enable the knockdown of suppressor genes, similar to standard approaches in mammalian cells [START_REF] Yang | Knockdown of P53 Combined with Expression of the Catalytic Subunit of Telomerase Is Sufficient to Immortalize Primary Human Ovarian Surface Epithelial Cells[END_REF]). One such advance is the successful induced stem cell neoplasia in the marine hydrozoan Hydractinia echinata by the ectopic expression of a POU domain transcription factor [START_REF] Millane | Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor[END_REF]). However, even immortalized tumor-like cells will need appropriate culturing conditions to proliferate properly. Lessons may be drawn from the failure to sustain in vitro the neoplastic hemocyte proliferation observed in vivo in spontaneously occurring clam leukemia, with research pointing to a role for the stress protein mortalin in the induction of apoptosis in cancerous hemocytes [START_REF] Walker | Mortalin in invertebrates and the induction of apoptosis by wild-type p53 following defeat of mortalin-based cytoplasmic sequestration in cancerous clam hemocytes[END_REF]. The RNA-seq approach may be applied to compare gene expression patterns in cultured cells and initial tissue, with a focus on essential cell proliferation and cell cycle arrest regulator genes, in order to develop future strategies for immortalization, as recently explored for developing shrimp cell lines [START_REF] Thammasorn | Investigation of essential cell cycle regulator genes as candidates for immortalized shrimp cell line establishment based on the effect of in vitro culturing on gene expression of shrimp primary cells[END_REF].

As a supplementary approach to support the development of cell lines from aquatic invertebrate stem cells, studies on metabolomes of cultured cells, and their secretomes in particular, could be considered. Such an approach may provide important insights into the requirements in media composition that support proliferative activities. Ample information has been gained on this issue in mammalian cell cultures [START_REF] Čuperlović-Culf | Cell Culture Metabolomics: Applications and Future Directions[END_REF][START_REF] Mohmad-Saberi | Metabolomics Profiling of Extracellular Metabolites in CHO-K1 Cells Cultured in Different Types of Growth Media[END_REF]). Yet, the study of the secretome of aquatic invertebrates has seldomly been undertaken [START_REF] Kocot | Sea Shell Diversity and Rapidly Evolving Secretomes: Insights into the Evolution of Biomineralization[END_REF], but data on the metabolome of whole organisms in the context of marine natural product discoveries are becoming quite common [START_REF] Reverter | Metabolomics and Marine Biotechnology: Coupling Metabolite Profiling and Organism Biology for the Discovery of New Compounds[END_REF]. Furthermore, high-precision tool development specific to seawater are now available [START_REF] Sogin | Marine Metabolomics: A Method for Nontargeted Measurement of Metabolites in Seawater by Gas Chromatography-Mass Spectrometry[END_REF].

Finally, future research should also address the still largely overlooked abiotic factors, such as testing hypoxia and pressure stimuli, on primary cultures.

Regarding hypoxia, parallel research in cultured mammalian cell models has highlighted the better survival and proliferation of stem cells in low oxygen environments [START_REF] Zhu | Effects of Hypoxia on the Proliferation and Differentiation of NSCs[END_REF][START_REF] Hung | Hypoxia Promotes Proliferation and Osteogenic Differentiation Potentials of Human Mesenchymal Stem Cells[END_REF][START_REF] Ramirez | Effect of Long-term Culture of Mouse Embryonic Stem Cells under Low Oxygen Concentration as Well as on Glycosaminoglycan Hyaluronan on Cell Proliferation and Differentiation[END_REF]. A shift from oxidative phosphorylation to aerobic glycolysis, known as the Warburg effect, has been documented in the context of proliferating cancer cells: the glucose consumed in high amounts to fuel the growing biomass of cancer cells is fermented to lactate rather than oxidized, even when there is sufficient oxygen to convert glucose to CO 2 , although the process is less efficient in terms of ATP synthesis (reviewed by DeBerardinis and Chandel 2020). Hyperactive glycolysis involving lactate supports the tumor energy metabolism of cancer stem cells in mostly hypoxic environments, and similar pathways might support the metabolism of aquatic invertebrate stem cells. A similar Warburg effect has indeed been documented in Crassostrea gigas oyster tissue. First discovered during the response to viral infection with ostreid herpesvirus-1 [START_REF] Corporeau | Proteomic Signatures of the Oyster Metabolic Response to Herpesvirus OsHV-1 Mvar Infection[END_REF], it is thought to be a mechanism to adapt the oyster metabolism to extreme (salinity and oxygen) changes in the intertidal environment [START_REF] Corporeau | The Oyster Crassostrea Gigas, a New Model against Cancer[END_REF]. In agreement with this finding, preliminary data obtained on oyster heart primary cell cultures showed transient increased proliferation between 2 and 4 weeks post-inoculation in a 2% O 2 atmosphere (obtained by incubation in a 95% N 2 /5% air incubator), compared with 20% O 2 atmosphere (air) (Domart-Coulon, unpublished), when medium was supplemented with growth factors, lipids and antioxidants [START_REF] Domart-Coulon | Identification of Media Supplements That Improve the Viability of Primarily Cell Cultures of Crassostrea Gigas Oysters[END_REF].

Regarding pressure, research on the primary cultures of vertebrate (Wharton Jelly's) mesenchymal stem cells has shown the combined positive effects of pressure and hypoxia [START_REF] Park | Pressure Stimuli Improve the Proliferation of Wharton's Jelly-Derived Mesenchymal Stem Cells under Hypoxic Culture Conditions[END_REF]. In response to pressure stimuli, cell proliferation was increased, and stemness was maintained. Cellular adhesion and confluency were higher in 5% O 2 hypoxia with 2.0 PSI pressure conditions relative to standard 5% CO 2 -95% air conditions, and hypoxia alone yielded a mild increase in stem cell adhesion and confluency. Thus, we propose the inclusion of these abiotic parameters in future invertebrate stem cell culture optimization efforts.

Fliegerová

  phototrophy (use light and inorganic C & N)

Figure 3 .

 3 Figure 3. Primary co-culture of squid gland cells with native bacteria. (A) Accessory nidamental gland tissue (white arrows) from Sepiola rondeletti is enzymatically dissociated by trypsine (0.2% 30 min at 25 • C). (B) Gland cells are enriched via centrifugation through a sucrose cushion (2% in seawater), and their seeding density is controlled by Malassez hemocytometer numeration. (C) Glandular cell types visualized via Fluorescence In Situ Hybridization (EUK, universal eukaryote probe, fluorescein, green) are covered with surface-associated symbiotic bacteria (EUB, universal bacterial probe, Cy3, red). (D) Four-week-old primary culture (without antibiotics) showing high bacterial density around the cultured glandular cell types, (E) which can be re-enriched via sucrose cushion centrifugation. (F) Gland cell viability (mitochondrial enzyme activity assessed by MTT reduction assay, DO 580/630) is higher in the absence than in the presence of antibiotics (AB) and increases in primary co-culture with native bacteria, along with cell density, indicating the beneficial effect of native bacteria on the survival of cell cultures. Times are given as days post-inoculation (dpi). Source: Graphic by authors.

Figure 4 .

 4 Figure 4. Aggregate vs. dissociated primary tissue and cell culture, on plastic dish-culture substrates. The figure shows micrographs of individual cells multicellular aggregates at the indicated days post-inoculation (dpi). (A) Scleractinian coral cell types and their Symbiodiniaceae endosymbionts (within coral gastrodermal host cell, or free-living in the culture medium). (B) Suspended coral multicellular aggregates spontaneously formed in explant culture of colonies of Pocillopora damicornis. (C) Spontaneous dissociation into multilayered, mixed-cell-type culture, containing translucent coral cells and brown-pigmented microalgal symbionts (Symbiodiniaceae). (D) Cephalopod hemocytes from Nautilus pompilius aggregate in cell culture when seeded at high seeding density (>10 6 cells/mL, 2 dpi). (E) Confluent primary culture 8 dpi, showing networks of adherent hemocytes and proliferating cell clusters (300-500 µm in diameter), which can be detached and transferred (passaged) to new culture dishes. (F) Subcultured hemocytes (14 days post transfer from cells detached from clusters in 8 days post-inoculation-primary culture) remain quiescent and do not grow to confluence. Source: Graphic by authors.

Table 1 .

 1 Established cell isolation techniques across taxa. Indicative examples were selected among the references listed in Appendix B TableA2.

		Reference		Rinkevich	and	Rabinowitz	(1993)	Rinkevich	and	Rabinowitz	(1994)	Rinkevich	and	Rabinowitz	(1997)	Moiseeva	et al. (2004)	Rabinowitz	and	Rinkevich	(2004)	Rabinowitz	et al. (2009)	Rabinowitz	and	Rinkevich	(2011)	Hyams et al.	(2017)
	Cell-Type	Enrichment	Strategy				none					none				none			none			none		none	none	none
	Enzyme	Digestion				none					none				collagenase			none			none		none	none	none
	Isolation	Medium	buffered	washing	solution	with HEPES	buffered	washing	solution	with HEPES		incubation	medium		Fisher's	medium	filter sea	water and	antibiotics	buffered	washing	solution	with HEPES	buffered	washing	solution	with HEPES	buffered	washing	solution	with HEPES
	Isolation	Technique	mechanical,	teasing	apart	colonial	zooids		mechanical,	chemical		mechanical,	enzymatic	dissociations	mechanical,	cell strainer			mechanical		mechanical,	cell strainer	mechanical,	cell strainer	mechanical
	Target	Cells			circulating	blood cells				all cell types				epithelial			cup cell	disease cells			epithelial		epithelial	epithelial
	Inoculum	Type				adult					embryo			zooids and	buds		zooids and	buds		zooids and	buds	zooids and	buds	paleal buds	adult,	hibernating	colonies	blood cells
		Species				Botryllus	schlosseri				Botryllus	schlosseri			Botryllus	schlosseri		Botryllus	schlosseri		Botryllus	schlosseri	Botryllus	schlosseri	Botryllus	schlosseri	Botrylloides	leachii
		Class					Ascidiacea					Ascidiacea				Ascidiacea			Ascidiacea			Ascidiacea		Ascidiacea	Ascidiacea	Ascidiacea
		Phylum				Chordata	(Tunicata)

Table 1 .

 1 Cont.

		Reference					Cai et al.	(2013)					Wang et al.	(2009)					Sakai (1960)	Fujita et al.	(1972)	Oppenheimer	and Meyer	(1982)	Kaneko	et al. (1995)
	Cell-Type	Enrichment	Strategy					none						none		sedimentation	by	centrifuging	at 1000	RPM	removal of	suspended	cells	two	successive	centrifugation	centrifugations
	Enzyme	Digestion			100 U/mL	type I	collagenase				none					none	none	trypsin 0.1	mg/mL	none
	Isolation	Medium					FSW						none				hypotonic	MaCI 2 (0.1 M)	solution	Ca 2+ and	Mg 2+ free SW	(CMF-SW) (1	h)	Ca 2+ and	Mg 2+ free SW	(CMF-SW)	1.2 M glycine	supplemented	with 1% (v/v)	nystatin-filtered	sea water, 6%	newborn	bovine serum
	Isolation	Technique	mechanical	and	enzymatic	for ovary	and	spermary		mechanical	dissociation					mechanical	dissociation	spontaneous	dissociation	mechanical	dissociation	mechanical	dissociation
	Target	Cells				all cell	types					all cell	types					egg cortex	cells	all cell	types	all cell	types	all cell	types
	Species Inoculum Type	Adult	Branchiostoma epidermis,	belcheri gill, gut,	Japanese spermary,	ovary	adult,	Branchiostoma buccal cirri, tail, gill, gut belcheri and tsingtauense metapleural	fold	Anthocidaris	crassispina,	Pseudocentrotus eggs depressus,	Hemicentrotus	pulcherrimus	Anthocidaris embryo (blastula crassispina stage)	purpuratus (blastula Strongylocentrotus embryo stage)	embryo	Asterias (mesenchyme	amurensis migration	stage)
		Class						Leptocardii						Leptocardii					Echinoidea	Echinoidea	Echinoidea	Asteroidea
		Phylum					Chordata	(Cephalochordata)											Echinodermata	(Ambulacraria)

Table 1 .

 1 Cont.

		Reference					Odintsova	et al. (2015)				Odintsova	et al. (2005)	De Mulder	et al. (2009)	Brody and	Chang	(1989)	Han et al.	(2013)	Toullec et al.	(1996)	Zhang et al.	(2011)	Ermakov	et al. (2012)
	Cell-Type	Enrichment	Strategy					none					none		none				none		sedimentation	none	none	FACS-sorting	(DNA-stained)
	Enzyme	Digestion				0.25%	collagenase				collagenase	CMF/1%	trypsin		type 2	collagenase	none	collagenase	pronase	none
	Isolation	Medium	Ca 2+ and	Mg 2+ -free	SW +	antibiotics	(100 IU/mL	penicillin	and	100 mg/mL	streptomycin)	sea water	nutrient-	enriched f/2	ASW			Medium 19		sea water	ASW	L-15	medium	citric acid	0.1 M with	0.5% Tween	20, 10 min	RT
	Target Isolation	Cells Technique				all cell spontaneous	types dissociation				guts Mechanical/ enzymatic	all cell enzymatic	types dissociation	hematopoietic	and mechanical	testicular dissociation	tissue	lymphoid mechanical organ	all cell Mechanical/	types enzymatic	larval Mechanical/	cells enzymatic	neoblasts chemical dissociation
	Phylum Class Species Inoculum Type				Echinoidea intermedius Strongylocentrotus embryo				Holothuroidea Apostichopus japonicus adult	Xenacoelomorpha Acoela Isodiametra pulchra adults	Pacifasticus	Arthropoda Malacostraca (Ecdysozoa) leniusculus, Homarus adults	americanus	Malacostraca M. ensis adults	Malacostraca Penaeus vannamei juvenile	Nematoda Chromadorea C. elegans (Ecdysozoa) eggs	Girardia adult	Platyhelminthes Turbellaria ystati, Schmidtea regenerating prepharyngeal	mediterranea zone

Table 1 .

 1 Cont.

		Reference				Hoshino	et al. (1991)					Asami et al.	(2002)						Hayashi	et al. (2006)		Schürmann	and Roland	(2001)	Lei et al.	(2019)
	Cell-Type	Enrichment	Strategy		FACS-sorting	(DNA	content)				FACS-sorting	(DNA-stained)				FACS-sorting	(X-ray	sensitive	cells)	centrifugation	(Percoll-density	gradient)	FACS	sorting (vital	dye	SiR-DNA;	size and	complexity	gating)
	Enzyme	Digestion				none						trypsin	0.25%						trypsin	0.25%		none	none
	Isolation	Medium	1/3 PBS	hypotonic	solution with	1% Triton	X100	5/8	Holtfreter's	solution	containing 30	µg/mL	trypsin	inhibitor	5/8	Holtfreter's	solution	containing 30	µg/mL	trypsin	inhibitor	1%	Digest-Eur in	hypotonic	solution	Ca-Mg-free	buffer, with	1% BSA
	Isolation	Technique			mechanical	dissociation					chemical	dissociation						chemical	dissociation		mechanical	dissociation	mechanical	dissociation
	Target	Cells			all cell	nuclei					neuronal	cells							neoblasts			neoblasts	neoblasts	and other	cell types
	Inoculum	Type	adult	(sexual and	asexual	strains)				adult head								adult			adult	adult tail
		Species				Dugesia	japonica					Dugesia	japonica						Dugesia	japonica		Schmidtea	polychroa	Schmidtea	mediterranea
		Class					Turbellaria							Turbellaria								Turbellaria			Turbellaria	Turbellaria
		Phylum

Table 1 .

 1 Cont.

		Reference			Bedi et al. (1998)		Schacher and	Proshansky	(1983)	Montgomery	et al. (2002)		Monnier and	Bride (1995)		Grimaldi et al.	(2009)	Bookman and Liu	(1990)	Masuda-Nakagawa	et al. (1994)	Przysiezniak and	Spencer (1989)	Schmid (1992)	Kingsley et al.	(1987)
	Cell-Type	Enrichment	Strategy		none			none		none		none			none		none	none	removal of	unattached	cells	microdissection	of striated	muscle	tissue	removal of	suspended	cells
	Enzyme	Digestion		none			pronase		collagenase		collagenase-	dispase			none		collagenase-	dispase	collagenase-	dispase	collagenase 100	U/mL (3-5 h)	collagenase 150	U/mL (6-8 h)	none
	Isolation	Medium	artificial	seawater	L-15	medium	L-15	medium		PBS			DMEM		L-15	medium	L-15	medium	Ca 2+ and	Mg 2+ free	ASW	Ca 2+ and	Mg 2+ free	ASW (15	min)	FSW with	antibiotics
	Isolation	Technique		mechanical		enzymatic	dissociation	enzymatic	dissociation		enzymatic	dissociation			mechanical		enzymatic	dissociation	enzymatic	dissociation	enzymatic	dissociation	mechanical	dissociation	mechanical	dissociation
		Target Cells			neurons			ganglia		ganglia		gonadal	cells		myoendothelial	cells	retzius cells	microglial	cells	neurons	smooth	muscle cells	scleroblasts
	Inoculum	Type	adult	pleural	ganglia	adult and	juvenile	adult		adult		adult,	Matrigel	implants	adult,	ganglia	adult	adult nerve	rings	adult	umbrella	adult
		Class Species			Gastropoda A. californica			Gastropoda A. californica		Gastropoda A. californica	Helix aspersa	Gastropoda aspersa, Helix aspersa	maxima	Clitellata Hirudo medicinalis	Clitellata Hirudo medicinalis	Clitellata Hirudo medicinalis	Hydrozoa Polyorchis penicillatus	Hydrozoa Podocoryne carnea	Anthozoa Leptogorgia virgulata
		Phylum			Mollusca												Annelida	(Spiralia)		Cnidaria

Table 1 .

 1 Cont.

	Phylum

Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference Anthozoa

			Rabinowitz et al.	(2016)					Apte et al. (1996)				Barnay-Verdier	et al. (2013)	Ventura et al.	(2018)	Gates and	Muscatine (1992)
	separation	of epithelial	layers by a	reducing	agent	Percoll	continuous	gradient	centrifugation	fragments	from 3d	post-cutting	tentacle	microdissected	epithelia	from 3d	post-cutting	tentacle	none
				none					papain 10 U/mL	(1.5 h)		0.05%	collagenase I (1	h)	0.15%	collagenase type	I (30 min)	none
		phosphate-	buffer-	saline		Ca 2+ and	Mg 2+ free	ASW	Ca 2+ and	Mg 2+ free	ASW then	ASW	Ca 2+ and	Mg 2+ free	ASW with 1	mM glycine,	then ASW	Ca 2+ free	ASW (3-4 h)
	mucolytic	agent (2%	NAC for 10	s)								enzymatic	dissociation	enzymatic	dissociation	mechanical	disruption
				ectodermis				epithelial	and	interstitial	cells	adherent	small round	cell types	gastrodermal	differentiated	cells	gastrodermal	cell	containing	zooxanthellae
			adult oral	fragment				adult	tentacle		adult	regenerating	tentacle	adult	regenerating	tentacle	adult
			Nematostella	vectensis				Anemonia	sulcata			Anemonia	viridis	Anemonia	viridis	Pocillopora	damicornis
											Anthozoa					Anthozoa		Anthozoa	Anthozoa

Table 1 .

 1 Cont.

	Phylum

Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference Anthozoa

									Frank et al. (1994)											Kopecky and	Ostrander (1999)			Domart-Coulon	et al. (2001)	Domart-Coulon	et al. (2004)	Helman et al.	(2008)	Helman et al.	(2008)
						Percoll	continuous	gradient	centrifugation								none		Percoll step	gradient	centrifugation	none	none	none
					collagenase	0.05% (4	h)/pronase 0.1%	(1 h)/EDTA	0.02% (2 h)							none				none	none	collagenase	0.15%	collagenase	0.15%
							Ca 2+ and	Mg 2+ free	ASW									Ca 2+ free	ASW (2-4 h)		Ca 2+ free	(and Mg 2+	free) ASW	(2-3 h)	Ca 2+ free	ASW (3 h)	Ca 2+ free	ASW (2.5 h)	Ca 2+ free	ASW (2.5 h)
						spontaneous	or	mechanical	fragmentation								spontaneous	dissociation			spontaneous	dissociation	spontaneous	dissociation	enzymatic	dissociation	enzymatic	dissociation
									all cell types											all cell types				all cell types	all cell types	all cell types	all cell types
								adult and	larva										adult				adult	adult	adult	adult
	Stylophora	pistillata,	Porites lutea,	Dendronephthya	hemprichi,	Paraerythropodium	fulvum fulvum,	Heteroxenia	fuscescence,	Clathraria	rubrinoides,	Plexaura sp.,	Millepora	dichotoma	Acropora	microphtalma,	Pocillopora	damicornis,	Montipora	digitata	Pocillopora	damicornis	Pocillopora	damicornis	Montipora	digitata	Xenia elongata
																					Anthozoa				Anthozoa	Anthozoa	Anthozoa	Anthozoa

Table 1 .

 1 Cont.

	Phylum

Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference Anthozoa

			none Khalesi (2008)		spontaneous	cell Nesa and Hidaka aggregation (2009) into	spheroids		two	successive Downs et al. Percoll step (2010) gradient	centrifugations	none Reyes-Bermudez and Miller (2009)	FACS-sorting	(size,	autofluorescence,	enzyme-activated Rosental et al.	fluorescence, (2017)	organelle-specific	fluo	markers)
		trypsine-EDTA	0.05%			none		dispase 2 U/mL,	lysozyme 3	U/mL, a	amylase 2 U/mL,	a glucosidase	0.5 U/mL, b	galactosidase	0.5 U/mL,	endoglycosidase	H 0.25 U/mL	none	none
	ASW with	1%	gentamycin-	streptomycin		FSW							ASW	spontaneous	dissociation	3.3× PBS	without Ca	and Mg, 2%	FCS, 20 mM	Hepes, pH	7.4
	mechanical/	enzymatic	dissociation	mechanical	dissociation						enzymatic	dissociation	spontaneous	dissociation	mechanical	dissociation
			all cell types				all cell types					coral cell	types	without	zooxanthellae	"interstitial"	cells	all cell types
			adult				adult							adult	larva	(planula)	adult
		Sinularia	flexibilis		ystatin,	Pavona	divaricata						Pocillopora	damicornis	Acropora	millepora	Pocillopora	damicornis
								Anthozoa							Anthozoa	Anthozoa	Anthozoa

Table 1 .

 1 Cont.

	Phylum

Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference Anthozoa

			Gardner et al.	(2015)							Rosental et al.	(2017)				Kawamura	et al. (2021)	Reyes-Bermudez	et al. (2021)	Vandepas et al.	(2017)
			spontaneous	spheroid	formation			FACS-sorting	(size,	autofluorescence,	enzyme-activated	fluorescence,	organelle-specific	fluo markers)	none	none	none
				none									none					trypsin-EDTA	+ collagenase	I (1-4 h)	none	0.25%	trypsin/EDTA
	FSW with	gentamicin	and	kanamycin	(both 50	µg/mL) for	2 days only	3.3× PBS	without Ca	and Mg, 2%	FCS, 20 mM	Hepes, pH	7.4	FSW	Ca 2+ free	ASW (2 h)	ctenophore	mesogleal	serum
			mechanical	dissociation							mechanical	dissociation				enzymatic	dissociation	spontaneous	dissociation	(after	thermal	bleaching)	spontaneous
			all cell types	(coral and	zooxanthellae)								all cell types					gastrodermal	secretory	cells,	undifferentiated	cells,	neuronal	cells, and	epidermal	cells	pluripotent	cells	all cell types
		adult	regenerating	tissue	(peeled off	skeleton)							adult					planula	adult	adult	healing lobe
			Fungia	granulosa							Pocillopora	damicornis				Acropora	tenuis	Acropora	digitifera	Mnemiopsis	leidyi
														Anthozoa					Anthozoa	Anthozoa	Ctenophora Tentaculata

Table 1 .

 1 Cont.

	Phylum

Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference

		Song et al.	(2021)	Lavrov and	Kosevich	(2016)	Robinson	(2015)		Schippers et al.	(2011)		Garcia	Camacho et al.	(2006)	Zhang et al.	(2004)	De Rosa et al.	(2001)
		none			none				sedimentation			none				none		none	none
		none			none				none			none				none		none	none
	Calcium-/	magnesium-free	ASW +	EDTA		FSW				CMFASW			FSW	sodium	hypochlorite	seawater,	gentamicin,	ystatin,	penicillin	CMFSW-E/AB	(penicillin,	streptomycin)	CMFSW,	ampicillin,	gentamycin,	kanamycin,	tylosin,	tetracyclin
		mechanical	dissociation		mechanical	dissociation		mechanical	dissociation		mechanical	dissociation			mechanical	dissociation	mechanical	dissociation	mechanical	dissociation
		all cell types			all cell types				all cell types			all cell types				all cell types		all cell types	all cell types
		adult			adult				adult			adult				adult		adult	adult
		Demospongiae Axinella sp.	H. panicea,	Demospongiae H. aquaeductus,	H. dujardinii	Spongosorites,	Demospongiae Cinachyrella,	Haliclona	H. oculata,	Demospongiae H. xena, D. avara, A.	polypoides		Demospongiae Crambe crambe	Demospongiae S. domuncula	Ircinia	muscarum,	Demospongiae Dysidea avara,	Suberites	domuncula
		Porifera

Table 1 . Cont. Phylum Class Species Inoculum Type Target Cells Isolation Technique Isolation Medium Enzyme Digestion Cell-Type Enrichment Strategy Reference Demospongiae

 1

	Müller et al.	(1999)			Koziol et al.	(1998)	Custodio et al.	(1998)
	none	density	gradient	centrifugation	using Percoll	solution	none
	none				none		none
	CMFSW	EDTA			PBSA-SW,	EDTA	CMFSW-E
	mechanical	dissociation			mechanical	dissociation	mechanical	dissociation
	all cell types			spherulous	cells	all cell types
	adult		Adult, outer	cortical	layer	adult
	Suberites	domuncula	Geodia	cydonium,	Suberites	domuncula	S.	domuncula
						Demospongiae		Demospongiae

Phylum Class Species Inoculum Type Target Cells Adherent/ Suspended Cell Culture Basal Formula Supplement Antioxidant Trace Elements and Vitamins Antimicrobials Substrate Light Atmosphere Temperature Medium Change Reference

	Rinkevich	and	Rabinowitz	(1993)		Rinkevich	and	Rabinowitz	(1994)		Rinkevich	and	Rabinowitz	(1997)	Moiseeva	et al.	(2004)	Rabinowitz	and	Rinkevich	(2004)	Rabinowitz	et al.	(2009)
			weekly						weekly				partly	replaced	every	other	day	partly	replaced	every 2w	partly	replaced	every 2w	unknown
		19 • C					20 • C						20 • C				20 • C	19-23 • C	20 • C
			air						air							air					air	air	air
			dark						dark							dark					dark	dark	dark
			plastic				plastic,	gelatin,	poly-l-lysin,	collagen					plastic					plastic	Plastic,	collagen,	Matrigel,	methocel,	fibronectin,	poly-l-lysine	plastic
	penicillin,	streptomycin,	nystatin		penicillin,	streptomycin,	nystatin			penicillin,	streptomycin,	nystatin			gentamycin	penicillin,	streptomycin,	amphotericin,	gentamycin	penicillin,	streptomycin,	amphotericin
		MgSO 4 , MgCl 2			MgSO 4 , MgCl 2				MgSO 4 , MgCl 2			salts	salts	salts
			none						none							none						none
		glutamine	solution	glutamine	solution,	hemolymph	from	Botrylloides,	chick	embryo	extract	fetal calf	serum,	HEPES		fetal calf	serum	fetal calf	serum,	HEPES,	L-glutamine,	EFG	fetal calf	serum,	HEPES,	L-glutamine
	DME	supplemented	with salts		DME	supplemented	with salts					RPMI-1640				Fisher's	medium	RPMI-1640,	DMEM,	HAM,	Fischer's,	Schnider's,	Grace,	Iscov's,	DCCM-1	L-15
		adherent/	suspended				adherent/	suspended					adherent/	suspended				suspended	adherent/	suspended	adherent
	circulatory	blood	cells			all cell	types						epithelium				cup cell	disease	epithelium	epithelium
		adult	zooids					embryo						colonial	buds			adult	zooids,	buds	palleal	buds	palleal	buds
		Botryllus	schlosseri				Botryllus	schlosseri					Botryllus	schlosseri			Botryllus	schlosseri	Botryllus	schlosseri	Botryllus	schlosseri
			Ascidiacea						Ascidiacea							Ascidiacea					Ascidiacea	Ascidiacea	Ascidiacea
		Chordata	(Tunicata)

Table 2 .

 2 Cont.

		Reference	Rabinowitz	and	Rinkevich	(2011)	Hyams	et al.	(2017)				Cai et al.	(2013)						Wang	et al.	(2009)		Kaneko	et al.	(1995)	Odintsova	et al.	(2015)	Odintsova	et al.	(2005)	De	Mulder	et al.	(2009)	Brody	and	Chang	(1989)
		Medium	Change			unknown				weekly						unknown							every	10-15	days		none	unknown	every 3	days	cells	used	directly	for	experimen-	tation	weekly
		Light Atmosphere Temperature		dark air 20 • C			dark air 19 • C					unknown unknown 25 • C							unknown air 25 • C			ambient air 18 • C	unknown unknown 17 • C	unknown unknown 15 • C	unknown unknown unknown	unknown air 20 • C
		Antimicrobials Substrate	penicillin,	streptomycin, plastic	amphotericin	penicillin,	streptomycin, plastic	nystatin	streptomycin	(100	U/mL), unknown rifampicin	(50	µg/mL)				none plastic			penicillin	G	potassium	(50 plastic units/mL)	/streptomycin	sulfate (50	µg/mL)	none glass	penicillin,	streptomycin, plastic	gentamycin	none none	penicillin, plastic streptomycin
	Trace	Elements	and	Vitamins		salts			MgSO 4 , MgCl 2					none								none			none	none	vitamin	E,	insulin	none	NaHCO 3
		Supplement Antioxidant	fetal calf	serum,	HEPES,	L-glutamine	glutamine none solution			10%	(FBS) + none	1% BPE						20% FBS none			4% (v/v)	newborn none bovine	albumin	2 or 8% none FCS	2% FCS none	none none	10% fetal	bovine none	serum
		Basal	Formula			L-15			DME	supplemented	with salts		2x	Leiboviz's	L-15	medium				L-15 and	F-12		Millipore-	filtered	seawater	seawater	L-15	ASW	Medium	199
	Adherent/	Suspended	Cell	Culture		adherent				adherent					suspended	cells							adherent			suspended	cells	adherent	cells	adherent	suspended	suspended
		Target	Cells			epithelium			blood	cells				all cell	types						all cell	types		all cell	types	all cell	types	guts	all cell	types	hematopoietic	and	testicular	tissue
		Inoculum	Type		palleal	buds		adult,	hibernating	colonies	Adult	epidermis,	gill, gut,	spermary,	ovary	adult,	buccal	cirri, tail,	gill, gut	and	metapleural	fold	embryo	(mesenchyme	migration	stage)	embryo	adult	adults	adults
		Species		Botryllus	schlosseri		Botrylloides	leachii			Branchiostoma	belcheri	Japanese					Branchiostoma	belcheri	tsingtauense		Asterias	amurensis	Strongy-	locentrotus	intermedius	Apostichopus	japonicus	Isodiametra	pulchra	Pacifasticus	leniusculus,	Homarus	americanus
		Class			Ascidiacea				Ascidiacea						Leptocardii								Leptocardii			Asteroidea	Echinoidea	Holothuroidea	Acoela	Malacostraca
		Phylum													Chordata	(Cephalo-	chordata)										Echinodermata	(Ambulacraria)	Xenacoelo-	morpha	Arthropoda	(Ecdysozoa)

Table 2 .

 2 Cont.

		Reference		Han et al.	(2013)	Toullec	et al.	(1996)	Zhang	et al.	(2011)		Asami	et al.	(2002)							Schürmann	and	Roland	(2001)	Lei et al.	(2019)	Bedi et al.	(1998)	Schacher	and	Proshansky	(1983)
		Medium	Change		unknown		unknown			unknown			none (4	d	culture)							every	3rd day	none (6	d	culture)	unknown	every 2	days
		Antimicrobials Substrate Light Atmosphere Temperature		Penicillin, plastic streptomycin dark 5% CO 2 26 • C	penicillin, plastic streptomycin unknown unknown unknown	penicillin, glass streptomycin dark without CO 2 20 • C	fibronectin	none or laminin-coated dark air 20 • C	glass			collagenI/	fibronectin	coated	neomycin glass 18 • sulfate 100 and dark air C	mg/L plastic;	planarian	homologous	matrix	poly-D-lysine 5%	none µg/mL) dark 2 -95% (50 CO air (for 22 • C	coated L-15:	plastic air)	none plastic dark unknown 18 • C	none Plastic, poly-l-lysine dark air RT
	Trace	Elements	and	Vitamins	NaCl,	NaHCO 3		none			none					unknown									d-biotin,	MEM	vitamins	unknown	none	dextrose,	glutamine
		Supplement Antioxidant		15% FBS, none glucose		FBS 10% none			15% FBS none					unknown unknown				FCS and	BSA	("BMS"),	MEM	amino	acids	(essential none and	non-essential),	glucose,	trehalose,	sodium	pyruvate,	glutamine	FBS 5% none	hemolymph none supplemented	1% FBS none
		Basal	Formula		L-15	L-15 or	M199		L-15	modified	TTP	medium;	Teshirogi	and	Tohya,	1998					isosmotic	saline	(Hepes-	buffered)	IPM,	KnockOut	DMEM,	diluted	L-15,	diluted	Grace's	medium	L-15	L-15
	Adherent/	Suspended	Cell	Culture	adherent		adherent			adherent				adherent	cells								adherent	cells	adherent	cells	adherent	adherent
		Target	Cells		lymphoid	organ	all cell	types	larval	cells			neuronal	cells								neoblasts	neoblasts	neuronal	cells	ganglia
		Inoculum	Type		adults		juvenile			eggs				adult	head								adult	adult tail	adult	pleural	ganglia	adult	and	juvenile
		Class Species		Malacostraca M. ensis	Malacostraca Penaeus vannamei	Chromadorea C. elegans			Turbellaria Dugesia japonica								Turbellaria Schmidtea polychroa	Turbellaria Schmidtea mediterranea	Gastropoda A. californica	Gastropoda A. californica
		Phylum								Nematoda	(Ecdysozoa)			Platyhe-	lminthes								Mollusca

Table 2 .

 2 Cont.

		Reference
		Medium	Change
		Atmosphere Temperature
		Light
		Antimicrobials Substrate
	Trace	Elements	and	Vitamins
		Supplement Antioxidant
		Basal	Formula
	Adherent/	Suspended	Cell	Culture
		Target	Cells
		Inoculum	Type
		Species
		Class	Gastropoda
		Phylum

Table 2 .

 2 Cont.

		Reference								Barnay-Verdier	et al. (2013)													Ventura et al.	(2018)	Gates and	Muscatine	(1992)
		Medium	Change									weekly															weekly	none
		Atmosphere Temperature								air 20 • C														air 20 • C	air 25 • C
		Light									dark															dark	NA
		Antimicrobials Substrate	1%	kanamycin	(100	µg/mL),	1%	Amphotericin	B (2.5 plastic µg/mL)	(after 3-7	d	MycoKill	1× initial	supple-	mentation)	1%	kanamycin	(100	µg/mL),	1%	Amphotericin plastic B (2.5	µg/mL),	1%	Antibiotics-	Antimycotics	(Sigma)	polylysine	none 0.1% coated	slides
	Trace	Elements	and	Vitamins								none															none	none
		Supplement Antioxidant									5% FBS none													5% FBS,	1% none	L-glutamate	none none
		Basal	Formula							80%	modified	DMEM												20%	GMIM	filtered	seawater
	Adherent/	Suspended	Cell	Culture						adherent	clusters	of cells													suspended	suspended	cells
		Target	Cells					anemone	small	round	cell	types									anemone	gastrodermal	differentiated	cells	gastrodermal	cell	containing	zooxanthellae
		Inoculum	Type							adult	regenerating	tentacle											adult	regenerating	(whole)	tentacle	adult
		Species								Anemonia	viridis													Anemonia	viridis	Pocillopora	damicornis
		Phylum Class									Anthozoa															Anthozoa	Anthozoa

Table 2 .

 2 Cont.

		Reference											Frank et al.	(1994)												Kopecky and	Ostrander	(1999)	Domart-Coulon	et al. (2001)	Domart-Coulon	et al. (2004)	Helman et al.	(2008)
		Medium	Change												weekly															none			once	every 2	weeks	weekly	weekly	(every	other	day	ascorbic	acid)
		Atmosphere Temperature											air 23-24 • C												5% 24 • CO 2 -95% air C	5% 24 • CO 2 -95% air C	air 27 • C	air 26 • C
		Light												NA														24 h	light		24 h	light	12	hL:12	hD	12	hL:12	hD
		Antimicrobials Substrate							1%	gentamycin,	penicillin, plastic streptomycin,	amphotericin,	nystatin								1%	Antibiotic-Primaria	Antimycotic plastic	Gibco	1%	Antibiotic-Primaria	Antimycotic plastic	Gibco	1% RGD Antibiotic-peptide-coated Antimycotic plastic/glass Gibco	1%	Antibiotic-Primaria	Antimycotic plastic	Gibco
	Trace	Elements	and	Vitamins											none													Mg 2+ ,	Fe 2+ ,	Mn 2+	Sr 2+	Sr 2+	MEM	vitamins
		Basal Supplement Antioxidant Formula									10%	+ taurine 10% FCS L-15/DMEM/M199 none	40 mg/L											25-50% 10% FBS DMEM none		50%	DMEM + 10% FBS taurine none	40 mg/L	12.5%	DMEM + 1.25% none taurine FCS	40 mg/L	ASW +	MEM	aminoacids	+ taurine	10 mM, ascorbic glutamine 2% FBS 2mM, acid 50 µg/mL aspartic	acid 20	µg/mL +	glucose	0.1-3 mM
	Adherent/	Suspended	Cell	Culture								adherent	and	suspended	cells										suspended	multicellular	isolates	suspended	cells and	adherent	multicellular	isolates	adherent	multicellular	isolates	adherent	cells and	cell	aggregates
		Target	Cells											all cell	types													all cell	types		all cell	types	all cell	types	all cell	types
		Inoculum	Type										adult	and	larva													adult			adult	adult	adult
		Species	Stylophora	pistillata,	Porites	lutea,	Dendron-	ephthya	hemprichi,	Paraerythro-	podium	fulvum	fulvum,	Heteroxenia	fuscescence,	Clathraria	rubrinoides,	Plexaura	sp.,	Millepora	dichotoma	Acropora	microphtalma,	Pocillopora	damicornis,	Montipora	digitata	Pocillopora	damicornis	Pocillopora	damicornis	Montipora	digitata,	Xenia	elongata
		Phylum Class												Anthozoa															Anthozoa			Anthozoa	Anthozoa	Anthozoa

Table 2 .

 2 Cont.

		Reference					Khalesi (2008)						Nesa and	Hidaka (2009)														Downs et al.	(2010)	Reyes-Bermudez	and Miller	(2009)	Gardner et al.	(2015)
		Medium	Change					weekly							daily															every	3rd day	every 2	weeks	every	3rd day
		Atmosphere Temperature			5% 24 • CO 2 -95% air C				air 25 • C															air 26 • C	air 23 • C	air 25 • C
		Light			12	hL:12	hD				24 h	light															light?	dark	10	hL:14	hD 100	µmol	photons	m -2 s -1
		Antimicrobials Substrate		1%	gentamycine-plastic 1%	streptomycin				none plastic															none plastic or Teflon	3%	Antibiotics-plastic Antimycotics	Sigma	none glass
	Trace	Elements	and	Vitamins				none							none									hydroxy-	cobalamin	0.01 mM,	Na-folate	0.001	mM,	succinate	0.05 g/L	2%	marine	enrichment	F/2	(Sigma)	none
		Supplement Antioxidant					none none			ascorbic	acid 125	µM +	none catalase 250	U/mL;	mannitol	10 mM											none ascorbate 0.25 mM	5% FBS none	coralline	algae none whole	fragment
		Basal	Formula		GIM,	GMIM	without	salt	addition				seawater				RPMI +	CaCl 2 1 mM,	NaPyruvate	1 mM,	glucose	2g/L,	galactose	2g/L,	Na-lactic	acid 0.25	g/L,	Bovine	Albumin	1 g/L,	cysteine	0.5 mM,	methionine	0.5 mM,	glutamine	0.25 g/L	10%	DMEM	FSW	without	antibiotics
	Adherent/	Suspended	Cell	Culture	suspended	cells and	cell	clusters		suspended	tissue	balls													suspended	cells	suspended	cells	suspended	spheroids
		Target	Cells				all cell	types					all cell	types												coral cell	types	without	zooxanthellae	"interstitial"	cells	all cell	types
		Inoculum	Type					adult							adult																adult	larva	(planula)	adult	regenerating	tissue	(peeled	off	skeleton)
		Species				Sinularia	flexibilis				Fungia	sp.,	Pavona	divaricata													Pocillopora	damicornis	Acropora	millepora	Fungia	granulosa
		Phylum Class					Anthozoa							Anthozoa																Anthozoa	Anthozoa	Anthozoa

Table 2 .

 2 Cont.

		Reference								Kawamura	et al. (2021)								Reyes-Bermudez	et al. (2021)							Vandepas et al.	(2017)	Song et al.	(2021)	Lavrov and	Kosevich	(2016)	Robinson	(2015)
		Medium	Change								every	3rd day								when	60%	confluence							every	3rd day	every	1-4 days	every 48	h	none
		Atmosphere Temperature								air 20 • C									air 23 • C								air 14-16 • C	air 24 • C	air 8-10 • C	reduced unknown air
		Light									dark										dark								dark	unknown	unknown	unknown
		Antimicrobials Substrate		penicillin	100 U/mL,	streptomycin	100 plastic µg/mL,	amphotericinB	(0.25	µg/mL)	1%	penicillin/	streptomycine, plastic	0.1%	fungizone					1% plastic, penicillin/ glass streptomycin	400 mg/L plastic gentamicin	none plastic	none plastic
	Trace	Elements	and	Vitamins					plasmin	2 µg/mL	(modular	protease)						1%	Glutamax							none	none	none	none
		Supplement Antioxidant									1.5% FBS none										10% FBS none					ctenophore	mesogleal	serum none	(CMS)	homogenate	none none	none none	none none
		Basal	Formula								10%	DMEM								30%	DMEM				10-50%	ctenophore	mesogleal	homogenate	(in	seawater)	NSW	(natural	see	water)	FSW	CMFASW
	Adherent/	Suspended	Cell	Culture					cell	aggregates	(brown-	pigmented)						suspended	cell	aggregates	round	ectodermal	cells/	adherent	giant	smooth	muscle	cell/	suspended	digestive	endodermal	cells	suspended	cells and	cell	clusters	suspended	cells	suspended	cells
		Target	Cells	gastrodermal	secretory	cells,	undifferen-	tiated	cells,	neuronal	cells,	and	epidermal	cells	pluripotent	cells							all cell	types	all cell	types	all cell	types	all cell	types
		Inoculum	Type									planula										adult						adult	wound-	healing	lobe	adult	adult	adult
		Species								Acropora	tenuis								Acropora	digitifera							Mnemiopsis	leidyi	Axinella	sp.	H.	panicea,	H.	aquaeductus,	H.	dujardinii	Spongosorites,	Cinachyrella,	Haliclona
		Class									Anthozoa										Anthozoa								Tentaculata	Demos-	pongiae	Demos-	pongiae	Demos-	pongiae
		Phylum																													Cteno-	phora	Pori-	fera

Table 2 .

 2 Cont.

		Reference				Schippers et al.	(2011)			Garcia	Camacho et al.	(2006)					Zhang et al.	(2004)		De Rosa et al.	(2001)	Müller et al.	(1999)	Koziol et al.	(1998)
		Medium	Change					unknown					1-4 per	week						none		Every 3	days	daily	none	daily,	once a	week	after the	2nd	week
		Light Atmosphere Temperature				unknown unknown 2 • C					unknown 4% CO 2 17-19 • C						unknown unknown 16 • C		Dark unknown or light 18 or 22 • C	unknown unknown 16 • C	unknown unknown 17 • C	unknown unknown 16 • C
		Antimicrobials Substrate					none plastic						none plastic							penicillin, plastic streptomycin		ampicillin,	gentamycin,	kanamycin, plastic	tylosin,	tetracyclin	penicillin, plastic streptomycin	none plastic	penicillin, plastic streptomycin
	Trace	Elements	and	Vitamins				none				inorganic	salts,	amino	acids	L-glutamine,	pyruvate,	ferric	citrate,	sodium	silicate,	RPMI	1640,	Marine	Broth	none	none	none	none
		Supplement Antioxidant					none none				octopus	extract none	5-20%					none none		sterols in none DMEM	none none	none none	none none
		Basal	Formula	Calcium-	and	magnesium-	free ASW	EDTA	RPMI	1640						SW-AB		CMFSW,	DMEM	after 3rd	week	CMFSW	EDTA	PBSA-SW	CMFSW-E
	Adherent/	Suspended	Cell	Culture			suspended	cells				suspended	cells						suspended	cells		suspended	cells	suspended	cells	suspended	cells	suspended	cells
		Target	Cells				all cell	types				all cell	types						all cell	types		all cell	types	all cell	types	spherulous	cells	all cell	types
		Inoculum	Type					adult						adult							adult		adult	adult	Adult,	outer	cortical	layer	adult
		Species	H.	oculata,	H. xena,	D. avara,	A.	polypoides	Crambe	crambe						S.	domuncula		Ircinia	muscarum,	Dysidea	avara,	Suberites	domuncula	Suberites	domuncula	Geodia	cydonium,	Suberites	domuncula	S.	domuncula
		Class				Demos-	pongiae					Demospongiae							Demospongiae		Demospongiae	Demospongiae	Demospongiae	Demospongiae
		Phylum

Table 3 .

 3 Molecular tools to detect the most frequently recorded unicellular microeucaryote contaminants. Sequences from cloned PCR products amplified with these eukaryote primers are blasted against National Center for Marine Biotechnology (NCBI) nucleotide databases to check the potential microbial nature of long-term cultured candidate aquatic invertebrate stem cells.

	Host Metazoan Taxon	Associated Microeukaryote Taxon	Opportunistic In Vitro Growth		Gene Marker	Amplicon Size (bp)	Primer Pairs	Oligonucleotide Sequences	Reference
			mixotrophy:
			use of
	Scleractinian coral	Chromeria velia (Chromerida: Alveolata)	medium-provided mono/disaccharides and aminoacids	psbA	~2000	psbAf6	5 -GARCAC AACATHYTNA TGCAYCC-3	Moore et al. (2003)
			(glutamate and
			glycine)
								5 -CRTGCA
							psbAL1	TWACTTC
								CATWCC-3
			use of C & N
			from Leibovitz
			L-15 formula					5 -TTCGG
	Scleractinian coral	Stramenopiles (Thraustochytriidae)	diluted in filtered		18S rRNA	1500	Lab-490F	TTCCGG AGAGGG	Siboni et al. (2010)
			seawater (and					AGCCTGAGAG-3
			organics from
			mucus of coral)
								5 -GCAGA
							Lab-2004R	ATCCGA AGATT
								CACCGG-3
	all aquatic invertebrates	unicellular holozoan Opisthokonts	predation on invertebrate cells and debris, osmotrophy	specific alpha-Amylases, alpha-glucosidases	variable	18SFU	5 -ATGC GCCATGC-3 TTGTC TCAAAG RYTAA	Tikhonenkov et al. (2020b)

Table 3 .

 3 Cont.

	Host Metazoan Taxon	Associated Microeukaryote Taxon	Opportunistic In Vitro Growth	Gene Marker	Amplicon Size (bp)	Primer Pairs	Oligonucleotide Sequences	Reference
							5 -CWGG
							TTCAC
						18SRU	CWACGG
							AAACC
							TTGTTACG-3
	all aquatic invertebrates	Fungi (including chytrids)	saprotrophy (feed on cellular debris)	18S rDNA	2000	SR1R	5'-TACCT GGTT GATQC TGCCAGT-3'

Acknowledgments:

We would like to thank Buki Rinkevich, Marta Wawrzyniak, Nathalie Weber and Aude Blanchoud for their help in compiling the data used for Tables 1 and2. We also thank Buki Rinkevich and Loriano Ballarin for constructive comments on this manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

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