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In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research. Graphical abstract

Pikeperch (Sander lucioperca) is a freshwater fish that has become increasingly popular as a food fish. Within this study, the influence of the spawning process on meat quality was investigated. For this purpose, adult pikeperch was examined directly before and after spawning, and compared regarding physical meat parameters and lipid composition. The results indicate that after spawning, the values of the pH, the electrical conductivity and the yellowness value of fillet were significantly higher than those of the animals sampled before spawning. Analysis of the sum of the total lipid content indicates no differences before and after closed season, but differences in the fatty acid profile were present. Despite significant lower MUFA concentrations, the EPA and DHA showed unaffected high contents. Therefore, the fish muscle indicated an equivalent meat quality. Nevertheless, the significant changes of some physical meat quality parameters after spawning season could have a particular impact on the shelf life and storage of the pikeperch fillet, highlighting the need for further research.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C. maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C. maranea.

There are still numerous difficulties in the successful farming of pikeperch in the anthropogenic environment of various aquaculture systems, especially during early developmental steps in the hatchery. To investigate the physiological processes involved on the molecular level, we determined the basal expression patterns of 21 genes involved in stress and immune responses and early ontogenesis of pikeperch between 0 and 175 days post hatch (dph). Their transcription patterns most likely reflect the challenges of growth and feed conversion. The gene coding for apolipoprotein A (APOE) was strongly expressed at 0 dph, indicating its importance for yolk sac utilization. Genes encoding bone morphogenetic proteins 4 and 7 (BMP4,BMP7), creatine kinase M (CKM), and SRY-box transcription factor 9 (SOX9) were highly abundant during the peak phases of morphological changes and acclimatization processes at 4–18 dph. The high expression of genes coding for peroxisome proliferator-activated receptors alpha and delta (PPARA,PPARD) at 121 and 175 dph, respectively, suggests their importance during this strong growth phase of juvenile stages. As an alternative experimental model to replace further in vivo investigations of ontogenetically important processes, we initiated the first approach towards a long-lasting primary cell culture from whole pikeperch embryos. The present study provides a set of possible biomarkers to support the monitoring of pikeperch farming and provides a first basis for the establishment of a suitable cell model of this emerging aquaculture species.

Exposure to elevated temperatures during embryogenesis can influence the plasticity of tissues in later-life. Despite these long-term changes in plasticity, few differentially expressed genes are ever identified, suggesting that the developmental programming of later-life plasticity may occur through the modulation of other aspects of the transcriptomic architecture, such as gene network function. Here, we use network modelling approaches to demonstrate that warm temperatures during embryonic development (developmental warming) have consistent effects in later-life on the organisation of transcriptomic networks across four diverse species of fishes:Scyliorhinus canicula, Danio rerio, Dicentrarchus labrax, andGasterosteus aculeatus. The transcriptomes of developmentally warmed fishes are characterised by an increased entropy of their pairwise gene interaction networks, implying a less structured, more ‘random’ set of gene interactions. We also show that, in zebrafish subject to developmental warming, the entropy of an individual gene within a network is associated with that gene’s probability of expression change during temperature acclimation in later-life. However, this association is absent in animals reared under ‘control’ conditions. Thus, the thermal environment experienced during embryogenesis can alter transcriptomic organisation in later-life, and these changes may influence an individual’s responsiveness to future temperature challenges.

Rising temperatures can affect fish survival, especially from shallower waters, as temperatures increase faster and more intensively in these areas, thus, species-specific temperature tolerance can be exceeded. Additionally, the amounts of anthropogenic pollutants are higher in coastal waters. Although increasing metabolic activity at higher temperatures could lead to stronger effects of toxins, there are hardly any studies on this topic. Subsequently, the aim was to investigate the response of fish cells upon exposure to industrial solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO)) in relation to a temperature increase (20 °C and 25 °C). Concerning the 3Rs (the replacement, reduction and refinement of animal experiments), in vitro tests were used for two threatened, vulnerable fish species: maraena whitefish (Coregonus maraena) and Atlantic sturgeon (Acipenser oxyrinchus). Both cell lines exhibited higher proliferation at 25 °C. However, ecotoxicological results indicated significant differences regarding the cell line, toxin, temperature and exposure time. The evolutionarily older fish lineage, Atlantic sturgeon, demonstrated lower mortality rates in the presence of isopropanol and recovered better during long-term ethanol exposure than the maraena whitefish. Atlantic sturgeon cells have higher adaptation potential for these alcohols. In summary, fish species respond very specifically to toxins and changes in temperature, and new ecotoxicological questions arise with increasing water temperatures.

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30 years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

The growth of fishes and their metabolism is highly variable in fish species and is an indicator for fish fitness. Therefore, somatic growth, as a main biological process, is ecologically and economically significant. The growth differences of two closely related salmonids, rainbow trout (Oncorhynchus mykiss) and maraena whitefsh (Coregonus maraena), have not been adequately studied as a comparative study and are therefore insufficiently understood. For this reason, our aim was to examine muscle growth in more detail and provide a first complex insight into the growth and muscle metabolism of these two fish species at slaughter size. In addition to skeletal muscle composition (including nuclear counting and staining of stem and progenitor cells), biochemical characteristics, and enzyme activity (creatine kinase, lactate dehydrogenase, isocitrate dehydrogenase) of rainbow trout and maraena whitefish were determined. Our results indicate that red muscle contains cells with a smaller diameter compared to white muscle and those fibres had more stem and progenitor cells as a proportion of total nuclei. Interestingly, numerous interspecies differences were identified; in rainbow trout muscle RNA content, intermediate fibres and fibre diameter and in whitefish red muscle cross-sectional area, creatine kinase activity were higher compared to the other species at slaughter weight. The proportional reduction in red muscle area, accompanied by an increase in DNA content and a lower activity of creatine kinase, exhibited a higher degree of hypertrophic growth in rainbow trout compared to maraena whitefish, which makes this species particularly successful as an aquaculture species.

Sander lucioperca is an organism of growing importance for the aquaculture industry. Nonetheless, the rearing of S. lucioperca larvae is proving to be a difficult task as it is facing a high mortality rate during hatching and the change to exogenous feeding. To gain insight into growth patterns during this period, the authors analysed pikeperch embryos and larvae from 9 days before hatching to 17 days after hatch. Hereby they were able to describe a natural development by using close to natural conditions based on using a direct flow-through supply of lake fresh water on specimens from a local wild population. The results show that between the early embryonic stages a steady growth was visible. Nonetheless, in between hatching and the start of exogenous feeding, a phase of growth stagnation took place. In the following larval stages, an increased growth with large size variations between individual specimens appeared. Both factors are conspicuous as they can indicate a starting point for cannibalism. With this analysis, the authors can provide a fundament to support the upcoming research on S. lucioperca and aid to optimize size-sorting procedures for a higher survival of pikeperch stock in aquaculture.

Although the renin-angiotensin system usually promotes oxidative stress and cell death, renin transcripts have been discovered, whose transcription product may be cardioprotective. These transcripts encode a non-secretory renin isoform that is localized in the cytosol and within mitochondria. Here we tested the hypotheses that cytosolic renin [ren(2-9)] expression promotes cell survival under hypoxia and glucose depletion by preserving the mitochondrial membrane potential (∆Ψm) and mitigating the accumulation of ROS. To simulate ischemic insults, we exposed H9c2 cells to glucose deprivation, anoxia or to combined oxygen-glucose deprivation (OGD) for 24 hours and determined renin expression. Furthermore, H9c2 cells transfected with the empty pIRES vector (pIRES cells) or ren(2-9) cDNA-containing vector [ren(2-9) cells] were analyzed for cell death, ∆Ψm, ATP levels, accumulation of ROS, and cytosolic Ca2+ content. In pIRES cells, expression of ren(1A-9) was stimulated under all three ischemia-related conditions. After OGD, the cells lost their ∆Ψm and exhibited enhanced ROS accumulation, increased cytosolic Ca2+ levels, decreased ATP levels as well as increased cell death. In contrast, ren(2-9) cells were markedly protected from these effects. Ren(2-9) appears to represent a protective response to OGD by reducing ROS generation and preserving mitochondrial functions. Therefore, it is a promising new target for the prevention of ischemia-induced myocardial damage.

The zebrafish (ZF) has become an essential model for biomedical, pharmacological and eco-toxicological heart research. Despite the anatomical differences between fish and human hearts, similarities in cellular structure and conservation of genes as well as pathways across vertebrates have led to an increase in the popularity of ZF as a model for human cardiac research. ZF research benefits from an entirely sequenced genome, which allows us to establish and study cardiovascular mutants to better understand cardiovascular diseases. In this review, we will discuss the importance of in vitro model systems for cardiac research and summarise results of in vitro 3D heart-like cell aggregates, consisting of myocardial tissue formed spontaneously from enzymatically digested whole embryonic ZF larvae (Zebrafish Heart Aggregate - ZFHA). We will give an overview of the similarities and differences of ZF versus human hearts and highlight why ZF complement established mammalian models (i.e. murine and large animal models) for cardiac research. At this stage, the ZFHA model system is being refined into a high-throughput (more ZFHA generated than larvae prepared) and stable in vitro test system to accomplish the same longevity of previously successful salmonid models. ZFHA have potential for the use of high-throughput-screenings of different factors like small molecules, nucleic acids, proteins and lipids which is difficult to achieve in the zebrafish in vivo screening models with lethal mutations as well as to explore ion channel disorders and to find appropriate drugs for safety screening.

The renin‐angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto‐renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto‐renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto‐renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto‐renin is a promising new target for the prevention of ischaemia‐induced myocardial damage.

Eighteen captive small-spotted catsharks Scyliorhinus canicula were successfully identified from hatching to 1 year of age using the free computer recognition software, I3S classic. The effect of increasing the time interval between recognition attempts on the accuracy of the software was investigated, revealing that recognition fiedelity decreases with increasing time intervals for younger (0 to 15 weeks), but not older (15 weeks onwards) sharks. Identification by I3S was validated using genetic analyses of seven microsatellite markers, revealing a 100% success rate. Thus, this non-invasive recognition method can be used as an inexpensive and effective alternative to invasive tagging, improving animal welfare and complementing ex-situ conservation methods.

This study compares the trunk skeletal muscle anatomy in 870- and 2900-degree-day-old lesser-spotted dogfish larvae (Scyliorhinus canicula) via haematoxylin/eosin staining as well as immunohistochemistry and Western blot analysis. The results showed poorly differentiated muscle formation in the trunk segments in the younger larvae and fully developed skeletal muscle with a division of red and white cells in the older larvae. The stem cell marker PAX7, which is present in all developmental stages of teleost fish, is only expressed in the younger dogfish. The results show the necessity of examining the skeletal muscle development in sharks to understand the evolutional changes from cartilaginous fishes to teleosts.

Elasmobranchs display various reproductive modes, which have been key to their evolutionary success. In recent decades there has been a rise in the number of reported cases of foetal abnormalities including fertilised, double-embryos held within one egg capsule, hereafter referred to as twins. Previously, the occurrences of twin egg cases have been reported in two batoid and one shark species. We report the first cases of twins in three species of oviparous elasmobranchs: the undulate ray (Raja undulata), the nursehound (Scyliorhinus stellaris), and the small-spotted catshark (Scyliorhinus canicula). We investigated the genetic relationships between the twins in S. stellaris, and S. canicula using microsatellite markers. Whilst the S. stellaris twins displayed the same genotypes, we found that the S. canicula twin individuals arose through heteropaternal superfecundation. This is the first reported incidence of such a paternity in elasmobranchs. The relationship between environmental change and reproductive strategy in elasmobranchs is unclear and further research is needed to determine its effect on the prevalence and mechanisms of formation of elasmobranch twins.

Despite improvements in imaging techniques during recent years, for many non- model systems the fixation of tissues followed by embedding and sectioning for histochemical or immunohistochemical staining remains an important technique in vertebrate histology. The pres- ent study sets out to explore the preservation of histological sections of fish tissues using different preparation techniques. The quality of transverse vibratome sections from trunk segments of the lesser-spotted dogfish Scyliorhinus canicula, Atlantic sturgeon Acipenser oxyrinchus and zebrafish Danio rerio were compared using different fixatives (formaldehyde, paraformaldehyde and zinc-formaldehyde) and embedding methods (gelatine, agarose and low-temperature melting agarose). Our data show that the quality of the vibratome sections for histochemical staining is strongly dependent upon fixation and embedding media. Although paraformaldehyde fixation results in a more pronounced shrinkage of the trunk segment than the other fixatives used , the quality of the sections and the histochemical staining was best with this fixative in zebrafish and dogfish. Additionally, the embedding methods have a strong influence on the quality of the sec- tions. In the dogfish and sturgeon samples, the preferred embedding media were agarose and low-temperature melting agarose, since gelatine often caused shrinkage of the tissues. In conclu- sion, for histochemical examinations, the processing protocols for vibratome sectioning need to be adapted individually to each study organism.

Background/Aims: Renin is known as a secretory glycoprotein that ultimately leads to angiotensin II generation. In this way renin exerts pro-inflammatory effects and promotes cardiac injury. Additional transcripts have been identified encoding for a cytosolic renin isoform that - in contrast to secretory renin - exhibits cardioprotective effects under ischemic conditions. The promoter of these transcripts is unknown. Methods: Using qRT-PCR and dual-luciferase reporter assay we examined the expression and promotor activity of cytosolic renin as well as the regulation by glucose starvation in H9c2 cardiomyoblasts. Results: We identified a promoter in intron1 of the rat renin gene with two glucose starvation-sensitive regions. One region contains a binding motif for serum response factor (SRF). Under glucose depletion expression of SRF increased prior to cytosolic renin. SRF knock down selectively decreased cytosolic renin expression and attenuated the increase of cytosolic renin expression under glucose depletion. Conclusions: Transcripts encoding for secretory and cytosolic renin are differentially expressed. The low basal expression of cytosolic renin as well as its induction under ischemia-related conditions represents an efficient system regulated in accordance with its previously identified unfavorable effects under control situations but protective effects seen after myocardial infarction or glucose depletion.

Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15-22 degrees. SCPCs production efficiency was not influenced by embryo's origin (0.75/farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15-22 degrees) or age (2-4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p

Background/Aims: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. Methods: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra – and extracellular recordings. Results: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. Conclusion: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

We describe here a novel, fast and inexpensive method for producing a 3D ‘heart’ structure that forms spontaneously, in vitro, from larval zebrafish (ZF). We have named these 3D ‘heart’ structures ‘zebrafish heart aggregate(s)’ (ZFHAs) and have characterised their basic morphology and structural composition using histology, immunohistochemistry, electron microscopy and mass spectrometry. After 2 days in culture the ZFHA spontaneously form and become a stable contractile syncytium consisting of cardiac tissue derived by in vitro maturation, which beats rhythmically and consistently for more than 8 days. We propose this model as a platform technology, which can be developed further to study in vitro cardiac maturation, regeneration, tissue engineering and safety pharmacological/toxicology testing.

c These authors contributed equally to this work Abstract Background/Aims: Safety pharmacology requires novel model systems for the detection of cardiac side effects. Ranging from cell-based systems to model organisms, no model available to date reflects the complexity of the human heart and evokes the great need for improved and more affordable systems. Many drugs interact with hERG potassium channels and consequently cause life threatening ventricular arrhythmias, further highlighting the importance of suitable model systems. Methods: Spontaneously Contracting Cell aggregates (SCC) as a 3D in vitro heart-syncytium obtained from rainbow trout larvae represent a novel model system for cardiac safety pharmacology. SCCs can be harvested cost-effectively and kept in culture for several weeks while retaining their functionality and displaying contraction rates similar to the human heart. Results: Extracellular field potential recordings with multielectrode arrays revealed significant prolongation of field potential duration upon administration of common hERG potassium channel blockers. Infusion of 1 µM Dofetilide and 10 µM Terfenadine prolonged field potentials 10 fold and 2 fold, respectively. In addition, SCCs enabled analysis of autonomous contraction frequencies. Conclusion: Thus, SCCs represent a novel and low- cost cardiac model system of the human heart for application in safety pharmacology.

Background: Beside its anti-proliferative, anti-hypertensive and anti-inflammatory effects, heparin has shown the apoptotic effect in lymphoblasts. In the study, it is aimed to show the apoptotic effect of heparin in lymphoblasts with measuring intracellular calcium and using DNA analysis by flow cytometry, in vitro. Methods: Twenty-three newly diagnosed acute lymphoblastic leukemia patients were included in the study. We added 10 and 20 U/ml heparin into the seperated lymphoblast samples and determined the percentages of apoptosis and intracellular Ca++ levels at 0, 1 and 2 hours by flow cytometry, in vitro. Results: The apoptotic effect on the lymphoblasts were established in 10 and 20 U/ml heparin concentrations at 0, 1 and 2 hours (p=0.005). The apoptotic effect of heparin in lymphoblasts was higher at the first hour than those at 0 and 2 hours in 10 and 20 U/ml heparin concentrations (p=0.005). The highest apoptosis was determined in 20 U/ml heparin concentration at the first hour. Statistically significant increase in intracellular Ca++ levels were determined in 10 and 20 U/ml heparin concentrations at 1 and 2 hours (p=0.005). In 10 and 20 U/ml heparin concentrations, intracellular Ca++ levels were significantly higher at the first hour than 0 and 2 hours (p=0.005). The highest intracellular Ca++ concentration was determined in 20 U/ml heparin concentration at the first hour. Conclusion: Heparin induces apoptosis in lymphoblasts and intracellular Ca++ levels of the lymphoblasts synchronously increase with apoptosis. The increase of intracellular Ca++ level supports a concept that the mitochondria plays a role heparin-induced apoptosis in lymphoblasts.

Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology.Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra - and extracellular recordings.Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed.Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

We describe a method for fast and easy isolation of cells via trypsin digestion from lar- vae of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus resulting in a stable, well-proliferating cell culture. The culture conditions for these cells were optimized with the aim of supporting the production of high amounts of biomass. To enhance cell growth and cell density, 4 different cultiva- tion temperatures as well as commercially available carp serum (CS) and fetal calf serum (FCS) at different concentrations were tested and evaluated. Cell growth was measured via an impedance- based online cell-monitoring system (xCELLigence). These results showed the best cultivation tem- perature to be at 25°C and a media composition of Dulbecco's modified Eagle's medium (DMEM) supplemented with either 10 or 20% FCS or 5% CS. The cells were stable in the process of long- term cultivation over 33 passages and could be cryo-preserved. Immunocytochemical analysis re- vealed that the cells expressed proteins of different blastodermic layers. Ectodermic glia fibrilliary acid protein, vigilin (mRNA transport protein), and pan cytokeratin were abundant. This fast- growing cell culture provides an important tool for research on Atlantic sturgeon populations.

In primary cell preparations from larvae of rainbow trout Oncorhynchus mykiss, the formation of autonomously contracting cell aggregates was observed after 7 days. These contracting elements could be propagated and some aggregates were maintained over a period of 35 days. Electron microscopical and immunocytochemical examination revealed the presence of cardiomyocytes.

Background/Aims: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. Methods: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra – and extracellular recordings. Results: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. Conclusion: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.