Fish Cell Research Articles

Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line.

Cells are equipped with intracellular RIG-like Receptors (RLRs) detecting double stranded (ds)RNA, a molecule with Pathogen-Associated Molecular Pattern (PAMPs) generated during the life cycle of many viruses. Melanoma Differentiation-Associated protein 5 (MDA5), a helicase enzyme member of the RLRs encoded by the ifih1 gene, binds to long dsRNA molecules during a viral infection and initiates production of type I interferon (IFN1) which orchestrates the antiviral response. In order to understand the contribution of MDA5 to viral resistance in fish cells, we have isolated a clonal Chinook salmon Oncorhynchus tshawytscha epithelial-like cell line invalidated for the ifih1 gene by CRISPR/Cas9 genome editing. We demonstrated that IFN1 induction is impaired in this cell line after infection with the Snakehead Rhabdovirus (SHRV), the Salmon Alphavirus (SAV) or Nervous Necrosis Virus (NNV). The cell line, however, did not show any increase in cytopathic effect when infected with SHRV or SAV. Similarly, no cytopathic effect was observed in the ifih1-/- cell line when infected with Infectious Pancreatic Necrosis Virus (IPNV), Infectious Haemorrhagic Necrotic Virus (IHNV). These results indicate the redundancy of the antiviral innate defence system in CHSE-derived cells, which helps with circumventing viral evasion strategies.

Mitochondrial dysfunction matures Ras-induced early senescence to full senescence with a proinflammatory senescence-associated secretory phenotype in the fish cell line, EPC

Fish cell lines differ from most mammalian diploid cell lines by the fact that cellular senescence is not readily induced. Previously, we demonstrated that the absence of the p16 gene in the fish genome prevents cells from reaching full senescence even when Ras is activated. Drosophila also lacks p16; however, early senescence triggered by Ras activation progresses to full senescence and is accompanied by a proinflammatory senescence-associated secretory phenotype (SASP), due to mitochondrial deficiency. It is unclear whether mitochondrial deficiency can also induce the maturation of Ras-induced early senescence (RIS) to full senescence along with a proinflammatory SASP in fish cell lines. Here, we investigated whether mitochondrial dysfunction induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in concert with activated Ras results in full senescence and whether this is accompanied by a proinflammatory SASPs in the EPC fish cell line. We found that although EPC cells with mitochondrial dysfunction exhibited a proinflammatory SASP, this did not result in permanent cell proliferation arrest or the upregulation of endogenous Ras expression. These findings suggest that other factors must act in concert with mitochondrial dysfunction to induce full senescence. The proliferation of EPC cells overexpressing a constitutively active mutant of H-Ras (H-RasV12) was markedly reduced, irrespective of CCCP treatment. These findings suggest that there are similarities between the cellular senescence observed in fish and Drosophila cells lacking the p16 gene. However, it should be noted that fish cells differ from Drosophila cells in that mitochondrial dysfunction alone can induce proinflammatory SASP factors.

Rapid and Accurate Ecotoxicological Assessment of Heavy Metals Using Cyprinus carpio Cells.

Heavy metals have serious negative effects on various aquatic organisms, and therefore rapid and accurate ecotoxicological assessments of heavy metals are necessary. Fish-derived cells sensitive to heavy metals have been used as valuable tools for ecotoxicological assessments. However, this method requires a minimum toxicity treatment time of 96 h, which limits its use when rapid ecotoxicological assessments are required or ecotoxicological assessments of a large number of toxicants are performed. In this study, these limitations were overcome by adjusting parameters including the concentration of fetal bovine serum (FBS) in the medium and the treatment time of the toxicant. Specifically, we found that the maximum time for fish cells to remain unstarved was 6 h when using a medium containing 1% FBS. We applied both parameters to the ecotoxicological assessment (using a medium containing 1% FBS for the toxicity assessment and treating the toxicant for only 6 h). Surprisingly, these adjusted parameters allowed us to obtain faster and more accurate data than the traditional assessment. This improvement was due to the new assessment conditions that minimized the possibility that the growth-inducing effects of nutrients present in excess in the medium could interfere with the cellular response to the toxicant. The accuracy of this assessment was not limited to measuring the toxicity of heavy metals. In conclusion, we have established an ecotoxicity assessment that can generate rapid and accurate data on heavy metals. This new platform will become the cornerstone of rapid and accurate ecotoxicity assessments of heavy metals.

Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.

Exploring the Effectiveness of Acyclovir against Ranaviral Thymidine Kinases: Molecular Docking and Experimental Validation in a Fish Cell Line.

The effectiveness of acyclovir, a selective anti-herpesvirus agent, was tested both in silico and in vitro against two ranaviruses, namely the European catfish virus (ECV) and Frog virus 3 (FV3). ECV can cause significant losses in catfish aquaculture, while FV3 poses a risk to vulnerable amphibian populations. The genome of ranaviruses encodes thymidine kinases (TKs) similar to those of herpesviruses. Molecular docking simulations demonstrated that the acyclovir molecule can bind to the active sites of both investigated viral TKs in an orientation conducive to phosphorylation. Subsequently, the antiviral effect of acyclovir was tested in vitro in Epithelioma Papulosum Cyprini (EPC) cells with endpoint titration and qPCR. Acyclovir was used at a concentration of 800 µM, which significantly reduced the viral loads and titers of the ranaviruses. A similar reduction rate was observed with Ictalurid herpesvirus 2, which was used as a positive control virus. These promising results indicate that acyclovir might have a wider range of uses; besides its effectiveness against herpesviruses, it could also be used against ranavirus infections.

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Toxicity evaluation of water-accommodated fraction of heavy and light oils on the rainbow trout fish cell line RTL-W1.

Fish are currently used models for the toxicity assessment of chemicals, including polycyclic aromatic hydrocarbons (PAHs). Alternative methods including fish cell lines are currently used to provide fast and reliable results on the toxic properties of chemicals while respecting ethical concerns about animal testing. The Rainbow trout liver cell line RTLW1 was used to analyze the effects of two water-accommodated fractions from two crude oils: Arabian Light crude oil (LO) and refined oil from Erika (HO). Several toxicity endpoints were assessed in this study, including cytotoxicity, EROD activity, DNA damage (comet and micronucleus assays), and ROS production. RTL-W1 cells were exposed for 24h at two or three dilutions of WAF at 1000µg/L (0.1% (1μg/L), 1% (10μg/L), and 10% (100μg/L)) for cytotoxicity and EROD activity and 1% and 10% for ROS production and genotoxicity). Exposure of RTL-W1 cells to LO WAF induced a significant increase of EROD activity and ROS production and altered DNA integrity as revealed by both the comet assay and the micronucleus test for 10µg/L of LO. On the other hand, HO WAF exhibited limited toxic effects except for an EROD induction for 1% WAF dilution. These results confirmed the usefulness of RTL-W1 cells for in vitro toxicological assessment of chemical mixtures.

Stable cytoactivity of piscine satellite cells in rice bran-gelatin hydrogel scaffold of cultured meat

In order to achieve high cell adhesion and growth efficiency on scaffolds for cultured meat, animal materials, especially gelatin, are necessary though the disadvantages of weak mechanical properties and poor stability of their hydrogel scaffolds are present during cell cultivation. Here, we use rice bran as a kind of filling and supporting materials to develop a composite scaffold with gelatin for fish cell cultivation, where rice bran is also inexpensive from high yield fibrous agricultural by-product. The rice bran (with a proportion of 1, 3, 5, 7, 10 to 3 of gelatin) could evenly distributed in the three-dimensional network composed of gelatin hydrogel. It contributed to delaying swelling and degradation rates, fixing water and improving elastic modulus. It is important that rice bran-gelatin hydrogel scaffolds (especially the hydrogel with 70 % rice bran, db) promoted piscine satellite cells (PSCs) proliferation effectively compared to the pure gelatin hydrogel, and the former could also support the differentiation of PSCs. Overall, this work showed a positive promotion to explore new source of scaffold materials like agricultural by-product for reducing the cost of cell cultured meat production.

Transcriptome Analysis of Transiently Reversible Cell Vacuolization Caused by Excessive Serum Concentration in Scophthalmus maximus.

As an important research tool, cell lines play a vital role in life science research, medical research, and drug development. During the culture of the Scophthalmus maximus head kidney (TK) cell line, we found a phenomenon of cell vacuolization caused by excessive serum concentration. Moreover, the vacuolization of the cells gradually disappeared after passage by trypsin digestion. In clarifying the formation mechanism of this reversible cellular vacuolation, transcriptomics was utilized to explore the mechanism of cell vacuolization caused by excessive serum concentration. Transcriptome analysis indicated that excessive serum concentration could cause the up-regulated expression of PORCN and other genes to promote cell proliferation. Compared with cells whose vacuolization disappeared after trypsin digestion and passage, the expression of mitosis-related genes (BUB1, ttk, Mad2, Cdc20, CDK1, CCNB1), nuclear stability-related genes LMNB1 and tissue stress and repair-related genes HMMR in vacuolated cells caused by excessive serum concentration was significantly up-regulated. There is a regulatory system related to adaptation and stress repair in the cells, which can maintain cell stability to a certain extent. This study provides a theoretical basis for the stable culture of fish cell lines and the solution to the problem of cell vacuolation.

Methods to Screen the Adhesion of Fish Cells on Plant-, Algal- and Fungal-Derived Biomaterials.

Cellular agriculture, an alternative and innovative approach to sustainable food production, has gained momentum in recent years. However, there is limited research into the production of cultivated seafood. Here, we investigated the ability of fish mackerel cells (Scomber scombrus) to adhere to plant, algal and fungal-based biomaterial scaffolds, aiming to optimize the cultivation of fish cells for use in cellular agriculture. A mackerel cell line was utilized, and metabolic assays and confocal imaging were utilized to track cell adhesion, growth, and differentiation on the different biomaterials. The mackerel cells adhered and grew on gelatin (positive control), zein, and soy proteins, as well as on alginate, chitosan, and cellulose polysaccharides. The highest adhesion and growth were on the zein and chitosan substrates, apart from the gelatin control. These findings provide a blueprint to enhance scaffold selection and design, contributing to the broader field of cellular agriculture through the development of scalable and eco-conscious solutions for meeting the growing global demand for seafood.

Generation of transgenic fish cell line with α-lactalbumin nanocarriers co-delivering Tol2 transposase mRNA and plasmids

Fish cells, such as grass carp (Ctenopharyngodon idella) kidney (CIK) cells, are harder to transfect than mammalian cells. There is a need for an efficient gene delivery system for fish cells. Here, we used CIK cell line as a model to develop a strategy to enhance RNA and plasmid DNA transfection efficiency using a nanocarrier generated from α-lactalbumin (α-NC). α-NC absorbed nucleic acid cargo efficiently and exhibited low cytotoxicity. Plasmid transfection was more efficient with α-NC than with liposomal transfection reagents. We used α-NC to co-transfect Tol2 transposase mRNA and a plasmid containing Cas9 and GFP, generating a stable transgenic CIK cell line. Genome and RNA sequencing revealed that the Cas9 and GFP fragments were successfully inserted into the genome of CIK cells and efficiently transcribed. In this study, we established an efficient transfection system for fish cells using α-NC, simplifying the process of generating stable transgenic fish cell lines.

Combined Effect of Gamma Radiation and Heavy Metals on Some Living Organisms.

The purpose of this study was to systematise scientific publications on the combined effect of gamma radiation and heavy metals on living organisms. For this purpose, the method of analysis was applied, by means of which scientific papers in PubMed, Google Scholar, and other related databases were analysed for compliance with the inclusion criteria, where the objects of research were toxic effects of radiation and heavy metals on cells and adaptation processes. The results revealed that the study of the problem was carried out on organisms such as microalgae, fungi, weed and agricultural plants, fish, laboratory rats, and human cell cultures. In most studies, an antagonistic effect between low doses of gamma radiation and heavy metal salts was reported, which was manifested by a reduction in the cytotoxicity of isolated exposure to each agent separately. However, there are studies showing additive effects, especially in heavy metals. At the molecular level, heavy metal accumulation in combination with low doses of radiation (typically defined as less than 0.1Gy or sievert) induces the expression of metallothionein proteins, which can bind free radicals. At the metabolic level, this is manifested by a decrease in lipid peroxidation products, activation of antioxidant enzymes, and a decrease in apoptosis. The study proved both a direct relationship between zinc and cadmium accumulation in cells and inhibition of caspases and an indirect one, by maintaining mitochondrial membrane integrity through metallothionein.

Rapid profiling of fish cell nitrogen metabolism with single-cell Raman spectroscopy: Unveiling enzyme's role in ammonia detoxification

Ammonia is a prevalent aquatic pollutant that disrupts cellular functions and energy metabolism in fish, posing significant environmental and health threats. This research investigates the critical role of arginase 2 (ARG2) in mitigating ammonia toxicity in fish cells and its implications in adapting to nitrogen metabolism under high ammonia exposure. Through a CRISPR-Cas9 engineered ARG2 knockdown (KD) in the Epithelioma Papulosum Cyprini (EPC) cell line, we first investigated the biochemical responses of ARG2 KD and wild-type (WT) EPC cells to ammonia stress (NH4Cl treatment), showing diminished urea production and decreased cell viability in ARG2 KD cells. Subsequently, single-cell Raman spectroscopy analysis revealed that ARG2 KD cells exhibited profound metabolic shifts, including changes in protein, nucleic acids, lipid and sugar levels, showing the adjusting role of ARG2 in the balance of carbohydrate and nitrogen metabolism. Furthermore, the upregulated responses of various amino acids, such as glutamine, arginine, alanine, glutamic acid, glycine, histidine, phenylalanine and valine, in WT cells after NH4Cl treatment diminished in ARG2 KD cells except for the decrease in aspartic acid, indicating a switching effect of ARG2 in nitrogen metabolism under ammonia stress. This study highlights ARG2's essential role in ammonia detoxification and emphasizes ARG2's protective function and its importance in metabolism, shedding light on the adaptive mechanisms fish cells deploy against high ammonia environments. These insights contribute to deep understanding of aquatic organisms' molecular responses to environmental ammonia pollution, offering potential strategies for their protection.

Application of Repetitive Sequences in Fish Cell Depletion as a Target for the CRISPR/Cas9 System.

Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.

Establishment and application of a brain cell line derived from Plectropomus leopardus

Plectropomus leopardus is an economically valuable marine farmed fish. However, diseases have seriously restricted the healthy development of its breeding industry. Cell line is an important in vitro research system in the fields of disease control, environmental toxicology, nanotechnology, and so on. However, the application of marine fish cell lines is far from the expected level, and many species have not yet been reported to establish cell lines. In our study, we developed a brain cell line derived from P. leopardus, designated as PLB, and investigated its susceptibility to bacterial agents and heavy metal exposure. The findings indicated that PLB cell lines grew fastest in L-15 medium supplemented with 20 % fetal bovine serum (FBS) at 26 °C. The established cell line was identified by 18 S rRNA gene sequencing as being derived from P. leopardus. Chromosome analysis revealed that the PLB cell line had a chromosome count of 48. The transfection efficiency of PLB cells was about 27 % by liposome transfection method, indicating that PLB cell lines offer utility for conducting functional investigations of exogenous genes. In addition, the bacterial sensitivity analysis results indicated that PLB cells exhibited susceptibility to both Vibrio harveyi and Edwardsiella tarda, furthermore, PLB cells displayed heightened sensitivity towards V. harveyi in comparison to E. tarda. The outcomes from the heavy metal toxicity experiments demonstrated significant toxicity of three heavy metals (Hg2+, Cu2+ and Cd2+) towards PLB cells, with Hg2+ exhibiting the highest degree of toxicity among them. Moreover, there was a dose-dependent relationship between cell survival rate of PLB and heavy metal concentration. In summary, PLB cell lines represent a promising tool for conducting in vitro analyses of foreign gene functionality, bacterial susceptibility, and heavy metal toxicity. It helps ensure the safety of aquatic breeding environments and also supports genetic enhancements in fish fry, promoting the development of traits such as increased growth and disease resistance, which are vital for aquaculture progress.

Growth and replication of infectious bursal disease virus in the fish cell line as an experimental vaccine

Recently, several attempts have been made to replace egg-based with cell-based vaccines to prevent and control Infectious Bursal Disease Virus (IBDV). This study aimed to evaluate a new fish cell line (M99) for culturing and replicating IBDV. After observing complete cytopathic effects (CPE) on the M99 cell line, virus titers were determined using the TCID50 test, and the presence of the virus was confirmed using an RT-PCR test. Subsequently, 135 broiler chickens (14 days old) were randomly divided into three equal groups for immune response measurements: G1: immunized with a commercial vaccine, G2: immunized with an experimental vaccine, and G3: control. Antibody responses, bursal index, and histopathological evaluations were examined on different days after immunization. Based on the results, CPE of the virus was noticeable from the first passage, becoming complete by the third passage. The infectious titer of the virus was log106.9. Antibody titer measured 21 days after immunization in both vaccinated groups were significantly differed from the control group (p < 0.05). The results obtained from examining the bursal index and histopathological evaluations showed no significant difference between the studied groups at different times. Overall, this research is the first report on the successful cultivation of infectious bursal virus on a permanent cell line of fish origin, with the advantages of tolerance to a wide temperature range (26–40 degrees Celsius). Therefore, this cell line has potential for use to attenuate, cultivate, and adapt other pathogens to cold temperatures in future studies.

Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

Essential features of in vitro fish cell culture: an overview

The reports of fish cell cultivation date back to the early 20th century, and since then, it has become a tool of great importance for different areas of ichthyology, such as immunology, toxicology, and biotechnology. The cell cultivation methodology can vary according to the species and cell type, requiring knowledge of the specificities and characteristics of the target cell for them to be efficiently cultured. In this review, we address the main characteristics of the in vitro culture of fish cells, presenting their purposes, uses, main parameters, and different characteristics necessary for the culture environment. Finally, we present the experiences in Brazil using cell culture techniques.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

New parameters for in vitro development of cell lines of the species Astyanax bimaculatus (Linnaeus, 1758) and Geophagus proximus (Castelnau, 1855).

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.