Name: Anord Charles Nkuba Nim; 141924153005 Introduction Cell lines provide an important biological method for physiological, virological, toxicological, carcinogenic and transgenic studies. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain and skin. This review summarizes the cell line from the brains of various teleost fishes. fish cell lines are becoming more and more important as a research tool in the aquaculture field, and they are suitable models to study fish physiology, immunology, virology, and toxicology among other aspects. In fact, the use of cell cultures instead of laboratory animals has become relevant to contribute to the 3Rs. principle of reduction, refinement and replacement. The physiology and blood plasma components of teleost fish are very similar to those of terrestrial vertebrates, so the cell culture approach is also similar. Nevertheless, the culture of fish cells varies somewhat from the culture of mammalian cells by providing a larger incubation temperature spectrum. For long periods of time, fish cells may be stored with little treatment. In comparison to mammalian cells, permanent fish cell lines are thus easier to sustain and manipulate and yield highly reproducible outcomes. Usually, formed cell lines are derived from malignant tumors or by random mutation or oncogenic immortalization, and these modifications allow continuous (immortal) cell lines to proliferate. There are several challenges in making a fish cell lines clonogenic. clonogenic assays using fish clonogenic cell lines are currently very time-consuming and labor-intensive because they can take up to 8 weeks with periodic fresh medium changes in order to complete one experiment. This is because cells from poikilothermic species like teleost have a much slower proliferation turnover rate than mammalian cells. As a result, many areas of veterinary medicine and fish research, especially pertained to environmental radioecology, are hindered or often heavily rely on data collected from a single particular clonogenic line derived from one fish species. Many fish cell lines have been established from fish tissues for the purpose of detection and isolation of fish viruses. For the analysis of species-specific responses to viral infection at the cellular level, cell lines from various tissues of different species would be useful. Some pathogenic viruses are considered to be organ or tissue-specific, making it necessary for the proper control of viral diseases to gather additional cell lines from various organs and tissues of a host species. While the origin of the tissue is diverse, most lineages of fish cells are formed from fins and embryos. There are very few cell lines originating from the nervous tissues despite the fact that the fish brain shows very high rates of neurogenesis and regeneration after injury. In this report we have reviewed the research work carried out since 2016 to 2020 and more that 15 new cell lines were reported for brain cell lines and characterization from different species of fish. 1 State of the Art Organisms Gilthead seabream (Sparus aurata) mummichog (Fundulus heteroclitus) Culture method/ cell culture brain-derived cell line (SaB1) Parameters Results References Cell characterization. SaB-1 cell growth curve. Susceptibility to fish virus. Antiviral immunity (RuizPalacios, Esteban, et al., 2020) brain cell line (FuB-1) FuB-1 cells characterization. Growth curve. Susceptibility to fish viruses and immune response. Toxicological effects of Nano-plastics and/or pharmaceutical agents they express both neural and glial cell markers, suggesting they are neural-stem cells, have a neuron-like morphology and show a rapid growth in culture. They were susceptible by (NNV), (SVCV), (IPNV) and (VHSV). SaB-1 cell line is continuous stable and could be useful, at least, for fish virology and immunity applications. FuB-1 cells show epithelial morphology and neural stem/astroglial origin. FuB-1 cells effectively supports the replication of both spring viremia carp (SVCV) and infectious pancreatic necrosis (IPNV)indicating its potential use for fish virology. polystyrene Nano plastics (PS-100) particles increase the antioxidant catalase and glutathione S-transferase activities and decrease the total non-protein thiols in FuB-1 cells. (RuizPalacios, Almeida, et al., 2020) Apteronotus leptorhynchus telencephalon, corpus cerebelli, and valvula cerebelli of growth factors on neurosphere development. cell density on neurosphere development. coating substrate on cellular differentiation. optimal fetal bovine serum concentration Immunocytochemistry Self-renewal potential ofneurospheres. Microscopic examination and image processing neurospheres developed that grew through cell proliferation and reached diameters of up to 140µm within 3 weeks. An increase in the number of developing neurospheres could be promoted by addition of epidermal growth factor or basic fibroblast growth factor. The cells isolated from the adult teleostean brain exhibited the ability for self-renewal, therefore it was hypothesized that they are true stem cells. (Hinsch & Zupanc, 2006) sea perch, Lateolabrax japonicus continuous cell line, LJB. subcultured for more than 60 times in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS) Growth curves. Cytogenetic analysis Transfection (Le et al., 2017) Epinephelus moara brain-cell line, EMB, more Effect of temperature and FBS LJB cells exhibited maximum growth rate at 28°C in DMEM supplemented with 20% FBS. Cytogenetic analysis indicated that the modal chromosome number was 48. Comparison of the 18S ribosomal RNA gene sequences of LJB cells and sea perch confirmed that LJB cells originated from sea perch. LJB cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein, indicating the potential application of LJB cells in gene expression studies. The LJB cell line might be used as an ideal in vitro tool for analyzing and understanding the mechanisms of nervous necrosis virus-host interaction. The cells could grow at 18–30∘ C, with the (Liu et al., 2018) than 60 passages. concentration on cell growth. Chromosome analysis. Cell transfection with pEGFP-N3 Virus challenge assay Chinese perch Siniperca chuatsi a continuous brain cell line (CPB) subculture >140 times 18s rRNA gene sequence analysis. Chromosome analysis. Cell growth characteristics. Tranfection ISKNV infection and viral replication efficiency. Virus challenge experiments koi (Cyprinus carpio L.) KB cell line Subcultured >100 times Effects of culture conditions on cell growth. Chromosome analysis. Cell transfection with GFP reporter gene. maximum growth between 24 and 30∘ C. The optimum FBS concentration for the cells growth ranged between 15 and 20%. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 45. Transfection with pEGFP-N3 plasmid shows the cells could successfully express green fluorescence protein (GFP). The cells were affected by Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) this suggested that these cells are potentials for viral studies CPB cell line predominantly are fibroblast-like cells that could grow better in Leibovitz’s L-15 supplemented with 10% foetal bovine serum at 28∘ C. The cryopreserved at different passage levels and revived successfully with 80–90% survival. CPB cells were highly susceptible to infectious spleen and kidney necrosis virus (ISKNV). CPB could be used as an in vitro tool for propagation of ISKNV and gene expression studies The KB cell line was optimally maintained at 27°Cin Leibovitz’s L-15 medium supplemented with 10% foetal bovine serum (FBS) KB cells at passage 80 maintained the abnormal (Fu et al., 2015) (Y. Wang et al., 2018) Viral susceptibility and confirmation. tilapia A cell line from the brain of tilapia, TiB Cryopreservation and thawing of cells. Effects of culture conditions on cell growth. Chromosome analysis. Viral susceptibility and confirmation. European sea bass (Dicentrarchus labrax) sea bass brain (DLB-1) cell line Characterization of the DLB-1 cell line. DLB-1 cell growth curve and doubling time. DLB-1 characterization by gene expression. Transfection with GFP reporter gene. Cytotoxicity assays DLB-1 diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The results of virus isolation demonstrated that KB cells were susceptible to KHV. The newly established KB cell line will serve as a useful tool to study KHV disease The TiB cell line was optimally maintained at 27°C using (M199) supplemented with 10% (FBS). Chromosome analysis revealed that 60% of TiB cells at passage 5 maintained the modal chromosome number 2n = 44. while at passage 60, there are 43% TiB cells with the diploid chromosome number 2n = 50. Significant cytopathic effect was observed onto TiB cells after infection with tilapia lake virus (TiLV‐2017A). The stable growth, susceptibility to fish viruses, makes TiB cells be a useful tool for fish virus–host cell interaction and for immune response of fish. There is alternation of cell cycle after exposure to metals. all the metals induce apoptosis as indicated by sub-Go/G1. exposure of DLB-1 cells to metals also produces significant alterations at gene expression level. (Yingying Wang et al., 2018) (Morcillo et al., 2017) European sea bass (Dicentrarchus labrax) European sea bass brain derived cell line (DLB-1) DLB-1 cells susceptibility to nodavirus. RNA-seq study (DLB-1) susceptibility to NNV genotypes and evaluated its transcriptomic profile. Differential expression analysis showed the upregulation of many genes related to immunity, heat-shock proteins or apoptosis but not to proteasome or autophagy processes. It also shows that the immune response, mainly the interferon (IFN) pathway, is not powerful enough to abrogate the infection, and cells finally suffer stress and die by apoptosis liberating infective particles. This study opens the way to understand key elements in sea bass brain and NNV interactions (ChavesPozo et al., 2019) tilapia (Oreochromis sp.) The effect of cold stress on tilapia genes. An immortal neural cell line designated as TBN was established from brain tissue of the GIFT Chromosome analysis RNA extraction, sequencing library construction and RNA sequencing RNA-seq data analysis. Effects of FBS concentration and temperature on cell growth. Cold sensitivity of TBN cells. Transfection The TBN cells demonstrate a neuronlike morphology at low density and form a fibroblast-like monolayer at high density. The TBN cells tolerate relatively high culture temperatures and the highest growth rate was observed for the cells cultured at 32 oC in comparison with those at 30 oC, 28 oC and 26 oC. However, this cell line is cold sensitive. Exposure of the cells to 16 oC or lower temperatures significantly decreased cell confluences and induced apoptosis. TBN cells can be efficiently transfected through electroporation. This study gives Understanding of the nature of cold sensitivity of tilapia, and to dissect (Long et al., 2020) the function and mechanism of genes in regulating cold tolerance of fish. Aequidens rivulatus (G€unther) a continuous cell line ARB8 (brown- marbled grouper Epinephelus fuscoguttatus continuous cell line designated BMGB susceptibility to fish viruses—including chum salmon reovirus (CSV), marbled eel infectious pancreative necrosis virus (MEIPNV), grouper nervous necrosis virus (GNNV), giant seaperch iri- dovirus (GSIV), red seabream iridovirus (RSIV), koi herpesvirus (KHV), herpesvirus anguilla (HVA) and marbled eel polyoma-like virus (MEPyV)—were examined. ARB8 Immunocytochemistry Detection of dead cells and viral infectivity. Viral susceptibility. RNA extraction, cDNA synthesis and PCR Uninfected. ARB8 were passaged >80 (Yeh et al., times grew well at 2018) temperatures ranging from 25°Cto 30°C in L15 medium containing 5%–15% FBS. The cells were highly susceptible to CSV, MEIPNV, GSIV and RSIV and showed the typical cytopathic effect (CPE). However, the cells were resistant to GNNV, KHV, HVA and MEPyV because no significant CPE was noted after infection. MGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic pro- tein and keratin and were persistently infected by betanodavirus, as confirmed through immunocyto- chemistry, polymerase chain reaction and immunoblot analyses. BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocy- tivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. The BMGB cells will be useful for investigating (Wen, 2016) virus and host cell interaction. American eel Anguilla rostrata continuous cell line brain endothelial cell line (eelB) goldfish and silver crucian carp two brain cell lines from goldfish and silver crucian carp subculture over 40 times. The effect of selenium deprivation and addition on the American eel brain endothelial cell line (eelB) was studied in three exposure media: complete growth medium (L15/ FBS), serum-free medium (L15), and minimal medium (L15/ex). L15/ex contains only galactose and pyruvate and allowed the deprivation of selenium on cells to be studied. In L15/ex, without any obvious source ofselenium, eelB cells survived for at least 7 d Adding selenite or selenate to eelB cell cultures for 24 h caused dose-dependent declines in cell viability selenite was approximately 70-fold more cytotoxic than selenate. Overall, the responses of eel cells to selenium depended on the selenium form, concentration, and exposure media, with responses to SeMet being most dependent on exposure media. Used to study viral The cells grew over the susceptibility to range of15 to 30°C, while Cyprinid herpesivirus- the optimal temperature 2. for culture was 30°C. Growth studies Following Cryopreservation of cryopreservation in liquid cells nitrogen, thawed cells Viral susceptibility. exhibit viability of> 90% after a 13-mo period of storage. The chromosome count of two cell lines were determined to be 154 and 110, respectively, which agreed well with triploid crucian carp brain cells and diploid goldfish brain cells. Polymerase chain reaction amplification and sequence analysis indicated 100 % and 94% match with known crucian carp (Bloch et al., 2017) (Xu et al., 2019) mitochondrial DNA sequences. These newly established cell lines could be a diagnostic tool for viral diseases in fish species. Conclusion Cell cultures, in particular those originating from fish, have been effectively utilized as a biological alternative to the use of whole animals. The increasing usage and value of fish cell lines indicate that cell culturists should be encouraged to position these lines with the international cell repositories like ATCC, EACC or other suitable repository in order to provide a dependable, high-quality supply of cells for the benefit of all Fish brain cell are potential for the study of various relationship with other pathogenic organisms such as viruses, bacteria and fungi, also brain cells shows the response to the environmental toxicology which marks it as a potential tissue to study various mechanism within the cells. Reference Bloch, S. R., Kim, J. J., Pham, P. H., Hodson, P. V., Lee, L. E. J., & Bols, N. C. (2017). Responses of an American eel brain endothelial-like cell line to selenium deprivation and to selenite, selenate, and selenomethionine additions in different exposure media. 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