Development and characterization of a new epithelial cell line PSF from caudal fin of Green chromide, <Emphasis Type="Italic">Etroplus suratensis</Emphasis> (Bloch, 1790)

A new cell line [pearlspot fin (PSF)] has been developed from caudal fin of Etroplus suratensis, a brackish/freshwater fish cultivated in India. The cell line was maintained in Leibovitz’s L-15 supplemented with 10% fetal bovine serum (FBS). The PSF cell line consisted predominantly of epithelial-like cells. The cells were able to grow at temperatures between 25°C and 32°C with optimum temperature of 28°C. The growth rate of PSF cells increased as the FBS proportion increased from 2% to 20% at 28°C with optimum growth at the concentration of 10% FBS. One marine fish virus (fish nodavirus) was tested on this cell line and found not susceptible. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the third d after subculture. Polymerase chain reaction amplification of mitochondrial 16S rRNA and COI indicated identity of this cell line with those reported from this fish species, confirming that the cell line was of pearlspot origin. The cells were successfully cryopreserved and revived at the tenth, 25th, and 35th passages. The bacterial extracellular products from Vibrio cholerae MTCC 3904 were found to be toxic to PSF. Karyotyping analysis indicated that the modal chromosome number was 48.     

A new epithelial-like cell line from eye muscle of catla Catla catla (Hamilton): development and characterization

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major carp ( Catla catla ), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% foetal bovine serum (FBS). These cells have been subcultured >80 times over a period of 1·5 years. This cell line has been designated SICE. The SICE cell line consists predominantly of epithelial-like cells. These cells are strongly positive for epithelial markers such as pancytokeratin and cytokeratin 19. The cells were able to grow at temperatures between 25 and 32° C with optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or 20% FBS. Six marine fish viruses (fish nervous necrosis virus, marine birnavirus-NC1, chum salmon virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus-Sp and hirame rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the fourth day after subculture. Polymerase chain reaction amplification of mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence-activated cell sorter revealed that most of the cells on the second day of culture were in S-phase, indicating a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the SICE cell line can be a useful tool for transgenic and genetic manipulation studies.     

Development and characterization of a new cell line CF from caudal fin of knifefish, Chitala chitala (Hamilton-Buchanan)

A new cell line was successfully obtained from caudal fin tissue of the economically important freshwater fish Chitala chitala. The cell line was optimally maintained at 28°C in Leibovitz’s L-15 supplemented with 20% fetal bovine serum (FBS). The effects of temperature and concentration of FBS on the growth of CF cells were examined. The CF cell line consisted predominantly of fibroblastic-like cells. Moderately low plating efficiencies 8%, 11%, and 17% were observed, with CF cell line in L-15 Medium with 20% FBS. Chromosomal analysis of the cell line revealed a diploid number of 42 chromosomes in C. chitala. Molecular characterization of mitochondrial 16S rRNA and cytochrome oxidase subunit I confirmed the origin of the cell line. The cells were successfully cryopreserved in liquid nitrogen (?196°C) for 6 mo, and more than 85–90% of CF cells were revived.     

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.     

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r ²?=?0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.     

Development of a continuous cell line, PBLE, from an American eel peripheral blood leukocyte preparation

Summary A continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitz's L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18° C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36° C, and proliferated at temperatures from 5° C to at least 30° C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.     

Establishment of a cell line with high transfection efficiency from zebrafish Danio rerio embryos and its susceptibility to fish viruses

A cell line ZBE3 isolated from a continuous cell culture derived from zebrafish Danio rerio blastomeres by clonal growth was characterized. ZBE3 cells had been subcultured for >120 passages since the initial primary culture of the blastomeres. The ZBE3 cells grow stably at temperature from 20 to 32° C with an optimum temperature of 28° C in ESM2 or ESM4 medium with 15% foetal bovine serum (FBS). The optimum FBS concentration for ZBE3 cell growth ranged from 15 to 20%. Cytogenetical analysis indicated that the modal chromosome number of ZBE3 cells was 50, the same as the diploid chromosome number of D. rerio. Significant cytopathic effect was observed in ZBE3 cells after infection with redspotted grouper nervous necrosis virus, Singapore grouper iridovirus and grass carp reovirus, and the viral replication in the cells was confirmed by real‐time quantitative PCR and transmission electron microscopy, indicating the susceptibility of ZBE3 cells to the three fish viruses. After transfected with pEGFP‐N3 plasmid, ZBE3 cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein. The stable growth, susceptibility to fish viruses as well as high transfection efficiency make ZBE3 cells be a useful tool in transgenic manipulation, fish virus‐host cell interaction and immune response in fish.     

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.)

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28°C in Leibovitz‐15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Establishment and characterization of a brain‐cell line from kelp grouper Epinephelus moara

A new brain‐cell line, EMB, was developed from kelp grouper Epinephelus moara, a cultured marine fish. The EMB cells were subcultured for more than 60 passages. The cells were cultured in Leibovitz's L‐15 medium (L15) supplemented with antibiotics, foetal bovine serum (FBS), 2‐mercaptoethanol (2‐ME) and basic fibroblast growth factor (bFGF). The cells could grow at 18–30° C, with the 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. After being transfected with pEGFP‐N3 plasmid, the cells could successfully express green fluorescence protein (GFP), implying that this cell line can be used for transgenic studies. A significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV) and the viral replication was confirmed by quantitative real‐time PCR (qrt‐PCR) assay, which suggested EMB's application potential for studies of SGIV and RGNNV.     

Unidentified cells reside in fish skeletal muscle

Cell cultures were established from the skeletal muscle tissue of 6–13 months old rainbow trout and 12–14 months old yellow perch. Approximately 27,000 ± 5,000 cells/g (trout; N = 5) and 5,000 ± 1,200 cells/g of tissue (perch; N = 4) were obtained. Isolation and propagation were qualitatively greater for both species when the cells (younger fish producer more cells than older fish) were exposed to DMEM + 15% FBS, rather than L-15 + 15% FBS, at 20 °C (trout) and at 24 °C (yellow perch). Two morphologically distinct cell types were observed in cultures of both species, some of which eventually formed very small myotubes, which displayed immunocytological reactivity for myogenin, myosin heavy chain, and α-actinin; the second population of cells remained unstained. Successful cryopreservation was achieved using a 5% DMSO and 95% serum mixture, but post-thawing viabilities were low 5–27% (trout) and 14–30% (perch). Further research is needed in order to determine cell type specificity of isolated cells.     

Maintaining mosquito cell lines at high tempetatures: Effects on the replication of flaviviruses

Summary The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32°C to 34°C,Anopheles stephensi cell line could be maintained at 37°C. At higher temperatures initial growth rate was higher, but yield of cells after about a week of incubation was lower than at the standard temperature (28°C). Replication of several flaviviruses inAedes albopictus cell cultures adapted to 34.5°C was faster, and viral titers were higher than at 28°C.     

A new epithelial cell line,HBF from caudal fin of endangered yellow catfish,Horabagrus brachysoma (Gunther, 1864)

A new epithelial cell line, Horabagrus brachysoma fin (HBF), was established from the caudal fin tissue of yellow catfish, H. brachysoma and characterized. This HBF cell line was maintained in Leibovitz’s-15 medium supplemented with 15 % fetal bovine serum (FBS) and subcultured more than 62 times over a period of 20 months. The HBF cell line consists predominantly of epithelial cells and is able to grow at temperatures between 20 and 35 °C with an optimum temperature of 28 °C. The growth rate of these cells increased as the proportion of FBS increased from 5 to 20 % at 28 °C with optimum growth at the concentrations of 15 % FBS. Partial amplification and sequencing of fragments of two mitochondrial genes 16S rRNA and COI confirmed that HBF cell line originated from yellow catfish. The HBF cells showed strong positive reaction to the cytokeratin marker, indicating that it was epithelial in nature. HBF cell line was inoculated with tissue homogenate from juveniles of Sea bass, Lates calcarifer infected with viral nervous necrosis virus (VNNV) and found not susceptible to VNNV. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the HBF cells. These cells were confirmed for the absence of Mycoplasma sp by PCR.     

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32°C for good growth of the cells. The growth rate of cells was higher in medium containing 10% FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72%, respectively, after six months of storage in liquid nitrogen (-196° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.     

Fish cell culture: Establishment of two fibroblast-like cell lines (OL-17 and OL-32) from fins of the medaka,Oryzias latipes

Summary Two fibroblast-like cell lines were obtained from fins of adults of the medaka,Oryzias latipes, and serially cultured at 27° C. One cell line, which was derived from a fish of the orange-red variety, reached to population doubling level (PDL) 434 on Day 840 by 120 passages. The other, which was derived from a fish of the inbred strain HB32C, reached to PDL 294 on Day 551 by 80 passages. Any symptom of crisis was not detected. The cell lines were named OL-17 and OL-32, respectively. Population doubling time was 29 h for OL-17 cells, and 32 h for OL-32 cells. Density-dependent inhibition of growth was clear in OL-32 cells, but not so obvious in OL-17 cells. Modal chromosome number of OL-17 cells was 50, and that of OL-32 cells was 47 (2N=48). Plating efficiency of OL-17 cells was about 10%, whereas that of OL-32 cells was about 5%. The use of conditioned medium (80% concentration) increased the plating efficiency of OL-32 cells to more than 25%.     

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26°C to 28°°C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.     

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland)

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Activation of an Aquareovirus,Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal‐lysosomal system and released in a more infectious form. When allowed to swim in CSV‐infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.     

Production of ginkgolides and bilobalide from optimized the<Emphasis Type="Italic">Ginkgo biloba</Emphasis> cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.