The piscine SAF-1 cell line: genetic stability and labeling

Fish cell lines are increasingly important research tools. The SAF-1 cell line, fibroblast-like culture derived from the marine fish gilthead seabream (Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their properties and authentication are essential for deeper understanding of their performance and thus more precise experimental design and applicability. In this study we characterized the SAF-1 cell line in terms of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal RNA regions, and DNA content. For genetic labeling 12 microsatellite loci were used. The results indicate that telomerase has been activated in the course of SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell proliferation, thus supporting a permanent cell line. This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA content values, and the genotypic profile allows SAF-1 authentication at the single individual level. This study increases the value of SAF-1 as an in vitro system, which is now one of the few well-characterized cell lines from a marine fish.     

Characterisation of the cell line HC-AFW1 derived from a pediatric hepatocellular carcinoma

Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 μg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.     

Establishment and characterization of JHUCS-1 cell line derived from carcinosarcoma of the human uterus

The cell line designed JHUCS-1 was established from a carcinosarcoma (malignant mixed mesodermal tumor) of the uterus that was surgically removed from a 57-year-old Japanese woman. We carefully examined the histopathology of the original tumor after the cell line was established and noted differentiation into a neuroendocrine carcinoma within the tumor's epithelial components. Immunohistochemical staining of the tumorous tissue that had been heterotransplanted was positive for Leu7. Additionally, secretary granules were observed in the grafted cells as determined by electron microscopy. These results support the existence of neuroendocrine cells within the JHUCS-1 cell line.     

New cell line derived from a human chondrosarcoma

Summary The morphological, chromosomal, and biological characteristics of a cell line derived from a human chondrosarcoma have been described. At the time of this report, the cell line has undergone 102 passages and continues to exhibit an epithelioid appearance, a modal number of 69 chromosomes, and has retained malignant properties. This cell line is easily cultivated in vitro or in vivo and may prove useful in human cancer research.     

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's ?15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell‐line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti‐cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5–20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell‐line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 μg of pAcGFP1‐N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.     

Cloning of the fish cell line SSN-1 for piscine nodaviruses

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and *BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degrees C for strain SGWak97 (RGNNV), 20 to 25 degrees C for strain SJNag93 (SJNNV), 20 degrees C for strain TPKag93 (TPNNV), and 15 to 20 degrees C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line.     

Biological characterization of a cell line derived from the pig oviduct

Summary A cell line has been derived from the inner lining of the pig oviduct. During 33 months in continous culture, the cells have been subcutured 145 times. Early passage cells had an epithelial-like morphology with clear and abundant cytoplasm. A spontaneous morphological alteration occurred at the 50th subculture by emergence of fibroblast-like cells. Evolution towards establishment of a cell line was suggested by the spontaneous morphological alteration, increase of the maximum population density without a significant change in the diploid state (92nd subculture), increase, in size and number of nucleoli, and increase in the nuclear-cytoplasmic ratio and of cytoplasmic basophilia. Evidence of pig origins is subtantiated by the karyotype (38, XX), the persistence of Barr bodies indicating female origin (140th subculture) and the persistence of species specificity by fluorescence (141th subculture). Preliminary studies indicate that nine viruses will replicate in both epithelial-like and fibroblast-like cells.     

A rat cell line derived from DMBA-induced mammary carcinoma

A new cell line, designated UHKBR-01, was successfully established from a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumour. DMBA was administered orally at a dose of 4 mg/ml per rat on the first day of the experiment and thereafter at weekly intervals of same dosage, until the rats have reached a weight of around 150-200 g. The tumours grew rapidly after the injection, and were transplanted into nude mice one the harvest size (2.5 x 2 x 1 mm(3)) was reached, it was transplanted onto nude mice. We have developed a cell line from a portion of the DMBA-induced carcinoma of the nude mice. The UHKBR-01 cell exhibited a slow increase in growth rate during the time of culture and was highly tumourigenic in nude mice. The cells have been grown in culture for over 40 passages. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, tumour antigen expression and xenograft implantation into nude mice. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. The above analyses also demonstrated that UHKBR-01 cells were oestrogen- and progesterone-receptor positive, in likeness to other established breast cancer cell lines such as MDA-MB-231 and MCF-7. The cell line grows as monolayers of oval-shaped cells with large folded nuclei accompanied by a rich supply of mitochondria. This report describes the first in vitro cell line from transplantable DMBA-induced mammary carcinoma of nude mice, which presents unique characteristics that may prove to be a good experimental model for investigating breast cancer biology.     

Establishment of a new fish cell line from the caudal fin of golden pompano Trachinotus ovatus and its susceptibility to iridovirus

A new cell line derived from the caudal fin of golden pompano Trachinotus ovatus (TOCF) was successfully established and characterized. TOCF cells grew well at 28° C in L‐15 medium supplemented with 10% foetal bovine serum (FBS). The cell line has been subcultured in more than 100 passages. Molecular characterization of 18S ribosomal (r)RNA and cytochrome oxidase subunit 1 (COI) confirmed that the TOCF cells were derived indeed from T. ovatus. TOCF cells have a modal chromosome number of 54. It was further showed that TOCF cells were transfected successfully with pEGFP‐N3 and pDsRED‐N1 plasmid, suggesting that TOCF cells could be used to research gene functions in vitro. Viral susceptibility tests showed that TOCF cells were susceptible to Singapore grouper iridovirus (SGIV), observed by the occurrence of the cytopathic effect (CPE) with the formation of inclusion bodies. In addition, the expression of major capsid protein (MCP) gene of SGIV changed during virus infection in TOCF cells. Thus, our present results described the characteristic of a TOCF cell line that could be a valuable tool for genetic manipulation, as well as isolation and propagation of iridovirus studies.     

RAPD-PCR analysis for molecular characterization and genotoxic studies of a new marine fish cell line derived from Dicentrarchus labrax

Continuous cell lines could provide an important tool for studying epidemiology, toxicology, cellular physiology and the host–pathogen interactions. Random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) was used for the molecular characterization of Dicentrarchus labrax embryonic cells (DLEC) as a possible tool to detect DNA alterations in environmental genotoxic studies. We studied the DNA pattern of the DLEC fish cell line, a fibroblast-like cell line derived from European sea bass. From a total of 15 primers only six showed good discriminatory power for the amplification process on DNA samples collected from cells by three different methods (organic extraction, salting-out method and chelating agent extraction). The results obtained show that the cell line chosen for this study could be used as a possible tool for the detection of potential genotoxicity of numerous chemical compounds.     

Pluripotency of bovine embryonic cell line derived from precompacting embryos

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.     

Growth characteristics of a cell line derived from the pig oviduct.

Further evidence for the establishment of the pig fallopian tube (PFT) cell line as a continuous cell line was shown by an increase in the maximum population density as the number of subcultures increased. The optimal pH and temperature-growth ranges appeared to be 7.4-7.8 and 37-41 degrees C respectively, and the population doubling time was 20-25 h under optimal growth conditions. With progressive subculture, the serum requirements dropped from 20 to 2%. A plating efficiency of 2 to 4% was found in all serial subcultures. Colonies were observed in agar suspension culture at the 146th subculture and thereafter. Chromosomal alterations were found in the 100th subculture and thereafter.