A new continuous cell line from larval hemocytes of Spodoptera litura (F.).

A new cell line has been established from larval hemocytes of the moth, S. litura (tobacco cut worm). It took 147 days to form a monolayer and one year for the first 17 passages. At present, the culture is at 86th passage level and is designated NIV-SU-1095. Three cell types could be distinguished, viz. plasmatocytes (53%), prohemocytes (36%) and granular hemocytes (11%). The chromosome number was very high, 74% metaphase cells showed more than 100 chromosomes. The cells could be cryopreserved. The cells were susceptible to the baculoviruses, Autographa californica nuclear polyhedrosis virus and S. litura nuclear polyhedrosis virus (SLNPV). Plaques could be observed on 7th post infection day with SLNPV. Six cloned cell lines have been developed of which clone II-1F was more sensitive to both the baculoviruses compared to the original cell line.     

Fish cell culture: A new cell line fromCynoscion nebulosus

Summary A new fibroblast-like cell line, CyN, has been developed from muscle tissue of the marine fishCynoscion nebulosus (the spotted weakfish). A subline, CyN-1, was characterized fully and carried through 100 subcultures. CyN-1 is heteroploid and has a modal chromosome number of 49. The line is susceptible to LT-1, lymphocystis, eastern equine encephalitis and vesicular stomatitis viruses. Optimal growth occurred at 25° to 34° C in growth medium adjusted to 0.150m NaCl. This study was supported in part by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58.     

Characterization of a new continuous cell line from the flood water mosquito,Aedes vexans

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (=2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.     

A new cell line derived from nickel sulfide-induced rat rhabdomyosarcoma

Summary The morphological and chromosome characteristics of a cell line derived from a nickel sulfide-induced rhabdomyosarcoma in the Fisher strain of rats have been described. At the time of this report the cell line has undergone 59 passages and continues to exhibit the heteroploid trait and has retained the malignant properties. This cell line, which lends itself to in vitro and in vivo (in rat) cultivation and carries easily recognizable chromosome markers, may prove to be useful in further research on tumor cells. This investigation was supported in part by a grant in aid of research from the National Cancer Institute of Canada. Department of Biomedical Sciences.     

Karyological characteristics of a new continuous cell line of human hypernephroma

A new stable human hypernephroma cell line has been characterized by differential staining R-, C- and Ag-I-techniques. The karyotype of the cultivated hypernephroma cells has been designed using the statistical analysis and karyotype reconstruction methods. The specific chromosomal markers of the cell line are described.     

Turtle urinary bladder epithelial cell line

1. We compared enzymatic and functional properties of a turtle bladder cell line to those of turtle bladder epithelial cells. Like the original epithelium, the cell line displays carbonic anhydrase activity and acetazolamide inhibits O2 consumption in isolated cells and acidification by cells grown in monolayers. 2. Staining with acridine orange revealed the presence of cytoplasmic orange red vesicles which could be dissipated by NH4Cl or protonophores indicating that these vesicles represent areas of low pH. 3. Addition of ATP to cells permeabilized by digitonin led to reappearance of the red granules suggesting that acidification of the vesicles is mediated by H+-ATPase.     

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.     

Establishment and characterization of a new squamous cell carcinoma cell line, TMUU-08, derived from human bladder cancer

Human squamous cell carcinoma (SCC) of the bladder is a rare malignancy that represents less than 5% of bladder tumors. In contrast to non-bilharzial SCC, bilharzial SCC is a distinct pathological disease that is rarely encountered in Japan. The majority of patients with non-bilharzial SCC present with a poorly differentiated, muscle-invasive tumor with no previous episode of urothelial carcinoma (UC). Even in the absence of distant metastases, the prognosis of patients with non-bilharzial SCC of the bladder remains dismal because patients die of localized recurrence. This is in contrast to UC in which distant metastasis accounts for the great majority of recurrence. The 5-year survival rate of the patients treated for non-bilharzial SCC of the bladder was only about 10%. To date, large numbers of reports have examined the establishment of a human bladder cancer cell line with UC. However, few reports exist regarding the establishment of the human bladder cancer cell line with SCC. In the present study, we established a new cell line (TMUU-08) from the metastatic lymph node of a patient with SCC of the bladder. The TMUU-08 cell line of human bladder SCC was characterized. These results indicate that TMUU-08 cells might be useful in basic studies not only in the treatment but also etiology of human bladder SCC.     

OMEC II: A new ovine mammary epithelial cell line

We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow rapidly on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO-1, a tight-junction associated protein, but negative immunostaining for an anti-vimentin antibody. In confluent cell monolayers ‘domes’ became visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end-buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of β-lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line.     

A new myxozoan from feral goldfish (Carassius auratus)

In February 2004, a mass die-off of common goldfish Carassius auratus L., presumptively caused by bacterial coldwater disease (Flavobacterium psychrophilum), occurred at Fern Ridge Reservoir, Oregon. A range of size classes was affected, but all mature fish were female and all fish were infected with a single myxozoan, Chloromyxum auratum n. sp. No histological changes were observed associated with the parasite. Infection was represented by mictosporic plasmodia and free-floating spores in the gall bladder. Parasite spores were nearly spherical, 13.6 microm long x 12.6 microm wide x 13.1 microm thick, and possessed 4 equal-sized polar capsules. Spores had a coglike appearance in apical view because of distinct ridges 2.1 microm high protruding from the valve cells. There were 6-9 extrasutural ridges per valve (15-20 ridges per spore), aligned along the longitudinal axis, with some branching, and convergence at both poles. Morphologically, spores identified most closely with Chloromyxum cristatum Léger, 1906; however, 18S rDNA sequence data indicated only 97.5% similarity over 2,076 bp with Chloromyxum cyprini, the only synonym of C. cristatum for which DNA data are available; additional sequence data may reveal the other synonyms to be distinct species. This is the first record of a species of Chloromyxum from goldfish.     

A continuous alveolar macrophage cell line: Comparisons with freshly derived alveolar macrophages

Summary Responses of a recently developed rat alveolar macrophage cell (NR8383.1) line were compared to those of freshly derived alveolar macrophages in vitro. Marked inter- and intraspecies heterogeneity in levels of phagocytosis of unopsonizedPseudomonas aeruginosa or zymosan was noted among freshly derived alveolar macrophages from rats, rabbits, and baboons. In contrast, phagocytic responses of alveolar macrophage cell line were predictable and highly reproducible. Similar results were obtained in measuring oxidative burst, as indicated by the production of H₂O₂ and luminol-enhanced chemiluminescence. Responses were again highly variable in freshly derived alveolar macrophages stimulated with zymosan or phorbol myristic acetate; moreover, freshly derived alveolar macrophages exhibited a wide range of chemiluminescence activity in unstimulated cultures. Results strongly suggest that data derived from the continuous alveolar macrophage culture NR8383.1 can be extrapolated to freshly derived alveolar macrophages of various species, and in many experiments will be useful in avoiding the significant animal-to-animal variance observed among freshly derived cell preparations. This work was supported in part by grant A119811 and SCOR HL23578, from the National Institutes of Health, Bethesda, MD. Portions of these studies appeared as a poster presentation at the American Society for Microbiology, Atlanta, GA, 1987.     

Development of an attached strain from a continuous insect cell line

Summary A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm,Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line. Contribution No.: 329.     

Purification and characterization of interferons from a continuous myeloblastic cell line

Several leukocyte interferon species have been purified from a continuous human myeloblast cell line. The purification procedure involving selective precipitations, gel chromatography, and several steps of high performance liquid chromatography results in interferons with specific activities of 1 to 4 X 10(8) units/mg on bovine MDBK cells. The total yield of interferon is 23%, with the yield of the individual fractions ranging from 0.2 to 11.4%. Five fractions are homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weights of the interferons were estimated by mobility on the sodium dodecyl sulfate gels and range from 17,600 to 26,200. The species differ in their relative antiviral activities on two cell lines, bovine MDBK and human AG-1732. In addition, the pure species have similar, but distinct, amino acid compositions and tryptic peptide profiles. These result support the conclusion that leukocyte interferon consists of several homologous proteins.     

A new principle of continuous flow cell cultivation

Summary A completely liquid-filled culture chamber with gas exchange across a synthetic membrane (Larsen andNilsson 1985) was incorporated into an automatic continuous flow system. The absence of an airliquid interface in the system permits removal of cell samples, and addition of fresh medium, under strictly sterile conditions. In this system,Tetrahymena pyriformis can be kept under optimal growth conditions in a rich nutrient medium and any defined cell density may be maintained for extended periods of time by varying the dilution rate of the culture. Furthermore, it has been possible to demonstrate, in the slope of the growth curve, even small changes which are difficult to detect in batch cultures since the duration of these changes is short. In the continuous flow system, the relative cell volume distribution and the food vacuole forming capacity of the cells were unaltered; however, all cells contained small refractive granules. The system permits the culture volume to be varied, but a standard volume of 20 ml was maintained in most experiments. Since the culture volume is small, the system requires less than one liter of fresh medium per week to maintain the cells in the exponentially multiplying growth phase.     

Isolation of melanized cell lines with stable phenotypes from a goldfish erythrophoroma cell line and cryopreservation of these cells by the use of autologous serum

Summary Cell lines with stable melanized phenotypes were isolated from a goldfish erythrophoroma cell line. These cell lines include several interesting phenotypes: a) reversible dedifferentiation and redifferentiation in response to withdrawal and addition of fish serum; b) irreversible dedifferentiation in response to withdrawal of fish serum; c) independence on fish serum for melanization; d) dependence on fish serum for growth; and e) pigment aggregation in response to epinephrine. We also report that cryopreservation of all the melanized cell lines, but not any of the unmelanized cell lines, requires the presence of fish serum. This raises the possibility that there may be advantages to use autologous serum for the cryopreservation of sensitive cell lines. This research was supported by grant DK 13724 from the National Institutes of Health, Bethesda, MD     

Isolation of a transforming sequence from a human bladder carcinoma cell line

We have isolated the component of human bladder carcinoma cell DNA that is able to transform mouse fibroblasts. The oncogenic sequence was isolated initially from a lambda phage genomic library made from DNA of a transfected mouse cell carrying the human oncogene. A subcloned insert of 6.6 kb that carried transforming activity was amplified in the plasmid vector pBR322. The subcloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has failed so far to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human tumor cell lines derived from colonic and lung carcinoma and from neuroblastoma. In contrast, the EJ bladder oncogene appears closely related to one that is active in the human T24 bladder carcinoma cell line. The oncogene appears to have undergone little, if any, amplification in several bladder carcinoma cell lines.     

A continuous cell line of a nonhematophagous mosquito,Toxorhynchites amboinensis

Summary A continuous cell line of a nonhematophagous mosquito was obtained by using partially incapacitated but living larvae as the source The cells were predominantly epithelioid and diploid (2N=6). Syncytia were occasionally observed in the normal cell cultures.