CHANGES IN CELL SURFACE MORPHOLOGY DURING DIFFERENTIATION OF MICROMERE- AND MESOMERE-DERIVED CELLS OF THE SEA URCHIN EMBRYO

Micromeres and mesomeres isolated from 16-cell embryos of the sea urchin, Strongylocentrotus intermedius , were cultured in vitro , and changes in the cells surface architecture during the differentiation of the micromere- and mesomere-derived cells were observed using scanning electron microscopy. Two types of the distribution of the surface microvilli were observed in both blastomere-derived cell masses. One type showed a uniform distribution of the microvilli and the other type showed an uneven one. Though many microvilli were observed in most of both mesomere and micromere-derived cells at the 64-cell stage and the early blastula stage (16 hr after the 16-cell stage at 6°C) respectively, the microvilli decreased in number at the later stages in both blastomere-derived cell masses as compared with the 64-cell stage and the early blastula stage respectively. Rapid disappearance of the surface microvilli was observed in the micromere-derived cells in contrast with the mesomere-derived cells which still had many microvilli even at the midmesenchyme stage.     

Establishment and characterization of a cell line from the head kidney of golden pompano Trachinotus ovatus and its application in toxicology and virus susceptibility

A cell line derived from the head kidney of golden pompano Trachinotus ovatus (TOHK) was established and characterized in this study. The TOHK cells grew most rapidly at 28° C and the optimum foetal bovine serum concentration in L‐15 medium was 10%. The TOHK cells have a diploid chromosome number of 2N = 54. The transfection efficiency of TOHK cells was 7·5% at the 15th passage and 72% at the 40th passage. The transfection efficiency in TOHK cells was high, so these cells are suitable for foreign gene expression. The cytotoxic effects of heavy metals and extracellular products from Vibrio anguillarum and Vibrio alginolyticus were demonstrated in TOHK cells, so this TOHK cell line could also be applied in environmental monitoring of heavy metals and pathogenic bacteria. TOHK cell line showed high virus susceptibility, such as grouper nervous necrosis virus (GNNV) and Singapore grouper iridovirus (SGIV). Then, TOHK cell line could be used for the study of viral pathogenesis and the development of antiviral strategies.     

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.     

Fish cell culture characteristics of a cell line from the silver perch Bairdiella chrysura.

A cell designated SP-1 was established from tissue of the silver perch, Bairdiella chrysura. Cells were fibroblast-like and grew best at 26 degrees C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150 M sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses.     

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.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

Human renal cell carcinoma: Establishment and characterization of a new cell line (OS-RC-2)

Summary A cell line, designated as OS-RC-2, has been established from a renal cell carcinoma in a 52-yr-old Japanese male patient and maintained for 23 mo. through 60 in vitro passages. The OS-RC-2 formed monolayers of polygonal epithelial cells and lacked contact inhibition. Doubling time of cells was about 60 h at the 30th passage. Electron microscopic findings indicated numerous long microvilli on the cell surface and many glycogen granules in the cytoplasm of this cell line, which are characteristic structures of renal cell carcinoma. Chromosomal analysis revealed that a small portion of this cell line had a hypodiploid modal number of 40 and a large portion had a hypotetraploid modal number of 75. The characteristics of the karyotype were one detected marker chromosome and the translocation between the Chromosomes 2 and 13. Cell line OS-RC-2 was serially transplantable into nude mice, and histopathological findings of heterotransplanted tumor showed a close similarity to those of the original tumor. Histocompatibility antigens of OS-RC-2 were HLA-A9, Bw52, which were identical to those of the peripheral blood lymphocytes of the patient.     

A new cell line from the wax moth Galleria mellonella Linne (lepidoptera: Pyralididae)

Summary A cell line derived from the larval-fat body tissues of the wax moth, Galleria mellonella Linne, was established in MGM-450 medium. The cells grew in suspension and were mainly spherical in shape. Population doubling time was between 1.4 and 1.7 d over a range of 15 to 35°C, and the maximum growth rate was at 25°C. The chromosome number ranged from 70–239, with a mode of 170. The cells were sensitive to 20-hydroxyecdysone, which stimulated their growth and induced morphological changes. The cell line was designated GaMe-LF1.     

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.     

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 °C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

Chromosome condensation may enhance X-ray-related cell lethality in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells (BHK21)

In the tsBN2 cell line, which has a temperature-sensitive defect in the regulatory mechanism for chromosome condensation, the lethal effect of X rays was enhanced by incubating the cells at a nonpermissive temperature (40 degrees C) following X irradiation. This enhancement was suppressed in the presence of cycloheximide, which inhibits induction of premature chromosome condensation. The findings obtained in the case of delayed incubation at 40 degrees C and in synchronized cells indicate that X-ray-related potentially lethal damage, which can be expressed by chromosome condensation, is produced in the cells at any stage of the cell cycle, but it is repairable for all cells except those at around the late G2-M phase, where chromosome condensation occurs at a permissive temperature (33.5 degrees C). These observations suggest that the high sensitivity of late G2-M cells to X rays is caused by the events associated with chromosome condensation.     

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.     

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 an embryonic cell line from Gampsocleis gratiosa (Orthoptera: Tettigoniidae)

The first continuous cell line from the embryo of Gampsocleis gratiosa (Orthoptera: Tettigoniidae), designated as RIRI-GG1, was established. This cell line was serially subcultured in modified Grace medium. The cells were grown adherent to a culture flask and had spindle-like and polygonal shapes. The chromosome number ranged from 26 to 79 at the 50th passage, and 68% of cells had a diploid chromosome number. The growth rate was determined at the 53rd passage, and the population doubling time was calculated to be 122.1 h. The rDNA internal transcribed spacer and the mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-GG1 cell line was derived from G. gratiosa. This cell line had no apparent susceptibility to Autographa californica nucleopolyhedrovirus and Bombyx mori nucleopolyhedrovirus.     

A temperature-sensitive mutant of cultured mouse cells defective in chromosome condensation

A temperature-sensitive mutant, designated ts85, was isolated from a mouse mammary carcinoma cell line, FM3A. The ts85 cells grew at 33 °C (permissive temperature) with a doubling time of 18 h, which was almost the same as with wild-type cells, whereas the cell number scarcely increased at all at 39 °C (non-permissive temperature). When the ts85 cells were shifted from 33 to 39 °C, their DNA synthesis fell to below 1% of the initial value in 14 h. RNA or protein synthesis, however, was maintained at the initial levels for at least 14 h at 39 °C. Cytofluorometric analysis of asynchronous cultures and studies with synchronous cultures suggested that the bulk of the cells cultured at 39 °C for 12–18 h were arrested in late S and G2 phases. Electron microscopic observations revealed that chromatin was abnormally condensed into fragmented and compact forms, particularly around nucleoli, in about 80% of cells of an asynchronous culture incubated at 39 °C for 16 h. Cells in mitosis were not detected in such cultures and nuclear membrane and nucleoli were still intact. Such abnormal chromosome condensation was not observed in the ts85 cells at 33 °C or in wild-type cells at either temperature. Since these findings suggest that a ts gene product of ts85 cells is necessary for chromosome condensation, ts85 cells may represent a useful tool for establishing the mechanisms of chromosome condensation. The interrelationship between abnormal chromosome condensation and reduction in DNA synthesis of the ts85 cells is discussed.     

Establishment and characterization of a brain cell line from sea perch, <Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>

A continuous cell line, designated LJB, derived from the brain of sea perch (Lateolabrax japonicus) was established. LJB cells have been subcultured for more than 60 times in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS) since the initial primary culture. 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, which was identical with the chromosome number of embryonic stem-like cells of sea perch. Comparison of the 18S ribosomal RNA gene sequences of LJB cells and sea perch confirmed that LJB cells originated from sea perch. After transfected with pEGFP-N3 plasmid, 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. Cytopathic effect was clearly observed, and RNA-dependent RNA polymerase gene was also detected in LJB cells post red-spotted grouper nervous necrosis virus (RGNNV) infection. Furthermore, virus replication was confirmed by quantitative RT-PCR, virus titer, and transmission electron microscopy assay in RGNNV-infected LJB cells. 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.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.