Establishment and partial characterization of a human tumor cell line,GBM-HSF,from a glioblastoma multiforme

This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10 % fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5 % of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, β-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

Effects of autocrine vascular endothelial growth factor (VEGF) in non-small cell lung cancer cell line A549

It is reported that the autocrine loop of the vascular endothelial growth factor (VEGF) is crucial for the survival and proliferation of non-small cell lung cancer (NSCLC) tumors. In this study we aimed to systematically investigate the role of autocrine vascular VEGF in NSCLC cell line A549 through inhibition of endogenous VEGF. A549 cells were transfected with florescence-labeled VEGF oligodeoxynucleotide with lipofectamine. For the experimental group, cells were transfected with VEGF anti-sense oligodeoxynucleotide (ASODN), sense oligodeoxynucleotide (SODN) and mutant oligodeoxynuleotide (MODN) respectively. For the control group cells were mock transfected with lipofectamine or culture medium. At indicated time point after transfection, the expression levels of VEGF mRNA and protein in A549 cells were analyzed by RT-PCR and ELISA respectively. Cell viability was measured by the MTT assay. Cell cycle distribution was detected by flow cytometry. As revealed by RT-PCR assay, the mRNA level of VEGF in cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 and 48 h after transfection. ELISA assay yielded similar result with significantly decreased level of VEGF protein expression (P < 0.05). The survival fraction of A549 cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 h after transfection. Also the percentage of G2 phase cells of ASODN group was significantly lower than other four groups. Our data indicate that VEGF expression is efficiently inhibited in A549 cells by ASODN transfection and this inhibition leads to inhibited cell growth and impaired cell cycle distribution.     

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.     

Establishment and characterization of the RMG-V cell line from human ovarian clear cell adenocarcinoma

A cell line, designated as RMG-V, was established from a patient with clear cell adenocarcinoma of the ovary. The cell line has grown without interruption and has been propagated continuously by serial passaging (more than 36 times) over 5 years. The cells are spindle-shaped, display neoplastic and pleomorphic features, and grow in a jigsaw puzzle-like arrangement while forming monolayers without contact inhibition. These cells proliferate rapidly, and the population doubling time is about 15.5 hours. The number of chromosomes ranges between 77 and 85, with a modal number of 83.     

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 of the AU-bek cell line and comparison with two other bovine cell lines

Summary Establishment of a new bovine cell line, AU-BEK, is reported. The cell line developed in a culture initiated from bovine embryonic kidneys by spontaneous cultural alteration to epithelioid cells that are indefinitely propagable. Epithelioid cells gradually increased to become the predominant cell. Whereas normal bovine cells have a diploid number of 60 chromosomes, of which only the two sex chromosomes are biarmed, AU-BEK cells at the 80th passage had a modal chromosome number of 84 and an average of 30 biarmed chromosomes per cell. AU-BEK cells are now in their 220th passage. Of the AU-BEK, MDBK, and CKT-1 bovine cell lines, the CKT-1 cell line had a karyotype closest to that of normal bovine cells. Their modal chromosome number was 57, and only three biarmed chromosomes were usually present. The bovine character of AU-BEK and CKT-1 cells was established by cytotoxic and viral susceptibility tests. Supported by the Alabama Agricultural Experiment Station. Publication No. 1115, School of Veterinary Medicine, Auburn University.     

Establishment and characterization of a novel ovarian clear cell adenocarcinoma cell line, TU-OC-1, with a mutation in the PIK3CA gene

A new cell line of human ovarian clear cell adenocarcinoma (CCC), TU-OC-1, was established and characterized. The cells showed a polygonal-shaped morphology and grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 64 to 90. A low rate of proliferation was observed, similar to other CCC cell lines tested (OVTOKO, RMG-I, and OVAS), and the doubling time was 38.4 h. The respective IC₅₀ values of cisplatin and paclitaxel were 12.2 μM and 58.3 nM. Mutational analysis revealed that TU-OC-1 cells harbored a PIK3CA mutation at codon 542 (E542K) in exon 9, which is a mutation hot spot on this gene. We observed that phosphorylated Akt protein was overexpressed in TU-OC-1 cells by western blot analysis. Heterotransplantation to nude mice produced tumors that reflected the original. This cell line may be useful to study the chemoresistant mechanisms of CCC and contribute to novel treatment strategies.     

Comparative analysis of the karyotype of new human cell line 4BL at long-term cultivation: Ploidy of the chromosomal set

Long-term cultivation of human cells, including stem cells, can lead to essential transformations of the karyotype and genetic instability. The aim of this research was a comparative cytogenetic study of the karyotype of new human stem cell line 4BL at 160 and 205 passages. During a standard cytogenetic examination, the nullisomy and monosomy of chromosomes 10 and 13, monosomy of chromosomes 4, 8, 11, 15, 17, 21, and X; and t(1, 11), t (5, 15), t(12, 15), and t(16, 21) were observed; also, six regular marker chromosomes were detected. At 160 and 205 passages, the modal class of the karyotype was 42–43 chromosomes. While passaging increased frequency of polyploidy cells (from 2.8 to 36%), disappearance of nearhaploid cells (22.1% at the 160th passage) and a decreased level of early division of chromatids (from 5 to 1.5%) were observed. We assume the stabilization of the karyotype of cell line 4BL at 205 passages and consider that it is necessary to conduct an additional molecular and cytogenetic study for the objective identification of the number of chromosomes of the modal class, as well as the number of chromosomal anomalies, and for forecasting the direction of the karyotype evolution of human cells 4BL in vitro.     

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.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.     

Development and characterization of a cell line WAF from freshwater shark Wallago attu

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.     

Development and characterization of a continuous macrophage cell line,LRTM, derived from thymus of Labeo rohita (Hamilton 1822)

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco’s modified Eagle’s medium at 37°C with 5% CO₂ and showed 85+?% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.     

Establishment and characterization of a human gestational choriocarcinoma cell line (HOCC)

Human gestational choriocarcinoma cell line (HOCC) was established from the mouse graft of choriocarcinorna. The HOCC cells were spindle or polygonal in shape and multi-nucleated giant cells, showing neoplastic and pleomorphic features. The cell proliferated stably, and the population doubling time was about 32 hours. The chromosome numbers showed a wide distribution of aneuploidy, the mode was in hypertriploid range and the marker chromosomes were recognized in the several generations. Heterotransplantation was easy, and subcutaneous transplantation of 1 × 10⁷ cells in nude mouse formed a tumor composed of choriocarcinoma. It is most noteworthy characteristic of the cell line that it produced human chorionic gonadotropin (hCG) in an in vitro culture system and in vivo in nude mice.     

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle,Chelonia mydas

Summary A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle,Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30°C although replication occurred between 16 and 37°C. These cells may be held as monolayers at 8°C or stored frozen in growth medium containing 10% dimethylsulfoxide at −70 or −196°C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.     

Genetic characterization of parental cell lines and biochemical analysis of somatic cell hybrids between mouse (RAG) cells and fibroblasts of Ateles paniscus chamek (primates,platyrrhini)

Mouse (RAG) cells, (deficient in hypoxanthine-phosphoribosyl-transferase), and Ateles paniscus chamek primary fibroblasts were used in fusion experiments to generate somatic cell hybrids. Both parental cell lines were genetically characterized by karyological and biochemical analyses with 27 isozyme systems. These procedures were useful for monitoring primate chromosome segregation in somatic cell hybrids, for detecting chromosome rearrangements of primate chromosomes, and for identifying individual primate chromosomes. These characterizations are necessary to distinguish between different hybrid cell lines and to generate a panel for gene mapping studies. This is achieved by selecting cell lines that segregate different sets of relatively few primate isozymes and chromosomes. Conversely, we eliminated hybrid cell lines either showing: (1) rearrangements between primate and mouse chromosomes, (2) extensive rearrangements of primate chromosomes, or (3) a large number of primate biochemical markers. © 1993 Wiley-Liss, Inc.     

A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.