A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.     

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers.     

The selection of somatic cell hybrids between human lymphoma cell lines.

Somatic cell hybrids have been selected between three pairs of established human lymphoid cell lines producing pure lines of proliferating hybrid cells: Raji/Namalwa, Raji/Daudi, and Raji/BJAB. The hybrid cell lines have been characterized with respect to isozyme pattern, volume, and karyotype.     

Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.     

Tooth regeneration from newly established cell lines from a molar tooth germ epithelium

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.     

Characterization and differentiation of human embryonic stem cells

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.     

Plasma membrane studies on drug-sensitive and -resistant cell lines. II. Ouabain sensitivity of (Na+ +K+)-stimulated Mg2+-ATPase.

Mutant cell lines have been selected from the murine plasmocytoma MOPC 173 for their resistance to ouabain, dibutyryl cyclic AMP, theophyllin and concanavalin A. We have compared three wild-type cell lines with their seven resistant counterparts. All resistant mutants exhibited a (Na+ + K+)-stimulated Mg2+-ATPase resistance to ouabain inhibition when measured in microsomes. The homogeneity of ouabain binding sites has been found in most of the cell lines; however, two different populations of sites have been detected in one wild-type and in one resistant cell lines. These results led us to hypothetise the (Na+ + K+)-ATPase-ouabain interaction being modulated by a non-specific membrane structure.     

Development and characerization of cell lines from subhuman primates

Summary Seven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro. Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence. All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties. These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells. The research upon which this publication is based was performed pursuant to Contract No. NIH-69-100 with the Division of Biologics Standards of the National Institutes of Health.     

Zebra finch cell lines from naturally occurring tumors

The zebra finch (Taeniopygia guttata) has been intensively studied in many research fields including neuroscience, behavioral neurobiology, and evolution of the genome. Although numerous molecular and genomic resources are available for this model species, immortalized cell lines have been lacking. We have established two zebra finch cell lines derived from spontaneous tumors. ZFTMA is a tetraploid female cell line and G266 as a diploid male cell line. These first zebra finch cell lines should facilitate development of research on this model species.     

Preferential expression of a common T cell receptor structure by T cells induced to proliferate in vitro with a murine retrovirus.

We have successfully isolated continuous T cell lines from murine spleen which have been induced to proliferate after in vitro exposure to the murine leukemia virus RadLV. Cell lines isolated from several strains of mice have an "immature" phenotype and are immortalized CD4- CD8- CD3+ cell lines. Cell lines of similar phenotype have now been derived from many individual mice, after spleens have been infected with two different RadLV viruses, a leukemogenic and a nonleukemogenic isolate. Among cell lines induced with RadLV/C6VL, an unusually high proportion of cells was found to bind the 124-40 anticlonotypic antibody specific for the alpha beta TCR expressed by C6VL/1 cells which produces RadLV/C6VL. This was not reflected in cell lines induced with the RadLV/V13 isolate nor in various lymphocyte subsets freshly isolated from normal mice, or induced to proliferate in culture. Cells expressing a common TCR structure would appear to be appropriate targets for in vitro proliferation and transformation induced by RadLV.     

Establishment of epithelial cell lines from adult mouse regenerating liver

A simple technique for developing epithelial cell lines from regenerating mouse liver has been described. Twenty-one epithelial cell lines have been developed and can be divided into four groups according to their morphology. All these near diploid cell lines have the capacity to metabolize diverse classes of chemical carcinogens (3-methylcholanthrene, 2-acetylaminofluorene, dimethylnitrosamine and aflatoxin B1) to cytotoxic metabolites. It is not yet possible to determine which ones of these cell lines originated from hepatocytes. Studies are in progress to further characterize and to use these cell lines as lethally irradiated feeder layers for cell-mediated activation of various classes of chemical carcinogens and mutagens with C3H/10T1/2 mouse embryo fibroblasts as indicator cells.     

Survey of ATCC stocks of human cell lines for hela contamination

Summary Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsabanded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.     

Survey of ATCC stocks of human cell lines for HeLa contamination

Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsabanded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.     

昆虫细胞系的培养和建立技术

迄今已经报道的昆虫细胞系有800株以上。昆虫细胞系在昆虫病理学、寄生虫学、内分泌学、遗传学和分子生物学等基础和应用研究中得到越来越广泛的应用。本文结合我们研究的结果和实践经验,概括了国内外昆虫细胞系建立技术的研究进展,包括昆虫细胞培养的发展、昆虫细胞系建立技术、不同昆虫组织来源细胞系的建立方法和过程,以及对昆虫细胞系特征的鉴定等方面。     

Glial Cell Lines: An Overview

The importance and essential functions of glial cells in the nervous system are now beginning to be understood and appreciated. Glial cell lines have been instrumental in the elucidation of many of these properties. In this Overview, the origin and properties of most of the existing cell lines for the major glial types: oligodendroglia, astroglia, microglia and Schwann cells, are documented. Particular emphasis is given to the culture conditions for each cell line and the degree to which the line can differentiate in vitro and in vivo. The major molecular markers for each glial cell lines are indicated. Finally, methods by which the glial cell lines have been developed are noted and the future directions of glial cell line research are discussed.     

Effect of Glutamate Analogues on Brain Tumor Cell Lines

Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.     

Interaction of porphyrins with heme proteins – a brief review

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.