A transitional cell carcinoma cell line

Summary A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies. Presented in part at the 28th Annual Meeting of the Tissue Culture Association, June 7, 1977. This work was supported by Grant No. CA 15018 awarded by the National Cancer Institute, DHEW, and the Mary B. and L. H. Marshall Fund.     

Mouse cell line authentication

The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.     

A transitional cell carcinoma cell line.

A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies.     

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process. A. Salmén and K. Lindgren contributed equally to the work.     

A human liposarcoma cell line

Summary The properties of a new cell line derived from a human retroperitoneal liposarcoma are described. The cells do not grow at low cell concentrations and contain in their cytoplasm large numbers of droplets that stain with Oil Red O. No indication of the presence of endogenous virus particles could be found. The mean chromosome number per cell is 36, with several constant markers, the most conspicuous of which is a large submetacentric marker due to a translocation between chromosomes 4 and 11, which is present in every cell. The liposarcoma cells show an enhanced uptake of [¹⁴C]acetate compared to normal human fibroblasts. This work was supported by funds from the Poliomyelitis Research Foundation, the National Cancer Association of South Africa, and the State health Department. The authors thank Mrs. D. Bower and Mrs. M. Orchard for their excellent technical assistance.     

哺乳动物细胞系(株)的应用

哺乳动物细胞系(株)已在细胞遗传学、分子遗传学和生物医学等方面得到越来越广泛的应用。其已被用于基因定位、检测理化因素的毒性和细胞因子活性、生产单克隆抗体、制备组织工程化人工器官和转基因动物等方面,更是研究发育生物学、肿瘤发生学、肿瘤治疗等不可或缺的材料。综述了哺乳动物细胞系(株)的建立方法及其在以上各方面的应用。     

Comparative characterization of a human large cell lung carcinoma cell line and the xenograft derived cell line.

The HK-1 cell line established from a human large cell lung carcinoma shows a high transformed phenotype and undifferentiated characteristics. This cell line grows as an adherent monolayer in fetal calf serum-supplemented medium, shows a high proliferation index, is able to grow in semi-solid agar and is tumorigenic in athymic nude rats. The cell line HK-2 derived from the HK-1 xenograft in athymic nude rats shows basically the same features found in the original HK-1 cell line, which include aneuploid nuclear DNA content, abnormal chromosomal number. rearranged marker chromosomes and abnormally localised nucleolar organizer regions. Cytokeratin and vimentin intermediary-sized filaments were found in both cell lines as well as in the original and induced tumour, while neither oncofetal antigens (alphafeto-protein, carcinoembryonic antigen, chorionic gonatropin and human placental lactogen) nor neural differentiation markers (neurofilament and neural specific enolase) were expressed. Analysis of sulphated glycosaminoglycans in the cell cultures and in the nude rat induced tumour showed high expression of chondroitin sulphate.     

Generation of cell hybrids via a fusogenic cell line

BACKGROUND: Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult. METHODS: We have established a stable cell line expressing a viral fusogenic membrane glycoprotein (FMG) that is not itself susceptible to fusion. This cell line has been used to generate hybrids and to evaluate the relevance of tools used for hybrid detection. RESULTS: This FMG-expressing cell line promotes fusion between autologous or allogeneic TC and DC in any combination, generating 'tri-parental hybrids'. At least 20% of TC are found to be integrated into hybrids. CONCLUSIONS: It is speculated that this tri-parental hybrid approach offers new possibilities to further modulate the anti-tumour effect of the DC/TC hybrids since it allows the expression of relevant immunostimulatory molecules by appropriate engineering of the fusogenic cell line.     

New cell line. Epithelial cell line and subline established from premalignant mouse mammary tissue.

A cell line and subline with epithelial characteristics were established from mouse mammary hyperplastic alveolar nodules (HAN). The cells do not grow in suspension cultures in vitro or form tumors in vivo. The cells do produce significant amounts of C-type and A-type virus and low amounts of plasminogen activator.     

Proteomic analysis of cholangiocarcinoma cell line

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countries, such as Thailand. Distinguishing CCA from hepatocellular carcinoma (HCC) of the liver often requires the use of histochemistry, so molecular markers for diagnosis and prognosis are still required. In this study, the two-dimensional (2-D) protein map of a Thai human bile duct epithelial carcinoma cell line (HuCCA-1) has been compared to human hepatocellular carcinoma cell lines (HepG2 and HCC-S102) and a human breast epithelial cancer cell line (MCF-7). Our results show that HuCCA-1 expressed a unique pattern of proteins. Forty-three major proteins were identified by matching to the map of MCF-7, and by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). Cytokeratins CK8 and CK18 were overexpressed in both HuCCA-1 and HCC, while CK7 and CK19 were only expressed in HuCCA-1. Four specific proteins with MW/pI 57.2/5.21 (U1, vimentin), 42.2/6.20 (U2), 43.2/6.20 (U3, EF-TU), and 42.2/6.40 (U4, unidentified) were absent from HepG2. U2 showed high expression in HuCCA-1, while U1 and U4 showed high expression in HCC-S102. U2 could be separated in 2 proteins, U2/1 (alpha-enolase) and U2/2 (not identified) by using IPG pH 4-7. Galectin-3 showed high expression level in HuCCA-1 by 1-DE immunodetection, and gave only one spot with MW 32.9 kDa and pI 8.29 on 2-DE immunoblotting, Thus, certain proteins, namely CK7, CK19, U2/2 and galectin-3, may be good markers useful for differential diagnosis of cholangiocarcinoma compared to hepatocellular carcinoma.     

New cell line of mouse ependymoblastoma

A new cell line derived from transplanted mouse ependimoblastoma consists of typical fibroblast-like cells with well-defined pleomorphism. The line is characterized by a rapid increase and decrease of proliferative activity, by a short stationary phase and early degeneration. The average time of themitotic cycle was determined as being 22-24, tG1-11-12, ts-8-9, tG2-3 and tM-near 0.9 hours. The cytophotometrical evaluation of DNA content revealed two cell subpopulations with different ploidy, both in culture and in tumor in vivo. As compared to the original tumor cells, the cultured cells differ in wider variability of DNA content and a higher average ploidy per nucleus.     

Telomerase-transduced osteoarthritic fibroblast-like synoviocyte cell line

To examine whether the life span of fibroblast-like synoviocytes (FLSs) can be extended and to establish FLS cell lines that preserve the characteristics of primary FLSs, we introduced human catalytic subunit of telomerase (hTERT) gene into human osteoarthritic (OA) FLSs. Two hTERT-transduced clonal cell lines were established and one line, hTERT-OA FLS 13A, was characterized. The hTERT-OA FLS 13A cells have a morphology similar to that of the parental untransduced cells and a population-doubling time similar to that of the parental cells of early passages. While the parental untransduced OA FLSs reached senescence after 100 days in culture, the hTERT-OA FLS 13A cells continued to grow at a population-doubling rate of once in about every 2-3 days. The hTERT-OA 13A cells have so far grown in culture beyond 450 days and maintained the same growth rate. Furthermore, the hTERT-OA FLS 13A cells preserved their sensitivity and response to the treatment with basic calcium phosphate crystals and interleukin-1beta. In conclusion, exogenous expression of telomerase represents a way to extend the life span of human FLSs and telomerase-transduced FLS cells offer a promising tool for gene regulation, cell-based assay, cell transplantation-based gene therapy, and tissue engineering research and development.     

CRISPR-interceded CHO cell line development approaches

For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high-quality biologics with human-like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long-term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR-Cas systems, the use of CRISPR-Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast-track product development process with consistent, improved product yield and quality.     

A possible mammary stem cell line

The cell line Rama 25 is derived from a mammary tumor induced in a rat by dimethylbenzanthracene. During rapid proliferation, Rama 25 cells appear as a single undifferentiated epithelial type; at high cell densities, however, small numbers of two other cell types are formed, which respectively resemble secretory and myoepithelial cells of the mammary gland, as judged by light and electron microscopy and immunofluorescent staining for casein (milk proteins). These additional cell types cannot be explained as contaminating cell populations since the cell line has been cloned several times; furthermore, the proportion of either can be increased by dimethylsulphoxide under different conditions. Specific epithelial features are seen by histological and ultrastructural examination of tumors formed by Rama 25 cells in immunodeficient mice. A line of the myoepithelial-like cells, Rama 29, isolated from a Rama 25 culture by cloning, is also described.We propose that the undifferentiated cell type is a form of mammary stem cell which can differentiate in culture.