Journal articles: 'Immortalized cell line'

1

Nitta, K., and N. Horiba. "An immortalized rat mesangial cell line." In Vitro Cellular & Developmental Biology - Animal 33, no. 3 (March 1997): 156–57. http://dx.doi.org/10.1007/s11626-997-0134-y.

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Nagai, Atsushi, Seiji Mishima, Yuri Ishida, Hiroto Ishikura, Takayuki Harada, Shotai Kobayashi, and Seung U. Kim. "Immortalized human microglial cell line: Phenotypic expression." Journal of Neuroscience Research 81, no. 3 (August 1, 2005): 342–48. http://dx.doi.org/10.1002/jnr.20478.

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Nakamura, Yukio, Takashi Hiroyama, Kenichi Miharada, and Ryo Kurita. "Red blood cell production from immortalized progenitor cell line." International Journal of Hematology 93, no. 1 (December 25, 2010): 5–9. http://dx.doi.org/10.1007/s12185-010-0742-2.

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Velazquez, Jessica, Amel Sengal, Carl E. Allen, and Rikhia Chakraborty. "Creating and Testing a CD207+ Langerhans Cell Histiocytosis-like Cell Line." Blood 134, Supplement_1 (November 13, 2019): 5399. http://dx.doi.org/10.1182/blood-2019-130588.

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Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) activation. Investigations into LCH have historically been challenged by the small percentage of pathologic CD207+ DCs. No cell lines with morphology or function representing LCH lesion CD207+ DCs currently exist. We utilized four strategies to generate cell line(s) mimicking differentiated LCH pathogenic cells. First, CD207+ cells were isolated from the lesion of an LCH patient and cultured in a cytokine cocktail. The cells maintained CD1a and CD207 expression for two weeks, after which there were significant changes in cell morphology and progression to cell death. When a similar approach was implemented to isolate and culture skin CD207+ cells, the cells were viable for only three days, supporting the potential role for somatic activating MAPK mutations in LCH lesion DCs to prolong viability. CD207+ cells were then isolated from HLA-DR+ (lineage negative) cells from the lymphoid stroma of healthy tonsils (tCD207+). The tCD207+ cells were transduced with a lentivirus encoding human telomerase reverse transcriptase (hTERT). These cells, also lacking MAPK activating mutations, died two weeks post-transduction. A fourth approach has been more successful in which tCD207+ cells were cultured in a cytokine cocktail which provided MAPK pathway stimulation. The cells retained CD207 expression and survived in culture for over two weeks. The cells were then immortalized using a lentivirus encoding HOXA9. Immortalized cells maintained CD207 expression. Allele specific MAPK pathway mutations (BRAF and MAP2K1) are being generated by class II CRISPR/Cpf1 genome editing as Cpf1 has been shown to have robust activity to induce specific disruption of only mutant, but not wild-type, BRAF allele. The phenotypic and genomic characteristics of the immortalized cells expressing the different MAPK pathway mutations will be assessed by RHG-banding cytogenetic analysis, fluorescence in situ hybridization, gene expression analysis, and immuno‐cytochemistry and results will be compared to cells isolated from LCH lesions to confirm whether the established cell line may be a viable in vitro mimic of the LCH lesion DC. Disclosures No relevant conflicts of interest to declare.

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Steele, Stacy L., Yongren Wu, Robert J. Kolb, Monika Gooz, Courtney J. Haycraft, Kent T. Keyser, Lisa Guay-Woodford, Hai Yao, and P. Darwin Bell. "Telomerase immortalization of principal cells from mouse collecting duct." American Journal of Physiology-Renal Physiology 299, no. 6 (December 2010): F1507—F1514. http://dx.doi.org/10.1152/ajprenal.00183.2010.

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Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

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Tan, Jia-Jie, Lu Wang, Ting-Ting Mo, Yuan-Feng Dai, Juan Lu, Xiong Liu, Huai-Hong Chen, Wen-Dong Tian, and Xiang-Ping Li. "Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1." Cell Transplantation 29 (January 1, 2020): 096368972090819. http://dx.doi.org/10.1177/0963689720908198.

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Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence β-galactosidase staining, 5-ethynyl-2′-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

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Winn, Shelley R., Gannon Randolph, Hasan Uludag, Shou C. Wong, Gregory A. Hair, and Jeffrey O. Hollinger. "Establishing an Immortalized Human Osteoprecursor Cell Line: OPC1." Journal of Bone and Mineral Research 14, no. 10 (October 1, 1999): 1721–33. http://dx.doi.org/10.1359/jbmr.1999.14.10.1721.

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Zhang, H., Y. Zhao, Y. Li, and F. Tian. "Establishment of immortalized epithelial cell line of endometrium." Fertility and Sterility 77 (February 2002): S32. http://dx.doi.org/10.1016/s0015-0282(01)03112-0.

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Levashova, Zoia B., Sergei Y. Plisov, and Alan O. Perantoni. "Conditionally immortalized cell line of inducible metanephric mesenchyme." Kidney International 63, no. 6 (June 2003): 2075–87. http://dx.doi.org/10.1046/j.1523-1755.2003.00010.x.

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10

Newman, S. L., A. A. Weikle, T. J. Neuberger, and J. W. Bigbee. "Myelinogenic potential of an immortalized oligodendrocyte cell line." Journal of Neuroscience Research 40, no. 5 (April 1, 1995): 680–93. http://dx.doi.org/10.1002/jnr.490400514.

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11

Bocchini, V., R. Mazzolla, R. Barluzzi, E. Blasi, P. Sick, and H. Kettenmann. "An immortalized cell line expresses properties of activated microglial cells." Journal of Neuroscience Research 31, no. 4 (April 1992): 616–21. http://dx.doi.org/10.1002/jnr.490310405.

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12

Joo, Hyun Woo, Min Kyu Kim, Soon Sun Bak, and Young Kwan Sung. "Bioengineering of Hair Follicle-like Structure for Validation of Hair Growth Promoting Compounds." Bioengineering 9, no. 11 (November 3, 2022): 645. http://dx.doi.org/10.3390/bioengineering9110645.

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We aimed to establish screening and efficacy test techniques for use in the development of hair-promoting agents. To this end, we used the dermal papilla cell (DPc)-derived immortalized cell line (SV40T-hTERT DPc) and neonatal foreskin-derived keratinocyte cell line (Ker-CT) to form an immortalized cell-based hair follicle-like structure. The SV40T-hTERT DPc spheroids exhibited a higher cell ratio in the spheroids than primary DPc spheroids, and SV40T-hTERT DPc aggregated with spheroids larger in diameter than primary DPc when the same cell number was seeded into the low-adhesion plate. Microscopic imaging and fluorescence staining results indicated that both primary and immortalized cell combinations form a hair follicle-like structure with a long-stretched keratinocyte layer under the condition that the spheroids have the same diameter as that of in vivo dermal papillary tissue in the hair follicle. The hair follicle-like structure elongation was increased upon treatment with three known hair follicle growth-promoting compounds (minoxidil, tofacitinib, and ascorbic acid) compared with that in the control group. Therefore, using immortalized cells to generate a coherent follicle-like structure, we have developed models for screening and evaluating hair-care materials commonly used in the industry.

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Choi, Sung S., Seung-Bin Yoon, Sang-Rae Lee, Sun-Uk Kim, Young Joo Cha, Daniel Lee, Seung U. Kim, Kyu-Tae Chang, and Hong J. Lee. "Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line." Cell Transplantation 26, no. 2 (February 2017): 271–81. http://dx.doi.org/10.3727/096368916x692852.

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Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.

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Manin, V. L., I. V. Vologina, and Ye A. Trofimova. "Preparation of rabbit kidney immortalized cell culture." Veterinary Science Today, no. 4 (January 13, 2021): 297–303. http://dx.doi.org/10.29326/2304-196x-2020-4-35-298-303.

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Preparation of immortalized cell lines obtained from organs and tissues of farm animals is an essential area of biotechnology. The paper presents results of continuous (immortalized) cell line preparation from a primary trypsinized cell culture of an adult rabbit kidney. Cytomorphologic analysis and karyotyping were performed during the process of subcultivation in the cell culture at passages 1, 3, 24, 31, 38, 56, 66, 75, 86, 101. Dynamics of spontaneous continuous cell line formation during long-term serial passaging was examined using standard nutrient media and fetal serum. Contrary to the known cell lines of rabbit origin (Oryctolagus cuniculus L.), immortalization was not accompanied with enhanced cell production and cell size reduction. The prepared continuous cell line in its adhesive phase was up to 200 µm in size and its productivity was about 7,000 cells/cm2. Significant differences (compared to the known cell lines) in the karyotype were detected during passaging. The formed genotype was found to be near-tetrapioid when the CCL cultural properties were stabilized at passages 66–101. The known cell lines – rabbit kidney (RK-13) and rabbit cornea (SIRC) – can be characterized as pseudotriploid basing on their karyotype. This culture demonstrated low sensitivity to viruses – causative agents of rabbit diseases and sensitivity to heterologous porcine and bovine viruses.

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15

Isono, Mitsuo, Herbert M. Geller, Maciej Poltorak, and William J. Freed. "Intracerebral transplantation of the A7 immortalized astrocytic cell line." Restorative Neurology and Neuroscience 4, no. 5 (1992): 301–9. http://dx.doi.org/10.3233/rnn-1992-4501.

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16

Loghman-Adham, Mahmoud, Andreas Rohrwasser, Catherine Helin, Shuhua Zhang, Daniel Terreros, Ituro Inoue, and Jean-Marc Lalouel. "A conditionally immortalized cell line from murine proximal tubule." Kidney International 52, no. 1 (July 1997): 229–39. http://dx.doi.org/10.1038/ki.1997.325.

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McDuffee, Laurie, Rodolfo Nino-Fong, Blanca Esparza, Juan Rodriguez-Lecompte, and William Montelpare. "Development of a Biologically Immortalized Equine Stem Cell Line." Veterinary and Comparative Orthopaedics and Traumatology 31, S 02 (July 2018): A1—A25. http://dx.doi.org/10.1055/s-0038-1668241.

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18

Zhang, H. "Characterization of an immortalized human granulosa cell line (COV434)." Molecular Human Reproduction 6, no. 2 (February 1, 2000): 146–53. http://dx.doi.org/10.1093/molehr/6.2.146.

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19

Nagamoto-Combs, Kumi, Joshua Kulas, and Colin K. Combs. "A novel cell line from spontaneously immortalized murine microglia." Journal of Neuroscience Methods 233 (August 2014): 187–98. http://dx.doi.org/10.1016/j.jneumeth.2014.05.021.

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20

Hida, Kyoko, Nako Maishi, Dorcas Akuba-Muhyia Annan, Miyako Kondoh, Takayuki Hojo, Umma Habiba, Noritaka Ohga, et al. "Aneuploidy of a murine immortalized endothelial cell line, MS1." Journal of Oral Biosciences 59, no. 1 (February 2017): 50–54. http://dx.doi.org/10.1016/j.job.2016.10.004.

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RAJAN, NITHYA, DENISE L. PRUDEN, HATTORI KAZNARI, QING CAO, BYRON E. ANDERSON, JAMES L. DUNCAN, and ANTHONY J. SCHAEFFER. "CHARACTERIZATION OF AN IMMORTALIZED HUMAN VAGINAL EPITHELIAL CELL LINE." Journal of Urology 163, no. 2 (February 2000): 616–22. http://dx.doi.org/10.1016/s0022-5347(05)67946-3.

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Narushima, M., N. Kobayashi, K. A. Westerman, T. Fukazawa, M. Sakaguchi, Y. Tanaka, Jonathan RT Lakey, Philippe Leboulch, and N. Tanaka. "ESTABLISHMENT OF AN IMMORTALIZED HUMAN PANCREATIC BETA CELL LINE." Transplantation 78 (July 2004): 620. http://dx.doi.org/10.1097/00007890-200407271-01669.

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23

Powell, Jason, Bernard Verdon, Janet A. Wilson, A. John Simpson, Jeffery Pearson, and Chris Ward. "Establishment of an immortalized human subglottic epithelial cell line." Laryngoscope 129, no. 11 (January 8, 2019): 2640–45. http://dx.doi.org/10.1002/lary.27761.

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24

Alliot, Françoise, Marie-Chantal Marty, Danielle Cambier, and Bernard Pessac. "A spontaneously immortalized mouse microglial cell line expressing CD4." Developmental Brain Research 95, no. 1 (August 1996): 140–43. http://dx.doi.org/10.1016/0165-3806(96)00101-0.

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25

Geller, H. M., and M. Dubois-Dalcq. "Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector." Journal of Cell Biology 107, no. 5 (November 1, 1988): 1977–86. http://dx.doi.org/10.1083/jcb.107.5.1977.

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We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.

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Pace, Margaret C., Ken L. Chambliss, Zohre German, Ivan S. Yuhanna, Michael E. Mendelsohn, and Philip W. Shaul. "Establishment of an immortalized fetal intrapulmonary artery endothelial cell line." American Journal of Physiology-Lung Cellular and Molecular Physiology 277, no. 1 (July 1, 1999): L106—L112. http://dx.doi.org/10.1152/ajplung.1999.277.1.l106.

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The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218–255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs ( passage 6). eNOS was also acutely activated by estradiol to levels 197–309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-α, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.

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Li, Zhe, Tuan T. Nguyen, and Alan Valaperti. "Human cardiac fibroblasts produce pro-inflammatory cytokines upon TLRs and RLRs stimulation." Molecular and Cellular Biochemistry 476, no. 9 (April 21, 2021): 3241–52. http://dx.doi.org/10.1007/s11010-021-04157-7.

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AbstractHeart inflammation is one of the major causes of heart damage that leads to dilated cardiomyopathy and often progresses to end-stage heart failure. In the present study, we aimed to assess whether human cardiac cells could release immune mediators upon stimulation of Toll-like receptors (TLRs) and Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs).Commercially available human cardiac fibroblasts and an immortalized human cardiomyocyte cell line were stimulated in vitro with TLR2, TLR3, and TLR4 agonists. In addition, cytosolic RLRs were activated in cardiac cells after transfection of polyinosinic-polycytidylic acid (PolyIC). Upon stimulation of TLR3, TLR4, MDA5, and RIG-I, but not upon stimulation of TLR2, human cardiac fibroblasts produced high amounts of the pro-inflammatory cytokines IL-6 and IL-8. On the contrary, the immortalized human cardiomyocyte cell line was unresponsive to the tested TLRs agonists. Upon RLRs stimulation, cardiac fibroblasts, and to a lesser extent the cardiomyocyte cell line, induced anti-viral IFN-β expression.These data demonstrate that human cardiac fibroblasts and an immortalized human cardiomyocyte cell line differently respond to various TLRs and RLRs ligands. In particular, human cardiac fibroblasts were able to induce pro-inflammatory and anti-viral cytokines on their own. These aspects will contribute to better understand the immunological function of the different cell populations that make up the cardiac tissue.

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DenBesten, Pamela K., Cen Gao, Wu Li, Catharina H. E. Mathews, and Dieter C. Gruenert. "Development and characterization of an SV40 immortalized porcine ameloblast-like cell line." European Journal of Oral Sciences 107, no. 4 (August 1999): 276–81. http://dx.doi.org/10.1046/j.0909-8836.1999.eos107407.x.

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Chen, Hu, Wang, Zhao, Yang, Li, Chen, et al. "Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen." International Journal of Molecular Sciences 20, no. 19 (September 30, 2019): 4874. http://dx.doi.org/10.3390/ijms20194874.

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Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

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Bagchi, Abhirup, Aneesha Nath, Vignesh Rajendiran, Madhavi Maddali, Ekta Jajodia, Mohankumar Kumarasamypet Murugesan, Yukio Nakamura, Alok Srivastava, and Shaji Ramachandran Velayudhan. "Generation of an Immortalized Erythroid Progenitor Cell Line Directly from Peripheral Blood Mononuclear Cells without Using Mobilized CD34+ Cells." Blood 132, Supplement 1 (November 29, 2018): 1032. http://dx.doi.org/10.1182/blood-2018-99-111340.

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Abstract A reliable stable human erythroid progenitor cell line that can differentiate to the later stages of erythropoiesis is an important cellular model for studying molecular mechanisms of human erythropoiesis in physiological and pathological situations. An erythroid progenitor cell line (HUDEP) was derived from cord blood haematopoietic stem cells (HSCs) by doxycycline inducible expression of HPV E6/E7 gene (Kurita et al., 2013). This cell line could be differentiated further to terminally differentiated red cells, and it has been extensively used for studying transcriptional regulation of human erythropoiesis. Using the same strategy, immortalized erythroid progenitors could also be generated from adult HSCs (Trakarnsanga et al, 2017). However, generation of immortalized erythroid cells from patients using this protocol is challenging as obtaining sufficient number of adult HSCs requires mobilization of HSCs using GCSF. Peripheral blood mononuclear cells (PBMNCs) contain a small number of erythroid progenitors, which can be expanded and differentiated in culture. Till date, there are no reports on the generation of immortalized erythroid progenitors directly from PBMNCs. In this study, we established a protocol for the generation of immortalized erythroid progenitors from PBMNCs of a normal donor. The PBMNCs isolated from 10ml of blood from a normal donor were cultured for 24 hours in the primary erythroid expansion medium as described earlier (Trakarnsanga et al, 2017). These cells were then transduced with lentiviruses to express HPV E6/E7 gene and a fluorescent protein hKO1. After 3 days, the cells were cultured in a serum free medium containing the cytokines (stem cell factor and erythropoietin) and dexamethasone in the presence of doxycycline for 15 days. The cells that expressed hKO1 were sorted by FACS, and they were cultured in the same medium till the immortalization was complete. We continuously monitored the cells for the kinetics in the expression of erythroid cell surface markers, CD36, CD71 and CD235a, till >95% of the cells expressed all these markers. On day 50, all the cells expressed high levels of the erythroid markers and the cell morphology analysis using Giemsa staining showed that 65% of the cells were pronormoblasts, 22% were basophilic normoblasts and the rest of the cells were at the later stages of differentiation. To evaluate the differentiation potential of these cells, the cells were cultured using the media and conditions described by Hawksworth et al, 2018. After the removal of doxycycline from the culture medium, the cells showed haemoglobinization and the morphology analysis showed that 10% of the cells were in the polychromatic stage and 88% of the cells were in the orthochromatic stage, suggesting robust erythroid differentiation of the immortalized erythroid progenitors with the suitable cell culture conditions. These data showed that immortalized erythroid progenitors with differentiation potential could be generated directly from peripheral blood without using mobilized haematopoietic stem cells. This protocol is suitable for the generation of immortalized erythroid cells from the patients with rare red cell genetic disorders for studying disease mechanisms. Disclosures No relevant conflicts of interest to declare.

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Wang, JunMei, Rui Hu, Zhisheng Wang, Yixin Guo, Sen Wang, Huawei Zou, Quanhui Peng, and Yahui Jiang. "Establishment of Immortalized Yak Ruminal Epithelial Cell Lines by Lentivirus-Mediated SV40T and hTERT Gene Transduction." Oxidative Medicine and Cellular Longevity 2022 (March 25, 2022): 1–17. http://dx.doi.org/10.1155/2022/8128028.

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Yak is a unique species of cattle that is adapted to the harsh natural environment of the Qinghai-Tibet Plateau. Research on the function of the yak rumen is limited to animal experiments, and the cell molecular mechanism is very limited. The high cost of isolation and culture of adult yak rumen epithelial cells (YRECs), low success rate, and limited cell life limit the scope of long-term physiological functions and nutrient absorption mechanisms of yak rumen epithelium in vitro studies. This study aimed to explore the isolation and immortal culture methods of primary YRECs and establish a new cell line model for studying cell molecular mechanisms. The human telomerase reverse transcriptase gene (hTERT) and simian virus 40 large T antigen (SV40T) were transferred into primary YDECs using mammalian gene expression lentiviral vectors. The immortalized cell line (SV40T-YREC-hTERT) retains the morphological and functional characteristics of primary cells. The epithelial cell marker protein cytokeratin 18 of the immortalized cell lines was positive, and the cell proliferation and karyotype were normal. The SV40T and hTERT genes were successfully transferred into immortalized cell lines and maintained high expression. Simultaneously, the immortalized cell lines had normal function of short-chain fatty acid (SCFA) transport and absorption, and the immortalized yak rumen epithelial cell lines were successfully established. In addition, the transepithelial electrical resistance value gradually increased with culture time, and the permeability of epithelial cells decreased by culturing epithelial cells in Transwell culture chambers. Transmission electron microscopy demonstrated the submicroscopic structure of cells in the integrity barrier model and established the YREC barrier model in vitro.

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Zhang, Yufei, Jing Shi, and Shuying Liu. "Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line." BioMed Research International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/5808575.

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The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-termin vitrostudies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunitβ(CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes.

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Spera, Iolanda, Ricardo Sánchez-Rodríguez, Maria Favia, Alessio Menga, Francisca C. Venegas, Roberta Angioni, Fabio Munari, et al. "The J2-Immortalized Murine Macrophage Cell Line Displays Phenotypical and Metabolic Features of Primary BMDMs in Their M1 and M2 Polarization State." Cancers 13, no. 21 (October 31, 2021): 5478. http://dx.doi.org/10.3390/cancers13215478.

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Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

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Tokiwa, Takayoshi, Taisuke Yamazaki, and Shin Enosawa. "Side Population Cells from an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties." Cell Medicine 3, no. 1-3 (January 2012): 127–35. http://dx.doi.org/10.3727/215517912x639450.

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Alexander, Dorothea, Regina Biller, Melanie Rieger, Nina Ardjomandi, and Siegmar Reinert. "Phenotypic Characterization of a Human Immortalized Cranial Periosteal Cell Line." Cellular Physiology and Biochemistry 35, no. 6 (2015): 2244–54. http://dx.doi.org/10.1159/000374029.

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Background/Aims: Human cell material for basic research work is limited due to restricted patient numbers and occurring cell senescence during prolonged in vitro cell cultivation. In the present study, we established for the first time a human immortalized cranial periosteal cell line and characterized its phenotype in detail in comparison to that of parental cells. Methods: For this purpose, human primary cranial periosteal cells were stably transduced with the large T antigen cDNA from polyomavirus SV40 (TAg cells). Results: The functional activity of the large T antigen was demonstrated by human telomerase gene expression. Whereas TAg cells maintained long-term cell proliferation, immortalization did not compromise their osteogenic differentiation potential. In contrast, TAg cells showed an earlier and stronger mineralization compared to parental cells. Among the analysed stem cell surface markers, CD146 and MSCA-1 (mesenchymal stem cell antigen-1) were shown to be elevated in Tag cells. Gene expression analyses revealed in general higher constitutive m-RNA levels of key factors of osteogenesis than in parental cells. Conclusion: We conclude that the herein generated cell line represents a suitable cell source for basic science research studying bone biology, the osteogenesis process or biomaterial tests for bone regeneration purposes.

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Delarue, Françoise, Angela Virone, Jacqueline Hagege, Roger Lacave, Marie-Nëlle Peraldi, Colette Adida, Eric Rondeau, Jean Feunteun, and Jean-Daniel Sraer. "Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells." Kidney International 40, no. 5 (November 1991): 906–12. http://dx.doi.org/10.1038/ki.1991.292.

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Blum, Walter, László Pecze, Emanuela Felley-Bosco, Janine Worthmüller-Rodriguez, Licun Wu, Bart Vrugt, Marc de Perrot, and Beat Schwaller. "Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line." In Vitro Cellular & Developmental Biology - Animal 51, no. 7 (April 15, 2015): 714–21. http://dx.doi.org/10.1007/s11626-015-9885-z.

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Randell, S. H., J. Y. Liu, P. C. Ferriola, L. Kaartinen, M. M. Doherty, C. W. Davis, and P. Nettesheim. "Mucin production by SPOC1 cells--an immortalized rat tracheal epithelial cell line." American Journal of Respiratory Cell and Molecular Biology 14, no. 2 (February 1996): 146–54. http://dx.doi.org/10.1165/ajrcmb.14.2.8630264.

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Gobel, Jeff, Margaret Gartland, Sarah Harris Gurley, Sue Kadwell, Dan Gillie, Chris Moore, and Aaron Goetz. "A Phenotypic High-Throughput Screen with RSV-Infected Primary Human Small Airway Epithelial Cells (SAECs)." Journal of Biomolecular Screening 20, no. 6 (April 10, 2015): 729–38. http://dx.doi.org/10.1177/1087057115580271.

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Respiratory syncytial virus (RSV) is a commonly occurring pathogen that can cause severe disease in children, the elderly, and immunocompromised individuals with a large, unmet clinical need. We developed a high-throughput, primary cell-based antiviral RSV assay to enable identification of small molecules using cytopathic effect (CPE) as a phenotypic end point. To provide increased biological relevance, we developed our assay with primary human small airway epithelial cells (SAECs), which originate from known sites of RSV infection and replication instead of a more traditional immortalized cell line. Using purchased low-passage cells, cost-effective large-scale culture methods were developed to provide assay-ready frozen SAECs. A high-throughput screening campaign using the GSK Screening Collection was performed. The screen was executed in 384-well plates over a 12-week period with an average Z′ of 0.5. The screen yielded 17 post-entry hits with activity in the primary cells, which were not active in immortalized cells. Potencies for this class of compounds were equal between the primary and immortalize cell lines. For entry inhibitors, the number was much lower, with increased potency observed in immortalized cells. This is the first known use of frozen primary human cells for an RSV high-throughput screening phenotypic campaign.

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Perrone, R. D., C. Johns, S. A. Grubman, E. Moy, D. W. Lee, J. Alroy, G. R. Sant, and D. M. Jefferson. "Immortalized human bladder cell line exhibits amiloride-sensitive sodium absorption." American Journal of Physiology-Renal Physiology 270, no. 1 (January 1, 1996): F148—F153. http://dx.doi.org/10.1152/ajprenal.1996.270.1.f148.

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We have produced a continuous cell line using retroviral transduction of simian virus 40 (SV40) large T antigen into epithelial cells grown from a cystoscopic bladder biopsy from a female patient with interstitial cystitis. Immortalized urothelial cells are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH-3T3 fibroblast coculture. They maintain their epithelial appearance and are positive for cytokeratins. SV40 large T antigen is localized to the cell nucleus. When grown on Anocell permeable supports, the cells form a complex epithelium with scalloped luminal membranes, apical junctional complexes containing tight junctions, stratification, transepithelial resistance of 500–1,000 omega.cm2, amiloride-sensitive short-circuit current indicative of active transepithelial Na+ absorption, and functional evidence for basolateral Na-K-adenosinetriphosphatase. This immortalized bladder cell line will facilitate the study of human bladder epithelial function and the response to diverse physiological and pathophysiological stimuli.

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Huyghe, Pauline, Laurent Dassonneville, Pierre Fenaux, and Christian Bailly. "Hydroxyurea-Induced Apoptosis in an EBV-Immortalized Lymphoblastoid Cell Line." Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics 14, no. 4 (January 1, 2003): 235–45. http://dx.doi.org/10.3727/000000003772505362.

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Iatsyshyna, A. P., O. V. Pidpala, T. P. Kochubey, and L. L. Lukash. "Cytogenetic analysis of the spontaneously immortalized mouse cell line G1." Biopolymers and Cell 22, no. 4 (July 20, 2006): 299–306. http://dx.doi.org/10.7124/bc.00073c.

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Fantauzzo, Katherine A., and Philippe Soriano. "Generation of an immortalized mouse embryonic palatal mesenchyme cell line." PLOS ONE 12, no. 6 (June 5, 2017): e0179078. http://dx.doi.org/10.1371/journal.pone.0179078.

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Scharfmann, Raphaël, Severine Pechberty, Yasmine Hazhouz, Manon von Bülow, Emilie Bricout-Neveu, Maud Grenier-Godard, Fanny Guez, et al. "Development of a conditionally immortalized human pancreatic β cell line." Journal of Clinical Investigation 124, no. 5 (March 25, 2014): 2087–98. http://dx.doi.org/10.1172/jci72674.

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DELOS SANTOS, NOEL M., RUOHONG ZHOU, and RADHAKRISHNA K. RAO. "EGF Antioxidant Signaling in an Immortalized Murine Podocyte Cell Line." FASEB Journal 22, S2 (April 2008): 166. http://dx.doi.org/10.1096/fasebj.22.2_supplement.166.

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Seigel, G. M., S. Manohar, Y. Y. Bai, D. Ding, and R. Salvi. "An immortalized microglial cell line (Mocha) derived from rat cochlea." Molecular and Cellular Neuroscience 85 (December 2017): 202–10. http://dx.doi.org/10.1016/j.mcn.2017.11.001.

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Sonti, Shilpa, Mansi Tolia, Richard I. Duclos, Ralph H. Loring, and Samuel J. Gatley. "Metabolic studies of synaptamide in an immortalized dopaminergic cell line." Prostaglandins & Other Lipid Mediators 141 (April 2019): 25–33. http://dx.doi.org/10.1016/j.prostaglandins.2019.02.002.

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Mao, Weiming, Yang Liu, Avani Mody, Michela Montecchi-Palmer, Robert J. Wordinger, and Abbot F. Clark. "Characterization of a spontaneously immortalized bovine trabecular meshwork cell line." Experimental Eye Research 105 (December 2012): 53–59. http://dx.doi.org/10.1016/j.exer.2012.10.007.

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Pálfi, A., Mónika Tarcsa, Szilvia Várszegi, and K. Gulya. "Calmodulin gene expression in an immortalized striatal GABAergic cell line." Acta Biologica Hungarica 51, no. 1 (March 2000): 65–71. http://dx.doi.org/10.1007/bf03542966.

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Ades, Edwin W., John C. Hierholzer, Velma George, Jodi Black, and Francisco Candal. "Viral susceptibility of an immortalized human microvascular endothelial cell line." Journal of Virological Methods 39, no. 1-2 (September 1992): 83–90. http://dx.doi.org/10.1016/0166-0934(92)90127-y.

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Journal articles on the topic 'Immortalized cell line'

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