Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro
To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary hum...
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Published in | Scientific reports Vol. 10; no. 1; pp. 20486 - 13 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
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24.11.2020
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Abstract | To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology. |
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AbstractList | Abstract To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology. Abstract To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology. To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology. To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology. |
ArticleNumber | 20486 |
Author | Thorley, Andrew J. Cajaraville, Miren P. Ruenraroengsak, Pakatip Katsumiti, Alberto Tetley, Teresa D. |
Author_xml | – sequence: 1 givenname: Alberto surname: Katsumiti fullname: Katsumiti, Alberto email: alberto.katsumiti@ehu.eus organization: CBET Research Group, Department of Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PiE, University of the Basque Country UPV/EHU, National Heart and Lung Institute, Imperial College London – sequence: 2 givenname: Pakatip surname: Ruenraroengsak fullname: Ruenraroengsak, Pakatip organization: Department of Materials and London Centre for Nanotechnology, Imperial College London, Department of Pharmacy, Faculty of Pharmacy, Mahidol University – sequence: 3 givenname: Miren P. surname: Cajaraville fullname: Cajaraville, Miren P. organization: CBET Research Group, Department of Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PiE, University of the Basque Country UPV/EHU – sequence: 4 givenname: Andrew J. surname: Thorley fullname: Thorley, Andrew J. organization: National Heart and Lung Institute, Imperial College London – sequence: 5 givenname: Teresa D. surname: Tetley fullname: Tetley, Teresa D. email: t.tetley@imperial.ac.uk organization: National Heart and Lung Institute, Imperial College London |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/33235275$$D View this record in MEDLINE/PubMed |
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CitedBy_id | crossref_primary_10_1002_adhm_202100633 crossref_primary_10_1089_hum_2022_130 crossref_primary_10_2174_0115680096266700231107071222 crossref_primary_10_1016_j_isci_2022_103780 crossref_primary_10_3389_ftox_2022_840606 crossref_primary_10_3390_ijms25126522 crossref_primary_10_1016_j_ejps_2023_106596 crossref_primary_10_1186_s13287_024_03763_8 crossref_primary_10_1371_journal_ppat_1010171 |
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Snippet | To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype,... Abstract To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the... Abstract To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the... |
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SubjectTerms | 631/1647 631/532 631/80 692/4017 Advanced glycosylation end products Alkaline phosphatase Alkaline Phosphatase - metabolism Alveolar Epithelial Cells - metabolism Alveolar Epithelial Cells - ultrastructure Alveoli Caveolin Caveolin-1 Cell Line, Transformed Cell Proliferation Cell Respiration Cell Survival Cells, Cultured Epithelial cells Epithelium Genetic Vectors - metabolism Glycosylation Humanities and Social Sciences Humans Hydrodynamics Lipids - chemistry multidisciplinary Nanoparticles Nanoparticles - ultrastructure Particle Size Phenotypes Polystyrene Protein C RNA, Messenger - genetics RNA, Messenger - metabolism Science Science (multidisciplinary) Static Electricity Strains (organisms) Surfactant protein C Telomerase Telomerase reverse transcriptase Transcription, Genetic Transfection Tumors Vectors (Biology) |
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Title | Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro |
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