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 inScientific reports Vol. 10; no. 1; pp. 20486 - 13
Main Authors Katsumiti, Alberto, Ruenraroengsak, Pakatip, Cajaraville, Miren P., Thorley, Andrew J., Tetley, Teresa D.
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 24.11.2020
Nature Publishing Group
Nature Portfolio
Subjects
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)
Online AccessGet full text

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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.
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.
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  givenname: Teresa D.
  surname: Tetley
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  email: t.tetley@imperial.ac.uk
  organization: National Heart and Lung Institute, Imperial College London
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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
URI https://link.springer.com/article/10.1038/s41598-020-77191-y
https://www.ncbi.nlm.nih.gov/pubmed/33235275
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https://pubmed.ncbi.nlm.nih.gov/PMC7686381
https://doaj.org/article/937c8d854bab44f99579b0458b77380f
Volume 10
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