Maintenance of Differentiated Murine Clara Cells in Microdissected Airway Cultures

Laura S. Van Winkle, Alan R. Buckpitt, Charles G. Plopper

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36 Citations (Scopus)

Abstract

Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary cytotoxicants and the progenitor during repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. The role of Clara cells in normal lung function is poorly understood partly because their abundance, sensitivity to cytotoxicants, and expression of differentiation markers vary by airway level and species. This study defines a strategy for maintenance in vitro of differentiated Clara cells within their local microenvironment. Lungs from adult mice were inflated with 1% agarose and distal airways were isolated by microdissection. Explants were cultured for 7 days in serum-free medium. Preservation of Clara cell morphology after 7 days in culture (DIC) was demonstrated using light and electron microscopy. Ciliated cells were also present. Cytochrome P450 monooxygenase activity, as measured by naphthalene epoxidation, was decreased 50% between O and 7 DIC, but the apparent stereoselectivity of metabolism was unchanged at 7 days. Marker proteins for differentiated Clara cells (secretory protein, CYP2F2 and CYP2B4) were detectable immunochemically throughout time in culture. Glutathione S-transferase activity and levels of reduced glutathione were unchanged over 7 DIC. We conclude that differentiated Clara cells can be maintained in cultures of explants from defined airway regions. Bronchiolar epithelial cells in this system are viable, synthesize and secrete secretory protein, metabolize xenobiotics via the cytochrome P450 system, have a stable phase II enzyme system, and maintain glutathione pools.

Original languageEnglish (US)
Pages (from-to)586-598
Number of pages13
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume14
Issue number6
StatePublished - 1996

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Maintenance
Cytochrome P-450 Enzyme System
Glutathione
Microdissection
Lung
Uteroglobin
Stereoselectivity
Epithelial Cells
Epoxidation
Differentiation Antigens
Serum-Free Culture Media
Xenobiotics
Mixed Function Oxygenases
Glutathione Transferase
Metabolism
Sepharose
Electron microscopy
Optical microscopy
Proteins
Repair

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

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title = "Maintenance of Differentiated Murine Clara Cells in Microdissected Airway Cultures",
abstract = "Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary cytotoxicants and the progenitor during repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. The role of Clara cells in normal lung function is poorly understood partly because their abundance, sensitivity to cytotoxicants, and expression of differentiation markers vary by airway level and species. This study defines a strategy for maintenance in vitro of differentiated Clara cells within their local microenvironment. Lungs from adult mice were inflated with 1{\%} agarose and distal airways were isolated by microdissection. Explants were cultured for 7 days in serum-free medium. Preservation of Clara cell morphology after 7 days in culture (DIC) was demonstrated using light and electron microscopy. Ciliated cells were also present. Cytochrome P450 monooxygenase activity, as measured by naphthalene epoxidation, was decreased 50{\%} between O and 7 DIC, but the apparent stereoselectivity of metabolism was unchanged at 7 days. Marker proteins for differentiated Clara cells (secretory protein, CYP2F2 and CYP2B4) were detectable immunochemically throughout time in culture. Glutathione S-transferase activity and levels of reduced glutathione were unchanged over 7 DIC. We conclude that differentiated Clara cells can be maintained in cultures of explants from defined airway regions. Bronchiolar epithelial cells in this system are viable, synthesize and secrete secretory protein, metabolize xenobiotics via the cytochrome P450 system, have a stable phase II enzyme system, and maintain glutathione pools.",
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N2 - Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary cytotoxicants and the progenitor during repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. The role of Clara cells in normal lung function is poorly understood partly because their abundance, sensitivity to cytotoxicants, and expression of differentiation markers vary by airway level and species. This study defines a strategy for maintenance in vitro of differentiated Clara cells within their local microenvironment. Lungs from adult mice were inflated with 1% agarose and distal airways were isolated by microdissection. Explants were cultured for 7 days in serum-free medium. Preservation of Clara cell morphology after 7 days in culture (DIC) was demonstrated using light and electron microscopy. Ciliated cells were also present. Cytochrome P450 monooxygenase activity, as measured by naphthalene epoxidation, was decreased 50% between O and 7 DIC, but the apparent stereoselectivity of metabolism was unchanged at 7 days. Marker proteins for differentiated Clara cells (secretory protein, CYP2F2 and CYP2B4) were detectable immunochemically throughout time in culture. Glutathione S-transferase activity and levels of reduced glutathione were unchanged over 7 DIC. We conclude that differentiated Clara cells can be maintained in cultures of explants from defined airway regions. Bronchiolar epithelial cells in this system are viable, synthesize and secrete secretory protein, metabolize xenobiotics via the cytochrome P450 system, have a stable phase II enzyme system, and maintain glutathione pools.

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