Separation of bovine bronchial epithelial cell subpopulations by density centrifugation: a method to isolate ciliated and nonciliated cell fractions.

H. Takizawa, Debra Romberger, J. D. Beckmann, T. Matsuda, C. Eccleston-Joyner, S. Shoji, K. A. Rickard, L. R. Claassen, R. F. Ertl, James Linder

Research output: Contribution to journalArticle

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Abstract

Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.

Original languageEnglish (US)
Pages (from-to)553-562
Number of pages10
JournalAmerican journal of respiratory cell and molecular biology
Volume3
Issue number6
DOIs
StatePublished - Dec 1990

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Centrifugation
Cell Count
Epithelial Cells
Cells
Fibronectins
Peptide Hydrolases
Cytology
Immunosorbents
Trypan Blue
Cell proliferation
Silicon Dioxide
Thymidine
Assays
Suspensions
Microscopic examination
Coloring Agents
Availability
Electrons
DNA
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Separation of bovine bronchial epithelial cell subpopulations by density centrifugation : a method to isolate ciliated and nonciliated cell fractions. / Takizawa, H.; Romberger, Debra; Beckmann, J. D.; Matsuda, T.; Eccleston-Joyner, C.; Shoji, S.; Rickard, K. A.; Claassen, L. R.; Ertl, R. F.; Linder, James.

In: American journal of respiratory cell and molecular biology, Vol. 3, No. 6, 12.1990, p. 553-562.

Research output: Contribution to journalArticle

Takizawa, H. ; Romberger, Debra ; Beckmann, J. D. ; Matsuda, T. ; Eccleston-Joyner, C. ; Shoji, S. ; Rickard, K. A. ; Claassen, L. R. ; Ertl, R. F. ; Linder, James. / Separation of bovine bronchial epithelial cell subpopulations by density centrifugation : a method to isolate ciliated and nonciliated cell fractions. In: American journal of respiratory cell and molecular biology. 1990 ; Vol. 3, No. 6. pp. 553-562.
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abstract = "Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95{\%} ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88{\%} nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2{\%} fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.",
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T2 - a method to isolate ciliated and nonciliated cell fractions.

AU - Takizawa, H.

AU - Romberger, Debra

AU - Beckmann, J. D.

AU - Matsuda, T.

AU - Eccleston-Joyner, C.

AU - Shoji, S.

AU - Rickard, K. A.

AU - Claassen, L. R.

AU - Ertl, R. F.

AU - Linder, James

PY - 1990/12

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N2 - Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.

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