Simple technique for culture of highly differentiated cells from dog tracheal epithelium

M. Kondo, W. E. Finkbeiner, Jonathan Widdicombe

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66 Citations (Scopus)

Abstract

Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (I(sc)), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 105 cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5% fetal calf serum (FCS medium). Transepithelial resistance (R(te)) and the increases in I(sc) and intracellular Ca in response to isoproterenol were also increased. However, baseline I(sc) and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in R(te), I(sc), and changes in I(sc) in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of >500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume261
Issue number2 5-1
StatePublished - 1991
Externally publishedYes

Fingerprint

Culture Techniques
Epithelium
Dogs
Intercellular Signaling Peptides and Proteins
Isoproterenol
Collagen
Cell Count
Air
Membranes
Cilia
Amiloride
Ion Transport
Growth
Microvilli
Trachea
Serum
Cyclic AMP
Cultured Cells

Keywords

  • Adenosine 3',5'-cyclic monophosphate
  • Air interface
  • Chloride secretion
  • Intracellular calcium
  • Serum-free culture medium
  • Ussing chambers

ASJC Scopus subject areas

  • Cell Biology
  • Physiology
  • Pulmonary and Respiratory Medicine

Cite this

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abstract = "Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (I(sc)), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 105 cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5{\%} fetal calf serum (FCS medium). Transepithelial resistance (R(te)) and the increases in I(sc) and intracellular Ca in response to isoproterenol were also increased. However, baseline I(sc) and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in R(te), I(sc), and changes in I(sc) in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of >500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.",
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AU - Kondo, M.

AU - Finkbeiner, W. E.

AU - Widdicombe, Jonathan

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N2 - Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (I(sc)), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 105 cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5% fetal calf serum (FCS medium). Transepithelial resistance (R(te)) and the increases in I(sc) and intracellular Ca in response to isoproterenol were also increased. However, baseline I(sc) and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in R(te), I(sc), and changes in I(sc) in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of >500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.

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