Isolation and Culture of Pulmonary Alveolar Epithelial Type II Cells

Dobbs, Leland G.; Gonzalez, Robert

doi:10.1002/0471221201.ch9

Cited by 17 publications

(15 citation statements)

References 47 publications

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“…The TI cell preparations used for these experiments contained 80 -91% TI cells and Ͻ1.5% TII cells, as analyzed by cytocentrifuged cell preparations stained with monoclonal antibodies specific for apical membranes of each cell type. The remaining cells consisted of alveolar macrophages and lymphocytes, as identified by their typical morphology after modified Papanicolaou staining (14). A total of seven different preparations of TI cells were used in these experiments.…”

Section: Cell Isolationmentioning

confidence: 99%

“…TII cells were isolated by previously described methods (14); TI cells were removed by negative selection with magnetic beads (28). The TII cell preparation used for these experiments contained 84 -92% TII cells and Ͻ0.1% TI cells.…”

Section: Cell Isolationmentioning

confidence: 99%

“…TII cells were cultured in the presence of 10% fetal bovine serum [University of California San Francisco (UCSF) Cell Culture Facility] as previously described (14) on tissue culture plastic previously coated with bovine fibronectin (200 g/ml) for 7 days. A total of eight separate TII cell isolations, four of which were for the cultured TII cells, were used in these experiments.…”

Section: Cell Isolationmentioning

confidence: 99%

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Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

Gonzalez¹,

Yang²,

Griffin³

et al. 2005

American Journal of Physiology-Lung Cellular and Molecular Phys

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Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes. We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained Ͻ2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells. gene expression profiling; transdifferentiation THE ALVEOLAR EPITHELIUM, which covers Ͼ99% of the very large internal surface area of the lung, comprises two cell types, type I (TI) and type II (TII) cells. TI cells are very large squamous cells with calculated diameters of 50 -100 m and volumes of 3,000 m 3 (56). Two or more very thin (ϳ50 nm) cytoplasmic sheets extend from the nucleus of the TI cell to cover the basement membrane that separates the epithelium from the interstitium. TI cells cover Ͼ98% of the internal surface area of the lungs. In contrast, TII cells are smaller, cuboidal cells (diameter ϳ10 m) characterized morphologically by surfactant-containing secretory granules called lamellar bodies. TI cells have an extremely high water permeability (13) and are capable of transporting ions (4, 33), suggesting that TI cells may play a role in lung liquid homeostasis. Additional functions of TI cells have been proposed, based on the known properties of several genes expressed in this cell type (9, 49, 62). TII cells, which cover the remainder of the alveolar surface, synthesize, secrete, and recycle surfactant components (reviewed in Ref. 51); TII cells also have the capacity to transport ions (26, 45), synthesize immune effector molecules (47, 57, 60), and act as progenitor cells following injury to the ...

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Section: Cell Isolationmentioning

confidence: 99%

Section: Cell Isolationmentioning

confidence: 99%

Section: Cell Isolationmentioning

confidence: 99%

See 1 more Smart Citation

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

Gonzalez¹,

Yang²,

Griffin³

et al. 2005

American Journal of Physiology-Lung Cellular and Molecular Phys

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show abstract

“…Although the primary culture of epithelial cells has disadvantages in that it involves repeated cell isolations and in that cells undergo different extents of phenotypic drift in culture, much has been learned from the study of primary cultures of both TI and TII cells. We have previously published a detailed method for the isolation of TII cells from rat lungs using the “panning method” to purify cells using IgG-coated plates (6–8). In this communication, we focus on methods using TI or TII cell-specific antibodies to isolate these cell types from rat lungs.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Culture of Alveolar Epithelial Type I and Type II Cells from Rat Lungs

Gonzalez

Dobbs

2012

Methods in Molecular Biology

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The pulmonary alveolar epithelium, comprised of alveolar Type I (TI) and Type II (TII) cells, covers more than 99% of the internal surface area of the lungs. The study of isolated and cultured alveolar epithelial TI and TII cells has provided a large amount of information about the functions of both cell types. This chapter provides information about methods for isolating and culturing both of these cell types from rat lungs.

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“…In this context, cell lines have been screened for their suitability to serve as in vitro respiratory cell models (Florea et al 2003;Foster et al 1998) and, with the aim of preserving the differentiated phenotype of the original tissue, airway epithelial cell lines have been obtained by immortalisation (Ehrhardt et al 2002Manford et al 2005). Furthermore, protocols have been established for the isolation and primary cultivation of respiratory epithelial cells, from various lung zones across several species (Gruenert et al 1995;Wise and Lechner 2002;Dobbs and Gonzales 2002). The differing properties of respiratory cell cultures used within the pharmaceutical sciences have recently been reviewed (Forbes and Ehrhardt 2005;Steimer et al 2005).…”

Section: Introductionmentioning

confidence: 99%

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

et al. 2006

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The distribution of the P-glycoprotein (P-gp/MDR1) efflux transporter at mucosal barriers has defined it as a functionally important element in limiting drug absorption into the systemic circulation. However, little is known about the distribution and functionality of P-gp/MDR1 in the human lung. Here, the presence of P-gp/MDR1 was investigated immunohistochemically in distal human lung tissue and at mRNA and protein levels in human alveolar epithelial cells (hAEpC) in primary culture. We studied the presence and activity of P-gp/MDR1 in hAEpC monolayers by Western blotting, by immunofluorescence microscopy and by conducting bi-directional transport studies employing a P-gp substrate (rhodamine 123) with and without a P-gp inhibitor (verapamil). The flux of fluorescein sodium was also examined as a paracellular transport marker. Alveolar tissue specimens showed P-gp localised at the luminal membranes of type I pneumocytes. Reverse transcription-polymerase chain reaction revealed the presence of mRNA encoding for P-gp/MDR1 in freshly isolated (i.e. type II) hAEpC and in monolayers of hAEpC cultured for 8 days (i.e. type I-like morphology). At the protein level, P-gp could be detected in hAEpC monolayers after 8 days in culture but not in freshly isolated type II pneumocytes. The flux of rhodamine 123 across hAEpC monolayers on day 8 in culture exhibited net secretion, which disappeared in the presence of verapamil. Fluorescein sodium fluxes showed no distinct directionality. Our findings indicate that P-gp is functionally active in the human alveolar airspace and that hAEpC monolayers might provide a suitable in vitro model for studying P-gp function mechanistically in the distal human lung.

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Isolation and Culture of Pulmonary Alveolar Epithelial Type II Cells

Cited by 17 publications

References 47 publications

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

Isolation and Culture of Alveolar Epithelial Type I and Type II Cells from Rat Lungs

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

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