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Porcine Alveolar Epithelial Cells in Primary Culture: Morphological, Bioelectrical and Immunocytochemical Characterization

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Abstract

Purpose

The purpose of this study was to establish a primary culture of porcine lung epithelial cells as an alternative to the currently existing cell cultures from other species, such as e.g., rat or human. Primary porcine lung epithelial cells were isolated, cultivated and analyzed at distinct time points after isolation.

Materials and Methods

The main part of the work focused on the morphology of the cells and the detection of alveolar epithelial cell markers by using electron microscopy, immunofluorescence microscopy and immunoblotting. Regarding a later use for in vitro pulmonary drug absorption studies the barrier properties of the cell monolayer were evaluated by monitoring bioelectrical parameters and by marker transport.

Results

Epithelial cells isolated from porcine lung grew to confluent monolayers with typical intercellular junctions within a few days. Maximum transepithelial resistance of about 2,000 Ωcm2 was achieved and demonstrated the formation of a tight epithelial barrier. Permeability data of sodium fluorescein recommended a minimal transepithelial resistance of 600 Ωcm2 for transport studies. The cell population changed from a heterogeneous morphology and marker distribution (caveolin-1, pro-SP-C, surface sugars) towards a monolayer consisting of two cell types resembling type I and type II pneumocytes.

Conclusions

The porcine alveolar epithelial primary cell culture holds promise for drug transport studies, because it shares major hallmarks of the mammalian alveolar epithelium and it is easily available and scaled up for drug screening.

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Abbreviations

(ab):

transport direction from apical to basolateral

AP:

alkaline phosphatase

ATCC:

american type culture collection

Flu-Na:

sodium fluorescein

hAEpC:

human alveolar epithelial cells

kDa:

kilo dalton

MPA:

maclura pomifera lectin

pAEpC:

porcine alveolar epithelial cells

pAEpC-n :

pAEpC isolation number n (cell batch)

P app :

apparent permeability coefficient

PD:

potential difference

rAEpC:

rat alveolar epithelial cells

RCA:

ricinus communis lectin

rpm:

revolutions per minute

SAGM:

small airway growth medium

SDS:

sodium dodecyl sufate

TEER:

transepithelial electrical resistance

TEERmax :

maximum transepithelial electrical resistance

TRIS:

Tris(hydroxymethyl)methylamine (buffer)

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Acknowledgments

We would like to thank Dr. Carsten Ehrhardt for helpful discussions and cooperation in immunostaining and confocal laser scanning microscopy, Birgit Leis and Norbert Pütz for technical assistance in electron microscopy and Manuel Birke (Symbiotec) for his kind introduction to the BioRad Scanner.

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Authors and Affiliations

  1. Across Barriers GmbH, Department R&D Cell & Tissue based Systems, Science Park Saar, Saarbrücken, Germany

    Anne Steimer, Helmut Franke & Eleonore Haltner-Ukomado

  2. Center for Electron Microscopy, Department of Anatomy and Cell Biology, Medical Faculty, Saarland University, Homburg, Germany

    Michael Laue

  3. Biopharmaceutics and Pharmaceutical Technology, Saarland University, PO Box 151150, 66041, Saarbrücken, Germany

    Anne Steimer & Claus-Michael Lehr

  4. Present Address: Robert Koch-Institute, Berlin, Germany

    Michael Laue

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  1. Anne Steimer
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Correspondence to Claus-Michael Lehr.

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Steimer, A., Laue, M., Franke, H. et al. Porcine Alveolar Epithelial Cells in Primary Culture: Morphological, Bioelectrical and Immunocytochemical Characterization. Pharm Res 23, 2078–2093 (2006). https://doi.org/10.1007/s11095-006-9057-7

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