Characterisation of the R3/1 cell line as an alveolar epithelial cell model for drug disposition studies

https://doi.org/10.1016/j.ejps.2008.11.010Get rights and content

Abstract

The rat cell line R3/1 displays several phenotypical features of alveolar epithelial type I cells. In order to evaluate this cell line as potential in vitro model for drug disposition studies, R3/1 cells were cultured on Transwell filters and the transepithelial electrical resistance (TEER) was measured to test the integrity of cell layers. The mRNA expression of cell junctional components including E-cadherin, occludin, ZO-1 and ZO-2 was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) and the corresponding proteins by immunofluorescence microscopy (IFM). Moreover, the expression pattern of catabolic peptidases, carboxypeptidase M, aminopeptidases (AP): A, B, N and P, γ-glutamyltransferase (GGT), dipeptidylpeptidase IV, angiotensin-converting enzyme (ACE), and endopeptidases (EP) 24.11 and 24.15 was analysed in R3/1 cells and compared to rat alveolar epithelial I-like cells in primary culture.

TEER peaked at 99 ± 17 Ω cm2 after 5 days in culture. Addition of 0.1 μM dexamethasone (DEX) with 20% foetal bovine serum further increased TEER by 65%. However, none of the culture conditions used in our study yielded monolayers with TEER values comparable to those of primary cultures of rat pneumocytes. No transcripts encoding for E-cadherin and occludin were detected by RT-PCR. However, ZO-1 and -2 mRNA transcripts were found. IFM using a monoclonal antibody against occludin confirmed the absence of the protein in R3/1 cells. Of the investigated proteolytic enzymes, mRNA transcripts encoding APA and APB as well as EP 24.11 and EP 24.15 were detected; a pattern similar to that of rat alveolar epithelial I-like cells in primary culture.

Thus, although R3/1 cells express certain markers typical for type I pneumocytes (e.g., T1α, ICAM-1, connexin-43, caveolins-1 and -2) they do not form electrically tight monolayers. This excludes R3/1 cells from being used as an in vitro model for alveolar absorption. However, the cell line may be suitable to study stability of inhaled and endogenous proteins.

Introduction

Drug delivery via the pulmonary route represents an attractive avenue for the non-invasive delivery of many clinically relevant compounds. However, limitations need to be acknowledged, among which are the potential impact of active transport mechanisms (e.g., membrane transporters and vesicular transport) upon the epithelial absorption. Moreover, the influence of metabolic enzymes and catabolic peptidases on the drug disposition process (particularly of biopharmaceuticals) is poorly understood (Patton, 1996).

Cell-based in vitro models are useful tools for investigations of drug transport and stability at the various epithelia of the lung (Sporty et al., 2008, Forbes and Ehrhardt, 2005). Due to the lack of availability of human lung tissue and ethical constrains pertaining to use of human tissues, most studies have been based on isolation and culture of alveolar epithelial cells (AECs) from the lungs of animals including mouse (Corti et al., 1996), rat (Goodman and Crandall, 1982), rabbit (Shen et al., 1999) and pig (Steimer et al., 2007). Since species differences between human and rodents might be more significant than once assumed (King and Agre, 2001), confirmation of relevance of rodent data to human using human pneumocyte cultures is crucial (Wang et al., 2007, Bur et al., 2006, Ehrhardt et al., 2005).

Primary culture techniques of AECs which involve isolation, purification, and culture of alveolar epithelial type II (ATII) cells from tissues obtained after lung resections or from isolated perfused lungs, are used for most in vitro studies of alveolar epithelial function due to the paucity of appropriate alveolar epithelial cell lines that form functional tight junctions (Kim et al., 2001). These ATII cells, when plated on permeable supports or plastic under appropriate culture conditions, acquire type I cell-like phenotypes and morphologies (Demling et al., 2006, Fuchs et al., 2003, Wang et al., 2007). Although isolation of alveolar epithelial type I (ATI) pneumocytes from rat lungs has recently been reported with some success (Borok et al., 2002, Johnson et al., 2002, Chen et al., 2004), development of confluent ATI cell monolayers with electrically tight characteristics has not been reported thus far. It should be noted that unlike many other cells in primary culture, AEC generally exhibit a very limited proliferation profile and are therefore not suitable for passaging (Sporty et al., 2008). Thus, a new preparation of cells must be generated and used for each data set which is tremendously costly and time consuming.

While a number of immortalised cell lines emanating from different cell types of the airway (i.e., tracheo-bronchial) epithelium of lungs from various mammalian species are available (Sporty et al., 2008, Kemp et al., 2008), reliable and continuously growing cell lines that possess alveolar epithelial cell morphology and phenotype have not been reported to date. Most studies have relied on the use of cell lines of alveolar epithelial origin, e.g., A549 cells, for drug absorption studies with observations that are meaningless or hard to extend to humans (Sporty et al., 2008, Lieber et al., 1976, Foster et al., 1998, Elbert et al., 1999).

The R3/1 cell line was established from pulmonary tissue of foetuses of Han-Wistar rats on day 20 of gestation by explant-replica techniques (Knebel et al., 1994). It has been reported to display several phenotypical features of alveolar epithelial type I cells (Koslowski et al., 2004, Barth et al., 2005, Reynolds et al., 2008). In order to characterise the cell line as an in vitro model for drug disposition studies, R3/1 cells were cultured on filter inserts at different seeding densities, in various cell culture media, as well as under liquid-covered vs. air-interfaced conditions. The transepithelial electrical resistance (TEER) was measured as a parameter for the integrity of cell layers. Presence of cell junctional proteins (E-cadherin, occludin, ZO-1, ZO-2) in R3/1 cells was studied as well as the expression pattern of catabolic peptidases. The following peptidases were investigated: carboxypeptidase M (CPM); aminopeptidases (AP): A, B, N, P; γ-glutamyl transpeptidase (GGT); angiotensin-converting enzyme (ACE); endopeptidases (EP): 24.11 (neprilysin), 24.15 (thimet oligopeptidase 1). Results were compared with data obtained from rat alveolar epithelial type I-like and type II cells in primary culture.

Section snippets

Continuous cell line

R3/1 cells of passage numbers 40–59 were grown in an 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (Sigma, Dublin, Ireland) at 37 °C in 5% CO2 atmosphere. The DMEM/Ham's F-12 was supplemented with 10% (v/v) foetal bovine serum (FBS), 1% (v/v) non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES. Moreover, RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin was used as an alternative medium. The media

R3/1 monolayer integrity

When cultured on Transwell Clear filters, R3/1 cells did not form monolayers with TEER values comparable to those of primary cultures of rat pneumocytes that typically reach epithelial resistances of >1000 Ω cm2 (Cheek et al., 1989) (Fig. 1). The peak TEER value of R3/1 monolayers was 99 ± 17 Ω cm2, after 5 days in culture. The changes in the seeding density from 50,000 to 800,000 cells/cm2 as well as culturing under LCC vs. AIC conditions did not significantly affect TEER of R3/1 monolayers (Fig. 1A

Discussion

We have shown that, R3/1 cells express some markers typical for type I pneumocytes (22) including T1α, ICAM-1, connexin-43, caveolins-1 and -2. In contrast to primary rat pneumocytes, these cells do not form electrically tight monolayers. Therefore, R3/1 cells cannot be considered as a reliable in vitro model for alveolar absorption studies.

However, our data indicate that R3/1 cells may be suitable for stability assays of inhaled proteins.

Pulmonary delivery of macromolecule aerosols has been

Acknowledgements

This study was funded by grants from the EU (Lenka Horálková and Sibylle Endter, personal GALENOS Marie-Curie Early Stage Research Training Fellowships, MEST CT-2004-504992), DFG (Roland Koslowski, KO 2219/4-1) and the National Development Plan co-funded by EU Structural Funds and Science Foundation Ireland (Carsten Ehrhardt, Strategic Research Cluster grant 07/SRC/B1154; Marek W. Radomski and Aneta Radziwon are supported by Science Foundation Ireland). We appreciate the critical reading of the

References (35)

  • K. Barth et al.

    Distribution of caveolin-1 and connexin43 in normal and injured alveolar epithelial R3/1 cells

    Histochem. Cell Biol.

    (2005)
  • Z. Borok et al.

    Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro

    Am. J. Respir. Cell Mol. Biol.

    (1995)
  • Z. Borok et al.

    Alveolar epithelial ion and fluid transport: Na transport proteins are expressed by rat alveolar epithelial type I cells

    Am. J. Physiol.

    (2002)
  • J.M. Cheek et al.

    Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport

    Am. J. Physiol.

    (1989)
  • M. Corti et al.

    Isolation and primary culture of murine alveolar type II cells

    Am. J. Respir. Cell Mol. Biol.

    (1996)
  • S.I. Danto et al.

    Reversible transdifferentiation of alveolar epithelial cells

    Am. J. Respir. Cell Mol. Biol.

    (1995)
  • N. Demling et al.

    Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells

    Cell Tissue Res.

    (2006)
  • Cited by (5)

    View full text