Co-culture of type I and type II pneumocytes as a model of alveolar epithelium

Abstract

The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

A Monoclonal Human Alveolar Epithelial Cell Line (“Arlo”) with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections

“…[50,95] Similarly, multilayer formation by the polyclonal cell line hAELVi was demonstrated in our study supporting the findings of others. [50,96,97] The single-cell clone "Arlo," however, demonstrated the formation of a polarized monolayer in a direct comparison with the polyclonal hAELVi cell line under equal culture conditions. The evaluation of the tissue morphology, which is based on quantitative image analysis, demonstrates that the monolayer morphology of "Arlo" is not limited to certain areas within selected sections-it represents the dominant tissue architecture.…”

“…Another study looked at culture times of 30 d, but only reported maximum TEER values of ≈140 Ω cm 2 at day 10 of culture for NCI-H441 cultured under ALI conditions, which also declined rapidly after this maximum. [50] In addition, multilayer formation was reported for NCI-H441 in several studies, further complicating the assessment of TEER development when no quantitative assessment of multilayer formation is performed in parallel. [50,95] Similarly, multilayer formation by the polyclonal cell line hAELVi was demonstrated in our study supporting the findings of others.…”

“…[50] In addition, multilayer formation was reported for NCI-H441 in several studies, further complicating the assessment of TEER development when no quantitative assessment of multilayer formation is performed in parallel. [50,95] Similarly, multilayer formation by the polyclonal cell line hAELVi was demonstrated in our study supporting the findings of others. [50,96,97] The single-cell clone "Arlo," however, demonstrated the formation of a polarized monolayer in a direct comparison with the polyclonal hAELVi cell line under equal culture conditions.…”

“…[45,48] This led to inconsistencies in the development of barrier properties depending on the used cell culture vial, experienced by the authors of this study as well as by other laboratories leading to irreproducible experimental results. [49][50][51] We here report the development as well as the characterization of a new monoclonal cell line named "Arlo." The cell line is based on a single-cell clone of the polyclonal hAELVi cell line generated by single-cell printing and shows enhanced barrier properties while preserving a monolayer morphology.…”

3D Lung Tissue Models for Studies on SARS-CoV-2 Pathophysiology and Therapeutics

“…In addition, unlike airway epithelial cells, a consistent method to passage and expand primary ATII cells under 2D conditions has not yet been established. A few studies have investigated the culture of alveolar epithelial cell lines (such as A549 and NCI-H441) or inducible pluripotent stem cells (iPSC)-derived ATII cells under ALI, showing increased cell polarization, barrier formation, ion transport, and cell maturation [ 38 , 39 , 40 ]. However, none of these models supports both the maintenance of the ATII phenotype and the differentiation into ATI cells.…”