AlveoMPU: Bridging the Gap in Lung Model Interactions Using a Novel Alveolar Bilayer Film

Abstract

The alveoli, critical sites for gas exchange in the lungs, comprise alveolar epithelial cells and pulmonary capillary endothelial cells. Traditional experimental models rely on porous polyethylene terephthalate or polycarbonate membranes, which restrict direct cell-to-cell contact. To address this limitation, we developed AlveoMPU, a new foam-based mortar-like polyurethane-formed alveolar model that facilitates direct cell-cell interactions. AlveoMPU features a unique anisotropic mortar-shaped configuration with larger pores at the top and smaller pores at the bottom, allowing the alveolar epithelial cells to gradually extend toward the bottom. The underside of the film is remarkably thin, enabling seeded pulmonary microvascular endothelial cells to interact with alveolar epithelial cells. Using AlveoMPU, it is possible to construct a bilayer structure mimicking the alveoli, potentially serving as a model that accurately simulates the actual alveoli. This innovative model can be utilized as a drug-screening tool for measuring transepithelial electrical resistance, assessing substance permeability, observing cytokine secretion during inflammation, and evaluating drug efficacy and pharmacokinetics.

Keywords: alveolar; drug screening; in vitro; polyurethane; porous membrane.

Figure 1

Conceptual diagram of AlveoMPU simulating the actual structure of the lungs and the interaction between alveolar epithelial cells and microvascular endothelial cells.

Figure 2

Characterization of the physicochemical properties of the porous polyurethane membrane (P-PU) used in AlveoMPU. (a) Scanning electron microscopy (SEM) image viewed from an oblique angle. (b) Atomic force microscopy (AFM) height image of the AlveoMPU membrane. (c) Distribution of pore diameters for the upper and lower sides of the AlveoMPU membrane. Red curved line: Gaussian distribution obtained from the frequency data of pore sizes. (d) Stress–strain curve for flat-polyurethane (F-PU), porous PU (P-PU; AlveoMPU), polycarbonate with 3 µm pores (PC3) and polyethylene terephthalate with 3 µm pores (PET3). (e) Time dependency of contact angles of F-PU and the P-PU. Two independent samples for F-PU and P-PU were replicated and are denoted by the blue and red curved lines, respectively.

Figure 3

Formation of alveolar model and barrier function on AlveoMPU membrane. (a) The temporal progression of trans-epithelial electrical resistance (TEER). (b) Scanning electron microscopy (SEM) images of the top surface of AlveoMPU; (b-ii): zoomed-in view of the image in (b-i); AT1 and AT2: type 1 and 2 alveolar epithelial cells, respectively. (c) Transmission electron microscopy (TEM) images of a cross section of AlveoMPU; P-PU: porous polyurethane of AlveoMPU, AE: alveolar epithelial cell, MV: microvascular endothelial cell; (c-i): The formation of sheets by alveolar epithelial and vascular endothelial cells across P-PU; (c-ii,c-iii): magnified views within white and black frames in (c-i); white arrowhead: collagen thin layer, black arrowhead: hemidesmosome-like structure, TJ: tight junction, DS: desmosome. (d) Immunostaining of alveolar epithelium (green: cytokeratine A1/A3) and vascular endothelium (red: vimentin)-specific markers and (e) tight junction marker proteins (green: Occludin, red: ZO-1, blue: nuclei) and (f) collagen backing layer (green: Type I Collagen, red: plasma membrane).

Figure 4

Evaluation of inflammatory cytokine secretion and pharmacokinetic testing utilizing AlveoMPU. Cytokine production of AlveoMPU models into apical (a,d,g), basolateral sides (b,e,h) or HLMVECs culture supernatant (c,f,i); IL-6: (a–c), IL-8: (d–f) and TNFα: (g–i). (j) Apparent permeability coefficient (Papp) of 4 kDa dextran-FITC (FD4) and fluticasone propionate. (k) Papp of FD4 after pretreatment of fluticasone propionate; white bar: control; gray bar: poly (I:C) (PIC); black bar: lipopolysaccharide (LPS); *: statistically significant difference (p < 0.05).