A miniaturized multicellular platform to mimic the 3D structure of the alveolar-capillary barrier

Abstract

Several diseases affect the alveoli, and the efficacy of medical treatments and pharmaceutical therapies is hampered by the lack of pre-clinical models able to recreate in vitro the diseases. Microfluidic devices, mimicking the key structural and compositional features of the alveoli, offer several advantages to medium and high-throughput analysis of new candidate therapies. Here, we developed an alveolus-on-a-chip recapitulating the microanatomy of the physiological tissue by including the epithelium, the fibrous interstitial layer and the capillary endothelium. A PDMS device was obtained assembling a top layer and a bottom layer obtained by replica molding. A polycaprolactone/gelatin (PCL-Gel) electrospun membrane was included within the two layers supporting the seeding of 3 cell phenotypes. Epithelial cells were grown on a fibroblast-laden collagen hydrogel located on the top side of the PCL-Gel mats while endothelial cells were seeded on the basolateral side of the membrane. The innovative design of the microfluidic device allows to replicate both cell-cell and cell-extracellular matrix interactions according to the in vivo cell arrangement along with the establishment of physiologically relevant air-liquid interface conditions. Indeed, high cell viability was confirmed for up to 10 days and the formation of a tight endothelial and epithelial barrier was assessed by immunofluorescence assays.

Keywords: ECM-like substrate; alveolar-capillary barrier; alveolus-on-a-chip; cell tri-culture; in vitro models.

FIGURE 1

Bottom and top layer of the alveolus-on-a-chip. (A) CAD drawing of top layer and bottom layer (B) Photograph of the top layer mold and replica and optical microscope images of their characteristic elements. (C) Photograph of the bottom layer mold and replica and optical microscope images of their characteristic elements.

FIGURE 2

Assembled alveolus-on-a-chip. (A) Photograph and an optical microscope image of a section of the assembled device (i) and PCL-Gel membrane (ii). (B) Static leakage tests performed in the device layout without (i) and with (ii) valves.

FIGURE 3

Optimization of HVEC cell density on PCL-Gel membrane. (A) Quantitative evaluation of HVEC cell viability with CellTiter-Blue^® assay. Statistical difference (**p < 0.01; ****p < 0.0001). (B) Representative LIVE/DEAD images (n = 3) of HVEC seeded at different cell densities (5 × 10⁵ and 5 × 10⁵) at 1 day, 3 days and 7 days after seeding. ×10 magnification images (scale bar = 100 μm).

FIGURE 4

Evaluation of cell viability after 1 day and 10 days (7 days at ALI) of culture in the alveolus-on-a-chip. Representative fluorescence images (n = 3) of LIVE/DEAD assay in HVEC (in the basolateral chamber), A549 and MRC5 (in the apical chamber) tri-culture. Live and dead cells exhibited green and red fluorescence, respectively. ×10 magnification images (scale bar = 100 μm).

FIGURE 5

Evaluation of cell behavior in the alveolus-on-a-chip. Bright-field image of the apical and basolateral chambers of the chip (left). Representative fluorescence images (n = 3) of cytoskeleton staining in a549 and MRC5 (in the apical chamber), and HVEC (in the basolateral chamber) tri-culture after 3 days and 10 days (7 days at ALI). 4 x magnification images (scale bar = 500 μm).

FIGURE 6

Immunofluorescence staining for E-cadherin and Vimentin in the apical chamber of the alveolus-on-a-chip. Expression of E-cadherin (green) in A549 cells (A, B) and Vimentin (red) in MRC-5 fibroblasts (C, D) after 10 days (7 days at ALI) from the same optical field imaged at a different z stack position. (A, C) ×20 magnification images (scale bar = 10 μm) and (B, D) ×60 magnification images (scale bar = 20 μm) (n = 3).

FIGURE 7

Immunofluorescence staining of A549 and HVEC cells in the apical and basolateral chamber of the alveolus-on-a-chip. Expression of AQP-5 (magenta) and SP-C (green) in A549 cells (A) and ZO-1 (magenta) in A549 (left) and HVEC (right) (B) after 10 days (7 days at ALI). ×60 magnification images (scale bar = 20 μm) (n = 3).