3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes

Abstract

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Keywords: alveolar epithelial cells; biomimetics; curvature; ion track-etched membranes; microthermoforming; organ on a chip (OoC).

Figure 1

Concept of the 3D lung-on-a-chip model based on biomimetically microcurved culture membranes. (A) We approached a structure similar to a cut and flipped open alveolar sac as a cell-populated membrane with the microcurved shape, size, and also arrangement of its bioartificial alveoli mimicking the ones of the adult organ. Integrated in microfluidic chips/OoC devices where the microcurved membranes separate a top from a bottom compartment, they can be seeded by infusion with lung epithelial and microvascular endothelial cells on the top and bottom side of the membrane. The spatial cell distribution is then similar to the alveolar–capillary barrier. (B) Potential future applications of the model include 3D ALI culture (following submerged culture), modeling of disease and repair/regeneration, and toxicity and pharmaceutical efficacy testing (temporarily under submerged conditions or exposed to vapors or aerosols).

Figure 2

Microfabrication of the 3D lung-on-chip device. (A) Fabrication of the biomimetically microcurved culture membrane by thermoforming. An additional (dense) PP sealing film allows the micro pressure (thermo)forming of the porous PC film/membrane using compressed nitrogen. (B) Microfluidic chip construction and design. The OoC device consists of a top and a bottom housing half from polydimethylsiloxane (PDMS) containing microfluidic structures and the biomimetic membrane sandwiched in between. (C) Fabrication of the housing halves of the PDMS chip body by casting of the PDMS precursor over an SU-8-silicon mold, curing of the precursor, and peeling off the structured PDMS layer. (D) Assembly and chemical−thermal bonding of the housing halves and the microcurved membrane.

Figure 3

Visual inspection, geometrical characterization, and permeability testing of the biomimetically microcurved culture membrane from PC. (A) Top and bottom view of the microwell array (upper and lower image, respectively; SEM images; scale bar represents 500 μm). (B) Mixed top and side view of a section of the microwell array (SEM image; scale bar represents 50 μm). (C) Mixed bottom and side view of a section of the microwell array (SEM image; scale bar represents 50 μm). (D) Graph of microwell depths of formed membranes from three subsequent forming cycles (n = 3) (* and *** indicate p-values smaller than 0.05 and 0.001, respectively). Graph of (E) pore diameters and (F) densities of unformed membranes, and between and in the horizontal centers of the microwells of formed membranes from three subsequent forming cycles (n = 3) (** and **** indicate p-values smaller than 0.01 and 0.0001, respectively). (G) Graph of the apparent permeability of the unformed, flat membrane semifinished product and the formed, curved membrane (n = 3).

Figure 4

Geometrical characterization of the (bottom) housing half of the chip from PDMS, and perfusion and leak test of the assembled OoC device. (A) Each housing half of the microfluidic chip contained one of the two central circular culture chambers with a diameter of 8 mm for receiving the hexagonal microwell array. This chamber was on either side connected to an inlet and an outlet channel with in each case a width of 500 μm and a length of 4 mm. At their lateral/peripheral ends across the culture chamber, the two channels in turn were connected to in each case one smaller chamber with a diameter of 1 mm located in two opposite corners of the chip. The height/depth of the microfluidic compartments was around 400 μm. (B) Cross-section of an assembled 3D lung-on-chip device (stitched image; housing halves that during cell culture host epithelial and endothelial cells are colored blue and pink/purple, respectively; scale bar represents 500 μm). (C) Assembled lung-on-chip device with its top and bottom chip compartment perfused through press-fitted tubing with water colored with green and blue (food) dye, respectively (scale bar represents 8 mm).

Figure 5

Epithelialization of the microcurved membrane in the chip. HAECs cultured submerged under flow for 7 days and stained for cell nuclei and (A) F-actin, (B) tight junctions, (C) vimentin, and (D) CK8 (fluorescent microscopy images; nuclei not shown in the right halves of the images for better visibility of the individual stains; scale bars represent 100 μm).

Figure 6

ALI culture on the microcurved membrane in the chip. HAECs cultured at the ALI under perfusion for 14 days and stained for cell nuclei and (A) F-actin, (B) CK8, (C) aquaporin 5, and (D) pSPC (fluorescent microscopy images; scale bars represent 100 μm).

Figure 7

Thickness of the formed, curved alveolar epithelial layer. The thickness of the HAEC lining was measured (A) in two perpendicular cross-sections and there in each case in five different locations: at the horizontal center of the bottom of the microwell, at the left and right sidewall of the microwell directly under its convex rim, and at the left and right sidewall roughly halfway between, in each case perpendicular to the microwell wall. (B) Representative vertical and horizontal cross-sectional images of the epithelial layer (image planes “x” and “y”, and “z₁” to “z₃”, respectively; scale bars represent 50 μm). (C) Graph of the HAEC layer thickness as a function of the measurement location as stated in (A) (n = 3).

Figure 8

Lung epithelial and endothelial coculture on the microcurved membrane in the chip. (A) Top views of sections of the microcurved membrane with Calu-3 cells cultured for 11 days and stained for cell nuclei and tight junctions (fluorescent microscopy image; scale bars represent 100 μm). (B) Bottom views of sections of the same microcurved membrane with HLMVECs cultured for 11 days and stained for nuclei and CD31 (fluorescent microscopy image; scale bars represent 100 μm). (C) Cross-section of the microcurved membrane from (A) and (B) (scale bar represents 100 μm). (D) Graph of the count of Calu-3 cells (n = 4) and HLMVECs per square microwell unit (n = 3) (**** indicates a p-value smaller than 0.0001).