A multiplex inhalation platform to model in situ like aerosol delivery in a breathing lung-on-chip

Abstract

Prolonged exposure to environmental respirable toxicants can lead to the development and worsening of severe respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and fibrosis. The limited number of FDA-approved inhaled drugs for these serious lung conditions has led to a shift from in vivo towards the use of alternative in vitro human-relevant models to better predict the toxicity of inhaled particles in preclinical research. While there are several inhalation exposure models for the upper airways, the fragile and dynamic nature of the alveolar microenvironment has limited the development of reproducible exposure models for the distal lung. Here, we present a mechanistic approach using a new generation of exposure systems, the Cloud α AX12. This novel in vitro inhalation tool consists of a cloud-based exposure chamber (VITROCELL) that integrates the breathing ^AXLung-on-chip system (AlveoliX). The ultrathin and porous membrane of the AX12 plate was used to create a complex multicellular model that enables key physiological culture conditions: the air-liquid interface (ALI) and the three-dimensional cyclic stretch (CS). Human-relevant cellular models were established for a) the distal alveolar-capillary interface using primary cell-derived immortalized alveolar epithelial cells (^AXiAECs), macrophages (THP-1) and endothelial (HLMVEC) cells, and b) the upper-airways using Calu3 cells. Primary human alveolar epithelial cells (^AXhAEpCs) were used to validate the toxicity results obtained from the immortalized cell lines. To mimic in vivo relevant aerosol exposures with the Cloud α AX12, three different models were established using: a) titanium dioxide (TiO2) and zinc oxide nanoparticles b) polyhexamethylene guanidine a toxic chemical and c) an anti-inflammatory inhaled corticosteroid, fluticasone propionate (FL). Our results suggest an important synergistic effect on the air-blood barrier sensitivity, cytotoxicity and inflammation, when air-liquid interface and cyclic stretch culture conditions are combined. To the best of our knowledge, this is the first time that an in vitro inhalation exposure system for the distal lung has been described with a breathing lung-on-chip technology. The Cloud α AX12 model thus represents a state-of-the-art pre-clinical tool to study inhalation toxicity risks, drug safety and efficacy.

Keywords: COPD; aerosolized drug delivery; air-liquid interface; cyclic stretch; inhalation therapeutics; lung-on-chip; nanoparticles; toxicity assessments.

FIGURE 1

Overview of the Cloud α AX12 platform (A) An illustration showing the alveolar barrier in situ. The alveolar epithelial cells are situated on a thin basement membrane and are in close contact with the endothelial cells (ECs) on the basal side. The alveolar epithelium is comprised of squamous and thin alveolar type 1 (AT1) and cuboidal alveolar type 2 (AT2) cells along with resident alveolar macrophages (AMs). Inhaled toxic particles are represented as black and grey spheres. Inhalation of such airborne toxic particles result in lung inflammation, toxicity and progression to COPD. (B) Schematic representation of the ^AXLung-on-chip (including ^AXBreather, ^AXExchanger, ^AXDock platform and the lung-on-chip consumable AX12) and Cloud α AX12 platform to model healthy and inflamed alveolar barrier in vitro. Primary and immortalized cells isolated from human sources were used in the model. Inflammation on-chip was mimicked using nebulized compounds with the Cloud α AX12. Created with BioRender.com.

FIGURE 2

Homogenous dose distribution using the Cloud α AX12 (A) Design of the Cloud α AX12 comprised of a stainless-steel base module maintained at 37°C containing the AX12 plate placed on top covered by a steel plate. The holes on the steel base-plate are aligned to the wells within the AX12 plate. The QCM 6 placed inside the polycarbonate removable exposure hood quantify real-time cell delivered dose and are recorded and analyzed with the Vitrocell software. The nebulizer on top relies on a piezoelectric vibrating mesh to form a consistent aerosol cloud from the solubilized substance nebulized. (B) Detailed closer inspection of the orientation and placement for the AX12 plate and the QCM 6 sensor. (C) Comparison between deposited mass as measured from the QCM 6 readings (in green) and theoretically calculated (in blue). (D) O.R. (mL/min) was measured for different volumes (100–500 µL) for two SBLs (0.9% NaCl in distilled water and 1% PBS in distilled water) (n = 3). Data shown as mean ± SEM. (E) PHMG compound was dissolved in NaCl suspension base liquid as a reference. O.R. was measured for SBL without and with compound dilution using the Cloud α AX12 (n = 3). (F) Dose distribution measured from fluorescence signals [A.U.] of nebulized Fluorescein deposited on-chip. Signals from each well depicted in color scheme (white to blue; low fluorescence signal to high) from two individual rounds in AX12 plates (N = 2; n = 12).

FIGURE 3

Nanoparticles (NPs)-induced inflammation and barrier disruption in ^AXiAECs mono-culture on-chip (A) The brown dotted corner bracket suggests the distinction between different trigger-induced models. On day 21 of the mono culture experiment, the ^AXiAECs were exposed to the ZnO NPs on-chip. Timeline and schematic provide further details of the experiment. (B) TER values (normalized to 0 h timepoint) for ALI and ALI + CS cell cultures exposed to ZnO NPs (n = 3/condition/timepoint). (C) Relative LDH release (NPs exposed with respect to Control; CTRL) shown for 48 h timepoint for ALI and ALI + CS cells (n = 4). (D) mRNA was isolated from CTRL and ZnO exposed cells (ALI + CS) at 48 h exposure timepoint. qPCR studies were performed with n = 4/conditions and exposure significance were measured in relation to CTRL expression levels. (E) Results for ^AXiAECs mono-cell culture model with TiO2 NPs is denoted by opening another brown dotted corner bracket. Timeline and schematic of TiO2 NPs nebulization on day 21 in monoculture (^AXiAECs) on-chip. (F) Absolute TER values (Ohm-cm2) compared pre-TiO2 NPs exposure (at 0 h) with 24h and 48 h after exposure (N = 2; n = 6). (G) Significance for cytotoxicity was calculated relative to respective ALI or ALI+CS CTRL. Cytotoxicity was calculated from LDH release at 24h and 48 h after TiO2 NPs nebulization from both ALI and ALI + CS samples (N = 2; n = 4/time-point). (H) ROS generation was measured using the H2-DCFDA assay (N = 2; n = 4). Fluorescence intensity for exposed cells were normalized with respective healthy CTRL. (I) Representative immunofluorescence staining for alveolar barrier after 48 h of TiO2 NPs exposure. Cells were probed for tight junction, ZO1 (green) and nuclei with Hoechst (blue). Scale bar is 20 µm. Data are shown as mean ± SEM.

FIGURE 4

TiO2 NPs-induced inflammation and barrier disruption in triple-cell culture model on-chip (A) Overview of the timeline for cell seeding and NPs exposure on-chip. (B) Representative immunofluorescent (maximum projection intensity) images for alveolar apical and basal endothelial barrier stained with ZO1 (in green) and nuclei with Hoechst (in blue) under ALI + CS. Scale bar here is 20 µm. (C) TER (Ohm-cm2) measured before at 0 h and after TiO2 NPs exposure at 24 h and 48 h (N = 2; n = 6). (D) LDH release was measured from 24 h to 48 h samples in ALI and ALI + CS conditions (N = 2; n = 4/conditions/time-point). Significance was measured relative to the respective ALI or ALI+CS CTRL. (E) mRNA was harvested at 48 h time-point. Gene expression for epithelial, inflammation and macrophage related markers were measured in both ALI and ALI + CS conditions (N = 2, n = 4/conditions). Data are shown as mean ± SEM.

FIGURE 5

NPs-induced inflammation in primary human alveolar epithelial cells (^AXhAEpC) on-chip (A) A schematic representation ^AXhAEpCs seeded in the AX12 plate. The timeline shows the cell culture and NP exposure process on-chip. (B) TER values (Ohm-cm2) were recorded before NPs nebulization (at 0 h) and at 6h and 24 h after TiO2 and TiO2+ZnO mixed NPs exposure (N = 2; n = 6). The significance of exposure at 6h and 24 h was measured compared with the values at 0 h. (C) The levels of LDH at 24 h were normalized using untreated control samples (ALI and ALI + CS) (N = 2, n = 4). (D) Gene expression was analyzed 24 h after NPs exposure using mRNA harvested from ALI + CS cell cultures. The fold change values were normalized to healthy controls (N = 2, n = 4). Data are shown as mean ± SEM.

FIGURE 6

Aerosolized PHMG induced barrier disruption and cytotoxicity in triple-cell culture on-chip (A) Timeline and schematic of PHMG nebulization at day 21 in tri-cell culture (^AXiAECs/d-THP1/HLMVEC) model on-chip. (B) Representative immunofluorescence staining for apical alveolar and basal endothelial barrier after 24 h of PHMG exposure. Cells were analyzed for tight junction, ZO1 (green) and nuclei with Hoechst (blue). Scale bar is 50 µm. (C) TER values (Ohm-cm²) were recorded before PHMG nebulization (at 0 h) and at 4 h, 24 h and 48 h after exposure (N = 3; n = 12). (D) Cytotoxicity was calculated from LDH release at 24 h and 48 h after PHMG nebulization from both ALI and ALI + CS conditions. LDH release was normalized with respective untreated controls. Data shown as mean ± SEM (N = 1; n = 3/condition/timepoint). (E) Gene expression after 48 h of exposure was assessed for cells in ALI + CS condition. Fold change values were normalized with respective untreated controls. Data presented as mean ± SEM (N = 3; n = 6).

FIGURE 7

Inhaled Fluticasone reduced PHMG-induced inflammation and EMT (A) Schematic timeline of the PHMG exposure and subsequent FL treatment on ^AXiAECs mono-culture in 96- well plates. (B) Representative stainings of ^AXiAECs exposed to PHMG with/without FL treatment under ALI with αMA (in red) and Hoechst (in blue). Scale bar is 100 µm. (C) The mean fluorescent intensity (MFI) was calculated and the proprotion of αSMA expression was calculated by normalizing with the nuclei intensity. PHMG exposed cells (+PHMG, -FL) was compared with healthy untreated controls (-PHMG, -FL) to check inflammation by PHMG. Next, PHMG exposed cells (+PHMG, -FL) were compared with PHMG and FL treated cells (+PHMG, +100nM/500nM/1000 nM FL) to check anti-inflammatory effect of FL. Region of interest (ROI or n = 6/conditions). Data are shown as mean ± SEM. (D) Brown dotted brackets represent on-chip experiments. Timeline and schematic for PHMG and FL treatment in tri-cell culture (^AXiAECs/d-THP1s/HLMVECs) on-chip. (E) Selected genes for epithelial cells, inflammation-associated, macrophage and endothelial cells were measured under both ALI and ALI + CS conditions on-chip. Significance associated with FL treated cells were estimated by comparing inflamed cells (+PHMG, -FL) with 100 nM (+PHMG, +100 nM FL) and 500 nM (+PHMG, +500 nM FL) FL nebulized cells (N = 2, n = 8). Data are shown as mean ± SEM normalized to PHMG induced but without subsequent FL (+PHMG -FL) treatment.