147 Human Lung Organoids Predict Response to Carbon-Based Nanomaterials and Model Pulmonary Fibrosis

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Abstract Advances in in-vitro culture systems have stimulated an interest in more reliable testing approaches that predict nanomaterial toxicity. Lung organoids are one example, and display functions consistent with their in-vivo counterparts. Organoids have been used in a variety of applications, but studies have yet to interrogate their suitability for nanomaterial testing. Here, we report the step-wise development of multilineage lung organoids, and validate their potential as tools for predicting pulmonary toxicity of carbon-based nanomaterials. After 50-days of differentiation, the lung organoids exhibited the six major proximal (goblet/basal/club/ciliated) and distal (AECI/II) epithelial cell types of the adult lung. Using microinjection, we developed an exposure model to exploit the airspace-like lumen of the organoids for cargo delivery. We compared the impact of small nanometric (50-950nm, sGO) and large micrometric (4-41µm, lGO) graphene oxide sheets, with multi-walled carbon nanotubes (1-20µm, MWCNT) on lung organoids after 1/7/28d exposure. Consistent with in-vivo data, sGO and lGO exhibited a transient interaction with the alveolar epithelium, and did not induce any histoarchitectural changes indicative of fibrosis. Organoids did however produce 2.5-fold more mucins in response to lGO than sGO exposure, suggesting that a thicker mucin barrier is required to prevent lGO-induced damage. In contrast, MWCNT caused detrimental effects on organoid architecture. Its persistent interaction with the alveolar epithelium led to a pro-fibrotic phenotype, where MWCNT caused AECII to undergo epithelial-mesenchymal transition. With further development, our model may reduce the need for rodent inhalation studies in the assessment of lung toxicity.