Highly Expandable Human Alveolar Organoids to Study Respiratory Diseases

Abstract

Organoids are emerging to be an excellent tool for studying human development and disease. The COVID-19 pandemic has highlighted the importance of physiologically relevant alveolar infection models that include both alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. To address the need for an alveolar organoid culture system for respiratory research, we developed the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media for the highly efficient expansion of isolated primary human AT2 cells and subsequent differentiation into AT1 cells. Alveolar organoids were established from a panel of various donors (n=5) by culturing purified human AT2 cells in Corning® Matrigel® domes with serum-free PneumaCult™ Alveolar Organoid Expansion Medium. Typically by day 10-14 the organoids are fully established and display a spherical morphology. Alveolar organoids can then be either expanded long-term by passaging cultures as single cells in Expansion Medium or differentiated into AT1 cells using the PneumaCult™ Alveolar Organoid Differentiation Medium. Organoids in PneumaCult™ Alveolar Organoid Expansion Medium contain self-renewing AT2 cells marked by the expression of HT2-280 in 89.9 +/- 14.5 (mean +/- SD;n=5 donors) of cells and the presence of Pro-SPC, demonstrate a great expansion potential of > 10,000-fold with more than 13 population doublings within 10 passages (n=5 donors). Alveolar organoids differentiated for 10 days in the PneumaCult™ Alveolar Organoid Differentiation Medium downregulate AT2 markers HT2-280 and Pro-SPC and start expressing AT1 markers HT1-56 in 93.8 +/- 7.2 (mean +/- SD;n=5 donors) of cells and are positive for RAGE and GPRC5a. Furthermore, we assessed the expression of SARS-CoV-2 entry receptor ACE2, which is present in both undifferentiated and differentiated alveolar organoids.To investigate the use of these alveolar organoids for infectious disease modeling, AT2-containing alveolar organoids were transduced with a GFP-labelled Respiratory Syncytial Virus (RSV). Alveolar organoids were susceptible to viral infection and replication was confirmed by fluorescence microscopy and quantitative PCR. In summary, the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media are highly efficient and reproducible tools for the feeder-free expansion of AT2 cells and robust differentiation into AT1 cells, which can be used for infectious disease modeling.