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Decellularized extracellular matrix hydrogels for human airway organoid culture

Background and aims: The majority of current organoids are grown within Matrigel, a protein mixture secreted by mouse sarcoma cells, due to the fact that it mimics the basement membrane and contains growth factors that support organoid formation. However, concerns have arisen as to the suitability of Matrigel for studying normal regeneration and stem cell behavior due to its cancerous origin. Hydrogels generated from pepsin extracted, decellularized extracellular matrix (dECM) solutions have recently emerged as a potential option for culturing organoids for other tissues, such as gut, but have not been evaluated for culturing primary lung stem or progenitor cells. Here, we developed protocols to generate dECM hydrogels from porcine lung and evaluated their potential for culturing organoids from human bronchial epithelial cells (HBECs) as an alternative matrix to Matrigel. Methods and results: HBECs were isolated from discarded surgical waste following resizing of airways for lung transplantation and seeded in Matrigel or dECM hydrogels. After 14 days of cell culture, we found that organoids formed in both Matrigel and in dECM hydrogels, with similar differentiation capacity, as assessed by immunofluorescence. Next, we performed bulk RNA sequencing of HBEC derived organoids from Matrigel or dECM to assess differences imposed by the surrounding matrix. We found that organoids formed in Matrigel expressed upregulation of oncogenic markers and pathways known to be aberrant in cancer as compared to organoids formed in dECM. Conclusion: We successfully developed decellularized porcine lung derived ECM gels which supported airway organoid formation. dECM hydrogels may be an alternative to Matrigel to study lung or airway regenerative capacity in a more physiologically relevant environment.