RESUMO
Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air-Liquid Interface (ALI) models, yet the limited accessibility of the organoids' apical side has hindered their applications in studies focusing on host-pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed. These apical-out airway organoids are generated in an Extracellular Matrix (ECM)-free environment from 2D-expanded bronchial epithelial cells and differentiated in suspension to develop uniformly-sized organoid cultures with robust ciliogenesis. Differentiated apical-out airway organoids are susceptible to infection with common respiratory viruses and show varying responses upon treatment with antivirals. In addition to the ease of apical accessibility, these apical-out airway organoids offer an alternative in vitro model to study host-pathogen interactions in higher throughput than the traditional air-liquid interface model.
Assuntos
Organoides , Viroses , Antivirais/farmacologia , Diferenciação Celular , Células Epiteliais , HumanosRESUMO
The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.