Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. Thus, extrusion of live, virus-infected cells from intact epithelial tissue is likely to benefit both the integrity of host tissues and the protected spread of this faecal-oral pathogen within and between hosts. In contrast, when mechanical force sensing is inhibited, large amounts of free virus are released. When isolated and used as inoculum, intact virus-containing extruded cells can initiate new infections. Differential sensitivity to chemical and peptide inhibitors demonstrated that extrusion of virus-infected cells is dependent on force sensing via mechanosensitive ion channels rather than apoptotic cell death. Here we report that entire Enterovirus A71-infected cells are specifically extruded from the apical surface of differentiated human colon organoids, as observed by confocal microscopy. Picornaviruses have previously been shown to exit infected cells via either cell lysis or secretion of vesicles. However, in most cases infection is asymptomatic and limited to the gastrointestinal tract, where virus is amplified for transmission. Enterovirus A71 causes severe disease upon systemic infection, sometimes leading to life-threatening neurological dysfunction.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |