Stem cell-derived organoids have become an increasingly important model to study normal and pathological states of the mucosa. Intestinal organoids preserve several critical functions of the gut epithelium including barrier integrity, polarized secretion and absorption, innate immune responses, and the presence of multi-lineage differentiation. However, traditional 3D culture methods using basement membrane extracts (BME) produce an “apical-in” phenotype, making access to the luminal inner surface of the epithelium difficult. Advanced microinjection techniques have been used to interrogate these interior apical cell types which are not conducive to high-throughput assays or imaging. A recent study described a protocol to reverse the polarity of 3D organoids, making them “apical-out”.1 Apical-out organoids facilitate assay of the luminal compartment of cells and expand the possibilities of organoid use in gastrointestinal health, drug screening, ADME/toxicity studies, and disease research. We have successfully generated apical-out gastric, ileum, and colon patient-derived organoids (PDOs) following this culture protocol and created a co-culture of gastric organoids with H. pylori to model host/pathogen interactions.
Figure 1.Generation of Apical-Out Human Colon Organoids. Epithelial cell polarity of 3dGRO™ Human Colon Intestinal Organoids (SCC321) were reversed using the apical-out organoid culture protocol. Polarity reversion was analyzed by immunocytochemical staining with Dapi (Blue) and ZO-1 (MABT339, Green), a marker of cell-to-cell tight junctions within luminal apical cells.
Immunofluorescence staining was carried out according to the established Protocol Guide: Immunofluorescent Staining of Whole-Mount Organoids using Antibodies.
Figure 2.Cell Viability Staining of Cryopreserved Apical-out Organoids. 3dGRO™ Human Ileum Intestinal Organoids (SCC339) were cryopreserved and cell viability was analyzed using a Calcein-AM staining dye (17783) at day 8 and 13 following thaw. Apical-out organoids remain highly viable after cryopreservation and thaw when cultured in organoid growth media such as L-WRN conditioned media (SCM105).
Figure 4.Generation of Apical-out Gastric Organoids. Epithelial cell polarity of 3dGRO™ Human Gastric Organoids (SCC344) were reversed using apical-out culture protocol. Organoid polarity reversion was analyzed by immunocytochemical staining with Dapi (Blue), Actin (Red) and ZO-1 (MABT339, Green), a marker of cell-to-cell tight junctions within apical luminal cells.
Figure 5.H. pylori Infection of Apical-out Gastric Organoids. Apical-out 3dGRO™ Human Gastric Organoids (SCC344) were infected with H. pylori for 24 hr followed by immunocytochemical staining with Dapi (Blue), Actin (Red) and H. pylori antibody (Dako Cat. B0471).
To continue reading please sign in or create an account.Don't Have An Account?