Background and aim Reliable in vitro derivation of mature subsets of human gastric epithelial cells has been challenging to achieve in 3D culture. Small intestinal organoids have been shown to self-organise into more complex structures in floating ring culture and express mature cell types in vitro.¹We examined the effect of culturing human gastric epithelial organoids (HGEO) in floating hydrogel rings on the expression of mature epithelial cell subsets.

Methods Hydrogels of collagen I and decellularised porcine small intestinal mucosa/submucosa were prepared and tested for cytocompatibility and gelation. Human gastric epithelial organoids (HGEO) from 11 week and 20 week foetuses and 11 year old patient were expanded in Matrigel®, then released and encapsulated in collagen or ECM hydrogels. The HGEO-loaded gels were plated as a rings (200µL/well) in 24-well plates, and following gelation were detached by forceful addition of human gastric organoid culture medium. The rings were maintained in floating culture for 13 days before analysis by immunofluorescence and RT-PCR, and comparison to tissue of origin.

Results By Day 6, each condition (except week 11 in ECM) showed spontaneous organisation of HGEO into a tube by growth and fusion of adjacent organoids. Formation of the tubes occurred together with contraction of the hydrogel. The tubes showed smooth and budding areas, with buds resembling Matrigel®-cultured organoids. Immunofluorescence demonstrated mature gastric epithelial cell subtypes including parietal (ATP4B), chief (pepsinogen), enteroendocrine (pepsinogen), and mucus secreting cells (MUC5AC). Expression of these cell types corresponded to the age of tissue of origin of the HGEO. Similarly, transcriptional analysis demonstrated developmental stage specificity.

Conclusions Collagen I and porcine small intestinal submucosal hydrogels support growth of HGEO. HGEO spontaneously organise into more complex tube structures in floating hydrogel culture with evidence of presence of mature epithelial cell subtypes at appropriate developmental time points.

Reference

1. Sachs, Development, 2017.