Abstract
PurposeKnowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies.MethodsFull-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue.ResultsGastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells.ConclusionGastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.
Highlights
The microscopic morphology of gastric glands in humans has been well described with surface, neck, and basal domains [1]
Gastric organoids can be successfully derived from paediatric gastric biopsies Gastric tissue samples were obtained from six patients ranging in age from 4 months to 16 years
Non-gland cells encapsulated in the Matrigel® died during the first 7 days in culture. These cells were likely either non-epithelial or non-progenitor epithelial cells, and as such were not supported by the gastric organoid-specific medium
Summary
The microscopic morphology of gastric glands in humans has been well described with surface (pit), neck, and basal domains [1]. While much is known about intestinal development and epithelial homeostasis in humans [2, 3], our knowledge of these processes in the gastric epithelium remains incomplete. Studies of stomach development have relied on animal models, usually in rodents [4,5,6]. Rodent stomach contains a forestomach lined by squamous epithelium, a different structure from humans, forcing caution in generalising these data to human development and disease [1]. Morphological studies of human embryos, diseased gastric tissue, and gastric epithelial cell lines derived from malignant tissue provide some human-specific insights but have significant ethical limitations or fail to represent normal homeostasis, respectively. A high-fidelity, reproducible in vitro system is preferable for understanding human development and homeostasis
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