Abstract
Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host–pathogen interactions. We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host–pathogen, food science and pharmaceutical research.
Highlights
Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host–pathogen interactions
Avian intestinal 18 embryonic day (ED) villi seeded in Matrigel quickly formed spheroid structures that increased in size over 7 days of culture, the architecture remained basic with no obviously defined crypt-villus development (Fig. 1a, b)
Multiple budding enteroid numbers increased significantly in the first day of culture decreased between 5–7 days (Fig. 1j) but the expression of apoptosis markers was relatively stable over the culture period
Summary
Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host–pathogen interactions. We report the development of avian enteroids with multiple villus-crypt structures that maintain the cellular diversity, polarity and barrier function present within the chicken intestinal epithelium in vivo. Phalloidin staining to detect F-actin showed that the epithelial cells of embryonic floating chicken enteroids at 2 and 7 days after cultivation had an atypical reversed polarity (Fig. 2a, b).
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