Recently organoids have become widely used in vitro models of many tissue and organs. These type of structures, originated from embryonic or adult mammalian intestines, are called “mini guts”. They organize spontaneously when intestinal crypts or stem cells are embedded in the extracellular matrix proteins preparation scaffold (Matrigel). This approach has some disadvantages, as Matrigel is undefined (the concentrations of growth factors and other biologically active components in it may vary from batch to batch), difficult to handle and expensive. Here we show that the organoids derived from chicken embryo intestine are formed in a hanging drop without embedding, providing an attractive alternative for currently used protocols. Using this technique we obtained compact structures composed of contiguous organoids, which were generally similar to chicken organoids cultured in Matrigel in terms of morphology and expression of intestinal epithelial markers. Due to the simplicity, high reproducibility and throughput capacity of hanging drop technique our model may be applied in various studies concerning the gut biology.
The intestinal epithelium isolated from chicken embryos in last 3 days of development can be used to establish the 3D culture of intestinal organoids. When fragments of epithelial tissue released by incubation with EGTA (2.5 mM, 2 h) are embedded in Matrigel matrix on cell culture inserts the formation of empty spheres covered by epithelial cells is observed in first 24 h of culture. The growth and survival of organoids are supported by the addition of R-spondin 1, Noggin, and prostaglandin E to the culture medium. The organoids are accompanied by myofibroblasts which become visible in the next 2 days of culture. The intestinal enteroids (free of myofibroblasts) can be obtained from adult chicken intestine.
The method of organoid culture has become a tool widely used in gastrointestinal research, but so far, the migration of organoids derived from gut epithelium and formed in 3D Matrigel matrix has not been reported and studied. The intestinal epithelial tissue derived from 19-day-old chicken embryo was cultured in Matrigel and the dynamic properties of the forming organoids were analyzed by time-lapse image analysis. It was observed that about one in ten organoids actively moved through the matrix, at a speed of 10–20 μm/h. Moreover, rotation was observed in the majority of organoids that did not migrate long distances. The fusion events took place between organoids, which collided during the movement or growth. In our previous paper, we showed that the presence of Toll-like receptor 4 ligand,
Escherichia coli
lipopolysaccharide (LPS, 1 μg/ml), increased the mean organoid diameter. Here, we confirm this result and demonstrate that the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (10 μM) did not completely abolish organoid migration, but prevented the fusion events, in both LPS-treated and untreated cultures. In consequence, in the presence of Y-27632, the differences between cultures incubated with and without LPS were not visible. We conclude that migration and fusion of organoids may influence their morphology and suggest that these phenomena should be taken into account during the design of experimental settings.
Electronic supplementary material
The online version of this article (10.1007/s00709-018-1312-3) contains supplementary material, which is available to authorized users.
In Figure 4 Section A, the upper right corner should read "3d", whereas it was incorrectly printed as "4d." Also, the caption of Figure 5 should read: "The cultures were photographed on the first, second and fifth day of culture (1d, 2d, 5d) with differential interference contrast optics." rather than the incorrect "The cultures were photographed on second, third, and fifth day of culture (2d, 3d, 5d) with differential interference contrast optics". These issues have now been corrected.
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