An crypts are isolated from healthy human jejunum that

An IR model in organoids will be set up to investigate
mechanisms underlying the intestinal ischemia-reperfusion. For the purpose of modelling IR in
vitro, patient-derived intestinal organoids will be used.
Intestinal organoids are three-dimensional self-organizing structures which
resemble the intestinal epithelium in
vivo but are cultured in vitro.
Intestinal organoids are organized in crypt and villus compartments, and
contain all the different cell types of the intestinal epithelium. 36

These organoids are of value because they give
the possibility to study tissue development, regeneration, gene function and to
study disease modelling and drug validation. Since intestinal organoids only have epithelial
cells, influence of immune cells and bacteria cannot be examined with this
model.

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1.1.1       
Intestinal
organoid culture

First of all, intestinal crypts are isolated from healthy human jejunum that
is resected as part of the standard surgical procedure. The mucosa layer is
first stripped of the muscle layer, followed by stripping the villi of the
mucosa layer to eventually isolate the crypts. The crypts are embedded in a
bubble of Geltrex extracellular matrix, which is submersed in medium containing
specific growth factors. Figure 4 shows the organoid culture process. When
cultured, the organoids form an epithelial monolayer surrounding the central
lumen. Paneth and stem cells are located in the budding crypt domains of the
organoid. Paneth cells are important because
they express components (EGF, Wnt3 and the Notch-ligand Dll4) which are
essential for stem cell maintains and therefore improve organoid formation. 8
27 Further, factors such as
R-spondin and Noggin are used for organoid culture since they improve organoid
growth and formation. In addition, Jagged-1 peptide adding to the support
matrix stimulates the Notch signalling which will be discussed later. 35 Equal to the in vivo intestinal epithelium, epithelial cells are shed into the
lumen after a few days in culture. The cellular debris within the lumen reduces
viability of the organoids, therefore periodically passaging is required.