Preparation of PCIS
PCIS were prepared as previously published in detail by de Graaf et al
with
modifications.20,22In short, a piece of full-thickness human intestinal tissue
(approximately 1.0 x 0.5 x 0.2cm) was cut from the healthy region of a
surgically resected specimen by a trained pathologist directly following
surgical removal. The sample was immediately placed into oxygenated,
chilled (4oC, 95% O2, 5%
CO2) Krebs Henseleit Buffer (KHB) and transported to the
laboratory within one hour. The sample was then dissected into strips
approximately 0.5 x 0.2cm and embedded in low melting temperature
agarose (37oC, 3% w/v, 0.9% NaCl; MilliporeSigma,
Darmstadt, Germany). Once the agarose solidified, the tissue was cut
into 400µm thick slices using a Krumdieck Tissue Slicer (Alabama
Research and Development, Munford, AL, USA), or a VT1200S Vibratome
(Leica Microsystems, Nussloch, Germany). PCIS were then incubated in
12-well culture plates in 1.2ml Williams’ medium E (WME) containing
L-glutamine and supplements (D-glucose 14mM, gentamycin 50µg/ml; Gibco,
MA, USA) in an oxygenated incubator
(37oC, 90%
O2, 5% CO2; Thermo Fisher Scientific,
MA, USA) with gentle shaking (Figure 1A).