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).