Introduction: Ischemia-reperfusion (IR) injury is inevitable during intestinal transplantation. IR damages the intestinal epithelium, which functions as a physical and immunological barrier and is therefore crucial in maintaining intestinal homeostasis. In order to investigate potential therapeutic targets to protect the epithelium during intestinal IR and promote a regenerative response, we aim to validate a model to study IR in human intestinal organoids. Intestinal organoids have been shown to closely resemble self-renewal kinetics, 3D architecture, and cell-type composition of the intestinal epithelium in vivo.

Methods: A well-established human experimental model to study IR was used for temporal expression profiling of the in vivo intestinal response to IR. The top perturbed pathway was further validated using qPCR. Intestinal epithelial organoids were cultured from crypts isolated from surgical specimens of normal human small intestine. To simulate IR, organoids were subjected to 12 hours of hypoxia (1% O₂) followed by 30 and 120 minutes of reoxygenation. Activation of the UPR response was assessed by qPCR for CHOP, GADD34, BiP and XBP1 splicing, and, in addition, signs of endoplasmic reticulum (ER) stress were evaluated using electron microscopy (EM).

Results: The unfolded protein response (UPR) was the top perturbed pathway during reperfusion of the ischemically injured human small intestine in vivo. Subjecting small intestinal organoids to 12 hours of hypoxia followed by 30 minutes of reoxygenation significantly increased expression of UPR-related genes CHOP and GADD34 and splicing of XBP1 mRNA compared to organoids not subjected to hypoxia. In addition, EM showed dilated ER after reoxygenation which is indicative of ER stress.

Conclusion: In line with findings in the in vivo human IR model, revealing the response to unfolded protein as a highly regulated process during reperfusion, hypoxia-reoxygenation in intestinal organoids induces significant activation of the UPR. Intestinal organoids can be used to improve insight in the pathophysiology of epithelial IR injury and regeneration, which could lead to new therapeutic targets.