Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Department of Clinical Pharmacy and Pharmacology University Medical Center Groningen Groningen The Netherlands

Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen The Netherlands

Department of Hepato Pancreato Biliary Surgery and Liver Transplantation University of Groningen University Medical Centre Groningen Groningen The Netherlands

Department of Medical Biochemistry Faculty of Medicine in Hradec Kralove Charles University Prague Czech Republic

Department of Pharmaceutical Analysis Groningen Research Institute of Pharmacy University of Groningen Groningen The Netherlands

Department of Pharmaceutical Technology and Biopharmacy Groningen Research Institute of Pharmacy University of Groningen Groningen The Netherlands

Department of Pharmacokinetics Toxicology and Targeting Groningen Research Institute of Pharmacy University of Groningen Groningen The Netherlands

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Kralove Charles University Prague Czech Republic

Innovative Testing in Life Science and Chemistry Group Utrecht University of Applied Science Utrecht The Netherlands

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