Optimized air–liquid interface cultivation enhances survival and integrity of mouse colon precision-cut tissue slices

Abstract Mouse colon precision-cut tissue slices (cPCTS) serve as valuable models for studying physiology, toxicology, and immunology. They retain a three-dimensional structure with physiological cell composition and intact metabolic and immune functions. Additionally, cPCTS support the 3R principle by reducing animal use. This study aimed to optimize cPCTS cultivation to maintain cell integrity, minimize damage, and enhance survival of the multilayer intestinal structure. We established an air–liquid interface (ALI) cultivation system, where cPCTS rest on a semi-permeable insert membrane with medium beneath and direct air exposure above. We compared the effects of different oxygen concentrations on cPCTS survival using LDH assays, TUNEL staining, qPCR, and immunohistochemistry. cPCTS preserved the characteristic colonic architecture for up to 120 h, with optimal tissue integrity maintained for at least 96 h. Compared with 80% O 2 , cultivation at atmospheric O 2 significantly reduced oxidative stress and DNA damage, as demonstrated by lower expression of oxidative stress-associated genes and fewer γH2AX-positive cells. Reduced apoptosis and cytotoxicity were confirmed by less TUNEL-positive cells and lower LDH release. Lower oxygen further preserved crypt morphology, increased Lgr5 and Mki67 expression, and resulted in higher numbers of Ki67-positive cells, indicating improved stemness and proliferation. Histological analyses additionally revealed a more physiological extracellular matrix remodeling response under atmospheric O 2 . ALI cultivation simplifies medium changes and facilitates experimental manipulation of the system. Lower oxygen conditions improve long-term survival and structural integrity of cPCTS providing a robust platform for extended mechanistic and co-culture studies.