Arian Christopher, O'Mahony Eimear, MacDonald James W, Bammler Theo K, Donowitz Mark, Kelly Edward J, Thummel Kenneth E
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington.
Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, Washington.
Drug Metab Dispos. 2025 Jan;53(1):100002. doi: 10.1124/dmd.124.001551. Epub 2024 Nov 22.
To further the development of an in vitro model that faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from 3 donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell inserts, and confirmed transformation into a largely enterocyte population via RNA sequencing analysis and immunocytochemistry (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH) vitamin D-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, because, to our knowledge, this is the first study to assess drug metabolizing enzymes and transporters expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. SIGNIFICANCE STATEMENT: This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers that maintain barrier function and metabolic functionality for up to 42 days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.
为了进一步开发能够如实地概括口服药物的药物处置情况的体外模型，我们研究了人肠类器官单层同时评估肠道药物吸收和首过代谢过程的效用。我们从3名供体培养了人肠类器官单层，这些供体通过含有十二指肠干细胞的活检组织获得，将其增殖后在可渗透的Transwell小室上进行分化，并通过RNA测序分析和免疫细胞化学（ICC）测定法确认其转变为主要的肠上皮细胞群体。通过明场显微镜以及紧密连接蛋白和其他顶端和基底外侧定位蛋白的ICC成像评估并确认了合适的细胞形态。培养10天后，经上皮电阻升高证明了肠类器官单层屏障的完整性，该电阻在培养10天后稳定，并持续了42天。培养7天和21天时低细胞旁转运探针通透性证实了这些结果。在基础、1α,25(OH)维生素D诱导和6',7'-二羟基佛手柑素抑制条件下，培养7天、21天和42天时确认了一种主要的药物代谢酶CYP3A的活性。这些实验的持续时间特别值得注意，因为据我们所知，这是第一项评估培养超过12天的肠类器官的药物代谢酶和转运蛋白表达/功能的研究。这些结果共同表明，肠类器官单层是一种有前途的体外模型，可用于研究和定量预测口服药物的肠道吸收和/或代谢。意义声明：本研究提出了一种新型的人肠道体外模型，即人肠道类器官（肠类器官）单层，其在培养中可维持屏障功能和代谢功能长达42天。在同一模型中长时间纳入屏障完整性和代谢功能是对历史上使用的体外系统的一种进步，后者要么缺乏这些属性中的一个或两个，要么生存能力有限。
