Plenge Masina, Schnepel Nadine, Müsken Mathias, Rohde Judith, Goethe Ralph, Breves Gerhard, Mazzuoli-Weber Gemma, Benz Pascal
Institute for Physiology and Cell Biology, University of Veterinary Medicine Hannover, Hannover, Germany.
Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
PLoS One. 2025 May 7;20(5):e0312989. doi: 10.1371/journal.pone.0312989. eCollection 2025.
The porcine colon epithelium plays a crucial role in nutrient absorption, ion transport, and barrier function. However ethical concerns necessitate the development of alternatives to animal models for its study. The objective of this study was to develop and characterize a two-dimensional (2D) in vitro model of porcine colonic organoids that closely mimics native colon tissue, thereby supporting in vitro research in gastrointestinal physiology, pathology, and pharmacology. Porcine colonic crypts were isolated and cultured in three-dimensional (3D) organoid systems, which were subsequently disaggregated to form 2D monolayers on transwell inserts. The integrity of the monolayers was evaluated through the measurement of transepithelial electrical resistance (TEER) and electron microscopy. The functional prerequisites of the model were evaluated through the measurement of the mRNA expression of key ion channels and transporters, using quantitative RT-PCR. Ussing chamber experiments were performed to verify physiological activity. The 2D monolayer displayed robust TEER values and retained structural characteristics, including microvilli and mucus-secreting goblet cells, comparable to those observed in native colon tissue. Gene expression analysis revealed no significant differences between the 2D organoid model and native tissue with regard to critical transporters. Ussing chamber experiments demonstrated physiological responses that were consistent with those observed in native colonic tissue. In conclusion, 2D porcine colonic organoid model can be recommended as an accurate representation of the physiological and functional attributes of the native colon epithelium. This model offers a valuable tool for investigating intestinal barrier properties, ion transport, and the pathophysiology of gastrointestinal diseases, while adhering to the 3R principles.
猪结肠上皮在营养吸收、离子转运和屏障功能中起着关键作用。然而，出于伦理考虑，有必要开发动物模型的替代物来进行相关研究。本研究的目的是开发并表征一种二维（2D）猪结肠类器官体外模型，该模型能紧密模拟天然结肠组织，从而支持胃肠道生理学、病理学和药理学的体外研究。分离猪结肠隐窝并在三维（3D）类器官系统中培养，随后将其解离以在Transwell小室上形成二维单层。通过测量跨上皮电阻（TEER）和电子显微镜评估单层的完整性。使用定量RT-PCR通过测量关键离子通道和转运蛋白的mRNA表达来评估该模型的功能前提条件。进行尤斯灌流小室实验以验证生理活性。二维单层显示出强大的TEER值，并保留了包括微绒毛和分泌黏液的杯状细胞在内的结构特征，与天然结肠组织中观察到的特征相当。基因表达分析显示，二维类器官模型与天然组织在关键转运蛋白方面无显著差异。尤斯灌流小室实验证明了与天然结肠组织中观察到的一致的生理反应。总之，二维猪结肠类器官模型可被推荐为天然结肠上皮生理和功能属性的准确代表。该模型为研究肠道屏障特性、离子转运和胃肠道疾病的病理生理学提供了一个有价值的工具，同时遵循3R原则。
