Department of Gastroenterology, IDIBAPS, Hospital Clínic, CIBER-EHD, Barcelona, Spain.
PLoS One. 2022 Oct 27;17(10):e0276195. doi: 10.1371/journal.pone.0276195. eCollection 2022.
Ulcerative colitis and Crohn's disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD. Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used. With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.
溃疡性结肠炎和克罗恩病是病因不明的慢性炎症性肠病（IBD），其特征为反复发作缓解。越来越多的证据支持上皮屏障在 IBD 的发病机制及其随时间演变中起核心作用的观点，因此代表了新型治疗选择的潜在靶点。在过去十年中，从体外扩增的肠成体干细胞（ASCs）中生成 3D 上皮培养物的方法，已经改变了我们研究包括 IBD 在内的多种胃肠道疾病中上皮功能的能力。在这里，我们详细描述了一种使用从小肠活检获得的非 IBD 和 IBD 供体的 ASC 生成 Matrigel 包埋的上皮类器官的可重复方案，包括对其进行优化的步骤。如果使用手术样本，也提供了该方案的略有修改版本。通过这种方法，可以在多个传代中扩增上皮类器官，从而产生大量可用于多种下游分析的活细胞，包括遗传、转录组、蛋白质组学和/或功能研究。此外，使用我们的方案生成的 3D 培养物适合建立 2D 培养物，该培养物可以模拟 IBD 黏膜中发生的相关细胞间相互作用。
