Discovery Immunology, Merck & Co., Inc., Cambridge, MA, 02141, USA.
Quantitative Biosciences, Merck & Co., Inc., Boston, MA, 02115, USA.
Sci Rep. 2023 Sep 29;13(1):16357. doi: 10.1038/s41598-023-43656-z.
Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.
肠道上皮屏障的破坏可导致慢性疾病的发生，如炎症性肠病（IBD）。传统上，我们在不包含所有人类肠道细胞类型的癌细胞系中评估屏障功能，导致其转化能力较差。为了弥补这一差距，我们将单层培养的人类原代肠道类器官适应化，以定量测定转录因子磷酸化、基因表达、细胞因子产生和屏障功能。在这项工作中，我们描述并表征了一种新型的 96 孔人类肠道类器官衍生的单层系统，该系统可用于定量评估候选治疗药物。我们对正常人类肠道的分化模式和屏障功能进行了特征描述和验证，以重现体内生物学的关键方面。接下来，我们使用多种定量检测方法来确定细胞对 TNF-α（IBD 的主要驱动因素）的反应。我们表明，TNF-α 通路拮抗剂以剂量依赖性方式挽救了 TNF-α 造成的损伤，表明该系统适合定量评估调节屏障的因素。总之，我们已经建立了一个强大的基于原代细胞的 96 孔系统，能够研究黏膜反应相关问题。该系统非常适合提供关键的功能数据，以支持转化靶标和药物发现工作。
