Organoids被认为是生理相关的细胞模型，并且在药物开发的化合物筛选中非常有用；然而，由于培养费用较高，它们的应用目前受到限制。我们以前成功地使用L细胞共表达Wnt3a、R-spondin1和Noggin的条件培养基（CM）减少了人类肠道器官培养的成本。在这里，我们通过用CM替换重组肝细胞生长因子，进一步降低了成本。此外，我们还表明，将器官体嵌入胶原凝胶中，比Matrigel更便宜的基质，维持器官体增殖和标记基因表达的方式类似于使用Matrigel。这些替换的组合也使得器官定向单层细胞培养成为可能。此外，使用精细方法扩增的器官体，筛选数千个化合物，鉴定出几种对器官体衍生细胞比Caco-2细胞具有更高选择性细胞毒性的化合物。其中一种化合物YC-1作用机制得到了进一步阐明。我们表明YC-1通过丝裂原活化蛋白激酶/细胞外信号调节激酶途径诱导细胞凋亡，其机制与其他命中化合物引起的细胞死亡不同。我们的成本削减方法使得大规模肠道器官培养和随后的化合物筛选成为可能，这可以扩展肠道器官在各种研究领域中的应用。©2023年 作者

Organoids are regarded as physiologically relevant cell models and useful for compound screening for drug development; however, their applications are currently limited because of the high cost of their culture. We previously succeeded in reducing the cost of human intestinal organoid culture using conditioned medium (CM) of L cells co-expressing Wnt3a, R-spondin1, and Noggin. Here, we further reduced the cost by replacing recombinant hepatocyte growth factor with CM. Moreover, we showed that embedding organoids in collagen gel, a more inexpensive matrix than Matrigel, maintains organoid proliferation and marker gene expression similarly when using Matrigel. The combination of these replacements also enabled the organoid-oriented monolayer cell culture. Furthermore, screening thousands of compounds using organoids expanded with the refined method identified several compounds with more selective cytotoxicity against organoid-derived cells than Caco-2 cells. The mechanism of action of one of these compounds, YC-1, was further elucidated. We showed that YC-1 induces apoptosis through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, the mechanism of which was distinct from cell death caused by other hit compounds. Our cost-cutting methodology enables large-scale intestinal organoid culture and subsequent compound screening, which could expand the application of intestinal organoids in various research fields.© 2023. The Author(s).