The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line. We developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time. The Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

中文翻译：

---

用于测量肠道胆固醇转运的 Caco-2 细胞 - 可能性和局限性。

人 Caco-2 细胞系是肠上皮屏障的常见体外模型。由于肠道是胆固醇转换的主要界面，并且代表了胆固醇排泄的非胆道途径，因此 Caco-2 细胞也是研究胆固醇稳态（包括胆固醇摄取和流出）的有价值模型。然而，目前可用的协议在不同实验室之间要么是粗略的，要么是不一致的。因此，我们的目标是生成一组优化的方案，考虑到不同实验室的不同方法，并强调用这种细胞系测量胆固醇转运的可能性和局限性。我们开发了全面和质量控制的协议，用于在单个紧密单层的过滤器插件上培养 Caco-2 细胞。详细描述了胆固醇摄取和胆固醇流出测定，包括合适的阳性对照。我们进一步表明，Caco-2 细胞可以有效地转染用于荧光素酶报告基因测定，以确定核受体激活、胆固醇转运蛋白的主要转录调节因子（ABCA1、ABCB1、ABCG5/8、NPC1L1）。检测在过滤器插件上生长的细胞中胆固醇转运蛋白的蛋白质和 mRNA 水平可能会带来挑战，我们强调需要考虑的基本步骤和替代方法。首次提供了一种在滤芯上分化的细胞进行活力测定的方案。Caco-2 细胞系在科学界被广泛用作肠上皮的模型，尽管其方案差异很大。