Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsThe 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.
Highlights
The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier
Liver X receptors (LXRs) activation in the intestine further upregulates ABCA1, leading to increased cholesterol efflux to apolipoprotein A1, which results in the formation of high density lipoprotein (HDL), highlighting the importance of the intestine in cholesterol turnover
The intestine plays a central role in cholesterol homeostasis, highlighting the importance of the Caco-2 cell model for cholesterol research
Summary
The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. Our aim was 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. The human Caco-2 cell line is a widely used in vitro model of the intestinal epithelial barrier. The available descriptions of the used methodology, are either undetailed or not consistent [1, 4, 9,10,11,12], as described in more detail in the results part This hampers the kick-off or reproduction of experiments with this cell line. Our objective was to generate a detailed and optimized protocol collection, considering the different approaches of the different laboratories and evaluating and modifying the conditions to achieve a reliable and valid outcome
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