Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

Abstract

Background: 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. Results: 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. Conclusions: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.

Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells

“…In this study, fetal bovine serum is used for cultivation of the cells according to established culture conditions of the cell lines and to ensure comparability to earlier studys [ 21 , 34 , 35 ]. However, it is desirable to replace this serum by an animal-free alternative while the comparability to the serum-using system must be verified and compared with other studies already using serum-free medium [ 87 ].…”

Defining the Cholesterol Lowering Mechanism of Bergamot (Citrus bergamia) Extract in HepG2 and Caco-2 Cells

“…Caco-2 cells are suitable for the study of cholesterol absorption as they can absorb cholesterol and share morphological and physical similarities to mature enterocytes in the small intestine. However, enterocytes are only one of the four main cell types found in the intestine, indicating that it does not entirely reflect cholesterol handling in vivo [ 40 ]. Further analysis of an in vivo study will provide a more thorough understanding of how cholesterol absorption can be modified.…”

Intestinal and Hepatic Uptake of Dietary Peroxidized Lipids and Their Decomposition Products, and Their Subsequent Effects on Apolipoprotein A1 and Paraoxonase1

“…As the only enterocyte model capable of differentiation to the point of physiologically relevant lipoprotein processing [ 62 ], Caco-2 cells are a popular tissue culture model for intestinal lipoprotein studies [ 63 , 64 ]. This cell model has established important physiology relating to fatty acid post-transcriptional regulation of ApoB production, lipoprotein production, and transintestinal cholesterol efflux (TICE), a pathway by which intestinal cholesterol uptake is limited, ApoA1 and HDL levels are increased, and cholesterol efflux into the intestinal lumen is increased [ 65 ].…”