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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.

SUBMITTER: Hiebl V

PROVIDER: S-EPMC7149936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene 'click chemistry' aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9-10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices.

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

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