A method for rapid generation of model intestinal barriers in vitro

About authors

1 National Research University Higher School of Economics, Moscow, Russia

2 SRC Bioclinicum, Moscow, Russia

Correspondence should be addressed: Sergey V. Nikulin
Vavilova, 7, Moscow, 117321; moc.liamg@b.c.nilukin

About paper

Funding: the study was supported by the Russian Science Foundation, project № 16-19-10597.

Author contribution: Nikulin SV — cell culture work, preparation of samples for transcriptome analysis, data analysis, manuscript preparation; Poloznikov AA — analysis of transcriptome data, manuscript preparation; Sakharov DA — supervision, manuscript preparation.

Compliance with ethical standards: the study complies with the 1964 Helsinki Declaration and its later amendments.

Received: 2020-11-09 Accepted: 2020-12-03 Published online: 2020-12-15

To increase the efficiency of drug development process, it is important to improve performance of preclinical experiments. A major drawback of the currently used in vitro intestinal barrier models is that it takes a significant time to obtain functional enterocyte monolayers with formed tight junctions. In this work, we have optimized various parameters such as cell density and different coatings, for a more rapid and efficient producing Caco-2 cell monolayers suitable for further experiments. In vivo microscopy and impedance spectroscopy were used to monitor cells state under various conditions. To determine possible biological mechanisms affected by exposure to various protein substrates, the transcriptomic analysis was applied. It was shown that collagen IV coating of the cell growth substrate significantly increased the rate of proliferation and migration of Caco-2 cells. This effect allows forming a functional monolayer of epithelial cells with tight junctions within 24 hours. Optimally, the initial cell density should be 90,000 to 200,000 cells/cm2. It was observed that collagen IV was poorly expressed by Caco-2 cells while the collagen IV receptor was expressed at a relatively high level in these cells. Laminin-332, another basement membrane component, was found to have no significant effect on times of formation of functional epithelial monolayers. Thus, using the optimal parameters determined in this study allows to significantly improve efficiency of using the in vitro intestinal barrier models.

Keywords: Caco-2, collagen IV, barrier tissues, laminin-332, TEER, extracellular matrix, impedance spectroscopy