Abstract
Three-dimensional (3D) complex in vitro cell systems are well suited to providing meaningful and translatable results in drug screening, toxicity measurements, and biological studies. Reliable complex gastrointestinal in vitro models as a testbed for oral drug administration and toxicity are very valuable in achieving predictive results for clinical trials and reducing animal testing. However, producing these models is time-consuming due to the lengthy differentiation of HT29 or other cells into mucus-producing goblet cells or other intestinal cell lineages. In the present work, HT29 cells were grown on an inorganic topographic surface decorated with a periodic pattern of micrometre-sized amorphous SiO2 structures for up to 35 days. HT29 cells on topographic surfaces were compared to undifferentiated HT29 in glucose-containing medium on glass or culture dish and with HT29 cells differentiated for 30 days in the presence of methotrexate (HT29-MTX). The cells were stained with Alcian blue for mucus, antibodies for mucus 2 (goblet cells), villin (enterocytes), lysozyme (Paneth cells), and FITC-labeled lectins to identify different cells, glycomic profiles, and cell features. We observed that HT29 cells on topographic surfaces showed more similarities with the differentiated HT29-MTX than with undifferentiated HT29. They formed islands of cell clusters, as observed for HT29-MTX. Already after 2 days, the first mucus secretion was shown by Alcian blue stain and FITC-wheat germ agglutinin. After 4–6 days, mucus was observed on the cell surface and in the intercellular space. The cell layer was undulated, and in 3D reconstruction, the cells showed a clear polarisation with a strong actin signal to one membrane. The lectins and the antibody-staining confirmed the heterogeneous composition of differentiated HT29 cells on topographic surfaces after 6–8 days, or after 6–8 days following MTX differentiation (30 days).
Highlights
Three-dimensional (3D) cell culture allows for a clearer understanding of disease mechanisms, e.g., for inflammatory bowel disease or colorectal cancer
HT29 cells were seeded in glucose-containing medium on topographic structures of
The results on the topographic surfaces were compared with HT29 cells either grown in the 2D cell culture on glass or pre-treated culture dishes
Summary
Three-dimensional (3D) cell culture allows for a clearer understanding of disease mechanisms, e.g., for inflammatory bowel disease or colorectal cancer. It provides more reliable results in drug screening, especially for orally administered drugs and their uptake by the gut [1–7] due to the presence of mucus and different cells and cell layers. It is still not yet frequently used for routine lab work, as producing realistic complex intestinal in vitro models is time-consuming. The standard procedure is the maintenance of the cells for several weeks under a constant regime of different chemical agents (sodium butyrate, suramin, trehalose, 120-tetradecanoylphorbol13-acetate, galactose) or in glucose-free medium to induce the 4.0/).
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