Key events in the life cycle of intestinal epithelial cells, such as proliferation, differentiation, apoptosis and migration, are controlled by organising principles that are determined by the spatial context of the cells. Taking into account that tissues in vivo function in a three-dimensional (3-D) environment while cell culture systems are generally two-dimensional, it obviously follows that culture models that more closely mimic essential aspects of the in vivo organisation of intestinal epithelial cells are invaluable. Evidently, such models more reliably reflect the proper physiological conditions that are essential for appreciating and understanding how processes of proliferation, differentiation and migration act in concert, and how this harmony is maintained and influenced by endogenous and exogenous factors, based on a correct cellular architecture.
Current in vitro approaches to study the integrity and permeability of intestinal epithelial monolayers predominantly employ cell culture systems in which epithelial cells are grown on flat Transwell filter membranes. Although such 2-D cell cultures may produce tight epithelial cell monolayers, important microenvironmental cues that in a coordinated manner promote key signaling pathways, thereby influencing and enabling cell proliferation, differentiation, and monolayer permeability, are lost. As a consequence, 2-D cell culture systems fail to capture physiologically-significant and three-dimensional aspects of tissue biology (reviewed in [1-2]). Many of these apects (which include in vivo morphology, proliferation rates, contact geometries, and trans- and para-epithelial transport properties) can be restored when cells are cultured in 3-D matrices.
Thus far cell biological and physiological characterization of such intestinal epithelial 3-D culture model system has been rudimentary and in order to employ the system as a reliable high throughput screening model to discover modifiers of intestinal epithelial integrity with non-invasive read-outs of cell signalling activity, proliferation and permeability. Therefore, further technical development and improvement is required.
Research question / problem definition
To develop a three-dimensional intestinal epithelial cell culture model system that allows integrated analyses of epithelial cell biology, including proliferation, differentiation and permeability).
Workplan
1. Different (human) intestinal cell lines will be tested for their capacity to form 3-D single-cell layer lumenal spheres (see picture)
2. human intestinal epithelial T84 cells (which will be used first to study paracellular permeability) will be modified to express red fluorescently tagged (mCherry) tight junction proteins (f.e. claudins, occludins) that will enable the simultaneous analysis of monolayer permeability and tight junction integrity. Alternatively, reporter genes will be expressed, such as a red fluorescent protein under control of the topoisomerase II alpha promoter or beta-catenin-responsive TCF promotor, which allows for the study of monolayer permeability in conjunction with a non-invasive read-out of cell proliferation or Wnt signalling activity, respectively
3. For examining the basolateral to apical flow of small molecules, low molecular weight fluorescent dyes or proteins are simply added to the basolateral milieu of the single cell-layer lumenal spheres. For examining the apical to basolateral flow of small molecules, low molecular weight fluorescent dyes or proteins of varying molecular weight (f.e. 0-50 kDa) can be microinjected into the lumenal space. We will determine the appropriate experimental procedures (f.e. needle size, compensation and injection pressure, injection time and buffer, etc.) that ensures optimal prevention of cell and monolayer damage and maximum recovery after microinjection
top