Resumen de Fabrication and characterisation of a micropattern-based platform to model epithelial tubulogenesis and to perform nephrotoxicity assays
Some epithelial tubes, such as the gastrointestinal tract and the pancreas, form through a process known as ‘de novo’ lumen formation. This mechanism of tubulogenesis requires a first step of multiple lumen initiation (generating various foci) along the anteroposterior axis, which eventually expand and fuse to adjacent lumens to form a single cavity along the whole tubular structure, that later differentiates to accomplish its biological function. A system with the potential to recapitulate this mechanism in vitro would emerge as a very powerful device to study the molecular mechanisms involved in organ tube formation, providing a more physiological model of epithelial organs. We have developed a micropattern-based approach that allows renal epithelial cells to grow in a three-dimensional architecture and form tubes in vitro through a process that closely resembles in vivo tubulogenesis.
We are able to modify tube architecture by changing micropattern shape, which can impact lumen formation and morphology, proliferation and spindle orientation. The device also allows the modulation of extracellular matrix composition and matrix stiffness to analyse the morphological response to different environmental scenarios. Tubes cultured on laminin display different features from those grown on fibronectin. Laminin promotes the formation of tubes with more opened lumens and with a rounded shape whereas fibronectin coating favours a higher spreading and cell adhesion. Furthermore, matrix stiffness is controlling the assembly of focal adhesions as tubes grown on stiffer substrates show higher expression of focal adhesion proteins and the formation of focal adhesion clusters compared to tubes grown on softer substrates like silicone. Mechanotransduction processes are also being affected as shown by the down-modulation of YAP and lower actomyosin contractility in more compliant gels.
The mechanisms controlling lumen resolution are poorly understood and these tubular platforms provide a very potent tool to study these events. We have analysed the role of planar cell polarity, actomyosin contractility and lumen expansion in lumen coalescence. Planar cell polarity proteins, although higher expressed in tubes compared to epithelial monolayers, do not seem to modulate lumen coalescence and actomyosin-driven junctional shrinking is not controlling lumen fusion. However, a very significant role of lumen expansion, either by fluid accumulation or by membrane repulsion, in the achievement of a single lumen has been reported using this in vitro system.
Besides its potential use to study morphogenetic processes, growing renal tubes in vitro provides a new platform for drug discovery and nephrotoxicity assays. We have found that some proximal tubule drug transporters are upregulated in micropatterns compared to epithelial monolayers, making them more functional and favouring drug transport, which in turn makes them highly sensitive to nephrotoxic effects. Consistent with this, treatment with low doses of gentamicin, a known nephrotoxic drug, only induced increased apoptosis in tubes on micropatterns but not in epithelial monolayers. Similarly, because of the particular tubular shape, we are able to observe further morphological defects in micropatterns that we are not able to see in monolayers.
Altogether, we have developed an epithelial tube model system able to mimic tubulogenesis in vitro. Besides it can highly accelerate research as it is easy to handle and manipulate and, moreover, it presents a way to circumvent animal experimentation. This platform is very interesting since it does not involve the use of porous filter supports and the resulting tubules are well suited for high-throughput screening, high-content imaging and analysis and chemical modifications.
