Abstract
Liver organoids (LOs) are of interest in tissue replacement, hepatotoxicity and pathophysiological studies. However, it is still unclear what triggers LO self-assembly and what the optimal environment is for their culture. Hypothesizing that LO formation occurs as a result of a fine balance between cell-substrate adhesion and cell-cell cohesion, we used 3 cell types (hepatocytes, liver sinusoidal endothelial cells and mesenchymal stem cells) to investigate LO self-assembly on different substrates keeping the culture parameters (e.g. culture media, cell types/number) and substrate stiffness constant. As cellular spheroids may suffer from oxygen depletion in the core, we also sought to identify the optimal culture conditions for LOs in order to guarantee an adequate supply of oxygen during proliferation and differentiation. The oxygen consumption characteristics of LOs were measured using an O2 sensor and used to model the O2 concentration gradient in the organoids. We show that no LO formation occurs on highly adhesive hepatic extra-cellular matrix-based substrates, suggesting that cellular aggregation requires an optimal trade-off between the adhesiveness of a substrate and the cohesive forces between cells and that this balance is modulated by substrate mechanics. Thus, in addition to substrate stiffness, physicochemical properties, which are also critical for cell adhesion, play a role in LO self-assembly.
Highlights
The growing evidence that three-dimensional (3D) microenvironments contribute critically to tissue function has led to the rapid development of cellular organoids
Since no gel formation was obtained at agarose concentrations below 0.1% w/v, this concentration represented the best compromise to mimic Matrigel-liver sinusoid endothelial cells (LSECs) stiffness and was chosen as an inert substrate to decouple the effect of mechanical properties from other Matrigel-related signals in triggering liver organoid formation
As the ECM gels derive from a decellularised matrix preparation rich with basement membrane proteins [8], cellsubstrate adhesion is likely to be dominant over cell-cell cohesion on these gels, so cell adhesion is promoted while Liver organoids (LOs) formation is inhibited
Summary
The growing evidence that three-dimensional (3D) microenvironments contribute critically to tissue function has led to the rapid development of cellular organoids. That cells aggregate spontaneously in vitro has been known for decades, only recently have scientists begun to manipulate stem cells and different parenchymal cell types in different conditions to generate mini-functional organs. We know that in the right conditions (cell number, cell types, substrate, agitation) and with the right timing (addition of differentiating media), stem cells proliferate and self-organize to form tissue proxies known as organoids [1,2,3]. Since they recapitulate the in vivo micro-environment to a large degree, self-assembled tissue organoids. The specific roles of these authors are articulated in the ‘author contributions’ section
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