Tissue Engineering of Liver Organoids

Researchers have demonstrated the ability to build organoids of many different tissue types, starting with just a cell sample. The open access paper linked here is one example of many at the present time. These organoids are tiny sections of functional or partially functional organ tissue, limited in size because the research community has yet to develop a reliable means of incorporating the intricate branching vasculature needed to support thicker and larger masses. Still, this is enough to build useful products, either for research such as drug development or even for transplantation in some cases. For organs that are essentially chemical factories, that function doesn't require the original organ to be exactly replicated in shape and size - transplanting a few dozen organoids grown from the patient's own cells might be good enough to form the basis for a viable therapy.

The idea to construct in vitro 3D tissue-like structures to be used as model system for the respective organ is an appealing experimental approach. The main focus hereby is to exploit the in vivo physiological mechanism that occurs during organ development or healing (regeneration) and to implement similar mechanisms to develop a functional tissue in vitro. Such 3D liver-like structures would for example meet the needs of the pharmacological and toxicological industry for drug screening. The main techniques to generate 3D cellular constructs are either the formation of spheroids or building of tissue-like structures by placing sheets of cells and extracellular matrix components on top of each other. The disadvantage of spheroids is that the cells are distributed randomly without formation of spatial organization i.e., liver spheroids neither possess typical hepatic cord-like alignment of polarized hepatocytes nor sinusoids lined with endothelial cells reflecting the in vivo situation. Similarly, 3D liver models generated using sandwich cultures can never fully recapitulate the true in vivo architecture of the organ. Such ex vivo formation of tissue for most complex organs such as heart, kidney or brain would be very challenging.

However, the liver is exceptional in its ability to regenerate. It is well established that fully differentiated adult liver is capable of regeneration as long as a sufficient amount of intact liver remains after damage. Therefore, in principle any differentiated adult liver cell should harbor the potential to proliferate and regenerate to a complex and functional organ under suitable conditions. Indeed, induced-pluripotent stem cells (iPSC) stimulated to become hepatic endoderm-like cells (iPSC-HE) together with mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (HUVEC), self-organize in vitro into macroscopically visible 3D cell clusters by an intrinsic organizing capacity. When these structures were transplanted into mice, they became vascularized, engrafted into the recipient's tissue and produced hepatic factors like albumin. Possible applications of such an organoid structure include replacement therapy but also the possibility to study hepatotoxic effects of new compounds. They could as well be used as simplified model system to investigate processes like liver regeneration, fibrogenesis or malignant transformation. Due to the fact that these organoids are formed out of different cell types, which are in 3D contact to each other, they can be expected to represent a system which is much closer to the depicted in vivo situation than conventional approaches.

In the present work we analyzed if liver organoids could also be generated from adult, differentiated cells and if these organoids can be cultured for long-term to study liver functions. Instead of stem cells, hepatocytes were used to reflect the parenchymal cells of the liver. In order to depict the native physiological condition of liver, we further used liver sinusoidal endothelial cells (LSECs) instead of conventional endothelial cells like HUVECs. LSECs are a specialized type of scavenger endothelial cells that are able to endocytose an array of physiological and foreign macromolecules and colloids from the blood. The generation of these cells involves stable transduction of primary cells with lentiviral constructs carrying sequences which code for certain proliferation-inducing factors. These cells are cultured in medium containing a defined mixture of growth factors, allowing tighter control over proliferation (up to 40 population doublings). Employing this process, almost unlimited numbers of cells from one donor can be obtained. Our results show that liver organoids can be generated and these organoids after culturing them for a period of 10 days, express several marker proteins, genes and enzymes to a degree that is comparable to adult human liver. Furthermore, the architecture of these liver organoids to some degree resembles typical hepatic structures.

Link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619350/

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