Real time monitoring of metabolism of cells encapsulated in 3D bioprinted hydrogels to bioengineer a liver sinusoid model

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Extrusion based 3D bioprinting methods have been envisioned as most suitable methods for bioengineering a transplantable liver tissue. In the recent years many conducive hydrogels have/are been developed for encapsulation and 3D bioprinting of liver specific cells. These hydrogels are routinely characterized for their physical properties such as viscosity, stiffness and extrudability. However, diffusion of oxygen in the newly developed hydrogels is seldom characterized. In case of hydrogels developed for liver tissue engineering, oxygen diffusion properties will play an important role, as the liver sinusoid exhibits highly heterogenous distribution of oxygen along its vascular channel. Thus a hydrogel employed for liver tissue engineering should be able to support attaining the heterogenous oxygen distribution. Aim of the proposed work is to fabricate liver lobule structures using 3D bioprinting with the focus on mimicking native liver sinusoid microenvironment – especially attaining different metabolic zonations of liver by establishing heterogenous oxygen distribution along the lumen of the sinusoid. To achieve this goal, real time oxygen monitoring method (established by us) will be systematically employed to develop and characterize novel/existing hydrogels for their oxygen diffusion properties. Suitable hydrogels will then be used to encapsulate liver specific cell types (hepatocytes, liver derived endothelial cells and fibroblasts) and will be used to fabricate liver sinusoid structures by core/shell 3D bioprinting method. The lumen of the 3D bioprinted liver sinusoid construct will be connected to a microperfusion system to establish dynamic culture conditions. The constructs will be then analyzed for their oxygen consumption rate using real time oxygen monitoring method. Simultaneously, liver specific cell markers such as cytokeratin 19 (CK19), alpha 1-antitrypsin (AAT) and multi drug resistance protein (MRP) expression, along with albumin secretion will be studied by ELISA and immunohistochemical methods. Thus providing information about cell viability and phenotypic activity with respect to oxygen consumption/distribution in 3D bioprinted liver sinusoid constructs. The established liver sinusoid model will be used to assess its response towards liver toxic drugs. The proposed investigations are envisioned to help in establishing an in vitro liver sinusoid model and will pave way for fabricating functional transplantable liver tissues. Also, the model could be employed in drug development processes by assessing new drug toxicity, thus circumventing animal experiments.