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

基于挤出的3D生物打印方法已经被设想为用于生物工程化可移植肝组织的最合适的方法。近年来，已经开发了许多有益的水凝胶用于肝特异性细胞的包封和3D生物打印。这些水凝胶通常表征其物理性质，如粘度、刚度和可挤出性。然而，氧在新开发的水凝胶中的扩散很少被表征。在开发用于肝组织工程的水凝胶的情况下，氧扩散性质将起重要作用，因为肝窦状隙呈现氧沿着其血管通道的高度不均匀分布。因此，用于肝组织工程的水凝胶应该能够支持获得异质氧分布。所提出的工作的目的是使用3D生物打印来制造肝小叶结构，重点是模仿天然肝窦微环境-特别是通过沿着窦腔建立异质氧分布来获得肝脏的不同代谢区带。为了实现这一目标，真实的时间氧监测方法（由我们建立）将系统地用于开发和表征新的/现有的水凝胶的氧扩散性能。然后，合适的水凝胶将用于封装肝脏特定细胞类型（肝细胞、肝脏衍生的内皮细胞和成纤维细胞），并将用于通过核/壳3D生物打印方法制造肝脏窦结构。3D生物打印的肝窦结构的管腔将连接到微灌注系统以建立动态培养条件。然后使用真实的时间氧监测方法分析构建体的耗氧率。同时，将通过ELISA和免疫组织化学方法研究肝脏特异性细胞标志物，如细胞角蛋白19（CK 19）、α 1-抗胰蛋白酶（AAT）和多药耐药蛋白（MRP）表达沿着白蛋白分泌。从而提供关于3D生物打印的肝窦状隙构建体中关于氧消耗/分布的细胞活力和表型活性的信息。建立的肝窦模型将用于评估其对肝毒性药物的反应。本研究将有助于建立体外肝窦模型，并为制备功能性可移植肝组织铺平道路。此外，该模型可以用于药物开发过程中评估新药的毒性，从而避免动物实验。

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.