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

基于挤压的3D生物打印方法被认为是最适合于生物工程可移植肝组织的方法。近年来，许多有益的水凝胶已经被开发出来，用于肝脏特异性细胞的包裹和3D生物打印。这些水凝胶通常根据其物理特性进行表征，如粘度、硬度和挤出性。然而，氧在新开发的水凝胶中的扩散很少被表征。在为肝脏组织工程开发的水凝胶中，氧的扩散特性将发挥重要作用，因为肝窦显示出高度不均匀的氧气沿其血管通道的分布。因此，用于肝脏组织工程的水凝胶应该能够支持异质氧分布。这项拟议的工作的目的是利用3D生物打印来制造肝小叶结构，重点是模拟天然的肝窦微环境-特别是通过建立沿肝窦管腔的异质氧分布来获得不同的肝脏代谢区带。为了实现这一目标，我们将系统地利用我们建立的实时氧监测方法来开发和表征新的/现有的水凝胶的氧扩散特性。合适的水凝胶将被用来包裹肝脏特定类型的细胞(肝细胞、肝源性内皮细胞和成纤维细胞)，并将被用于通过核/壳3D生物打印方法构建肝窦结构。3D生物打印的肝窦结构的管腔将连接到微灌流系统，以建立动态培养条件。然后，将使用实时氧气监测方法分析这些构造的耗氧率。同时，采用酶联免疫吸附试验和免疫组织化学方法检测肝脏特异性细胞标志物细胞角蛋白19(CK19)、α1抗胰蛋白酶(AAT)和多药耐药蛋白(MRP)的表达及白蛋白分泌情况。从而提供关于3D生物打印的肝窦结构中氧气消耗/分布的细胞活性和表型活性的信息。已建立的肝窦模型将用于评估其对肝脏毒性药物的反应。这些研究将有助于建立体外肝窦模型，并为构建功能性可移植肝组织铺平道路。此外，该模型还可以通过评估新药毒性来应用于药物开发过程，从而绕过动物实验。

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.