(19f) Culturing the Co-Encapsulated Primary Hepatocytes with Mesenchymal Stem Cells: Study on Effect of Co-Encapsulation and Perfusion on Hepatocyte Metabolic Activity

Liver is one of the most sophisticated organs in the body which is responsible for a wide spectrum of tasks including drug detoxification and metabolism, hormone production and protein synthesis. In addition, liver physiology and geometry exert another level of complexity for fabrication of a suitable in-vitro model to analyze the hepatocyte metabolic activity. To do so, there is an immense demand to study the hepatocytes activity over a longer period. One of the great challenges in the field of liver bioengineering is the loss of hepatocyte morphology and functionality in all in-vitro models in a short period unlike the in-vivo conditions which the liver can thoroughly regenerate up to 80% of the lost hepatocytes due to disease. Therefore, disparate methods including collagen sandwich cultures, co-culture with nonparenchymal cells (NPCs), hepatocyte spheroids and perfusion bioreactors have been studied to overcome this limitation. Collagen sandwich cultures have been used for long time due to the proper microenvironment they provide for the hepatocyte in-vitro. Though sandwich cultures have improved the metabolic activity and functionality of primary hepatocytes compared to the normal culture techniques, they still have drawbacks including bath to batch variation of the collagen used and the barrier effects the collagen sandwich culture inputs on the transfer of oxygen to the cells. To overcome these drawbacks, here we propose microencapsulation of hepatocytes in an ECM protein. The ECM proteins used are chondroitin sulfate and hyaluronic acid which were mixed with hepatocytes and electrosprayed into a chitosan solution. These capsules provide structural organization, zonation, shear protection, and scalability. In addition, the transport phenomena inside the microcapsules can be controlled easily by changing the capsule size and ECM concentrations used in making the microcapsules. Another key parameter in sustaining the hepatocyte metabolic activity is to co-culture with NPCs. It has been shown that neighbor NPCs inside the liver play a major role in regulation of hepatocyte activity and functionality. Liver sinusoid endothelial cells (LSECs) are one of these cells which are responsible for vascularization and angiogenesis inside the liver sinusoid. Mesenchymal stem cells (MSCs) are also multipotent stem cells which are widely used in stem cell therapies and have high proliferation capacity. Alongside, they can be differentiated to vascular endothelial cells (VECs) and hepatocyte under certain conditions. Due to all these characteristics, in this research we have co encapsulated hepatocytes with MSCs and analyzed its effect on hepatocyte metabolic activity including albumin, urea secretion and CYP450 activity. In all tests, coculturing hepatocytes with MSCs improved the metabolic activity and viability of hepatocytes compared to monoculture hepatocytes. High oxygen demand is one of the major characteristics of hepatocytes. Traditional static 2D and even 3D culture systems are not able to fulfill this requirement, therefore, perfusion culture systems have been proposed by various research groups to overcome this limitation. In this research, the coencapsulated capsules were used in a perfusion chip system which consists of a culture chamber with inlet and outlet for the medium. The bottom of the chip was coated with collagen and the input medium was oxygenated. The bioreactor is a pump free which lets precise achieve low flowrates inside the chip to prevent cell damage due to shear stress. The results were compared to the microcapsules inside the static culture plates which revealed higher albumin and urea secretion in the perfusion system. These results suggest that coencapsulation of rat primary hepatocyte with MSC in a GAG-chitosan microenvironment inside the perfusion system sustains the viability and metabolic activity better than other culture techniques. These microcapsules can be used in various modular tissue engineering and regenerative medicine applications.