Sferoid Yüklü Ecm-Mimetik Peptit Amfifil Hidrojellerle Çip-Üstü-Karaciğer Geliştirilmesi

Abstract

This study was financially supported by Hacettepe University Scientific Research Projects Coordination Unit with a project entitled “Development of the liver-on-chip to be used in determining the toxicity of drugs” (project number FBA-2017-12820) and The Scientific and Technological Research Council of Turkey (TUBITAK) under 2210-C Primary Subject National Scholarship Program for MSc Students. The aim of this study is to develop a liver-on-chip system, which can effectively perform liver tasks, for drug screening and toxicity tests. In the first step of the study, spheroid production was carried out by hanging drop method from hepatocyte (HepG2 cell line) and endothelial cells (HUVEC cell line), which are located in the smallest functional unit of the liver. Spheroids containing 1500 cells, in which hepatocytes were combined in 2:1 ratio with endothelial cells, were found to have a diameter of 428 ± 45 μm after 5 days of incubation and these spheroids were used within the scope of the thesis. Scanning Electron Microscopy (SEM) images demonstrated that the pores with a diameter of 2-3 μm on the outer surface of the spheroid are similar to the bile canals in the liver (1-3 μm). Immunocytochemical staining of hepatocytes that were performed with the aim of investigating the function of cells in spheroids showed that the cells maintained the liver-specific function of albumin synthesis and drug conversion. Von Willebrand Factor staining was performed to examine the HepG2 and HUVEC cell organization and vascularization in spheroids, and resulting confocal microscopy images showed that HUVEC cells covered HepG2 cells and formed tubular structures from the external surface to the center of the spheroid in 35 μm depth. Confocal images have proved that the self-assembly and cell arrangement process between HepG2 and HUVEC has been achieved. As a result it is found that the spheroids are vascular and functional. In the second part of the thesis, collagen-mimetic (PA-GFOGER) and fibronectin-mimetic (PA-RGDS) peptide amphiphiles were used to construct artificial microenvironments that support cellular behavior in spheroids. Since PA-RGDS has a net negative charge (-1) and PA-GFOGER is positively charged (+4), ECM-mimetic (PA-GFOGER + RGDS) hydrogel was obtained by mixing these two hydrogels in the ratio of 4:1. After the addition of calcium ions, it was found that the nanofiber organization occurred via β-sheet secondary structure. PA-GFOGER and PA-GFOGER + RGDS hydrogels were found to form fiber bundles with a diameter of 75-100 nm after self-assembly and gelation. Additionally, PA-GFOGER+RGDS hydrogel provided a hierarchical organization similar to ECM components. As a result of the rheology analysis, it was observed that all the hydrogels were viscoelastic and the storage module is decreased by collagen deposition. In the final part of the thesis, a "liver-on-chip" system was established consisting of a syringe pump, culture medium reservoir, polydimethylsiloxane (PDMS) chip and fitting elements. The culture of hydrogel-encapsulated spheroids under static and dynamic conditions was evaluated in terms of gene expression, albumin and urea release in comparison with hydrogel-free spheroids. At the end of 7 days, it was found that 2023 ± 409 ng albumin and 31003 ± 3595 nmol urea was released under dynamic culture conditions of spheroids, whereas under static conditions 1444 ± 92 ng albumin and 1279 ± 40 nmol urea was released. In particular, dynamic culture of hydrogel encapsulated spheroids significantly increased ALBUMIN and CYP 2E1 gene expressions by 4 and 28343 folds, respectively. In the light of these findings, it can be stated that the dynamic culture with the use of a perfusion bioreactor at 5 μL/min flow rate improved spheroid performance and hydrogel use in the organ-on-chip systems gave better results in terms of cell viability and functionality. However, it is concluded that the operating conditions in the present organ-on-chip system should be optimized for advanced applications.