İlaç Araştırmaları İçin 3-Boyutlu Mikroakışkan Damar Modelinin Geliştirilmesi

Abstract

There is a need for a 3-dimensional (3D) vessel model that can be used to simulate in vivo vessel physiology, anatomy, and blood flow-induced fluid tension force that can be used in the investigation of vascular diseases, screening, and detection of new drug candidate molecules and investigating their vascular toxicities. In recent years, microfluidic organ chips modeled to mimic the physiological responses of human tissues and/or organs have been the focus of attention in in vitro modeling of 3D tissues/organs. This thesis aims to develop a perfusable 3D vessel chip model that mimics physiological conditions and in vivo vascular structure (tunica externa, tunica media, and tunica intima layers). For this purpose, channel with 1.8 mm diameter, 20 mm length and 1.1 cm2 lateral area was created with Pluronic F-127 copolymer on a 35 mm diameter PDMS base with a 3D printer. The channel was coated first with dopamine hydrochloride (polyDOPA, 2 mg/mL), then with collagen or fibronectin. After that, isolated and characterized vascular smooth muscle cells (vSMC), vascular endothelial cells (vEC), and human (hFB) or mouse (L-929) fibroblast cells were cultured in PDMS channel under static or dynamic condition (60 μL/min.). Cell behavior was investigated by testing the culture time, substrate, cell number (50,000 cells/chip or 25,000 cells/chip) to create the 3D vessel structure. Subsequently, different CellTracker® labeled fibroblast cells, and vSMC were iv co-cultured and perfused in PDMS channels under dynamic conditions; cell presence, cell localization pattern, and formation of cell layers were examined. Finally, hFB, vSMC and vEC (1:1:1) cells were co-cultured in polyDOPA or polyDOPA+collagen coated PDMS channel, respectively, and a 3D vessel model was developed under dynamic conditions. This model was characterized for the expression of endothelial and smooth muscle cellspecific proteins. The contact angle and FTIR results showed that the PDMS surface was coated with polydopamine and obtained a hydrophilic surface. Microscopic analysis showed that vSMCs could adhere to the surface by showing similar behavior in different cell number, substrate type and culture time tested under static conditions, and formed micro tissue with high cell viability in all substrates and cell numbers tested under dynamic conditions. In dynamic conditions, it was determined that vEC and fibroblast cells preserve the cell layer structure they formed under static conditions, vECs tend to lose their layer structure, and fibroblast cells tend to develop a denser layer depending on the exposure time to shear stress. vSMC-fibroblast co-culture studies showed that fibroblast-vSMC direct co-culture was successfully performed, and the cells were preserved in culture. Confocal and immunofluorescence analyzes showed that the 3D vessel model, including fibroblast, smooth muscle, and endothelial layers was formed by successful triple co-culture in PDMS channel coated with polyDOPA+collagen under dynamic conditions.