Production and characterization of three-dimensional (3D) in vitro Parkinson's disease models

In this thesis, a three-dimensional (3D) in vitro Parkinson's Disease (PD) model was designed and produced in order to test pre-clinical in vitro studies rapidly and simulating in vivo microenvironment conditions closer. For this purpose, we compared the ability of PC12 and SH-SY5Y cell lines' microtissue forming abilities and characterized the 3D neurotoxic model formed by 6-hydroxy dopamine (6-OHDA) toxicity on these microtissues. Cells were seeded at different numbers in the microtextures of agar plates formed using the 3D Petri Dish® technique to compare microdissection capabilities and the resulting microtissue sizes were observed with inverted phase contrast light microscopy. Effective doses of 6-OHDA neurotoxin were determined by MTT assay in 2D and 3D culture conditions for both PC12 and SH-SY5Y cells. Live & dead cell analyses were performed for 3D models and cytotoxicity (LDH) analyses were performed on both 2D and 3D PD models. The expression of tyrosine hydroxylase (TH), substance P (Substance P, SP), vasoactive intestinal peptide (VIP) and synaptophysin (SYN) markers in the PC12 and SH-SY5Y PD models generated in 2D and 3D culture conditions were evaluated by immunocytochemical (ICC) and immunohistochemical (IHC) staining. Finally, the expression ratios of Tyrosine Hydroxylase (TH), Dopamine Transporter-1 (DAT1), Parkin (PARK2) and alpha-synuclein (α-Syn) genes were identified by real-time quantitative polymeric chain reactions (qRT-PCR). As a result of LDH analysis after induction of 6-OHDA with active doses, no cell membrane damage was observed in SH-SY5Y cells, while PC12 cells' membrane integrity deteriorated in both 2D and 3D culture conditions. It has been observed that the toxicity of 6-OHDA on PC12 cells was due to the increased amount of ROS produced and the activation of the apoptotic pathway as well as the deterioration of membrane integrity. Thus, we have determined that PC12 cells have weaker membrane properties than SH-SY5Y cells. In addition, it has been identified that PC12 cells in 3D culture conditions are more likely to protect membrane integrity compared to 2D conditions. It was determined that the dead cells tend to move away from microtissues due to their low cohesive properties after 6-OHDA induction. In both 2D and 3D SH-SY5Y experimental groups, the expression of TH, VIP and SP markers was lower than the control groups that did not receive 6-OHDA induction, while the expression of the SYN was higher in the 3D culture conditions than in the 2D culture conditions. This could be because of the tendencey of SH-SY5H cells' increased synaptic activity in order to increase their interactions after stress that was caused by induction with 6-OHDA. In the 3D PC12 model, it was not possible to compare the TH, VIP, SP and SYN markers because the microtissues were dispersed. qRT-PCR analysis in 2D models revealed that 6-OHDA caused a decline in expression of TH and PARK2 genes compared to the control group while causing an increase in expression of α-Syn gene. DAT1 gene was expressed slightly in SH-SY5Y cells, however PC12 cells didn't express DAT1 at all. Under 3D culture conditions, all studied genes' expressions were observed in SH-SY5Y cells, while PC12 microtissues did not show any expression of DAT1 and α-Syn, even in control groups, while PARK2 was only slightly expressed in the control group. However, following the 6-OHDA induction, PARK2 gene expression was lost. No expression of the control group of PC12 cells's certain genes in 3D conditions might be the proof that PC12 cells lost their dopaminergic properties completely in 3D conditions. As a result of all the data obtained from comparison of tested 3D PD models, it has been shown that SH-SY5Y microtissues can be used as a 6-OHDA toxicity-induced 3D in vitro PD model and can be an alternative model to fill the gap between 2D in vitro and in vivo studies in the diagnosis and treatment of PD. Keywords: Parkinson's disease (PD), in vitro model, 3-dimensional (3B) cell culture