Akademik Çıktılar

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Development and characterization of electrospun polymer scaffolds for 3D cell culture model to mimic human intestine system
(01. Izmir Institute of Technology, 2020-12) Firik, Tuğba; Arslan Yıldız, Ahu,; 01. Izmir Institute of Technology
Intestinal tissue engineering is a subfield of tissue engineering, which utilizes biomaterials to mimic the extracellular matrix (ECM) for tissue regeneration, repairment, simulating intestine function in 3D models. It combines scaffolds, which are obtained from natural, semisynthetic or synthetic polymer by using different fabrication methods, with various intestinal cells. In this thesis, polycarbonate (PC), polycaprolactone (PCL) and Polyvinylidene fluoride (PVDF), which are synthetic polymers, were used for fabrication of scaffolds by electrospinning to obtain an alternative material against commercial Transwell supports. Thus, electrospun PC, PCL and PVDF scaffold containing a 12-well Transwell insert was designed by sandwiching between two PMMA circles. To observe cellular behavior on scaffolds Caco-2 cells, which are most used human colorectal cancer cells in the intestinal tissue engineering system, were cultured on electrospun PC, PCL and PVDF scaffolds. According to cell viability analysis, toxic effect was not observed at the end of 21 days culturing period. Additionally, DAPI/Actin and collagen staining was performed for 21-days and results proved that intra/intercellular components and proteins were expressed successfully. As known in literature, Caco-2 cells make a tight junction that is formed by paracellular interactions after 3-4 weeks culturing. To analyze tight junction integrity, phenol red transport and transepithelial resistance were measured. The permeability of phenol red for PCL scaffold exhibited a significant decrease from 50% to 12% after 21 days on the contrary of PC and PVDF. In parallel, TEER measurements supported the phenol red transport assay because in PCL results showed that on the 7th day TEER was measured 468 Ω.cm2 in average and 21st day measurement was calculated approximately 1133 Ω.cm2.
Development and characterization of magnesium alginate hydrogels for 3d cell culture formation
(01. Izmir Institute of Technology, 2021-07) Çoban, Başak; Arslan Yıldız, Ahu; 01. Izmir Institute of Technology
Cell culture is an important tool for biological research. Two-dimensional (2D) cell culture is still used but growing cells on plastic surfaces offering unnatural growth kinetics and cell attachment. Three-dimensional (3D) cell culture allows cells to growth in their 3D physical shape and interact with their surroundings which represent the natural microenvironment. Hydrogels are crosslinked networks, have become increasingly used biomaterial for 3D cell culture with their ability to simulate the nature of most soft tissues. In this thesis, a new methodology based on bio-patterning was developed to fabricate (3D) cellular structures by using Mg-alginate hydrogel and fabricated 3D cellular structures was utilized for drug screening studies. Mg-alginate hydrogel has a specific gelation/de-gelation characteristics compared to other types of hydrogels due to its weak polymer-ion interaction. In this study slow gelation and de-gelation property of Mg-alginate hydrogel was used for biopatterning of 3D cellular structures. Plackett-Burman and Box-Behnken design models were used to optimize parameters of Mg alginate-based biopatterning method while using HeLa cells as a model cell line. Then, the applicability of newly developed methodology was successfully demonstrated by using SaOS-2 and SH-SY5Y cells to fabricate 3D cellular structures. Cell proliferation and migration profiles were observed during long-term culturing with time-dependent light microscopy images. Also cell proliferation and viability of long-term cultured tumor models were analyzed by using Alamar Blue and Live/Dead assays. Moreover, F-actin, Collagen I, and DAPI staining/immunostaining was done to investigate cellular and extracellular components of 3D cellular structures for short and long-term culture times. Finally, the dose-response of fabricated 3D structures was evaluated and compared with standard 2D cell culture by applying doxorubicin (DOX). The IC50 values were calculated for 3D cellular structure of HeLa, SaOS-2 and SH-SY5Y cells as 8.2, 7.8, and 2.1 µM respectively while IC50 values of 2D controls obtained as 3.2, 4.4, and 0.2 µM respectively. These results were also statistically analyzed and dose responses were found significantly different according to t-test, which means 3D cellular structures were more resistant to drug exposure compared to 2D cell culture.
Development and use of contactless magnetic manipulation methodologies for the formation of 3D cardiac models
(01. Izmir Institute of Technology, 2022-12) Önbaş, Rabia; Arslan Yıldız, Ahu
In this thesis, two contactless magnetic manipulation methodologies were introduced, which are magnetic levitation (MagLev) and biopatterning techniques. The optimization steps of both techniques were completed with NIH/3T3 mouse fibroblast cells. Later, 3D cardiac models were developed using H9c2 rat cardiomyocytes. For the MagLev technique, tunable 3D spheroids were obtained with changing initial cell seeding number, gadobutrol concentrations, and culturing time. For the biopatterning approach, a new bio-ink formulation, which comprises alginate, magnetic nanoparticles, and cells, was developed. Further, biopatterned cellular structures were fabricated in different shapes such as discs, rings, and rectangles under an external magnetic field. Later, characterization was done successfully via immunostaining of collagen I, F-actin, and DAPI. Moreover, cardiac-specific markers; cardiac troponin T and MYH6 were analyzed for both 3D cardiac spheroids and patterned 3D cardiac structures. Finally, doxorubicin was applied to evaluate the drug responses. IC50 values were calculated as 14.7 μM and 8.1 μM for 3D cardiac spheroids and 3D cellular structures respectively, while standard 2D cell culture was 3.5 μM which indicated 3D cardiac models were more resistant to drug exposure. In the last part of thesis, patterned 3D cardiac structures were fabricated using co-cultured hiPSC-derived cardiomyocytes and cardiac fibroblast cells via biopatterning methodology. Characterization was carried out successfully by immunostaining of α-actinin, collagen I, Cx-43, Troponin T, and DAPI. Taken together, to fabricate 3D cell culture models, MagLev and biopatterning-based contactless manipulation methodologies may be good alternatives to conventional 2D cell culture methods for tissue engineering applications, especially for drug screening.
Generation and characterization of three dimensional organotypic KID syndrome skin model
(Izmir Institute of Technology, 2017-07) Öztürk, Özgür; Meşe Özçivici, Gülistan
Keratitis, ichthyosis, deafness (KID) syndrome is a rare genetic disorder caused by connexin 26 gene mutation that shows quite debilitating and horrific effects on patients. Syndrome itself is complex and 2D culture methods fail to provide complex and close to real conditions to investigate KID syndrome. Our goal is to construct organotypic 3D skin model for the KID syndrome. First of all, stable cell lines expressing wild type and D50Y mutant Cx26 protein were generated. Immunostaining, Western blot and qRT-PCR analysis confirmed Cx26 expression in stable cell lines, meaning these cell lines can be utilized to construct 3D skin model. In order to generate organotypic KID syndrome skin models, commercially available transwell inserts were used. Constructs were prepared by plating fibroblast-collagen mixture in inserts and then plating generated stable cell lines on top of the fibroblast-collagen layer. Then immunostaining was performed on generated skin constructs. Immunostaining of cytokeratin 14 confirmed that 3D model has basal layer of the epidermis. Also, KID skin model with Whatman paper was conducted as an alternative to transwell inserts. Phalloidin staining results showed that generated cell lines formed 3D structures within cellulose fibers. Furthermore; Cx43-Cx26 interaction and cell viability were investigated in stable HaCaT cells. Western blot results showed that increase in Cx26 protein, wild type or mutant, caused an increase in Cx43 levels. According to MTT assay, increase of wild type or D50Y mutant Cx26 did not change cell viability. Overall with these findings, provides a new point of view for KID syndrome mechanism and treatment.
Lab-on-a-chip devices for drug screening
(Izmir Institute of Technology, 2019-05) Gökçe, Begüm; Pesen Okvur, Devrim; Çağır, Ali
Breast cancer is one of the cancers with the highest incidence and mortality rates in women in Turkey as well as in the world. Tumor micro environment comprises of cancer and normal cells, extracellular matrix, soluble biological and chemical factors. Research has shown that cell shape, adhesion, migration, response to growth factors and drugs are different in 2D and 3D culture. Today, only 8 out of 100 anti-cancer clinical trial gives effective results. 3D cell culture systems have shown to be a necessary step between in vitro, in vivo and clinical studies. Therefore, it is necessary to better understand the interactions of cancer cells with their micro environment, for which new cell culture setups are required. The most apparent disadvantage of widely used 3D cell culture setups is the lack of stromal cells. The systems to be developed should both provide a 3D environment and comprise multiple cell types. The drug screen in 3D tri-culture method with a lab-on-a-chip device, that will be developed in this study will be able to answer these needs. Cell lines that represent different breast cancer types alone or together with stromal cells were cultured in 3D in the to be developed lab-on-a-chip; by determining the effects of drugs with different targets on the viability and distribution of cells, a drug screening method is developed.

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Browsing Akademik Çıktılar by Subject "3D cell culture"