A Cell Culture Chip with Transparent, Micropillar-Decorated Bottom for Live Cell Imaging and Screening of Breast Cancer Cells

In the recent years, microfabrication technologies have been widely used in cell biology, tissue engineering, and regenerative medicine studies. Today, the implementation of microfabricated devices in cancer research is frequent and advantageous because it enables the study of cancer cells in controlled microenvironments provided by the microchips. Breast cancer is one of the most common cancers in women, and the way breast cancer cells interact with their physical microenvironment is still under investigation. In this study, we developed a transparent cell culture chip (Ch-Pattern) with a micropillar-decorated bottom that makes live imaging and monitoring of the metabolic, proliferative, apoptotic, and morphological behavior of breast cancer cells possible. The reason for the use of micropatterned surfaces is because cancer cells deform and lose their shape and acto-myosin integrity on micropatterned substrates, and this allows the quantification of the changes in morphology and through that identification of the cancerous cells. In the last decade, cancer cells were studied on micropatterned substrates of varying sizes and with a variety of biomaterials. These studies were conducted using conventional cell culture plates carrying patterned films. In the present study, cell culture protocols were conducted in the clear-bottom micropatterned chip. This approach adds significantly to the current knowledge and applications by enabling low-volume and high-throughput processing of the cell behavior, especially the cell-micropattern interactions. In this study, two different breast cancer cell lines, MDA-MB-231 and MCF-7, were used. MDA-MB-231 cells are invasive and metastatic, while MCF-7 cells are not metastatic. The nuclei of these two cell types deformed to distinctly different levels on the micropatterns, had different metabolic and proliferation rates, and their cell cycles were affected. The Ch-Pattern chips developed in this study proved to have significant advantages when used in the biological analysis of live cells and highly beneficial in the study of screening breast cancer cell-substrate interactions in vitro

Photo-crosslinked gelatin methacrylate hydrogels with mesenchymal stem cell and endothelial cell spheroids as soft tissue substitutes

Tumors, trauma, and congenital defects require volume restoration of soft tissues. Tissue engineering provides an alternative source for substituting these defects. Cell encapsulation into hydrogels provides a three-dimensional microenvironment. Spheroids of cells provide close packing and increase cell-to-cell contacts resulting in differentiation. Gelatin is a natural polymer with low immunogenicity and preserved amino acid motifs for cell adhesion and proliferation. In the present study, a soft photo-crosslinked gelatin methacrylate (GelMA) hydrogel with long in vitro lifetime was synthesized. Stem cells (dental pulp derived, DPSC) and endothelial cells (umbilical cord derived, HUVEC) were formed into spheroids to induce prevascular network formation and encapsulated into GelMA (10% weight/volume). Results showed high cell viability, better gel mechanical properties, and longer HUVEC sprouting with spheroids compared to the same combination of cells. Altogether, the photo-crosslinked GelMA hydrogels with DPSC and HUVEC spheroids provided a promising tissue engineering and vascularization strategy in vitro

Mikrodesenli polimerik sübstratların kanser hücresi davranışı üzerine etkisi

The aim of this study was to develop micropatterned surfaces on biodegradable polymers such as PLGA and PLLA and non-degradable polymer PMMA to study cellular responses including proliferation, cellular morphology, nucleus morphology and deformation, focal adhesions and related pathways, cell division and cycle, and epithelial to mesenchymal transition in cancer cells. An array of nine surfaces decorated with micron sized micropillars were produced using photolithography. Saos-2 osteosarcoma and hOB human osteoblast-like cells were cultured on the micropillar array made from PLGA, PLLA or their blends for focal adhesion and micropillar bending studies. Deformations of nuclei on the micropatterned surfaces were studied with Saos-2, hOB, L-929 mouse fibroblast, SH-SY5Y neuroblastoma, and MCF-7 breast carcinoma cells. Cell division and cycle studies were conducted with Saos-2 cells on PLGA and MDA-MB231 and MCF-7 cells on PMMA. All surfaces induced nucleus deformations but smaller interpillar distances were found to be most effective. Of all the cells tested for nucleus deformations, cancer cells (Saos-2, MCF-7, SH-SY5Y) deformed most prominently. Both Saos-2 and hOB cells were found to apply similar forces to bend pillars and highest bending forces were applied on PLGA and PLLA substrates rather than their blends. Micropatterned PMMA substrates were found to effect cell cycle and induce an arrest at G0/G1 phase. RT-qPCR and RNA sequencing analysis demonstrated that Micropatterned PMMA surfaces induced EMT in epithelial breast cancer cells. Micropatterned substrates were proven to affect many cellular processes and intracellular signaling pathways. Cancer cells were found to be more prone to these changes.Ph.D. - Doctoral Progra

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

MİKRODESENLERİN IN VIVO IMPLANT YÜZEYİ-HÜCRE ETKİLEŞİLERİ ÜZERİNE ETKİSİNİN DEĞERLENDİRİLEMES

In situ silver nanoparticle synthesis on 3D-printed polylactic acid scaffolds for biomedical applications

An ultraviolet (UV) irradiation-based in situ silver nanoparticle (AgNP) synthesis approach has drawn significant attention for functionalizing a great variety of biomaterials. Here, we designed an AgNP-functionalized 3D-printed polylactic acid (PLA) composite scaffold with a green physical approach by employing the UV irradiation (1, 2, and 3 h) method without using any reducing agent or heat treatments. In situ AgNP synthesis was performed under different UV exposure times. The zeta sizer analysis results demonstrated that AgNPs were highly monodisperse with the particle size of 20 +/- 2.2, 30 +/- 3.6, and 50 +/- 4.8 nm under various UV light exposure times. In situ synthesis of AgNPs on 3D-printed PLA scaffolds significantly changed the surface hydrophilicity of the 3D-printed scaffolds. These results showed that UV irradiation-based in situ AgNP synthesis on 3D-printed PLA scaffolds can be useful in various biomedical applications, such as cell culture scaffolds, biosensors, and wound healing applications

MİKRO DESENLİ POLİMERİK YÜZEYLER KULLANARAK SAĞLIKLI VE NEOPLASTİK HÜCRELERİN DAVRANIŞARININ SAPTANMASI

Bu projenin amacı, mikro desenli polimerik filmler üreterek, desenlerin sağlıklı ve neoplastik hücre tipleri üzerine fiziksel etkisinin araştırılması ve kanser tanısında kullanılabilirliğinin saptanmasıdır. Mikro-elektro-mekanik sistemler (MEMS) teknolojisi kullanılarak geometrik desenli mikro kalıplar üretilecek, polimer filmler bu kalıplardan üretilen polidimetilsiloksan (PDMS) negatif kalıplarda çoğaltılacaktır. Bu çalışmada farklı hücre gruplarında hem hücre çekirdeklerinin şekillerinin ve fokal adezyonlarda görev alan önemli proteinlerin yoğunluk farklarının saptanması, hem de yüzey desenlerindeki boyutsal farklılıkların hücre davranışı üzerine etkisinin araştırılması planlanmaktadır. Bu çalışmadan elde edilecek veriler ışığında, kanser tanı testi olmaya en uygun yüzey geometrisinin belirleneceği düşünülmektedir

Modified chitosan scaffolds: Proliferative, cytotoxic, apoptotic, and necrotic effects on Saos-2 cells and antimicrobial effect on Escherichia coli

Scaffolds used in tissue engineering applications should have high biocompatibility with minimum allergic, toxic, apoptotic, or necrotic effects on the growing cells and newly forming tissue and, if possible, have antimicrobial property to prevent infection at the host site. In this study, novel micro-fibrous chitosan scaffolds, having mineralized bioactive surface to enhance cell adhesion and a model antibiotic (gentamicin) to prevent bacterial attack, were prepared. The effects of the scaffolds on proliferation, viability, apoptosis, and necrosis of Saos-2 cells are reported for the first time. Wet spinning technique was used in the scaffold preparation and biomineralization was achieved by incubating them in five-time concentrated simulated body fluid for 2, 7, or 14days (coded as CH-BM/2, CH-BM/7, and CH-BM/14, respectively). Gentamicin, an effectively used antibiotic in bone treatments, was loaded by vacuum-pressure cycle. Energy-dispersive X-ray results demonstrated that Ca/P ratio of the mineral phase varies depending on the incubation period. When the scaffolds were cultured with Saos-2 cells, cell adhesion and extracellular matrix formation occurred on all types of scaffolds. Alamar Blue cytotoxicity tests showed correlation among mineral concentration and cytotoxicity where CH-BM/2 had significantly more favorable properties. For all types of scaffolds, apoptosis and necrosis were less than 10%, meaning the samples are biocompatible. Gentamicin-loaded scaffolds showed high antimicrobial efficacy against Escherichia coli. The presence of mineral phase enhanced the adhesive capacity of cells and entrapment efficiency of antibiotic. These results suggest that the bioactive and antimicrobial scaffolds prepared in this study can act as promising matrices in bone tissue engineering applications

Micro and Nanofabrication methods to control cell-substrate interactions and cell behavior: A review from the tissue engineering perspective

Cell-substrate interactions play a crucial role in the design of better biomaterials and integration of implants with the tissues. Adhesion is the binding process of the cells to the substrate through interactions between the surface molecules of the cell membrane and the substrate. There are several factors that affect cell adhesion including substrate surface chemistry, topography, and stiffness. These factors physically and chemically guide and influence the adhesion strength, spreading, shape and fate of the cell. Recently, technological advances enabled us to precisely engineer the geometry and chemistry of substrate surfaces enabling the control of the interaction cells with the substrate. Some of the most commonly used surface engineering methods for eliciting the desired cellular responses on biomaterials are photolithography, electron beam lithography, microcontact printing, and microfluidics. These methods allow production of nano-and micron level substrate features that can control cell adhesion, migration, differentiation, shape of the cells and the nuclei as well as measurement of the forces involved in such activities. This review aims to summarize the current techniques and associate these techniques with cellular responses in order to emphasize the effect of chemistry, dimensions, density and design of surface patterns on cell-substrate interactions. We conclude with future projections in the field of cell-substrate interactions in the hope of providing an outlook for the future studies. (c) 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)