Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Citation - Scopus: 11Μdacs Platform: a Hybrid Microfluidic Platform Using Magnetic Levitation Technique and Integrating Magnetic, Gravitational, and Drag Forces for Density-Based Rare Cancer Cell Sorting(Elsevier, 2023) Keçili, Seren; Yılmaz, Esra; Özçelik, Özge Solmaz; Anıl İnevi, Müge; Günyüz, Zehra Elif; Yalçın Özuysal, Özden; Özçivici, Engin; Tekin, Hüseyin CumhurCirculating tumor cells (CTCs) are crucial indicators of cancer metastasis. However, their rarity in the bloodstream and the heterogeneity of their surface biomarkers present challenges for their isolation. Here, we developed a hybrid microfluidic platform (microfluidic-based density-associated cell sorting (µDACS) platform) that utilizes density as a biophysical marker to sort cancer cells from the population of white blood cells (WBCs). The platform utilizes the magnetic levitation technique on a microfluidic chip to sort cells based on their specific density ranges, operating under a continuous flow condition. By harnessing magnetic, gravitational, and drag forces, the platform efficiently separates cells. This approach involves a microfluidic chip equipped with a microseparator, which directs cells into top and bottom outlets depending on their levitation heights, which are inversely proportional to their densities. Hence, low-density cancer cells are collected from the top outlet, while high-density WBCs are collected from the bottom outlet. We optimized the sorting efficiency by varying the flow rates, and concentrations of the sorting medium's paramagnetic properties using standard densities of polymeric microspheres. To demonstrate the platform's applicability, we performed hybrid microfluidic sorting on MDA-MB-231 human breast cancer cells and U-937 human monocytes. The results showed efficient sorting of rare cancer cells (≥100 cells/mL) from serum samples, achieving a sorting efficiency of ∼70% at a fast-processing speed of 1 mL h−1. This label-free approach holds promise for rapid and cost-effective CTC sorting, facilitating in-vitro diagnosis and prognosis of cancer. © 2023 The Author(s)Article Citation - WoS: 7Citation - Scopus: 7Connexin 32 Overexpression Increases Proliferation, Reduces Gap Junctional Intercellular Communication, Motility and Epithelial-To Transition in Hs578t Breast Cancer Cells(Springer, 2022) Uğur, Deniz; Güngül, Taha Buğra; Yücel, Simge; Özçivici, Engin; Yalçın Özuysal, Özden; Meşe Özçivici, GülistanConnexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32's effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.Article Citation - WoS: 17Citation - Scopus: 22Protein Corona Formation on Silver Nanoparticles Under Different Conditions(Elsevier, 2022) Tomak, Aysel; Yılancıoğlu, Buket; Winkler, David; Öksel Karakuş, CeydaThe surfaces of nanoparticles become covered by biomolecules in biological fluids. This protein ‘corona’ modifies materials’ characteristics and biological activity. The composition of the protein corona is dynamic, abundant biomolecules that bind first are subsequently replaced by less abundant but more tightly bound ones. Here, we explore the formation of the silver nanoparticle protein corona on exposure to cell culture media containing 10 % fetal bovine serum supplemented Dulbecco's Modified Eagle's medium. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry analysis were used to monitor how different parameters such as incubation time, heating duration, cell culture medium, incubation temperature, and the number of washes affect the nanoparticle–protein corona complex. silver nanoparticles with and without bound proteins were characterized by electron microscopy, dynamic light scattering, and ultraviolet-visible-near-IR spectroscopy. The tetrazolium-based MTT assay was used to determine viability of A549 human lung adenocarcinoma cells treated with silver nanoparticles. Characterization of the nanoparticles before and after protein binding provided insights into their changing morphology on corona formation. Our results confirmed that the physiological environment directly affects protein corona formation on nanoparticle surfaces. In particular, incubation condition-dependent differences in the amount of bound proteins were observed. This work highlights the importance of environmental drivers of protein adsorption, which should be considered when predicting and/or controlling protein targets of silver nanoparticles.Article Citation - WoS: 22Citation - Scopus: 26Magnetic Levitation Assisted Biofabrication, Culture, and Manipulation of 3d Cellular Structures Using a Ring Magnet Based Setup(Wiley, 2021) Anıl İnevi, Müge; Delikoyun, Kerem; Meşe Özçivici, Gülistan; Tekin, Hüseyin Cumhur; Özçivici, EnginDiamagnetic levitation is an emerging technology for remote manipulation of cells in cell and tissue level applications. Low-cost magnetic levitation configurations using permanent magnets are commonly composed of a culture chamber physically sandwiched between two block magnets that limit working volume and applicability. This work describes a single ring magnet-based magnetic levitation system to eliminate physical limitations for biofabrication. Developed configuration utilizes sample culture volume for construct size manipulation and long-term maintenance. Furthermore, our configuration enables convenient transfer of liquid or solid phases during the levitation. Before biofabrication, we first calibrated/ the platform for levitation with polymeric beads, considering the single cell density range of viable cells. By taking advantage of magnetic focusing and cellular self-assembly, millimeter-sized 3D structures were formed and maintained in the system allowing easy and on-site intervention in cell culture with an open operational space. We demonstrated that the levitation protocol could be adapted for levitation of various cell types (i.e., stem cell, adipocyte and cancer cell) representing cells of different densities by modifying the paramagnetic ion concentration that could be also reduced by manipulating the density of the medium. This technique allowed the manipulation and merging of separately formed 3D biological units, as well as the hybrid biofabrication with biopolymers. In conclusion, we believe that this platform will serve as an important tool in broad fields such as bottom-up tissue engineering, drug discovery and developmental biology.Article Citation - WoS: 5Citation - Scopus: 5Low Magnitude High Frequency Vibrations Expedite the Osteogenesis of Bone Marrow Stem Cells on Paper Based 3d Scaffolds(Springer, 2020) Karadaş, Özge; Meşe, Gülistan; Özçivici, EnginAnabolic effects of low magnitude high frequency (LMHF) vibrations on bone tissue were consistently shown in the literature in vivo, however in vitro efforts to elucidate underlying mechanisms are generally limited to 2D cell culture studies. Three dimensional cell culture platforms better mimic the natural microenvironment and biological processes usually differ in 3D compared to 2D culture. In this study, we used laboratory grade filter paper as a scaffold material for studying the effects of LHMF vibrations on osteogenesis of bone marrow mesenchymal stem cells in a 3D system. LMHF vibrations were applied 15 min/day at 0.1 g acceleration and 90 Hz frequency for 21 days to residing cells under quiescent and osteogenic conditions. mRNA expression analysis was performed for alkaline phosphatase (ALP) and osteocalcin (OCN) genes, Alizarin red S staining was performed for mineral nodule formation and infrared spectroscopy was performed for determination of extracellular matrix composition. The highest osteocalcin expression, mineral nodule formation and the phosphate bands arising from the inorganic phase was observed for the cells incubated in osteogenic induction medium with vibration. Our results showed that filter paper can be used as a model scaffold system for studying the effects of mechanical loads on cells, and LMHF vibrations induced the osteogenic differentiation of stem cells.Article Citation - WoS: 22Citation - Scopus: 24Scaffold-Free Biofabrication of Adipocyte Structures With Magnetic Levitation(John Wiley and Sons Inc., 2021) Sarıgil, Öykü; Yalçın Özuysal, Özden; Anıl İnevi, Müge; Meşe Özçivici, Gülistan; Fıratlıgil Yıldırır, Burcu; Fıratlıgil Yıldırır, Burcu; Ünal, Yağmur Ceren; Ünal, Yağmur Ceren; Yalçın Özuysal, Özden; Özçivici, Engin; Meşe, Gülistan; Sarıgil, Öykü; Özçivici, Engin; Anıl İnevi, Müge; Meşe Özçivici, GülistanTissue engineering research aims to repair the form and/or function of impaired tissues. Tissue engineering studies mostly rely on scaffold-based techniques. However, these techniques have certain challenges, such as the selection of proper scaffold material, including mechanical properties, sterilization, and fabrication processes. As an alternative, we propose a novel scaffold-free adipose tissue biofabrication technique based on magnetic levitation. In this study, a label-free magnetic levitation technique was used to form three-dimensional (3D) scaffold-free adipocyte structures with various fabrication strategies in a microcapillary-based setup. Adipogenic-differentiated 7F2 cells and growth D1 ORL UVA stem cells were used as model cells. The morphological properties of the 3D structures of single and cocultured cells were analyzed. The developed procedure leads to the formation of different patterns of single and cocultured adipocytes without a scaffold. Our results indicated that adipocytes formed loose structures while growth cells were tightly packed during 3D culture in the magnetic levitation platform. This system has potential for ex vivo modeling of adipose tissue for drug testing and transplantation applications for cell therapy in soft tissue damage. Also, it will be possible to extend this technique to other cell and tissue types.Article Citation - WoS: 3Citation - Scopus: 4Applicability of Low-Intensity Vibrations as a Regulatory Factor on Stem and Progenitor Cell Populations(Bentham Science Publishers, 2020) Baskan, Öznur; Karadaş, Özge; Meşe, Gülistan; Özçivici, EnginPersistent and transient mechanical loads can act as biological signals on all levels of an organism. It is therefore not surprising that most cell types can sense and respond to mechanical loads, similar to their interaction with biochemical and electrical signals. The presence or absence of mechanical forces can be an important determinant of form, function and health of many tissue types. Along with naturally occurring mechanical loads, it is possible to manipulate and apply external physical loads on tissues in biomedical sciences, either for prevention or treatment of catabolism related to many factors, including aging, paralysis, sedentary lifestyles and spaceflight. Mechanical loads consist of many components in their applied signal form such as magnitude, frequency, duration and intervals. Even though high magnitude mechanical loads with low frequencies (e.g. running or weight lifting) induce anabolism in musculoskeletal tissues, their applicability as anabolic agents is limited because of the required compliance and physical health of the target population. On the other hand, it is possible to use low magnitude and high frequency (e.g. in a vibratory form) mechanical loads for anabolism as well. Cells, including stem cells of the musculoskeletal tissue, are sensitive to high frequency, low-intensity mechanical signals. This sensitivity can be utilized not only for the targeted treatment of tissues, but also for stem cell expansion, differentiation and biomaterial interaction in tissue engineering applications. In this review, we reported recent advances in the application of low-intensity vibrations on stem and progenitor cell populations. Modulation of cellular behavior with low-intensity vibrations as an alternative or complementary factor to biochemical and scaffold induced signals may represent an increase of capabilities in studies related to tissue engineering.Book Part Citation - Scopus: 15Stem Cell Culture Under Simulated Microgravity(Springer, 2020) Anıl İnevi, Müge; Sarıgil, Öykü; Kızılkaya, Melike; Meşe, Gülistan; Tekin, Hüseyin Cumhur; Özçivici, EnginChallenging environment of space causes several pivotal alterations in living systems, especially due to microgravity. The possibility of simulating microgravity by ground-based systems provides research opportunities that may lead to the understanding of in vitro biological effects of microgravity by eliminating the challenges inherent to spaceflight experiments. Stem cells are one of the most prominent cell types, due to their self-renewal and differentiation capabilities. Research on stem cells under simulated microgravity has generated many important findings, enlightening the impact of microgravity on molecular and cellular processes of stem cells with varying potencies. Simulation techniques including clinostat, random positioning machine, rotating wall vessel and magnetic levitation-based systems have improved our knowledge on the effects of microgravity on morphology, migration, proliferation and differentiation of stem cells. Clarification of the mechanisms underlying such changes offers exciting potential for various applications such as identification of putative therapeutic targets to modulate stem cell function and stem cell based regenerative medicine. © Springer Nature Switzerland AG 2020.Article Citation - WoS: 13Citation - Scopus: 15Cytotoxic Tolerance of Healthy and Cancerous Bone Cells To Anti-Microbial Phenolic Compounds Depend on Culture Conditions(Humana Press, 2019) Karadaş, Özge; Meşe, Gülistan; Özçivici, EnginCarnosol and carnosic acid are polyphenolic compounds found in rosemary and sage with known anti-oxidant, anti-inflammatory, and anti-microbial properties. Here, we addressed the potential use of carnosol and carnosic acid for in vitro bone tissue engineering applications, specifically depending on their cytotoxic effects on bone marrow stromal and stem cells, and osteosarcoma cells in monolayer and 3D cultures. Carnosol and carnosic acid displayed a bacteriostatic effect on Gram-positive bacteria, especially on S. aureus. The viability results indicated that bone marrow stromal cells and bone marrow stem cells were more tolerant to the presence of carnosol compared to osteosarcoma cells. 3D culture conditions increased this tolerance further for healthy cells, while not affecting the cytotoxic potential of carnosol for osteosarcoma cells. Carnosic acid was found to be more cytotoxic for all cell types used in the study. Results suggest that phenolic compounds might have potential use as anti-microbial and anti-carcinogenic agents for bone tissue engineering with further optimization for controlled release.Article Citation - WoS: 34Citation - Scopus: 43Label-Free Density-Based Detection of Adipocytes of Bone Marrow Origin Using Magnetic Levitation(Royal Society of Chemistry, 2019) Sarıgil, Öykü; Anıl İnevi, Müge; Yılmaz, Esra; Meşe, Gülistan; Tekin, Hüseyin Cumhur; Özçivici, EnginAdipocyte hypertrophy and hyperplasia are important parameters in describing abnormalities in adipogenesis that are concomitant to diseases such as obesity, diabetes, anorexia nervosa and osteoporosis. Therefore, technical developments in the detection of adipocytes become an important driving factor in adipogenesis research. Current techniques such as optical microscopy and flow cytometry are available in detection and examination of adipocytes, driving cell- and molecular-based research of adipogenesis. Even though microscopy techniques are common and straightforward, they are restricted in terms of manipulation and separation of the cells. Flow cytometry is an alternative, but mature adipocytes are fragile and cannot withstand the flow process. Other separation methods usually require labeling of the cells or usage of microfluidic platforms that utilize fluids with different densities. Magnetic levitation is a novel label-free technology with the principle of movement of cells towards the lower magnetic field in a paramagnetic medium depending on their individual densities. In this study, we used a magnetic levitation device for density-based single cell detection of differentiated adipogenic cells in heterogeneous populations. Results showed that the magnetic levitation platform was sensitive to changes in the lipid content of mesenchymal stem cells committed to adipogenesis and it could be successfully used to detect the adipogenic differentiation of the cells.