Umut A. Gurkan

Three‐Dimensional Magnetic Assembly of Microscale Hydrogels

Directed assembly of nano and microscale particles is of great interest and has widespread applications in various fields including electronics, nanomaterials and tissue engineering. Bottom-up tissue engineering is motivated by the occurrence of repeating functional units in vivo. The bottom-up approach requires novel techniques to assemble engineered functional units as building blocks at a high speed with spatial control over three-dimensional (3D) micro-architecture. Here, we report a magnetic assembler that utilizes nanoparticles and microscale hydrogels as building blocks to create 3D complex multi-layer constructs via external magnetic fields using different concentrations of magnetic nanoparticles. This approach holds potential for 3D assembly processes that could be utilized in various tissue engineering and regenerative medicine applications.

An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles

Controlled assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues. Here we present a novel electrochemical alignment technique to control the assembly of type-I collagen molecules into highly oriented and densely packed elongated bundles at the macroscale. The process involves application of electric currents to collagen solutions which in turn generate a pH gradient. Through an isoelectric focusing process, the molecules migrate and congregate within a plane. It was possible to fabricate collagen bundles with 50-400 microm diameter and several inches length via this process. The current study assessed the orientational order, and the presence of fibrillar assembly in such electrochemically oriented constructs by polarized optical microscopy, small angle X-ray scattering, second harmonic generation, and electron microscopy. The mechanical strength of the aligned crosslinked collagen bundles was 30-fold greater than its randomly oriented-crosslinked counterpart. Aligned crosslinked collagen bundles had about half the strength of the native tendon. Tendon-derived fibroblast cells were able to migrate and populate multiple macroscopic bundles at a rate of 0.5mm/day. The anisotropic order within biocompatible collagenous constructs was conferred upon the nuclear morphology of cells as well. These results indicate that the electrochemically oriented collagen scaffolds carry baseline characteristics to be considered for tendon/ligament repair.

Extreme sensitivity biosensing platform based on hyperbolic metamaterials.

Optical sensor technology offers significant opportunities in the field of medical research and clinical diagnostics, particularly for the detection of small numbers of molecules in highly diluted solutions. Several methods have been developed for this purpose, including label-free plasmonic biosensors based on metamaterials. However, the detection of lower-molecular-weight (<500 Da) biomolecules in highly diluted solutions is still a challenging issue owing to their lower polarizability. In this context, we have developed a miniaturized plasmonic biosensor platform based on a hyperbolic metamaterial that can support highly confined bulk plasmon guided modes over a broad wavelength range from visible to near infrared. By exciting these modes using a grating-coupling technique, we achieved different extreme sensitivity modes with a maximum of 30,000 nm per refractive index unit (RIU) and a record figure of merit (FOM) of 590. We report the ability of the metamaterial platform to detect ultralow-molecular-weight (244 Da) biomolecules at picomolar concentrations using a standard affinity model streptavidin-biotin.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

Nanoplasmonic quantitative detection of intact viruses from unprocessed whole blood.

Infectious diseases such as HIV and hepatitis B pose an omnipresent threat to global health. Reliable, fast, accurate, and sensitive platforms that can be deployed at the point-of-care (POC) in multiple settings, such as airports and offices, for detection of infectious pathogens are essential for the management of epidemics and possible biological attacks. To the best of our knowledge, no viral load technology adaptable to the POC settings exists today due to critical technical and biological challenges. Here, we present for the first time a broadly applicable technology for quantitative, nanoplasmonic-based intact virus detection at clinically relevant concentrations. The sensing platform is based on unique nanoplasmonic properties of nanoparticles utilizing immobilized antibodies to selectively capture rapidly evolving viral subtypes. We demonstrate the capture, detection, and quantification of multiple HIV subtypes (A, B, C, D, E, G, and subtype panel) with high repeatability, sensitivity, and specificity down to 98 ± 39 copies/mL (i.e., HIV subtype D) using spiked whole blood samples and clinical discarded HIV-infected patient whole blood samples validated by the gold standard, i.e., RT-qPCR. This platform technology offers an assay time of 1 h and 10 min (1 h for capture, 10 min for detection and data analysis). The presented platform is also able to capture intact viruses at high efficiency using immuno-surface chemistry approaches directly from whole blood samples without any sample preprocessing steps such as spin-down or sorting. Evidence is presented showing the system to be accurate, repeatable, and reliable. Additionally, the presented platform technology can be broadly adapted to detect other pathogens having reasonably well-described biomarkers by adapting the surface chemistry. Thus, this broadly applicable detection platform holds great promise to be implemented at POC settings, hospitals, and primary care settings.

Paper and Flexible Substrates as Materials for Biosensing Platforms to Detect Multiple Biotargets

The need for sensitive, robust, portable, and inexpensive biosensing platforms is of significant interest in clinical applications for disease diagnosis and treatment monitoring at the point-of-care (POC) settings. Rapid, accurate POC diagnostic assays play a crucial role in developing countries, where there are limited laboratory infrastructure, trained personnel, and financial support. However, current diagnostic assays commonly require long assay time, sophisticated infrastructure and expensive reagents that are not compatible with resource-constrained settings. Although paper and flexible material-based platform technologies provide alternative approaches to develop POC diagnostic assays for broad applications in medicine, they have technical challenges integrating to different detection modalities. Here, we address the limited capability of current paper and flexible material-based platforms by integrating cellulose paper and flexible polyester films as diagnostic biosensing materials with various detection modalities through the development and validation of new widely applicable electrical and optical sensing mechanisms using antibodies and peptides. By incorporating these different detection modalities, we present selective and accurate capture and detection of multiple biotargets including viruses (Human Immunodeficieny Virus-1), bacteria (Escherichia coli and Staphylococcus aureus), and cells (CD4+ T lymphocytes) from fingerprick volume equivalent of multiple biological specimens such as whole blood, plasma, and peritoneal dialysis effluent with clinically relevant detection and sensitivity.

Manipulating biological agents and cells in micro-scale volumes for applications in medicine

Recent technological advances provide new tools to manipulate cells and biological agents in micro/nano-liter volumes. With precise control over small volumes, the cell microenvironment and other biological agents can be bioengineered; interactions between cells and external stimuli can be monitored; and the fundamental mechanisms such as cancer metastasis and stem cell differentiation can be elucidated. Technological advances based on the principles of electrical, magnetic, chemical, optical, acoustic, and mechanical forces lead to novel applications in point-of-care diagnostics, regenerative medicine, in vitro drug testing, cryopreservation, and cell isolation/purification. In this review, we first focus on the underlying mechanisms of emerging examples for cell manipulation in small volumes targeting applications such as tissue engineering. Then, we illustrate how these mechanisms impact the aforementioned biomedical applications, discuss the associated challenges, and provide perspectives for further development.

Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules

Seventy-five percent of patients with epithelial ovarian cancer present with advanced-stage disease that is extensively disseminated intraperitoneally and prognosticates the poorest outcomes. Primarily metastatic within the abdominal cavity, ovarian carcinomas initially spread to adjacent organs by direct extension and then disseminate via the transcoelomic route to distant sites. Natural fluidic streams of malignant ascites triggered by physiological factors, including gravity and negative subdiaphragmatic pressure, carry metastatic cells throughout the peritoneum. We investigated the role of fluidic forces as modulators of metastatic cancer biology in a customizable microfluidic platform using 3D ovarian cancer nodules. Changes in the morphological, genetic, and protein profiles of biomarkers associated with aggressive disease were evaluated in the 3D cultures grown under controlled and continuous laminar flow. A modulation of biomarker expression and tumor morphology consistent with increased epithelial–mesenchymal transition, a critical step in metastatic progression and an indicator of aggressive disease, is observed because of hydrodynamic forces. The increase in epithelial–mesenchymal transition is driven in part by a posttranslational up-regulation of epidermal growth factor receptor (EGFR) expression and activation, which is associated with the worst prognosis in ovarian cancer. A flow-induced, transcriptionally regulated decrease in E-cadherin protein expression and a simultaneous increase in vimentin is observed, indicating increased metastatic potential. These findings demonstrate that fluidic streams induce a motile and aggressive tumor phenotype. The microfluidic platform developed here potentially provides a flow-informed framework complementary to conventional mechanism-based therapeutic strategies, with broad applicability to other lethal malignancies.

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor β1 (TGF- β1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation

Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.