UTILIZING DIELECTROPHORESIS TO DETERMINE THE PHYSIOLOGICAL DIFFERENCES OF EUKARYOTIC CELLS

Type 1 diabetes affects over 108,000 children, and this number is steadily increasing. Current insulin therapies help manage the disease but are not a cure. Over a child’s lifetime they can develop kidney disease, blindness, cardiovascular disease and many other issues due to the complications of type 1 diabetes. This autoimmune disease destroys beta cells located in the pancreas, which are used to regulate glucose levels in the body. Because there is no cure and many children are affected by the disease there is a need for alternative therapeutic options that can lead to a cure. Human mesenchymal stem cells (hMSCs) are an important cell source for stem cell therapeutics due to their differentiation capacity, self-renewal, and trophic activity. hMSCs are readily available in the bone marrow, and act as an internal repair system within the body, and they have been shown to differentiate into insulin producing cells. However, after isolation hMSCs are a heterogeneous cell population, which requires secondary processing. To resolve the heterogeneity issue hMSCs are separated using fluorescent- and magnetic-activate cell sorting with antigen labeling. These techniques are efficient but reduce cell viability after separation due to the cell labeling. Therefore, to make hMSCs more readily available for type 1 diabetes therapeutics, they should be separated without diminishing there functional capabilities. Dielectrophoresis is an alternative separation technique that has the capability to separated hMSCs. This dissertation uses dielectrophoresis to characterize the dielectric properties of hMSCs. The goal is to use hMSCs dielectric signature as a separation criteria rather than the antigen labeling implemented with FACS and MACS. DEP has been used to characterize other cell systems, and is a viable separation technique for hMSCs

Towards Rapid Label-free Enrichment of Specific Stem Cell Populations for Autologous Cell Therapies

Autologous mesenchymal stem cell (MSC) therapies have huge potential in addressing clinical challenges for otherwise intractable diseases. Label-free, intra-operative separation and enrichment of MSC subpopulations would provide a step change in delivery of such therapies. The long term goal of this research is to use binding proteins to provide a surface with switchable affinity, coupled with microfluidics to selectively bind and subsequently collect released cells. The specific aim of this thesis was to take the first steps towards achieving this goal, by identifying the most suitable binding proteins for cell capture and release in a prototype device and determining the feasibility of cell enrichment from complex clinical samples such as bone marrow aspirate. A prototype device was developed exploiting the cell surface marker CD271 to select for MSCs. Affimer binding proteins and a commercially available antibody were investigated for specific cell capture and release. Specificity for CD271+ cells was demonstrated via flow cytometry using two different cell types. CD271 binding proteins were immobilised to a low-fouling substrate in a microfluidic channel and known mixtures of the two cell populations used to demonstrate specific cell capture. Increased flow rates allowed for bound cells to be released, collected and analysed, providing evidence that cells remained viable and minimally manipulated after enrichment. Clinical samples of bone marrow aspirate were then used in the same way and the results compared to gold standard methods of cell sorting. Results showed that the percentage of CD271+ cells selected from bone marrow mononuclear cell populations using the prototype device was similar to results obtained using established cell sorting methodologies. This work demonstrated that affinity capture via antibody technology, together with a surface designed to provide a controlled release mechanism, offers a high-throughput, minimally manipulative approach to select and enrich MSC populations for therapeutic applications

Microfluidics as efficient technology for the isolation and characterization of stem cells.

The recent years have been passed with significant progressions in the utilization of microfluidic technologies for cellular investigations. The aim of microfluidics is to mimic small-scale body environment with features like optical transparency. Microfluidics can screen and monitor different cell types during culture and study cell function in response to stimuli in a fully controlled environment. No matter how the microfluidic environment is similar to in vivo environment, it is not possible to fully investigate stem cells behavior in response to stimuli during cell proliferation and differentiation. Researchers have used stem cells in different fields from fundamental researches to clinical applications. Many cells in the body possess particular functions, but stem cells do not have a specific task and can turn into almost any type of cells. Stem cells are undifferentiated cells with the ability of changing into specific cells that can be essential for the body. Researchers and physicians are interested in stem cells to use them in testing the function of the body's systems and solving their complications. This review discusses the recent advances in utilizing microfluidic techniques for the analysis of stem cells, and mentions the advantages and disadvantages of using microfluidic technology for stem cell research

Rapid cell separation with minimal manipulation for autologous cell therapies

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of > 98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities

Skeletal stem cell isolation and differentiation: Interdisciplinary strategies for skeletal tissue engineering

Stem cell based tissue engineering is viewed as a promising approach for orthopaedic reparative medicine and the application of microfluidic techniques for isolation and characterisation of individual skeletal stem cells is considered a potential source of cells for regenerative medicine. The studies described in this thesis aim to develop original techniques for isolation and characterisation of mesenchymal stem cells and to examine their possible uses in skeletal tissue engineering. These studies developed novel microfluidic technology using dielectrophoretic ring traps and sorting gates for isolation and recovery of specific cells according to immunofluorescent intensity. To date, the devices outlined in this work are limited by the small number of cells that can be isolated, but are capable of recovering established and primary cell populations with 100% purity for specific markers such as STRO-1, while also displaying potential for on-chip analysis and culture due to the ability to precisely control the device's microenvironment. This study has also investigated 28 day organotypic culture of 3D fetal femur-derived cell pellets at an air-liquid interface. It was demonstrated that addition of serum, ascorbate, dexamethasone and BMP-2 resulted in mimicry of in vivo femur development, while addition of ascorbate and TGF- phenotype, thus offering potential models for both cartilage and early bone development. Analysis of pellets demonstrated that significant pellet diameter at day 1 (greater than 0.8mm) is crucial for maintaining reproducible results in osteogenic and chondrogenic conditions. Furthermore, addition of BMP-2 to fetal femur-derived cells cultured in chemically defined media induced formation of a novel cobblestone cell morphology. Characterisation of the cobblestone cells demonstrated a primitive adipogenic phenotype as indicated by the lack of endothelial and haematopoietic marker expression including CD146, TIE2, CD34, and CD105 and upregulation of mesenchymal differ lipid. Overall these studies have offered a novel approach to stem cell isolation for characterisation and have furthered the knowledge of fetal femur-derived cell and their potential as an alternative cell source for skeletal tissue engineerin

A scalable label-free approach to separate human pluripotent cells from differentiated derivatives

The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)–10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells

Development of a cell enrichment device for bone repair utilising tissue non-specific alkaline phosphatase on the surface of dental pulp stromal cells

The overall aim of this thesis was to develop a minimally manipulative, label-free microfluidic cell separator device which is able to deliver an enriched population of autologous cells, positive for tissue non-specific alkaline phosphatase (TNAP) via cell capture using either antibody or non-antibody protein binding. TNAP is a promineralising cell surface marker and is potentially useful as a marker for isolation of stem cell populations for use in regenerative therapies. For clinical applications, cells would be isolated from bone marrow aspirate or orthopaedic surgical waste within intraoperative time of less than two hours, then paired with an osteoconductive scaffold to provide an alternative treatment option with potentially accelerated bone repair and regeneration. Dental Pulp Stomal Cells (DPSCs) which were used as a model system throughout this work, were shown to express 2.8 ± 1.3 x 10^5 TNAP molecules on the cells’ surface and the number of TNAP molecules per TNAP+ cell was not altered by factors such as passage number, seeding density and cell donor. Following this, a microfluidc cell separation device was designed and developed for the enrichment of TNAP+ cells, by capture and subsequent release of TNAP+ DPSCs via a surface functionalised with anti-TNAP antibodies. The recovered cells demonstrated a TNAP+ enriched population with up to a two fold enrichment of TNAP+ cells. The device also begun to meet the requirements for a minimally manipulated cell separation as minimal antibody could be detected on the surface of the recovered cells. As well, the capture and release mechanism had minimal effect on the cells’ biological characteristics, as the recovered enriched population retained a high viability and retained their osteogenic differentiation potential. The specificity to TNAP on the cells’ surface of previously identified non-antibody TNAP binding proteins, known as Affimers, was investigated for potential use within the cell separation technology. Affimer proteins were identified for expression and purification, and demonstrated specificity to recombinant TNAP protein. However, there was minimal evidence of specificity to TNAP on the cells’ surface and therefore subsequent development of the device utilised an anti-TNAP antibody instead. This thesis demonstrated a novel cell separation technology capable of providing an enriched population of viable TNAP+ cells with no obvious alterations in their biological characteristics. This provides a platform technology for potential future clinical use in bone regenerative therapies

Single-cell microfluidic impedance cytometry: From raw signals to cell phenotypes using data analytics

The biophysical analysis of single-cells by microfluidic impedance cytometry is emerging as a label-free and high-throughput means to stratify the heterogeneity of cellular systems based on their electrophysiology. Emerging applications range from fundamental life-science and drug assessment research to point-of-care diagnostics and precision medicine. Recently, novel chip designs and data analytic strategies are laying the foundation for multiparametric cell characterization and subpopulation distinction, which are essential to understand biological function, follow disease progression and monitor cell behaviour in microsystems. In this tutorial review, we present a comparative survey of the approaches to elucidate cellular and subcellular features from impedance cytometry data, covering the related subjects of device design, data analytics (i.e., signal processing, dielectric modelling, population clustering), and phenotyping applications. We give special emphasis to the exciting recent developments of the technique (timeframe 2017-2020) and provide our perspective on future challenges and directions. Its synergistic application with microfluidic separation, sensor science and machine learning can form an essential tool-kit for label-free quantification and isolation of subpopulations to stratify heterogeneous biosystems

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Construction of artificial stem cell microniches

Artificial embryonic stem cell niches were made from murine embryonic stem cells (ESCs) and SAOS-2 osteoblast-like cells (a human osteosarcoma cell line) by constructing aggregates with well-defined architectures with dielectrophoresis between the castellations of interdigitated oppositely castellated electrodes. This combination of the cells was chosen to mimic the bone marrow endosteal niche that harbours haematopoietic stem cells in a quiescent stage, with the aim of transforming the embryonic stem cells into hematopoietic precursor cells. Within aggregates made with dielectrophoresis cells are in very close contact, allowing strong cell-cell interactions to occur. Puramatrix gel was used to immobilize the cells; a concentration of 25% Puramatrix was found to be optimal. Aggregates consisting of only ESCs formed embryoid bodies upon aggregation with dielectrophoresis within 24 hours. The size of the electrodes determines the size of embryoid bodies. Embryonic bodies formed at electrodes with a characteristic size larger than 100 μm tended to split; electrodes smaller than 75 μm gave embryonic bodies which tended to merge. 75 to 100 μm was optimal. When aggregates were made containing both SAOS-2 and ESCs, the reorganization of the two cell types after their aggregation was found to be controlled by the different adhesive-cohesive properties of the two cell types and their initial position. Optimum cell-cell interaction was obtained in an aggregate with a layered architecture with the osteoblasts initially in bottom position, and the ESCs in top position. The study of differentiation in ESCs was made by conducting experiments with Bry ESCs, which mark the onset of differentiation along mesenchymal lineage with the production of GFP. The results indicated that aggregation with dielectrophoresis causes the ESCs to take the first steps towards differentiation along the mesenchymal lineage, and that the differentiation is stronger in aggregates formed at electrodes of 75 μm than at electrodes of 100 and 50 μm. Co-culture with SAOS-2 cells did not lead to differentiation along the mesenchymal lineage. Lastly it was shown that optical tweezers could be combined with dielectrophoresis to move individual cells between niches