An Integrative Approach to Elucidating the Governing Mechanisms of Particles Movement under Dielectrophoretic and Other Electrokinetic Phenomena

Dielectrophoresis (DEP) has been a subject of active research in the past decades and has shown promising applications in Lab-on-Chip devices. Currently researchers use the point dipole method to predict the movement of particles under DEP and guide their experimental designs. For studying the interaction between particles, the Maxwell Stress Tensor (MST) method has been widely used and treated as providing the most robust and accurate solution. By examining the derivation processes, it became clear that both methods have inherent limitations and will yield incorrect results in certain occasions. To overcome these limitations and advance the theory of DEP, a new numerical approach based on volumetric-integration has been established. The new method has been proved to be valid in quantifying the DEP forces with both homogeneous and non-homogeneous particles as well as particle-particle interaction through comparison with the other two methods. Based on the new method, a new model characterizing the structure of electric double layer (EDL) was developed to explain the crossover behavior of nanoparticles in medium. For bioengineering applications, this new method has been further expanded to construct a complete cell model. The cell model not only captures the common crossover behavior exhibited by cells, it also explains why cells would initiate self-rotation under DEP, a phenomenon we first observed in our experiments. To take a step further, the new method has also been applied to investigate the interaction between multiple particles. In particular, this new method has been proved to be powerful in elucidating the underlying mechanism of the tumbling motion of pearl chains in a flow condition as we observed in our experiments. Moreover, it also helps shed some new insight into the formation of different alignments and configurations of ellipsoidal particles. Finally, with the consideration of the Faradic current from water electrolysis and effect of pH, a new model has been developed to explain the causes for the intriguing flow reversal phenomenon commonly observed (but not at all understood) in AC-electroosmosis (ACEO) with reasonable outcomes

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

PROBLEMS IN THE STUDY AND USE OF AC DIELECTROPHORESIS AND THEIR CONSEQUENCES: A STUDY BASED ON COMSOL MULTIPHYSICS MODELING

Dielectrophoresis (or DEP) is an important phenomenon which is induced when a dielectric particle is placed in a non-uniform electric field. The force generated by DEP has been exploited for various micro and nano fluidics applications like positioning, sorting and separation of particles involved in medical diagnostics, drug discovery, cell therapeutics, biosensors, microfluidics, nanoassembly, particle filtration etc. The integration of DEP systems into the microfluidics enables inexpensive, fast, highly sensitive, highly selective, label-free detection and also the analysis of target bioparticles. This work aims to provide a complete compilation of the factors affecting the DEP force. It elucidates the underlying mechanisms using COMSOL Multiphysics and sheds new insight into the mechanisms for the separation and sorting of different types of particles. This research identifies the problems in the literature and uses COMSOL to analyze the impact of these problems on the end results. It examines four factors that affect the DEP force: physical conditions, electrode setup, properties of the particles and suspension medium. Moreover, it analyzes the influence of the Clausius-Mossotti factor (CM factor) and its cross-over upon the magnitude and direction of the DEP force. From the analysis, it becomes clear that particle size not only affects the magnitude of the DEP force but also the conductivity of the particle. This factor, which is largely ignored, could lead to a shift in the crossover frequency. Shell model plays an important role in determining the dielectric properties of particles that are not homogenous. In such a situation assuming uniform dielectric properties may lead to inconclusive results. The presence of an electric double layer surrounding a particle affects the conductivity of the particle. Also, assuming DEP force to be the only force acting on a particle suspended in a non-uniform electric field leads to errors in the end results. This research provides knowledge on the basic characteristics of the DEP force and its mechanism. It provides a better understanding by examining numerous works carried out in the past and brings out the problems and their consequences

Cell Culture on MEMS Platforms: A Review

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bioincompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bioincompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.Institute of Bioengineering and Nanotechnology (Singapore)Singapore. Biomedical Research CouncilSingapore. Agency for Science, Technology and ResearchSingapore. Agency for Science, Technology and Research (R-185-001-045-305)Singapore. Ministry of EducationSingapore. Ministry of Education (Grant R-185- 000-135-112)Singapore. National Medical Research CouncilSingapore. National Medical Research Council (Grant R-185-000-099-213)Jassen Cilag (Firm)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Global Enterprise for Micro-Mechanics and Molecular Medicin

Construction of artificial skin tissue with placode-like structures in well-defined patterns using dielectrophoresis

During embryonic development of animal skin tissue, the skin cells form regular patterns of high cell density (placodes) where hair or feathers will be formed. These placodes are thought to be formed by the aggregation of dermal cells into condensates. The aggregation process is thought to be controlled by a reaction-diffusion mechanism of activator and inhibitor molecules, and involve mechanical forces between cells and cells with the matrix. In this project, placode formation in chicken embryonic skin cells was used as a model system for the study of the mechanism by which the placodes are formed. Artificial aggregates of chicken embryonic skin cells were created by suspending them in a 300 mM low conductivity sorbitol solution and attracting them by positive dielectrophoresis to high field regions within microelectrode arrays by applying a 10 - 20 Vpk-pk 1 MHz signal across the microelectrodes. It was demonstrated that using this method aggregates can be produced in a large variety of patterns and that the distance between the aggregates and aggregate size and shape within the pattern can be controlled effectively. Custom-built image analysis tools were developed in LabVIEW to analyze the patterns formed. The formation of aggregates by dielectrophoresis was followed by an immobilization phase of the resulting patterns inside a gel matrix, forming an artificial skin. Nutrients and oxygen were supplied externally. Long-term incubation of the artificial skin shows that embryonic skin cells in the aggregates were viable and showed behavior similar to that of developing embryonic skin, including further aggregation of the cells and the formation of cell condensates. The domain size was shown to have an influence on the condensation process, with cells in small aggregates forming only one condensate near the centre of the aggregate, and several condensates in larger aggregates. Whilst the distribution of cell condensates within the aggregates in round large aggregates is predominantly random, some line formation could be observed in linear aggregations, indicating some self-organization may be occurring

Microfluidic devices for cell cultivation and proliferation

Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined

Living Cell Microarrays: An Overview of Concepts

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.Niedersächsische Krebsgesellschaft e.V.BIOFABRICATION FOR NIFE InitiativeLower Saxony ministry of Science and CultureVolkswagen Stiftun

모세관 현상 기반의 패터닝 기법을 활용한 고효율 삼차원 면역세포 항암효능 평가 플랫폼

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 기계공학부, 2020. 8. 전누리.Organs-on-chips have been developed for recapitulating human organ functions in in vitro as microfabrication techniques meet biology since the early 2000s. Specifically, polydimethylsiloxane (PDMS) based microfluidic devices enabled to mimic organ functions by providing spatially compartmented cell patterning for culturing cells with in vivo like layout. The selective cell patterning enabled 3D cell culture and spatiotemporal analysis which were challenging to conduct with conventional cell culturewares such as petri-dishes, flasks, and well-plates. However, traditional organs-on-chips have limitations in salability, experimental throughput, and absence of standard due to their closed channel designs based on PDMS. Here, we introduce two capillarity guided patterning (CGP) methods by integrating microstructures with conventional cell culturewares. First, we fabricated micropillar arrays on open polystyrene (PS) surfaces and the micropillars can capture liquids swept over the surface. Using the devices, we demonstrated 3D culture applications, single cell capturing and retrieval and multiple cell co-culture. Second, we integrated rail-structures with microplate. Beneath a rail-structure, hydrogel precursors can selectively remain according to meniscus dynamics when the pre-loaded precursors are aspirated. These two CGP methods can be produced with injection molding and provide enhanced experimental throughput. Using the rail-based CGP method, we developed a 3D cytotoxicity assay for cancer immunotherapy based on an injection molded plastic culture (CACI-IMPACT) device to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Owing to the aspiration-mediated patterning, hydrogel precursors can be patterned in 12 wells within 30 s. For functional evaluation of the cytotoxic lymphocytes engineered for cancer immunotherapy, HeLa cells encapsulated by collagen matrix were patterned beneath low rails and NK-92 cells were loaded into the channel formed by the collagen matrix. We observed infiltration, migration and killing activity of NK-92 cells against HeLa cells in collagen matrix. Through image-based analysis, we found ECM significantly influences migration and cytotoxicity of lymphocytes. Hence, the CACI-IMPACT platform has the potential to be used for pre-clinical evaluation of ex vivo engineered cytotoxic lymphocytes for cancer immunotherapy against solid tumors, and the CGP methods are expected to accelerate the commercialization of organs-on-chips.장기모사칩은 2000년대 초부터 마이크로 공정 기술이 생물학적 연구에 활용됨에 따라 인간 장기 기능을 모사하기 위해 개발되었다. 구체적으로, polydimethylsiloxane (PDMS) 기반 미세유체 장치는 공간적으로 구분된 세포 패터닝을 가능케 함으로써 생체와 유사한 구조로 세포를 배양할 수 있게 해주었다. 이러한 세포 패터닝은 페트리 디쉬, 플라스크, 혹은 웰플레이트와 같은 기존의 세포 배양 도구에서는 수행하기 어려운 삼차원 세포 배양과 그 안에서의 시공간적 분석을 가능하게 하였다. 하지만, 종래의 장기모사칩은 PDMS에 기반한 닫힌 형태의 채널 설계로 인해 낮은 생산성, 낮은 실험 효율, 낮은 장비 호환성을 갖는다. 따라서, 본 연구는 대중적인 세포 배양 장치들에 마이크로 구조물을 통합한 두가지 모세관 현상 기반의 패터닝 방법을 제시한다. 첫번째 방법은 페트리 디쉬나 polystyrene (PS) 필름과 같이 개방된 PS 표면에 마이크로 기둥 어레이를 제작하여 그 위에서 액체가 쓸려 지나갈 때 기둥 구조물들 사이에 액체를 포획하는 방식이다. 마이크로 기둥 어레이의 배치에 따라 나노리터부터 마이크로리터에 이르는 액체를 빠르게 패터닝할 수 있게 한다. 이러한 기둥 구조를 활용하면 다양한 세포의 배치 및 배양이 가능하여, 본 연구에서는 삼차원 환경에서의 단일세포 배양과 다세포 공배양 플랫폼으로의 활용 가능성을 제시하였다. 두번째 방법은 마이크로 레일 형태의 마이크로구조물을 표준화된 마이크로 플레이트의 웰과 통합하여 고효율 삼차원 배양 플랫폼을 제시한다. 레일 구조의 아래에 주입된 액체가 빨아들여질 때 구조물에 의해 형성된 액체-기체 계면들의 순차적 이동을 활용하여 특정 레일의 아래에만 액체를 남기는 기술을 개발하였다. 이 두가지 모세관 현상 기반 패터닝 방법을 위한 장치들은 사출성형으로 대량생산이 가능하고 우수한 실험 효율을 갖는다. 이 중 레일 구조를 활용한 흡인 기반의 패터닝 방법을 이용하여 면역세포치료제의 성능 평가를 위한 사출 성형된 플라스틱 어레이 배양 장치 (CACI-IMPACT)를 개발하였다. 흡인 기반 패터닝 덕분에 20 μl 파이펫으로 빨아들인 하이드로젤 용액을 30 초 이내에 12개의 웰에 패터닝 할 수 있었다. 면역세포치료제의 기능적 평가를 위해, 콜라겐 젤에 포함된 HeLa 세포를 패터닝하고 NK-92 세포의 콜라겐 매트릭스 내부로의 침투, 매트릭스 내부에서의 이동 및 암세포 살해 활동을 관찰하였다. 이를 통해 세포외기질이 세포 독성 림프구의 이동 및 세포 독성에 상당히 영향을 미친다는 것을 확인할 수 있었다. 따라서, 암세포와 세포 독성 림프구의 고효율 삼차원 공동 배양을 가능하게 하는 본 플랫폼은 고형 종양에 대한 면역 치료를 위해 개발된 세포 독성 림프구의 전임상 평가에 사용될 가능성이 있으며, 본 연구에서 개발 및 사용된 모세관 현상 기반 패터닝 기술들은 장기모사칩의 상용화를 가속화시킬 것으로 기대한다.Chapter 1. Introduction 1 1.1. History of organs-on-chips 1 1.2. Challenges in current organs-on-chips 4 1.3. Models for cancer immunotherapy 7 1.4. Purpose of research 8 Chapter 2. Microstructure-guided multi-scale liquid patterning on open surface 11 2.1. Introduction 11 2.2. Materials and Methods 13 2.2.1. Fabrication of the microstructured PS surface 13 2.2.2. Single cell isolation and retrieval of single colony 16 2.2.3. In vitro vasculogenesis 17 2.2.4. Visualization of the in vitro blood vessel 19 2.3. Results and discussion 18 2.3.1. Liquid patterning process 18 2.3.2. Comparison of microliquid trapping with a micropillar array and microwells 30 2.3.3. Arrangement of micropillars for controlling the volume and shape of patterned liquids 33 2.3.4. Single cell culture & recovery platform 37 2.3.5. Sequential patterning for co-culture in a 3D microenvironment 42 2.4. Conclusions 46 Chapter 3. Aspiration-mediated microliquid patterning using rail-based open microfluidics 47 3.1. Introduction 47 3.2 Materials and Methods 50 3.2.1. Fabrication of open microfluidic devices 50 3.2.2. Cell culture 50 3.2.3. Hydrogel micropatterning 51 3.2.4. Image analysis 52 3.3. Results 53 3.3.1. Microstructures for aspiration-mediated patterning 53 3.3.2. Theoretical analysis of microchannel formation 56 3.3.3. Formation of multiple discrete microchannels 63 3.3.4. An application for screening vasculogenic capacities 70 3.4. Conclusions 75 Chapter 4. High-throughput microfluidic 3D cytotoxicity assay for cancer immunotherapy 77 4.1. Introduction 77 4.2. Materials and Methods 81 4.2.1. Cell culture 81 4.2.2. Fluorescent labeling of live and dead cells 81 4.2.3. 3D cytotoxicity assay using gel patterned device 82 4.2.4. Image analysis 83 4.2.5. 2D cytotoxicity assay 84 4.3. Results 84 4.3.1. Design and fabrication of devices 84 4.3.2. Cytotoxicity assay in 3D ECM environment 89 4.3.3. 3D ECM reduce cytotoxicity 94 4.3.4. Dense ECM impede migration of CLs 98 4.4. Conclusions 104 Chapter 5. Concluding Remarks 110 Bibliography 113 Abstract in Korean 124Docto

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