Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

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

Cell patterning via optimized dielectrophoretic force within hexagonal electrodes in vitro for skin tissue engineering

Abstract(#br)Tissue reconstruction through in vitro cell seeding is a popular method for tissue engineering. In this paper, we proposed a thin-layer structure consisting of multiple hexagons for the regeneration of skin tissue. Cells could be seeded and cultured within the structure via dielectrophoresis (DEP) actively. A thin layer of the structure was fabricated with biocompatible medical-grade stainless steel via precise laser cutting. The fabricated layers were stacked together to form a 3D electrode pair, which could be used to generate a 3D electric field. Thus, the suspended cells within the structure could be patterned via DEP manipulation. The input voltage was examined and optimized to ensure cell viability and patterning efficiency during the DEP manipulation process. As soon..

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient

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

3D microfabrication of biological machines

The burgeoning field of additive manufacturing, or “3D printing”, centers on the idea of creating three-dimensional objects from digital models. While conventional manufacturing approaches rely on modifying a base material via subtractive processes such as drilling or cutting, 3D printing creates three-dimensional objects through successive deposition of two- dimensional layers. By enabling rapid fabrication of complex objects, 3D printing is revolutionizing the fields of engineering design and manufacturing. This thesis details the development of a projection-based stereolithographic 3D printing apparatus capable of high- resolution patterning of living cells and cell signals dispersed in an absorbent hydrogel polymer matrix in vitro. This novel enabling technology can be used to create model cellular systems that lead to a quantitative understanding of the way cells sense, process, and respond to signals in their environment. The ability to pattern cells and instructive biomaterials into complex 3D patterns has many applications in the field of tissue engineering, or “reverse engineering” of cellular systems that replicate the structure and function of native tissue. While the goal of reverse engineering native tissue is promising for medical applications, this idea of building with biological components concurrently brings about a new discipline: “forward engineering” of biological machines and systems. In addition to rebuilding existing systems with cells, this technology enables the design and forward engineering of novel systems that harness the innate dynamic abilities of cells to self-organize, self-heal, and self-replicate in response to environmental cues. This thesis details the development of skeletal and cardiac muscle based bioactuators that can sense external electrical and optical signals and demonstrate controlled locomotive behavior in response to them. Such machines, which can sense, process, and respond to signals in a dynamic environment, have a myriad array of applications including toxin neutralization and high throughput drug testing in vitro and drug delivery and programmable tissue engineered implants in vivo. A synthesis of two fields, 3D printing and tissue engineering, has brought about a new discipline: using microfabrication technologies to forward engineer biological machines and systems capable of complex functional behavior. By introducing a new set of “building blocks” into the engineer’s toolbox, this new era of design and manufacturing promises to open up a field of research that will redefine our world

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

Additive nanomanufacturing: a review

Additive manufacturing has provided a pathway for inexpensive and flexible manufacturing of specialized components and one-off parts. At the nanoscale, such techniques are less ubiquitous. Manufacturing at the nanoscale is dominated by lithography tools that are too expensive for small- and medium-sized enterprises (SMEs) to invest in. Additive nanomanufacturing (ANM) empowers smaller facilities to design, create, and manufacture on their own while providing a wider material selection and flexible design. This is especially important as nanomanufacturing thus far is largely constrained to 2-dimensional patterning techniques and being able to manufacture in 3-dimensions could open up new concepts. In this review, we outline the state-of-the-art within ANM technologies such as electrohydrodynamic jet printing, dip-pen lithography, direct laser writing, and several single particle placement methods such as optical tweezers and electrokinetic nanomanipulation. The ANM technologies are compared in terms of deposition speed, resolution, and material selection and finally the future prospects of ANM are discussed. This review is up-to-date until April 2014

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

Droplet microfluidics: a tool for biology, chemistry and nanotechnology

The ability to perform laboratory operations on small scales using miniaturized devices provides numerous benefits, including reduced quantities of reagents and waste as well as increased portability and controllability of assays. These operations can involve reaction components in the solution phase and as a result, their miniaturization can be accomplished through microfluidic approaches. One such approach, droplet microfluidics, provides a high-throughput platform for a wide range of assays and approaches in chemistry, biology and nanotechnology. We highlight recent advances in the application of droplet microfluidics in chip-based technologies, such as single-cell analysis tools, small-scale cell cultures, in-droplet chemical synthesis, high-throughput drug screening, and nanodevice fabrication