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

On Chip Isolation and Enrichment of Tumor Initiating Cells

We report for the first time a microdevice that enables the selective enrichment and culture of breast cancer stem cells using the principles of mammosphere culture. For nearly a decade, researchers have identified breast cancer stem cells within heterogeneous populations of cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for cancer stem cells. While this assay has proven to be useful for identifying cancer stem cells from a bulk population, ultimately its utility is limited by difficulties in combining the mammosphere technique with other useful cellular and molecular analyses. However, integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, including cell sorting and analysis, as well as popular molecular assays. In this work, we demonstrate mammosphere culture within a polydimethylsiloxane (PDMS) microsystem. We first prove that hydrophobic PDMS surfaces are as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then demonstrate the culture of mammospheres as large as 0.25 mm within a PDMS microsystem. Finally, we verify that reagents can be delivered to the cell culture wells exclusively by diffusion-based transport, which is necessary because the cells are unattached. This microsystem component can be integrated with other microfluidic functions, such as cell separation, sorting, and recovery, as well as molecular assays, to enable new discoveries in the biology of cancer stem cells that are not possible today

Investigations into Convective Deposition from Fundamental and Application-Driven Perspectives

Crystalline particle coatings can provide critical enhancement to wide-ranging energy and biomedical device applications. One method by which ordered particle arrays can be assembled is convective deposition. In convective deposition, particles flow to a surface via evaporation-driven convection, then order through capillary interactions. This thesis will serve to investigate convective deposition from fundamental and application-driven perspectives. Motivations for this work include the development of point-of-care diagnostic devices, macroporous membranes, and various energy applications. Immunoaffinity cell capture devices display enhanced diagnostic capabilities with intelligently varied surface roughness in the form of particle coatings. Relatedly, highly crystalline particle coatings can be used to template the fabrication of macroporous polymer membranes. These membranes display highly monodisperse pores at particle contact points. In addition, ordered areas of particles, acting as microlenses, can enhance LED performance by 2.66-fold and DSSC efficiency by 30%. Previous research has targeted the formation of crystalline monolayers of particles. However, much insight can be gleaned from imperfect coatings. The analysis of submonolayer coatings, exhibiting significant void spaces, provides insight as to the specific mechanisms and timescales for flow and crystallization. A pair of competing deposition modes, termed ballistic and locally-ordered, enables the intelligent design of experiments and enables significant enhancement in control of resultant thin film morphology. Surface tension-driven particle assembly is subject to a number of native instabilities and macroscale defects that can irreversibly compromise coating uniformity. These include the formation of three-dimensional streaks, where surface tension-driven flow spurs on the nucleation of large imperfections. These imperfections, once nucleated, exhibit a feedback loop of dramatically enhanced evaporation and resultant flow. In addition, thick nanoparticle coatings, subject to enormous drying stresses, exhibit highly uniform crack formation and spacing in an attempt to minimize system energy. Both these imperfections yield insight on convective deposition as a fundamental phenomenon, and intelligent design of experiments moving forward. Cracking can be suppressed through layer-by-layer particle assembly, whereas streaking can be controlled via several significant process enhancements. Process enhancements include the addition of smaller constituent, as packing aids, to suspension, the application of lateral vibration, and the reversal of relevant surface tension gradients. The transition from unary to binary suspensions represents a significant improvement to convective deposition as a process. Nanoparticles act as packing, and flow, aids, wholly suppress macroscale defects under ideal conditions. A relative deficiency or excess of nanoparticles can generate complex coating morphologies including multilayers and transverse stripes. The application of lateral vibration to convective deposition allows the assembly of monolayer particle coatings under a larger range of operating conditions and at a faster rate. Macroscale defect formation can increased through an enhancement of the natural condition, where evaporative cooling generates a thermal gradient in drying droplets. Conversely, these defects can be suppressed with a reversal of this gradient, which will reverse the direction of surface tension-driven recirculation. These fundamental developments in understanding, and associated process enhancements, are critical in current efforts to scale up convective deposition. As convective deposition evolves from laboratory-scale batch experiments to continuous, large scale, coatings, repeatability and robustness, as well as an ability to controllably change thin film morphology, will be essential

Lab-on-a-chip Thermoelectric and Solid-phase Immunodetection of Biochemical Analytes and Extracellular Vesicles: Experimental and Computational Analysis

Microfluidics is the technology of controlling and manipulating fluids at the microscale. Microfluidic platforms provide precise fluidic control coupled with low sample volume and an increase in the speed of biochemical reactions. Lab-on-a-chip platforms are used for detection and quantification of biochemical analytes, capture, and characterization of various proteins, sensitive analysis of cytokines, and isolation and detection of extracellular vesicles (EVs). This study focuses on the development of microfluidic and solid-phase capture pin platforms for the detection of cytokines, extracellular vesicles, and cell co-culture. The fabrication processes of the devices, experimental workflows, numerical analysis to identify optimal design parameters, and reproducibility studies have been discussed. Layer-by-layer assembly of polyelectrolytes has been developed to functionalize glass and stainless-steel substrates with biotin for the immobilization of streptavidinconjugated antibodies for selective capture of cytokines or EVs. Microstructure characterization techniques (SEM, EDX, and fluorescence microscopy) have been implemented to assess the efficiency of substrate functionalization. A detailed overview of current methods for purification and analysis of EVs is discussed as well. Additionally, the dissertation demonstrates the feasibility of a calorimetric microfluidic immunosensor with an integrated antimony-bismuth (Sb/Bi) thermopile sensor for the detection of cytokines with picomolar sensitivity. The developed platform can be used for the universal detection of both exothermic or endothermic reactions. A three-dimensional numerical model was developed to define the critical design parameters that enhance the sensitivity of the platform. Mathematical analyses identified the optimal combinations of substrate material and dimensions that will maximize the heat transfer to the sensor. Lab-on-a-chip cell co-culture platform with integrated pneumatic valve was designed, numerically characterized, and fabricated. This device enables the reversible separation of two cell culture chambers and serves as a tool for the effective analysis of cell-to-cell communication. Intercellular communication is mediated by extracellular vesicles. A protocol for the functionalization of stainless-steel probe with exosomespecific CD63 antibody was developed. The efficiency of the layer-by-layer deposition of polyelectrolytes and the effectiveness of biotin and streptavidin covalent boding were characterized using fluorescent and scanning electron microscopy

Development of Multiscale Materials in Microfluidic Devices: Case Study for Viral Separation from Whole Blood

Separation and concentration of nanoscale species play an important role in various fields such as biotechnology, nanotechnology and environmental science. Inevitably, the separation efficiency strongly affects the quality of downstream detections or productions. For biotechnology and diagnostic applications, conventional separation techniques such as centrifugation, chromatography, filtration, and electrophoresis have been well established and the related instruments and reagents are readily available commercially. However, other factors such as cost, processing time, bulky instruments, infrastructure, and well trained technicians limit their applications in resource-limited settings. Consequently, innovations in materials science that can separate bionanoparticles efficiently and do not require complex setups, reagents or external fields are highly demanded. This work focuses on developing new materials for the affinity separation of bio-nanoparticles such as viruses or macromolecules from a complex mixture, such as whole blood. To enhance the interaction between target nanoparticles and the capture bed, methods to produce porous matrices with a uniform pore size matching the dimension of targets are studied. Furthermore, regarding viral separation from whole blood, macroporous materials are further patterned into microarrays to allow multiscale separation. Considering the needs in resource-limited settings, these materials are integrated with microfluidic technologies to reduce the volume of samples and reagents, simplify operating processes, and enable the use of inexpensive and portable components. Beyond the application of viral separation as demonstrated in the work, the fundamental study of macroporous material formation and transport in these materials also shed light to the separation of many other nanospecies in multiscale materials.Specifically, two macroporous materials, based on template synthesis, are created in this work. The first type employs porous anodic aluminum oxide (AAO) films as the template to create hexagonal arrays of nanoposts. However, pore sizes and interpore distances (cell size) of ordered porous AAO films are limited by the conventional fabrication process. Moreover, the process usually yields defective pore morphologies and large pore and cell size distributions. To overcome these limitations, a patterning method using nanobead indentation on aluminum substrate prior to anodization is evaluated to control the growth of AAO. Together with controlled anodizing voltages and electrolytic concentrations, AAO pore and cell sizes are shown to be tunable and controllable with narrow size distributions within submicron range. A high degree of order of AAO pore arrangement is also demonstrated. In addition, overall anodization becomes more time-efficient and stable at high anodizing voltages. Secondly, a three-dimensional (3D) assembly of microbeads is used as a template to fabricate a spherical pore network with small interconnected openings. After depositing and drying a suspension containing both micro- and nanobeads, the microbeads assemble into a 3D close-packed structure while the nanobeads fill the interstitial space. When the nanobeads are melted and microbeads are removed, a spherical pore matrix then form with small interconnected openings. Such the opening size is in submicron range can be adjusted depending on the size of microbead. The advantages of the two macroporous materials are not only controllable and tunable pore size, but also high surface-to-volume ratio due to the nanoscale features. With a ratio on the order of ~1 µm-1, the porous materials provide a significantly large binding surface. Computational and experimental results reveal that porous materials with a pore size matching the nanoparticle size are suitable for their capture. Separation of human immunodeficiency virus (HIV) is used as a model and capture yields of ~99 % and ~80 % are achieved in the nanopost structure and spherical pore network, respectively, after treated with a functional chemistry. Hence, the properties of these two macroporous materials are suitable as a size-exclusion and affinity separation for viral particles.To further explore multiscale separation, i.e. capturing viruses from whole blood, micropatterned arrays of macroporous materials have been designed. In this design, a microscale gap allows the passage of microparticles such as blood cells, and the nanoscale pores promote permeation for affinity capture of bionanoparticles. Consequently, particles with a size difference of 3-4 orders of magnitude can be separated in a simple flow-through process. Computational analyses are employed to study the effect of micropattern shape and layout. A half-ring pattern is shown to reduce flow resistance and promote fluid permeation compared to a circular pattern. In the experiment, the micropatterned porous arrays yield around 4 times higher viral capture from whole blood compared with a micropatterned solid array. The micropatterned porous devices are capable of handling a large volume of fluid sample without clogging by cells. Therefore they can be used for nanoparticle concentration. Our study also indicates that the layout of micropatterns can be adjusted to improve the capture yield. For example, an increase in pattern radius, or a decrease in gap distance between each post and in width of half ring will enhance fluid permeation in the porous structure. When combined with downstream detection, these materials integrated into microfluidic platforms can be created as point-of-care diagnostics, as well as other applications for particle separation and analysis

Diffusion studies of nanomedicines within increasing complexity tissue models

Many solid tumors develop biological characteristics different from those which characterize the healthy tissues; compared to normal tissues, tumoral main features include blood vessels with fenestration and a higher rigidity of extracellular matrix (ECM) that, with its architecture, influence drug delivery and diffusion to the tumoral mass playing a leading role on the effectiveness of the therapy. Living cells are always surrounded by extracellular matrix, which can be understood as a three-dimensional structured filter; no substance can pass directly from the bloodstream to cell and vice versa, but must reach the cell over the ECM. The nanocarriers are the most important drug transporters to whom the researchers always pose attention for overcoming biological barriers to enabling the drug reach the pathological site. They can carry hydrophilic and/or hydrophobic drugs, protecting them from degradation, providing a drug controlled release and reducing toxic effects to the healthy tissues. Particles movement in tissues depends on their size, charge, and configuration and these features can be modified in order to optimize particles delivery to cancer cells. As well as from particle features, particle movements depend also on ECM properties; it is necessary to understand the best way how these particles diffuse in the ECM. Drug and particles transport through interstitial tissue is ruled by a diffusive flux due to concentration gradient and a convective flux due to fluid movement even if high interstitial fluid pressure makes the transport of drugs dependent only by the diffusion. Drug delivery depends also on the cells that form the tumor mass and on the matrix structure. It is of fundamental importance to understand how these barriers interfere with the drugs transport to improve even more the transport of therapeutic molecules. For this purpose, in this work it has been developed a Tissue Chamber Chip that represents a tool to investigate the diffusion of different nanoparticles (NPs) in an extravascular space modeled by collagen, the main component of the extracellular matrix. Before clinical trials and food and drug administration (FDA) approval, drugs and delivery mechanisms need to be tested to determine their effectiveness and toxicity. Here, six different nanocarriers, almost similar in size but with different surface decoration were tested. The found results highlight that the surface PEGylation promotes diffusion by acting as a lubricant agent. In particular, it has been found that the greater the percentage of PEG on the surface, the greater the mobility of these nanovectors within the ECM. The particles covered with hyaluronic acid, instead, showed a different behavior: their diffusion was hampered proportionally to the molecular weight of this glycosaminoglycan. To demonstrate the generality of our approach, the same NPs were tested on murine brain tissue. The results obtained provide the same trend that can be seen from collagen alone, even if the order of magnitude of the diffusivity is different because of the tissue architecture and complexity. Collectively, these results suggest that the procedure adopted for the nanomedicine diffusion studies, regardless of the tissue, is solid. And, in particular, this suggests that the Tissue Chamber chip can be used as a predictive model of NPs behavior within a biological environment. Finally, to further increase the translational characteristic of our platform, the same collagen matrix was used as a nutritional environment for a 3D culture of cells derived from colorectal cancer. The in vivo tumor tissue has been recreated in vitro in order to potentially allow patient-specific drug screening and the development of personalized treatment. This work demonstrated that our device can be efficiently used to test the extravascular transport of NPs and, moreover, it can be modified increasing its complexity to get closer to a real model. In addition, this project could continue using patient-derived 3D culture to effectively test drugs and NPs to make clinical trials increasingly oriented and well targeted

Microfluidic Devices with Engineered Micro-/Nanostructures for Cell Isolation

Isolation of cells from blood is critical for vast biomedical applications. The focus of this dissertation is on the isolation of circulating tumor cells (CTCs) from patient blood, which contains important prognostic and diagnostic information. Challenges in this field originates from the striking contrast between the rare amount of CTCs (1-10 per mL) and vast other normal cells (millions of white blood cells (WBCs) and billions of red blood cells per mL). Various techniques have been developed to isolate CTCs in the recent decades, while the most demanding clinical requirements lie in two aspects: higher capture efficiency meaning the strong ability to isolate the rare CTCs and higher purity meaning the strong ability to repel all other normal cells. In order to better serve clinical practice, we developed four microfluidic platforms aiming at high capture efficiency and high purity, thus advancing the cancer patient care. By extending the concept of the hallmark immunoaffinity based grooved-herringbone (HB) chip, we first developed a wavy-HB chip by smoothing the grooved patterns to wavy patterns. The wavy-HB chip was demonstrated to not only achieve high capture efficiency (up to 85.0%) by micro-vortexes induced by HB structures, but achieve high purity (up to 39.4%) due to the smooth wavy microstructures. The HB structures were then further optimized through a refined computational model implemented with cell adhesion probability. The particulate cell transport dynamics was shown to be crucial in determining the optimized geometry for CTC capture. To further enhance the CTC capture, integration of nanostructures was examined due to their intrinsic large surface area-to-volume ratio. By exploring the geometric effects of nanopillars on CTC capture, we unraveled an interesting linear relationship between CTC capture efficiency and effective nanopillar contact area. We then developed a fabrication approach to deposit nanoparticles on the wavy-HB patterns to form hierarchical micro/nanostructures. The hierarchical wavy-HB chip was demonstrated to achieve a capture efficiency up to ~98% and a high purity performance (only ~680 WBCs per 1 mL blood). Over the course of the above-mentioned work, there emerges another clinical need which requires captured CTCs to be released and re-cultured for post-analysis such as drug screening. We thus developed two microfluidic chips attempting to achieve this goal. The first platform is an integration of immunomagnetic particles on the developed wavy-HB chip. In addition to the good device performance brought by the wavy-HB patterns, CTCs were able to be released from the capture bed by removing the magnetic field. The collected CTCs labeled with magnetic particles were able to be re-cultured and it was found that these magnetic particles were subject to self-removal during cell proliferations. The second platform was an inclined wavy patterns coated with E-selectin, which was able to form weak adhesion forces with WBCs and CTCs. A proof-of-concept work was performed to demonstrate that WBCs and CTCs were able to be separated along different pathways due to the different adhesion forces and the inclined direction guidance. With all these developed cancer cell isolation microfluidic chips, we showed our contributions toward effective cancer cell isolation and eventually cancer treatment

Southwest Research Institute assistance to NASA in biomedical areas of the technology utilization program Final report, 1 Feb. 1969 - 24 Aug. 1970

Research progress in technology transfer by NASA Biomedical Application Tea

Novel devices and protocols enabling isolation and enumeration of low abundant biological cells from complex matrices

The dimensions of microfluidic devices closely parallel those of biological cells; thusly, they are excellent platforms for the speciation, transport, manipulation, and analysis of cells. Electrokinetic transport of Escherichia coli and Saccharomyces cerevisiae was evaluated in microfluidic devices fabricated in pristine and UV-modified poly(methylmethacrylate) and polycarbonate. The magnitude and direction of transport of the cells was dictated by the buffer composition, conduit surface chemistry, and intrinsic cellular electrical properties. Baker’s yeast in all devices migrated toward the cathode, because of their smaller electrophoretic mobility compared to the electroosmotic flow of the polymer. E. coli cells suspended in 20 mM PBS migrated toward the anode, which indicated that the apparent mobility of the E. coli cells changed direction at higher ionic strengths. The observed differential migrations were exploited to sort cells, whereby judicious choice of the buffer concentration and the polymeric material in which the cell sorting was performed was controlled, allowed for cell enumeration via laser-based backscatter signals. A novel microfluidic device that selectively and specifically isolated the exceedingly small numbers of circulating tumor cells (CTCs) from whole blood through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (\u3c37 min) was designed, manufactured and implemented. Upon processing, the CTCs were concentrated into small volumes (190 nL) and the number of cells captured were read without the need for labeling by using an integrated conductivity sensor following an enzyme mediated release of the captured CTCs from the microchannel surface. The microchannel walls were covalently decorated with mABs directed toward breast cancer cells that over-express epithelial cell adhesion molecules. The released CTCs were then enumerated on-device using conductivity detection with 100% detection efficiency and exquisite specificity for CTCs. The CTC capture efficiency was made highly quantitative (\u3e97%) by designing capture channels with the appropriate widths and heights. Extension of the technique to environmental samples was performed using analogously patterned polyclonal anti-E. coli O157:H7 antibodies directed towards the virolent bacterial strain were used to isolate the enterohemorrhagic bacteria while E. coli K12 were not adsorbed to the antibody containing surface

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