The goal of cancer immunotherapies is to boost the immune system's ability to detect tumor antigens and mount an effective anti-tumor immune response. Currently, adoptive T cell transfer therapy (ACT), the administration of ex vivo expanded autologous tumor-specific T cells, is one of the most promising immunotherapies under development; however, its efficacy has been limited so far with a mere 10% complete remission rate in the most successful clinical trials. The prolonged ex vivo culture process is a potential reason for this ineffectiveness because the transfused cells may reach replicative senescence and immunosenescence prior to patient transfer. The objective of this thesis is to offer two approaches towards an improvement of treatment efficacy. First, we generated a 'senescence metric' from the identification of biomarkers that can be used in the clinic towards predicting age and responsiveness of ex vivo expanded T cells. The second approach is to understand at the molecular level the changes that occur during ex vivo expansion to devise improved ACT protocols. In particular, we focused on the shift towards a pro-oxidizing environment and its potential effects on calcium signaling. The combined development and application of microfluidic technologies and computational models in this thesis facilitated our investigations of the phenotypic and signaling changes occurring in T cells during the progression towards immunosenescence. Our findings of altered T cell properties over long term culture provide insight for the design of future cancer immunotherapy protocols.

Endothelial cells (ECs) form the inner lining of all blood vessels in the body, and coat the outer surfaces of heart valves. Because ECs are anchored to extracellular matrix proteins and are positioned between flowing blood and underlying interstitium, ECs are constantly exposed to hemodynamic shear, and act as a semi-permeable barrier to blood-borne factors. In vitro cell culture flow (ICF) systems have been employed as laboratory tools for testing endothelial properties such as adhesion strength, shear response, and permeability. Recently, advances in microscale technology have introduced microfluidic systems as alternatives to conventional ICF devices, with a multitude of practical advantages not available at the macroscale. However, acceptance of microfluidics as a viable platform has thus far been reserved because utility of microfluidics has yet to be fully demonstrated. For biologists to embrace microfluidics, engineers must validate microscale systems and prove their practicality as tools for cell biology. Microfluidic devices were designed, fabricated, and implemented to study properties of two EC types: aortic ECs and valve ECs. The objective was to streamline experimentation to reveal phenotypic traits of the two types and in the process demonstrate the usefulness of microfluidics. The first task was to develop a protocol to isolate pure populations of valve ECs because reported methods were inadequate. Dispase and collagenase in combination for leaflet digestion followed by clonal expansion of cell isolates was optimal for obtaining pure valve EC populations. Using a parallel microfluidic network, we discovered that valve ECs adhered strongly and spread well only on fibronectin and not on type I collagen. In contrast, aortic ECs adhered strongly on both proteins. Both aortic and valve ECs were then exposed to shear and analyzed for cell orientation. Morphological analyses showed aortic and valve ECs both aligned parallel to flow when sheared in a macroscale flow chamber, but aortic ECs aligned perpendicular to flow when sheared in a microchannel. Finally, a microfluidic membrane device was designed and characterized as a potential tool for measuring albumin permeability through sheared endothelial monolayers. Overall, these studies revealed novel EC characteristics and phenomena, and demonstrated accessibility of microfluidics for EC studies.

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nL-mL droplets containing samples and reagents are controlled(i.e., moved, merged, mixed, and dispensed from reservoirs) by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquid and solid) in heterogeneous systems with no need for complex networks of microvalves. In this thesis, digital microfluidics has been applied to address key challenges in the fields of chemistry, biology and medicine. For applications in chemistry, the first two-plate digital microfluidic platform for synchronized chemical synthesis is reported. The new method, which was applied to synthesizing peptide macrocycles, is fast and amenable to automation, and is convenient for parallel scale fluid handling in a straightforward manner. For applications in biology, I present the first DMF-based method for extraction of proteins (via precipitation) in serum and cell lysate. The performance of the new method was comparable to that of conventional techniques, with the advantages of automation and reduced analysis time. The results suggest great potential for digital microfluidics for proteomic biomarker discovery. Furthermore, I integrated DMF with microchannels for in-line biological sample processing and separations. Finally, for applications in medicine, I developed the first microfluidic method for sample clean-up and extraction of estrogen from one-microliter droplets of breast tissue homogenates, blood, and serum. The new method is fast and automated, and features >1000x reduction in sample use relative to conventional techniques. This method has significant potential for applications in endocrinology and breast cancer risk reduction. In addition, I describe a new microfluidic system incorporating a digital microfluidic platform for on-chip blood spotting and processing, and a microchannel emitter for direct analysis by mass spectrometry. The new method is fast, robust, precise, and is capable of quantifying analytes associated with common congenital disorders such as homocystinuria, phenylketonuria, and tyrosinemia.

Endothelial cells (ECs) form the inner lining of all blood vessels in the body, and coat the outer surfaces of heart valves. Because ECs are anchored to extracellular matrix proteins and are positioned between flowing blood and underlying interstitium, ECs are constantly exposed to hemodynamic shear, and act as a semi-permeable barrier to blood-borne factors. In vitro cell culture flow (ICF) systems have been employed as laboratory tools for testing endothelial properties such as adhesion strength, shear response, and permeability. Recently, advances in microscale technology have introduced microfluidic systems as alternatives to conventional ICF devices, with a multitude of practical advantages not available at the macroscale. However, acceptance of microfluidics as a viable platform has thus far been reserved because utility of microfluidics has yet to be fully demonstrated. For biologists to embrace microfluidics, engineers must validate microscale systems and prove their practicality as tools for cell biology. Microfluidic devices were designed, fabricated, and implemented to study properties of two EC types: aortic ECs and valve ECs. The objective was to streamline experimentation to reveal phenotypic traits of the two types and in the process demonstrate the usefulness of microfluidics. The first task was to develop a protocol to isolate pure populations of valve ECs because reported methods were inadequate. Dispase and collagenase in combination for leaflet digestion followed by clonal expansion of cell isolates was optimal for obtaining pure valve EC populations. Using a parallel microfluidic network, we discovered that valve ECs adhered strongly and spread well only on fibronectin and not on type I collagen. In contrast, aortic ECs adhered strongly on both proteins. Both aortic and valve ECs were then exposed to shear and analyzed for cell orientation. Morphological analyses showed aortic and valve ECs both aligned parallel to flow when sheared in a macroscale flow chamber, but aortic ECs aligned perpendicular to flow when sheared in a microchannel. Finally, a microfluidic membrane device was designed and characterized as a potential tool for measuring albumin permeability through sheared endothelial monolayers. Overall, these studies revealed novel EC characteristics and phenomena, and demonstrated accessibility of microfluidics for EC studies.

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nL-mL droplets containing samples and reagents are controlled(i.e., moved, merged, mixed, and dispensed from reservoirs) by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquid and solid) in heterogeneous systems with no need for complex networks of microvalves. In this thesis, digital microfluidics has been applied to address key challenges in the fields of chemistry, biology and medicine. For applications in chemistry, the first two-plate digital microfluidic platform for synchronized chemical synthesis is reported. The new method, which was applied to synthesizing peptide macrocycles, is fast and amenable to automation, and is convenient for parallel scale fluid handling in a straightforward manner. For applications in biology, I present the first DMF-based method for extraction of proteins (via precipitation) in serum and cell lysate. The performance of the new method was comparable to that of conventional techniques, with the advantages of automation and reduced analysis time. The results suggest great potential for digital microfluidics for proteomic biomarker discovery. Furthermore, I integrated DMF with microchannels for in-line biological sample processing and separations. Finally, for applications in medicine, I developed the first microfluidic method for sample clean-up and extraction of estrogen from one-microliter droplets of breast tissue homogenates, blood, and serum. The new method is fast and automated, and features >1000x reduction in sample use relative to conventional techniques. This method has significant potential for applications in endocrinology and breast cancer risk reduction. In addition, I describe a new microfluidic system incorporating a digital microfluidic platform for on-chip blood spotting and processing, and a microchannel emitter for direct analysis by mass spectrometry. The new method is fast, robust, precise, and is capable of quantifying analytes associated with common congenital disorders such as homocystinuria, phenylketonuria, and tyrosinemia.

The embryonic stem cell (ESC) microenvironment contains various localized physical and biochemical cues to direct cell fate. Current approaches for microenvironmental regulation rely on restricting cell behaviour to control endogenous signals such as secreted ligands. This report presents a microfluidic device that can directly manipulate the removal of autoregulatory ligands from culture and control the activation of Signal Transducer and Activator of Transcription-3 (Stat3) in ESCs. Specifically, the response of Stat3 was measured under diffusive and convective mass transfer regimes. A Brownian dynamics algorithm was also developed to simulate ligand transport and predict cellular response under these conditions. Stat3 activation under perfusion culture was found to depend on flow rate and axial distance in the flow direction. Long-term perfusion also allowed for the formation of a sustained gradient of Stat3 activation that led to selective loss of ESC pluripotency. These results demonstrate the utility of microfluidic culture for stem cell bioengineering applications.

Microfluidics is a very recent branch of science and technology. The development and the success, it has had in the last 15 years, is mainly due to the concept of lab-on-a-chip. Those miniaturized devices, integrating one or more laboratory functions, have aroused great interest among several research areas as physics, chemistry, biology and bioengineering. When a fluid is confined in a micro or nano scale structure, its behaviour is strongly affected by its interactions with the surrounding surfaces. In this context, the theme of fluid/solid slippage has been widely studied both theoretically and experimentally. Innovative technologies to enhance the surface slippage by specifically designing the solid interfaces have reportedly demonstrated to be an effective way to reduce the fluid/solid friction. To this end, superhydrophobic surfaces have increasingly attracted the interest of the scientific and technological community thanks to the large wall-slippage they present for liquid water. Though their behaviour has been extensively investigated through several theoretical and numerical methods, the experimental approaches are still indispensable to test and understand the properties of these surfaces. However, the lack of a general predicting model is also due to the fact that no one of the several existing experimental techniques has shown up as a very reliable one. Indeed, the reported measurements of slippage still depends on the specific adopted method, thwarting attempts to corroborate the proposed theoretical and numerical schemes. Therefore, it is evident that a more sensitive and effective experimental technique is still missing. This thesis began and developed inside the wider project of setting up an innovative technique to investigate the fluid-solid slippage on superhydrophobic surfaces by means of optical tweezers. Even though this project is still going on, this work reports the steps performed along the long way towards this main goal and it consists of a collection of several researches involving different scientific fields as optics, microscopy, surface science, microhydrodynamics, microfluidics and microfabrication. The researches presented in this work can be separated in two main categories: i) holographic micromanipulation and microscopy, ii) superhydrophobicity.

Recent advances in micro technologies have equipped researches with novel tools for interacting with biological molecules and cells. This thesis focuses on the design, fabrication and application of microfluidic platforms for stimuli-responsive drug delivery and the electromechanical characterization of single cells. Stimuli-responsive hydrogels are promising materials for controlled drug delivery due to their ability to respond to changes in local environmental conditions. In particular, nanohydrogel particles have been a topic of considerable interest due to their rapid response times compared to micro and macro-scale counterparts. Owing to their small size and thus low drug-loading capacity, these materials are unsuitable for prolonged drug delivery. To address this issue, stimuli-responsive implantable drug delivery micro devices by integrating microfabricated drug reservoirs with smart nano-hydrogel particles embedded composite membranes have been proposed. In one proposed glucose-responsive micro device, crosslinked glucose oxidase enables the oxidation of glucose into gluconic acid, producing a microenvironment with lower pH values to modulate the pH-responsive nanoparticles. In vitro glucose-responsive drug release profiles were characterized and normoglycemic glucose levels in diabetic rats with device implantation were also recorded. The biophysical properties of single cells have recently been demonstrated as an important indicator of disease diagnosis. Existing technologies are capable of characterizing single parameter either electrical or mechanical rapidly, but not both, which could only collect limited information for cell status evaluation. To address this issue, two microfluidic platforms capable of simultaneously characterizing both the electrical and mechanical properties of single cells based on electrodeformation and integrated impedance spectroscopy with micropipette aspiration have been proposed. In one proposed microfluidic device, a negative pressure was used to suck cells continuously through the aspiration channel with impedance profiles measured. By interpreting impedance profiles, transit time and impedance amplitude ratio can be quantified as cellular mechanical and electrical property indicators. Neural network based cell classification was conducted, demonstrating that two biophysical parameters could provide a higher cell classification success rate than using electrical or mechanical parameter alone.

This thesis is an investigation of methods to remediate methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilms through conventional and microfluidic-based in vitro assays. MRSP biofilm related infections are a major concern for veterinary clinicians as they may complicate remediation by the immune system or antimicrobials. Novel antimicrobials that have been found to reduce biofilm growth in other staphylococci were assessed in both mono- and combination therapy against MRSP biofilm. Quantitative assay results (p < 0.05) suggest fosfomycin alone and in combination with clarithromycin can significantly reduce biofilm formation. Morphological examination using scanning electron microscopy and atomic force microscopy further demonstrated the effectiveness of fosfomycin alone on biofilm formation on orthopaedic screws and mica sheets. Fabricated microfluidic assays were utilized to assess multiple concentrations of antimicrobial therapy against pre-formed biofilm under physiologically relevant conditions in a quick and repeatable manner. Results demonstrated the usefulness of microfluidic platforms in determining minimum biofilm eradication concentrations.

This thesis focuses on the development of two new applications using ion concentration polarization (ICP): an out-of-plane microfluidic approach for water desalination and a method for concentration and transportation of charged analytes in paper-based biomedical diagnostic device. In the first work, we present an out-of-plane desalination approach using ICP. A depletion boundary separates salt ions and purified water into distinct vertically stacked layers. The out-of-plane design enables multiplexing in three dimensions, providing the functional density required for practical applications. The second work demonstrates an active control mechanism of target analytes in paper using ICP. Both external devices (with all functional units on one side of paper) and integrated paper microfluidic devices (by embedding all functional units in paper) were developed to concentrate and transport charged analyte molecules in the paper. We also demonstrate a new fabrication method of nanofluidic and hydrophobic barriers (nanoporous membrane patterning) in paper microfluidic device.