Numerous applications in biology and medicine requires efficient and reliable separation of cellular or sub-cellular components from blood for disease diagnosis, genetic analysis, drug screening, and therapeutics. However, analyzing undiluted whole human blood is challenging due to its complex composition of hematopoietic cellular populations, plasma and sub-cellular components such as nucleic acids, metabolites, and proteins. In addition, current state-of-art for blood fractionation relies on multi-step density-based centrifugation or flow cytometry which are manual, time-consuming and unable to process whole blood. In this dissertation, we aim to demonstrate a multi-functional, low-cost microfluidic acoustic streaming platform to enrich and characterize cellular components from whole blood and utilize blood’s sub-cellular components for simultaneous detection of multiple infections.

First, we present Lateral Cavity Acoustic Transducers (LCATs) that enables (1) the sorting of undiluted donor whole blood into its cellular subsets (platelets, RBCs, and WBCs), (2) the enrichment and retrieval of breast cancer cells (MCF-7) spiked in donor whole blood at rare cell relevant concentrations (10/ml), and (3) on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns. Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment, to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling.

Second, we integrated a passive (spiral inertial microfluidic device) and an active device (LCAT) for high-throughput separation, enrichment and release of specific target cellular population (such as larger side-population (SP) of DU-145 cells from tissue biopsy or larger monocytic cell population from blood sample). After optimization with particles spiked in blood, this platform removed >90% of the smaller cells, such as RBCs in a blood sample or smaller cancer cells in a heterogeneous cell line, and provided 44,000x enrichment from remaining sample within 5 minutes of device operation.

Third, since whole blood comprises not only cellular subsets, but also sub-cellular components, we utilized the antibodies in both blood serum and saliva for the multiplexed screening of HIV, HPV and HSV within 17.5 minutes. We integrated this acoustic microstreaming platform with a protein microarray and verified reactivity of patient antibodies with HIV, HPV and HSV antigens. This integrated microfluidic protein array platform is the basis of a potent strategy to delay progression of primary infection, reduce the risk of co-infections and prevent onward transmission of infections by point-of-care detection of multiple pathogens.

Synthetic biology is the study and engineering of organisms with novel, unique functions. Specifically, bottom-up synthetic biology involves assembling complex cells or structures from simpler units. One such component of this field involves the production of artificial cells. By employing a bottom-up approach, researchers can investigate individual properties of the cell such as lipid membrane characteristics, specific biological pathways, and encapsulation phenomena.

Microfluidic methods enable the uniform generation of artificial cell precursors, otherwise known as Double Emulsions Droplets (DEDs). Although DEDs are versatile in both composition and application, they can be limited by their instability and susceptibility to rupture. This is observed by a reduction in DED concentration over time. Here we present a study for the optimization of the external aqueous phase resulting in the most stable and highest concentration of DEDs over a period of two weeks. By post-processing the DEDs in squeezing channels, the oil layer is thinned resulting in less depletion of the sample.

The immunological network comprises of complex and interconnected system of cells, tissues and molecules that work together to defend the body against foreign invaders, such as bacteria, viruses, and cancer cells. This network includes different types of immune cells, such as T cells, B cells, natural killer cells, and dendritic cells, as well as various molecules, such as cytokines, chemokines, and antibodies. The immunological network functions through series of interactions and feedback loops, in which different components of the immune systems communicate with each other to coordinate their activities and mount an effective immune response. In my Ph.D. dissertation, I will be focusing on three essential components for therapeutic discovery and development: T cells, cancer cells, and antibodies. These three components initiate reciprocal communications and coordinate with each other within the human body to maintain homeostasis. However, genetic mutation or uncooperative behavior in their relationships can lead to the initiation of cancer. To better understand and defend against cancer, it’s crucial to detect the disease, develop targeted cell therapies and screen drugs effectively. Three specific platforms that aid in these efforts have been developed. The first is a 3D confinement enabled system that activates and expands human primary T cells. The second is a rapid acoustic microstreaming platform to isolate tumor cells from patient blood samples, aiding in the diagnosis of cancer. The third platform is an acoustic microstreaming platform that screens antibody/drug viscosity. Autologous cell therapy depends on T lymphocyte expansion efficiency and is hindered by suboptimal interactions between T cell receptors and peptide-MHC molecules. Various artificial antigen presenting cell systems that enhance these interactions are often labor-intensive, fabrication costly, highly variable, and potentially unscalable towards clinical setting. Here, 3D confinement-enabled priming of T cell immune-synapse junctions was performed to generate tight T cell–Dynabead skeletons at a rate 200-fold faster than that of conventional 24-h bulk shaking. Furthermore, by forming T cell–Dynabead skeletons in the starting culture, two- to six-fold greater T cell expansion was achieved over conventional T cell expansion for cancer patient-derived primary T cells, and excessive cell exhaustion was not induced. Creating 3D T cell–Dynabead skeletons as the “booster” material enables highly efficient polyclonal T cell expansion without the need for complex surface modification of artificial antigen-presenting cells. This method can be modularly adapted to existing T cell expansion processes for various applications, including adoptive cell therapies (ACTs). The second platform is based on an acoustic microstreaming microfluidic platform. We demonstrate a label free and high-throughput microbubble-based acoustic microstreaming technique to isolate rare circulating cells such as circulating cancer associated fibroblasts (cCAFs) in addition to circulating tumor cells (CTCs) and immune cells (i.e. leukocytes) from clinically diagnosed patients with a capture efficiency of 94% while preserving cell functional integrity within 8 minutes. The microfluidic device is self-pumping and was optimized to increase flow rate and achieve near perfect capturing of rare cells enabled by having a trapping capacity above the acoustic vortex saturation concentration threshold. Our approach enables rapid isolation of CTCs, cCAFs and their associated clusters from blood samples of cancer patients at different stages. By examining the combined role of cCAFs and CTCs in early cancer onset and metastasis progression, the device accurately diagnoses both cancer and the metastatic propensity of breast cancer patients. Our LCAT-based approach can thus be developed into a metastatic propensity assay for clinical usage by elucidating cancer immunological responses and the complex relationships between CTCs and its companion tumor microenvironment. Lastly, we demonstrate an acoustic microstreaming platform termed as microfluidic viscometer by acoustic streaming transducers (µVAST) that induces fluid transport from second-order microstreaming to measure viscosity. Measurement of fluid viscosity represents a huge need for many biomedical and materials processing applications. Sample fluids containing DNA, antibodies, protein-based drugs, and even cells have become important therapeutic options. The physical properties, including viscosity, of these biologics are critical factors in the optimization of the biomanufacturing processes and delivery of therapeutics to patients. Validation of our platform is achieved with different glycerol content mixtures to reflect different viscosities and show that viscosity can be estimated based on the maximum speed of the second-order acoustic microstreaming. The µVAST platform requires only a small volume of fluid sample (~ 1.2 uL), which is 16 – 30 times smaller than that of commercial viscometers. In addition, µVAST can be scaled up for ultra-high throughput measurements of viscosity. Here we demonstrate 16 samples within 3 seconds, which is an attractive feature for automating the process flows in drug development and materials manufacturing and production.

Single-cell analysis is of critical importance in revealing population heterogeneity, identifying minority sub-populations of interest, as well as discovering unique characteristics of individual cells. Conventional bench-top methods are limited by their high cost, low throughput, and inadequacy in analyzing small amount of material. Microfluidic platforms, on the contrary, work at the scale comparable to cell diameter, and recent advances in microfluidics have made it possible to automate the processing and analysis of single cells in a high-throughput and low-cost manner.

In this dissertation, a system of microfluidic platforms enabling high-throughput single-cell analysis from phenotype to genotype is developed. The system is built upon a microfluidic trapping array for rapid and deterministic single-cell trapping in highly-packed microwells. It is a serpentine channel with microwells arrayed along each row, wherein cells are delivered to the traps sequentially by the horizontal flow and pushed into traps by the perpendicular stream through the gap area at each trap. 1600 microwells are filled within 3 min with a single-cell occupying efficiency > 80%, while smaller cells/debris are filtered out simultaneously.

Two innovative single-cell analyses from phenotype to genotype are established on this single-cell array: live-cell real-time metabolic imaging via fluorescence-lifetime-imaging-microscopy (FLIM), and single-cell mRNA live-probing by dielectrophoretic nanotweezers (DENT). Rapid trapping and identification (both by FLIM and mRNA probing) of single circulating-tumor-cells (CTCs) from blood have been successfully demonstrated. After characterizing the individual trapped cells, in order to edit aberrant genes for the identified cells of interest, a droplet-microfluidic platform has been developed for efficient single-cell transfection (10X higher efficiency for suspension cells compared to the bulk approach). To explore cell-cell interaction at single-cell level, the single-cell array has also been modified into an easy-to-operate cell-pairing array.

As the trapping efficiency is determined by the channel parameter instead of the flow rate, the single-cell array can be integrated with various sample-processing units operating at different flow rates. The presented microfluidic system enables high-throughput single-cell trapping, label-free metabolic imaging, mRNA extraction without cell lysing, efficient gene transfection, and cell-cell interaction analysis. It is expected to have myriad applications in cancer diagnostics, gene therapy, immunology, etc.

Human aging is often linked to the gradual decline of the immune system. This age-related decline of the immune system, or immunosenescence, is often attributed to the increased frequency of elder morbidity and mortality. One prominent immunosenescence feature is that of a low grade chronic inflammation. Dendritic cells (DC) play a major role in inducing adaptive immunity by presenting pathogenic antigens at T-Cells to initiate immune response. Once activated, dendritic cells undergo physiological transformation and express a host of costimulatory and pro-inflammatory molecules. This transformation requires a shift in metabolic demand to meet the bioenergetic and biosynthetic needs of activated dendritic cells. We hypothesized that aging initiates excessive dendritic cell activation which could in turn triggers chronic inflammation observed in elderly subjects. As such, through studying known cellular metabolic markers from microfluidic aided fluorescence lifetime Imaging microscopy (FLIM) and flow cytometry, we report the preliminary results on the metabolic effects of aging on dendritic cells.

Early cancer detection results in higher survival rates for patients. Circulating tumor cells (CTCs) traveling through the blood can be used for early cancer diagnosis. Unfortunately, these CTCs are rare (as low as 1 CTC per billion red blood cells), limiting their use in early diagnostic applications. A substantial amount of research suggests a correlation between the presence of cancer and the circulation of EVs, but detailed knowledge on the role of EVs is still lacking due to inefficient methods in EV isolation. The current “gold standard” for EV isolation is ultracentrifugation. Ultracentrifugation produces low yield, contain damages cells, has poor reproducibility and low purity.

Microfluidic technologies are an attractive alternative for EV isolation due to their low-cost, low sample and rapid throughput. We propose a quick, low-sample microfluidic solution to EV isolation with efficiencies that rival the gold standard of ultracentrifugation. Using acoustic microstreaming and immunoaffinity capture methods, we report isolation of EVs for downstream analysis at high efficiencies and faster turnaround times, fewer steps and less equipment than UC.

The bottleneck for drug discovery and drug screening has prompted the recent surge for organ-on-a-chip 3D in vitro models that recapitulate physiological and disease mechanisms. Among those models, it is critical to incorporate the vascular capillary networks into micro tissue to better recapitulate the in vivo microenvironment. Microfluidics technology has become an important tool to provide better control of mass transport in the microenvironment for nutrient supply and waste removal. However, the current models only mimic certain aspects of in vivo situations. As in vitro models continue to be developed, more complex on-chip vascularized tissues are necessary to provide more precise physiological systems towards the ultimate goal of ‘human-on-a-chip’.

In this dissertation, a series of microfluidic platforms are developed to enable the culturing of large scale vascularized micro tissues. The system is built on a high throughput format integrated with 96-well plate that contains multiple units for parallelization of experiments. The microfluidics channel design of each unit in this system allows co-culture of multiple types of tissue. Here, complex tissues are formed by retaining certain types of tissue in designated regions, and the interaction of different tissue types can be studied by controlling the tissue formation and mass transport of each region. In this dissertation, a novel 3D hydrogel/medium interface and a siphon-based passive pump are developed to overcome these difficulties. Furthermore, oxygen, one of the important factors affecting tissue growth, is controlled to be at a more physiological level. Various physiological or disease models can be built by selecting and combining these microfluidics components, such as compartmentalized micro tumors co-cultured with vasculature, hereditary hemorrhagic telangiectasia induced by high shear stress, vascularized liver model with in vivo liver structure and oxygen gradient, etc.

Giant unilamellar vesicles (GUVs) show great potential as biosensors and artificial cells and have been utilized extensively as cellular models for studies on dynamic membrane properties, mechanical measurements, phospholipid domain formation, and membrane protein integration. Macroscale methods to make GUVs are widespread, but suffer from polydispersity, multilamellarity, and low encapsulation efficiency. While the control of droplet production provided by microfluidic technologies have been able to overcome many of these limitations, widespread usage of GUVs outside of research settings has been hindered by their fragility and short shelf life, rendering them difficult to ship and commercialize. A similar structure, known collectively as multisomes, can be suitable for the same applications as GUVs, but their potential has been undervalued and they also suffer similar storage limitations as GUVs. In order for GUVs and multisomes to realize their vast potential, it is paramount that they can be produced in bulk, and stored for a reasonable amount of time without structural degradation.

Numerous microfluidic methods to produce GUVs employ water-in-oil-in-water (W/O/W) double emulsion templates that quickly convert into lipid vesicles via dewetting and/or solvent extraction. These methods generally use harsh, volatile solvents in the sheath (oil) phase that can potentially harm both cells and encapsulated biological materials. Furthermore, the immediate removal of the solvent to form GUVs limits their useful lifetime. Nearly all the work to improve GUV shelf life has been focused on modifying their storage conditions after production, which can be damaging.

In this work, we present a method to circumvent the storage limitations and fragility of GUVs and multisomes. Double emulsions templates are produced utilizing a single junction co-focusing microfluidic device to ensure monodispersity, and are composed of only biocompatible surfactants and fatty acids. The design allows for double emulsions to be produced with diameters ranging from 9-50µm and generation rates up to 3 kHz by simply varying the fluid flow rates. The templates can be stored for up to a year and converted into multisomes or GUVs on demand within minutes by a simple solution change that alters the spreading coefficients of the system. We have also uncovered the interfacial parameters that control the stability of double emulsions for storage, and their subsequent dewetting to form multisomes or GUVs. Furthermore, multisomes were surface functionalized to demonstrate their applicability as artificial cells. In particular, they were surface modified to promote insulin secretion in pancreatic B cells through surface interactions with neuroligin-2. Ultimately, we envision that these multisomes and GUVs can be utilized as blank templates for a variety of applications, including drug delivery vehicles, artificial antigen presenting cells, and biosensors.