archives@tulane.edu / Fibrotic diseases comprise up to 45% of deaths in the industrialized world. Few effective anti-fibrotic therapeutics exist, due in part to the lack of human-relevant preclinical models. The goal of this research was to improve the modeling of fibrotic diseases in microphysiological systems (MPS) by engineering quiescence in cultured human fibroblasts prior to MPS incorporation. To create an assay for testing this approach, a versatile organ chip was designed while optimizing workflow for production of the organ chip molds with an SLA 3D printer. After identifying 2D culture conditions that repress fibroblast activation, we tested the hypothesis that the 2D culture protocol would impact the fibrotic baseline in our MPS. 3D confocal microscopy and multi-metric image analysis of immunostaining for cellular and extracellular matrix (ECM) components via intensity and pattern quantification revealed the establishment of more physiological baseline for MPS fibrosis models. To test in a disease-relevant context, we created a model of the stromal reaction in lung cancer using our organ chip and demonstrated that our integrated MPS can be used to quantify the fibrosis-inducing effects of cancer cells that drive stromal reactions. / 1 / Max Wendell

abstract: This dissertation studies two outstanding microscale fluid mechanics problems: 1) mechanisms of gas production from the nanopores of shale; 2) enhanced mass flow rate in steady compressible gas flow through a micro-conduit. The dissertation starts with a study of a volumetric expansion driven drainage flow of a viscous compressible fluid from a small capillary and channel in the low Mach number limit. An analysis based on the linearized compressible Navier-Stokes equations with no-slip condition shows that fluid drainage is controlled by the slow decay of the acoustic wave inside the capillary and the no-slip flow exhibits a slip-like mass flow rate. Numerical simulations are also carried out for drainage from a small capillary to a reservoir or a contraction of finite size. By allowing the density wave to escape the capillary, two wave leakage mechanisms are identified, which are dependent on the capillary length to radius ratio, reservoir size and acoustic Reynolds number. Empirical functions are generated for an effective diffusive coefficient which allows simple calculations of the drainage rate using a diffusion model without the presence of the reservoir or contraction. In the second part of the dissertation, steady viscous compressible flow through a micro-conduit is studied using compressible Navier-Stokes equations with no-slip condition. The mathematical theory of Klainerman and Majda for low Mach number flow is employed to derive asymptotic equations in the limit of small Mach number. The overall flow, a combination of the Hagen-Poiseuille flow and a diffusive velocity shows a slip-like mass flow rate even through the overall velocity satisfies the no-slip condition. The result indicates that the classical formulation includes self-diffusion effect and it embeds the Extended Navier-Stokes equation theory (ENSE) without the need of introducing additional constitutive hypothesis or assuming slip on the boundary. Contrary to most ENSE publications, the predicted mass flow rate is still significantly below the measured data based on an extensive comparison with thirty-five experiments. / Dissertation/Thesis / Doctoral Dissertation Mechanical Engineering 2019

Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

No description available.

This work describes the use of microfluidic technology and biomaterials in cancer research by mimicking the extracellular matrix (ECM) and development of drug delivery system. Initially, biomaterials such as Gelatin methacryloyl (GelMA) and collagen type I were combined to create a hydrogel composite able to mimic both healthy and cancerous ECM. The impact of the tumor microenvironment was analyzed by using the hydrogel inside of a pressurized microfluidic device and by tracking the movement of gold nanoparticles (GNPs). The GNPs showed a decrease in diffusion coefficient of 77% when analyzed in cancerous conditions. This investigation was further explored by analyzing the diffusion of charged GNPs in the same system, while also tracking cellular uptake. An inverse correlation between diffusion and cellular uptake was obtained for charged GNPs in breast cancer cells. Due to the tunable properties and biocompatibility of GelMA, this hydrogel was also employed in the development of pH-responsive drug delivery systems. Since GelMA contains a gelatin backbone, two responsive polymers (Polymers A and B) were synthesized. Microspheres of ~40 μm were fabricated in flow focusing microfluidic devices. Polymer A microspheres displayed a swelling increase of 167% in pH 6.0, while polymer B spheres showed a 296% swelling in pH 10. Considering the unique properties of the tumor microenvironment such as leaky vasculature and acid pH environment, polymer A was selected to be used in the production of nanocarriers. The behavior of this polymer in acidic environment illustrated its potential applicability as drug delivery systems to the tumor area. Polymer A nanogels displayed a uniform size of 74 ± 7 nm. Lastly, GNPs were added to the solution of polymer A, leading to the fabrication of GNPs-loaded nanogels, presenting a homogenous distribution of gold particles inside nanogels. / Graduate / 2023-07-05

The goal of this study is to design a protocol for the adherent cell culture within a novel microfluidic device. Microscale cell culture protocols were developed for loading cells using poly-L-lysine to enhance adherent cell culture of murine derived NIH 3T3 fibroblasts. This work sought to develop a method for adherent microculture by examining various sterilization, surface treatment, and seeding techniques. Using a vacuum suction loading technique, air plasma treatment and a poly-L-lysine surface treatment adherent cell culture was observed within the device. The work presented here is part of a collaborative effort that aims to develop protocols for the electrical and optical characterization of cell culture within a novel microfluidic device.

Building upon previous research on Digital Light Projection (DLP) 3D printing for microfluidics, in this thesis I performed the detailed design and fabrication of a novel DLP 3D printer to increase resolution and device footprint flexibility. This new printer has a pixel resolution twice that of our group’s previous printers (3.8 μm vs 7.6 μm). I demonstrated a new state of the art for minimum channel width, reducing the minimum width to 15 μm wide (and 30 μm tall). This is an improvement over the previous smallest width of 20 μm. This printer also has the capacity to perform multiple spatially distinct exposures per printed layer and stitch them into one interconnected device. Image stitching enables printing devices with identical build areas to previous printers, and with smaller pixel pitch. I pursued validation of this stitching capacity by fabricating channel devices with features crossing the stitched image boundary, with the goal of printing channels that would flow fluid consistently and without leaking. To accomplish this, I began by characterizing the print parameters for successfully printing single microfluidics channels across the stitched image boundary, and then I explored the sensitivity of my method to multiple crossings of the image boundary by printing a stacked serpentine channel that crossed the stitched image boundary 392 times. This demonstrated that an arbitrary number of stitched boundary crossings are feasible and thus a high degree of complex device component integration across these boundaries is also possible. These developments will be useful in future research and design of 3D printed microfluidic devices.

Recent technologies in micro-devices for investigation of functional biology in a controlled microenvironment are continually growing and evolving. In particular, electric-field mediated microfluidic platforms are evolving technologies that have significant applications in drug delivery and cell migration investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge; in recent years, the positive impact of electric fields is being explored. The primary objectives of the dissertation are to design, fabricate, and employ two novel microfluidic platforms for drug delivery and cell migration in the presence of electric fields. Description of iontophoretic carboplatin delivery into the MDA-MB-231 triple-negative breast cancer cells and investigation of neutrophil electro taxis are two main aims of the dissertation. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemotherapeutics for tumor treatment not only because of their non-invasiveness but also due to their lower systematic toxicity compared to other drug delivery systems. While iontophoresis animal models are commonly being used for the development of new cancer therapies, there are some obstacles for precise control of the tumor microenvironment's chemoresistance and scaffold in the animal models. We employed experimental and computational approaches, the iontophoresis-on-chip and the fraction of tumor killed mathematical model, for predicting the outcome of iontophoresis treatment in a controlled microenvironment. Also, precise control over the cell electromigration is a challenging investigation which we will address in the second aim of the dissertation. Here, we developed a microfluidic platform to study the consequences of DC electric fields on neutrophil electromigration (electrotaxis), which has an application of directing neutrophils away from healthy tissue by suppressing the migration of neutrophils toward pro-inflammatory chemoattractant. / Doctor of Philosophy / Recent technologies in the micro-scale medical devices for diagnosis and treatment purposes are continually growing and evolving. Microfluidic platforms are reproducible devices with the dimensions from tens to hundreds of micrometers for manipulating and controlling fluids. In particular, electric-field mediated microfluidic platforms, are developing technologies that have significant applications in drug delivery and biological cell directional movement investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge. In recent years, the positive impact of electric fields is a significant advancement in drug delivery techniques. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemo drugs for tumor treatment not only because of their sensitivity but also to their lower systematic toxicity compared to injection or oral drug delivery. While iontophoresis animal models are conventional for the development of new cancer therapies, there are some obstacles to precise control of the tumor scaffold in the animal models. We also developed a novel microfluidic platform to study the consequences of DC electric fields on white blood cells' (WBC) directional movement, which has an application of directing WBC away from healthy tissue by suppressing the damage of WBC accumulation in healthy organs.

This research presents a novel capacitive-sensing-based method to measure fluid velocity for microfluidics devices. To illustrate the importance of fluid velocity measurement, a case study was first conducted for a split and recombine micromixer. The study underscored the influence of fluid velocity on micromixer efficiency and mixing quality. The proposed fluid velocity measurement method employs two capacitance sensing electrodes placed along the fluid channel, capable of detecting small capacitance changes as fluid passing through the sensing area. The relation between capacitance changes and fluid velocity in the proposed sensing structures was developed and hence used to estimate fluid velocity. The proposed technique does not require extensive bench equipment and is suitable for point-of-care applications. To validate our approach, we implemented a two-step 3D printing process, creating a Polylactic acid (PLA) micro platform with embedded graphene–PLA composite electrodes. The accuracy of the developed method was investigated by cross-verifying the obtained velocities with an optical measurement method. Most absolute percentage discrepancies between the results from the proposed method and the optical method are under 12%, validating the precision of the proposed method. Future research will focus on integrating this velocity measurement method into microfluidic devices produced using advanced microfabrication technologies.

Key Words: Thrombosis, Vascular Graft, Microfluidics, Wall Shear Stress / Vascular grafts are essential for the management of cardiovascular disease. However, the lifesaving potential of these devices is undermined by thrombosis arising from material and flow interactions on the blood contacting surface. To combat this issue, the use of antithrombogenic coatings has emerged as a promising strategy for modulating blood and graft interaction in vivo. Although an important determinant of graft performance, hemodynamics are frequently overlooked in the in vitro testing of coatings and their translatability remains poorly understood. We address this limitation with a microscale platform that incorporates vascular prosthesis and coatings with tuneable flow and surface conditions in vitro. As a proof of concept, we use the platform to test the thrombogenic performance of a novel class of lubricant infused (LIS) and antibody lubricant infused (anti-CD34 LIS) coated ePTFE vascular grafts in the presence of arterial wall shear stress, with and without the presence of endothelial cells. Our findings suggest lubricant infused coated ePTFE vascular grafts are thromboresistant under flow and may have potential for in vivo arterial grafting applications. It is moreover apparent that the microscale properties of the device could be advantageous for the testing and translation of novel antithrombogenic coatings or blood contacting prosthesis in general. / Thesis / Master of Applied Science (MASc)