USE OF MICROSCALE HYDROPHOBIC SURFACE FEATURES FOR INTEGRATION OF 3D CELL CULTURE INTO MULTI-FUNCTIONAL MICROFLUIDIC DEVICES

3D cell culture and microfluidics both represent powerful tools for replicating critical components of the cell microenvironment; however, challenges involved in integration of the two and compatibility with standard tissue culture protocols still represent a steep barrier to widespread adoption. Here we demonstrate the use of engineered surface roughness in the form of microfluidic channels to integrate 3D cell-laden hydrogels and microfluidic fluid delivery. When a liquid hydrogel precursor solution is pipetted onto a surface containing open microfluidic channels, the solid/liquid/air interface becomes pinned at sharp edges such that the hydrogel forms the “fourth wall” of the channels upon solidification. We designed Cassie-Baxter microfluidic surfaces that leverage this phenomenon, making it possible to have barrier-free diffusion between the channels and hydrogel; in addition, sealing is robust enough to prevent leakage between the two components during fluid flow, but the sealing can also be reversed to facilitate recovery of the cell/hydrogel material after culture. This method was used to culture MDA-MB-231 cells in collagen, which remained viable and proliferated while receiving media exclusively through the microfluidic channels over the course of several days. Further modifications were made to create a multi-functional 3D cell culture platform. Gas impermeable polymer structure and deoxygenated flow were used to lower the oxygen content in the device, and the oxygen content was monitored in real-time using embedded oxygen sensors. This is particularly useful in replication of the tumor microenvironment where hypoxic conditions affect the cellular behavior and morphology. Also, by incorporating two inlets in the microfluidic device, binary concentrations of solutes were introduced into the system which created a lateral concentration gradient across the fluidic path. This allows studying of cell migration and response to various chemoattractant and drug doses. And finally, two high throughput designs to create 4-well and eight-well microfluidic devices were proposed and tested. This enables conducting more replicates of an experiment and even comparative studies on a single chip

Thermo-hygro-chemo-mechanical model of concrete at early ages and its extension to tumor growth numerical analysis.

The aim of the PhD thesis is the development of two multiphase models from a common theoretical basis, applied to two very different research fields: i) the study of the behavior of concrete at early ages, essentially for the prevention of cracking and related issues; ii) the analysis of the physical, chemical and biological processes that govern the growth of cancer. The modeling of concrete at early ages is very important and useful for the design of durable and sustainable structures. The model developed during the PhD thesis has been implemented on the finite element code Cast3M and then validated via the simulation of experimental cases. Nowadays this model allows for several applications: study of stresses and cracking in young concrete, analysis of thermal and hygral gradients, predictions of autogenous and drying shrinkage, creep strain, stress redistribution, study of the inhibition of hydration caused by drying, study of repairs, etc.. In the fight against cancer, the advance of medical strategies based on numerical analysis has a crucial scientific interest and can have a great social impact. The equations which govern the thermo-hygro-chemo-mechanical behavior of concrete at early ages have many formal analogies with those typically used to model tumor growth. Hence, these equations have been readapted and a novel mathematical model for tumor growth has been developed. This model has been implemented in Cast3M and the first numerical results are encouraging since qualitatively close to the experimental data present in the scientific bibliography

Role of extracellular matrix in development and cancer progression

The immense diversity of extracellular matrix (ECM) proteins confers distinct biochemical and biophysical properties that influence cell phenotype. The ECM is highly dynamic as it is constantly deposited, remodelled, and degraded during development until maturity to maintain tissue homeostasis. The ECM’s composition and organization are spatiotemporally regulated to control cell behaviour and differentiation, but dysregulation of ECM dynamics leads to the development of diseases such as cancer. The chemical cues presented by the ECM have been appreciated as key drivers for both development and cancer progression. However, the mechanical forces present due to the ECM have been largely ignored but recently recognized to play critical roles in disease progression and malignant cell behaviour. Here, we review the ways in which biophysical forces of the microenvironment influence biochemical regulation and cell phenotype during key stages of human development and cancer progression

Mechanotransduction in Breast and Ovarian Cancers: Using Bioreactors to Study the Cellular Response to Physiological Mechanical Stimuli

Cells within the body experience a wide range of dynamic mechanical stimuli. These stimuli are exacerbated in cancers and can alter the progression of the disease. As the tumor grows and expands, it presses out against the surrounding matrix and cell environment, creating internal compressive forces. The growing tumor also alters interstitial and vascular blood flow thereby enhancing shear stress exposure. How cells translate this mechano-environment into downstream signaling is known as mechanotransduction. Though preliminary research has touched on the influence physiological mechanical stimulus can have on cancer progression, the work remains erratic on cell metastasis, gene expression, proliferation, and chemotherapeutic response. In order to address this unknown effect on cellular phenotypes and treatment response, two bioreactors capable of tunable three-dimensional stimulus with either shear stress or compressive stress were developed. Breast and ovarian cancer cells were first encapsulated within a 3D combination agarose/collagen-I hydrogel and then exposed to physiological mechanical stimuli relevant to the unique microenvironments of pleural effusions and ascitic environment respectively for 24 to 72 hours. Stimulated cells were then assessed for morphological alterations, altered gene expression (RT-qPCR), proliferation (ki67 expression), and drug resistance via standard chemotherapeutic treatment (cell death via casp-3 expression). Breast cancer cells exposed to varying levels of shear stimulus occurring within the pleural effusion microenvironment showed stimulus aided in cancer cell proliferation, invasive potential, and survival in the presence of paclitaxel treatment concurrent with the activation of the PLAU and COX2 pathways. Next, ovarian cancer cells were subjected to compressive forces found within the solid tumor microenvironment and as consequence of the hydrostatic pressure caused via ascitic fluid retainment. Ovarian cancer proliferation, morphological elongation, and enhanced survival was observed under physiological compressive stimulus alongside chemoresistance and upregulation and activation of CDC42. Finally, ovarian cancer cells were stimulated with shear stresses representative of ascitic fluid buildup in the peritoneal cavity in ovarian cancer patients. This shear stress conditioning altered cellular morphology, enhanced proliferation as well as chemoresistance to dual chemotherapeutic drug treatment with paclitaxel and carboplatin. This alteration in cellular phenotype was found alongside consistent downregulation of MUC15, a potential protein of interest for future mechanotransduction studies. Overall, findings suggest that this dynamic mechanical environment aids in the advancement of cancer migration, proliferation, and chemoresistance which may be mitigated by targeting various mechanotransduction pathways. This is the first reported tie of shear stress stimulation to PLAU and COX2 pathway activation in breast cancer. Additionally, this is the first time mechanotransduction has been tied to CDC42 activation and MUC15 downregulation in ovarian cancer. The bioreactors constructed and utilized for this study provide 3D platforms ideal for understanding the influence of compressive and shear stress stimulus on cellular behavior, a critical component to our understanding and improvement of cancer patient treatments.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155032/1/cmnovak_1.pd

A strategy for integrating essential three-dimensional microphysiological systems of human organs for realistic anticancer drug screening

Cancer is one of the leading causes of morbidity and mortality around the world. Despite some success, traditional anticancer drugs developed to reduce tumor growth face important limitations primarily due to undesirable bone marrow and cardiovascular toxicity. Many drugs fail in clinical development after showing promise in preclinical trials, suggesting that the available in vitro and animal models are poor predictors of drug efficacy and toxicity in humans. Thus, novel models that more accurately mimic the biology of human organs are necessary for high-throughput drug screening. Three-dimensional (3D) microphysiological systems can utilize induced pluripotent stem cell technology, tissue engineering, and microfabrication techniques to develop tissue models of human tumors, cardiac muscle, and bone marrow on the order of 1 mm3 in size. A functional network of human capillaries and microvessels to overcome diffusion limitations in nutrient delivery and waste removal can also nourish the 3D microphysiological tissues. Importantly, the 3D microphysiological tissues are grown on optically clear platforms that offer non-invasive and non-destructive image acquisition with subcellular resolution in real time. Such systems offer a new paradigm for high-throughput drug screening and will significantly improve the efficiency of identifying new drugs for cancer treatment that minimize cardiac and bone marrow toxicity

Cancer Models on Chip:Paving the Way to Large Scale Trial Applications

Cancer kills millions of individuals every year all over the world (Global Cancer Observatory). The physiological and biomechanical processes underlying the tumor are still poorly understood, hindering researchers from creating new, effective therapies. Inconsistent results of preclinical research, in vivo testing, and clinical trials decrease drug approval rates. Three-dimensional tumor-on-chip models integrate biomaterials, tissue engineering, fabrication of microarchitectures, and sensory and actuation systems in a single device, enabling reliable studies in fundamental oncology and pharmacology. This review includes a critical discussion about their ability to reproduce the tumor microenvironment, the advantages and drawbacks of existing tumor models and architectures, and major components and fabrication techniques. The focus is on current materials and micro/nanofabrication techniques used to manufacture reliable and reproducible microfluidic tumor-on-chip models for large-scale trial applications.</p

Experimentation and Multiphysical Modeling of Bioanalytical Microdevices

Bioanalytics involves quantitative measurements of complex biological samples that contain metabolites, DNA, RNA, and proteins. Efficient sample preparation for downstream analysis and sensitive detection of analytes can be achieved via bioanalytical microdevices. Fully realizing the potential of these devices requires tool characterization and bioprocess optimization, in addition to understanding device physics. Therefore, this thesis introduces multiphysical modeling and experimentation of microdevices, with applications to diabetes care and single-cell analysis. To understand the physics of viscometric glucose microsensors, this thesis presents a model of the sensor, which couples the fluid flow with vibrating diaphragms. The model is used to predict the sensor response to glucose via theory of squeeze-film damping and vibrations of pre-stressed plate. A first-principle-based model resulting from the theory can be evaluated from the device's geometric and material properties, and quantitatively determines the device response to vibrational excitations at varying glucose concentrations. Next, this thesis introduces a theoretical model for viscometric glucose microsensors that employ harmonic microcantilever oscillation in the sensing liquid. The presented model associates the unsteady Stokes equation with the motion of a bounded viscous liquid to understand the hydrodynamic impact on the cantilever. With a proper consideration of the viscosity and bounded geometry of liquid media, the model relaxes the thin-film assumption required for the diaphragm-based model, enabling an accurate representation of fluid-structure interactions based on fundamental structural vibration and fluid flow equations. Next, this thesis presents an experimental exploration of a hydrogel-based affinity microsensor for glucose monitoring via dielectric measurements. The microsensor incorporates a synthetic hydrogel that is attached to the device surface via in situ polymerization, which eliminates mechanical moving parts required in the viscometric glucose sensors. Changes in the dielectric properties of the hydrogel when binding reversibly with glucose molecules have been measured using a MEMS capacitive transducer to determine the glucose concentration. Experimental results demonstrate that in a glucose concentration range of 0–500 mg/dL and with a resolution of 0.35 mg/dL or better, the microsensor exhibits a repeatable and reversible response, and can potentially be useful for continuous glucose monitoring in diabetes care. Additionally, this thesis presents a microfluidic preprocessing method that integrates single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis at single-cell level for individual cells. The approach utilizes a photocleavable bead-based microfluidic device to synthesize and deliver stable complementary DNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. Finally, this thesis ends with concluding remarks and directions of future work towards continuous glucose monitoring and high-throughput single-cell genetic analysis

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application