Improving flow-induced hemolysis prediction models.

Partial or complete failure of red blood cell membrane, also known as hemolysis, is a persistent issue with almost all blood contacting devices. Many experimental and theoretical contributions over the last few decades have increased insight into the mechanisms of mechanical hemolysis in both laminar and turbulent flow regimes, with the ultimate goal of developing a comprehensive, mechanistic and universal hemolysis prediction model. My research is broadly divided into two sections: theoretical/analytical/Computational Fluid Dynamics (CFD) analyses and experimental tests. The first part of my research revolved entirely around analyzing the simplest and most popular hemolysis model, commonly called as the power-law model. This model was developed only for laminar pure shear flow within a limited range of exposure time. Subsequently, modified versions of this model have been developed to be used for more complex flows. Many of these modified models assume that hemolysis scales with a resultant, scalar stress representing all components of the fluid stress tensor. The most common representative stress used in the power-law model is a von-Mises-like stress. However, using membrane tension models for pure shear and pure extension in both laminar and turbulent flows, for some simple example cases, we have shown that scalar stress alone is inadequate for scaling hemolysis. Alternatively, the rate of viscous energy dissipation rate has also been proposed as the parameter to scale hemolysis with. Applying the same order-of-magnitude estimate as vi mentioned above, we have found that dissipation rate even behaves worse than the resultant scalar stress for hemolysis prediction. It is therefore concluded that energy dissipation rate alone is also not sufficient to universally scale blood damage across complex flows. These show that a realistic model of hemolysis must take into account different responses of the viscoelastic cell membrane to different stress type. Various discretized version of the power-law model has also been introduced for post-processing of the CFD results. The power law can be either discretized in space, Eulerian treatment, or in time, Lagrangian treatment. Our study on the Eulerian approach revealed that the current equations used in the literature has a missing term, and thus incorrect. We also examined the mathematical stability of the discretized power-law model, and found that it may introduce significant error in red cell damage prediction for certain pathlines with specific stress history. Experimental results on deformation of red cell in pure shear flow is present for a relatively wide range of shear rates. However, red cell deformation/elongation in pure laminar extensional flow is scarce, with only one publication reporting their results on red cell deformation for only up to stress level of 10 Pa. For the experimental part of my research, we conducted experiments to observe the difference in deformation of red cell in pure shear and pure extensional flows, for stresses beyond what has already been reported in the literature. This dissertation is composed of three chapters. Chapter I is the literature survey and introductory materials. Chapter II contains the discussion and results for the theoretical/analytical/CFD part of the research. Finally, discussion and results for the experimental tests are presented in Chapter III

Femtosecond laser microfabricated devices for biophotonic applications

Femtosecond Laser DirectWriting has emerged as a key enabling technology for realising miniaturised biophotonic applications offering clear advantages over competing soft-lithography, ion-exchange and sol-gel based fabrication techniques. Waveguide writing and selective etching with three-dimensional design flexibility allows the development of innovative and unprecedented optofluidic architectures using this technology. The work embodied in this thesis focuses on utilising the advantages offered by direct laser writing in fabricating integrated miniaturised devices tailored for biological analysis. The first application presented customised the selective etching phenomenon in fused silica by tailoring the femtosecond pulse properties during the writing process. A device with an embedded network of microchannels with a significant difference in aspect-ratio was fabricated, which was subsequently applied in achieving the high-throughput label-free sorting of mammalian cells based on cytoskeletal deformability. Analysis on the device output cell population revealed minimal effect of the device on cell viability. The second application incorporated an embedded microchannel in fused silica with a monolithically integrated near-infrared optical waveguide. This optofluidic device implemented the thermally sensitive emission spectrum of semiconductor nanocrystals in undertaking remote thermometry of the localised microchannel environment illuminated by the waveguide. Aspects relating to changing the wavelength of illumination from the waveguide were analysed. The effect of incorporating carbon nanotubes as efficient heaters within the microchannel was investigated. Spatio-thermal imaging of the microchannel illuminated by the waveguide revealed the thermal effects to extend over distances appreciably longer than the waveguide cross-section. On the material side of direct laser writing, ultra-high selective etching is demonstrated in the well-known laser crystal Nd:YAG. This work presents Nd:YAG as a material with the potential to develop next-generation optofluidic devices

Multiscale Modeling of Biological Flow using Lattice Boltzmann Method

In this dissertation, we have developed a fluid-structure interaction code specifically designed to simulate soft microparticle deformation in biological flow. We have used this tool for two different applications. First, we study red blood cell deformation under shear flow to evaluate stress distribution on membrane and subsequently pore formation on RBC membrane. Second, we utilized this code to show a proof of concept for an idea where we can separate soft particles based on their biophysical properties. In the following, these applications are discussed in more details.Under high shear rates, pores form on RBC membrane through which hemoglobin leaks out and increases free hemoglobin content of plasma leading to hemolysis. We hypothesize that local flow dynamics such as flow rate and shear stress determines blood cell damage. In this dissertation, a novel model is presented to study red blood cell (RBC) hemolysis at cellular level. The goal of the proposed work is to establish multiscale computational techniques to predict the blood cell dynamics and damage in complex flow conditions, i.e., blood-wetting biomedical devices. The cell membrane damage model will be coupled with local fluid flow to study cell deformation and rupture and a generalized cellular level blood cell damage model will be developed based on these simulations. By coupling Lattice Boltzmann and spring connected network models through immersed boundary method, we estimate hemolysis of a single red blood cell under various shear rates. First, we use adaptive meshing to find local strain distribution and critical sites on RBC membrane, then we apply underlying molecular dynamic simulations to evaluate damage. Our approach is comprised of three sub-models: defining criteria of pore formation, calculating pore size, and measuring Hb diffusive flux out of pores. Our damage model uses information of different scales to predict cellular level hemolysis. Results are compared with experimental studies and other models in literature. The developed cellular damage model can be used as a predictive tool for hydrodynamic and hematologic design optimization of blood-wetting medical devices.Isolating cells of interest from a heterogeneous mixture has been of critical importance in biological studies and clinical applications. In this dissertation, we have proposed to use ciliary system in microfluidic devices to isolate target subpopulation of soft particles based on their biophysical properties. In this model, the bottom of microchannel is covered with an equally spaced cilia array which can be magnetically actuated. A series of simulations are performed to study cilia-particle interaction and isolation dynamic. It is shown that these elastic hair-like filaments can influence particle’s trajectories differently depending on their biophysical properties. This modeling study also uses immersed boundary (IB) method coupled with lattice Boltzmann method. Soft particles are simulated by connected network of nonlinear springs. Moreover, cilia is modeled by point-particle scheme. It is demonstrated that active ciliary system is able to continuously and non-destructively sort cells based on their size, shape and stiffness. Ultimately, a design map for fabrication of a programmable microfluidic device capable of isolating various subpopulation of cells is developed. This biocompatible, label-free design can separate cells/soft microparticles with high throughput which can greatly complement existing separation technologies

WIDE-RANGE COMPRESSION FORCES TO INVESTIGATE SINGLE-CELL IN-FLOW MOTIONS, MECHANOBIOLOGICAL RESPONSES AND INTRACELLULAR DELIVERY

The aim of the PhD work is to create a new microfluidic approach to finely tune applied in-flow forces in order to explore controlled single-cell deformation. In fact, we propose a microfluidic device based on compression forces arising from a viscoelastic fluid solution that firstly align cells and then deform them. By simply changing the rheological properties and the imposed fluid-flow conditions, our approach represents an easy-to-use and versatile tool to collect a comprehensive mapping of single-cell properties, investigating both biophysical and biomechanical characteristics. In a wide-range of applied compression, we observe how different degrees of deformation lead to cell-specific deformation-dependent in-flow dynamics, which correlate the classical deformation parameters (e.g. cell aspect-ratio), with dynamic quantities (e.g. revolution time of rotation during in-flow motion). Thus, a precise in-flow label-free cell phenotyping is achieved allowing the distinction of different cell classes. The observation of different degrees of deformation corresponding to variable compression, lead us to interrogate the inner cell structures possibly involved into the mechanical responses. We demonstrate that re-organization phenomena of actin cortex and microtubules as well as of nuclear envelope and chromatin content, occur. Also in this case, cell-specific responses are collected, allowing us to distinguish healthy from pathological cells depending on the structural mechanical reaction. Furthermore, by playing with the high levels of compression, we show preliminary results about the possibility to induce a nanoparticle intracellular delivery process by escaping physiological endocytosis. In fact, cells result to be able to incorporate nanoparticles into the cytoplasm, without involving a vesicle formation for the entry. These outcome open up new interesting scenarios about the possibility to use the microfluidic device as a platform for cell phenotyping and intracellular delivery, properly engineered for both diagnostic and therapeutic purposes

Simultaneous real-time viscoelasticity, mass and cell cycle monitoring for single adherent cancer cells

Cancer is a complex disease caused by the combined effects of genetic and environmental factors. Evidently, there exists a correlation between the surrounding environment of a cell, its biophysical properties and health. Information gained from biomechanics has led to an improved understanding of the way diseases evolve and their progression cycle, providing methods targeted towards curing these diseases. Countless studies have been carried out on the mechanisms underlying cell cycle progression. More particularly, these studies on the mechanics of individual cells have pointed to their coordination, which helps us understand cellular metabolic and physiological process better. Development of more precise, versatile and reliable measurement tools and techniques will provide a greater understanding of cellular behavior and biophysical properties. Micromechanical systems (MEMS) technology can provide these tools – for analyzing single cells and providing important and useful information of their biophysical properties. In modern research, the ability to reliably investigate and understand these cellular properties requires measurement devices that provide high sensitivity, high throughput, and adaptability to include multiple on-chip functionalities. Many MEMS-based resonant sensors have been extensively studied and used as biological and chemical sensors. However, previous works have shown that there are several technology limitations that inhibit application of various MEMS-sensors to mechanical measurement and analysis, including insufficient cell capture efficiency, media perfusion for long term growth, cell adhesion and cell movement/spreading and cell-sensor modelling. Cellular mechanics and viscoelastic properties are known to play a role in biological processes such as cell growth, stem cell differentiation, cell crawling, wound healing, protein regulation, cell malignancy and even apoptosis (programmed cell death). Thus, an accurate measurement of stiffness and growth is fundamental to understanding cellular proliferation in cancer. Capturing these biophysical properties of cancer cells over the duration of their growth cycle through MEMS devices can help provide a better insight into the mechanics of the metastasis of cancer cells. Meanwhile, many MEMS sensing devices still require further development and characterization to reliably investigate long-term cell behaviors. This dissertation focuses on characterization of our MEMS resonant sensors to address current challenges in the measurement of long-term biophysical behaviors of cells across its cell cycle. The amplitude and frequency of MEMS resonant pedestal sensors were used in conjunction with a vibration induced and optically-sensed phase shift of target light incident on an adhering sample to extract the loss tangent - a measure of the relative viscoelasticity of soft materials. This observed phase shift, combined with a representative two-degree-of-freedom Kelvin-Voigt model, is used to simultaneously obtain the elasticity (stiffness), viscosity and mass associated with individual adherent cancer cells. The research is unique as it decouples the heterogeneity of individual cells in our population and further refines our viscoelastic solution space. This novel development enables long-term simultaneous measurement of changes in stiffness and mass of normal and cancerous cells over time. This is the first investigation of the time-varying simultaneous measurement of viscoelasticity and mass for individual adherent cells using our MEMS resonant sensors

Microfluidics for the detection of Cryptosporidium

This thesis details the development of microfluidics for the label-free sorting and/ or identification of waterborne pathogens which are commonly detected in contaminated drinking-water supplies using the United States Environmental Protection Agency method 1623.1 (USEPA 1623.1). This method recovers and detects pathogens of the Cryptosporidium and Giardia species, which can cause human gastroenteritis upon ingestion. USEPA 1623.1 is employed universally in developed regions (e.g., Europe, North America, Australia, New Zealand). Specifically, this thesis describes microfluidic systems that were developed with the objective of rapidly discriminating viable (i.e., intact and apparently infectious), humanpathogenic Cryptosporidium oocysts from non-viable, human-pathogenic oocysts and/ or species which are considered non-hazardous to human health. Such a system would reduce the overall detection time and allow a more accurate assessment of the risk posed to human health. A microfluidic setup incorporating dielectrophoresis was designed and employed for the viability-based sorting and enumeration of a human-pathogenic species of Cryptosporidium. This device enabled the sorting of untreated (live) and heat-inactivated (non-viable) sub-populations of the human pathogenic Cryptosporidium parvum with over 80% efficiency. Existing Microfluidic Impedance Cytometry (MIC) and Microfluidic-enabled Force Spectroscopy (MeFS) technologies were adapted for the enumeration, detection and viability determination of human-pathogenic Cryptosporidium oocysts, plus the discrimination of Cryptosporidium species which pose a major risk to human health from those which pose little to no risk. Using MIC, it was possible to discriminate untreated and heat-inactivated C. parvum with over 90% certainty. Furthermore, populations of C. parvum, Cryptosporidium muris (low-risk, human pathogen) and Giardia lamblia (also recovered using USEPA 1623.1) were discriminated from one another with over 90% certainty. Using MeFS, it was possible to differentiate temperature-inactivated (either by freeze- or heat-treatment) C. parvum from live C. parvum with a minimum of 78% efficiency. Finally, the high-risk, human pathogenic C. parvum was discriminated from C. muris with over 85% efficiency. Upon further validation, i.e., the analysis of other Cryptosporidium species and of oocysts which have been inactivated by other means (e.g., ozonation, ultraviolet radiaton), it is hoped that water utilities will employ such method(s) to more accurately characterise the human risk associated with contaminated supplies