Modelling and control of dynamic platelet aggregation under disturbed blood flow

Diagnosis of platelet function is fundamental for identifying blood disorders of patients, assessing the impact of antiplatelet agents, and enabling the appropriate titration of individual antithrombotic treatments. Following the advancement of new technologies such as microfluidic devices and the use of control engineering methods, new devices have the potential to offer new opportunities in point-of-care diagnosis of platelet function. Such new devices may have significant utility in the development of more tailored antiplatelet therapies. The aim of this thesis is to investigate modelling and control systems which support the study of the dynamic relationship between newly discovered mechanisms of platelet aggregation and disturbed blood flow, using state-of-the-art micro-engineered technologies. In order to observe the dynamics of platelet aggregation under disturbed blood flow, blood perfusion experiments carried out on a device mimicking a scenario of severe vessel narrowing are presented. The resulting biological response, that is the aggregation of platelets, is monitored in real-time and synthesised through novel measures developed using image processing techniques. A mechanistic model identifying four distinct stages observed in the formation of the aggregate is formulated, describing the nonlinear relationship between blood flow dynamics and platelet aggregation. The observed effect of disturbed blood flow on the aggregation of platelets is then modelled mathematically employing System Identification methods. A detailed account of a novel approach for the generation of experimental data is presented, as well as the formulation of tailored mathematical model structures and the calculation of their parameters using collected data. The proposed models replicate experimental results with low variation, and the reduced number of model parameters is suggested as a novel systematic measure of platelet aggregation dynamics in the presence of blood flow disturbances. In order to stabilise, optimise, and automate the measurement of platelet function in response to disturbed blood flow, custom-made control algorithms based on principles of Sliding Mode Control and Pulse-Width Modulation are developed. Moreover, the control algorithms are developed to handle the large variability of the aggregation responses from blood types with platelet hyper- and hypo-function. Simulation results illustrate the robustness of the control algorithms in the presence of time-varying nonlinearities and model uncertainty, and indicate the possibility to regulate the extent of aggregation in the device through modulation of the blood flow rate in the microchannel. The main contribution of this thesis is the development of dynamic models and control systems that allow a systematic measurement of platelet function in response to rapid changes in the blood flow (shear rate micro-gradients), in a microfluidics device containing a scenario of disturbed blood flow. Analysis of the platelet aggregation dynamics revealed that although the aggregate growth appears to be constant at times, measuring its mean fluorescence intensity indicates an increase in the dynamics of platelet density. This densification process appears fundamental for the development of an amplification phase in the aggregation response. The proposed mathematical models and control algorithms facilitate the systematic measurement of platelet function in vitro, pioneering the development of a novel framework for automated blood disorder diagnosis

Simplified fabrication of complex multilayer microfluidics: enabling sophisticated lab-on-a-chip and point-of-care platforms

Complex multilayer microfluidics have generated a lot of interest in recent years. Early research introduced elastomer microvalves and postulated they would bring about a revolution for microfluidic systems, similar in scale to introduction of the transistor for electronic systems. In the following years, many researchers have been active in the use of complex multilayer microfluidic systems, with numerous high impact research outcomes using these systems as precise and active control components, providing fluidic isolation, switching or fluidic actuation, and allowing unprecedented sophistication and precise control and automation of experimental conditions. While application of complex multilayer microfluidic platforms has been demonstrated in numerous research settings, there is little evidence that the technology has become ubiquitously accepted, with a lack of evidence for point-of-care application, or widespread acceptance within the research community. While the advantages that the technology offers have been well documented, the field seems to have failed to gain traction, or facilitate the revolution that was predicted on its introduction. There are various possible explanations for this lack of acceptance, as with any technology, there are caveats to the application of complex multilayer microfluidic systems, however given the broad range of demonstrated applications, it is unlikely that the bottleneck in their application is related to a fundamental application related limitation. In contrast, fabrication technology utilised in realisation of complex multilayer microfluidic systems, has not advanced at the same rate to the multitude of application-based publications in the past decade. This thesis explores the hypothesis that one of the fundamental limiting factors in widespread application of complex multilayer microfluidic systems, is related to the challenges associated with fabrication of these systems. To explore this hypothesis, firstly, a new fabrication approach is introduced which aims to eliminate many of the challenges associated with traditional multilayer fabrication methods, this technique is demonstrated in a proof of concept capacity, fabricating common multilayer microfluidic structures and doing so with surprising ease. Having developed method with simpler fabrication, it is possible to explore whether overcoming the multilayer fabrication bottleneck would allow the advantages inherent to complex multilayer microfluidic systems to be applied to fields which would otherwise be considered prohibitively difficult, if reliant on traditional fabrication methods. This hypothesis is investigated through harnessing the new, simplified fabrication technique to advance point-of-care photonic biosensor research through short term collaborative engagements.  It is found that the use of modular building blocks and the simple, rapid fabrication enables sophisticated microfluidic chip prototypes to be developed in a very short period of time achieving multiple iterations over a matter of weeks and even facilitating collaboration on these integrated platforms remotely. The outcomes of these short-term collaborations have produced publications automating the fluid handling of highly sensitive interferometric waveguide biosensors and environmental control for a single cell analysis platform utilising integrated plasmonic biosensors.       Having demonstrated that simplifying complex microfluidic fabrication can accelerate the development and deployment of these systems to enhance research platforms, the next step was to explore whether this simplified system could also lower the barrier to deployment in a clinical setting. The ability for the fluidic system to handle whole blood was chosen as a deliberately challenging target with great sensitivity to fluid dynamics and large variability in patient samples and environmental factors, requiring large number of replicate devices to determine statistical significance. Here the fabrication technique is applied to enable a study investigating the hemocompatibility of common multilayer control components, paving the way for point of care blood handling devices.  It is shown that not only can the technique be used to rapidly develop platforms that can be used with blood, but the same technique can produce even hundreds of replicates required for limited clinical trials, leading the collaborating clinicians to seriously consider these complex microfluidics for future point of care diagnostics. In Summary, it has been demonstrated that access to complex multilayer microfluidic systems without the fabrication overheads generally associated with these systems can allow their application to areas that would otherwise be prohibitively difficult. The fabrication method presented can allow rapid development, and rapid and reliable deployment to various research applications, while allowing the consistency and throughput required enabling large volume fabrication required for clinical investigations.  The fact that such a large advancement toward real world application within the scope of a single PhD is possible, supports the hypothesis that lowering the barrier to fabricating complex microfluidic devices has the potential to significantly increase their scope of application

Multiscale Modelling Of Platelet Aggregation

During clotting under flow, platelets bind and activate on collagen and release autocrinic factors such ADP and thromboxane, while tissue factor (TF) on the damaged wall leads to localized thrombin generation. Toward patient-specific simulation of thrombosis, a multiscale approach was developed to account for: platelet signaling (neural network trained by pairwise agonist scanning, PAS-NN), platelet positions (lattice kinetic Monte Carlo, LKMC), wall-generated thrombin and platelet-released ADP/thromboxane convection-diffusion (PDE), and flow over a growing clot (lattice Boltzmann). LKMC included shear-driven platelet aggregate restructuring. The PDEs for thrombin, ADP, and thromboxane were solved by finite element method using cell activation-driven adaptive triangular meshing. At all times, intracellular calcium was known for each platelet by PAS-NN in response to its unique exposure to local collagen, ADP, thromboxane, and thrombin. The model accurately predicted clot morphology and growth with time on collagen/TF surface as compared to microfluidic blood perfusion experiments. The model also predicted the complete occlusion of the blood channel under pressure relief settings. Prior to occlusion, intrathrombus concentrations reached 50 nM thrombin, ~1 μM thromboxane, and ~10 μM ADP, while the wall shear rate on the rough clot peaked at ~1000-2000 sec-1. Additionally, clotting on TF/collagen was accurately simulated for modulators of platelet cyclooxygenase-1, P2Y1, and IP-receptor. The model was then extended to a rectangular channel with symmetric Gaussian obstacles representative of a coronary artery with severe stenosis. The upgraded stenosis model was able to predict platelet deposition dynamics at the post-stenotic segment corresponding to development of artery thrombosis prior to severe myocardial infarction. The presence of stenosis conditions alters the hemodynamics of normal hemostasis, showing a different thrombus growth mechanism. The model was able to recreate the platelet aggregation process under the complex recirculating flow features and make reasonable prediction on the clot morphology with flow separation. The model also detected recirculating transport dynamics for diffusible species in response to vortex features, posing interesting questions on the interplay between biological signaling and prevailing hemodynamics. In future work, the model will be extended to clot growth with a patient cardio-vasculature under pulsatile flow conditions

Microfluidic-based analysis of 3D cell migration under different biophysical and chemical gradients

Several mechanochemical factors are involved in cell migration, fundamental to establish and maintain the proper organization of multicellular organisms. The alteration of migratory patterns of cells could be related to the development of several pathologies. Focusing this work on tissue regeneration, more specifically wound healing, bone regeneration and blood vessel formation, the main aim of this work is to advance in the understanding of how chemical or physical factors present in the cell niche can regulate the cell movement (fibroblasts, osteoblasts and endothelial cells respectively). In an effort to understand what mechanisms are involved, it has been seen that both the extracellular matrix surrounding tissue cells and the biomolecules present in the cellular microenvironment can affect the behavior of cells [1–3]. In turn, interstitial fluid flow, defined as the convective transport of liquids through the extracellular matrix of tissue, is also capable of altering the morphology and cellular movement. Similarly, biomolecules, such as growth factors or drugs, modify the migration pattern. The main mechanisms studied throughout this thesis have been chemotaxis, durotaxis and rheotaxis. The biological processes for which these analyses have been performed were angiogenesis, wound healing and bone regeneration respectively. For the in vitro study of these variables, and making use of novel microfabrication techniques such as microfluidics, new platforms for 3D cell culture have been developed [4,5]. The microfluidic chips used allow replication of the ex vivo tissue microenvironment through the use of hydrogels and the generation of concentration gradients and controlled fluid flows. It should be noted that the versatility of this technology has allowed us to simultaneously study several microenvironmental factors, such as chemical gradients and matrix stiffness applied to fibroblast culture to understand its behavior in the wound area. In addition, these types of systems allow the visualization and/or monitoring of the cellular response in real time, being able to quantify the cellular migration. For the application of fluid flow, a novel system was designed to avoid the rupture of the hydrogels, allowing to obtain a stable interstitial flow inside the chip chamber. Throughout this thesis, it has been seen that there are several factors involved in 3D cell migration. Not only variables such as the chemical gradient (studied in endothelial cells and fibroblasts) or the rigidity of the extracellular matrix (analyzed in fibroblasts and osteoblasts) affect cells [6,7]. The architecture of the matrix, more specifically the disposition of the fibers that conform this matrix, has been identified as playing an important role in cell migration, also altering the morphology of cells, in this case osteoblasts.[1] N. Movilla, C. Borau, C. Valero, J.M. García-Aznar, Degradation of extracellular matrix regulates osteoblast migration : a microfluidic-based study, Bone. 107 (2018) 10–17.[2] O. Moreno-Arotzena, C. Borau, N. Movilla, M. Vicente-Manzanares, J.M. García-Aznar, Fibroblast Migration in 3D is Controlled by Haptotaxis in a Non-muscle Myosin II-Dependent Manner, Ann. Biomed. Eng. (2015). doi:10.1007/s10439-015-1343-2.[3] O. Moreno-Arotzena, G. Mendoza, M. Cóndor, T. Rüberg, J.M. García-Aznar, Inducing chemotactic and haptotactic cues in microfluidic devices for three-dimensional in vitro assays, Biomicrofluidics. 64122 (2014). doi:10.1063/1.4903948.[4] W.A. Farahat, L.B. Wood, I.K. Zervantonakis, A. Schor, S. Ong, D. Neal, R.D. Kamm, H.H. Asada, Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures., PLoS One. 7 (2012) e37333. doi:10.1371/journal.pone.0037333.[5] Y. Shin, S. Han, J.S. Jeon, K. Yamamoto, I.K. Zervantonakis, R. Sudo, R.D. Kamm, S. Chung, Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels, Nat Protoc. 7 (2012) 1247–1259. doi:10.1038/nprot.2012.051.[6] C. Del Amo, C. Borau, R. Gutiérrez, J. Asín, J.M. García-Aznar, Quantification of angiogenic sprouting under different growth factors in a microfluidic platform, J. Biomech. 49 (2016) 1340–1346. doi:10.1016/j.jbiomech.2015.10.026.[7] C. Del Amo, C. Borau, N. Movilla, J. Asín, J.M. Garcia-Aznar, Quantifying 3D chemotaxis in microfluidic-based chips with step gradients of collagen hydrogel concentrations, Integr. Biol. (2017) 1–27. doi:10.1039/C7IB00022G.<br /

Innovative approaches to study basophil function in inflammation

Basophils are circulating granulocytes. They are very rare and represent less than 1% of peripheral blood leukocytes. Basophils connect the innate and adaptive immune responses by the secretion of a variety of immune-mediators involved in the pathogenesis of many inflammatory diseases mainly allergic reactions and autoimmune diseases. For many years it was difficult to study basophil function due to their rareness in peripheral blood which resulted in scant yields and purity when isolated. This thesis focuses on studies of the basophil function in two inflammation-driven diseases: chronic kidney disease (CKD) and allergy. The investigations have been done using newly developed microfluidic-based lab-on-chip technology and conventional immunological methods. In paper I, we investigated the impact of blood-membrane interaction on circulating basophils and neutrophils in hemodialysis patients (stage 5D), using high-flux and low-flux dialyzers. Passage through the low-flux dialyzer, as opposed to high-flux, induced a significant upregulation of CD63 on formyl-methyinoyl-leucyl-phenylalanine (fMLP) and anti-FcεRI antibody stimulated basophils. Furthermore, (fMLP) stimulated basophils significantly upregulated CD63, in patients compared to healthy controls. There were no significant differences in the expression of neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88), when comparing the two dialyzers, or when compared to healthy controls. In paper II, we analyzed the expression of activator markers on basophils related to two crucial functions (transmigration and degranulation) in CKD (stage 5D). The CD300a expression was significantly higher in patients following activation by fMLP and anti-FcɛRI-ab and the expression of the active epitope of CD11b was significantly higher in patients after lipopolysaccharide (LPS) activation. The CD62L expression was significantly downregulated in anti-FcɛRI activated basophils from healthy controls. In paper III, we developed a novel microfluidic immuno-affinity based basophil activation test (miBAT) assay. The microfluidic device is capable of isolating basophils directly from whole blood and we analyzed the regulation of CD203c and CD63 in anti-FcεRI activated basophils in healthy and allergic individuals. The microfluidic chip was able to capture basophils from whole blood with an efficiency of 65% and the CD63 expression detected via fluorescent microscope was significantly higher in activated basophils compared to non-activated basophils (negative control), as well as in allergic patients compared to healthy controls in microfluidic chip. The result was comparable to flow cytometry data. In paper IV we validated that the miBAT platform can be used for allergy diagnosis. CD63 expression on basophils activated with allergens was detected in microfluidic chip and flow cytometry. The activation was significantly higher compared to non-activated basophils in allergic patients. Basophils from non-allergic individuals did not respond to allergen activation. The microfluidic chip analysis was comparable with flow cytometry data. In conclusion, this thesis presents new insights on the role of basophils in the inflammatory responses, mainly related to innate immune responses in CKD patients. Moreover, we introduced a novel microfluidics based method (miBAT) to quantify basophil activation in allergic patients. The method has great potential to be used as a point of care for allergy diagnosis

Microfluidic devices for cell cultivation and proliferation

Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices