Desarrollo de dispositivo microfluídico para la visualización del fenómeno de la extravasación leucocitaria

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA INFORMÁTICA Grado en Ingeniería de la Salud Mención en Ingeniería Biomédica Desarrollo de dispositivo microfluídico para la visualización del fenómeno de la extravasación leucocitaria Development of flow focusing device for the visualization of leukocyte rolling adhesion Realizado por Pablo Morales Navarrete Tutorizado por José Antonio Andrades Gómez* Co-tutorizado por Joaquín Ortega Casanova** Departamento * Biología Celular, Genética y Fisiología ** Ingeniería Mecánica, Térmica y de Fluidos UNIVERSIDAD DE MÁLAGA MÁLAGA, Junio 2018 Fecha defensa: El Secretario del Tribunal Resumen La microfluídica es un área de la microtecnología basada en chips de PDMS que está siendo utilizada cada vez más en multitud de aplicaciones. Una de estas aplicaciones es la investigación biomédica. La microfluídica o “Lab on a Chip” se ha convertido en una manera de realizar experimentos biomédicos y diagnósticos de una manera barata, rápida y eficaz. Cuando se realizan estudios sobre la extravasación leucocitaria utilizando chips microfluídicos, podemos observar la inconsistencia en la trayectoria de rodadura de los leucocitos debido a un flujo laminar. En este trabajo de fin de grado presentamos un método para centrar la interfaz de células en el centro de canal microfluídico. Cuando las células circulan por los sistemas microfluídicos, las células tienden a circular de manera aleatoria por los canales. Por tanto, con el sistema propuesto en este trabajo, dichas células serán redirigidas a la porción central del canal con el fin de recrear el fenómeno de rodadura presente en nuestro sistema circulatorio y así obtener información más detallada. Los resultados de este trabajo muestran la utilidad y la versatilidad de este dispositivo para experimentos relacionados. Palabras clave: Microfluídica, Lab on a Chip, Point of Care, Extravasación Leucocitaria, Litografía blanda, Inmunología, Modelado Multifísico, Método de elementos finitos. Abstract Microfluidics is an area of technology based on PDMS chips that is being increasingly used for many applications. One of said applications is biomedical research. Microfluidics or Lab on a chip poses as a great way of cutting costs, time, space and overall improving efficiency in medical diagnosis and biomedical experiments. When studying the rolling cell adhesion behaviour of leukocytes on microfluidic devices, we can observe the inconsistency in the manner cells roll due to laminar flow. In this final year dissertation we present a method to focus the flow of leukocytes on a rectangular microfluidic channel for rolling cell adhesion assays. When flowing in microfluidic channels, cells tend to circulate in a random manner, therefore with the system proposed, said cells will circulate in the central area of the channel, in order to optimally recreate the rolling cell adhesion phenomenon present in our circulatory system and achieve precise information regarding this cellular behaviour. The results of this work show the viability of the system and the versatility it may have with other related experiments. Keywords: Microfluidics, Lab on a Chip, Point of Care, Rolling Cell Adhesion, Hydrodynamic flow focusing, Soft Lithography, Immunology, Multiphysics Modelling, Finite Element Method. Acknowledgements To my parents for making the trip to Japan, where this dissertation was developed, possible, for never losing faith in me and for always supporting my decisions. To my sister, for encouraging me to try my hardest in every step along the way. To the Professors José Antonio Andrades Gómez and Joaquín Ortega Casanova, for getting involved in this project and for their help developing the content of this dissertation. To Masahiro Motosuke, for allowing me to work at his lab, where I learnt so much. To Sayaka Nomura and Takuya Ichikawa, for being great colleagues and helping me with this research. Contents Chapter 1: Introduction ..................................................................................... 13 1.1 Background and Motivation ........................................................................ 13 1.2 Objectives ................................................................................................... 14 1.3 Content Outline .......................................................................................... 15 Chapter 2: The Immune System ....................................................................... 17 2.1 The Immune System .................................................................................. 17 2.1.1 Haematopoiesis ................................................................................... 18 2.1.2 Immune System Organs ...................................................................... 20 2.2 Leukocyte Activation and Migration ............................................................ 22 2.2.1 Cell Adhesion Molecules ...................................................................... 22 2.2.2 Leukocyte Extravasation ...................................................................... 24 2.2.3 Leukocyte Adhesion Deficiency ........................................................... 25 Chapter 3: Microfluidics .................................................................................... 27 3.1 What is Microfluidics? ................................................................................. 27 3.2 Properties of Fluidics .................................................................................. 28 3.3 Shear Stress ............................................................................................... 29 3.4 Types of Fluids ........................................................................................... 29 3.5 Types of Flows ........................................................................................... 30 3.5.1 Laminar flow ........................................................................................ 30 3.6 Hydrodynamic Focusing ............................................................................. 32 3.6.1 Conservation of Energy ....................................................................... 33 3.7 Lab on a Chip ............................................................................................. 34 3.7.1 Organ on a chip ................................................................................... 35 17 Chapter 2: The Immune System 2.1 The Immune System Like all mammals, humans possess a specialized system destined for the protection and the defence of our body; we refer to this mechanism as the Immune System. This complex is composed by very specialized cells that function inside very well organized anatomical structures. Therefore to understand the importance of these specialized cells we must study them in conjunction with the tissues and organs in which these cells interact with. If we go back in history, evolutionary speaking, our immune system was developed in order to combat the infections caused by viruses, bacteria, protozoa, fungus and helminths. These pathogens can be responsible for many infections both intracellular and extracellular, for which the response must be different. The immune system has developed a variety of appropriate defence mechanisms in order to fight off each of the different pathogens that infect us. In order to eradicate a pathogen that has infected its host, the immune system must first detect its presence and then take the necessary steps to destroy it. For the destruction of pathogens, the human body has developed diverse immune mechanisms which are both innate and adaptive; the difference rests in the fact that they use different methods to detect threats and infections. In order for the innate response to work, a series of molecular patterns that are present in same group pathogens must be detected. All members of a same animal species are born with an innate and immediate capacity of detecting and destroying numerous pathogens which we have not been in contact before. The innate immune response is very effective; however, it cannot protect us from all threats. The majority of pathogenic agents are single celled organisms that divide very quickly; therefore, they evolve at higher rates and evade the body’s innate response. In order to avoid this, vertebrates have developed a pathogen reconnaissance strategy which enables the detection of foreign agents that have never been encountered before. This defence mechanism is referred to as 18 the adaptive immune system. Lymphocytes are the cells which will be responsible for this adaptive immunity. Immune cells are the key players in our immune system, enabling detection and destruction of foreign pathogens. There are many types of cells, each with different functions and characteristics. However, their origin is similar. Immune cells, commonly referred to as white blood cells or leukocytes, originate from the bone marrow, a semi-solid tissue found in the trabecular bone tissue. In this myeloid tissue, leukocyte precursors are created. These “parent” cells will then differentiate through a process called haematopoiesis to create white blood cells. There are many types of cells in our immune system as seen in Figure 2.1, all with different characteristics but same objective: to destroy foreign pathogens. Figure 2.1: Leukocyte development, from progenitor to mature differentiated cells (Mikael Häggström 2009). 2.1.1 Haematopoiesis All blood cells arise from a type of cell called the hematopoietic stem cell (HSC). Stem cells are undifferentiated cells which have the unique capacity of differentiating into many other types of cells (see Figure 2.1). Also, they possess many different characteristics which enable them to self-renew themselves indefinitely and regulate their population by cell divisions (Kuby, et al. 1992). In humans, haematopoiesis begins in the embryonic yolk sac during 19 the first two weeks of development, by the third month, hematopoietic stem cells have migrated to the foetal liver; after that, the differentiation of HSCs in the bone marrow becomes the mayor factor in haematopoiesis. By birth there is little or no haematopoiesis in the liver and spleen (Kuby, et al. 1992, 23). The first step to haematopoiesis consists in the differentiation of a multipotent stem cell, creating either a myeloid progenitor cell or a lymphoid progenitor cell. These cells have now lost the capacity of self-renewal and are therefore destined to give rise to cells of its particular cell lineage. Lymphoid progenitors will then differentiate into Natural Killers (NK) cells, T lymphocytes and B lymphocytes. Analogously, myeloid progenitors will give rise to erythrocytes (red blood cells, RBCs), platelets, basophils, neutrophils and other immune cells commonly referred to as white blood cells. Each of these cells are created and induced under different circumstances, different signals and microenvironments will promote growth and differentiation of these cells. These microenvironments are occasioned by scaffold like structures created by stromal cells and by growth factors that arrive to their target cells by diffusion. To summarise, it can be said that the different cells that descend from the HSCs will come to be due to the different interactions of the different factors related with its growth and differentiation. Haematopoiesis is regulated at genetic level, the development of the stem cells into the different cell types requires the expression of different sets of lineagedetermining and lineage-specific genes at appropriate times and in correct order (Kuby, et al. 1992, 24). Therefore, we can say with certainty that the proteins expressed by these genes are of utmost importance and will determine the outcome of cellular differentiation. Much remains to be learnt in this field; however, there are some discoveries that have provided a very useful insight into understanding how this process works. One of these advances in the immune-genetic research led to the discovery of Leukocyte Adhesion Deficiency (LAD), a heritable genetic disease that we will discuss in the following pages due to the close relationship with our research. 20 2.1.2 Immune System Organs Like many other systems, the Immune system has distinct organs and tissues with specialized functions, destined to contribute to the development of immune responses. Immunologists classify these organs by function, dividing them into primary and secondary lymphoid organs. The thymus and bone marrow belong to the central o primary lymphoid organs, whereas the secondary lymphoid organs are comprised of lymph nodes, the spleen and other tissues. Maturation of lymphocytes takes place in the primary lymphoid organs, whereas in the previously mentioned secondary lymphoid organs, the matured lymphocytes interact with antigens. These lymphocytes will then circulate around the body through the circulatory and lymphatic system (Figure 2.2), detecting and fighting off possible pathogens. Primary lymphoid Organs Lymphocytes generated in haematopoiesis are yet to be matured and become committed to antigenic specificity. Only after the cell has matured within a primary organ, is the cell immunocompetent. T-lymphocytes are called as such due to the fact that they mature in the thymus, a primary lymphoid organ. The thymus is a specialized organ formed by specialized cells called thymocytes separated by connective tissue. This organ is the site for tlymphocyte development and maturation. The thymus is flat and bi-lobed, being located above the heart (see Figure 2.2), it is very complex and of utmost importance. Its role in the immune system can be studied in mice by examining the effects of neonatal thymectomy. Thymectomized mice show a dramatic decrease in circulating lymphocytes of the T-cell lineage and an absence of cellmediated immunity (Kuby, et al. 1992, 41). 21 Figure 2.2: Schematic of the Lymphatic System (Blaus 2013). The other primary lymphoid organ is the bone marrow. The bone marrow is a sponge-like tissue situated inside of bones; most of the cells of our immune system are produced and developed here and, once they develop and mature, they migrate to the bloodstream to circulate the body. At birth, many bones contain red bone marrow, which actively builds defence cells. During the course of life, more and more of this red bone marrow turns into fat tissue. Eventually, all the red bone marrow transforms, except in a few bones, such as the ribs, the breast bone and the pelvic bone (Schmidt, Lang and Heckmann 2010). Lymphocytes that originate from this organ are referred to as B-lymphocytes. Lymphatic System The circulatory system is a pressurised system, because of this as blood circulates the body, the plasma seeps through the capillary’s endothelium, this fluid is called interstitial fluid and bathes all the surrounding cells. Due to the high volumes of this fluid being permeated through the capillary walls, a lymphatic system needs to collect it to prevent swelling of the nearby areas (edema) which would slowly become life threatening. This lymphatic system forms a network of lymphatic vessels which collect all the lymph (plasma) and then drains it back into the blood. When a foreign antigen gains entrance to the 22 tissues, it is picked up by the lymphatic system and is carried to the lymph nodes where it is trapped and treated accordingly. 2.2 Leukocyte Activation and Migration When we experience an infection from an external agent, our bodies react in many different ways to signal our immune system that the integrity of a tissue has been compromised. An inflammatory response is generated as a response to a local injury or trauma, which the immune system detects as a distress signal. This inflammation is a complex response that involves several immune system cells and numerous mediators. Once the innate immune system reaches the area of infection, it mounts the initial attack against the foreign pathogen. However this initial defence may not suffice and lymphocytes are recruited and become activated in response to antigens with the goal of helping in the battle against foreign pathogens. This process is very complex therefore we shall study it in more detail in the following pages. 2.2.1 Cell Adhesion Molecules The human body is made out of many cells and tissues joint together forming bigger and more functional structures. All these cells are bonded together by what we call cell adhesion molecules (CAMs), and it is these same molecules that help the leukocytes and the immune system cells interact with other tissues. Leukocytes are circulating around the body waiting for an infection, ready to attack foreign pathogens. Therefore, vascular endothelium serves as an important entry point for these leukocytes to arrive at the targeted tissue. Due to the high pressure in the circulatory system, cells are under high mechanical stresses and find it impossible to attach themselves to the vascular endothelium and trespass the vascular barrier. Therefore, they need some sort of aid to migrate from the blood to the lymphoid organs or surrounding tissues. This process is called extravasation and it happens partly due to the presence of leukocyte specific CAMs that will bind white blood cells to the vascular walls. 23 Endothelial cells express leukocyte adhesion molecules, some are cell specific and others are response specific, e.g. cytokine produced during an inflammatory process. All in all, we can say that regardless the origin of the response or specificity; these CAMs are paramount in the migration and movement of white blood cells across the body. In addition to their role in leukocyte adhesion to vascular endothelial cells, CAMs on leukocytes serve to increase the strength of the functional interactions between cells of the immune system. Various adhesion molecules have been shown to contribute to the interactions between TH cells and APCs, TH and B cells, and CTLs and target cells (Stein and Nombela-Arrieta 2005). Most of these CAMs belong to four families of adhesion proteins: selectin 1 , mucin-like, integrin and the immunoglobulin family. Selectins The selectin family of cell adhesion protein are a group of glycoproteins that has a specific domain that enables biding with other molecules due to the affinity of this lectin-like domain to specific carbohydrates. The selectin protein family groups three molecules: L-, Eand P-selectin. Eand P-selectin are mostly found on vascular endothelium whereas L-selectin is expressed on the surface of most leukocytes. E-selectin is a very interesting protein that needs synthesis of new proteins for it to express itself on the vascular walls. These new proteins commonly appear after the stimulation of inflammatory cytokines. Selectins are the initial bonders of leukocytes to vascular endothelium; therefore they play a prominent role in leukocyte extravasation. Chemokines are another family of proteins relevant in leukocyte extravasation, these molecules are responsible for certain CAMs activation, e.g. E-selectin. Consequently they are a mayor regulator in lymphocyte and leukocyte traffic (Stein and Nombela-Arrieta 2005). Chemokines are typically induced due to an inflammation process, commonly as a response for tissue infection. These molecules are of high importance because they essentially activate E-selectin 1 Due to the importance of selectin family adhesion protein in our research we shall only focus on this protein for the time being. 24 molecules. In the event of chemokines not being expressed, leukocytes would not be correctly recruited due to E-selectin not being activated (Collins, et al. 1991). 2.2.2 Leukocyte Extravasation Lymphocyte and Leukocyte extravasation as a whole is a multistep process that occurs in blood vessels and regulates the traffic of white blood cells in the body. This process is normally initiated by an inflammatory response due to the infection of a tissue by a foreign agent. Once this inflammatory response develops, cytokines and other inflammatory mediators act on the local blood vessel walls, inducing the expression on endothelial CAMs. Once the site is activated, leukocytes can then proceed to extravasate to the site of infection. For this to happen, leukocytes will have to strongly attach themselves to the vessel wall to avoid being swept away by the highly pressurised flow of blood. Leukocyte extravasation can be divided into four steps: leukocyte rolling, activation, arrest and migration. We shall focus on the rolling phase of the cell’s movement. Rolling is mediated by selectins; this movement is comprised of a loosely attached bond that will bind the cell to the endothelium by a low-affinity selectin-surface carbohydrate interaction. As we’ve mentioned previously, Eselectin is activated by chemokines, therefore once they are properly expressed in the endothelium they can adhere to carbohydrates expressed in leukocyte membranes. Being such a weak adhesion, leukocytes will only bond briefly and soon detach itself due to the shear force of the circulating blood. Once this happens it will rapidly attach itself to another endothelial cell. This tumbling of the cell is repeated over and over, creating a rolling motion (see Figure 2.3). This process is fairly slow; therefore it slows down the speed of the cell just enough to allow interactions between chemokines present in vessel wall and the receptors on the leukocyte surface to interact and generate a tighter bond that will completely adhere the cell to the wall. Once the movement of the leukocyte has been stopped by this tight adhesion, the cell will squeeze through two neighbouring endothelial cells and traverse the endothelial barrier and enter the infected tissue (Anderson and Anderson 1976). 25 Figure 2.3: Steps in Leukocyte (Neutrophil) extravasation (H. Shaz, R. Stowell y D. Hillyer 2011). 2.2.3 Leukocyte Adhesion Deficiency Leukocyte Adhesion Deficiency or LAD is a genetic disorder that is characterized by a poor leukocyte adhesion to the capillary walls, making it impossible for the immune system cells to adhere properly and therefore not extravasate to the infected tissue. This disease results in the immunodeficiency of the patient and concludes in the patient having recurrent infections. LAD affected patients will suffer from bacterial infections from a very early age and can become a life threatening condition due to the infant’s inability to combat foreign pathogens. This inability to combat bacteria and other agents is due to the non-existing leukocyte migration. Besides the notable absence of a defence system, LAD patients will also have high counts of leukocytes in the blood and will not be able to form pus (Abbas, Lichtmann and Pillai 2012). As we speak there is no cure for this disease, the only current curative therapy is intensive antibiotherapy or haemopoietic stem cell transplant. I wanted to include this section in this chapter because although there is no cure, the prototype and experiment that we propose in this work could be used as a tool to conduct experiments and assays to design a novel drug and, eventually, test its suitability for curing this disease. 32 3.2, we can clearly see this behaviour in a straight channel. In this figure, the flow in the central section has a maximum velocity as opposed to the lateral sections where a substantial decrease in velocity is observed. Figure 3.2: Parabolic profile in a fluid flow simulation (COMSOL). This behaviour disrupts the way in which we calculate the speed of the flow. However through mathematical analysis we can extrapolate the equation that governs the flow. 𝑢(𝑟)=2 𝑉𝑎𝑣𝑔 (1 −𝑟2 𝑅2) , (3.6) 𝑉𝑎𝑣𝑔= −𝑅2 8𝜇 (𝑑𝑃 𝑑𝑥) , (3.7) where 𝑟 equals the distance from the middle point and 𝑅 is the radius of the pipe the fluid flows through. 3.6 Hydrodynamic Focusing Hydrodynamic focusing is also called flow focusing or sheath flow, however many names it may have, they all have one underlining function: to force a fluid or cell to pass through a specified section. This technique has been used for many years in the realm of fluid dynamics to control the way we interact with the different phases of a fluid in a closed environment. Hydrodynamic focusing has 33 been widely used in many applications as in flow cytometry (Shuler, Aris and Tsuchiya 1972). Figure 3.3: Flow focusing structure. A flow focusing device is mainly composed of three inlets and one outlet as seen in Figure 3.3. The flow focusing method has the goal of placing a stream of fluid or solid phase materials in the central portion of the channel by the use of the auxiliary side channels. When the central channel which flows at a lower pressure, arrives at the junction point, the fluid that comes in from the auxiliary inlets pushes the central interface into a determined section, which will be fixed by the pressure at which the focusing fluid is flowing at. This phenomenon and tool widely used in microfluidics is due to the conservation of Energy and Mass law. 3.6.1 Conservation of Energy Energy within a fluidic system remains constant in such a way that energy acting on the system is continuously changed in to other forms such as work. For fluids the equation that governs this principle is very complicated, due to fact that is taken in consideration several forms of energy acting and dissipating out of a defined body. In its simplest form it can be described as: ∆𝐸=𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 . (3.8) This has certain ramification such as the conservation of volumetric flow rate. 𝑄(𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒)=𝑣· 𝐴 , (3.9) where 𝑣 is the velocity of the flow in 𝑚/𝑠 and 𝐴 is the cross sectional area. 34 This formula explains that in the presence of a bottleneck, the cross sectional area will decrease and therefore increase the speed at which the flow is flowing. 3.7 Lab on a Chip Lab on a Chip or “LOC” is a term used to describe a specific use of microfluidics. As explained in the introductory section of this chapter, microfluidics came to be due to the need of having a quick and small field deployable device to detect certain chemical agents in the air. This initial goal of miniaturization has been extrapolated to other applications creating the field of LOC. LOC tries to synthesize all the necessary analytical tests that would be needed to perform certain laboratory analysis on a silicone chip the size of a coin as seen in Figure 3.4. Lab on a chip microfluidic devices are being developed to be used in many different industries, with the goal of making chemical analysis much faster, cheaper and precise (Whitesides 2006). One of the said industries is healthcare. Hospitals perform thousands of tests on a daily basis, costing them a lot of resources and money on personnel and equipment. LOC devices offer a very promising future where certain clinical testing can be made accessible and affordable to those healthcare facilities that cannot afford traditional glassware laboratories. Figure 3.4: Example of microfluidic lab on a chip device. This device aims to perform chemical analysis using microfluidic and MEM technology (Shutterstock). 35 3.7.1 Organ on a chip Another emerging area where microfluidics is being used would be biological emulation. Laboratories worldwide are trying to replicate the phenomena that occur in our bodies with the use of microfluidic technology (see Figure 3.5). These devices are referred to as “Organs on a chip”. With these organs on a chip, researchers aim to study how our body works and how they could behave under the use of certain drugs and diseases. Figure 3.5: Emulate Lung on a chip microfluidic device. This device aims to provide a medium in which we test new therapy drugs. 37 Chapter 4: Simulations 4.1 Introduction Computer simulations and CAD software has become an increasingly used technology in engineering, due to the advantages of working in a virtual environment. This chapter contains the simulations performed on COMSOL that were necessary for the development of this research. Simulations were used and are needed in this research because they pose a cheap and accessible environment where we can perform tests on our prototypes before building the physical models; these models give a deep understanding of the possible behaviour of our device. The software of choice was COMSOL, a Multiphysics modelling software which allows simulating microfluidic and creeping flows. 4.2 Simulations 4.2.1 Fluid Flow Simulation This section corresponds to the simulation of a viscous fluid along a microfluidic channel in a laminar flow. The cross section of the channel is 50 μm x 100 μm. 38 Figure 4.1: Fluid Flow simulation 50 μL/h XZ plane visualization. The units expressed in the legend are m/s. As we can observe in Figure 4.1, where velocity contours at different longitudinal locations are show, the flow is faster in the central portion rather than on the sides, this is the well-known effect of the symmetrical parabolic fluid profile. The average velocity is given as: 𝑣𝑚=1 4𝜂 ∆𝑃 ∆𝑥 𝑅2 , (4.1) where, 𝜂 is the dynamic viscosity, ∆𝑃 the difference of fluid pressure, ∆𝑥 the distance covered and 𝑅 half the width of the channel. While the velocity at a given point is defined by the following formula: 𝑣(𝑥)=𝑣𝑚 [1 −𝑥2 𝑅2] , (4.2) where 𝑣𝑚 is calculated in Formula 4.1, 𝑅 is half of the width of the channel and 𝑥 is the position along the perpendicular axis to the flow of the fluid. This effect of faster fluid at the centre of the geometry is given by the wall friction with the flowing fluid. 39 Figure 4.2: Fluid Flow simulation 50 μL/h YX plane visualization. The units expressed in the legend are m/s. As we can observe in Figure 4.2 and Figure 4.1 where the flow is faster in the central section, this effect is given in both the 𝑥 and the 𝑧 direction, this is due to the presence of side walls in both these axes. In the following figure (see Figure 4.3) we can perfectly observe how the speed varies in the different 𝑥, 𝑦 and 𝑧 coordinates. Due to the laminar flow and dynamic viscosity the profile of our flow will be parabolic. For this simulation we used particle tracing physics to clearly represent the different speed and their distribution along the channel. 40 Figure 4.3: Parabolic profile of a laminar flow in a rectangular channel. 4.2.2 Sheath Flow Simulation These following simulations try to show the effect of the sheath flow in a microfluidic channel. In Figure 4, the total flow in the central, post focusing stage is 50 μL/h; in this plot all three inlets have the same flow rate: 16.67 μL/h, as given by the following formula. 𝑄𝑡𝑜𝑡𝑎𝑙 =𝑄1+𝑄2+𝑄3 . (4.3) 𝑄1, 𝑄2 and 𝑄3 correspond to the different inlets of the microfluidic hydrodynamic focusing device; by altering the different inflow rates of these inlets we will have different focusing widths in the post-focusing section. 41 Figure 4.4: Surface plot of the fluid velocity. Speed measured in m/s. In Figure 4, we can observe the surface plot of the fluid velocity. Each of the inlets have the same flow rate: 4.63 x 10-12 m3/s or 16.67 μL/h. The total flow rate in the central channel is 50 μL/h. We immediately see that in this central channel, the velocity magnitude is much greater. Flow rate in a pipe is given by fluid speed multiplied by the area of the channel cross-section: 𝑄=𝑣𝑎𝑣𝑒𝑟𝑎𝑔𝑒 ×𝐴𝑐𝑟𝑜𝑠𝑠−𝑠𝑒𝑐𝑡𝑖𝑜𝑛 . (4.4) 𝑣𝑎𝑣𝑒𝑟𝑎𝑔𝑒 =𝑄 𝐴𝑐𝑟𝑜𝑠𝑠−𝑠𝑒𝑐𝑡𝑖𝑜𝑛 . (4.5) Therefore, while the cross sectional area remains constant we see a threefold increase in volumetric fluid flow rate. 48 5.3 Microfabrication Technology For the development of this microfluidic device, the standard microfluidic protocol was used. Firstly, a photomask was developed using microfabrication technology, then several Silicon wafers were produced by photolithography, lastly, PDMS was poured on onto the Silicon wafers and cured to produce the resulting microfluidic channel. 5.3.1 Electron Beam Lithography As it is the case with almost all of microfluidic technology, this technology was firstly developed for electronics applications, however due to the capacity of achieving very high resolutions it may be used in several different applications. This technique consists in performing patterns on a substrate using an electron beam. This photomask is then used in the successive stages of the development of microfluidic channels. The photomask was developed at the University of Tokyo, Takeda Tipezaki Building Clean Room by the student Sayaka Nomura of the Tokyo University of Science. 5.3.1.1 Photomask Creation Figure 5.2: Electron Beam Schematic showing the process of characterization of a photomask. As illustrated in Figure 5.2, the different steps to making the photomask are: 1. Initially, the photomask (see Figure 5.2) is covered in a special photoresist that when in contact with the electron beam will draw the desired pattern onto the mask (see Table 5.1). This part is critical in the development of the mask, 49 an incorrect exposure to the photoresist will conclude in an inaccurate master photomask that will then influence in a negative manner the rest of the process. For this section of our process the machine shown in Figure 5.3 was used, with the parameters shown in Table 5.2, respectively. Each resist has specific curing parameters and a required energy per unit area, therefore the amount of light and exposure time must be adjusted accordingly to the resist used. An incorrect adjustment of these specifications could result in an insufficient chromium removal of the underlying layers. Manufacturer Toppan Model ST-TLR6-TQZ-5009(5T) Mask depth 2.3 mm Resist ZEP7000 Resist depth 3000 Å Prebaking 190 ºC for 30 minutes Metal layer Cr / CrO2 Metal Layer depth 100 nm Table 5.1: Photomask Specification. Figure 5.3: Electron Beam Lithography equipment used for the creation of the photomask. This system is located in Tokyo University. Manufacturer Advantest Model F5112 Smallest dimension possible 100 nm Dimension deviation 3σ ≤ 15 nm Superposition precision |mean value| + 3 σ ≤ 40 nm Table 5.2: Specification of the Advantest Electron Beam System. 50 2. Once the resist has been exposed to the electron beam, it has to be developed and then rinsed off. Isopropyl alcohol (IPA) and methyl isobutyl ketone (MIBK) are the chemicals used in this process. The machine EVG 101D Automatic Developer was used for this step (see Figure 5.4) (see Table 5.3). Figure 5.4: Automatic photomask developing system. Located in Tokyo University. Manufacturer EVG Model 101D Spin module max. deployment num. 1 Types of Development Pressure tank, flow control, nitrogen nozzle for spay developing, paddle developing, rinse developing Table 5.3: Automatic Photomask Developer technical Specifications. 3. This step is called Etching. Etching consists in using strong acids and chemicals to cut or produce a pattern into the unprotected parts of a metal surface. In our case, we immerse the mask into a chemical solution with the goal of removing the exposed metal and thus forming our pattern. In some cases Plasma Ashing is used as a method to pre-treat the surface for a better etching process. Using a plasma source, Plasma Ashing is performed onto the photoresist to enable a better penetration of the chemicals used in the later etching process. The ashing apparatus used was the FA-1 manufactured by Samco (see Figure 5.5) (see Table 5.4). The etching apparatus used was the Fairchild 1140 etching machine (see Figure 5.7) (see Table 5.5) 51 Figure 5.5: Ashing apparatus located in Tokyo University. Manufacturer Samco Model FA-1 Gas CF4 (50 sccm) O2 (50 sccm) Maximum RF 200 W Table 5.4: Ashing apparatus technical specifications. 4. Finally, we wash off the resist with an ashing device (see Figure 5.6); consequently, all the resist is removed from our mask leaving behind a Chromium Oxide layer on top of a glass substrate, with a pattern that will then be used throughout the whole microfluidic device fabrication. This process previously explained will lead to the creation of a photomask (see Figure 5.6). The photomask has a series of patterns etched into the glass-metal surface which are transparent, this will enable light to pass through and thus create the shape of the device we desire. Figure 5.6: Photomask used in the microfluidic channel development, created by Sayaka Nomura in 2015. 52 Figure 5.7: Etching apparatus, located in Tokyo University. Manufacturer Fairchild Model 1140 Output Power 3.6 kW Supply Pressure CDA 80PSI N2 60 PSI D1water 30PSI Discharge Condition 80SCFM 20SCFM Table 5.5: Technical specifications of the etching machine. 5.3.2 Photolithography We call photolithography the process in which we use UV light to cure photosensitive chemicals in order to make patters on a wafer. Just like the previous technology, this proceeding was borrowed from the electronics field. In electric and electronic engineering, photolithography is used in the manufacturing of transistors and complex integrated circuits. In this process, we shall use the photomask fabricated in the previous section and use it for patterning of a silicon wafer, this silicon wafer will be the master mould we will then pour our PDMS solution on to build the channel. 53 5.3.2.1 Silicon patterning procedure Figure 5.8: Master mould lithography process. As illustrated in Figure 5.8, the different steps to manufacturing the silicon wafer microfluidic channel master mould: 1. The first step in photolithography is to apply a coating of photoresist to our silicon wafer. In our case we used SU-8 photoresist (see Figure 5.9) due to its chemical attributes (see Table 5.6). Figure 5.9: SU-8 photoresist. Manufacturer Micro Chem. Model SU-8 3000 Adhesive strength 71 MPa Glass transition point 198 ºC, DMA tan δ Volume Resistivity 1.8 x 1016 Ω·cm Water Absorption 0.5 % 85 ºC / 85 %RH, 120h Table 5.6: Chemical Specifications of Su-8 photoresist. 54 In order to apply the photoresist we first pour the solution onto a silicon wafer and we spin it at a rate of 2250 rpm (see Table 5.7) with the use of the spin coater (see Figure 5.10), this spinning will extend the SU-8 over the entire silicon wafer achieving a thickness of 50 μm. We then prebake the silicon wafer using a hot plate at 60 ºC for 1 minute and then at 90 ºC for 15 minutes. After heating, we leave to cool down in room temperature. Figure 5.10: Spin coating apparatus located in Motlab, Tokyo University of Science. Manufacturer Active Model ACT-220D Rotation range 200 ~ 8000 rpm Rotation revolutions ± 3 rpm Vacuum 350 Torr Steps / patterns 10 steps / 100 patterns Table 5.7: Technical specifications for spin coater. 2. The next step consists of exposing the spin coated silicon wafer to a UV light to generate the pattern on our wafer. For this we use the photomask aligner (see Figure 5.11). This machine will expose our silicon wafer to a UV source with the shape of the photomask, for this to happen UV light is emitted through the source (see Table 5.8) and only passes through the photomask in the areas where the Chromium Oxide has been etched out in the previous process. Once the photoresist is cured with UV light, we can proceed to the development of the SU-8. After the exposure, we perform a post-bake by heating on a hot plate the Si wafer at 60 ºC for 1 minute and 90 ºC for 15 minutes. 55 Figure 5.11: Mask Aligner apparatus, located in Tokyo University of Science. Manufacturer Nanotec Model ES20tr Exposure area 105 mm Illumination uniformity ±5 % Illumination distance 200 mm Dominant Wavelength 365, 405, 436 nm UV irradiation strength More than 35m W/cm2 (at 365 nm) Table 5.8: Technical specifications of Nanotec mask aligner. 3. Finally, we proceed to develop the silicon wafer by rinsing and immersing the wafer into SU-8 developer. In some cases, SU-8 will remain; therefore, we will wash the wafer with isopropanol, acetone, ethanol and distilled water. Once we have completed all this process we will have correctly developed a silicon wafer with our pattern drawn on it (see Figure 5.12). Figure 5.12: Silicon wafer microfluidic channel master mould. 56 5.3.3 PDMS Channel For the characterisation of the microfluidic channel, we used PDMS (Figure 5.13). PDMS is a widely used silicon-based organic polymer in microfluidics; this polymer is used due to its many advantages. As a result of its flexibility and its mechanical properties this type of technological procedure is also referred to as soft lithography. Figure 5.13: PDMS. In order to create our PDMS channel, we will simply pour the mixture of PDMS on our SU-8 mould and leave to dry up until forming a solid block. The mixture we pour is a 10:1 PDMS to curing agent ratio (Table 5.9). Note that before pouring the PDMS onto the wafer we should place the mixture into a vacuum for 40 minutes to remove all bubbles. Manufacturer Dow Corning Toray Model Silpot 184 Viscosity 5000 cSt Density 1.11 g/cm3 Mixing Ratio Main Agent: Curing Agent 10:1 Heating temperature and time 60 ºC 80 minutes, 150 ºC 30 minutes Refractive Index 1.41 Table 5.9: PDMS chemical specifications. Once we pour the PDMS we heat at 60ºC for 80 minutes and 150 ºC for 30 minutes respectively. When dry (see Figure 5.14), we can simply cut with a razor blade, punch holes into the inlet and outlets and place on a clean glass slide to perform our experiments and tests. 57 Figure 5.14: Final microfluidic PDMS channel before assembly. 5.4 E-Selectin substrate preparation The cell adhesion molecule used in this work is the E-selectin (see Figure 5.15) therefore we must coat the substrate of the channel for our setup to work properly. The preparation method is fairly simple due to the characteristics of the materials used (see Table 5.10). Figure 5.15: E-selectin. First we coat our glass slide with a fine layer of PDMS, for this we must spin coat the PDMS on the glass substrate. Thanks to the functional amino group of the E-selectin, we can easily bond the CAM to the PDMS forming a peptide bond, which strongly binds the molecule to the substrate of out microfluidic channel. Therefore, we must simply pour the E-selectin (500 mg) dissolved in 64 6.1.3 Discussion As we’ve seen previously, the setup and execution of this experiment show a promising hydrodynamic focusing. We have demonstrated that by varying the flow rates in each of the inputs we can modify the width of out focused channel. This is a fundamental first step in the development of our device, considering that we can now control the amount of fluid and cells that will pass through the central portion of our channel. We must take a minute to point out the possible errors in calculating the size of the sheath. The error in the calculations may be of ±1 μm; this is not significant as the size of the leukocyte is generally 10 μm. When using the final iteration of our design, the user may want to change the conditions of flow in the channel therefore we can make many combinations of flow rates to fit our needs. In our case the since we want a single cell focused flow, we aim to have dimensions ranging from 11 μm to 15 μm therefore we could use the above tested combinations. When choosing said flow rates, we must elect appropriate rates bearing in mind the total flow downstream from focusing point. Once the three flows meet in the channel the total flow rate would be the sum of the three flow rates, as indicated in the following equation: 𝑄=𝑄𝑐𝑒𝑛𝑡𝑟𝑎𝑙 𝑐ℎ𝑎𝑛𝑛𝑒𝑙 +2×𝑄𝑠𝑖𝑑𝑒 𝑐ℎ𝑎𝑛𝑛𝑒𝑙𝑠 . (5.3) As we know, cells will behave differently with different flows; therefore, we must adjust these parameters accordingly. 6.2 Rolling Cell Experiment In this second experiment, we recreate the rolling cell experiment on a simple straight channel microfluidic device. The goal of this experiment is to test the rolling conditions in which the last experiment will take place. We shall experiment under the same conditions as in the last experiment and expose the problems we face when working with the absence of a hydrodynamic focusing module. 65 6.2.1 Experimental Setup Figure 6.4: Schematic image showing the experimental setup for this assay. The setup is very similar to the previous experiment, changing in aspects such as the number of pumps used and the nature of the fluid used. Due to the fact that we are testing live leukocytes on this assay, we used PBS as working fluid in order to keep the medium as stable as possible. As explained in chapter 6.4, this experiment needs an E-selectin coated surface for the leukocytes to bind correctly; therefore a previously treated substrate was used for this experiment. The conditions, as explained previously, do not change much from the previous experiment; however we used Calcein AM for this experiment to stain the cytoplasm of the cell. Calcein AM is a fluorescent dye with emission wavelengths of 495/515 nm. This dye is used in biology due to its ability to penetrate the cellular membrane in live cells, making it a very useful tool for testing cell viability and for short term cell labelling. 6.2.2 Results The following results were obtained at 0.2 Pa of wall shear stress and are depicted in Figure 6.5 and Figure 6.6. As we can observe only leukocytes on the side of the channels attach, where the fluid flow is slower, leaving the central section completely barren. 66 Figure 6.5: Top view of microfluidic channel. Leukocytes flow on the edges of the channel as opposed to the central area where we would expect them. Figure 6.6: Top view of microfluidic channel. Leukocytes flow path is scattered throughout the whole domain of the channel. 6.2.3 Discussion As we can see in the Figures 6.5 and 6.6, the conditions in which the measurements take place aren’t the ideal position for us to have an accurate reading on the results of the experiment. This is the reason we need a sheath flow, in order to focus the lymphocytes in the central domain to control all the variables in our future tests. Besides this inconvenience, the setup works as expected: the leukocytes bind to the substrate and roll at the appropriate speeds. flow 50 μm flow 50 μm 67 6.3 Rolling Cell Sheath Flow Experiment In this experiment we test the definitive version of our experiment in order to validate the prototype we have created. We shall introduce a syringe of PBS with live lymphocytes suspended and produce a sheath flow to centre the immune cells in the channel to have more controllable interface. 6.3.1 Experimental Setup The experimental setup is similar to those explained in the above experiments. The substrate was prepared the day before and left overnight in the incubator at 37.5 ºC, as explained in chapter 5. The channel used was prepared on the day of the experiment following the procedure of soft lithography explained in chapter 5. Once the channel was prepared it was sterilized accordingly with ethanol and distilled water following the same protocol as the experiments explained previously. The syringes used for the experiments contained PBS alone and PBS with lymphocytes suspended in them, respectively, prepared following the same steps as the previous experiments. The lymphocytes dyed with Calcein AM, in the same way we performed in the previous experiment. 6.3.2 Results The results observed in this experiment were as expected. When the lymphocytes were introduced into the microfluidic channel, the cells were focused into the central section of the posterior channel. This is due to the action of the hydrodynamic focusing module we have built. As we can see in the previous experiment and on Figure 6.5, we can observe that the flow of the leukocytes is random, the same happens in Figure 6.7(a) and Figure 6.7(b). However once the cells have been focused by the action of the sheath flow they circulate through the centre of the channel as observed in the following images of Figure 6.7(c)-(h). 68 Figure 6.7: Time-lapse visualizing the flow of the lymphocytes through the flow focusing module. The total time of this video lasts 1 second. The leukocyte can be observed as a white dot over a grey background. This image was extracted using fluorescence microscopy. We have to bear in mind, that although we are focusing the flow of the lymphocytes in the centre of the channel, due to the physics of the hydrodynamic focusing section, the speed at which these cells move along the channel will also increase. In this said section, we are pumping three times the volume of fluid as pumped in the initial section therefore as we explained in chapter 3, the increase of volume being pumped under the constant width of the channel will cause the speed of the particles to be increased. 6.3.3 Discussion The initial results were positive, and we managed to obtain the results we were looking for. The ultimate goal was to create a stream of focused cells in order to analyse them in a more controlled environment. However good the result may be, there are certain aspects we must talk about regarding this experiment. Firstly, as we have observed in the results, the speed of the cells increases by a certain factor depending on the flow in the inlets. The speed of the particle will see itself increased depending on how focused the flow of cells are. Meaning that the more focused we desire our model to be the higher the speed will be in our channel, due to the flow of higher volumes per unit of time pumped into the (a) (b) (c) (d) (e) (f) (g) (h) 69 system. This may pose certain problems, due to the limitations of the equipment used for the experiment. If we desire a certain low particle speed (where Q ≤ 20 μL/h) where we aim to perform an experiment with a specific pressure factor and we want a high focusing rate, the central inlets must pump the experimental fluid at very low flows, meaning that the microfluidic pumps used might have certain limitations. When performing high focusing rates and lower particle speed experiments, the microfluidic pumps exhibited technical limitations and generated a pulsation effect in the fluid. This pulsation is a very negative effect that we want to eradicate at all costs. Secondly, we observed that the binding with the E-selectin substrate was not always achieved and suspect this is due to the absence of contact with the substrate of the channel. Due to this most of the cells will flow along the channel never attaching to the substrate. However nothing can be done, because in the event of forcing the cells to attach we must exert a force onto the lymphocyte that would modify the behaviour of the cell significantly. All in all, one thing is clear, although the experiment has given us positive results, luck is also a factor when performing experiments because lymphocytes can fail to attach as discussed previously. 6.4 Pulsation Experiment In this experiment we aim to observe at what flow the microfluidic pump, Harvard Apparatus Pump 11 Elite, exhibits mechanical limitation and thus affect the flow of our fluid causing pulsation. 6.4.1 Experimental Setup For this experiment we fabricated a simple straight channel microfluidic chip and pumped into the channel micro particles suspended in a distilled water solution at flows under 5 μL/h as seen in Figure 6.8. We then observed the flow of the fluid under the fluorescence microscope and analysed the results. 70 Figure 6.8: Schematic image showing experimental setup for this section. 6.4.2 Results After testing a range of different flows, we observed pulsation in flows under 5 μL/h. 6.4.3 Discussion As pointed out in the results section, pulsation occurs in flows under 5 μL/h, indicating a limitation in the apparatus used in our experimentation. Therefore, when performing experiments that require low flow rates, we must be very careful not to surpass this value because it could introduce undesired alterations to our experiment that could change the results of our tests. 71 Chapter 7: Conclusion 7.1 Conclusions After analysing all the relevant results from the different experiments we can conclude the following:  The potentiality of microfluidics for biomedical research has been proved. Microfluidics poses as an excellent technology that is widely used in many labs around the world.  The importance of multiphysics simulation software in microfluidic research has been confirmed.  A microfluidic system for leukocyte experimentation or Lab on a Chip that overcomes the initial non central rolling problem has been successfully developed.  Although the system solves many problems, in cases of low flow rates, the system presents limitations due to the equipment used. 7.2 Future Work Although the system that has been designed is fully functional and could be used in future research, there is still room for improvement. 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