Microfluidic system for cell separation and deformation assessment by using passive methods

Tese de doutoramento em Biomedical EngineeringOs sistemas microfluídicos têm sido usados com sucesso em muitas aplicações biomédicas. As principais vantagens destes sistemas consistem na utilização de volumes de amostras reduzidos e com tempos de ensaios curtos. Além disso, os sistemas microfluídicos possibilitam a execução de várias tarefas em paralelo numa única plataforma microfluídica, como por exemplo a separação e medição da deformabilidade de células/partículas. Em dispositivos microfluídicos, existem dois métodos principais para separar células: métodos passivos, baseados em microestruturas e escoamentos laminares, e métodos ativos, baseados em campos de forças externos. Muitos estudos têm sido realizados com métodos passivos, pois estes não necessitam de forças externas. Nesta tese serão apresentadas diferentes geometrias passivas para um dispositivo microfluídico, constituído por vários filtros de fluxo cruzado e multiníveis com o intuito de separar células/partículas em função do seu tamanho. Outra característica importante é a implementação de microcanais hiperbólicos a montante das saídas por forma a criar um escoamento extensional homogéneo e consequentemente medir a deformabilidade das células de forma controlada. Após a separação e avaliação da deformação, a quantidade de glóbulos vermelhos será quantificada por um método de espectrofotometria. Os resultados indicam que várias geometrias mostraram uma boa taxa de separação, confirmada pelas medidas de camada livre de células e pela espectrofotometria. Verificou-se também que os sistemas microfluídicos testados são capazes de separar amostras patológicas de sangue, demostrando assim o seu potencial em realizar simultaneamente a separação e deformação de células patológicas, como por exemplo células provenientes de pacientes diagnosticados com malária e/ou diabetes.Microfluidic systems have been successfully used at many biomedical applications. Their great advantages allow working with minimal sample volumes and with short assays times. Additionally, microfluidic systems allow parallel operations in a single microfluidic platform such as separation and measurement of single cell/particles deformability. In microfluidic devices, there are two main methods for cells separation: passive methods, based on microstructures and laminar flow, and active methods, based on external force fields. Many studies have been made using passive methods because they do not require external forces. In this thesis it will be presented different geometrical passive approaches for a microfluidic device, that will have crossflow filters and multilevel steps that will separate the cells/particles by their size. Another important feature is the implementation of hyperbolic microchannels upstream the outlets in order to create a homogeneous extensional flow and consequently to measure the cells deformability in a controlled way. After the separation and deformation assessment, the amount of RBCs will be quantified by a spectrophotometry method. The results indicate that several geometries have shown a good separation rate, confirmed by the cell free layer and spectrophotometry measurements. It was also verified that the tested microfluidic systems are able to separate pathological blood samples, showing its potential to perform simultaneously separation and deformation assessments of blood diseases, such as malaria and diabetes.I want to acknowledge the financial support provided by scholarship SFRH/BD/99696/2014 from FCT (Science and Technology Foundation), COMPETE 2020, Portugal 2020 and POCH, that allow the successful development of this PhD project

The study of cell behaviour using biomedical microdevices

The cell culture has become over the years an important discipline for the thorough knowledge of cells behaviour and migration, as also cell-cell interaction. A few years ago it was only possible to create culture systems in vitro systems, however, with the advance of new technologies it has become possible to create culture systems in vivo closer to reality. The microfabrication processes such as photolithography were used in this work for the design of a microdevice which is aimed to study the migration of cells into nanofibers. Beyond the cell studies, microdevices were also used for rheological studies. The behaviour of blood cells is very important, regarding the transport of oxygen in our body as well as nutrients. The knowledge about the factors that influence changes in mechanical properties of erythrocytes is of great importance because it is their deformability that allows them to carry oxygen and pass through smaller arteries and veins. Therefore, another microdevice was used for the purpose of observing the deformation of the erythrocytes after contact with human tumour cell lines (NCI-H460- non-small cell lung cancer and HCT-15 - colon carcinoma) up to two consecutive days. With the flow rate of 500 nl/min, the flowing erythrocytes through the microchannel were captured by a high speed camera and their deformability was examined. The erythrocytes that contacted tumour cells show less deformability than ―healthy‖ erythrocytes. It was found that the tests were insufficient to conclude clearly the effect of the tumour cells in the deformation of erythrocytes. We have found evidence that erythrocytes also influence the growth of tumour cells, however, further testing would be needed to clarify this observation. A cultura de células tornou-se ao longo dos anos uma importante disciplina para o conhecimento profundo do seu comportamento, migração e interação entre células. Com o avanço de novas tecnologias tornou-se possível criar sistemas de cultura in vivo mais próximos da realidade, quando anteriormente apenas existiam sistemas in vitro. Neste trabalho, foram aplicados processos de microfabricação, nomeadamente a fotolitografia, para a conceção de um microdispositivo que teve como objetivo principal o estudo da migração de células em nanofibras. Para além de estudos celulares, os microdispositivos também foram usados em estudos reológicos. O comportamento do sangue é muito importante, pois é ele que transporta o oxigénio no nosso organismo assim como nutrientes. Perceber o que influencia a alteração das características mecânicas dos eritrócitos é de grande importância pois é a sua deformabilidade que lhes permite transportar o oxigénio e passar nas artérias e veias mais pequenas. Assim, utilizou-se um outro microdispositivo, com o propósito de observar a deformação dos eritrócitos após contacto com linhas celulares tumorais humanas (NCI-H460 – cancro do pulmão e HCT-15 – carcinoma do cólon) até um máximo de dois dias consecutivos. Os eritrócitos fluíram pelo microcanal com uma taxa de fluxo de 500 nl/min e foram capturados por uma câmara de alta velocidade para ver a sua capacidade de deformação. Os eritrócitos que estiveram em contacto com as células tumorais revelam menos deformação que os eritrócitos ―saudáveis‖. Verificou-se que os ensaios realizados foram insuficientes para concluir com clareza o efeito das células tumorais na deformação dos eritrócitos. Encontraram-se também indícios de os eritrócitos influenciarem o crescimento das células tumorais, no entanto, mais ensaios também seriam necessários para clarificar essas observações.This Final Project Report was a collaboration of the University of Navarra and research centre, CEI

Técnicas de separação biomimética em microfluídica

A separação e a identificação de células são essenciais em várias aplicações biomédicas, incluindo a biologia celular e os métodos de diagnóstico e terapêuticos. O sangue é um fluido não-Newtoniano contendo inúmeras informações preciosas sobre o estado fisiológico e patológico do corpo humano. No entanto, devido à sua complexidade, existem actualmente poucos métodos de análise precisos. A maioria das técnicas convencionais usadas na separação e contagem de células são dispendiosas e normalmente é necessário usar agentes externos adicionais para identificar as células. As técnicas biomiméticas de separação em microfluídica usam fenómenos microfluídicos, que ocorrem à microescala, para realizar a separação de determinadas células sanguíneas. Estes fenómenos incluem a separação do plasma, a camada livre de células (CLC), a migração dos glóbulos brancos (GBs), também designados por leucócitos, e a lei da bifurcação. Recentemente, vários investigadores têm replicado estes efeitos, que acontecem em ambientes in vivo, em sistemas microfluídicos, ou seja, em ambientes in vitro. Nos microcanais, os glóbulos vermelhos (GVs), devido à sua deformabilidade e ao gradiente de velocidades (às forças de sustentação), tendem a concentrar-se na zona central dos microcanais, enquanto os GBs e os GVs rígidos (tal como, os GVs infectados com malária) tendem a migrar para a CLC que se forma nas zonas próximas das paredes. A lei da bifurcação estabelece, relativamente ao comportamento dos GVs, que em microcanais com bifurcações eles tendem a escolher o microcanal de secção maior.Os autores agradecem à FCT, COMPETE, QREN e União Europeia (FEDER) pelo financiamento através dos seguintes projetos: PTDC/SAU-BEB/105650/2008, PTDC/SAU-BEB/108728/2008, PTDC/EME-MFE/099109/2008 ePTDC/SAU-ENB/116929/2010

Proposal for improving of paddy rice drying process in dryers inclined pools: Propuesta de mejoramiento del proceso de secado de arroz paddy en secaderos tipo albercas inclinadas

The operation of inclined type dryers for paddy rice is studied to improve their operation and efficiency, considering the variables of drying time, humidity distribution in the rice layer after drying and air velocities within the plenum. Simulating the distribution and velocities of the air flow before crossing the rice layer, the outlet ducts to the plenum are relocated and dampers are placed to distribute the flow evenly. On the other hand, it is proposed to replace the flat-type mesh with a zigzag-type mesh.  To verify the proposed modifications, a scale model was built to determine the humidity and drying time variation inside the rice layer, obtaining more uniform moisture percentages within the rice layer and a decrease in drying time.  Se estudia el funcionamiento de los secaderos tipo inclinado para arroz paddy, con el fin de mejorar su operación y eficiencia, considerando las variables de tiempo de secado, distribución de la humedad en la capa de arroz después del secado y velocidades del aire dentro del plenum. Simulando la distribución y velocidades del flujo de aire antes de cruzar la capa de arroz se reubican los conductos de salida al plenum y se colocan dampers que distribuyan uniformemente el flujo. Por otro lado, se propone la sustitución de la malla tipo plana por una malla tipo zigzag.  Para verificar las modificaciones propuestas se construyó un modelo a escala para medir la variación del tiempo de secado y la humedad dentro de la capa de arroz paddy, obteniendo porcentajes de humedad más uniformes dentro de la capa de arroz y disminución del tiempo de secado

A simple microfluidic device for the deformability assessment of blood cells in a continuous flow

Blood flow presents several interesting phenomena in microcirculation that can be used to develop microfluidic devices capable to promote blood cells separation and analysis in continuous flow. In the last decade there have been numerous microfluidic studies focused on the deformation of red blood cells (RBCs) flowing through geometries mimicking microvessels. In contrast, studies focusing on the deformation of white blood cells (WBCs) are scarce despite this phenomenon often happens in the microcirculation. In this work, we present a novel integrative microfluidic device able to perform continuous separation of a desired amount of blood cells, without clogging or jamming, and at the same time, capable to assess the deformation index (DI) of both WBCs and RBCs. To determine the DI of both WBCs and RBCs, a hyperbolic converging microchannel was used, as well as a suitable image analysis technique to measure the DIs of these blood cells along the regions of interest. The results show that the WBCs have a much lower deformability than RBCs when subjected to the same in vitro flow conditions, which is directly related to their cytoskeleton and nucleus contents. The proposed strategy can be easily transformed into a simple and inexpensive diagnostic microfluidic system to simultaneously separate and assess blood cells deformability.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/2010 and EXPL/EMS-SIS/ 2215/2013 from FCT (Fundação para a Ciência e a Tecnologia), COMPETE, QREN and European Union (FEDER). R. O. Rodrigues, D. Pinho and V. Faustino acknowledge respectively, the PhD scholarships SFRH/BD/97658/2013, SFRH/BD/89077/2012 and SFRH/BD/99696/ 2014 granted by FCT. The authors would also like to thank Dr. Ângela Fernandes for providing the blood samples and Dr. Ricardo Calhelha for supplying the tissue culture medium used in this work.info:eu-repo/semantics/publishedVersio

Proposal for improving of paddy rice drying process in dryers inclined pools: Propuesta de mejoramiento del proceso de secado de arroz paddy en secaderos tipo albercas inclinadas

The operation of inclined type dryers for paddy rice is studied to improve their operation and efficiency, considering the variables of drying time, humidity distribution in the rice layer after drying and air velocities within the plenum. Simulating the distribution and velocities of the air flow before crossing the rice layer, the outlet ducts to the plenum are relocated and dampers are placed to distribute the flow evenly. On the other hand, it is proposed to replace the flat-type mesh with a zigzag-type mesh.  To verify the proposed modifications, a scale model was built to determine the humidity and drying time variation inside the rice layer, obtaining more uniform moisture percentages within the rice layer and a decrease in drying time.  Se estudia el funcionamiento de los secaderos tipo inclinado para arroz paddy, con el fin de mejorar su operación y eficiencia, considerando las variables de tiempo de secado, distribución de la humedad en la capa de arroz después del secado y velocidades del aire dentro del plenum. Simulando la distribución y velocidades del flujo de aire antes de cruzar la capa de arroz se reubican los conductos de salida al plenum y se colocan dampers que distribuyan uniformemente el flujo. Por otro lado, se propone la sustitución de la malla tipo plana por una malla tipo zigzag.  Para verificar las modificaciones propuestas se construyó un modelo a escala para medir la variación del tiempo de secado y la humedad dentro de la capa de arroz paddy, obteniendo porcentajes de humedad más uniformes dentro de la capa de arroz y disminución del tiempo de secado

Usos, limitaciones y prospectiva de la evaluación por pares

The peer review process is a mechanism instituted by the academic and scientific communities around the world to guarantee the quality of the articles published in journals. In this paper, the general foundations of this practice are analyzed along with some of its aspects identified as strengths and weakness having as a starting point the results of a documental research. Some proposals to improve the process are also analyzed; these are mainly focused in reducing the bias and the time that the process currently takes

Erythrocyte deformability in a hyperbolic microchannel

Serious diseases such as diabetes and malaria are deeply related to microcirculation disorders caused by abnormal erythrocytes deformability [1]. Therefore, various studies on red blood cells (RBCs) deformation have been carried out but those studies rather focus on shear ow or extensional ow [2-6]. In this study, a relatively low aspect ratio (AR) hyperbolic microchannel was used in order to create an extensional ow combined with a shear ow, where AR= h w , h means the depth and w means the width of the microchannel. The objective of the study is to investigate the degree of RBC deformation throughout the microchannel at the centerline (y = 0). The blood samples were RBCs diluted with the Hank's Balanced Salt Solution (HBSS) with the hematocrit (Hct) level set to be 2%. A polydimethylsiloxane (PDMS) microchannel, having a hyperbolic contraction region followed by an abrupt expansion shape, was fabricated by a soft-lithography technique. The dimensions of the microchannel: width of the inlet (w), length of contraction region (l), width of the exit of contraction (wc) and depth (h) were 400 m, 580 m, 20 m and 14 m, respectively. Hence, the AR was 0:035. The experimental equipment consisted of an inverted microscope (Diaphot 300, Nikon), a high-speed camera (FASTCAM SA3, Photron) connected to a computer and a 1mL syringe (TERUMO R SYRING) controlled by a syringe pump (PHD ULTRA). The manufactured PDMS microchannel was placed on the stage of the microscope where the ow rate Q of the working uids was kept constant at 0:5 L=min. The owing RBCs were recorded by a high speed camera at a frame rate of 7500 frames/s and analyzed. The DI was obtained by DI= a b a + b where a refers to the major axis and b refers to the minor axis of the ellipse best tted to RBCs. High DI values in the contraction region were observed. It is possible to say that the RBCs highly elongate with both extensional and shear dominated ows achieved by a hyperbolic microchannel with a relatively low aspect ratio AR

Cell-free layer analysis in a polydimethysiloxane microchannel: A global approach

The cell-free layer (CFL) is a hemodynamic phenomenon that has an important contribution to the rheological properti es of blood flowing in microvessels. The present work aims to find the closest function describing RBCs flowing around the cell depleted layer in a polydimethysiloxane (PDMS) microchannel with a diverging and a converging bifurcation. The flow behaviour of the CFL was investigated by using a high-speed video microscopy system where special attention was devoted to its behaviour before the bifurcation and after the confluence of the microchannel. The numerical data was first obtained by using a manual tracking plugin and then analysed using the genetic algorithm approach. The results show that for the majority of the cases the function that more closely resembles the CFL boundary is the sum of trigonometric functions.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/ 2010 and EXPL/EMS-SIS/2215/2013 from FCT (Science and Technology Foundation), COMPETE, QREN and European Union (FEDER). R.O. Rodrigues, D. Pinho and V. Faustino acknowledge respectively, the PhD scholarships SFRH/BD/97658/2013, SFRH/BD/89077/2012 and SFRH/BD/99696/2014 granted by FCT.info:eu-repo/semantics/publishedVersio