A versatile cancer cell trapping and 1D migration assay in a microfluidic device

Highly migratory cancer cells often lead to metastasis and recurrence and are responsible for the high mortality rates in many cancers despite aggressive treatment. Recently, the migratory behavior of patient-derived glioblastoma multiforme cells on microtracks has shown potential in predicting the likelihood of recurrence, while at the same time, antimetastasis drugs have been developed which require simple yet relevant high-throughput screening systems. However, robust in vitro platforms which can reliably seed single cells and measure their migration while mimicking the physiological tumor microenvironment have not been demonstrated. In this study, we demonstrate a microfluidic device which hydrodynamically seeds single cancer cells onto stamped or femtosecond laser ablated polystyrene microtracks, promoting 1D migratory behavior due to the cells' tendency to follow topographical cues. Using time-lapse microscopy, we found that single U87 glioblastoma multiforme cells migrated more slowly on laser ablated microtracks compared to stamped microtracks of equal width and spacing (p < 0.05) and exhibited greater directional persistence on both 1D patterns compared to flat polystyrene (p < 0.05). Single-cell morphologies also differed significantly between flat and 1D patterns, with cells on 1D substrates exhibiting higher aspect ratios and less circularity (p < 0.05). This microfluidic platform could lead to automated quantification of single-cell migratory behavior due to the high predictability of hydrodynamic seeding and guided 1D migration, an important step to realizing the potential of microfluidic migration assays for drug screening and individualized medicine. Published under license by AIP Publishing

Design, fabrication and optimization of a multifunctional microfluidic platform for single-cell analyses.

La manipulación celular es clave en el desarrollo de la investigación y aplicaciones para diagnóstico clínico. Hoy en día, es bien conocido que las células individuales, incluso aquellas con la misma apariencia, pueden mostrar diferentes fenotipos. Estas disparidades hacen que cada célula, aún encontrándose en las mismas condiciones, responda de manera diferente frente a un mismo estímulo. Por lo tanto, el estudio celular a nivel individual proporciona información más precisa que los datos promediados obtenidos tradicionalmente en estudios poblacionales, abriendo nuevas oportunidades en el desarrollo de nuevos fármacos y en la medicina personalizada. La microfluídica se ha constituido como una tecnología con gran capacidad para investigar la complejidad inherente de los sistemas celulares. Los canales microfluídicos poseen dimensiones en el rango de las decenas a cientos de micras siendo comparables con el tamaño de una célula. Esto permite la realización de numerosos estudios biomédicos con gran resolución espacial y temporal. Además, el desarrollo de plataformas microfluídicas cuenta con numerosas ventajas inherentes a su escala micrométrica como son: la minimización en el consumo de reactivos y producción de desechos, su alta sensibilidad y rápida respuesta o, su alta capacidad de integración. Todo ello se traduce en gran versatilidad a bajo coste. Dentro de este marco, esta tesis presenta una aproximación multidisciplinar para el desarrollo de dispositivos microfluídicos capaces de capturar, tratar y/o analizar células de forma individual con el consiguiente valor añadido. Para ello, los diferentes dispositivos microfluídicos han sido diseñados utilizando un programa de diseño asistido por ordenador. La fabricación y desarrollo de los mismos se ha llevado a cabo mediante técnicas de microfabricación como fotolitografía o micromoldeo de polímeros. Para su posterior caracterización y validación estructural, se ha utilizado tanto la perfilometría como diversas técnicas de microscopía. Además, se ha estudiado la dinámica de fluidos dentro de la plataforma desarrollada de manera teórica, mediante simulaciones computacionales, así como de forma empírica. Finalmente, la versatilidad de los dispositivos se ha validado llevando a cabo ensayos biológicos entre los que cabe destacar: estudios de viabilidad celular, citotoxicidad y migración celular tanto de células sanas como cancerígenas. Como resultado de este trabajo, se presenta una plataforma microfluídica de alto rendimiento y multifuncional de bajo coste. En concreto, el mecanismo de atrapamiento utilizado se basa en trampas hidrodinámicas con un manejo simple y mediante el control del fenómeno de co-flujo laminar. Gracias a su naturaleza modular, diferentes tipos de microestructuras pueden ser adaptadas a la plataforma principal permitiendo, por ejemplo, el análisis de la migración celular o de comunicaciones intercelulares. En conclusión, la plataforma microfluídica desarrollada permitirá una mejor comprensión de procesos biológicos fundamentales proporcionando microambientes celulares altamente adaptables y controlables.Cell handling is essential for research and application development in clinical diagnostics. In numerous biological assays thousands of cells are cultured without considering that the interaction between them may interfere in the resulting data. Nowadays, it is well known that individual cells, even those with identical appearance may display different phenotypes. These disparities make individual cells have different responses to equal stimulus in the same conditions. Therefore, single-cell studies provide more precise information, than average responses traditionally obtained from cell populations, and open new opportunities for drug discovery and personalized medicine. Microfluidics has emerged as a powerful enabling technology to investigate the natural complexity of cellular systems. Microfluidic channels have dimensions ranging from tens to hundreds of microns which are comparable to the size of a cell. This allows the realization of large numbers of biomedical studies with high spatial and temporal resolution. In addition, the microfluidic platforms have numerous advantages inherent to the micrometer scale, such as: minimized reagent consumption and waste production, high sensitivity and fast response or high integration capabilities. Overall, this is translated to versatile and cost-effective solutions. In this framework, this thesis presents a multidisciplinary approach for the development of microfluidic platforms capable of capturing, treating and/or analyzing single-cells with the corresponding added value. In order to achieve this, different microfluidic platforms have been designed using computer aided design (CAD) software. The manufacture and development of these microdevices have been carried out by means of microfabrication techniques such as photolithography and polymer micromolding processes. The fabricated microfluidic platforms have been structurally characterized and validated by means of profilometry and various microscopic techniques. In addition, theoretical (computational fluid dynamics simulations) and experimental fluid dynamics characterizations inside the microfluidic platform have been performed. Finally, the versatility of the devices has been highlighted through several cell-based assays including cell viability, cytotoxicity and cell migration studies accounting for healthy and cancer cells. As a result of this work, a multifunctional, low cost and high-throughput microfluidic platform has been devised. In particular, the trapping mechanism relies on hydrodynamic traps and the accurate and simple handling is based on the control of the laminar co-flow phenomenon. Thanks to its modular nature, different microstructures can be easily coupled to the main platform enabling cell migration and co-culture analyses. Overall, the developed microfluidic platform will facilitate a better understanding of key biological processes by providing well-controlled and versatile microenvironments

Design, fabrication and optimization of a multifunctional microfluidic platform for single-cell analyses.

La manipulación celular es clave en el desarrollo de la investigación y aplicaciones para diagnóstico clínico. Hoy en día, es bien conocido que las células individuales, incluso aquellas con la misma apariencia, pueden mostrar diferentes fenotipos. Estas disparidades hacen que cada célula, aún encontrándose en las mismas condiciones, responda de manera diferente frente a un mismo estímulo. Por lo tanto, el estudio celular a nivel individual proporciona información más precisa que los datos promediados obtenidos tradicionalmente en estudios poblacionales, abriendo nuevas oportunidades en el desarrollo de nuevos fármacos y en la medicina personalizada. La microfluídica se ha constituido como una tecnología con gran capacidad para investigar la complejidad inherente de los sistemas celulares. Los canales microfluídicos poseen dimensiones en el rango de las decenas a cientos de micras siendo comparables con el tamaño de una célula. Esto permite la realización de numerosos estudios biomédicos con gran resolución espacial y temporal. Además, el desarrollo de plataformas microfluídicas cuenta con numerosas ventajas inherentes a su escala micrométrica como son: la minimización en el consumo de reactivos y producción de desechos, su alta sensibilidad y rápida respuesta o, su alta capacidad de integración. Todo ello se traduce en gran versatilidad a bajo coste. Dentro de este marco, esta tesis presenta una aproximación multidisciplinar para el desarrollo de dispositivos microfluídicos capaces de capturar, tratar y/o analizar células de forma individual con el consiguiente valor añadido. Para ello, los diferentes dispositivos microfluídicos han sido diseñados utilizando un programa de diseño asistido por ordenador. La fabricación y desarrollo de los mismos se ha llevado a cabo mediante técnicas de microfabricación como fotolitografía o micromoldeo de polímeros. Para su posterior caracterización y validación estructural, se ha utilizado tanto la perfilometría como diversas técnicas de microscopía. Además, se ha estudiado la dinámica de fluidos dentro de la plataforma desarrollada de manera teórica, mediante simulaciones computacionales, así como de forma empírica. Finalmente, la versatilidad de los dispositivos se ha validado llevando a cabo ensayos biológicos entre los que cabe destacar: estudios de viabilidad celular, citotoxicidad y migración celular tanto de células sanas como cancerígenas. Como resultado de este trabajo, se presenta una plataforma microfluídica de alto rendimiento y multifuncional de bajo coste. En concreto, el mecanismo de atrapamiento utilizado se basa en trampas hidrodinámicas con un manejo simple y mediante el control del fenómeno de co-flujo laminar. Gracias a su naturaleza modular, diferentes tipos de microestructuras pueden ser adaptadas a la plataforma principal permitiendo, por ejemplo, el análisis de la migración celular o de comunicaciones intercelulares. En conclusión, la plataforma microfluídica desarrollada permitirá una mejor comprensión de procesos biológicos fundamentales proporcionando microambientes celulares altamente adaptables y controlables.Cell handling is essential for research and application development in clinical diagnostics. In numerous biological assays thousands of cells are cultured without considering that the interaction between them may interfere in the resulting data. Nowadays, it is well known that individual cells, even those with identical appearance may display different phenotypes. These disparities make individual cells have different responses to equal stimulus in the same conditions. Therefore, single-cell studies provide more precise information, than average responses traditionally obtained from cell populations, and open new opportunities for drug discovery and personalized medicine. Microfluidics has emerged as a powerful enabling technology to investigate the natural complexity of cellular systems. Microfluidic channels have dimensions ranging from tens to hundreds of microns which are comparable to the size of a cell. This allows the realization of large numbers of biomedical studies with high spatial and temporal resolution. In addition, the microfluidic platforms have numerous advantages inherent to the micrometer scale, such as: minimized reagent consumption and waste production, high sensitivity and fast response or high integration capabilities. Overall, this is translated to versatile and cost-effective solutions. In this framework, this thesis presents a multidisciplinary approach for the development of microfluidic platforms capable of capturing, treating and/or analyzing single-cells with the corresponding added value. In order to achieve this, different microfluidic platforms have been designed using computer aided design (CAD) software. The manufacture and development of these microdevices have been carried out by means of microfabrication techniques such as photolithography and polymer micromolding processes. The fabricated microfluidic platforms have been structurally characterized and validated by means of profilometry and various microscopic techniques. In addition, theoretical (computational fluid dynamics simulations) and experimental fluid dynamics characterizations inside the microfluidic platform have been performed. Finally, the versatility of the devices has been highlighted through several cell-based assays including cell viability, cytotoxicity and cell migration studies accounting for healthy and cancer cells. As a result of this work, a multifunctional, low cost and high-throughput microfluidic platform has been devised. In particular, the trapping mechanism relies on hydrodynamic traps and the accurate and simple handling is based on the control of the laminar co-flow phenomenon. Thanks to its modular nature, different microstructures can be easily coupled to the main platform enabling cell migration and co-culture analyses. Overall, the developed microfluidic platform will facilitate a better understanding of key biological processes by providing well-controlled and versatile microenvironments

A versatile cancer cell trapping and 1D migration assay in a microfluidic device

Highly migratory cancer cells often lead to metastasis and recurrence and are responsible for the high mortality rates in many cancers despite aggressive treatment. Recently, the migratory behavior of patient-derived glioblastoma multiforme cells on microtracks has shown potential in predicting the likelihood of recurrence, while at the same time, antimetastasis drugs have been developed which require simple yet relevant high-throughput screening systems. However, robust in vitro platforms which can reliably seed single cells and measure their migration while mimicking the physiological tumor microenvironment have not been demonstrated. In this study, we demonstrate a microfluidic device which hydrodynamically seeds single cancer cells onto stamped or femtosecond laser ablated polystyrene microtracks, promoting 1D migratory behavior due to the cells' tendency to follow topographical cues. Using time-lapse microscopy, we found that single U87 glioblastoma multiforme cells migrated more slowly on laser ablated microtracks compared to stamped microtracks of equal width and spacing (p < 0.05) and exhibited greater directional persistence on both 1D patterns compared to flat polystyrene (p < 0.05). Single-cell morphologies also differed significantly between flat and 1D patterns, with cells on 1D substrates exhibiting higher aspect ratios and less circularity (p < 0.05). This microfluidic platform could lead to automated quantification of single-cell migratory behavior due to the high predictability of hydrodynamic seeding and guided 1D migration, an important step to realizing the potential of microfluidic migration assays for drug screening and individualized medicine. Published under license by AIP Publishing