Overlooked? Underestimated? Effects of Substrate Curvature on Cell Behavior

In biological systems, form and function are inherently correlated. Despite this strong interdependence, the biological effect of curvature has been largely overlooked or underestimated, and consequently it has rarely been considered in the design of new cell–material interfaces. This review summarizes current understanding of the interplay between the curvature of a cell substrate and the related morphological and functional cellular response. In this context, we also discuss what is currently known about how, in the process of such a response, cells recognize curvature and accordingly reshape their membrane. Beyond this, we highlight state-of-the-art microtechnologies for engineering curved biomaterials at cell-scale, and describe aspects that impair or improve readouts of the pure effect of curvature on cells

Development of microfluidic tools to reproduce and characterize the tumor microenvironment

A pesar de que la incidencia del cáncer está en aumento, sobre todo en los países desarrollados, el desarrollo de nuevos fármacos contra esta enfermedad es cada vez menos efectivo. Para revertir esta tendencia, aparece la necesidad de desarrollar mejores herramientas para reproducir y caracterizar el microentorno tumoral. Una de ellas son modelos in vitro más precisos.En este contexto, la microfluídica se presenta como una potente alternativa para el desarrollo de estos nuevos modelos in vitro más precisos, que puedan emplearse para un desarrollo y selección de fármacos más racional y efectivo. No obstante, se trata de un conjunto de técnicas poco extendido en los laboratorios de biología molecular. Así, en la presente tesis se desarrollan dos modelos microfluídicos del microentorno tumoral para tumores sólidos, junto a las herramientas necesarias para su caracterización, todo ello de fácil uso para tratar de generalizar la aplicación de los mismos.En el capítulo 1 se realiza una revisión del estado de la cuestión en lo referente a modelos de cáncer in vitro y su caracterización. En el capítulo 2 se desarrolla un modelo microfluídico de co-cultivo que permite estudiar las interacciones endotelio-tumor, así como la capacidad de penetración y erradicación de células tumorales de nuevos fármacos. En el capítulo 3 se presenta una herramienta para caracterizar los niveles de oxígeno molecular en cualquier punto de un cultivo in vitro 3D. En el capítulo 4 se presenta un modelo de tumor centrado en la generación y caracterización de gradientes biológicos, así como su adaptación a las técnicas tradicionales de biología molecular para el análisis del perfil genético del microentorno tumoral a lo largo del tiempo. Para generar los sistemas microfluídicos descritos anteriormente, se emplearon dispositivos fabricados mediante distintas técnicas y materiales. En los dispositivos se sembraron distintas poblaciones celulares, intentando así reproducir la estructura y organización de los tejidos biológicos. Mediante diferentes técnicas de microscopía (óptica, fluorescencia, confocal, imagen en tiempo real) y sondas fluorescentes se monitorizó la evolución y comportamiento celular. La caracterización del hidrogel sensible al oxígeno se realizó a través de las técnicas ya citadas, así como espectrofotometría, microscopía de fuerza atómica y electrónica de barrido en condiciones ambientales. Finalmente, la extracción de las células de los hidrogeles se realizó por medio de degradaciones enzimáticas, y la cuantificación de la expresión génica mediante extracción de RNA, retrotranscripción y reacción en cadena de la polimerasa cuantitativa.La conclusión general de la tesis, es que la utilización de modelos biomiméticos cambia dramáticamente el resultado de los ensayos realizados in vitro, por lo que su uso es necesario para obtener resultados relevantes y trasladables a la clínica. Asimismo, el desarrollo de sistemas biomiméticos in vitro del microentorno tumoral de uso generalizado es posible mediante el desarrollo de dispositivos de fácil uso, así como del establecimiento de métodos robustos de caracterización de los mismos, tanto in situ como “aguas abajo” del establecimiento de los modelos. Bibliografía: 1. Balkwill FR, Capasso M, Hagemann T (2012) The tumor microenvironment at a glance. J Cell Sci 125: 5591-5596.2. Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501: 346-354.3. Scannell JW, Blanckley A, Boldon H, Warrington B (2012) Diagnosing the decline in pharmaceutical R&D efficiency. Nat Rev Drug Discov 11: 191-200.4. Adriani G, Pavesi A, Tan AT, Bertoletti A, Thiery JP, et al. (2016) Microfluidic models for adoptive cell-mediated cancer immunotherapies. Drug Discov Today 21: 1472-1478.5. Ayuso JM, Virumbrales-Munoz M, Lacueva A, Lanuza PM, Checa-Chavarria E, et al. (2016) Development and characterization of a microfluidic model of the tumour microenvironment. Sci Rep 6: 36086.6. Ayuso JM, Monge R, Martínez-González A, Virumbrales-Muñoz M, Llamazares GA, et al. (2017) Glioblastoma on a microfluidic chip: Generating pseudopalisades and enhancing aggressiveness through blood vessel obstruction events. Neuro-Oncology: now230.<br /

Leveraging Microtechnology to Study Multicellular Microvascular Systems and Macromolecular Interaction

Biological systems are large-scale, complex systems comprised of many hierarchical subsystems interacting physico-chemically in a dynamic and coordinated fashion. The complex interactions of subsystems (in micro-scale) lead to the formation of emergent properties (in macro-scale); these are properties that are not visible if individual subsystems are studied. The inherent high-throughput characteristics of microfabrication technology (microtechnology) along with its ability to manipulate biological species at the micro-scale makes it an ideal tool to elucidate the mechanisms leading to the formation of emergent properties at the macro-scale. In this dissertation, by combining microtechnologies with advanced computational algorithms, we demonstrate system-level analysis of biological systems in development and disease. The abundance of high quality molecular and genetic data along with the drastic increase in computational power resulted in considerable progress in genomics, epigenomics and proteomics, but not for the so-called cellomics as we define it here: high-throughput study of single-cell phenotype and heterotypic cell-cell interaction via micromanipulation and bioinformatics analysis. Lack of high-throughput robust experimental tools is the major roadblock to cellomics. Using microtechnologies, in the context of developmental biology we studied vascular tissue morphogenesis (vasculogenesis). Formation of microvessels is of critical significance in development and for vascularizing newly engineered tissues in regenerative medicine. First, we sought to map the heterogeneous morphodynamic behavior of individual clonal cells in the process of capillary-like structure (CLS) formation (Chapter 2 and 3). Then we looked into deciphering the role of extracellular matrix (ECM) mediated mechanical signals in deriving the process of CLS formation (Chapter 4). In the second half of this thesis, we demonstrated the capabilities of microtechnologies and advanced computational algorithms in tackling the challenging problems in disease: global health diagnostics and cancer drug screening. First, we studied the performance of microfluidic-based diagnostic as a large-scale complex system under real-world constraints (Chapter 5). Then, we present the development of two microfluidic-based platforms to study the heterotypic interaction of cells in both a biomimetic in vitro and a realistic in vivo setting. We developed an implantable construct carrying a densely-packed heterogeneous panel of tumor cells. This platform could ultimately be used to test anti-cancer drug efficacy against a large number of genotypes in an in vivo setting (Appendices A and B). Together, these methods provide a powerful suite of tools for high-throughput analysis of biological species at the micro-scale and could potentially unlock the mysteries behind the emergent properties observed at the macro-scale

Simulation of the spatial structure and cellular organization evolution of cell aggregates arranged in various simple geometries, using a kinetic monte carlo method applied to a lattice model

ilustraciones, graficasEsta tesis trata los modelos de morfogénesis, en particular los modelos de evolución guiada por contacto que son coherentes con la hipótesis de la adhesión diferencial. Se presenta una revisión de algunos modelos, sus principios biológicos subyacentes, la relevancia y aplicaciones en el marco de la bioimpresión, la ingeniería de tejidos y la bioconvergencia. Luego, se presentan los detalles de los modelos basados en métodos de Monte Carlo para profundizar más adelante en el modelo basados en algoritmos Kinetic Monte Carlo (KMC) , más específicamente, se describe en detalle un modelo KMC de autoaprendizaje (SL-KMC). Se presenta y explica la estructura algorítmica del código implementado, se evalúa el rendimiento del modelo y se compara con un modelo KMC tradicional. Finalmente, se realizan los procesos de calibración y validación, se observó que el modelo es capaz de replicar la evolución del sistema multicelular cuando las condiciones de energía interfacial del sistema simulado son similares a las del sistema de calibraciones. (Texto tomado de la fuente)This thesis treats the models for morphogenesis, in particular the contact-guided evolution models that are coherent with the differential adhesion hypothesis. A review of some models, their biological underpinning principles, the relevance and applications in the framework of bioprinting, tissue engineering and bioconvergence are presented. Then the details for the Monte Carlo methods-based models are presented to later deep dive into the Kinetic Monte Carlo (KMC) based model, and more specifically a Self-Learning KMC (SL-KMC) model is described to detail. The algorithmic structure of the implemented code is presented and explained, the model performance is assessed and compared with a traditional KMC model. Finally, the calibration and validation processes have been carried out, it was observed that the model is able to replicate the multicellular system evolution when the interfacial energy conditions of the simulated system are similar to those of the calibrations system.MaestríaMagíster en Ingeniería - Ingeniería Químic

Designing a Scaffold-Free Bio-Orthogonal Click Chemistry Method of Cell Assembly for Application in Tissue Engineering

Tissue engineering is a growing field of science that relies on the use of material chemistry, engineering, genetics, and cell biology to produce functional tissues for use in transplantation, drug testing and disease modelling. Presently, there is an urgent need for a technology which would enable assembly of cells into 3-dimensional multilayered tissues. Current cell-assembly technologies rely on biodegradable polymer scaffolds to assemble cells into 3D structures and to support the cell mass of the growing tissue. The presence of these materials in tissues, however, lowers the cell density and the process of scaffold biodegradation results in accumulation of monomer byproducts within the tissue. To overcome these issues we developed a scaffold free method of cell-assembly based on bio-orthogonal ligation reactions between oxyamine and ketone groups to form a stable oxime bond. The reaction is quick, specific and occurs under physiological conditions without a catalyst. To deliver the bio-orthogonal functionalities onto cell surfaces, ketone- and oxyamine- functionalized lipids were incorporated into liposomes which were subsequently fused with cell membranes. The surface engineered cells were assembled into three-dimensional tissues. Using this approach, we were able to produce functional cardiac and liver tissues with variable thicknesses and cell orientations for drug testing as well as the complex 3D co-cultures of stem cells to study stem cell differentiation. The rapid bio-orthogonal cell ligation process also enables assembly of cells into co-culture spheroids in flow, inside a microchannel. The introduction of a bi-functional oxyamine crosslinker molecule allowed for the rapid crosslinking of ketone-functionalized cells into 3D tissues. This bio-orthogonal click chemistry technology can be used with different cell types to produce customized tissues for applications in drug development and regenerative medicine

HEPATOCYTE POLARITY AND FUNCTION ENHANCEMENT THROUGH SCALABLE COMPACTION

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Inverted Adipose Mammary Microtissues for Early-Stage Tumor Progression Testing

Stromal adipocytes have been increasingly shown to play a role in multiple stages of breast cancer progression. While new culture models are being developed to test hypotheses about interplay between adipose tissue and tumors, none currently exist that recapitulate invasion from the lumen of the mammary duct through a cell-constructed basement membrane into the stromal compartment. This dissertation describes a hanging drop culture-based method for producing novel adipocyte-laden mammary microtissues with a basal side-in geometry, that show potential in their application to early-stage tumor invasion testing. By seeding cancer cells into co-culture with the self-organized microtissue, invasion into the tissue is observable through acquisition of confocal image stacks of whole mount specimens. Interestingly, tissues containing adipocytes in a 1:1 ratio with normal epithelium enhance invasion by a triple-negative three-fold compared to epithelium-only controls. Testing was also conducted using experimental therapies for disrupting CCL5-mediated paracrine chemotaxis and local fibrotic shifts in the microenvironment. The anti-fibrotic drug pirfenidone showed efficacy in the invasion model by slowing invasion at non-toxic concentrations. The use of primary adipocytes in the current model iteration potentially enables its use in a variety of microenvironment models, including for studying early-stage breast cancer with comorbidities like obesity and diabetes which may require more precise therapeutic testing.Ph.D