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The mechanical microenvironment plays a crucial role in the evolution of colorectal cancer, a complex disease characterized by heterogeneous tumors with varying elasticity. Toward setting up distinct scenarios, herein, we describe the preparation and characterization of gelatin methacrylamide (GelMA)-based hydrogels via two different mechanisms: free-radical photopolymerization and photo-induced thiol-ene reaction. A precise stiffness modulation of covalently crosslinked scaffolds was achieved through the application of well-defined irradiation times while keeping the intensity constant. Besides, the incorporation of thiol chemistry strongly increased stiffness with low to moderate curing times. This wide range of finely tuned mechanical properties successfully covered from healthy tissue to colorectal cancer stages. Hydrogels prepared in phosphate-buffered saline or Dulbecco’s modified Eagle’s medium resulted in different mechanical and swelling properties, although a similar trend was observed for both conditions: thiol-ene systems exhibited higher stiffness and, at the same time, higher swelling capacity than free-radical photopolymerized networks. In terms of biological behavior, three of the substrates showed good cell proliferation rates according to the formation of a confluent monolayer of Caco-2 cells after 14 days of cell culture. Likewise, a characteristic apical-basal polarization of cells was observed for these three hydrogels. These results demonstrate the versatility of the presented platform of biomimetic materials as in vitro cell culture scaffolds.

The tissue microenvironment plays a crucial role in tissue homeostasis and disease progression. However, the in vitro simulation has been limited by the lack of adequate biomimetic models in the last decades. Thanks to the advent of microfluidic technology for cell culture applications, these complex microenvironments can be recreated by combining hydrogels, cells and microfluidic devices. Nevertheless, this advance has several limitations. When cultured in three-dimensional (3D) hydrogels inside microfluidic devices, contractile cells may exert forces that eventually collapse the 3D structure. Disrupting the compartmentalisation creates an obstacle to long-term or highly cell-concentrated assays, which are extremely relevant for multiple applications such as fibrosis or ischaemia. Therefore, we tested surface treatments on cyclic-olefin polymer-based microfluidic devices (COP-MD) to promote the immobilisation of collagen as a 3D matrix protein. Thus, we compared three surface treatments in COP devices for culturing human cardiac fibroblasts (HCF) embedded in collagen hydrogels. We determined the immobilisation efficiency of collagen hydrogel by quantifying the hydrogel transversal area within the devices at the studied time points. Altogether, our results indicated that surface modification with polyacrylic acid photografting (PAA-PG) of COP-MD is the most effective treatment to avoid the quick collapse of collagen hydrogels. As a proof-of-concept experiment, and taking advantage of the low-gas permeability properties of COP-MD, we studied the application of PAA-PG pre-treatment to generate a self-induced ischaemia model. Different necrotic core sizes were developed depending on initial HCF density seeding with no noticeable gel collapse. We conclude that PAA-PG allows long-term culture, gradient generation and necrotic core formation of contractile cell types such as myofibroblasts. This novel approach will pave the way for new relevant in vitro co-culture models where fibroblasts play a key role such as wound healing, tumour microenvironment and ischaemia within microfluidic devices.

Hydrogel confinement is critical in microfluidics to generate gradients, as cell remodelling process boundaries the development of biological models such as ischemia or fibrosis. Here, we test the immobilization efficiency of collagen hydrogels cells embedded to treated COP-based microfluidic devices to preserve 3D structure and avoid gel contraction.

La combinación de modelos matemáticos y dispositivos microfluídicos permite recrear procesos biológicos complejos como la evolución del glioblastoma (GBM). En este trabajo, se utiliza un enfoque multidisciplinar para evaluar las diferencias en el comportamiento tumoral en distintos sustratos, con el objetivo de estudiar su influencia en la progresión del tumor.

A novel fluorinated soft thermoplastic elastomer (sTPE) for microfluidics is presented. It allows the rapid fabrication of microfluidic devices through a 30‐second hot embossing cycle at 220°C followed by self‐sealing through simple conformal contact at room temperature, or with baking. The material shows high chemical resistance, particularly in comparison to polydimethylsiloxane (PDMS), to many common organic solvents and can be rapidly micropatterned with high fidelity using a variety of microfluidic master molds thanks to its low mechanical stiffness. Self‐sealing of the material is reversible and withstands pressures of up to 2.8 bar with room temperature sealing and four bar with baking at 185°C for 2 hours. The elastomeric, transparent sTPE exhibits material characteristics that make it suited for use as a microreactor, such as low absorption, surface roughness and oxygen permeability, while also allowing a facile and scalable fabrication process. Modular microfluidic devices, leveraging the fast and reversible room temperature self‐sealing, are demonstrated for the generation of water droplets in a toluene continuous phase using T‐junctions of variable size. The sTPE offers an alternative to common microfluidic materials, overcoming some of their key drawbacks, and giving scope for low‐cost and high‐throughput devices for flow chemistry applications.

During the second half of the twentieth century, oncology adopted a tumor-centric approach to cancer treatment, focusing primarily on the tumor cell to identify new therapeutic targets [...]

Microencapsulation of cells in hydrogel-based porous matrices is an approach that has demonstrated great success in regenerative cell therapy. These microcapsules work by concealing the exogenous cells and materials in a robust biomaterial that prevents their recognition by the immune system. A vast number of formulations and additives are continuously being tested to optimize cell viability and mechanical properties of the hydrogel. Determining the effects of new microcapsule additives is a lengthy process that usually requires extensive in vitro and in vivo testing. In this paper, we developed a workflow using nanoindentation (i.e., indentation with a nanoprobe in an atomic force microscope) and a custom-built microfluidic constriction device to characterize the effect of graphene oxide (GO) on three microcapsule formulations. With our workflow, we determined that GO modifies the microcapsule stiffness and surface properties in a formulation-dependent manner. Our results also suggest, for the first time, that GO alters the conformation of the microcapsule hydrogel and its interaction with subsequent coatings. Overall, our workflow can infer the effects of new additives on microcapsule surfaces. Thus, our workflow can contribute to diminishing the time required for the validation of new microcapsule formulations and accelerate their clinical translation.

Spheroids are in vitro spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. In this paper, we propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroids evolution under in vitro suspension, namely, nutrients diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. This preliminary validation allowed us to conclude that glioblastoma tumor spheroids evolution is mainly driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. In particular, it is concluded that our model is able to explain quite different setups, such as spheroids growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. Indeed, the main contribution of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease.Author summarySpheroids structures of cell aggregates are an available experimental platform to analyze the evolution and drug response of solid tumors. In particular, the dynamics of different glioblastoma cell lines have been studied in this work using spheroids. Interestingly, very different behaviors were observed, from a half of the initial configuration shrinking for U-251 MG cell line to six times the initial configuration growth for U-87 MG cell line. These results were replicated by means of a coupled mathematical model which accounts for nutrients diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. Tumor growth or shrinkage can be explained from a continuum mechanics view driven by cell activity and nutrients availability. This modeling put the focus on mechanistic effects and is aligned with novel experimental techniques to quantify the mechanical microenvironment in tumors. These techniques may be combined with the approach presented in this work to further investigate the role of mechanics in cancer disease.

espanolLa dinamica expansiva del turismo alrededor del mundo, del centro hacia la periferia, ha propiciado que muchas poblaciones nativas de sitios remotos participen en el sector, lo que afecta su modo de vida y sus relaciones sociales. Mucha atencion se ha prestado al control de agentes externos sobre estas poblaciones. Este articulo propone un avance conceptual al enfatizar en las formas de coordinacion. Como abordaje metodologico, el articulo avanza sobre las variables de la coordinacion del enfoque de cadenas globales de valor aplicadas a un caso de turismo en la Amazonia colombiana. La informacion se genero por medio de entrevistas, observacion participativa y talleres con funcionarios de un parque nacional, seis comunidades indigenas y un concesionario privado. Se propone la coordinacion diversificada basada en el dialogo de saberes y se sugiere que procesos de largo plazo que respeten los modos de vida tradicionales y las expectativas de las poblaciones nativas pueden mejorar su participacion en el turismo y disminuir su impacto. EnglishThe global expansion of tourism industry has forced several indigenous populations to participate in the sector. Such participation affected negatively their traditional livelihood and internal relations. Several studies have focused on the analysis of control that lead firms exert over the whole chain. However, the coordination, as the mechanism that allows the transmission of information between agents, has receive little attention. The article expands over the variables of coordination as a methodological tool for analyze a case study of tourism in a National Park. We gather information with interviews, workshops, participant observation at institutional level and with indigenous populations. The participation of native populations in global tourism can be improve through long-term processes based on intensive communication between tourism agents and the implementation of diversified coordination that respect their own livelihoods.

En este trabajo se analiza la incidencia de ciertos factores individuales, académicos y socio-económicos sobre el tiempo de permanencia y deserción de los estudiantes en el Programa Especial de Admisión y Movilidad Académica PEAMA de la Universidad Nacional de Colombia - Sede Amazonia. Se aplicó la metodología de modelos de duración, con técnicas paramétricas y no-paramétricas. Para el análisis estadístico se consideran datos de estudiantes que hicieron parte de este programa entre los años 2008 y 2014, de todas las carreras de pregrado del programa y las diferentes sedes a las que se movilizaron los estudiantes. Entre los factores que más inciden en la deserción de esta población están: carrera y número de periodos académicos cursados, sede de movilidad y variables socio-económicas como género e ingreso familiar. La condición étnica no tendría ninguna incidencia sobre la tasa de deserción.

En el presente ensayo, Pensar la realidad humana es darle sentido a todo lo inherente a vida; por tanto, es necesario una cultura que propicie espacios de reflexión y acción que debe ser realidad como hecho significativo para reconocer al semejante como sujeto en cosmovisión, para cosificar la diversidad cognitiva, saber, reflexión, reconocimiento de autonomía y trascendencia, entre el otro en relación con el otros y la mismicidad como yo, como punto de partida de la singularidad cognitiva, en existencia de dignidad plural, dialogo y reconocimiento en relaciones realmente humanas.

[EN]Cardiosphere-derived cells (CDCs) encapsulated within alginate-poly-L-lysine-alginate (APA) microcapsules present a promising treatment alternative for myocardial infarction. However, clinical translatability of encapsulated CDCs requires robust long-term preservation of microcapsule and cell stability, since cell culture at 37 degrees C for long periods prior to patient implantation involve high resource, space and manpower costs, sometimes unaffordable for clinical facilities. Cryopreservation in liquid nitrogen is a well-established procedure to easily store cells with good recovery rate, but its effects on encapsulated cells are understudied. In this work, we assess both the biological response of CDCs and the mechanical stability of microcapsules after long-term (i.e., 60 days) cryopreservation and compare them to encapsulated CDCs cultured at 37 degrees C. We investigate for the first time the effects of cryopreservation on stiffness and topographical features of microcapsules for cell therapy. Our results show that functionality of encapsulated CDCs is optimum during 7 days at 37 degrees C, while cryopreservation seems to better guarantee the stability of both CDCs and APA microcapsules properties during longer storage than 15 days. These results point out cryopreservation as a suitable technique for long-term storage of encapsulated cells to be translated from the bench to the clinic. This work has been supported by the European Union's H2020 Framework Program (H2020/2014-2020) and National Authorities through the Electronic Components and Systems for European Leadership Joint Undertaking (ECSEL JU) program under grant agreement Ecsel-78132-Position-II-2017-IA. The regional Government of Aragon provided L.P. studentship. This research was partially funded by Instituto de Salud Carlos III (PI20/00247) and Agencia Estatal de Investigacion (PID2019-107329RA-C22), cofunded by European Regional Development Fund "A way to make Europe."

Background and objectives Spheroids are the most widely used 3D models for studying the effects of different micro-environmental characteristics on tumour behaviour, and for testing different preclinical and clinical treatments. In order to speed up the study of spheroids, imaging methods that automatically segment and measure spheroids are instrumental; and, several approaches for automatic segmentation of spheroid images exist in the literature. However, those methods fail to generalise to a diversity of experimental conditions. The aim of this work is the development of a set of tools for spheroid segmentation that works in a diversity of settings. Methods In this work, we have tackled the spheroid segmentation task by first developing a generic segmentation algorithm that can be easily adapted to different scenarios. This generic algorithm has been employed to reduce the burden of annotating a dataset of images that, in turn, has been employed to train several deep learning architectures for semantic segmentation. Both our generic algorithm and the constructed deep learning models have been tested with several datasets of spheroid images where the spheroids were grown under several experimental conditions, and the images acquired using different equipment. Results The developed generic algorithm can be particularised to different scenarios; however, those particular algorithms fail to generalise to different conditions. By contrast, the best deep learning model, constructed using the HRNet-Seg architecture, generalises properly to a diversity of scenarios. In order to facilitate the dissemination and use of our algorithms and models, we present SpheroidJ, a set of open-source tools for spheroid segmentation. Conclusions In this work, we have developed an algorithm and trained several models for spheroid segmentation that can be employed with images acquired under different conditions. Thanks to this work, the analysis of spheroids acquired under different conditions will be more reliable and comparable; and, the developed tools will help to advance our understanding of tumour behaviour.

Cell encapsulation in hydrogel-based microspheres has demonstrated successes in regenerative cell therapy. We developed a workflow based on Atomic Force Microscopy (AFM) and a microfluidic constriction system to characterize stiffness and surface properties of microcapsules.

An effective-medium theory for the electrical conductivity of Ohmic dispersions taking explicit account of particle shape and spatial distribution independently is available from the work of Ponte Castaneda and Willis [J Mech Phys Solids 43:1919–1951, 1996]. When both shape and distribution take particular “ellipsoidal” forms, the theory provides analytically explicit estimates. The purpose of the present work is to evaluate the predictive capabilities of these estimates when dispersions exhibit dissimilar particle shape and distribution. To this end, comparisons are made with numerical bounds for coated ellipsoid assemblages computed via the finite element method. It is found that estimates and bounds exhibit good agreement for the entire range of volume fractions, aspect ratios, and conductivity contrasts considered, including those limiting values corresponding to an isotropic distribution of circular cracks. The fact that the explicit estimates lie systematically within the numerical bounds hints at their possible realizability beyond the class of isotropic dispersions., Facultad de Ingeniería

The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.

The complexity of cancer biology and its clinical manifestation are driven by genetic, epigenetic, transcriptomic, proteomic and metabolomic alterations, supported by genomic instability as well as by environmental conditions and lifestyle factors. Although novel therapeutic modalities are being introduced, efficacious cancer therapy is not achieved due to the frequent emergence of distinct mechanisms of multidrug resistance (MDR). Advanced technologies with the potential to identify and characterize cancer MDR could aid in selecting the most efficacious therapeutic regimens and prevent inappropriate treatments of cancer patients. Herein, we aim to present technological tools that will enhance our ability to surmount drug resistance in cancer in the upcoming decade. Some of these tools are already in practice such as next-generation sequencing. Identification of genes and different types of RNAs contributing to the MDR phenotype, as well as their molecular targets, are of paramount importance for the development of new therapeutic strategies aimed to enhance drug response in resistant tumors. Other techniques known for many decades are in the process of adaptation and improvement to study cancer cells’ characteristics and biological behavior including atomic force microscopy (AFM) and live-cell imaging. AFM can monitor in real-time single molecules or molecular complexes as well as structural alterations occurring in cancer cells induced upon treatment with various antitumor agents. Cell tracking methodologies and software tools recently progressed towards quantitative analysis of the spatio-temporal dynamics of heterogeneous cancer cell populations and enabled direct monitoring of cells and their descendants in 3D cultures. Besides, novel 3D systems with the advanced mimicking of the in vivo tumor microenvironment are applicable to study different cancer biology phenotypes, particularly drug-resistant and aggressive ones. They are also suitable for investigating new anticancer treatment modalities. The ultimate goal of using phenotype-driven 3D cultures for the investigation of patient biopsies as the most appropriate in vivo mimicking model, can be achieved in the near future.

In silico models and computer simulation are invaluable tools to better understand complex biological processes such as cancer evolution. However, the complexity of the biological environment, with many cell mechanisms in response to changing physical and chemical external stimuli, makes the associated mathematical models highly non-linear and multiparametric. One of the main problems of these models is the determination of the parameters’ values, which are usually fitted for specific conditions, making the conclusions drawn difficult to generalise. We analyse here an important biological problem: the evolution of hypoxia-driven migratory structures in Glioblastoma Multiforme (GBM), the most aggressive and lethal primary brain tumour. We establish a mathematical model considering the interaction of the tumour cells with oxygen concentration in what is called the go or grow paradigm. We reproduce in this work three different experiments, showing the main GBM structures (pseudopalisade and necrotic core formation), only changing the initial and boundary conditions. We prove that it is possible to obtain versatile mathematical tools which, together with a sound parametric analysis, allow to explain complex biological phenomena. We show the utility of this hybrid “biomimetic in vitro-in silico” platform to help to elucidate the mechanisms involved in cancer processes, to better understand the role of the different phenomena, to test new scientific hypotheses and to design new data-driven experiments.

Resumen En Colombia el turismo es promovido como una estrategia de desarrollo económico y un instrumento para la conservación ambiental. Sin embargo, al ser una industria global, no contempla dinámicas locales como la percepción de las poblaciones en su territorio. El enfoque de cadenas globales de valor, aplicado a dos estudios de caso en la Amazonia colombiana, evidencia cómo los proyectos turísticos liderados por el gobierno nacional han modificado la configuración y la percepción social del territorio. La imposición de una idea de desarrollo económico sobre las necesidades de la población local genera unas relaciones de poder desiguales y una distribución de las rentas en la cadena valor de forma inequitativa, al tiempo que aumenta la fragilidad ambiental y social del territorio. No obstante, el turismo puede ser una alternativa económica y de conservación importante para las poblaciones locales siempre y cuando sean tenidas en cuenta como actores legítimos del sector. Abstract In Colombia tourism is promoted as a strategy for economic development, and as a tool for environmental conservation. However, the implementation of projects from the center to the periphery, sometimes ignores the cultural attributes and the social perceptions from the territory. The global value chain approach, applied to two case studies, put in evidence the modifications in the Amazon caused by tourism development projects lead by national government. The main results are that social perception of the territory changed for a more monetary one, and the projects had no changed the inequality in power relations in the value chain. The imposition of an external idea of economic development can increase the fragility of the territory. If tourism is based on a good planning process could be an important economic and conservational alternative for local populations and also for environmental conservation in the scenario of Peace Agreements in Colombia. Resumo Na Colômbia o turismo é considerado como uma estratégia de desenvolvimento econômico ou como uma ferramenta para a conservação ambiental. Mais, a implementação de projetos com visão centro-periferia muitas vezes ignora as percepções sociais das populações sobre o território. Uma aplicação do enfoque de cadeias globais de valor a dois experiências de desenvolvimento turístico na Amazônia colombiana permite que embora o turismo possa ser uma fonte de ingressos econômicos, e uma alternativa menos danada que outros setores extrativos, o artigo demonstra como foram transformadas as percepções sociais sobre o território sem nenhum câmbio nas desiguais relações de poder na cadeia de valor. A imposição de uma ideia de desenvolvimento econômico pode aumentar a fragilidade ambiental e social do território. Mas, se estas variáveis foram consideradas adequadamente, o turismo pode se constituir em um importante setor no apoio da implementação dos Acordos de Paz na Amazônia colombiana. Résumé En Colombie le tourisme est encouragé comme stratégie économique en même temps qu'il serait un moyen pour la conservation de l'environnement. Toutefois, étant une entreprise globale, le tourisme prend peu en compte, ou pas du tout, les dynamiques locales et la perception qu'en ont les populations sur leur environnement. L'impact d'une telle chaîne de valeurs sera analysé à partir de deux études de cas réalisées en Amazonie colombienne. De tels projets à finalité économique ont en effet modifié la configuration et la perception de leur territoire par ces populations, sans affecter pour autant les relations inégalitaires du pouvoir ni assurer une distribution équitable des ressources. Cependant, si le développement du tourisme est mis en place en coordination avec une participation active de la population, il pourrait être une alternative économique et de la préservation de l'environnement, tout en prenant en compte les défis posés par les Accords de Paix signés récemment dans le pays.

Islet transplantation has the potential of reestablishing naturally-regulated insulin production in Type 1 diabetic patients. Nevertheless, this procedure is limited due to the low islet survival after transplantation and the lifelong immunosuppression to avoid rejection. Islet embedding within a biocompatible matrix provides mechanical protection and a physical barrier against the immune system thus, increasing islet survival. Alginate is the preferred biomaterial used for embedding insulin-producing cells because of its biocompatibility, low toxicity and ease of gelation. However, alginate gelation is poorly controlled, affecting its physicochemical properties as an injectable biomaterial. Including different concentrations of the phosphate salt Na2HPO4 in alginate hydrogels, we can modulate their gelation time, tuning their physicochemical properties like stiffness and porosity while maintaining an appropriate injectability. Moreover, these hydrogels showed good biocompatibility when embedding a rat insulinoma cell line, especially at low Na2HPO4 concentrations, indicating that these hydrogels have potential as injectable biomaterials for Type 1 Diabetes Mellitus treatment.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

Microfluidic systems have emerged as a technique with the potential to recreate biomimetic microenvironments that permit in vitro modeling of several important organ functions (organ-on-chip) as well as to analyze cellular processes. This allows designing experiments for validating scientific hypotheses and better understand complex biological processes, and determine biological parameters in an unprecedented way. The crosstalk between this new experimental capacity and mathematical models within a general framework is extensively discussed in this chapter, reviewing the formulation of cell processes, together with their interaction with the surrounding environment. Also, its particularization to a particular problem of interest such as the analysis of tumor evolution, which can be modeled in organ-on-chip devices. One of the important cases of the solid tumors is glioblastoma (GBM), the most lethal primary brain tumor. One of the characteristics in such types of tumors is the appearance of hypercellularized regions, named as pseudopalisades. These structures have been hypothesized to be produced by oxygen and nutrient depletion caused by tumor-induced blood vessel occlusion. However, despite the important instrumental role of these pseudopalisades in GBM's spread and invasion, the recreation of these in vitro has remained challenging. To analyze this problem, we designed a microdevice that comprises a central microchamber flanked by two lateral microchannels, separated by a series of projections. GBM cells were then embedded within a collagen hydrogel in the central microchamber thereby mimicking the three-dimensional structure and distribution. The lateral microchannels were used to perfuse different media or drugs, so normoxic, hypoxic, and necrotic regions may be naturally generated. In this chapter, we detail the model itself and the fitting of the model parameters. We compare the experimental and numerical results and we discuss the experimental trends and the underlying hypotheses.

The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.

Spin-coating process of a commercially available soft thermoplastic elastomer (sTPE) is demonstrated for the realization of microfluidic prototypes. It is a reliable, wrinkling-free, and high-yield holding and transfer methodology for single and multilayered devices fabrication. Complex microfluidic systems including embedded microlenses array, dilutor-like chip for stable capillary filling, and pneumatically drive valve confirms the performances and quality of the process. The dissolved sTPE, a soft styrenic-block polymer-based material, is flexible in its raw state as poly(dimethylsiloxane) and thermoformable as classical thermoplastics. Depending on the solvent evaporation rate, two stable morphologies of the block nanodomains are revealed: either a nanocylinder or hexagonal nanospheres pattern. Both are evenly distributed over the whole surface and account for a uniform and convenient surface at a “microfluidic scale device.” Plasma hydrophilization allows a stable and hydrophilic surface (≈20° after 7 d). The biocompatibility is assessed through the cell culture proliferation assay of progenitor endothelial cells. This sTPE solution displays fast and reliable fabrication and assembly performances, it can achieve strong and stable hydrophilicity within a low level of initial equipment investment.

The main reasons for the inefficiency of standard glioblastoma (GBM) therapy are the occurrence of chemoresistance and the invasion of GBM cells into surrounding brain tissues. New therapeutic approaches obstructing these processes may provide substantial survival improvements. The purpose of this study was to assess the potential of lipophilic antioxidant coenzyme Q10 (CoQ10) as a scavenger of reactive oxygen species (ROS) to increase sensitivity to temozolomide (TMZ) and suppress glioma cell invasion. To that end, we used a previously established TMZ-resistant RC6 rat glioma cell line, characterized by increased production of ROS, altered antioxidative capacity, and high invasion potential. CoQ10 in combination with TMZ exerted a synergistic antiproliferative effect. These results were confirmed in a 3D model of microfluidic devices showing that the CoQ10 and TMZ combination is more cytotoxic to RC6 cells than TMZ monotherapy. In addition, cotreatment with TMZ increased expression of mitochondrial antioxidant enzymes in RC6 cells. The anti-invasive potential of the combined treatment was shown by gelatin degradation, Matrigel invasion, and 3D spheroid invasion assays as well as in animal models. Inhibition of MMP9 gene expression as well as decreased N-cadherin and vimentin protein expression implied that CoQ10 can suppress invasiveness and the epithelial to mesenchymal transition in RC6 cells. Therefore, our data provide evidences in favor of CoQ10 supplementation to standard GBM treatment due to its potential to inhibit GBM invasion through modulation of the antioxidant capacity.

El efecto de las corporaciones transnacionales de turismo ha sido estudiado generalmente en mercados maduros de larga trayectoria. En las investigaciones se detallan interesantes mecanismos dentro y fuera del mercado que son utilizados por las corporaciones para controlar la cadena de valor ejerciendo poder sobre agentes y poblaciones locales. No obstante, poca atención se ha prestado al efecto/impacto que estas corporaciones tienen en destinos remotos de regiones periféricas. Este artículo evalúa los efectos de estas compañías en el turismo en la Amazonia con base en la aplicación del enfoque de cadenas globales de valor. El poder y el control se evalúan en un estudio de caso de la cadena Hoteles Decameron y su presencia en Leticia, en la Amazonia colombiana. Las alianzas estratégicas y la integración vertical, favorecidas por políticas gubernamentales de nivel nacional, permitieron la consolidación de la cadena hotelera como la fuerza motriz del turismo en la ciudad. Con la consolidación del turismo de masas en la región se pone en entredicho la sostenibilidad del sector.

Despite recent advances in the understanding and treatment options for osteoporosis, this condition remains a serious public health issue. Adrenomedullin (AM) is a regulatory peptide with reported activity on bone remodelling. To better understand this relationship we built an inducible knockout for AM. An outstanding feature of knockout mice is their heavier weight due, in part, to the presence of denser bones. The femur of knockout animals was denser, had more trabeculae, and a thicker growth plate than wild type littermates. The endocrine influence of AM on bone seems to be elicited through an indirect mechanism involving, at least, the regulation of insulin, glucose, ghrelin, and calcitonin gene-related peptide (CGRP). To confirm the data we performed a pharmacological approach using the AM inhibitor 16311 in a mouse model of osteoporosis. Ovariectomized females showed significant bone mass loss, whereas ovariectomized females treated with 16311 had similar bone density to sham operated females. In conclusion, we propose the use of AM inhibitors for the treatment of osteoporosis and other conditions leading to the loss of bone mass.

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Adherent cells exert contractile forces which play an important role in the spatial organization of the extracellular matrix (ECM). Due to these forces, the substrate experiments a volume reduction leading to a characteristic shape. ECM contraction is a key process in many biological processes such as embryogenesis, morphogenesis and wound healing. However, little is known about the specific parameters that control this process. With this aim, we present a 3D computational model able to predict the contraction process of a hydrogel matrix due to cell-substrate mechanical interaction. It considers cell-generated forces, substrate deformation, ECM density, cellular migration and proliferation. The model also predicts the cellular spatial distribution and concentration needed to reproduce the contraction process and confirms the minimum value of cellular concentration necessary to initiate the process observed experimentally. The obtained continuum formulation has been implemented in a finite element framework. In parallel, in vitro experiments have been performed to obtain the main model parameters and to validate it. The results demonstrate that cellular forces, migration and proliferation are acting simultaneously to display the ECM contraction.

Reseña del libro: Geraldo Andrello. 2006. Cidade do índio. Transformações e cotidiano em Iauaretê. São Paulo: Editora unesp – isa; Rio de Janeiro: nuti. 447 pp. isbn: 8571396590.

The tumour microenvironment is very complex, and essential in tumour development and drug resistance. The endothelium is critical in the tumour microenvironment: it provides nutrients and oxygen to the tumour and is essential for systemic drug delivery. Therefore, we report a simple, user-friendly microfluidic device for co-culture of a 3D breast tumour model and a 2D endothelium model for cross-talk and drug delivery studies. First, we demonstrated the endothelium was functional, whereas the tumour model exhibited in vivo features, e.g., oxygen gradients and preferential proliferation of cells with better access to nutrients and oxygen. Next, we observed the endothelium structure lost its integrity in the co-culture. Following this, we evaluated two drug formulations of TRAIL (TNF-related apoptosis inducing ligand): soluble and anchored to a LUV (large unilamellar vesicle). Both diffused through the endothelium, LUV-TRAIL being more efficient in killing tumour cells, showing no effect on the integrity of endothelium. Overall, we have developed a simple capillary force-based microfluidic device for 2D and 3D cell co-cultures. Our device allows high-throughput approaches, patterning different cell types and generating gradients without specialised equipment. We anticipate this microfluidic device will facilitate drug screening in a relevant microenvironment thanks to its simple, effective and user-friendly operation.

Background Glioblastoma (GBM) is one of the most lethal tumor types. Hypercellular regions, named pseudopalisades, are characteristic in these tumors and have been hypothesized to be waves of migrating glioblastoma cells. These "waves" of cells are thought to be induced by oxygen and nutrient depletion caused by tumor-induced blood vessel occlusion. Although the universal presence of these structures in GBM tumors suggests that they may play an instrumental role in GBM's spread and invasion, the recreation of these structures in vitro has remained challenging. Methods Here we present a new microfluidic model of GBM that mimics the dynamics of pseudopalisade formation. To do this, we embedded U-251 MG cells within a collagen hydrogel in a custom-designed microfluidic device. By controlling the medium flow through lateral microchannels, we can mimic and control blood-vessel obstruction events associated with this disease. Results Through the use of this new system, we show that nutrient and oxygen starvation triggers a strong migratory process leading to pseudopalisade generation in vitro. These results validate the hypothesis of pseudopalisade formation and show an excellent agreement with a systems-biology model based on a hypoxia-driven phenomenon. Conclusions This paper shows the potential of microfluidic devices as advanced artificial systems capable of modeling in vivo nutrient and oxygen gradients during tumor evolution.