Effects of substrate-derived cues in driving the selforganization of organoid-derived intestinal epithelia

[eng] Intestinal cells self-organize into 3-dimensional (3D) organoids that recapitulate the in vivo structural and functional characteristics when embedded in a 3D cell-derived protein mixture (Matrigel). However, these very same cells self-organize into 2-dimensional (2D) intestinal epithelial monolayers that recapitulate the in vivo-like cell organization when seeded on thin layers of the same cell-derived protein mixture. Moreover, in vivo, the intestine integrates regulation from paracrine signals to establish its characteristic crypt-villus axis self- organization. However, direct experimental manipulation of these paracrine signals, as well as their functional concentrations and effects at the cellular level, has been hampered by limitations of the in vivo and in vitro currently available systems. In general, changes in epithelial cell organization are characterized by a cross-talk between cell-substrate and cell-cell interactions, but the role of the ECM dimensionality, protein composition and spatial distribution in the intestinal epithelial cells’ organization is not fully understood. In this thesis, we show that intestinal epithelial cells self-organize in 2D-monolayers or 3D-tubular networks depending on the Matrigel protein concentration when the dimensionality is fixed. This self-assembles tubular networks have inner apical polarization and are similar to soap foams or de-wetted collagen networks. They have well defined topological and metrical properties and become spontaneously ordered at large length scales. Interestingly, stem cells have a particular dynamic during the formation of each self-organized patterns. On low Matrigel concentration, stem cells present a confined random movement to form a 2D-monolayer. In contrast, on higher Matrigel concentration, stem cells perform a direct motion towards a specific target to form the 3D- tubular networks. By reducing the proportion of stem cells in the culture, the formation of 3D-tubular networks is impaired. Instead, primary cells form aggregates when seeded above the transition protein concentration, similar to two other epithelial cell types (Caco-2 and MDCK cells). On the other hand, the 2D-monolayers formed on low Matrigel concentration contain crypt- and villus-like domains resembling those found in vivo. These compartments are randomly distributed and their shape is not uniform. However, by producing localized micropatterns of immobilized Wnt and ephrin factors on freeze-dried Matrigel-coated substrates by microcontact printing, we can drive the compartmentalization of the intestinal epithelial monolayers by spatially positioning the crypt- and villus-like domains. Finally, by changing the shape and dimension of the patterns we can control the distance between the crypt-like domains as well as their dimensions and shape. Overall, our experiments illustrate how Matrigel concentration regulates intestinal epithelial cell organization as a function of cell-substrate adhesion, and show that primary intestinal epithelial cells self-organize in structures with well-defined sizes and shapes independently of dimensionality or external signaling gradients. Also, we show that the amount of stem cells in the culture regulates the geometry of those self-organized structures. On the other hand, micropatterns of immobilized proteins to the ECM provides accurate control of the crypt-villus domain positioning in our epithelial monolayers. Thus, our work could yield insights about the roles of stem cells and protein concentration in tissue morphogenesis and their influence in the in vivo tissue morphological features such as the dimension of the crypts. In addition, we believe our platform will allow an easy and reliable manner to analyze the effect of relevant proteins on the epithelial cell compartmentalization, as well as the study of important intestinal epithelial processes such as stem cells proliferation, cell migration and differentiation both in homeostasis and pathological processes.[spa] Las células intestinales cultivadas dentro de Matrigel se auto-organizan en organoides tridimensionales (3D) que recapitulan la organización del tejido in vivo. Sin embargo, estas mismas células cultivadas sobre láminas del mismo sustrato se auto-organizan en monocapas bidimensionales (2D) que también recapitulan la organización del tejido in vivo. Además, in vivo, el intestino integra señales paracrinas para establecer su característica auto- organización en criptas y vellosidades. Sin embargo, la manipulación de estás señalizaciones se ha visto obstaculizada por limitaciones en los sistemas in vivo e in vitro actuales. En general, la organización epitelial se caracteriza por interacciones célula-célula y célula-sustrato. Sin embargo, no se acaba de entender el rol de la dimensión, la composición y la distribución de la matriz extracelular (ECM) sobre la organización de las células epiteliales del intestino. En esta tesis, se analiza la organización de células epiteliales en monocapas 2D o redes tubulares 3D en función de la adhesión célula-sustrato. De esta manera, se ilustra la organización en estructuras con tamaños y formas bien definidas independientemente de la dimensionalidad o señalizaciones externas. Además, la proporción de células madre regula la geometría de dichas estructuras. Por otro lado, en contraste con lo que se observa in vivo, los dominios de cripta de las monocapas están desordenados y su forma no es uniforme. Mediante una plataforma que localiza micropatrones de proteínas sobre la ECM, controlamos el posicionamiento de los dominios de cripta-vellosidad. Para concluir, nuestro trabajo proporciona información sobre como influencia la composición y la distribución de la ECM y las células madre en la morfología del tejido in vivo, como la dimensión de las criptas. Además, la plataforma permite analizar el efecto de diferentes proteínas en la compartimentación de las células y en otros procesos epiteliales como proliferación, migración o diferenciación celular, tanto en homeostasis como en proceso patológicos

Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies

Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand–receptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique

New technologies and apps for the study, conservation and dissemination of Contemporary Art. The Basque Country University ́s Collection

Uno de los grandes retos a los que se enfrentan los futuros conservadores y conservadoras de arte del siglo XXI, cuya disciplina ha estado tradicionalmente vinculada al trabajo manual, es adaptarse a las nuevas metodologías de investigación y restauración ligadas al uso de herramientas y medios digitales. Las nuevas tecnologías y apps disponibles en el mercado pueden ayudar a llevar a cabo protocolos de estudio, análisis y diagnóstico que beneficien la preservación de las obras, convirtiéndose en un medio de conservación pasivo mediante la documentación y el registro. El presente texto recoge la experiencia y los primeros resultados de la aplicación de un proyecto de innovación cuyo objetivo principal es proporcionar a los futuros profesionales de la conservación los instrumentos necesarios para el desarrollo de su práctica profesional. El planteamiento busca para ello integrar diferentes dispositivos y recursos tecnológicos para catalogar, conservar y difundir el patrimonio artístico de la Universidad del País Vasco, así como evaluar su utilidad y efectividad como medio de estudio y monitorización de las obras de arte que componen su colección.One of the challenges faced by the future 21st century art conservators, whose profession has traditionally been linked to manual work, is to adapt to the new research and restoration methodologies linked to use of tools and digital media. The new technologies and apps available in the market can help carry out study, analysis and diagnostic protocols that benefit the preservation ofthe works, becoming a means of passive conservation through documentation and registration. This text gathers the experience and the first results of the application of an innovation project whose main objective is to provide future conservation professionals with the necessary instruments for the development of their professional practice. The approach seeks to integrate different devices and technological resources to catalog, conserve and disseminate the artistic heritage of the University of the Basque Country, as well as evaluate its usefulness and effectiveness for the study and monitoring of the works of art that make up its collection

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies

Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate with molecular sensitivity the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

New technologies and apps for the study, conservation and dissemination of Contemporary Art. The Basque Country University´s Collection

Uno de los grandes retos a los que se enfrentan los futuros conservadores y conservadoras de arte del siglo XXI, cuya disciplina ha estado tradicionalmente vinculada al trabajo manual, es adaptarse a las nuevas metodologías de investigación y restauración ligadas al uso de herramientas y medios digitales. Las nuevas tecnologías y apps disponibles en el mercado pueden ayudar a llevar a cabo protocolos de estudio, análisis y diagnóstico que beneficien la preservación de las obras, convirtiéndose en un medio de conservación pasivo mediante la documentación y el registro. El presente texto recoge la experiencia y los primeros resultados de la aplicación de un proyecto de innovación cuyo objetivo principal es proporcionar a los futuros profesionales de la conservación los instrumentos necesarios para el desarrollo de su práctica profesional. El planteamiento busca para ello integrar diferentes dispositivos y recursos tecnológicos para catalogar, conservar y difundir el patrimonio artístico de la Universidad del País Vasco, así como evaluar su utilidad y efectividad como medio de estudio y monitorización de las obras de arte que componen su colección.One of the challenges faced by the future 21st century art conservators, whose profession has traditionally been linked to manual work, is to adapt to the new research and restoration methodologies linked to use of tools and digital media. The new technologies and apps available in the market can help carry out study, analysis and diagnostic protocols that benefit the preservation of the works, becoming a means of passive conservation through documentation and registration. This text gathers the experience and the first results of the application of an innovation project whose main objective is to provide future conservation professionals with the necessary instruments for the development of their professional practice. The approach seeks to integrate different devices and technological resources to catalog, conserve and disseminate the artistic heritage of the University of the Basque Country, as well as evaluate its usefulness and effectiveness for the study and monitoring of the works of art that make up its collection

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies

Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies