Staff member

Elena Martínez Fraiz

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+34 934 039775
Staff member publications

Torras N, Zabalo J, Abril E, Carré A, García-Díaz M, Martínez E, (2023). A bioprinted 3D gut model with crypt-villus structures to mimic the intestinal epithelial-stromal microenvironment Biomaterials Advances 153, 213534

The intestine is a complex tissue with a characteristic three-dimensional (3D) crypt-villus architecture, which plays a key role in the intestinal function. This function is also regulated by the intestinal stroma that actively supports the intestinal epithelium, maintaining the homeostasis of the tissue. Efforts to account for the 3D complex structure of the intestinal tissue have been focused mainly in mimicking the epithelial barrier, while solutions to include the stromal compartment are scarce and unpractical to be used in routine experiments. Here we demonstrate that by employing an optimized bioink formulation and the suitable printing parameters it is possible to produce fibroblast-laden crypt-villus structures by means of digital light projection stereolithography (DLP-SLA). This process provides excellent cell viability, accurate spatial resolution, and high printing throughput, resulting in a robust biofabrication approach that yields functional gut mucosa tissues compatible with conventional testing techniques.Copyright © 2023 Elsevier B.V. All rights reserved.

JTD Keywords: 3d microstructure, barrier, cells, epithelial-stromal interactions, gelma-pegda soft hydrogels, growth, hydrogel, intestinal mucosa model, methacrylamide, microfabrication, proliferation, scaffold, stereolithography, 3d bioprinting, 3d microstructure, Epithelial-stromal interactions, Fibroblasts, Gelma-pegda soft hydrogels, Intestinal mucosa model

Macedo MH, Torras N, García-Díaz M, Barrias C, Sarmento B, Martínez E, (2023). The shape of our gut: Dissecting its impact on drug absorption in a 3D bioprinted intestinal model Biomaterials Advances 153, 213564

The small intestine is a complex organ with a characteristic architecture and a major site for drug and nutrient absorption. The three-dimensional (3D) topography organized in finger-like protrusions called villi increases surface area remarkably, granting a more efficient absorption process. The intestinal mucosa, where this process occurs, is a multilayered and multicell-type tissue barrier. In vitro intestinal models are routinely used to study different physiological and pathological processes in the gut, including compound absorption. Still, standard models are typically two-dimensional (2D) and represent only the epithelial barrier, lacking the cues offered by the 3D architecture and the stromal components present in vivo, often leading to inaccurate results. In this work, we studied the impact of the 3D architecture of the gut on drug transport using a bioprinted 3D model of the intestinal mucosa containing both the epithelial and the stromal compartments. Human intestinal fibroblasts were embedded in a previously optimized hydrogel bioink, and enterocytes and goblet cells were seeded on top to mimic the intestinal mucosa. The embedded fibroblasts thrived inside the hydrogel, remodeling the surrounding extracellular matrix. The epithelial cells fully covered the hydrogel scaffolds and formed a uniform cell layer with barrier properties close to in vivo. In particular, the villus-like model revealed overall increased permeability compared to a flat counterpart composed by the same hydrogel and cells. In addition, the efflux activity of the P-glycoprotein (P-gp) transporter was significantly reduced in the villus-like scaffold compared to a flat model, and the genetic expression of other drugs transporters was, in general, more relevant in the villus-like model. Globally, this study corroborates that the presence of the 3D architecture promotes a more physiological differentiation of the epithelial barrier, providing more accurate data on drug absorbance measurements.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: 3d architecture, alkaline-phosphatase, caco-2 cells, culture, drug development, efflux proteins, gene-expression, human-colon, intestinal absorption, intestinal models, microenvironment, paracellular transport, permeability, photopolymerization, villi, 3d architecture, 3d bioprinting, Drug development, In-vitro, Intestinal absorption, Intestinal models, Photopolymerization, Villi

Srinivasan, SY, Cler, M, Zapata-Arteaga, O, Dorling, B, Campoy-Quiles, M, Martinez, E, Engel, E, Perez-Amodio, S, Laromaine, A, (2023). Conductive Bacterial Nanocellulose-Polypyrrole Patches Promote Cardiomyocyte Differentiation Acs Applied Bio Materials 6, 2860-2874

The low endogenous regenerative capacity of the heart,added tothe prevalence of cardiovascular diseases, triggered the advent ofcardiac tissue engineering in the last decades. The myocardial nicheplays a critical role in directing the function and fate of cardiomyocytes;therefore, engineering a biomimetic scaffold holds excellent promise.We produced an electroconductive cardiac patch of bacterial nanocellulose(BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the naturalmyocardial microenvironment. BC offers a 3D interconnected fiber structurewith high flexibility, which is ideal for hosting Ppy nanoparticles.BC-Ppy composites were produced by decorating the network of BC fibers(65 & PLUSMN; 12 nm) with conductive Ppy nanoparticles (83 & PLUSMN; 8 nm).Ppy NPs effectively augment the conductivity, surface roughness, andthickness of BC composites despite reducing scaffolds' transparency.BC-Ppy composites were flexible (up to 10 mM Ppy), maintained theirintricate 3D extracellular matrix-like mesh structure in all Ppy concentrationstested, and displayed electrical conductivities in the range of nativecardiac tissue. Furthermore, these materials exhibit tensile strength,surface roughness, and wettability values appropriate for their finaluse as cardiac patches. In vitro experiments withcardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibilityof BC-Ppy composites. BC-Ppy scaffolds improved cell viability andattachment, promoting a desirable cardiomyoblast morphology. Biochemicalanalyses revealed that H9c2 cells showed different cardiomyocyte phenotypesand distinct levels of maturity depending on the amount of Ppy inthe substrate used. Specifically, the employment of BC-Ppy compositesdrives partial H9c2 differentiation toward a cardiomyocyte-like phenotype.The scaffolds increase the expression of functional cardiac markersin H9c2 cells, indicative of a higher differentiation efficiency,which is not observed with plain BC. Our results highlight the remarkablepotential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerativetherapies.

JTD Keywords: bacterial nanocellulose, cardiac patches, conducting polymers, polypyrrole, Arrhythmias, Bacterial nanocellulose, Biomaterials, Cardiac patches, Cell therapy, Cellulose, Conductingpolymers, H9c2, In-vitro, Polymer, Polypyrrole, Scaffolds, Tissue, Tissue engineering, Viability

Comelles, Jordi, Fernández-Majada, Vanesa, Acevedo, Verónica, Rebollo-Calderon, Beatriz, Martínez, Elena, (2023). Soft topographical patterns trigger a stiffness-dependent cellular response to contact guidance Materials Today Bio 19, 100593

Topographical patterns are a powerful tool to study directional migration. Grooved substrates have been extensively used as in vitro models of aligned extracellular matrix fibers because they induce cell elongation, alignment, and migration through a phenomenon known as contact guidance. This process, which involves the orientation of focal adhesions, F-actin, and microtubule cytoskeleton along the direction of the grooves, has been primarily studied on hard materials of non-physiological stiffness. But how it unfolds when the stiffness of the grooves varies within the physiological range is less known. Here we show that substrate stiffness modulates the cellular response to topographical contact guidance. We find that for fibroblasts, while focal adhesions and actin respond to topography independently of the stiffness, microtubules show a stiffness-dependent response that regulates contact guidance. On the other hand, both clusters and single breast carcinoma epithelial cells display stiffness-dependent contact guidance, leading to more directional and efficient migration when increasing substrate stiffness. These results suggest that both matrix stiffening and alignment of extracellular matrix fibers cooperate during directional cell migration, and that the outcome differs between cell types depending on how they organize their cytoskeletons.© 2023 The Authors.

JTD Keywords: actin, behavior, cell migration, contact guidance, cytoskeleton, fibroblasts, focal adhesions, matrix, microtubules, stiffness, stress fibers, topography, transduction, Contact guidance, Substrate stiffness, Topography

Larrañaga, Enara, Fernández‐Majada, Vanesa, Ojosnegros, Samuel, Comelles, Jordi, Martinez, Elena, (2022). Ephrin Micropatterns Exogenously Modulate Cell Organization in Organoid‐Derived Intestinal Epithelial Monolayers Advanced Materials Interfaces 9, 2201301

García-Díaz, María, Cendra, Maria del Mar, Alonso-Roman, Raquel, Urdániz, María, Torrents, Eduard, Martínez, Elena, (2022). Mimicking the Intestinal Host–Pathogen Interactions in a 3D In Vitro Model: The Role of the Mucus Layer Pharmaceutics 14, 1552

The intestinal mucus lines the luminal surface of the intestinal epithelium. This mucus is a dynamic semipermeable barrier and one of the first-line defense mechanisms against the outside environment, protecting the body against chemical, mechanical, or biological external insults. At the same time, the intestinal mucus accommodates the resident microbiota, providing nutrients and attachment sites, and therefore playing an essential role in the host–pathogen interactions and gut homeostasis. Underneath this mucus layer, the intestinal epithelium is organized into finger-like protrusions called villi and invaginations called crypts. This characteristic 3D architecture is known to influence the epithelial cell differentiation and function. However, when modelling in vitro the intestinal host–pathogen interactions, these two essential features, the intestinal mucus and the 3D topography are often not represented, thus limiting the relevance of the models. Here we present an in vitro model that mimics the small intestinal mucosa and its interactions with intestinal pathogens in a relevant manner, containing the secreted mucus layer and the epithelial barrier in a 3D villus-like hydrogel scaffold. This 3D architecture significantly enhanced the secretion of mucus. In infection with the pathogenic adherent invasive E. coli strain LF82, characteristic of Crohn’s disease, we observed that this secreted mucus promoted the adhesion of the pathogen and at the same time had a protective effect upon its invasion. This pathogenic strain was able to survive inside the epithelial cells and trigger an inflammatory response that was milder when a thick mucus layer was present. Thus, we demonstrated that our model faithfully mimics the key features of the intestinal mucosa necessary to study the interactions with intestinal pathogens.

JTD Keywords: 3d in vitro models, barrier function, bile-salts, cells, drug-delivery, host-pathogen interaction, host–pathogen interaction, hydrogels, ileal mucosa, infection, intestinal models, intestinal mucus, microbiome, patient, responses, 3d in vitro models, Intestinal mucus, Invasive escherichia-coli

Clua-Ferre, L, De Chiara, F, Rodriguez-Comas, J, Comelles, J, Martinez, E, Godeau, AL, Garcia-Alaman, A, Gasa, R, Ramon-Azcon, J, (2022). Collagen-Tannic Acid Spheroids for beta-Cell Encapsulation Fabricated Using a 3D Bioprinter Advanced Materials Technologies 7, 2101696

Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.© 2022 The Authors. Advanced Materials Technologies published by Wiley‐VCH GmbH.

JTD Keywords: 3d bioprinter, beta-cell, biomaterial, collagen, encapsulation, mechanics, microspheres, survival, 3d bioprinter, ?-cell, Advanced material technologies, Biocompatibility, Cell encapsulations, Cells, Collagen, Cross-linking, Cytology, Drug delivery, Encapsulation, Fabrication, Flavonoids, Gelation, In-vitro, Insulin injections, Insulin release, Microspheres, Tannic acid, Tannins, Throughput, Tissue grafts, Type 1 diabetes, Β‐cell

Comelles J, Castillo-Fernández Ó, Martínez E, (2022). How to Get Away with Gradients Advances In Experimental Medicine And Biology 1379, 31-54

Biomolecular gradients are widely present in multiple biological processes. Historically they were reproduced in vitro by using micropipettes, Boyden and Zigmond chambers, or hydrogels. Despite the great utility of these setups in the study of gradient-related problems such as chemotaxis, they face limitations when trying to translate more complex in vivo-like scenarios to in vitro systems. In the last 20 years, the advances in manufacturing of micromechanical systems (MEMS) had opened the possibility of applying this technology to biology (BioMEMS). In particular, microfluidics has proven extremely efficient in setting-up biomolecular gradients which are stable, controllable, reproducible and at length scales that are relevant to cells. In this chapter, we give an overview of different methods to generate molecular gradients using microfluidics, then we discuss the different steps of the pipeline to fabricate a gradient generator microfluidic device, and at the end, we show an application example of the fabrication of a microfluidic device that can be used to generate a surface-bound biomolecular gradient.© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.

JTD Keywords: biomems, gradient, microfluidics, model, nanotechnology, proteins, Biomems, Gradient, Mechanisms, Microfabrication, Microfluidics, Nanotechnology

The influence of the properties of different solid substrates on the tethering of two antibodies, IgG1-CR3022 and IgG1-S309, which were specifically engineered for the detection of SARS-CoV-2, has been examined at the molecular level using conventional and accelerated Molecular Dynamics (cMD and aMD, respectively). Two surfaces with very different properties and widely used in immunosensors for diagnosis, amorphous silica and the most stable facet of the face-centered cubic gold structure, have been considered. The effects of such surfaces on the structure and orientation of the immobilized antibodies have been determined by quantifying the tilt and hinge angles that describe the orientation and shape of the antibody, respectively, and the dihedrals that measure the relative position of the antibody arms with respect to the surface. Results show that the interactions with amorphous silica, which are mainly electrostatic due to the charged nature of the surface, help to preserve the orientation and structure of the antibodies, especially of the IgG1-CR3022, indicating that the primary sequence of those antibodies also plays some role. Instead, short-range van der Waals interactions with the inert gold surface cause a higher degree tilting and fraying of the antibodies with respect to amorphous silica. The interactions between the antibodies and the surface also affect the correlation among the different angles and dihedrals, which increases with their strength. Overall, results explain why amorphous silica substrates are frequently used to immobilize antibodies in immunosensors. © 2022 The Authors

JTD Keywords: amorphous silica, antibody immobilization, enzyme, gol d, gold, immobilization, immunosensor, molecu l a r dynamics, molecular dynamics, protein adsorption, sars-cov-2 immunosensor, simulations, spike protein, surface interactions, target, vaccine, Amorphous silica, Antibodies, Antibody engineering, Antibody immobilization, Antibody structure, Article, Chemical detection, Computer model, Controlled study, Dihedral angle, Gold, In-silico, Molecular dynamics, Molecular levels, Molecular-dynamics, Nonhuman, Property, Sars, Sars-cov-2 immunosensor, Severe acute respiratory syndrome coronavirus 2, Silica, Silico studies, Silicon dioxide, Solid substrates, Structure analysis, Substrate chemistry, Substrates, Van der waals forces, Virus detection

Torras Andres, N, Zabalo, J, Abril, E, Martinez, E, (2022). 3D-BIOPRINTING SYSTEM FOR ENGINEERING COMPLEX, SOFT HYDROGEL SCAFFOLDS (Abstract 775) Tissue Engineering Part a 28, S218-S219

There is an increasing evidence that the tissue architecture has astrong influence on cell behavior and it is key to obtain functionaltissue replicates (1). Hence, future improvements in the tissue en-gineering field will encompass tailoring three-dimensional (3D) cellmicroenvironments with the precision required to mimic in vivofeatures.3D printing techniques have been positioned as a feasible alter-native to conventional manufacturing techniques to achieve properspatial resolution in a high-throughput manner, mainly due to highadaptability and reduced costs, becoming one of the current hottopics in applied research (2,3). However, requirements in terms ofresolution, mechanical properties, or biocompatibility to obtainbiomimetic 3D tissues demand further innovative approaches.Here we present a fast, low cost and versatile printing methodbased on visible light polymerization for the fabrication of 3Dbioengineered substrates and cell-friendly interfaces using photo-polymerizable hydrogel-based bioinks. These bioinks are highlyS-218ABSTRACTStransparent and have low molecular content, mimicking the softmechanical properties of the tissues.With the appropriate combination of bioink composition, accurateselection of the printing parameters, and optimized designs we suc-ceeded with prints for different applications, including cell culturesubstrates, microfluidic channels and tissue constructs for in vitroassays, compatible with the standard cell culture techniques. As aproof-of-concept, we developed functional scaffolds mimicking the3D microstructure of the small intestine, containing both the epi-thelial and stromal compartments, using cell-laden bioinks.Through this versatile top-down technique we demonstrated thepotential of the light-based 3D bioprinting technology, contributingon providing alternatives beyond tissue engineering state-of-the-art.


Martinez-Hernandez, M, Ordono, J, Tuduri, L, Castano, O, Perez-Amodio, S, Martinez, E, Engel, E, (2022). MODULATION OF CARDIAC FIBROBLAST ACTIVATION THROUGH LACTATE-BASED BIOMATERIALS FOR IN SITU CARDIAC REGENERATION (Abstract 966) Tissue Engineering Part a 28, S271-S272

INTRODUCTION: Lactate, an important metabolite during car-diogenesis and cardiac development, has been recently shown topromote cardiomyocyte proliferation and reprogramming towards adedifferentiated stem cell-like state.1 Non-myocytic cardiac fibro-blasts (CFBs) have a key role during cardiac remodeling aftermyocardial infarction, where they become activated and releaseextracellular matrix proteins. Excessive extracellular matrix depo-sition or myofibroblast activation is detrimental to cardiac repair.The aim of this work is to elucidate the effect of lactate on CFBsfor a complete interpretation of its potential proregenerative cap-abilities. Then, we developed bioactive lactate-releasing scaffoldsfor cardiac regeneration.METHODS: CFBs metabolic activity and proliferation were as-sessed. The inflammatory and fibrotic responses to exogenous lactatewere investigated in terms of cytokine and collagen production,migration assays and myofibroblast differentiation. Then, polylactic-acid(PLA) scaffolds were fabricated by electrospinning and phy-siochemically characterized (SEM, DSC, lactate release,.).RESULTS: Our results indicate that lactate does not affect fi-broblast proliferation, migration, collagen production, or activationand significantly reduces the expression of detrimental cytokines forcardiac repair. Furthermore, our PLA scaffolds are amorphous,biodegradable, nanofibrous materials that release lactate in a sus-tained manner. They do not affect CFB viability and promote cellattachment.DISCUSSION: Altogether, this study further supports the pro-spective use of lactate as a bioactive signal in new endogenouscardiac regeneration therapies. Thus, PLA patches that releaselactate as a major product from its breakdown can then be used as anABSTRACTSS-271effective source of lactate for alleviating the aftermath of myo-cardial infarction.This project was funded by ’’LaCaixa’’ Foundation(LCF/BQ/DR19/11740025) and MINECO/FEDER(RTI2018-096320-B-C21).


Abad, A, Altay, G, Gualda, E, Garcia-Diaz, M, Torras, N, Folch, J, Tosi, S, Fernandez, V, Batlle, E, Loza, P, Martinez, E, (2022). MICROENGINEERED VILLUS-LIKE PEGDA HYDROGELS UNDER SPATIO-BIOCHEMICAL GRADIENTS FOR PRIMARY INTESTINAL EPITHELIUM IN VITRO MODEL (Abstract 874) Tissue Engineering Part a 28, S245

The intestinal epithelium is formed by villi and crypts. Intestinalstem cells (ISCs) located at the crypt base divide giving rise toproliferative cells that migrate up along the villi while differentiating,ultimately dying at the tips of the villi. This homeostasis is tightlycontrolled by biomolecular gradients of EGF, Wnt and BMP sig-naling pathways along the crypt-villus axis1. Intestinal organoids,despite including many physiologically relevant features, are notvalid cultures when access to the lumen is required. Here we present aculture platform that overcomes this limitation while comprising allkey features of the intestinal epithelium: 3D architecture, prolifera-tive and differentiated cell domains, and gradients of ISCs nichebiomolecules.Employing a simple photolithographic technique2, we fabricatedpoly(ethylene) glycol diacrylate (PEGDA) 3D villus-like scaffolds.We developed in silico models to simulate gradients of ISCs nichebiomolecules, we created them through the hydrogels by free diffusionand we characterized them by Light-sheet fluorescence microscopy.Organoid-derived intestinal epithelial cells covered the wholescaffold surface. The gradients profile and composition, constantover time, impacted on cell behavior by modifying the proportionand positioning of the different intestinal epithelial cell types alongthe vertical axis of our scaffold, faithfully recreating in vivo cellcompartmentalization.We have developed an apically accessible and 3D in vitro intes-tinal epithelial model, which bears biomolecular ISC niche gradientsand all relevant epithelial cell types. Therefore, we believe our modelcan be employed in many applications, particularly in the study ofintestinal epithelium biology in physiological and pathologicalconditions.


Fernandez-Garibay, X, Ortega, MA, Cerro-Herreros, E, Comelles, J, Martinez, E, Artero, R, Fernandez-Costa, JM, Ramon-Azcon, J, (2022). BIOENGINEERED IN VITRO 3D MODEL OF MYOTONIC DYSTROPHY TYPE 1 HUMAN SKELETAL MUSCLE (Abstract 2087) Tissue Engineering Part a 28, S591

Myotonic dystrophy type 1 (DM1) is the most common hereditarymyopathy in adults. The disease is characterized by progressiveskeletal muscle degeneration that produces severe disability. There isstill no effective treatment for DM1 patients, but new therapeuticstrategies are being tested. Animal models and in vitro 2D cell cul-tures have been essential for these advances. However, these modelscannot reproduce the biological complexity of the disease. Biofab-rication tools can be applied to engineer human 3D culture systemsthat complement current preclinical research models.Here, we describe the development of the first in vitro 3D model ofDM1 human skeletal muscle. Patient-derived cells were encapsulatedin micromolded gelatin methacryloyl-carboxymethyl cellulose meth-acrylate (GelMA-CMCMA) hydrogels through photomold patterning.These hydrogels present a microstructured topography that promotesmyoblast alignment and differentiation, resulting in highly alignedmyotubes from healthy and DM1 cells. The DM1 3D microtissuespresent the molecular alterations detected in patient biopsies. Im-portantly, fusion index analyses demonstrate that 3D micropatterningsignificantly improved DM1 cell differentiation into multinucleatedmyotubes compared to standard cell cultures. Moreover, character-ization of the 3D cultures of DM1 myotubes detects a reduced thick-ness of myotubes that can be used for drug screening. Therefore, weevaluated the therapeutic effect of antagomiR-23b administration onbioengineered DM1 skeletal muscle microtissues. AntagomiR-23btreatment rescues both molecular DM1 hallmarks and structural phe-notype, restoring myotube diameter to healthy control sizes. Overall,these new microtissues represent an improvement over conventionalmodels and can be used as biomimetic platforms to establish preclin-ical studies for myotonic dystrophy.


Breideband, L, Pampaloni, F, Martensson, G, Eklund, R, Wurst, H, Angres, B, Torras, N, Martinez, E, Shalom-Feuerstein, R, (2022). BIOPRINTING BY LIGHT SHEET LITHOGRAPHY: ENGINEERING COMPLEX TISSUES WITH HIGH RESOLUTION AT HIGH SPEED (Abstract 1581) Tissue Engineering Part a 28, S443

Three-dimensional bioprinting (3D bioprinting) has been at theforefront of tissue engineering research in the past years, with evermore efficient systems reaching the market(1). While existing 3Dbioprinting techniques are numerous and varied, they are limited bylong printing times when used at high resolution(2). The techniquedescribed in this work aims at enabling fast and accurate productionof monolayered skin constructs.To achieve shorter production times, a digital scanned light sheetis used to produce patterns of polymerized hydrogel, which enablesthe printing of a full three-dimensional plane in a matter of a fewhundred milliseconds. The high resolution resides in the properties ofthe light sheet itself – the width of the light sheet represents the z-axial resolution of the system (as low as 10mm) and the x-axialresolution is determined by the intensity profile of the gaussian beam(around 50mm). In order to fully exploit this system, the hydrogelused to encapsulate the cells must therefore be tailor-made for pho-toactivated cross-linking.As a proof of concept, a light sheet microscope is used as a po-lymerization source for novel photosensitive hydrogels. The up-coming hardware, software, chemical and biological improvementsneeded to reach the full potential of this system are expected toeventually be sufficient to print a complete skin construct, whichcould be used in the drug development industry, or as a graft forregenerative medicine therapy. Additionally, the constructs can beused to reduce and even replace animal testing for drug or cosmetictesting.

JTD Keywords: 3d bioprinting, Light sheet microscopy, Stereolithography

Garcia-Diaz, M, Abad, A, Torras, N, Martinez, E, (2022). MIMICKING THE INTESTINAL 3D TOPOGRAPHY WITH PHOTOCROSSLINKED HYDROGEL SCAFFOLDS (Abstract 1198) Tissue Engineering Part a 28, S335-S336

The intestinal epithelium is characterized by a complex micro-scale topography formed by finger-like protrusions called villi andinvaginations called crypts. This organized three-dimensional (3D)architecture together with the soft mechanical properties of the in-testinal tissue are essential for cell behavior and tissue function.However, the experimental in vitro modeling of the delicate intes-tinal architecture is limited due to the difficulty in microfabricatingsoft materials with complex 3D geometries, high aspect ratio andcurvature using efficient and simple methods. In this work, we fab-ricated soft hydrogel scaffolds that accurately mimic the villus andcrypt morphologies of the intestinal epithelium using a simple andmoldless approach (1).We used mixtures of polyethylene glycol diacrylate (PEGDA)and acrylic acid (AA), or PEGDA and gelatin methacryloyl (Gel-MA) to fabricate 3D hydrogels by a single-step photolithographyprocess. Different photomasks were used for the fabrication of themicropillars mimicking the intestinal villi and the crypt-like in-vaginations. The dimensions of the villi and crypts were easilyfine-tuned just by changing the energy dose. The scaffolds weredirectly microfabricated onto permeable membranes, enablingtheir assembly into cell culture inserts. Intestinal epithelial cellslines or organoid-derived primary cells covered the biomimeticscaffolds and form polarized monolayers with proper barrierproperties. The use of PEGDA-GelMA blends also allowed thefabrication of cell-laden scaffolds that mimic the intestinal stromalcompartment.Through these results, we demonstrated that this microfabricationtechnology is a promising tool to faithfully replicate the intestinalmucosa topography with improved performance, while keepingcompatibility with standard cell culture techniques.


Garcia-Diaz, M, Altay, G, Tosi, S, Martinez, E, (2022). CELL MORPHOLOGICAL RESPONSE TO 3D TOPOGRAPHY AND CURVATURE IN ENGINEERED INTESTINAL IN VITRO MODELS (Abstract 1200) Tissue Engineering Part a 28, S336

Many epithelial tissues have complex three-dimensional (3D)topographies that are inherently curved. Recent advances in mi-crofabrication techniques have allowed the development of so-phisticated in vitro models that replicate this complex architectureof the native tissues. In particular, engineered intestinal tissuesoften use hydrogels to mimic villi structures (1). These finger-likeprotrusions of a few hundred microns in height have a well-definedtopography and curvature. However, the characterization of theselarge tissue engineered constructs at single-cell resolution is tech-nically challenging. Confocal microscopy imaging is limited by thesample thickness, whereas routine histological procedures are notsuitable for high water content samples such as hydrogels. Wedeveloped a novel embedding method that allows for the histo-logical processing of these delicate hydrogel structures (2). Usinglow molecular weight poly(ethylene glycol) diacrylate (PEGDA) asembedding media, we obtained a block that could be further sec-tioned with vibrating microtome or cryotome faithfully preservingthe villi-like structures of the scaffold. We then examined the cellmorphological response to the villus-like microstructures by high-resolution imaging of the cross-sections. We analyzed, in a spatiallyresolved manner, the cellular and nuclear morphology along thevilli. We observed that cell morphological response was highlyinfluenced by the microstructures showing significant differences incell height and shape at different regions of the villi that presenteddistinguished curvatures. These findings, which are in goodagreement with the data reported for in vivo experiments on theintestinal native tissue, highlight the impact of the micron-scaletopography and curvature on epithelial cell behavior.


Macedo, MH, Barros, AS, Martinez, E, Barrias, CC, Sarmento, B, (2022). All layers matter: Innovative three-dimensional epithelium-stroma-endothelium intestinal model for reliable permeability outcomes Journal Of Controlled Release 341, 414-430

Drug development is an ever-growing field, increasingly requesting reliable in vitro tools to speed up early screening phases, reducing the need for animal experiments. In oral delivery, understanding the absorption pattern of a new drug in the small intestine is paramount. Classical two-dimensional (2D) in vitro models are generally too simplistic and do not accurately represent native tissues. The main goal of this work was to develop an advanced three-dimensional (3D) in vitro intestinal model to test absorption in a more reliable manner, by better mimicking the native environment. The 3D model is composed of a collagen-based stromal layer with embedded fibroblasts mimicking the intestinal lamina propria and providing support for the epithelium, composed of enterocytes and mucus-secreting cells. An endothelial layer, surrogating the absorptive capillary network, is also present. The cellular crosstalk between the different cells present in the model is unveiled, disclosing key players, namely those involved in the contraction of collagen by fibroblasts. The developed 3D model presents lower levels of P-glycoprotein (P-gp) and Multidrug Resistance Protein 2 (MRP2) efflux transporters, which are normally overexpressed in traditional Caco-2 models, and are paramount in the absorption of many compounds. This, allied with transepithelial electrical resistance (TEER) values closer to physiological ranges, leads to improved and more reliable permeability outcomes, which are observed when comparing our results with in vivo data.

JTD Keywords: 3d intestinal model, drug absorption, drug development, endothelium, hydrogel, 3d intestinal model, 3d modeling, 3d models, 3d-modeling, Alkaline-phosphatase, Animal experiments, Biopharmaceutics classification, Caco-2 cells, Cell culture, Collagen, Collagen gel, Drug absorption, Drug development, Endothelium, Fibroblasts, Glycoproteins, Hydrogel, In-vitro, Matrix metalloproteinases, Membrane-permeability, Paracellular transport, Permeability, Single-pass vs., Speed up

Fernández-Garibay X, Ortega MA, Cerro-Herreros E, Comelles J, Martínez E, Artero R, Fernández-Costa JM, Ramón-Azcón J, (2021). Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle Biofabrication 13, 035035

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.

JTD Keywords: 3d cell culture, hydrogel micropatterning, myotonic dystrophy, skeletal muscle, tissue engineering, 3d cell culture, Hydrogel micropatterning, Myotonic dystrophy, Skeletal muscle, Tissue engineering

De Goede M, Dijkstra M, Chang L, Acharyya N, Kozyreff G, Obregón R, Martínez E, García-Blanco SM, (2021). Mode-splitting in a microring resonator for self-referenced biosensing Optics Express 29, 346-358

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement. Self-referenced biosensing based on mode-splitting on a microring resonator is experimentally demonstrated. A Bragg grating integrated on the surface of the ring provides coupling between the clockwise and counterclockwise travelling modes of the pristine ring resonator lifting their degeneracy. The amount of mode-splitting is directly related to the reflectivity of the grating and it is only affected by structurally modifying the grating. Environmental perturbations to the surroundings of the gratings, such as temperature and bulk refractive index variations, have a minor effect on the amount of mode-splitting. This principle allows the realization of a self-referenced sensing scheme based on the detection of variations of the mode-splitting induced by structural changes to the grating. In this work, a polymethyl methacrylate (PMMA) Bragg grating is integrated onto a ring resonator in Al2O3. It is shown both theoretically and experimentally that the amount of splitting of a resonance varies minimally under temperature or bulk refractive index perturbations. However, the structural change of attaching a layer of biomolecules inside the grating does affect its reflectivity and the amount of mode splitting present. This result represents the first proof-of-concept demonstration of an integrated mode-splitting biosensor insensitive to temperature and refractive index variations of the liquid matrix where the molecules to be detected are embedded. The reported results pave the road towards the realization of truly self-referenced biosensors.


Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the 5309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

JTD Keywords: cr3022, igg1, molecular engineering, s309, Antibodies, Antibody engineering, Biosensors, Chemical detection, Clinical application, Cov, Cr3022, Crystal structure, Design, Diseases, Gold nanoparticles, Igg1, Igg1 antibody, Immobilization, Immunoglobulin g, Immunosensor, In-silico, Merging, Molecular dynamics, Molecular engineering, Orientation, Protein-based biosensors, Receptor-binding domains, S309, Sars, Sensor, Spike protein, Target, Vaccine, Viruses

Macedo, M.H., Martínez, Elena, Barrias, Cristina C., Sarmento, B., (2020). Development of an improved 3D in vitro intestinal model to perform permeability studies of paracellular compounds Frontiers in Bioengineering and Biotechnology 8, 524018

The small intestine is the primary site of drug absorption following oral administration, making paramount the proper monitoring of the absorption process. In vitro tools to predict intestinal absorption are particularly important in preclinical drug development since they are less laborious and cost-intensive and raise less ethical considerations compared to in vivo studies. The Caco-2 model is considered the gold standard of in vitro intestinal models regarding the prediction of absorption of orally delivered compounds. However, this model presents several drawbacks, such as the expression of tighter tight junctions, not being suitable to perform permeability of paracellular compounds. Besides, cells are representative of only one intestinal cell type, without considering the role of non-absorptive cells on the absorption pathway of drugs. In the present study, we developed a new three-dimensional (3D) intestinal model that aims to bridge the gap between in vitro tools and animal studies. Our 3D model comprises a collagen layer with human intestinal fibroblasts (HIFs) embedded, mimicking the intestinal lamina propria and providing 3D support for the epithelium, composed of Caco-2 cells and mucus-producing HT29-MTX cells, creating a model that can better resemble, both in terms of composition and regarding the outcomes of drug permeability when testing paracellular compounds, the human small intestine. The optimization of the collagen layer with HIFs was performed, testing different collagen concentrations and HIF seeding densities in order to avoid collagen contraction before day 14, maintaining HIF metabolically active inside the collagen disks during time in culture. HIF morphology and extracellular matrix (ECM) deposition were assessed, confirming that fibroblasts presented a normal and healthy elongated shape and secreted fibronectin and laminin, remodeling the collagen matrix. Regarding the epithelial layer, transepithelial electrical resistance (TEER) values decreased when cells were in the 3D configuration, comparing with the 2D analogs (Caco-2 and coculture of Caco-2+HT29-MTX models), becoming more similar with in vivo values. The permeability assay with fluorescein isothiocyanate (FITC)–Dextran 4 kDa showed that absorption in the 3D models is significantly higher than that in the 2D models, confirming the importance of using a more biorelevant model when testing the paracellular permeability of compounds.


Fernánez-Majada, V., García-Díaz, María, Torras, N., Raghunath, M., Martínez, Elena, (2020). Editorial: When the shape does matter: Three-dimensional in vitro models of epithelial barriers Frontiers in Bioengineering and Biotechnology 8, 617361

Vila, A., Torras, N., Castaño, Albert G., García-Díaz, María, Comelles, Jordi, Pérez-Berezo, T., Corregidor, C., Castaño, O., Engel, E., Fernández-Majada, Vanesa, Martínez, Elena, (2020). Hydrogel co-networks of gelatine methacrylate and poly(ethylene glycol) diacrylate sustain 3D functional in vitro models of intestinal mucosa Biofabrication 12, 025008

Mounting evidence supports the importance of the intestinal epithelial barrier and its permeability both in physiological and pathological conditions. Conventional in vitro models to evaluate intestinal permeability rely on the formation of tightly packed epithelial monolayers grown on hard substrates. These two-dimensional (2D) models lack the cellular and mechanical components of the non-epithelial compartment of the intestinal barrier, the stroma, which are key contributors to the barrier permeability in vivo. Thus, advanced in vitro models approaching the in vivo tissue composition are fundamental to improve precision in drug absorption predictions, to provide a better understanding of the intestinal biology, and to faithfully represent related diseases. Here, we generate photo-crosslinked gelatine methacrylate (GelMA) - poly(ethylene glycol) diacrylate (PEGDA) hydrogel co-networks that provide the required mechanical and biochemical features to mimic both the epithelial and stromal compartments of the intestinal mucosa, i.e., they are soft, cell adhesive and cell-loading friendly, and suitable for long-term culturing. We show that fibroblasts can be embedded in the GelMA-PEGDA hydrogels while epithelial cells can grow on top to form a mature epithelial monolayer that exhibits barrier properties which closely mimic those of the intestinal barrier in vivo, as shown by the physiologically relevant transepithelial electrical resistance (TEER) and permeability values. The presence of fibroblasts in the artificial stroma compartment accelerates the formation of the epithelial monolayer and boosts the recovery of the epithelial integrity upon temporary barrier disruption, demonstrating that our system is capable of successfully reproducing the interaction between different cellular compartments. As such, our hydrogel co-networks offer a technologically simple yet sophisticated approach to produce functional three-dimensional (3D) in vitro models of epithelial barriers with epithelial and stromal cells arranged in a spatially relevant manner and near-physiological functionality.


Altay, Gizem, Tosi, Sébastien, García-Díaz, María, Martínez, Elena, (2020). Imaging the cell morphological response to 3D topography and curvature in engineered intestinal tissues Frontiers in Bioengineering and Biotechnology 8, 294

While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.

JTD Keywords: Hydrogel scaffold, Confocal microscopy, Substrate curvature, Cell morphology, Cell orientation, Histological section, Small intestine, Villus

Steeves, A.J., Ho, W., Munisso, M.C., Lomboni, D.J., Larrañaga, E., Omelon, S., Martínez, Elena, Spinello, D., Variola, F., (2020). The implication of spatial statistics in human mesenchymal stem cell response to nanotubular architectures International Journal of Nanomedicine 15, 2151-2169

Introduction: In recent years there has been ample interest in nanoscale modifications of synthetic biomaterials to understand fundamental aspects of cell-surface interactions towards improved biological outcomes. In this study, we aimed at closing in on the effects of nanotubular TiO2 surfaces with variable nanotopography on the response on human mesenchymal stem cells (hMSCs). Although the influence of TiO2 nanotubes on the cellular response, and in particular on hMSC activity, has already been addressed in the past, previous studies overlooked critical morphological, structural and physical aspects that go beyond the simple nanotube diameter, such as spatial statistics. Methods: To bridge this gap, we implemented an extensive characterization of nanotubular surfaces generated by anodization of titanium with a focus on spatial structural variables including eccentricity, nearest neighbour distance (NND) and Voronoi entropy, and associated them to the hMSC response. In addition, we assessed the biological potential of a two-tiered honeycomb nanoarchitecture, which allowed the detection of combinatory effects that this hierarchical structure has on stem cells with respect to conventional nanotubular designs. We have combined experimental techniques, ranging from Scanning Electron (SEM) and Atomic Force (AFM) microscopy to Raman spectroscopy, with computational simulations to characterize and model nanotubular surfaces. We evaluated the cell response at 6 hrs, 1 and 2 days by fluorescence microscopy, as well as bone mineral deposition by Raman spectroscopy, demonstrating substrate-induced differential biological cueing at both the short- and long-term. Results: Our work demonstrates that the nanotube diameter is not sufficient to comprehensively characterize nanotubular surfaces and equally important parameters, such as eccentricity and wall thickness, ought to be included since they all contribute to the overall spatial disorder which, in turn, dictates the overall bioactive potential. We have also demonstrated that nanotubular surfaces affect the quality of bone mineral deposited by differentiated stem cells. Lastly, we closed in on the integrated effects exerted by the superimposition of two dissimilar nanotubular arrays in the honeycomb architecture. Discussion: This work delineates a novel approach for the characterization of TiO2 nanotubes which supports the incorporation of critical spatial structural aspects that have been overlooked in previous research. This is a crucial aspect to interpret cellular behaviour on nanotubular substrates. Consequently, we anticipate that this strategy will contribute to the unification of studies focused on the use of such powerful nanostructured surfaces not only for biomedical applications but also in other technology fields, such as catalysis.

JTD Keywords: Nanotubes, Nanotopography, Spatial statistics, Stem cells, Bone quality

Altay, Gizem, Batlle, Eduard, Fernández-Majada, Vanesa, Martínez, Elena, (2020). In vitro self-organized mouse small intestinal epithelial monolayer protocol Bio-protocol 10, (3), e3514

Developing protocols to obtain intestinal epithelial monolayers that recapitulate in vivo physiology to overcome the limitations of the organoids’ closed geometry has become of great interest during the last few years. Most of the developed culture models showed physiological-relevant cell composition but did not prove self-renewing capacities. Here, we show a simple method to obtain mouse small intestine-derived epithelial monolayers organized into proliferative crypt-like domains, containing stem cells, and differentiated villus-like regions, closely resembling the in vivo cell composition and distribution. In addition, we adapted our model to a tissue culture format compatible with functional studies and prove close to physiological barrier properties of our in vitro epithelial monolayers. Thus, we have set-up a protocol to generate physiologically relevant intestinal epithelial monolayers to be employed in assays where independent access to both luminal and basolateral compartments is needed, such as drug absorption, intracellular trafficking and microbiome-epithelium interaction assays.

JTD Keywords: Mouse intestinal organoids, Adult intestinal stem cells, Matrigel, Intestinal epithelial monolayer, In vitro intestinal epithelial model, Tissue-like functionality, TEER

Comelles, Jordi, Fernández-Majada, Vanesa, Berlanga-Navarro, Nuria, Acevedo, Verónica, Paszkowska, Karolina, Martínez, Elena, (2020). Microfabrication of poly(acrylamide) hydrogels with independently controlled topography and stiffness Biofabrication 12, (2), 025023

The stiffness and topography of a cell's extracellular matrix are physical cues that play a key role in regulating processes that determine cellular fate and function. While substrate stiffness can dictate cell differentiation lineage, migration, and self-organization, topographical features can change the cell's differentiation profile or migration ability. Although both physical cues are present and intrinsic to the native tissues in vivo, in vitro studies have been hampered by the lack of technological set-ups that would be compatible with cell culture and characterization. In vitro studies therefore either focused on screening stiffness effects in cells cultured on flat substrates or on determining topography effects in cells cultured onto hard materials. Here, we present a reliable, microfabrication method to obtain well defined topographical structures of micrometer size (5-10 µm) on soft polyacrylamide hydrogels with tunable mechanical stiffness (3-145 kPa) that closely mimic the in vivo situation. Topographically microstructured polyacrylamide hydrogels are polymerized by capillary force lithography using flexible materials as molds. The topographical microstructures are resistant to swelling, can be conformally functionalized by extracellular matrix proteins and sustain the growth of cell lines (fibroblasts and myoblasts) and primary cells (mouse intestinal epithelial cells). Our method can independently control stiffness and topography, which allows to individually assess the contribution of each physical cue to cell response or to explore potential synergistic effects. We anticipate that our fabrication method will be of great utility in tissue engineering and biophysics, especially for applications where the use of complex in vivo-like environments is of paramount importance.


Cutrale, Francesco, Rodriguez, Daniel, Hortigüela, Verónica, Chiu, Chi-Li, Otterstrom, Jason, Mieruszynski, Stephen, Seriola, Anna, Larrañaga, Enara, Raya, Angel, Lakadamyali, Melike, Fraser, Scott E., Martinez, Elena, Ojosnegros, Samuel, (2019). Using enhanced number and brightness to measure protein oligomerization dynamics in live cells Nature Protocols 14, 616-638

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.


Castaño, Albert G., García-Díaz, María, Torras, Núria, Altay, Gizem, Comelles, Jordi, Martínez, Elena, (2019). Dynamic photopolymerization produces complex microstructures on hydrogels in a moldless approach to generate a 3D intestinal tissue model Biofabrication 11, (2), 025007

Epithelial tissues contain three-dimensional (3D) complex microtopographies that are essential for proper performance. These microstructures provide cells with the physicochemical cues needed to guide their self-organization into functional tissue structures. However, most in vitro models do not implement these 3D architectural features. The main problem is the availability of simple fabrication techniques that can reproduce the complex geometries found in native tissues on the soft polymeric materials required as cell culture substrates. In this study reaction-diffusion mediated photolithography is used to fabricate 3D microstructures with complex geometries on poly(ethylene glycol)-based hydrogels in a single step and moldless approach. By controlling fabrication parameters such as the oxygen diffusion/depletion timescales, the distance to the light source and the exposure dose, the dimensions and geometry of the microstructures can be well-defined. In addition, copolymerization of poly(ethylene glycol) with acrylic acid improves control of the dynamic reaction-diffusion processes that govern the free-radical polymerization of highly-diluted polymeric solutions. Moreover, acrylic acid allows adjusting the density of cell adhesive ligands while preserving the mechanical properties of the hydrogels. The method proposed is a simple, single-step, and cost-effective strategy for producing models of intestinal epithelium that can be easily integrated into standard cell culture platforms.


Martinez, Elena, St-Pierre, Jean-Philippe, Variola, Fabio, (2019). Advanced bioengineering technologies for preclinical research Advances in Physics: X 4, (1), 1622451

Current in vitro practices must overcome important challenges to compare favorably with human studies. The limited applicability of conventional in vitro assays and strategies can be explained by the fact that standard approaches do not enable recapitulation of the complexity of human tissues and physiological functions. To address this challenge, novel bioengineering tools, techniques and technologies are rapidly emerging to advance current fundamental knowledge and innovate in vitro practices. For example, organs-on-a-chip have recently appeared as a small-scale solution to overcome the transability, financial and ethical concerns associated with animal studies in drug discovery and development. In parallel, biomimetic interfaces are increasingly recapitulating 3D structures with tissue-like dynamic properties to allow in-depth investigation of disease mechanisms. This review aims at highlighting current bioengineering approaches poised to address the shortcomings of conventional in vitro research practices towards the generation of more effective solutions for improving human health.


Valls-Margarit, M., Iglesias-García, O., Di Guglielmo, C., Sarlabous, L., Tadevosyan, K., Paoli, R., Comelles, J., Blanco-Almazán, D., Jiménez-Delgado, S., Castillo-Fernández, O., Samitier, J., Jané, R., Martínez, Elena, Raya, Á., (2019). Engineered macroscale cardiac constructs elicit human myocardial tissue-like functionality Stem Cell Reports 13, (1), 207-220

In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term “CardioSlice.” PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers. Continuous electrical stimulation for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include electrocardiogram-like signals that can be readily measured on the surface of CardioSlice constructs, and a response to proarrhythmic drugs that is predictive of their effect in human patients.

JTD Keywords: Cardiac tissue engineering, CardioSlice, ECG-like signals, Electrical stimulation, Heart physiology, Human induced pluripotent stem cells, Perfusion bioreactor, Tissue-like properties

Hortigüela, Verónica, Larrañaga, Enara, Lagunas, Anna, Acosta, Gerardo A., Albericio, Fernando, Andilla, Jordi, Loza-Alvarez, Pablo, Martínez, Elena, (2019). Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies Nanomaterials 9, (4), 579

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.


Altay, Gizem, Larrañaga, Enara, Tosi, Sébastien, Barriga, Francisco M., Batlle, Eduard, Fernández-Majada, Vanesa, Martínez, Elena, (2019). Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function Scientific Reports 9, (1), 10140

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.


de Goede, Michiel, Chang, Lantian, Mu, Jinfeng, Dijkstra, Meindert, Obregón, Raquel, Martínez, Elena, Padilla, Laura, Mitjans, Francesc, Garcia-Blanco, Sonia M., (2019). Al2O3:Yb3+ integrated microdisk laser label-free biosensor Optics Letters 44, (24), 5937-5940

Whispering gallery mode resonator lasers hold the promise of an ultralow intrinsic limit of detection. However, the widespread use of these devices for biosensing applications has been hindered by the complexity and lack of robustness of the proposed configurations. In this work, we demonstrate biosensing with an integrated microdisk laser. Al2O3doped with Yb3+ was utilized because of its low optical losses as well as its emission in the range 1020–1050 nm, outside the absorption band of water. Single-mode laser emission was obtained at a wavelength of 1024 nm with a linewidth of 250 kHz while the microdisk cavity was submerged in water. A limit of detection of 300 pM (3.6 ng/ml) of the protein rhS100A4 in urine was experimentally demonstrated, showing the potential of the proposed devices for biosensing.

JTD Keywords: Distributed feedback lasers, Fiber lasers, Laser modes, Microdisk lasers, Single mode lasers, Tunable lasers

de Goede, M., Dijkstra, M., Obregón, R., Ramón-Azcón, J., Martínez, Elena, Padilla, L., Mitjans, F., Garcia-Blanco, S. M., (2019). Al2O3 microring resonators for the detection of a cancer biomarker in undiluted urine Optics Express 27, (13), 18508-18521

Concentrations down to 3 nM of the rhS100A4 protein, associated with human tumor development, have been detected in undiluted urine using an integrated sensor based on microring resonators in the emerging Al2O3 photonic platform. The fabricated microrings were designed for operation in the C-band (λ = 1565 nm) and exhibited a high-quality factor in air of 3.2 × 105. The bulk refractive index sensitivity of the devices was ~100 nm/RIU (for TM polarization) with a limit of detection of ~10−6 RIU. A surface functionalization protocol was developed to allow for the selective binding of the monoclonal antibodies designed to capture the target biomarker to the surface of the Al2O3 microrings. The detection of rhS100A4 proteins at clinically relevant concentrations in urine is a big milestone towards the use of biosensors for the screening and early diagnosis of different cancers. Biosensors based on this microring technology can lead to portable, multiplexed and easy-to-use point of care devices.

JTD Keywords: Distributed feedback lasers, Effective refractive index, Laser coupling, Polarization maintaining fibers, Refractive index, Scanning electron microscopy

Hortigüela, Verónica, Larrañaga, Enara, Cutrale, Francesco, Seriola', Anna, García-Díaz, María, Lagunas, Anna, Andilla, Jordi, Loza-Alvarez, Pablo, Samitier, Josep, Ojosnegros', Samuel, Martinez, Elena, (2018). Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering Nano Letters 18, (1), 629-637

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 crosslinked 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.


Macedo, Maria Helena, Araújo, Francisca, Martínez, Elena, Barrias, Cristina, Sarmento, Bruno, (2018). iPSC-Derived enterocyte-like cells for drug absorption and metabolism studies Trends in Molecular Medicine 24, (8), 696-708

Intestinal cell models have been widely studied and used to evaluate absorption and metabolism of drugs in the small intestine, constituting valuable tools as a first approach to evaluate the behavior of new drugs. However, such cell models might not be able to fully predict the absorption mechanisms and metabolic pathways of the tested compounds. In recent years, induced pluripotent stem cells (iPSCs) differentiated into enterocyte-like cells have been proposed as more biorelevant intestinal models. In this review, we describe mechanisms underlying the differentiation of iPSCs into enterocyte-like cells, appraise the usefulness of these cells in tridimensional intestinal models, and discuss their suitability to be used in the future for drug screening.

JTD Keywords: iPSCs, Enterocytes, Differentiation, Small intestine, Drug absorption, Intestinal models

Torras, N., García-Díaz, M., Fernández-Majada, V., Martínez, Elena, (2018). Mimicking epithelial tissues in three-dimensional cell culture models Frontiers in Bioengineering and Biotechnology 6, Article 197

Epithelial tissues are composed of layers of tightly connected cells shaped into complex three-dimensional (3D) structures such as cysts, tubules, or invaginations. These complex 3D structures are important for organ-specific functions and often create biochemical gradients that guide cell positioning and compartmentalization within the organ. One of the main functions of epithelia is to act as physical barriers that protect the underlying tissues from external insults. In vitro, epithelial barriers are usually mimicked by oversimplified models based on cell lines grown as monolayers on flat surfaces. While useful to answer certain questions, these models cannot fully capture the in vivo organ physiology and often yield poor predictions. In order to progress further in basic and translational research, disease modeling, drug discovery, and regenerative medicine, it is essential to advance the development of new in vitro predictive models of epithelial tissues that are capable of representing the in vivo-like structures and organ functionality more accurately. Here, we review current strategies for obtaining biomimetic systems in the form of advanced in vitro models that allow for more reliable and safer preclinical tests. The current state of the art and potential applications of self-organized cell-based systems, organ-on-a-chip devices that incorporate sensors and monitoring capabilities, as well as microfabrication techniques including bioprinting and photolithography, are discussed. These techniques could be combined to help provide highly predictive drug tests for patient-specific conditions in the near future.

JTD Keywords: 3D cell culture models, Biofabrication, Disease modeling, Drug screening, Epithelial barriers, Microengineered tissues, Organ-on-a-chip, Organoids

de Goede, M., Chang, L., Dijkstra, M., Obregón, R., Ramón-Azcon, J., Martínez, Elena, Padilla, L., Adan, J., Mitjans, F., García-Blanco, S.M., (2018). Al2O3 Microresonator based passive and active biosensors ICTON 2018 20th International Conference on Transparent Optical Networks , IEEE Computer Society (Bucharest, Romania) , 8473820

Al2O3 microresonators were realized for sensing applications of both passive and active devices. Passive microring resonators exhibited quality factors up to 3.2×105 in air. A bulk refractive index sensitivity of 100 nm/RIU was demonstrated together with a limit of detection of 10-6 RIU. Functionalizing their surface allowed for the label-free detection of the biomarker rhS100A4 from urine with a limit of detection of 3 nM. Furthermore, single-mode Al2O3:Yb3+ microdisk lasers were realized that could operate in an aqueous environment. Upon varying the bulk refractive index their lasing wavelength could be tuned with a sensitivity of 20 nm/RIU and a LOD of 3×10-6 RIU.


de Goede, M., Chang, L., Dijkstra, M., Obregón, R., Ramón-Azcon, J., Martínez, Elena, Padilla, L., Adan, J., Mitjans, F., García-Blanco, S.M., (2018). Al2O3 Mmicroresonators for passive and active sensing applications Sensors 2018 Optical Sensors , OSA - The Optical Society (Zurich, Switzerland) Part F110, 1-2

The Al2O3 waveguide technology was explored for sensing applications. Passive microring resonators with a quality factor in air of 3.2×105 were developed with a bulk refractive index sensitivity of ~100 nm/RIU and limit of detection of ~10-6 RIU. These were functionalized to detect the biomarker rhS100A4 from urine down to concentrations of 3 nM. Furthermore, Al2O3:Yb3+ microdisk lasers were realized that exhibited single mode lasing operation in water. Their lasing wavelength was tuned by varying the bulk refractive index and a bulk refractive index sensitivity of ~20 nm/RIU with a LOD of ~3×10-6 was achieved.


de Goede, M., Dijkstra, M., Obregón, R., Martínez, Elena, García-Blanco, S.M., (2018). High quality factor Al2O3 microring resonators for on-chip sensing applications Proceedings SPIE. Integrated Optics: Devices, Materials, and Technologies XXII SPIE OPTO , SPIE (California, USA) 10535, 7

Microring resonators find many applications for on-chip integrated optical sensors. Their spectral response contains resonance dips that shift due to variations of the optical path length of the microring probed. Numerous examples of such microring resonator sensors in the SOI, Si3N4 and SiON waveguide technologies have been reported for the detection of bulk refractive index variations and the label-free detection of biomarkers. Al2O3 is an alternative waveguide technology that exhibits low optical propagation losses, is transparent over a large spectral range extending from the visible to the mid-IR and permits co-doping with active rare-earth ions, which enables the co-integration of active devices on the chip. In this work an Al2O3 microring resonator sensor was developed for the label-free detection of protein biomarkers. The uncladded microring with a radius of 200 μm had a measured quality factor of 3.2 × 105 at 1550 nm. Submerging the devices in water decreased the quality factor to 45 × 103. This corresponds with propagation losses in the rings of 0.6 dB/cm and 5.7 dB/cm respectively. The bulk refractive index sensitivity of the sensor was determined by flowing NaCl dissolved in water in different concentrations. A sensitivity of 102.3 ± 0.5 nm/RIU with a corresponding limit of detection of 1.6 × 10-6 RIU was demonstrated for TM polarized light. High affinity human monoclonal antibodies mAb S100A4 were immobilized on the sensor to detect the S100A4 protein biomarker down to 12 nM concentrations. These results demonstrate the feasibility of this material for label-free optical biosensors.


Ojosnegros', Samuel, Cutrale, Francesco, Rodríguez, Daniel, Otterstrom, Jason J., Chiu, Chi Li, Hortigüela, Verónica, Tarantino, Carolina, Seriola', Anna, Mieruszynski, Stephen, Martínez, Elena, Lakadamyali, Melike, Raya, Angel, Fraser, Scott E., (2017). Eph-ephrin signaling modulated by polymerization and condensation of receptors Proceedings of the National Academy of Sciences of the United States of America 114, (50), 13188-13193

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.

JTD Keywords: Eph, Ephrin, Receptor tyrosine kinase, Gradients, Cell communication

Garreta, E., de Oñate, L., Fernández-Santos, M. E., Oria, R., Tarantino, C., Climent, A. M., Marco, A., Samitier, M., Martínez, Elena, Valls-Margarit, M., Matesanz, R., Taylor, D. A., Fernández-Avilés, F., Izpisua Belmonte, J. C., Montserrat, N., (2016). Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts Biomaterials 98, 64-78

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands, targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features, and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall, the approach described here allows for the rapid generation of human cardiac grafts from hPSCs, in a total of 24 days, providing a suitable platform for cardiac engineering and disease modeling in the human setting.

JTD Keywords: Cardiac function, Extracellular matrix, Gene targeting, Pluripotent stem cells

Lagunas, A., Sasso, B., Tesson, N., Cantos, C., Martinez, Elena, Samitier, J., (2016). Synthesis of a polymethyl(methacrylate)-polystyrene-based diblock copolymer containing biotin for selective protein nanopatterning Polymer Chemistry 7, 212-218

Protein patterning is of interest in high-throughput screening. Due to an increase in demand for further miniaturization of protein assays, block copolymers (BCPs) that can undergo large-area phase separation into nanometer-size domains have attracted great attention as substrates for protein nanopatterning. Here we report the synthesis of a polymethyl(methacrylate)-polystyrene-based diblock copolymer which, once spin-coated, is capable of self-segregating into cylindrical polystyrene (PS) domains. In this copolymer, the PS block was modified to introduce biotin below 10% molar in order to achieve molecular recognition of streptavidin. The PMMA matrix used to introduce poly(ethylene glycol) enabled us to obtain an antifouling environment that prevents unspecific protein adsorption outside the domains. The use of the biotin-streptavidin pair in this BCP makes it suitable for nanopatterning of other biotinylated proteins of interest for the purposes of cell biology, biosensors, and tissue engineering.


Lagunas, Anna, Martinez, Elena, Samitier, Josep, (2015). Surface-bound molecular gradients for the high throughput screening of cell responses Frontiers in Bioengineering and Biotechnology 3, Article 132

Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. Continuous chemical gradients have recently been proposed as alternative to discrete microarrays for the high throughput screening of the effects of ligand concentration in cells. Here we review some of the most recent examples in which gradients have been used to evaluate the effect of a varying ligand concentration in cell adhesion, morphology, growth and differentiation of cells, including some of our recent findings. They show the importance of the organization of ligands at the nanoscale, which is highlighted by abrupt changes in cell behavior at critical concentration thresholds.

JTD Keywords: Cell Adhesion, Cell Differentiation, Cell growth, Cell morphology, Molecular gradient

Galan, Teresa, Lagunas, Anna, Martinez, Elena, Samitier, Josep, (2015). Fabrication of bioactive polypyrrole microelectrodes on insulating surfaces by surface-guided biocatalytical polymerization RSC Advances 5, (82), 67082-67088

Although promising, organic microelectronics lacks standard fabrication methods comparable to photolithography in terms of resolution. Here we propose a novel and easily scalable on-surface biocatalytical procedure for the fabrication of polypyrrole microelectrodes on insulating surfaces. Arrays of polypyrrole microelectrodes were obtained by surface-guided biocatalytical polymerization, achieving up to 5 [small micro]m in resolution and conductivities up to 3 S cm-1. The mild reaction conditions provided by the biocatalytical approach permit the entrapment of bioactive compounds during polymer synthesis. This system is convenient for drug release purposes, as demonstrated by the controlled release of entrapped biotin through electrical stimulation. These results pave the way for the application of polypyrrole microelectrodes produced through biocatalysis in the development of implantable devices for remotely controlled tissue interactions.


Estévez, M., Martínez, Elena, Yarwood, S. J., Dalby, M. J., Samitier, J., (2015). Adhesion and migration of cells responding to microtopography Journal of Biomedical Materials Research - Part A , 103, (5), 1659-1668

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

JTD Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

Comelles, J., Hortigüela, V., Martínez, Elena, Riveline, D., (2015). Methods for rectifying cell motions in vitro: Breaking symmetry using microfabrication and microfluidics Methods in Cell Biology - Biophysical Methods in Cell Biology (ed. Wilson, L., Tran, P.), Academic Press (Santa Barbara, USA) 125, 437-452

Cell motility is an important phenomenon in cell biology, developmental biology, and cancer. Here we report methods that we designed to identify and characterize external factors which direct cell motions by breaking locally the symmetry. We used microfabrication and microfluidics techniques to impose and combine mechanical and chemical cues to moving fibroblasts. Gradients can thereby be engineered at the cellular scale and this approach has allowed to disentangle roles of the nucleus and protrusion activity in setting cell directions.

JTD Keywords: Adhesion, Biological physics, Cell motility, Gradient, Ratchet

de Oñate, L., Garreta, E., Tarantino, C., Martínez, Elena, Capilla, E., Navarro, I., Gutiérrez, J., Samitier, J., Campistol, J.M., Muñoz-Cánovas, P., Montserrat, N., (2015). Research on skeletal muscle diseases using pluripotent stem cells Muscle Cell and Tissue (ed. Sakuma, K.), InTech (Rijeka, Croatia) , 333-357

The generation of induced pluripotent stem cells (iPSCs), especially the generation of patient-derived pluripotent stem cells (PSCs) suitable for disease modelling in vitro, opens the door for the potential translation of stem-cell related studies into the clinic. Successful replacement, or augmentation, of the function of damaged cells by patient-derived differentiated stem cells would provide a novel cell-based therapy for skeletal muscle-related diseases. Since iPSCs resemble human embryonic stem cells (hESCs) in their ability to generate cells of the three germ layers, patient-specific iPSCs offer definitive solutions for the ethical and histo-incompatibility issues related to hESCs. Indeed human iPSC (hiPSC)-based autologous transplantation is heralded as the future of regenerative medicine. Interestingly, during the last years intense research has been published on disease-specific hiPSCs derivation and differentiation into relevant tissues/organs providing a unique scenario for modelling disease progression, to screen patient-specific drugs and enabling immunosupression-free cell replacement therapies. Here, we revise the most relevant findings in skeletal muscle differentiation using mouse and human PSCs. Finally and in an effort to bring iPSC technology to the daily routine of the laboratory, we provide two different protocols for the generation of patient-derived iPSCs.

JTD Keywords: Pluripotent stem cells, Myogenic differentiation, Disease modelling, Patient-specific induced pluripotent stem cells, Muscular dystrophy

Comelles, J., Caballero, D., Voituriez, ., Hortigüela, V., Wollrab, V., Godeau, A. L., Samitier, J., Martínez, Elena, Riveline, D., (2014). Cells as active particles in asymmetric potentials: Motility under external gradients Biophysical Journal , 107, (7), 1513-1522

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.


Oberhansl, S., Garcia, A., Lagunas, A., Prats-Alfonso, E., Hirtz, M., Albericio, F., Fuchs, H., Samitier, J., Martinez, Elena, (2014). Mesopattern of immobilised bone morphogenetic protein-2 created by microcontact printing and dip-pen nanolithography influence C2C12 cell fate RSC Advances 4, (100), 56809-56815

Dip-pen nanolithography and microcontact printing were used to fabricate mesopatterned substrates for cell differentiation experiments. A biotin-thiol was patterned on gold substrates and subsequently functionalised with streptavidin and biotinylated bone morphogenetic protein-2 (BMP-2). The feasibility of mesopatterned substrates containing immobilised BMP-2 was proven by obtaining similar differentiation outcomes compared to the growth factor in solution. Therefore, these substrates might be suitable for replacing conventional experiments with BMP-2 in solution.

JTD Keywords: Bone morphogenetic protein-2, C2C12 cells, Dip-pen nanolithography, Micro contact printing

Garcia, A., Hortigüela, V., Lagunas, A., Cortina, C., Montserrat, N., Samitier, J., Martinez, Elena, (2014). Protein patterning on hydrogels by direct microcontact printing: application to cardiac differentiation RSC Advances 4, (55), 29120-29123

An extended microcontact printing technique to chemically pattern hydrogels is reported. The procedure employs standard polydimethylsiloxane stamps and requires minor pre-processing of the hydrogels by freeze-drying. Micropatterned Matrigel[trade mark sign] and gelatin hydrogels induce NIH-3T3 cell alignment and elongation. Furthermore, human embryonic stem cells cultured on fibronectin-patterned hydrogels display beating foci earlier than those cultured on non-patterned substrates.


Lagunas, A., Comelles, J., Oberhansl, S., Hortigüela, V., Martínez, Elena, Samitier, J., (2013). Continuous bone morphogenetic protein-2 gradients for concentration effect studies on C2C12 osteogenic fate Nanomedicine: Nanotechnology, Biology, and Medicine 9, (5), 694-701

Cells can respond to small changes in a varying concentration of exogenous signaling molecules. Here we propose the use of continuous surface chemical gradients for the in-depth study of dose-dependent effects on cells. A continuous surface gradient of bone morphogenetic protein-2 (BMP-2) is presented. The gradient covers a narrow range of surface densities (from 1.4 to 2.3 pmol/cm2) with a shallow slope (0.9 pmol/cm3). These characteristics represent a quasi-homogeneous surface concentration at the cell scale, which is crucial for cell screening studies. Cell fate evaluation at early stages of osteogenesis in C2C12 cells, indicates the potential of continuous gradients for in vitro screening applications.


Prats-Alfonso, E., Oberhansl, S., Lagunas, A., Martínez, Elena, Samitier, J., Albericio, F., (2013). Effective and versatile strategy for the total solid-phase synthesis of alkanethiols for biological applications European Journal of Organic Chemistry , 2013, (7), 1233-1239

Biological applications increasingly demand tailored surfaces with a range of functional groups. Herein we describe a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique overcomes all the difficulties encountered during the preparation of these molecules in solution. The procedure allows the use of ATs without further purification for the preparation of self-assembled monolayers on gold, typically used to achieve functional group diversity on this surface. This paper describes a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique allows a variety of ATs to be obtained in only three steps, overcoming the difficulties encountered during their preparation in solution.


Rodriguez-Segui, Santiago A., Ortuno, Maria Jose, Ventura, Francesc, Martinez, Elena, Samitier, Josep, (2013). Simplified microenvironments and reduced cell culture size influence the cell differentiation outcome in cellular microarrays Journal of Materials Science: Materials in Medicine , 24, (1), 189-198

Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.


Diéguez, Lorena, Caballero, David, Calderer, Josep, Moreno, Mauricio, Martínez, Elena, Samitier, Josep, (2012). Optical gratings coated with thin Si3N4 layer for efficient immunosensing by optical waveguide lightmode spectroscopy Biosensors , 2, (2), 114-126

New silicon nitride coated optical gratings were tested by means of Optical Waveguide Lightmode Spectroscopy (OWLS). A thin layer of 10 nm of transparent silicon nitride was deposited on commercial optical gratings by means of sputtering. The quality of the layer was tested by x-ray photoelectron spectroscopy and atomic force microscopy. As a proof of concept, the sensors were successfully tested with OWLS by monitoring the concentration dependence on the detection of an antibody-protein pair. The potential of the Si3N4 as functional layer in a real-time biosensor opens new ways for the integration of optical waveguides with microelectronics.

JTD Keywords: Silicon nitride, Optical gratings, Waveguide, Biosensor

Oberhansl, Sabine, Hirtz, Michael, Lagunas, Anna, Eritja, Ramon, Martinez, Elena, Fuchs, Harald, Samitier, Josep, (2012). Facile modification of silica substrates provides a platform for direct-writing surface click chemistry Small 8, (4), 541-545

Lagunas, Anna , Comelles, Jordi, Martínez, Elena, Prats-Alfonso, Elisabet , Acosta, Gerardo A., Albericio, Fernando , Samitier, Josep , (2012). Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects Nanomedicine: Nanotechnology, Biology and Medicine , 8, (4), 432-439

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell–adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm2), with a threshold value of 4.0 pmol/cm2 for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis.

JTD Keywords: RGD gradient, Cell adhesion, Poly(methyl methacrylate), Hydrolysis, Biotin-streptavidin

Tort, N., Salvador, J. P., Avino, A., Eritja, R., Comelles, J., Martinez, E., Samitier, J., Marco, M. P., (2012). Synthesis of steroid-oligonucleotide conjugates for a DNA site-encoded SPR immunosensor Bioconjugate Chemistry , 23, (11), 2183-2191

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N(1)up, N(2)up, and N(3)up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR).

JTD Keywords: Directed protein immobilization, Plasmon resonance biosensor, Self-assembled monolayers, Label-free, Serum samples, Assay, Immunoassays, Antibodies, Progress, Binding

Caballero, D., Martinez, E., Bausells, J., Errachid, A., Samitier, J., (2012). Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface Analytica Chimica Acta 720, 43-48

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si 3N 4) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si 3N 4-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO 2/Si 3N 4 structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10 -13-10 -7M were detected, showing a sensitivity of 0.128ΩμM -1 and a limit of detection of 10 -14M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.

JTD Keywords: Aldehyde, Electrochemical impedance spectroscopy, Human serum albumin, Immunosensor, Silicon nitride, Bovine serum albumins, Chemical reagents, Complex samples, Covalent binding, Detection capability, Electrochemical impedance, Electrochemical impedance spectroscopy measurements, Functionalizations, Human serum albumins, Impedimetric immunosensors, Label free, Limit of detection, Linear range, Protein concentrations, Silicon-based, Specific detection, Aldehydes

Comelles, J., Hortigüela, V., Samitier, J., Martinez, E., (2012). Versatile gradients of covalently bound proteins on microstructured substrates Langmuir 28, (38), 13688-13697

In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a rnicrofluidic approach to obtain linear gradients with high slope (0.5, relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.

JTD Keywords: Cell-migration, Microfluidic channel, Surface, Streptavidin, Molecules, Topography, Mechanisms, Generation, Responses, Guidance

Semaphorins are secreted or membrane-anchored proteins that play critical roles in neural development and adult brain plasticity. Sema4F is a transmembrane semaphorin found on glutamatergic synapses, in which it is attached to the PSD-95-scaffolding protein. Here we further examined the expression of Sema4F by raising specific antibodies. We show that Sema4F protein is widely expressed by neurons during neural development and in the adult brain. We also demonstrate a preferential localization of this protein in postsynaptic dendrites. Moreover, Sema4F is expressed not only by neurons but also by oligodendrocyte precursors in the optic nerve and along the migratory pathways of oligodendroglial cells, and also by subsets of postnatal oligodendroglial cells in the brain. Finally, in vitro experiments demonstrate that endogenous Sema4F expressed by brain cells of oligodendroglial lineage regulates the outgrowth migration of oligodendrocyte precursors and promotes their differentiation. The present data extend our knowledge about the expression of Sema4F and uncover a novel function in the control of oligodendrocyte precursor migration in the developing brain.

JTD Keywords: Semaphorin, Oligodendrocyte, Guidance, Optic nerve, Brain

Azevedo, S., Diéguez, L., Carvalho, P., Carneiro, J. O., Teixeira, V., Martínez, Elena, Samitier, J., (2012). Deposition of ITO thin films onto PMMA substrates for waveguide based biosensing devices Journal of Nano Research , 17, 75-83

Biosensors' research filed has clearly been changing towards the production of multifunctional and innovative design concepts to address the needs related with sensitivity and selectivity of the devices. More recently, waveguide biosensors, that do not require any label procedure to detect biomolecules adsorbed on its surface, have been pointed out as one of the most promising technologies for the production of biosensing devices with enhanced performance. Moreover the combination of optical and electrochemical measurements through the integration of transparent and conducting oxides in the multilayer structures can greatly enhance the biosensors' sensitivity. Furthermore, the integration of polymeric substrates may bring powerful advantages in comparison with silicon based ones. The biosensors will have a lower production costs being possible to disposable them after use ("one use sensor chip"). This research work represents a preliminary study about the influence of substrate temperature on the overall properties of ITO thin films deposited by DC magnetron sputtering onto 0,5 mm thick PMMA sheets.

JTD Keywords: ITO thin films, PMMA sheets, Waveguide biosensing devices, Biosensing devices, Conducting oxides, Dc magnetron sputtering, Electrochemical measurements, Enhanced performance, Innovative design, ITO thin films, Multilayer structures, Overall properties, PMMA sheets, Polymeric substrate, Production cost, Sensor chips, Silicon-based, Substrate temperature, Biosensors, Deposition, Design, Film preparation, Optical multilayers, Thin films, Vapor deposition, Waveguides, Substrates

Martínez, Elena, Pla, M., Samitier, J., (2012). Micro/nanopatterning of proteins using a nanoimprint-based contact printing technique Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 79-87

Micro and nanoscale protein patterning based on microcontact printing technique on large substrates have often resolution problems due to roof collapse of the poly(dimethylsiloxane) (PDMS) stamps used. Here, we describe a technique that overcomes these issues by using instead a stamp made of poly(methyl methacrylate) (PMMA), a much more rigid polymer that do not collapse even using stamps with very high aspect ratios (up to 300:1). Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density submicrometer scale patterns, or, alternatively, micro/nanopatterns spaced at large distances.

JTD Keywords: Microcontact printing, Nanoimprint lithography, Poly(methyl methacrylate), Protein

Rodriguez-Segui, Santiago A., Pons Ximenez, Jose Ignacio, Sevilla, Lidia, Ruiz, Ana, Colpo, Pascal, Rossi, Francois, Martinez, Elena, Samitier, Josep, (2011). Quantification of protein immobilization on substrates for cellular microarray applications Journal of Biomedical Materials Research - Part A , 98A, (2), 245-256

Cellular microarray developments and its applications are the next step after DNA and protein microarrays. The choice of the surface chemistry of the substrates used for the implementation of this technique, that must favor proper protein immobilization while avoiding cell adhesion on the nonspotted areas, presents a complex challenge. This is a key issue since usually the best nonfouling surfaces are also the ones that retain immobilized the smallest amounts of printed protein. To quantitatively assess the amount of protein immobilization, in this study several combinations of fluorescently labeled fibronectin (Fn*) and streptavidin (SA*) were microspotted, with and without glycerol addition in the printing buffer, on several substrates suitable for cellular microarrays. The substrates assayed included chemically activated surfaces as well as Poly ethylene oxide (PEO) films that are nonfouling in solution but accept adhesion of proteins in dry conditions. The results showed that the spotted Fn* was retained by all the surfaces, although the PEO surface did show smaller amounts of immobilization. The SA*, on the other hand, was only retained by the chemically activated surfaces. The inclusion of glycerol in the printing buffer significantly reduced the immobilization of both proteins. The results presented in this article provide quantitative evidence of the convenience of using a chemically activated surface to immobilize proteins relevant for cellular microarray applications, particularly when ECM proteins are cospotted with smaller factors which are more difficult to be retained by the surfaces.

JTD Keywords: Protein immobilization, Quantification, Microarray, Substrate, Surface chemistry

Martinez, Elena, Samitier, Josep, (2011). Soft lithography and variants Generating micro- and nanopatterns on polymeric materials (ed. del Campo, Aranzazu , Arzt, Eduard), Wiley-VCH Verlag GmbH&Co (Weinheim) , 57-66

Comelles, J., Estevez, M., Martinez, E., Samitier, J., (2010). The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion Nanomedicine: Nanotechnology Biology and Medicine , 6, (1), 44-51

Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly (methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. From the Clinical Editor: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).

JTD Keywords: Thin-films, Poly(methyl methacrylate), Osteoblast adhesion, Electron-microscopy, Fibronectin, Polystyrene, Oly(dimethylsiloxane), Biocompatibility, Hydroxyapatite, Behavior

Estevez, M., Fernandez-Ulibarri, I., Martinez, E., Egea, G., Samitier, J., (2010). Changes in the internal organization of the cell by microstructured substrates Soft Matter 6, (3), 582-590

Surface features at the micro and nanometre scale have been shown to influence and even determine cell behaviour and cytoskeleton organization through direct mechanotransductive pathways. Much less is known about the function and internal distribution of organelles of cells grown on topographically modified surfaces. In this study, the nanoimprint lithography technique was used to manufacture poly(methyl methacrylate) (PMMA) sheets with a variety of features in the micrometre size range. Normal rat kidney (NRK) fibroblasts were cultured on these substrates and immunofluorescence staining assays were performed to visualize cell adhesion, the organization of the cytoskeleton and the morphology and subcellular positioning of the Golgi complex. The results show that different topographic features at the micrometric scale induce different rearrangements of the cell cytoskeleton, which in turn alter the positioning and morphology of the Golgi complex. Microposts and microholes alter the mechanical stability of the Golgi complex by modifying the actin cytoskeleton organization leading to the compaction of the organelle. These findings prove that physically modified surfaces are a valuable tool with which to study the dynamics of cell cytoskeleton organization and its subsequent repercussion on internal cell organization and associated function.

JTD Keywords: Actin stress fibers, Golgi-complex, Focal adhesions, Cytoskeletal organization, Osteoblast adhesion, Mammalian-cells, Micron-scale, Nanoscale, Dynamics, Rho

Lagunas, A., Comelles, J., Martinez, E., Samitier, J., (2010). Universal chemical gradient platforms using poly(methyl methacrylate) based on the biotin streptavidin interaction for biological applications Langmuir 26, (17), 14154-14161

This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PM MA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToRSIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SA V) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.

JTD Keywords: Wettability gradient, Polyethylene surface, Combinatorial, Immobilization, Biomaterials, Fabrication, Deposition, Bradykinin, Monolayers, Discharge

Tort, N., Salvador, J. P., Eritja, R., Poch, M., Martinez, E., Samitier, J., Marco, M. P., (2009). Fluorescence site-encoded DNA addressable hapten microarray for anabolic androgenic steroids Trac-Trends in Analytical Chemistry , 28, (6), 718-728

We report a new strategy for immunochemical screening of small organic molecules based on the use of a hapten microarray. Using DNA-directed immobilization strategies, we have been able to convert a DNA chip into a hapten microarray by taking advantage of all the benefits of the structural and electrostatic homogeneous properties of DNA. The hapten microarray uses hapten-oligonucleotide probes instead of proteins, avoiding the limitations of preparing stochiometrically-defined protein-oligonucleotide bioconjugates. As proof of concept, we show here the development of a microarray for analysis of anabolic androgenic steroids. The microchip is able to detect several illegal substances with sufficient detectability to be used as a screening method, according to the regulations of the World Anti-Doping Agency for sport and the European Commision for food safety. The results that we show corroborate the universal possibilities of the DNA chip, and, in this case, they open the way to develop hapten microarrays for the immunochemical analysis of small organic molecules.

JTD Keywords: Anti-doping, DNA chip, DNA-directed immobilization (DDI), Fluorescence, Food safety, Hapten microarray, Immunochemical screening, Proof of concept, Small organic molecule, Steroid

Ruiz, A., Mills, C. A., Valsesia, A., Martinez, E., Ceccone, G., Samitier, J., Colpo, P., Rossi, F., (2009). Large-area, nanoimprint-assisted microcontact stripping for the fabrication of microarrays of fouling/nonfouling nanostructures Small 5, (10), 1133-1137

Methods for the accurate positioning of nanometric beads on a substrate have been developed over a number of years, and range from serial atomic force microscopy (AFM)techniques for single-bead positioning to parallel techniques for the positioning of large populations of beads in monolayer or multilayer architectures, typically from a liquid suspension. For example, topographic cues have been used for bead-based protein array production, although in this case, there is a random distribution of beads within the topography. Bead patterning has also been achieved in capillaries using a micromolding in capillaries (MIMIC) technique. Line patterns with micrometer widths are possible with this technique, achieving good multilayer organization. For monolayer bead patterning at micrometer dimensions, electrostatic forces and similar electrostatic assemblies using nanoxerography, as well as patterning by selective chemical functionalization, by transfer of particles from a liquid–liquid interface, and by subtracting top–down processes, are possible.

JTD Keywords: Microcontact stripping, Nanostructures, Poly(acrylic acid), Polystyrene, Surface patterning

Martinez, E., Lagunas, A., Mills, C. A., Rodriguez-Segui, S., Estevez, M., Oberhansl, S., Comelles, J., Samitier, J., (2009). Stem cell differentiation by functionalized micro- and nanostructured surfaces Nanomedicine 4, (1), 65-82

New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.

JTD Keywords: Cell pattering, Differentiation, Microcontact printing, Micropatterning, Microstructure, Nanoimprinting, Nanostructure, Stem cells

Rodriguez-Segui, S. A., Pla, M., Engel, E., Planell, J. A., Martinez, E., Samitier, J., (2009). Influence of fabrication parameters in cellular microarrays for stem cell studies Journal of Materials Science: Materials in Medicine , 20, (7), 1525-1533

Lately there has been an increasing interest in the development of tools that enable the high throughput analysis of combinations of surface-immobilized signaling factors and which examine their effect on stem cell biology and differentiation. These surface-immobilized factors function as artificial microenvironments that can be ordered in a microarray format. These microarrays could be useful for applications such as the study of stem cell biology to get a deeper understanding of their differentiation process. Here, the evaluation of several key process parameters affecting the cellular microarray fabrication is reported in terms of its effects on the mesenchymal stem cell culture time on these microarrays. Substrate and protein solution requirements, passivation strategies and cell culture conditions are investigated. The results described in this article serve as a basis for the future development of cellular microarrays aiming to provide a deeper understanding of the stem cell differentiation process.

JTD Keywords: Bone-marrow, Protein microarrays, Progenitor cells, Differentiation, Surfaces, Growth, Biomaterials, Commitment, Pathways, Culture media

Merolli, A., Rocchi, L., Catalano, F., Planell, J., Engel, E., Martinez, E., Sbernardori, M. C., Marceddu, S., Leali, P. T., (2009). In vivo regeneration of rat sciatic nerve in a double-halved stitch-less guide: a pilot-study Microsurgery , 29, (4), 310-318

It is about 20 years that tubular nerve guides have been introduced into clinical practice as a reliable alternative to autograft, in gaps not-longer-than 20 mm, bringing the advantage of avoiding donor site sacrifice and morbidity. There are limitations in the application of tubular guides. First, tubular structure in itself makes surgical implantation difficult; second, stitch sutures required to secure the guide may represent a site of unfavorable fibroblastic reaction; third, maximum length and diameter of the guide correlate with the occurrence of a poorer central vascularization of regenerated nerve. We report on the in vivo testing of a new concept of nerve-guide (named NeuroBox) which is double-halved, not-degradable, rigid, and does not require any stitch to be held in place, employing acrylate glue instead. Five male Wistar rats had the new guide implanted in a 4-mm sciatic nerve defect; two guides incorporated a surface constituted of microtrenches aligned longitudinally. Further five rats had the 4-mm gap left without repair. Contralateral intact nerves were used as controls. After 2 months, nerve regeneration occurred in all animals treated by the NeuroBox; fine blood vessels were well represented. There was no regeneration in the un-treated animals. Even if the limited number of animals does not allow to draw definitive conclusions, some result can be highlighted: an easy surgical technique was associated with the box-shaped guide and acrylate glue was easily applied; an adequate intraneural vascularization was found concurrently with the regeneration of the nerve and no adverse fibroblastic proliferation was present.

JTD Keywords: Peripheral-nerve, Polyglycolic acid, Guidance cues, Collagen tube, Median nerve, Repair, Growth, Cyanoacrylate, Complications, Anastomosis

Martinez, E., Engel, E., Planell, J. A., Samitier, J., (2009). Effects of artificial micro- and nano-structured surfaces on cell behaviour Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 126-135

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nanofeatures such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell. orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

JTD Keywords: Microstructure, Topography, Cell behaviour, Cell morphology, Cell orientation

Engel, E., Martinez, E., Mills, C. A., Funes, M., Planell, J. A., Samitier, J., (2009). Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 136-144

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

JTD Keywords: Mesenchymal stem cells, Osteoblasts, Topography, Microstructures

Barreiros dos Santos, M., Sporer, C., Sanvicens, N., Pascual, N., Errachid, A., Martinez, E., Marco, M. P., Teixeira, V., Samiter, J., (2009). Detection of pathogenic Bacteria by Electrochemical Impedance Spectroscopy: Influence of the immobilization strategies on the sensor performance Procedia Chemistry 23rd Eurosensors Conference (ed. Brugger, J., Briand, D.), Elsevier Science, BV (Lausanne, Switzerland) 1, 1291-1294

Electrochemical impedance spectroscopy (EIS) is applied to detect pathogenic E. coli O157:H7 bacteria via a label free immunoassay-based detection method. Polyclonal anti-E.coli antibodies (PAb) are immobilized onto gold electrodes following two different strategies, via chemical bond formation between antibody amino groups and a carboxylic acid containing self-assembled molecular monolayer (SAM) and alternatively by linking a biotinylated anti-E. coli to Neutravidin on a mixed-SAM. Impedance spectra for sensors of both designs for increasing concentrations of E. coli are recorded in phosphate buffered saline (PBS). The Nyquist plots can be modeled with a Randle equivalent circuit, identifying the charge transfer resistance RCT as the relevant concentration dependent parameter. Sensors fabricated from both designs are able to detect very low concentration of E. coli with limits of detection as low as 10-100 cfu/ml. The influence of the different immobilization protocols on the sensor performance is evaluated in terms of sensitivity, dynamic range and resistance against nonspecific absorption.

JTD Keywords: Bacteria detection, Biosensors, E-coli, Impedance spectroscopy

Diaguez, L., Darwish-, N., Mir, M., Martinez, E., Moreno, M., Samitier, J., (2009). Effect of the refractive index of buffer solutions in evanescent optical biosensors Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 851-855

Evanescent field optical biosensors are label free sensors that measure the variation of the refractive index of the adsorbed layer onto a chip surface and translate this variation into surface concentration of the adsorbed molecule. The evanescent field based techniques depend on a theoretical model of the waveguide to determine the desired parameters of the adsorbed layer. As this layer is not only composed by the biomolecules, but also by some amount of the buffer solution, in this study, we have developed a new calibration method to take into account the refractive index buffer changes. We report a new methodology to characterize each sensor chip before the measurements and we present the refractive indexes of different buffer solutions considering the most common ones used in biosensor applications. This work will set the calibration bases for any optical grating biosensor instrument.

JTD Keywords: -----

Issle, J., Pla, M., Martínez, Elena, Hartmann, U., (2008). Patterning of magnetic nanobeads on surfaces by poly(dimethylsiloxane) stamps Langmuir 24, (3), 888-893

Poly(dimethylsiloxane) (PDMS) stamps are widely used in soft lithographic methods. They are powerful tools for obtaining structures of soft material in the micrometer to nanometer range by printing techniques. In this contribution, a new application of h-PDMS stamps for nanobead deposition is introduced. Magnetite-polysaccharide particles of an average diameter of 150 nm are used. They can be biologically functionalized by attaching various molecular groups. Deposition of these particles on a carrier substrate results in well-reproducible structures. This is achieved by means of PDMS stamps with different patterns using a microfluidic approach on one hand and a printing approach on the other hand. Furthermore, magnetic substrates with particular domain structures have been used. The beads can then be arranged in rather complicated but well-defined geometrical structures along the domain walls. The magnetic interaction considerably increases the adhesion of the beads to the carrier substrate. All involved materials are biocompatible. Thus the setup can be used in cell culture experiments in order to investigate influences of different particle-bound proteins and particle patterns on cell growth and vitality.


Fernandez, Javier G., Mills, C. A., Martinez, E., Lopez-Bosque, M. J., Sisquella, X., Errachid, A., Samitier, J., (2008). Micro- and nanostructuring of freestanding, biodegradable, thin sheets of chitosan via soft lithography Journal of Biomedical Materials Research - Part A , 85A, (1), 242-247

A technique for imparting micro- and nano-structured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures similar to 60 nm tall and similar to 500 X 500 nm(2) to be produced on freestanding similar to 0.5 mm thick sheets of the polymer when cured at 293 K, and structures similar to 400 nm tall and 5 X 5 mu m(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (similar to 200 mu m thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

JTD Keywords: Chitin/chitosan, Microstructure, Nanotopography, Polymerization, Soft lithography

Martinez, E., Engel, E., Lopez-Iglesias, C., Mills, C. A., Planell, J. A., Samitier, J., (2008). Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-/nanopatterned substrates: A study of cell-substrate interactions Micron , 39, (2), 111-116

Topographic micro and nanostructures can play an interesting role in cell behaviour when cells are cultured on these kinds of patterned substrates. It is especially relevant to investigate the influence of the nanometric dimensions topographic features on cell morphology, proliferation, migration and differentiation. To this end, some of the most recent fabrication technologies, developed for the microelectronics industry, can be used to produce well-defined micro and nanopatterns on biocompatible polymer substrates. In this work, osteoblast-like cells are grown on poly(methyl methacrylate) substrates patterned by nanoimprint lithography techniques. Examination of the cell-substrate interface can reveal important details about the cell morphology and the distribution of the focal contacts on the substrate surface. For this purpose, a combination of focused ion beam milling and scanning electron microscopy techniques has been used to image the cell-substrate interface. This technique, if applied to samples prepared by freeze-drying methods, allows high-resolution imaging of cross-sections through the cell and the substrate, where the interactions between the nanopatterned substrate, the cell and the extracellular matrix, which are normally hidden by the bulk of the cell, can be studied.

JTD Keywords: Electron microscopy, Interface, Nanotopography, Osteoblast, Adhesion molecule, Cell morphology

Pla, M., Fernandez, Javier G., Mills, C. A., Martinez, E., Samitier, J., (2007). Micro/nanopatterning of proteins via contact printing using high aspect ratio PMMA stamps and NanoImprint apparatus Langmuir 23, (16), 8614-8618

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

JTD Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

Mills, C. A., Pla, M., Martin, C., Lee, M., Kuphal, M., Sisquella, X., Martinez, E., Errachid, A., Samitier, J., (2007). Structured thin organic active layers and their use in electrochemical biosensors Measurement & Control , 40, (3), 88-91