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Publications

by Keyword: fabrication

Webster-Wood, VA, Guix, M, Xu, NW, Behkam, B, Sato, H, Sarkar, D, Sanchez, S, Shimizu, M, Parker, KK, (2023). Biohybrid robots: recent progress, challenges, and perspectives Bioinspiration & Biomimetics 18, 015001

The past ten years have seen the rapid expansion of the field of biohybrid robotics. By combining engineered, synthetic components with living biological materials, new robotics solutions have been developed that harness the adaptability of living muscles, the sensitivity of living sensory cells, and even the computational abilities of living neurons. Biohybrid robotics has taken the popular and scientific media by storm with advances in the field, moving biohybrid robotics out of science fiction and into real science and engineering. So how did we get here, and where should the field of biohybrid robotics go next? In this perspective, we first provide the historical context of crucial subareas of biohybrid robotics by reviewing the past 10+ years of advances in microorganism-bots and sperm-bots, cyborgs, and tissue-based robots. We then present critical challenges facing the field and provide our perspectives on the vital future steps toward creating autonomous living machines.

JTD Keywords: Biohybrid, Cell, Cyborg, Delivery, Fabrication, Flight, Insect, Living machines, Muscle activities, Muscular thin-films, Nanoparticles, Stimulation, Tissue


Widhe, M, Diez-Escudero, A, Liu, YL, Ringstrom, N, Ginebra, MP, Persson, C, Hedhammar, M, Mestres, G, (2022). Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs Ceramics International 48, 31449-31460

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.

JTD Keywords: Bioceramics, Bone, Bone regeneration, Composites, Degradation, Fabrication, Hydroxyapatite, Hydroxyapatite scaffolds, Injectability, Porosity, Recombinant spider silk, Rgd motifs, Silk, Stem-cells


Mughal, S, Lopez-Munoz, GA, Fernandez-Costa, JM, Cortes-Resendiz, A, De Chiara, F, Ramon-Azcon, J, (2022). Organs-on-Chips: Trends and Challenges in Advanced Systems Integration Advanced Materials Interfaces 9, 2201618

Organ-on-chip platforms combined with high-throughput sensing technology allow bridging gaps in information presented by 2D cultures modeled on static microphysiological systems. While these platforms do not aim to replicate whole organ systems with all physiological nuances, they try to mimic relevant structural, physiological, and functional features of organoids and tissues to best model disease and/or healthy states. The advent of this platform has not only challenged animal testing but has also presented the opportunity to acquire real-time, high-throughput data about the pathophysiology of disease progression by employing biosensors. Biosensors allow monitoring of the release of relevant biomarkers and metabolites as a result of physicochemical stress. It, therefore, helps conduct quick lead validation to achieve personalized medicine objectives. The organ-on-chip industry is currently embarking on an exponential growth trajectory. Multiple pharmaceutical and biotechnology companies are adopting this technology to enable quick patient-specific data acquisition at substantially low costs.

JTD Keywords: A-chip, Biosensor, Biosensors, Cancer, Cells, Culture, Disease models, Epithelial electrical-resistance, Hydrogel, Microfabrication, Microphysiological systems, Models, Niches, Organ-on-a-chips, Platform


Casanellas I, Samitier J, Lagunas A, (2022). Recent advances in engineering nanotopographic substrates for cell studies Frontiers In Bioengineering And Biotechnology 10, 1002967

Cells sense their environment through the cell membrane receptors. Interaction with extracellular ligands induces receptor clustering at the nanoscale, assembly of the signaling complexes in the cytosol and activation of downstream signaling pathways, regulating cell response. Nanoclusters of receptors can be further organized hierarchically in the cell membrane at the meso- and micro-levels to exert different biological functions. To study and guide cell response, cell culture substrates have been engineered with features that can interact with the cells at different scales, eliciting controlled cell responses. In particular, nanoscale features of 1-100 nm in size allow direct interaction between the material and single cell receptors and their nanoclusters. Since the first "contact guidance" experiments on parallel microstructures, many other studies followed with increasing feature resolution and biological complexity. Here we present an overview of the advances in the field summarizing the biological scenario, substrate fabrication techniques and applications, highlighting the most recent developments.Copyright © 2022 Casanellas, Samitier and Lagunas.

JTD Keywords: cell response, density, differentiation, lithography, micro, nanofabrication, nanopatterning, nanopatterns, nanoscale, nanotopography, organization, photolithography, Cell response, Nanofabrication, Nanopatterning, Nanotopography, Plasma-membrane, Receptor nanoclustering


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

Herrero-Gomez, A, Azagra, M, Marco-Rius, I, (2022). A cryopreservation method for bioengineered 3D cell culture models Biomedical Materials 17, 045023

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

JTD Keywords: 3d cell culture, Biofabrication, Biomaterials, Carboxymethyl cellulose, Cryopreservation, Hepatocytes, Prevention, Scaffolds, Spheroids


Comelles J, Castillo-Fernández Ó, Martínez E, (2022). How to Get Away with Gradients Microfluidics And Biosensors In Cancer Research 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, Microfabrication, Microfluidics, Nanotechnology


Vilela, Diana, Guix, Maria, Parmar, Jemish, Blanco‐Blanes, Àngel, Sánchez, Samuel, (2022). Micromotor‐in‐Sponge Platform for Multicycle Large‐Volume Degradation of Organic Pollutants Small 18, 2107619

García-Torres, Jose, Lázaro, Carmen, Sylla, Dioulde, Lanzalaco, Sonia, Ginebra, Maria-Pau, Alemán, Carlos, (2022). Combining 2D organic and 1D inorganic nanoblocks to develop free-standing hybrid nanomembranes for conformable biosensors Journal Of Nanostructure In Chemistry ,

We report a simple approach to fabricate free-standing perforated 2D nanomembranes hosting well-ordered 1D metallic nanostructures to obtain hybrid materials with nanostructured surfaces for flexible electronics. Nanomembranes are formed by alternatively depositing perforated poly(lactic acid) (PLA) and poly(3,4-ethylenedioxythiophene) layers. Copper metallic nanowires (NWs) were incorporated into the nanoperforations of the top PLA layer by electrodeposition and further coated with silver via a transmetallation reaction. The combination of 2D polymeric nanomembranes and aligned 1D metallic NWs allows merging the flexibility and conformability of the ultrathin soft polymeric nanomembranes with the good electrical properties of metals for biointegrated electronic devices. Thus, we were able to tailor the nanomembrane surface chemistry as it was corroborated by SEM, EDX, XPS, CV, EIS and contact angle. The obtained hybrid nanomembranes were flexible and conformable showing sensing capacity towards H2O2 with good linear concentration range (0.35–10 mM), sensitivity (120 µA cm?2 mM?1) and limit of detection (7 ?m). Moreover, the membranes showed good stability, reproducibility and selectivity towards H2O2.

JTD Keywords: biointegrated sensors, designs, electronics, fabrication, free-standing films, h2o2, metallic nanowires, nanoparticles, nanowires, sensor, skin, Hydrogen-peroxide, Perforated nanomembranes


Raymond, Yago, Johansson, Linh, Thorel, Emilie, Ginebra, Maria-Pau, (2022). Translation of three-dimensional printing of ceramics in bone tissue engineering and drug delivery Mrs Bulletin 47, 59-69

del-Mazo-Barbara L, Ginebra MP, (2021). Rheological characterisation of ceramic inks for 3D direct ink writing: A review Journal Of The European Ceramic Society 41, 18-33

3D printing is a competitive manufacturing technology, which has opened up new possibilities for the fabrication of complex ceramic structures and customised parts. Extrusion-based technologies, also known as direct ink writing (DIW) or robocasting, are amongst the most used for ceramic materials. In them, the rheological properties of the ink play a crucial role, determining both the extrudability of the paste and the shape fidelity of the printed parts. However, comprehensive rheological studies of printable ceramic inks are scarce and may be difficult to understand for non-specialists. The aim of this review is to provide an overview of the main types of ceramic ink formulations developed for DIW and a detailed description of the more relevant rheological tests for assessing the printability of ceramic pastes. Moreover, the key rheological parameters are identified and linked to printability aspects, including the values reported in the literature for different ink compositions.

JTD Keywords: 3-dimensional structures, behavior, deposition, direct ink writing, freeform fabrication, gelation, glass scaffolds, mechanical-properties, printability, rheology, robocasting, suspensions, 3d printing, Direct ink writing, Phosphate scaffolds, Printability, Rheology, Robocasting


Chausse, Victor, Schieber, Romain, Raymond, Yago, Ségry, Brian, Sabaté, Ramon, Kolandaivelu, Kumaran, Ginebra, Maria-Pau, Pegueroles, Marta, (2021). Solvent-cast direct-writing as a fabrication strategy for radiopaque stents Additive Manufacturing 48,

López-Canosa A, Perez-Amodio S, Yanac-Huertas E, Ordoño J, Rodriguez-Trujillo R, Samitier J, Castaño O, Engel E, (2021). A microphysiological system combining electrospun fibers and electrical stimulation for the maturation of highly anisotropic cardiac tissue Biofabrication 13,

The creation of cardiac tissue models for preclinical testing is still a non-solved problem in drug discovery, due to the limitations related to thein vitroreplication of cardiac tissue complexity. Among these limitations, the difficulty of mimicking the functional properties of the myocardium due to the immaturity of the used cells hampers the obtention of reliable results that could be translated into human patients.In vivomodels are the current gold standard to test new treatments, although it is widely acknowledged that the used animals are unable to fully recapitulate human physiology, which often leads to failures during clinical trials. In the present work, we present a microfluidic platform that aims to provide a range of signaling cues to immature cardiac cells to drive them towards an adult phenotype. The device combines topographical electrospun nanofibers with electrical stimulation in a microfabricated system. We validated our platform using a co-culture of neonatal mouse cardiomyocytes and cardiac fibroblasts, showing that it allows us to control the degree of anisotropy of the cardiac tissue inside the microdevice in a cost-effective way. Moreover, a 3D computational model of the electrical field was created and validated to demonstrate that our platform is able to closely match the distribution obtained with the gold standard (planar electrode technology) using inexpensive rod-shaped biocompatible stainless-steel electrodes. The functionality of the electrical stimulation was shown to induce a higher expression of the tight junction protein Cx-43, as well as the upregulation of several key genes involved in conductive and structural cardiac properties. These results validate our platform as a powerful tool for the tissue engineering community due to its low cost, high imaging compatibility, versatility, and high-throughput configuration capabilities.

JTD Keywords: bioreactor, cardiac tissue engineering, cardiomyocytes, electrospinning, fabrication, fibroblasts, heart-on-a-chip, heart-tissue, in vitro models, myocardium, orientation, platform, scaffolds, Cardiac tissue engineering, Electrospinning, Field stimulation, Heart-on-a-chip, In vitro models, Microphysiological system


Olmo C, Franco L, Vidal A, Del Valle LJ, Puiggalí J, (2021). Ultrasound micromolding of porous polylactide/hydroxyapatite scaffolds Express Polymer Letters 15, 389-403

© BME-PT. Ultrasound micromolding (USM) preparation of hybrid scaffolds based on polylactide (PLA) and hydroxyapatite (HAp) particles has been evaluated. PLA was stable under the applied ultrasound source since a minimum degradation was detected. Porous materials were achieved using polyethylene glycol (PEG) and NaCl salts to the initial PLA and the subsequent leaching of the micromolded specimens. To avoid cavitation and decomposition problems during micromolding, it was necessary to use HAp free of typical synthesis impurities like carbonate and nitrate compounds. Compact PLA/HAp pieces allowed a maximum HAp load of 60 wt%, while porous specimens could be obtained with a maximum load of 38 wt%. Physical characterization of new scaffolds was performed by X-ray diffraction, spectroscopic and calorimetric techniques, stress-strain tests and contact angle measurements. Results indicated that a degree of porosity of 35% and relatively good mechanical properties could be achieved (i.e., 580 MPa, 4%, and 15.6 MPa for the Young modulus, elongation at break, and tensile strength, respectively). Scaffolds showed the positive effect of HAp and porosity on cell proliferation; this latter was 40% higher than that detected for non-porous PLA specimens.

JTD Keywords: apatite, conformation, fabrication, hydroxyapatite, micropieces, polymers, porous scaffolds, proliferation, tissue, ultrasound micromolding, vibration, Composite scaffolds, Hydroxyapatite, Micropieces, Porous scaffolds, Processing technologies, Ultrasound micromolding


Guix M, Mestre R, Patiño T, de Corato M, Fuentes J, Zarpellon G, Sánchez S, (2021). Biohybrid soft robots with self-stimulating skeletons Science Robotics 6,

Bioinspired hybrid soft robots that combine living and synthetic components are an emerging field in the development of advanced actuators and other robotic platforms (i.e., swimmers, crawlers, and walkers). The integration of biological components offers unique characteristics that artificial materials cannot precisely replicate, such as adaptability and response to external stimuli. Here, we present a skeletal muscle–based swimming biobot with a three-dimensional (3D)–printed serpentine spring skeleton that provides mechanical integrity and self-stimulation during the cell maturation process. The restoring force inherent to the spring system allows a dynamic skeleton compliance upon spontaneous muscle contraction, leading to a cyclic mechanical stimulation process that improves the muscle force output without external stimuli. Optimization of the 3D-printed skeletons is carried out by studying the geometrical stiffnesses of different designs via finite element analysis. Upon electrical actuation of the muscle tissue, two types of motion mechanisms are experimentally observed: directional swimming when the biobot is at the liquid-air interface and coasting motion when it is near the bottom surface. The integrated compliant skeleton provides both the mechanical self-stimulation and the required asymmetry for directional motion, displaying its maximum velocity at 5 hertz (800 micrometers per second, 3 body lengths per second). This skeletal muscle–based biohybrid swimmer attains speeds comparable with those of cardiac-based biohybrid robots and outperforms other muscle-based swimmers. The integration of serpentine-like structures in hybrid robotic systems allows self-stimulation processes that could lead to higher force outputs in current and future biomimetic robotic platforms. Copyright © 2021 The Authors, some rights reserved;

JTD Keywords: actuators, design, fabrication, mechanics, mems, myotubes, platform, tissue, 3d printers, Agricultural robots, Biological components, Biomimetic processes, Electrical actuation, Geometrical stiffness, Intelligent robots, Liquefied gases, Liquid-air interface, Mechanical integrity, Mechanical stimulation, Muscle, Muscle contractions, Phase interfaces, Robotics, Serpentine, Springs (components), Threedimensional (3-d)


Vera D, García-Díaz M, Torras N, Álvarez M, Villa R, Martinez E, (2021). Engineering Tissue Barrier Models on Hydrogel Microfluidic Platforms Acs Applied Materials & Interfaces 13, 13920-13933

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.

JTD Keywords: hydrogel, microfabrication, microfluidics, organ-on-chip, tissue barrier, Hydrogel, Microfabrication, Microfluidics, Organ-on-chip, Tissue barrier


Prat-Vidal, C., Rodríguez-Gómez, L., Aylagas, M., Nieto-Nicolau, N., Gastelurrutia, P., Agustí, E., Gálvez-Montón, C., Jorba, I., Teis, A., Monguió-Tortajada, M., Roura, S., Vives, J., Torrents-Zapata, S., Coca, M. I., Reales, L., Cámara-Rosell, M. L., Cediel, G., Coll, R., Farré, R., Navajas, D., Vilarrodona, A., García-López, J., Muñoz-Guijosa, C., Querol, S., Bayes-Genis, A., (2020). First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft EBioMedicine 54, 102729

Background Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. Methods The PeriCord is a clinical-size (12–16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth. Findings PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. Interpretation This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation. Funding La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, “La Caixa” Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.

JTD Keywords: Advanced therapy medicinal product (ATMP), Biofabrication, Cardiac tissue engineering, Myocardial infarction, Scaffold, Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs)


Cofiño, C., Perez-Amodio, S., Semino, C. E., Engel, E., Mateos-Timoneda, M. A., (2019). Development of a self-assembled peptide/methylcellulose-based bioink for 3D bioprinting Macromolecular Materials and Engineering 304, (11), 1900353

The introduction of 3D bioprinting to fabricate living constructs with tailored architecture has provided a new paradigm for biofabrication, with the potential to overcome several drawbacks of conventional scaffold-based tissue regeneration strategies. Hydrogel-based materials are suitable candidates regarding cell biocompatibility but often display poor mechanical properties. Self-assembling peptides are a promising source of biomaterials to be used as 3D scaffolds based on their similarity to extracellular matrices (structurally and mechanically). In this study, an advanced bioink for biofabrication is presented based on the optimization of a RAD16-I-based biomaterial. The strategy followed to build 3D predefined structures by 3D printing is based on an enhancement of bioink viscosity by adding methylcellulose (MC) to a RAD16-I solution. The resultant constructs display high shape fidelity and stability and embedded human mesenchymal stem cells present high viability after 7 days of culture. Moreover, cells are also able to differentiate to the adipogenic lineage, suggesting the suitability of this novel biomaterial for soft tissue engineering applications.

JTD Keywords: 3D bioprinting, Biofabrication, Bioinks, Self-assembling peptides, Tissue engineering


Samitier, Josep, Correia, A., (2019). Biomimetic Nanotechnology for Biomedical Applications (NanoBio&Med 2018) Biomimetics MDPI

Emerging nanobiotechnologies can offer solutions to the current and future challenges in medicine. By covering topics from regenerative medicine, tissue engineering, drug delivery, bionanofabrication, and molecular biorecognition, this Special Issue aims to provide an update on the trends in nanomedicine and drug delivery using biomimetic approaches, and the development of novel biologically inspired devices for the safe and effective diagnosis, prevention, and treatment of disease.

JTD Keywords: Bioinspired nanotechnologies, Bionanofabrication, Bio-nano measurement and microscopy, Nanomaterials for biological and medical applications, Nanoassemblies, Nanostructured surfaces, Drug delivery, Nanobioelectronics, Integrated systems/nanobiosensors, Nanotoxicology, Graphene-based applications


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


da Palma, R. K., Campillo, N., Uriarte, J. J., Oliveira, L. V. F., Navajas, D., Farré, R., (2015). Pressure- and flow-controlled media perfusion differently modify vascular mechanics in lung decellularization Journal of the Mechanical Behavior of Biomedical Materials , 49, 69-79

Organ biofabrication is a potential future alternative for obtaining viable organs for transplantation. Achieving intact scaffolds to be recellularized is a key step in lung bioengineering. Perfusion of decellularizing media through the pulmonary artery has shown to be effective. How vascular perfusion pressure and flow vary throughout lung decellularization, which is not well known, is important for optimizing the process (minimizing time) while ensuring scaffold integrity (no barotrauma). This work was aimed at characterizing the pressure/flow relationship at the pulmonary vasculature and at how effective vascular resistance depends on pressure- and flow-controlled variables when applying different methods of media perfusion for lung decellularization. Lungs from 43 healthy mice (C57BL/6; 7-8 weeks old) were investigated. After excision and tracheal cannulation, lungs were inflated at 10cmH2O airway pressure and subjected to conventional decellularization with a solution of 1% sodium dodecyl sulfate (SDS). Pressure (PPA) and flow (V'PA) at the pulmonary artery were continuously measured. Decellularization media was perfused through the pulmonary artery: (a) at constant PPA=20cmH2O or (b) at constant V'PA=0.5 and 0.2ml/min. Effective vascular resistance was computed as Rv=PPA/V'PA. Rv (in cmH2O/(ml/min)); mean±SE) considerably varied throughout lung decellularization, particularly for pressure-controlled perfusion (from 29.1±3.0 in baseline to a maximum of 664.1±164.3 (p<0.05), as compared with flow-controlled perfusion (from 49.9±3.3 and 79.5±5.1 in baseline to a maximum of 114.4±13.9 and 211.7±70.5 (p<0.05, both), for V'PA of 0.5 and 0.2ml/min respectively. Most of the media infused to the pulmonary artery throughout decellularization circulated to the airways compartment across the alveolar-capillary membrane. This study shows that monitoring perfusion mechanics throughout decellularization provides information relevant for optimizing the process time while ensuring that vascular pressure is kept within a safety range to preserve the organ scaffold integrity.

JTD Keywords: Acellular lung, Fluid mechanics, Lung bioengineering, Lung scaffold, Organ biofabrication, Tissue engineering, Vascular resistance


Tahirbegi, I. B., Alvira, M., Mir, M., Samitier, J., (2014). Simple and fast method for fabrication of endoscopic implantable sensor arrays Sensors 14, (7), 11416-11426

Here we have developed a simple method for the fabrication of disposable implantable all-solid-state ion-selective electrodes (ISE) in an array format without using complex fabrication equipment or clean room facilities. The electrodes were designed in a needle shape instead of planar electrodes for a full contact with the tissue. The needle-shape platform comprises 12 metallic pins which were functionalized with conductive inks and ISE membranes. The modified microelectrodes were characterized with cyclic voltammetry, scanning electron microscope (SEM), and optical interferometry. The surface area and roughness factor of each microelectrode were determined and reproducible values were obtained for all the microelectrodes on the array. In this work, the microelectrodes were modified with membranes for the detection of pH and nitrate ions to prove the reliability of the fabricated sensor array platform adapted to an endoscope.

JTD Keywords: Chemical sensors, Cyclic voltammetry, Electrochemistry, Endoscopy, Fabrication, Implants (surgical), Microelectrodes, Needles, Nitrates, Scanning electron microscopy, Biomedicine, Fabricated sensors, Fabrication equipment, Implantable devices, Implantable sensors, Optical interferometry, Planar electrode, Roughness factor, Ion selective electrodes


Mendes, A. C., Smith, K. H., Tejeda-Montes, E., Engel, E., Reis, R. L., Azevedo, H. S., Mata, Alvaro, (2013). Co-assembled and microfabricated bioactive membranes Advanced Functional Materials 23, (4), 430-438

The fabrication of hierarchical and bioactive self-supporting membranes, which integrate physical and biomolecular elements, using a single-step process that combines molecular self-assembly with soft lithography is reported. A positively charged multidomain peptide (with or without the cell-adhesive sequence arginine-glycine-aspartic acid-serine (RGDS)) self-assembles with hyaluronic acid (HA), an anionic biopolymer. Optimization of the assembling conditions enables the realization of membranes with well-controlled and easily tunable features at multiple size scales including peptide sequence, building-block co-assembly, membrane thickness, bioactive epitope availability, and topographical pattern morphology. Membrane structure, morphology, and bioactivity are investigated according to temperature, assembly time, and variations in the experimental setup. Furthermore, to evaluate the physical and biomolecular signaling of the self-assembled microfabricated membranes, rat mesenchymal stem cells are cultured on membranes exhibiting various densities of RGDS and different topographical patterns. Cell adhesion, spreading, and morphology are significantly affected by the surface topographical patterns and the different concentrations of RGDS. The versatility of the combined bottom-up and top-down fabrication processes described may permit the development of hierarchical macrostructures with precise biomolecular and physical properties and the opportunity to fine tune them with spatiotemporal control.

JTD Keywords: Membrane scaffolds, Mesenchymal stem cells, Microfabrication, Self-assembly, Topography


Ghassemi, S., Meacci, G., Liu, S., Gondarenko, A. A., Mathur, A., Roca-Cusachs, P., Sheetz, M. P., Hone, J., (2012). Cells test substrate rigidity by local contractions on submicrometer pillars Proceedings of the National Academy of Sciences of the United States of America 109, (14), 5328-5333

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell-matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 μm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-μm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-μm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-μm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.

JTD Keywords: Cell mechanics, Mechanotransduction, Nanofabrication


Fernandez, Javier G., Samitier, Josep, Mills, Christopher A., (2011). Simultaneous biochemical and topographical patterning on curved surfaces using biocompatible sacrificial molds Journal of Biomedical Materials Research - Part A , 98A, (2), 229-234

A method for the simultaneous (bio)chemical and topographical patterning of enclosed structures in poly(dimethyl siloxane) (PDMS) is presented. The simultaneous chemical and topography transference uses a water-soluble chitosan sacrificial mold to impart a predefined pattern with micrometric accuracy to a PDMS replica. The method is compared to conventional soft-lithography techniques on planar surfaces. Its functionality is demonstrated by the transference of streptavidin directly to the surface of the three-dimensional PDMS structures as well as indirectly using streptavidin-loaded latex nanoparticles. The streptavidin immobilized on the PDMS is tested for bioactivity by coupling with fluorescently labeled biotin. This proves that the streptavidin is immobilized on the PDMS surface, not in the bulk of the polymer, and is therefore accessible for use as signaling/binding element in micro and bioengineering. The use of a biocompatible polymer and processes enables the technique to be used for the chemical patterning of tissue constructions.

JTD Keywords: Biotechnology, Chitosan, Microfabrication, MEMs, Soft lithography


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


Caballero, D., Villanueva, G., Plaza, J. A., Mills, C. A., Samitier, J., Errachid, A., (2010). Sharp high-aspect-ratio AFM tips fabricated by a combination of deep reactive ion etching and focused ion beam techniques Journal of Nanoscience and Nanotechnology , 10, (1), 497-501

The shape and dimensions of an atomic force microscope tip are crucial factors to obtain high resolution images at the nanoscale. When measuring samples with narrow trenches, inclined sidewalls near 90 or nanoscaled structures, standard silicon atomic force microscopy (AFM) tips do not provide satisfactory results. We have combined deep reactive ion etching (DRIE) and focused ion beam (FIB) lithography techniques in order to produce probes with sharp rocket-shaped silicon AFM tips for high resolution imaging. The cantilevers were shaped and the bulk micromachining was performed using the same DRIE equipment. To improve the tip aspect ratio we used FIB nanolithography technique. The tips were tested on narrow silicon trenches and over biological samples showing a better resolution when compared with standard AFM tips, which enables nanocharacterization and nanometrology of high-aspect-ratio structures and nanoscaled biological elements to be completed, and provides an alternative to commercial high aspect ratio AFM tips.

JTD Keywords: Atomic-Force Microscope, Carbon nanotube tips, Probes, Roughness, Cells, Microfabrication, Calibration, Surfaces


Caballero, D., Samitier, J., Errachid, A., (2009). Submerged nanocontact printing (SnCP) of thiols Journal of Nanoscience and Nanotechnology , 9, (11), 6478-6482

Biological patterned surfaces having sub-micron scale resolution are of great importance in many fields of life science and biomedicine. Different techniques have been proposed for surface patterning at the nanoscale. However, most of them present some limitations regarding the patterned area size or are time-consuming. Micro/nanocontact printing is the most representative soft lithography-based technique for surface patterning at the nanoscale. Unfortunately, conventional micro/nanocontact printing also suffers from problems such as diffusion and stamp collapsing that limit pattern resolution. To overcome these problems, a simple way of patterning thiols under liquid media using submerged nanocontact printing (SnCP) over large areas (similar to cm(2)) achieving nanosize resolution is presented. The technique is also low cost and any special equipment neither laboratory conditions are required. Nanostructured poly(dimethyl siloxane) stamps are replicated from commercially available digital video disks. SnCP is used to stamp patterns of 200 nm 1-octadecanethiol lines in liquid media, avoiding ink diffusion and stamp collapsing, over large areas on gold substrates compared with conventional procedures. Atomic force microscopy measurements reveal that the patterns have been successfully transferred with high fidelity. This is an easy, direct, effective and low cost methodology for molecule patterning immobilization which is of interest in those areas that require nanoscale structures over large areas, such as tissue engineering or biosensor applications.

JTD Keywords: Submerged Nanocontact Printing, Replica Molding, Nanopatterning, Large Area, Dip-pen nanolithography, High-aspect-ratio, Soft lithography, Submicronscale, Nanoimprint lithography, Thin-film, Surfaces, Fabrication, Proteins, Nanofabrication


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