Javier Ramon got his PhD in the Department of Organic Chemistry at the University of Barcelona (UB). Posteriorly during their post-doc stay, he was working under the direction of professor Mizutani at Hyogo University in Japan on lithography fabrication, microfluidics and dielectrophoresis technic (2009-2011). After their post-doctoral stay (2011), he was hired by the Advanced Institute for Materials Research (AIMR) at Tohoku University as Assistant Researcher. The AIMR-WPI institute is Japan's third most relevant institute and one worldwide material science reference. He joined the group of Prof. Matsue in the device/systems group, and in April 2013, he was promoted to Assistant Professor. In 2015 he joined as Ramon y Cajal researcher IBEC. In 2020, he became an ICREA Research Professor and is now leading the Biosensors for Bioengineering group, focused on integrating fully functional tissues with microscale biosensor technology to obtain "organs-on-a-chip".
Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.
The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.
In recent years, metaplasmonic biosensors have emerged as a novel counterpart of well-established plasmonic biosensors based on thin metallic layers. Metaplasmonic biosensors offer high potential for sensor miniaturiza-tion, extreme sensitivity biosensing, and high multiplexing capabilities with detection methods free of coupling optical elements. These capabilities make metaplasmonic biosensors highly attractive for Point-of-Care and handled/portable devices or novel On-Chip devices; as a result, it has increased the number of prototypes and potential applications that emerged during the last years. One of the main challenges to achieving fully operative devices is the achievement of high-throughput biointerfaces for sensitive and selective biodetection in complex media. Despite the superior surface sensitivity achieved by metaplasmonic sensors compared to conventional plasmonic sensors based on metallic thin films, the main limitations to achieving high-throughput and multiplexed biosensing usually are associated with the sensitivity and selectivity of the bioin-terface and, as a consequence, their application to the direct analysis of real complex samples. This graphical review discusses the potential challenges and capabilities of different biofunctionalization strategies, biorecog-nition elements, and antifouling strategies to achieve scalable and high-throughput metaplasmonic biosensing for Point-of-Care devices and bioengineering applications like Organs-On-Chip.
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.
Abstract Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human platelet lysate reinforced with cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.
The liver neutralizes endogenous and exogenous toxins and metabolites, being metabolically interconnected with many organs. Numerous clinical and experimental studies show a strong association between Non-alcoholic fatty liver disease (NAFLD) and loss of skeletal muscle mass known as sarcopenia. Liver transplantation solves the hepatic-related insufficiencies, but it is unable to revert sarcopenia. Knowing the mechanism(s) by which different organs communicate with each other is crucial to improve the drug development that still relies on the two-dimensional models. However, those models fail to mimic the pathological features of the disease. Here, both liver and skeletal muscle cells were encapsulated in gelatin methacryloyl and carboxymethylcellulose to recreate the disease’s phenotype in vitro. The 3D hepatocytes were challenged with non-esterified fatty acids (NEFAs) inducing features of Non-alcoholic fatty liver (NAFL) such as lipid accumulation, metabolic activity impairment and apoptosis. The 3D skeletal muscle tissues incubated with supernatant from fatty hepatocytes displayed loss of maturation and atrophy. This study demonstrates the connection between the liver and the skeletal muscle in NAFL, narrowing down the players for potential treatments. The tool herein presented was employed as a customizable 3D in vitro platform to assess the protective effect of albumin on both hepatocytes and myotubes.
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.
Research into the development of therapeutic strategies is basedmainly on animal models and cell cultures. The ability to extrapolatedata from them is limited, and research on new drugs cannot beperformed efficiently. This is especially dramatic in rare diseases,which are intrinsically very heterogeneous. The generation of ad-vanced models using tissue engineering and patient-derived cellsallows fabricating new platforms for studying pathological processesand discovering new potential drugs. Here, we developed a patient-derived 3D functional skeletal muscle for Duchenne muscular dys-trophy (DMD). DMD is the most prevalent neuromuscular diseasediagnosed during childhood. The disease is characterized by pro-gressive degeneration of skeletal and cardiac muscle caused by thelack of dystrophin protein. Although there are several molecules indrug development for DMD, there is no treatment available for pa-tients to date. By using a 3D-printed casting mold, we encapsulatedpatient-derived myogenic precursor cells in a fibrin-composite ma-trix. This platform incorporated two flexible T-shaped pillars thatprovided continuous tension to the tissue, thus allowing the orien-tation of the muscle fibers. Our 3D muscle model expressed maturemuscle markers and responded to electric pulse stimulation (EPS).Besides, contraction dynamics between DMD and control tissueswere shown to be different. Moreover, an increase of damagemarkers after EPS was observed in DMD but not in healthy tissues.Finally, the tissues will be integrated into a microfluidic device tomonitor drug administration. Eventually, the microfluidic systemwill be connected to a biosensors system for the real-time detectionof biomarkers.
The limitations of obtaining pancreatic islets from differentsources as animal models or human donors complicate the study oftype 2 diabetes (T2D) in vitro. Immortalized cell lines as the in-sulin-producing INS1Eb-cells appeared as a valid alternative tomodel insulin-related diseases. The formation of 3D structures topromote cell aggregations from single cells is a handy tool togenerate resemblance islet-like pseudoislets. Traditionally usedhydrogel encapsulation methods induce a lack of nutrient and ox-ygen diffusion for pancreatic tissue engineering. Here, we usecryogelation technology to develop a more resemblance scaffoldwith the mechanical and physical properties needed to engineerpancreatic tissue. This study shows that carboxymethyl cellulose(CMC) cryogels prompted cells to generateb-cell clusters. Thehigh porosity achieved with this approach allowed us to createspecific range pseudoislets. However, gelatin-based scaffolds didnot induce this cell organization. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 days and responded betterto the glucose over conventional monolayer cultures. Overall, ourresults demonstrate that CMC-scaffolds can be used to control theorganization and function of insulin-producingb-cells, represent-ing a suitable technique to generateb-cell clusters to study pan-creatic islet function.
Diabetes mellitus is a significant public health problem worldwide. It encompasses a group of chronic disorders characterized by hyperglycemia, resulting from pancreatic islet dysfunction or as a consequence of insulin-producing ?-cell death. Organ-on-a-chip platforms have emerged as technological systems combining cell biology, engineering, and biomaterial technological advances with microfluidics to recapitulate a specific organ’s physiological or pathophysiological environment. These devices offer a novel model for the screening of pharmaceutical agents and to study a particular disease. In the field of diabetes, a variety of microfluidic devices have been introduced to recreate native islet microenvironments and to understand pancreatic ?-cell kinetics in vitro. This kind of platforms has been shown fundamental for the study of the islet function and to assess the quality of these islets for subsequent in vivo transplantation. However, islet physiological systems are still limited compared to other organs and tissues, evidencing the difficulty to study this “organ” and the need for further technological advances. In this review, we summarize the current state of islet-on-a-chip platforms that have been developed so far. We recapitulate the most relevant studies involving pancreatic islets and microfluidics, focusing on the molecular and cellular-scale activities that underlie pancreatic ?-cell function.
Over the last years, optical biosensors based on plasmonic nanomaterials have gained great scientific interest due to their unquestionable advantages compared to other biosensing technologies. They can achieve sensitive, direct, and label-free analysis with exceptional potential for multiplexing and miniaturization. Recently, it has been demonstrated the potential of using optical discs as high throughput nanotemplates for the development of plasmonic biosensors in a cost-effective way. This work is a pilot study focused on the development of an integrated plasmonic biosensor for the monitoring of cell adhesion and growth of human retinal pigmented cell line (ARPE-19) under different media conditions (0 and 2% of FBS). We observed an increase of the plasmonic band displacement under 2% FBS compared to 0% conditions over time (1, 3, and 5 h). These preliminary results show that the proposed plasmonic biosensing approach is a direct, non-destructive, and real-time tool that could be employed in the study of living cells behavior and culture conditions. Furthermore, this setup could assess the viability of the cells and their growth over time with low variability between the technical replicates improving the experimental replicability.
Abstract The development of nanostructured plasmonic biosensors has been widely widespread in the last years, motivated by the potential benefits they can offer in integration, miniaturization, multiplexing opportunities, and enhanced performance label-free biodetection in a wide field of applications. Between them, engineering tissues represent a novel, challenging, and prolific application field for nanostructured plasmonic biosensors considering the previously described benefits and the low levels of secreted biomarkers (?pM–nM) to detect. Here, we present an integrated plasmonic nanocrystals-based biosensor using high throughput nanostructured polycarbonate substrates. Metallic film thickness and incident angle of light for reflectance measurements were optimized to enhance the detection of antibody–antigen biorecognition events using numerical simulations. We achieved an enhancement in biodetection up to 3× as the incident angle of light decreases, which can be related to shorter evanescent decay lengths. We achieved a high reproducibility between channels with a coefficient of variation below 2% in bulk refractive index measurements, demonstrating a high potential for multiplexed sensing. Finally, biosensing potential was demonstrated by the direct and label-free detection of interleukin-6 biomarker in undiluted cell culture media supernatants from bioengineered 3D skeletal muscle tissues stimulated with different concentrations of endotoxins achieving a limit of detection (LOD) of ? 0.03 ng/mL (1.4 pM).
Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.
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.
In vitro research for the study of type 2 diabetes (T2D) is frequently limited by the availability of a functional model for islets of Langerhans. To overcome the limitations of obtaining pancreatic islets from different sources, such as animal models or human donors, immortalized cell lines as the insulin-producing INS1E beta-cells have appeared as a valid alternative to model insulin-related diseases. However, immortalized cell lines are mainly used in flat surfaces or monolayer distributions, not resembling the spheroid-like architecture of the pancreatic islets. To generate islet-like structures, the use of scaffolds appeared as a valid tool to promote cell aggregations. Traditionally-used hydrogel encapsulation methods do not accomplish all the requisites for pancreatic tissue engineering, as its poor nutrient and oxygen diffusion induces cell death. Here, we use cryogelation technology to develop a more resemblance scaffold with the mechanical and physical properties needed to engineer pancreatic tissue. This study shows that carboxymethyl cellulose (CMC) cryogels prompted cells to generate beta-cell clusters in comparison to gelatin-based scaffolds, that did not induce this cell organization. Moreover, the high porosity achieved with CMC cryogels allowed us to create specific range pseudoislets. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 d and a better response to glucose over conventional monolayer cultures. Overall, our results demonstrate that CMC-scaffolds can be used to control the organization and function of insulin-producing beta-cells, representing a suitable technique to generate beta-cell clusters to study pancreatic islet function.
In this work we have explored the use of a Surface Plasmon resonance (SPR) phenomenon for the detection of interleukin-6 (IL-6), a pro-inflammatory cytokine. It plays an important role in the muscle tissues, having direct relation with muscle contraction and, thus, it is considered a biomarker for some types of muscular dystrophies. Here we show that SPR can be used as a real-time monitoring of the shift of the reflectance dip of a gold diffraction grating in front to the antibody adhesion to gold.
Organ-on-a-chip (OOC) devices offer new approaches for metabolic disease modeling and drug discovery by providing biologically relevant models of tissues and organs in vitro with a high degree of control over experimental variables for high-content screening applications. Yet, to fully exploit the potential of these platforms, there is a need to interface them with integrated non-labeled sensing modules, capable of monitoring, in situ, their biochemical response to external stimuli, such as stress or drugs. In order to meet this need, we aim here to develop an integrated technology based on coupling a localized surface plasmon resonance (LSPR) sensing module to an OOC device to monitor the insulin in situ secretion in pancreatic islets, a key physiological event that is usually perturbed in metabolic diseases such as type 2 diabetes (T2D). As a proof of concept, we developed a biomimetic islet-on-a-chip (IOC) device composed of mouse pancreatic islets hosted in a cellulose-based scaffold as a novel approach. The IOC was interfaced with a state-of-the-art on-chip LSPR sensing platform to monitor the in situ insulin secretion. The developed platform offers a powerful tool to enable the in situ response study of microtissues to external stimuli for applications such as a drug-screening platform for human models, bypassing animal testing.
Non-alcoholic fatty liver affects about 25% of global adult population. On the long-term, it is associated with extra-hepatic compliances, multiorgan failure, and death. Various invasive and non-invasive methods are employed for its diagnosis such as liver biopsies, CT scan, MRI, and numerous scoring systems. However, the lack of accuracy and reproducibility represents one of the biggest limitations of evaluating the effectiveness of drug candidates in clinical trials. Organ-on-chips (OOC) are emerging as a cost-effective tool to reproduce in vitro the main NAFLD’s pathogenic features for drug screening purposes. Those platforms have reached a high degree of complexity that generate an unprecedented amount of both structured and unstructured data that outpaced our capacity to analyze the results. The addition of artificial intelligence (AI) layer for data analysis and interpretation enables those platforms to reach their full potential. Furthermore, the use of them do not require any ethic and legal regulation. In this review, we discuss the synergy between OOC and AI as one of the most promising ways to unveil potential therapeutic targets as well as the complex mechanism(s) underlying NAFLD.
Gómez-Domínguez, D., Epifano, C., Miguel, F., Castaño, A. G., Vilaplana-Martí, B., Martín, A., Amarilla-Quintana, S., Bertrand, A. T., Bonne, G., Ramón-Azcón, J., Rodríguez-Milla, M. A., Pérez de Castro, I., (2020). Consequences of Lmna exon 4 mutations in myoblast functionCells 9, (5), 1286
Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.
Non-alcoholic fatty liver (NAFLD) is a metabolic disorder related to a chronic lipid accumulation within the hepatocytes. This disease is the most common liver disorder worldwide, and it is estimated that it is present in up to 25% of the world’s population. However, the real prevalence of this disease and the associated disorders is unknown mainly because reliable and applicable diagnostic tools are lacking. It is known that the level of albumin, a pleiotropic protein synthesized by hepatocytes, is correlated with the correct function of the liver. The development of a complementary tool that allows direct, sensitive, and label-free monitoring of albumin secretion in hepatocyte cell culture can provide insight into NAFLD’s mechanism and drug action. With this aim, we have developed a simple integrated plasmonic biosensor based on gold nanogratings from periodic nanostructures present in commercial Blu-ray optical discs. This sensor allows the direct and label-free monitoring of albumin in a 2D fatty liver disease model under flow conditions using a highly-specific polyclonal antibody. This technology avoids both the amplification and blocking steps showing a limit of detection within pM range (≈0.26 ng/mL). Thanks to this technology, we identified the optimal fetal bovine serum (FBS) concentration to maximize the cells’ lipid accumulation. Moreover, we discovered that the hepatocytes increased the amount of albumin secreted on the third day from the lipids challenge. These data demonstrate the ability of hepatocytes to respond to the lipid stimulation releasing more albumin. Further investigation is needed to unveil the biological significance of that cell behavior.
Currently, the fabrication of scaffolds for engineered skeletal muscle tissue is unable to reach the millimeter size. The main drawbacks are the poor nutrients diffusion, lack of internal structure to align precursor cells as well as poor mechanical and electric properties. Herein, we present a combination of gelatin-carboxymethyl cellulose materials polymerised by a cryogelation process that allowed us to reach scaffold fabrication up to millimeters size and solve the main problems related with large size muscle tissue constructs. 1) By incorporating carbon nanotubes (CNT) we can improve the electrical properties of the scaffold, thereby enhancing tissue maturation when applying an electric pulse stimulus (EPS). 2) We have fabricated an anisotropic internal three-dimensional microarchitecture pore distribution with high aligned morphology to enhance cells alignment, cell fusion and myotubes formation. With this set up, we were able to generate a fully functional skeletal muscle tissue using a combination of EPS and our doped-biocomposite scaffold and obtain a mature tissue in a millimeter scale. We also characterized pore distribution, swelling, stiffness and conductivity of the scaffold. Moreover, we proved that the cells are viable and able to fuse in a three-dimensional (3D) functional myotubes throughout the scaffold. In conclusion, we fabricate a biocompatible and customizable scaffold for 3D cell culture suitable for a wide range of application such as organ-on-a-chip, drug screening, transplantation and disease modelling.
Understanding the protein-secretion dynamics from single, specific tissues is critical toward the advancement of disease detection and treatments. However, such secretion dynamics remain difficult to measure in vivo due to the uncontrolled contributions from other tissue populations. Here, we describe an integrated platform designed for the reliable, near real-time measurements of cytokines secreted from an in vitro single-tissue model. In our setup, we grow 3D biomimetic tissues to discretize cytokine source, and we separate them from a magnetic microbead-based biosensing system using a Transwell insert. This design integrates physiochemically controlled biological activity, high-sensitivity protein detection (LOD < 20 pg mL−1), and rapid protein diffusion to enable non-invasive, near real-time measurements. To showcase the specificity and sensitivity of the system, we use our setup to probe the inflammatory process related to the protein Interleukine 6 (IL-6) and to the Tumor Necrosis Factor (TNF-α). We show that our setup can monitor the time-dependence profile of IL-6 and TNF-α secretion that results from the electrical and chemical stimulation of 3D skeletal muscle tissues. We demonstrate a novel and affordable methodology for discretizing the secretion kinetics of specific tissues for advancing metabolic-disorder studies and drug-screening applications.
Despite the increasing number of organs-on-a-chip that have been developed in the past decade, limited efforts have been made to integrate a sensing system for in situ continual measurements of biomarkers from three-dimensional (3D) tissues. Here, we present a custom-made integrated platform for muscle cell stimulation under fluidic conditions connected with a multiplexed high-sensitivity electrochemical sensing system for in situ monitoring. To demonstrate this, we use our system to measure the release levels and release time of interleukin 6 and tumor necrosis factor alpha in vitro by 3D muscle microtissue under electrical and biological stimulations. Our experimental design has enabled us to perform multiple time point measurements using functionalized screen-printed gold electrodes with sensitivity in the ng mL−1 range. This affordable setup is uniquely suited for monitoring factors released by 3D single cell types upon external stimulation for metabolic studies.
Non-alcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation within the liver affecting 1 in 4 people worldwide. As the new silent killer of the twenty-first century, NAFLD impacts on both the request and the availability of new liver donors. The liver is the first line of defense against endogenous and exogenous metabolites and toxins. It also retains the ability to switch between different metabolic pathways according to food type and availability. This ability becomes a disadvantage in obesogenic societies where most people choose a diet based on fats and carbohydrates while ignoring vitamins and fiber. The chronic exposure to fats and carbohydrates induces dramatic changes in the liver zonation and triggers the development of insulin resistance. Common believes on NAFLD and different diets are based either on epidemiological studies, or meta-analysis, which are not controlled evidences; in most of the cases, they are biased on test-subject type and their lifestyles. The highest success in reverting NAFLD can be attributed to diets based on high protein instead of carbohydrates. In this review, we discuss the impact of NAFLD on body metabolic plasticity. We also present a detailed analysis of the most recent studies that evaluate high-protein diets in NAFLD with a special focus on the liver and the skeletal muscle protein metabolisms.
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.
Abstract New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment.
Animal models have been the main resources for drug discovery and prediction of drugs’ pharmacokinetic responses in the body. However, noticeable drawbacks associated with animal models include high cost, low reproducibility, low physiological similarity to humans, and ethical problems. Engineered tissue models have recently emerged as an alternative or substitute for animal models in drug discovery and testing and disease modeling. In this review, we focus on skeletal muscle and cardiac muscle tissues by first describing their characterization and physiology. Major fabrication technologies (i.e., electrospinning, bioprinting, dielectrophoresis, textile technology, and microfluidics) to make functional muscle tissues are then described. Finally, currently used muscle tissue models in drug screening are reviewed and discussed.
Obregón, R., Ramón-Azcón, J., Ahadian, S., (2017). Nanofiber composites in blood vessel tissue engineering Nanofiber Composites for Biomedical Applications (ed. Ramalingam, M., Ramakrishna, S.), Elsevier (Duxford, UK) Woodhead Publishing Series in Biomaterials, 483-506
Tissue engineering (TE) aims to restore function or replace damaged tissue through biological principles and engineering. Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Composite nanofibers, which are hybrid nanofibers blended from natural and synthetic polymers, represent a major advancement in TE and regenerative medicine, since they take advantage of the physical properties of the synthetic polymer and the bioactivity of the natural polymer while minimizing the disadvantages of both. Although various nanofibrous matrices have been applied to almost all the areas of TE, in this chapter we will focus on nanofiber composites scaffolds for vascular TE.
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Fernández-Costa JM, Tejedera-Vilafranca A, Fernández-Garibay X, Ramón-Azcón J, (2023). Muscle-on-a-chip devices: a new era for in vitro modelling of muscular dystrophies Disease Models & Mechanisms 16, dmm050107 
Current Pharmaceutical Design , 23, (20), 2991-3004