Biosensors for bioengineering

Our research is focused on multi tissues organs-on-a-chip (OOC) and more specifically in the metabolic crosstalk within tissues and their relationship with metabolic diseases. Our projects are focused on four key tissues regulating glucose homeostasis, namely, the pancreas, liver, skeletal muscle, and adipose tissue. To achieve this objective, it is necessary a combined interdisciplinary approach:

Human myotubes encapsulated in micropatterned hydrogel scaffold. Scale is 100 µm.

-Biomaterials and tissue engineering research

1) We have several lines of research related with skeletal muscle. Our first approach was with C2C12 mice cell line. We evaluated the influence of mechanical stiffness and geometrical confinement on the 3D culture of myoblast-laden chemically modified gelatin photo-cross linkable composite hydrogels in terms of in vitro myogenesis.

2) Encapsulation of beta-cells like from human skin fibroblast (collaboration with IDIBAPS). This work addresses two critical issues in the design of an efficient beta-cell replacement therapy: an accessible cell source for generation of substitute beta-cells and an adequate delivery device for transplantation. On one hand, we propose to generate transplantable functional insulin-producing beta-cells from fibroblasts through direct reprogramming strategies that bypass the pluripotent iPS stage. On a second objective, we are working in a new system of encapsulating beta-cells like in two steps, microencapsulation to protect cells from immune system and microencapsulation to mechanically protect them and manipulate them.

3) We are developing three-dimensional micro liver models using various biomaterials to recreate the in vivo-like mechanical properties and using hepatocytes and stellate cells. We are collaborating with Grifols company to test some drugs in our model.

4) We have a collaboration project with NovoNordisk to work in new approaches to encapsulate retinal cells.

-Biosensing technology:

Pancreas islet stained in blue for nuclei and red for actin inside a microporous microfibrillated cellulose-gelatin scaffold stained with green.

1) Integrating biosensors in an organ-on-a-chip. We are studying with in situ electrochemical biosensors the release of insulin under the effect of external stimuli, changes in glucose levels and myokines secreted by skeletal muscle (multi-OOC approach).

2) Related with this project we are implementing new biosensors systems. To fully exploit the potential of the organs-on-a-chip, there is a need to interface them to integrated sensing modules, capable to monitor in real-time their biochemical response to external stimuli, like stress or drugs.  The goal of this project is to answer this need, by developing a novel technology based on integrating localized surface plasmon resonance (LSPR) sensing module to organs-on-a-chip devices to monitor disease and evaluate drug response in organs-on-a-chip models.

3) Myotonic dystrophy type 1 (DM1) (collaboration with Hospital de la Fe and INCLIVA, Valencia, Spain). We have developed human skeletal muscle micro physiological tissues using micro molding technology and we have integrated them with amperometric biosensors to study the inflammatory process related with electrical and chemical stimuli. We have used transdifferentiated skin fibroblast human cells from DM1 patients and healthy human. Using this platform, we have started to evaluate different treatments, to screen drugs and to evaluate doses.

4) NMR integrated with OOC. The objective of this project is to develop a new technology based on magnetic resonance spectroscopy and imaging using dynamic nuclear polarisation (DNP-MR) integrated with OOC devices to monitor disease and evaluate drug response in OOC models. As a proof-of-concept, this project will fabricate a biomimetic multi OOC integrated device composed of liver spheroids and pancreatic islets and develop the necessary DNP-MR hardware and software to study metabolic diseases and for future drug screening applications. We are working in collaboration with Oxford instrument and Multiwave companies. 

Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies.

Keywords: Biomaterials, Drug screening platforms, Muscular dystrophy, Skeletal muscle, Tissue engineering

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.

Keywords: CRISPR, Laminopathy, LMNA, Nuclear envelope

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.

Keywords: 2D fatty liver in vitro model, Blu-Ray disc, Plasmonic nanomaterials, Label-Free Biosensing

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.

Keywords: Microphysiological tissues, Tissue engineering, Electrochemical, biosensors, Magnetic particles, Skeletal muscle, Electric stimulation

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.

Keywords: High protein diet, Low carbohydrates, NAFLD, NASH, Physical activity

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.

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

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.

Observing biochemical processes within living cell is imperative for biological and medical research. Fluoresce imaging is widely used for intracellular sensing of cell membranes, nuclei, lysosomes, and pH. Electrochemical assays have been proposed as an alternative to fluorescence-based assays because of excellent analytical features of electrochemical devices. Notably, thanks to the rapid progress of micro/nanotechnologies and electrochemical techniques, intracellular electrochemical sensing is making rapid progress, leading to a successful detection of intracellular components. Such insight can provide a deep understanding of cellular biological processes and, ultimately, define the human healthy and diseased states. In this review, we present an overview of recent research progress in intracellular electrochemical sensing. We focus on two main topics, electrochemical extraction of cytosolic contents from cells and intracellular electrochemical sensing in situ.

Keywords: Micro/nanoelectrode, Analytical electrochemistry, Intracellular sensing, Cell analysis

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.

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.

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.

Keywords: Cardiac muscle, Drug screening, Engineering muscle, Human pharmacological response, Physiological similarity, Skeletal muscle

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.

Keywords: Blood vessels, Nanofiber composite, Tissue engineering, Vascularized tissue