Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines.

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.

Ammonium quantification in human plasma by proton nuclear magnetic resonance for staging of liver fibrosis in alcohol-related liver disease and nonalcoholic fatty liver disease.

Liver fibrosis staging is a key element driving the prognosis of patients with chronic liver disease. Currently, biopsy is the only technique capable of diagnosing liver fibrosis in patients with alcohol-related liver disease (ArLD) and nonalcoholic fatty liver disease (NAFLD) unequivocally. Noninvasive (e.g. plasma-based) biomarker assays are attractive tools to diagnose and stage disease, yet must prove that they are reliable and sensitive to be used clinically. Here, we demonstrate proton nuclear magnetic resonance as a method to rapidly quantify the endogenous concentration of ammonium ions from human plasma extracts and show their ability to report upon early and advanced stages of ArLD and NAFLD. We show that, irrespective of the disease etiology, ammonium concentration is a more robust and informative marker of fibrosis stage than current clinically assessed blood hepatic biomarkers. Subject to validation in larger cohorts, the study indicates that the method can provide accurate and rapid staging of ArLD and NAFLD without the need for an invasive biopsy.

Sensors and Biosensors in Organs-on-a-Chip Platforms.

Biosensors represent a powerful analytical tool for analyzing biomolecular interactions with the potential to achieve real-time quantitative analysis with high accuracy using low sample volumes, minimum sample pretreatment with high potential for the development of in situ and highly integrated monitoring platforms. Considering these advantages, their use in cell-culture systems has increased over the last few years. Between the different technologies for cell culture, organs-on-a-chip (OOCs) represent a novel technology that tries to mimic an organ's functionality by combining tissue engineering/organoid with microfluidics. Although there are still challenges to achieving OOC models with high organ mimicking relevance, these devices can offer effective models for drug treatment development by identifying drug targets, screening toxicity, and determining the potential effects of drugs in living beings. Consequently, in the future, we might replace animal studies by offering more ethical test models. Considering the relevance that different physiological and biochemical parameters have in the correct functionality of cells, sensing and biosensing platforms can offer an effective way for the real-time monitoring of physiological parameters and, in our opinion, more relevant, the secretion of biomarkers such as cytokines, growth factors, and others related with the influence of drugs or other types of stimulus in cell metabolism. Keeping this concept in mind, in this chapter, we focus on describing the potential use of sensors and biosensors in OOC devices to achieve fully integrated platforms that monitor physiological parameters and cell metabolism.

Assessment of analytical methods to determine pyrethroids content of bednets.

OBJECTIVE: To present and evaluate simple, cost-effective tests to determine the amount of insecticide on treated materials. METHODS: We developed and evaluated a competitive immunoassay on two different platforms: a label-free impedimetric biosensor (EIS biosensor) and a lateral flow. Both approaches were validated by gas chromatography (GC) and ELISA, gold standards for analytical methods and immunoassays, respectively. Finally, commercially available pyrethroid-treated ITN samples were analysed. Different extraction methods were evaluated. RESULTS: Insecticide extraction by direct infusion of the ITN samples with dichloromethane and dioxane showed recovery efficiencies around 100% for insecticide-coated bednets, and >70% for insecticide-incorporated bednets. These results were comparable to those obtained with standard sonication methods. The competitive immunoassay characterisation with ELISA presented a dynamic range between 12 nm and 1.5 µm (coefficient of variation (CV) below 5%), with an IC50 at 138 nm, and a limit of detection (LOD) of 3.2 nm. EIS biosensor had a linear range between 1.7 nm and 61 nm (CV around 14%), with an IC50 at 10.4 nm, and a LOD of 0.6 nm. Finally, the lateral flow approach showed a dynamic range between 150 nm and 1.5 µm, an IC50 at 505 nm and a LOD of 67 nm. CONCLUSIONS: ELISA can replace chromatography as an accurate laboratory technique to determine insecticide concentration in bednets. The lateral flow approach developed can be used to estimate ITN insecticide concentration in the field. This new technology, coupled to the new extraction methods, should provide reliable guidelines for ITN use and replacement in the field.

Microtubule guiding in a multi-walled carbon nanotube circuit.

In nanotechnological devices, mass transport can be initiated by pressure driven flow, diffusion or by employing molecular motors. As the scale decreases, molecular motors can be helpful as they are not limited by increased viscous resistance. Moreover, molecular motors can move against diffusion gradients and are naturally fitted for nanoscale transportation. Among motor proteins, kinesin has particular potential for lab-on-a-chip applications. It can be used for sorting, concentrating or as a mechanical sensor. When bound to a surface, kinesin motors propel microtubules in random directions, depending on their landing orientation. In order to circumvent this complication, the microtubule motion should be confined or guided. To this end, dielectrophoretically aligned multi-walled-carbon nanotubes (MWCNT) can be employed as nanotracks. In order to control more precisely the spatial repartition of the MWCNTs, a screening method has been implemented and tested. Polygonal patterns have been fabricated with the aim of studying the guiding and the microtubule displacement between MWCNT segments. Microtubules are observed to transfer between MWCNT segments, a prerequisite for the guiding of microtubules in MWCNT circuit-based biodevices. The effect of the MWCNT organization (crenellated or hexagonal) on the MT travel distance has been investigated as well.

Molecular motor-powered shuttles along multi-walled carbon nanotube tracks.

As a complementary tool to nanofluidics, biomolecular-based transport is envisioned for nanotechnological devices. We report a new method for guiding microtubule shuttles on multi-walled carbon nanotube tracks, aligned by dielectrophoresis on a functionalized surface. In the absence of electric field and in fluid flow, alignment is maintained. The directed translocation of kinesin propelled microtubules has been investigated using fluorescence microscopy. To our knowledge, this is the first demonstration of microtubules gliding along carbon nanotubes.

The use of microtechnology and nanotechnology in fabricating vascularized tissues.

Tissue engineering (TE) is a multidisciplinary research area that combines medicine, biology, and material science. In recent decades, microtechnology and nanotechnology have also been gradually integrated into this field and have become essential components of TE research. Tissues and complex organs in the body depend on a branched blood vessel system. One of the main objectives for TE researchers is to replicate this vessel system and obtain functional vascularized structures within engineered tissues or organs. With the help of new nanotechnology and microtechnology, significant progress has been made. Achievements include the design of nanoscale-level scaffolds with new functionalities, development of integrated and rapid nanotechnology methods for biofabrication of vascular tissues, discovery of new composite materials to direct differentiation of stem and inducible pluripotent stem cells into the vascular phenotype. Although numerous challenges to replicating vascularized tissue for clinical uses remain, the combination of these new advances has yielded new tools for producing functional vascular tissues in the near future.

Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy.

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin-proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

A New Microfluidic Device to Facilitate Functional Precision Medicine Assays.

Functional precision medicine (FPM) has emerged as a new approach to improve cancer treatment. Despite its potential, FPM assays present important limitations such as the number of cells and trained personnel required. To overcome these impediments, here we describe a novel microfluidic platform that can be used to perform FPM assays, optimizing the use of primary cancer cells and simplifying the process by using microfluidics to automatize the process.

The Tissue Engineering Revolution: From Bench Research to Clinical Reality.

At its core, tissue engineering involves the use of a scaffold for the formation of new viable tissue for medical purposes [...].

A contactless electrical stimulator: application to fabricate functional skeletal muscle tissue.

Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields.

Mimicking sarcolemmal damagein vitro: a contractile 3D model of skeletal muscle for drug testing in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this crucial structural protein leads to sarcolemmal fragility, resulting in muscle fiber damage during contraction. Despite ongoing efforts, there is no cure available for DMD patients. One of the primary challenges is the limited efficacy of current preclinical tools, which fail in modeling the biological complexity of the disease. Human-based three-dimensional (3D) cell culture methods appear as a novel approach to accelerate preclinical research by enhancing the reproduction of pathophysiological processes in skeletal muscle. In this work, we developed a patient-derived functional 3D skeletal muscle model of DMD that reproduces the sarcolemmal damage found in the native DMD muscle. These bioengineered skeletal muscle tissues exhibit contractile functionality, as they responded to electrical pulse stimulation. Sustained contractile regimes induced the loss of myotube integrity, mirroring the pathological myotube breakdown inherent in DMD due to sarcolemmal instability. Moreover, damaged DMD tissues showed disease functional phenotypes, such as tetanic fatigue. We also evaluated the therapeutic effect of utrophin upregulator drug candidates on the functionality of the skeletal muscle tissues, thus providing deeper insight into the real impact of these treatments. Overall, our findings underscore the potential of bioengineered 3D skeletal muscle technology to advance DMD research and facilitate the development of novel therapies for DMD and related neuromuscular disorders.

Muscle-on-a-chip devices: a new era for in vitro modelling of muscular dystrophies.

Muscular dystrophies are a heterogeneous group of highly debilitating diseases that result in muscle atrophy and weakness. The lack of suitable cellular and animal models that reproduce specific aspects of their pathophysiology is one of the reasons why there are no curative treatments for these disorders. This highlights a considerable gap between current laboratory models and clinical practice. We strongly believe that organs-on-chip could help to fill this gap. Organs-on-chip, and in particular muscles-on-chip, are microfluidic devices that integrate functional skeletal muscle tissues. Biosensors in these systems allow monitoring of muscle homeostasis or drug responses in situ. This Perspective outlines the potential of organs-on-chip as advanced models for muscular dystrophies, as well as the current challenges and future opportunities for this technology.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

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.

Direct reprogramming of human fibroblasts into insulin-producing cells using transcription factors.

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing ß cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create ß-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into ß-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a ß-cell fate. Reprogrammed cells exhibit ß-cell features including ß-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.

Fatty Hepatocytes Induce Skeletal Muscle Atrophy In Vitro: A New 3D Platform to Study the Protective Effect of Albumin in Non-Alcoholic Fatty Liver.

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.

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels.

Bioengineered human skeletal muscle tissues have emerged in the last years as newin vitrosystems 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 (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-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.

Collagen-Tannic Acid Spheroids for ß-Cell Encapsulation Fabricated Using a 3D Bioprinter.

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

Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy.

Precision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events ('priming') and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology's predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.

Sensitive and spatially multiplexed detection system based on dielectrophoretic manipulation of DNA-encoded particles used as immunoreactions platform.

In this work, we designed a new immunodevice that combines competitive immunoreactions on the microparticles, accumulation of these particles by negative dielectrophoresis (n-DEP), and their subsequent capture through hybridization among single-stranded DNAs (ssDNAs). Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to test the resulting simultaneous detection system. For sensing, we prepared two different sets of microparticles: one modified with atrazine-conjugated bovine serum albumin (BSA-2d) and ssDNA-J1(up) and the other with bromopropylate-conjugated aminodextran (AD-155) and ssDNA-J2(up). The microparticles were incubated in a mixture of analyte-specific antibody and analyte at different concentrations to trap the unreacted antibodies prior to being labeled with antibodies conjugated with a fluorescence molecule. A suspension containing both types of microparticles was introduced into an n-DEP device consisting of an interdigitated microarray (IDA) electrode and channel modified with ssDNA-J1(down) and ssDNA-J2(down), which are complementary to ssDNA-J1(up) and ssDNA-J2(up), respectively. The n-DEP force generated by applying AC voltage to the IDA electrode displaced the microparticles toward the encoded areas, causing them to rapidly accumulate on the upper surfaces. Hybridization allowed us to distinguish the microparticles and sense multiple analytes by spatial recognition in the DNA-encoded areas. The fluorescence intensity of the captured particles, which depends on analyte concentrations, was measured selectively by focusing on specific areas. The strategy is advantageous for sensitivity due to the equivalent trapping efficiency by DNA hybridization and large surface area of the microparticle for immunoreactions. The rapidity and simplicity were still supported by particle manipulation. Using this concept, we detect atrazine and bromopropylate simultaneously with limits of detection (LODs) of 0.2 µg·L(-1), which covered the maximum residue level (MRL) in food samples established the European Union (EU) and Japan Ministry of Health, Labor and Welfare (MHLW).