On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry.

We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.

Kinetic Detection of Apoptosis Events Via Caspase 3/7 Activation in a Tumor-Immune Microenvironment on a Chip.

The development of advanced biological models like microphysiological systems, able to rebuild the complexity of the physiological and/or pathological environments at a single-cell detail level in an in-vivo-like approach, is proving to be a promising tool to understand the mechanisms of interactions between different cell populations and main features of several diseases. In this frame, the tumor-immune microenvironment on a chip represents a powerful tool to profile key aspects of cancer progression, immune activation, and response to therapy in several immuno-oncology applications. In the present chapter, we provide a protocol to identify and characterize the time evolution of apoptosis by time-lapse fluorescence and confocal imaging in a 3D microfluidic coculture murine model including cancer and spleen cells.

Dual-color core-shell silica nanosystems for advanced super-resolution biomedical imaging.

Fluorescent core-shell silica nanoparticles are largely employed in nanomedicine and life science thanks to the many advantages they offer. Among these, the enhancement of the stability of the fluorescent signal upon fluorophore encapsulation into the silica matrix and the possibility to combine in a single vehicle multiple functionalities, physically separated in different compartments. In this work, we present a new approach to the Stöber method as a two-cycle protocol for the tailored synthesis of dual-color fluorescent core-shell silicon dioxide nanoparticles (SiO2 NPs) using two commercial dyes as model. To facilitate the colloidal stability, the nanoparticle surface was functionalized with biotin by two approaches. The biotinylated nanosystems were characterized by several analytical and advanced microscopy techniques including Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), UV-vis, transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Moreover, advanced super-resolution based on structured illumination was used for the imaging of the double-fluorescent NPs, both on a substrate and in the cellular microenvironment, at nanometric resolution 100 nm, in view of their versatile potential employment in fluorescence optical nanoscopy as nanoscale calibration tools as well as in biomedical applications as biocompatible nanosystems for intracellular biosensing with high flexibility of use, being these nanoplatforms adaptable to the encapsulation of any couple of dyes with the desired function.

A 3D adipogenesis platform to study the fate of fibro/adipogenic progenitors in muscular dystrophies.

In human dystrophies, progressive muscle wasting is exacerbated by ectopic deposition of fat and fibrous tissue originating from fibro/adipogenic progenitors (FAPs). In degenerating muscles, the ability of these cells to promote successful healing is attenuated, and FAPs aberrantly expand and differentiate into adipocytes and fibroblasts. Thus, arresting the fibro/adipogenic fate of FAPs, without affecting their physiological role, represents a valuable therapeutic strategy for patients affected by muscle diseases. Here, using a panel of adipose progenitor cells, including human-derived FAPs, coupled with pharmacological perturbations and proteome profiling, we report that LY2090314 interferes with a genuine adipogenic program acting as WNT surrogate for the stabilization of a competent ß-catenin transcriptional complex. To predict the beneficial impact of LY2090314 in limiting ectopic deposition of fat in human muscles, we combined a poly-ethylene-glycol-fibrinogen biomimetic matrix with these progenitor cells to create a miniaturized 3D model of adipogenesis. Using this scalable system, we demonstrated that a two-digit nanomolar dose of this compound effectively represses adipogenesis at higher 3D scale, thus indicating the potential for LY2090314 to limit FAP-derived fat infiltrates in dystrophic muscles.

The interaction of ß-arrestin1 with talin1 driven by endothelin A receptor as a feature of α5ß1 integrin activation in high-grade serous ovarian cancer.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5ß1 integrin (Intα5ß1) activity. Although the binding of Intα5ß1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5ß1 activation and accelerates tumor cells toward invasive disease, involving the protein ß-arrestin1 (ß-arr1) and the activation of the endothelin A receptor (ETAR) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intß1 and downstream FAK/paxillin activation. Mechanistically, ß-arr1 directly interacts with talin1 and Intß1, promoting talin1 phosphorylation and its recruitment to Intß1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ETAR/ß-arr1-driven Intα5ß1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ETAR, Ambrisentan (AMB), and of Intα5ß1, ATN161, inhibits ET-1-driven Intα5ß1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intß1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ETAR/ß-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ETAR/ß-arr1 regulates Intα5ß1 functional pathway.

Droplet-based microfluidic synthesis of nanogels for controlled drug delivery: tailoring nanomaterial properties via pneumatically actuated flow-focusing junction.

Conventional batch syntheses of polymer-based nanoparticles show considerable shortcomings in terms of scarce control over nanomaterials morphology and limited lot-to-lot reproducibility. Droplet-based microfluidics represents a valuable strategy to overcome these constraints, exploiting the formation of nanoparticles within discrete microdroplets. In this work, we synthesized nanogels (NGs) composed of hyaluronic acid and polyethyleneimine using a microfluidic flow-focusing device endowed with a pressure-driven micro-actuator. The actuator achieves real-time modulation of the junction orifice width, thereby regulating the microdroplet diameter and, as a result, the NG size. Acting on process parameters, NG hydrodynamic diameter could be tuned in the range 92-190 nm while preserving an extremely low polydispersity (0.015); those values are hardly achievable in batch syntheses and underline the strength of our toolbox for the continuous in-flow synthesis of nanocarriers. Furthermore, NGs were validated in vitro as a drug delivery system in a representative case study still lacking an effective therapeutic treatment: ovarian cancer. Using doxorubicin as a chemotherapeutic agent, we show that NG-mediated release of the drug results in an enhanced antiblastic effect vs. the non-encapsulated administration route even at sublethal dosages, highlighting the wide applicability of our microfluidics-enabled nanomaterials in healthcare scenarios.

A Soft Zwitterionic Hydrogel as Potential Coating on a Polyimide Surface to Reduce Foreign Body Reaction to Intraneural Electrodes.

Invasive intraneural electrodes can control advanced neural-interfaced prostheses in human amputees. Nevertheless, in chronic implants, the progressive formation of a fibrotic capsule can gradually isolate the electrode surface from the surrounding tissue leading to loss of functionality. This is due to a nonspecific inflammatory response called foreign-body reaction (FBR). The commonly used poly(ethylene glycol) (PEG)-based low-fouling coatings of implantable devices can be easily encapsulated and are susceptible to oxidative damage in long-term in vivo applications. Recently, sulfobetaine-based zwitterionic hydrogels have emerged as an important class of robust ultra-low fouling biomaterials, holding great potential to mitigate FBR. The aim of this proof-of-principle in vitro work was to assess whether the organic zwitterionic-poly(sulfobetaine methacrylate) [poly(SBMA)]-hydrogel could be a suitable coating for Polyimide (PI)-based intraneural electrodes to reduce FBR. We first synthesized and analyzed the hydrogel through a mechanical characterization (i.e., Young's modulus). Then, we demonstrated reduced adhesion and activation of fibrogenic and pro-inflammatory cells (i.e., human myofibroblasts and macrophages) on the hydrogel compared with PEG-coated and polystyrene surfaces using cell viability assays, confocal fluorescence microscopy and high-content analysis of oxidative stress production. Interestingly, we successfully coated PI surfaces with a thin film of the hydrogel through covalent bond and demonstrated its high hydrophilicity via water contact angle measurement. Importantly, we showed the long-term release of an anti-fibrotic drug (i.e., Everolimus) from the hydrogel. Because of the low stiffness, biocompatibility, high hydration and ultra-low fouling characteristics, our zwitterionic hydrogel could be envisioned as long-term diffusion-based delivery system for slow and controlled anti-inflammatory and anti-fibrotic drug release in vivo.

Silicone-Textile Composite Resistive Strain Sensors for Human Motion-Related Parameters.

In recent years, soft and flexible strain sensors have found application in wearable devices for monitoring human motion and physiological parameters. Conductive textile-based sensors are good candidates for developing these sensors. However, their robust electro-mechanical connection and susceptibility to environmental factors are still an open challenge to date. In this work, the manufacturing process of a silicone-textile composite resistive strain sensor based on a conductive resistive textile encapsulated into a dual-layer of silicone rubber is reported. In the working range typical of biomedical applications (up to 50%), the proposed flexible, skin-safe and moisture resistant strain sensor exhibited high sensitivity (gauge factor of -1.1), low hysteresis (maximum hysteresis error 3.2%) and ease of shaping in custom designs through a facile manufacturing process. To test the developed flexible sensor, two applicative scenarios covering the whole working range have been considered: the recording of the chest wall expansion during respiratory activity and the capture of the elbow flexion/extension movements.

A primer to traction force microscopy.

Traction force microscopy (TFM) has emerged as a versatile technique for the measurement of single-cell-generated forces. TFM has gained wide use among mechanobiology laboratories, and several variants of the original methodology have been proposed. However, issues related to the experimental setup and, most importantly, data analysis of cell traction datasets may restrain the adoption of TFM by a wider community. In this review, we summarize the state of the art in TFM-related research, with a focus on the analytical methods underlying data analysis. We aim to provide the reader with a friendly compendium underlying the potential of TFM and emphasizing the methodological framework required for a thorough understanding of experimental data. We also compile a list of data analytics tools freely available to the scientific community for the furtherance of knowledge on this powerful technique.

Nano-encapsulation of hydroxytyrosol into formulated nanogels improves therapeutic effects against hepatic steatosis: An in vitro study.

Nanomaterials hold promise as a straightforward approach for enhancing the performance of bioactive compounds in several healthcare scenarios. Indeed, nanoencapsulation represents a valuable strategy to preserve the bioactives, maximizing their bioavailability. Here, a nanoencapsulation strategy for the treatment of nonalcoholic fatty liver disease (NAFLD) is presented. NAFLD represents the most common chronic liver disease in Western societies, and still lacks an effective therapy. Hydroxytyrosol (HT), a naturally occurring polyphenol, has been shown to protect against hepatic steatosis through its lipid-lowering, antioxidant and anti-inflammatory activities. However, the efficient delivery of HT to hepatocytes remains a crucial aspect: the design of smart nanogels appears as a promising tool to promote its intracellular uptake. In this paper, we disclose the synthesis of nanogel systems based on polyethylene glycol and polyethyleneimine which have been tested in an in vitro model of hepatic steatosis at two different concentrations (0.1 mg/mL and 0.5 mg/mL), taking advantage of high-content analysis tools. The proposed HT-loaded nanoscaffolds are non-toxic to cells, and their administration showed a significant decrease in the intracellular triglyceride levels, restoring cell viability and outperforming the results achievable with HT in its non-encapsulated form. Moreover, nanogels do not induce oxidative stress, thus demonstrating their biosafety. Overall, the formulated nanogel system achieves superior performance compared to conventional drug administration routes and hence represents a promising strategy for the management of NAFLD.

Synthesis of Nanogels: Current Trends and Future Outlook.

Nanogels represent an innovative platform for tunable drug release and targeted therapy in several biomedical applications, ranging from cancer to neurological disorders. The design of these nanocarriers is a pivotal topic investigated by the researchers over the years, with the aim to optimize the procedures and provide advanced nanomaterials. Chemical reactions, physical interactions and the developments of engineered devices are the three main areas explored to overcome the shortcomings of the traditional nanofabrication approaches. This review proposes a focus on the current techniques used in nanogel design, highlighting the upgrades in physico-chemical methodologies, microfluidics and 3D printing. Polymers and biomolecules can be combined to produce ad hoc nanonetworks according to the final curative aims, preserving the criteria of biocompatibility and biodegradability. Controlled polymerization, interfacial reactions, sol-gel transition, manipulation of the fluids at the nanoscale, lab-on-a-chip technology and 3D printing are the leading strategies to lean on in the next future and offer new solutions to the critical healthcare scenarios.

Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK.

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) drives invadopodia by a not fully explored coordinated function of ß-arrestin1 (ß-arr1). Here, we report that ß-arr1 links the integrin-linked kinase (ILK)/ßPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/ß-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.

Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration.

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo-substitutes support the formation of new blood vessels and neuromuscular junctions-pivotal aspects for cell survival and muscle contractile functionalities-together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo-substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.

Photocurable Biopolymers for Coaxial Bioprinting.

Thanks to their unique advantages, additive manufacturing technologies are revolutionizing almost all sectors of the industrial and academic worlds, including tissue engineering and regenerative medicine. In particular, 3D bioprinting is rapidly emerging as a first-choice approach for the fabrication-in one step-of advanced cell-laden hydrogel constructs to be used for in vitro and in vivo studies. This technique consists in the precise deposition layer-by-layer of sub-millimetric hydrogel strands in which living cells are embedded. A key factor of this process consists in the proper formulation of the hydrogel precursor solution, the so-called bioink. Ideal bioinks should be able, on the one side, to support cell growth and differentiation and, on the other, to allow the high-resolution deposition of cell-laden hydrogel strands. The latter feature requires the extruded solution to instantaneously undergo a sol-gel transition to avoid its collapse after deposition.To address this challenge, researchers are recently focusing their attention on the synthesis of several derivatives of natural biopolymers to enhance their printability. Here, we present an approach for the synthesis of photocurable derivatives of natural biopolymers-namely, gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen-that can be used to formulate tailored innovative bioinks for coaxial-based 3D bioprinting applications.

Quercetin and hydroxytyrosol as modulators of hepatic steatosis: A NAFLD-on-a-chip study.

Organs-on-chip (OoCs) are catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative. OoCs enable the creation of three-dimensional (3D) tissue microenvironments with physiological and pathological relevance at unparalleled precision and complexity, offering new opportunities to model human diseases and to test the potential therapeutic effect of drugs, while overcoming the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver-on-a-chip model to investigate the effects of two naturally occurring polyphenols, namely quercetin and hydroxytyrosol, on nonalcoholic fatty liver disease (NAFLD) using a high-content analysis readout methodology. NAFLD is currently the most common form of chronic liver disease; however, its complex pathogenesis is still far from being elucidated, and no definitive treatment has been established so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid-induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant and lipid-lowering properties. In conclusion, the findings of the present work could guide novel strategies to contrast the onset and progression of NAFLD.

Smoothened/AMP-Activated Protein Kinase Signaling in Oligodendroglial Cell Maturation.

The regeneration of myelin is known to restore axonal conduction velocity after a demyelinating event. Remyelination failure in the central nervous system contributes to the severity and progression of demyelinating diseases such as multiple sclerosis. Remyelination is controlled by many signaling pathways, such as the Sonic hedgehog (Shh) pathway, as shown by the canonical activation of its key effector Smoothened (Smo), which increases the proliferation of oligodendrocyte precursor cells via the upregulation of the transcription factor Gli1. On the other hand, the inhibition of Gli1 was also found to promote the recruitment of a subset of adult neural stem cells and their subsequent differentiation into oligodendrocytes. Since Smo is also able to transduce Shh signals via various non-canonical pathways such as the blockade of Gli1, we addressed the potential of non-canonical Smo signaling to contribute to oligodendroglial cell maturation in myelinating cells using the non-canonical Smo agonist GSA-10, which downregulates Gli1. Using the Oli-neuM cell line, we show that GSA-10 promotes Gli2 upregulation, MBP and MAL/OPALIN expression via Smo/AMP-activated Protein Kinase (AMPK) signaling, and efficiently increases the number of axonal contact/ensheathment for each oligodendroglial cell. Moreover, GSA-10 promotes the recruitment and differentiation of oligodendroglial progenitors into the demyelinated corpus callosum in vivo. Altogether, our data indicate that non-canonical signaling involving Smo/AMPK modulation and Gli1 downregulation promotes oligodendroglia maturation until axon engagement. Thus, GSA-10, by activation of this signaling pathway, represents a novel potential remyelinating agent.

Graphene-laden hydrogels: A strategy for thermally triggered drug delivery.

The synthesis of graphene-based materials has attracted considerable attention in drug delivery strategies. Indeed, the conductivity and mechanical stability of graphene have been investigated for controlled and tunable drug release via electric or mechanical stimuli. However, the design of a thermo-sensitive scaffold using pristine graphene (without distortions related to the oxidation processes) has not been deeply investigated yet, although it may represent a promising approach for several therapeutic treatments. Here, few-layer graphene was used as a nanofiller in a hydrogel system with a thermally tunable drug release profile. In particular, varying the temperature (25 °C, 37 °C and 44 °C), responsive drug releases were noticed and hypothesized depending on the formation and perturbation of π-π interactions involving graphene, the polymeric matrix and the model drug (diclofenac). As a result, these hybrid hydrogels show a potential application as thermally triggered drug release systems in several healthcare scenarios.

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1 (transforming growth factor ß1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification.

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

Biofabrication of Hepatic Constructs by 3D Bioprinting of a Cell-Laden Thermogel: An Effective Tool to Assess Drug-Induced Hepatotoxic Response.

A thermoresponsive Pluronic/alginate semisynthetic hydrogel is used to bioprint 3D hepatic constructs, with the aim to investigate liver-specific metabolic activity of the 3D constructs compared to traditional 2D adherent cultures. The bioprinting method relies on a bioinert hydrogel and is characterized by high-shape fidelity, mild depositing conditions and easily controllable gelation mechanism. Furthermore, the dissolution of the sacrificial Pluronic templating agent significantly ameliorates the diffusive properties of the printed hydrogel. The present findings demonstrate high viability and liver-specific metabolic activity, as assessed by synthesis of urea, albumin, and expression levels of the detoxifying CYP1A2 enzyme of cells embedded in the 3D hydrogel system. A markedly increased sensitivity to a well-known hepatotoxic drug (acetaminophen) is observed for cells in 3D constructs compared to 2D cultures. Therefore, the 3D model developed herein may represent an in vitro alternative to animal models for investigating drug-induced hepatotoxicity.