Nanoscopic gel particle for intra-articular injection formulation.

Hyaluronic acid (HA) based nanogels showed effective intracellular delivery efficacy for anti-cancer and anti-inflammatory drugs, characterized by their ability targeting relevant cell receptors. In the present study, we demonstrate the ability of hyaluronic acid-polyethyleneimine (HA-PEI) nanogels as a promising dual-functional interfacial active for intra-articular injection to intervene arthritis. Nanomechanical measurements on both model substrates and human cartilage samples confirm that the HA-PEI nanogels can significantly improve interfacial lubrication, in comparison to HA molecules, or silica-based nanoparticles. We show that the Coefficient of Friction significantly decreases with a decreasing nanogel size. The exceptional lubricating performance, coupled with the proven drug delivery capability, evidences the great potential of nanoscopic hydrogels for early-stage arthritis treatment. The flexibility in choosing the chemical nature, molecular architecture, and structural characteristics of nanogels makes it possible to modulate both drug delivery kinetics and interfacial lubrication, thus representing an innovative approach to treat degenerative joint diseases.

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.

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.

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.

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.

Morphological and Molecular Assessment in Thyroid Cytology Using Cell-Capturing Scaffolds.

The increased frequency of thyroid nodules is paralleled by the rise of thyroid cancer diagnosis. To define the nature of most thyroid nodules, fine needle aspiration (FNA) followed by cytological evaluation is considered the method of choice. About 20% of FNA biopsies on thyroid nodules, however, show indeterminate cytological features and may require diagnostic surgery. Several immunocytochemical and molecular markers have been proposed to improve classification of thyroid nodules, but these tests require adequate cell amount and cytological paraffin inclusion. Polymeric matrices were recently proposed for the collection of cells for diagnostic purposes. In this study, we evaluated the diagnostic use of a new matrix (CytoMatrix). Morphological, molecular and immunohistochemical investigations were carried out on 23 FNA samples included in CytoMatrix and compared with data obtained from the definitive histology of surgical samples. Our results showed that CytoMatrix is suitable to capture and preserve the cellularity of the samples harvested by FNA and that its paraffin sections mimic the morphology of those obtained from real histological tissue. Immunohistochemistry on CytoMatrix samples was consistent with the immunophenotypical profile of the corresponding histological surgical specimens. Mutational analysis of the BRAF (V600E) gene performed on CytoMatrix inclusions and paired surgical tissue matched in all but one cases while matrix immunohistochemistry identified 91.6% of BRAF mutated samples. In conclusion, we suggest that CytoMatrix could be a reliable tool to overcome the current limits of traditional collection methods for the study of thyroid cytology, thereby improving their reliability for a conclusive diagnostic interpretation.

Ester coupling of ibuprofen in hydrogel matrix: A facile one-step strategy for controlled anti-inflammatory drug release.

Ibuprofen (IBU) is a non-steroidal anti-inflammatory drug (NSAID) commonly used in the treatment of pain, fever and inflammation. However, the administration of IBU in its free carboxylic acid form is strongly dependent on its limited solubility in aqueous solution. This mandates for an increased drug concentration to reach the therapeutic window, and promotes the alternative use of IBU sodium salt, even if this latter form poses significant constraints in terms of tunable release due to its uncontrolled and rapid diffusion. A potential solution is represented by oral administration through physical encapsulation of ibuprofen in designed carriers, despite this route limits the application of this therapeutic agent. In this work, we propose the covalent tethering of ibuprofen to a hydrogel matrix via esterification reaction. Exploiting the cleavability of the ester bond under physiological conditions, we propose a controlled drug delivery system where the whole drug payload can be released, thus overcoming the questioned aspects of over-dosage and solubility-dependent administration. In particular, we tested the biological activity of cleaved ibuprofen in terms of cyclooxygenase inhibition, reporting that chemical tethering did not alter the efficiency of the NSAID. Moreover, due to the sol-gel transition of the hydrogel matrix, these ibuprofen-functionalized hydrogels could be used as injectable tools in several clinical scenarios, performing a localized drug release and opening advanced avenues for in situ treatments.

EGFR/ErbB Inhibition Promotes OPC Maturation up to Axon Engagement by Co-Regulating PIP2 and MBP.

Remyelination in the adult brain relies on the reactivation of the Neuronal Precursor Cell (NPC) niche and differentiation into Oligodendrocyte Precursor Cells (OPCs) as well as on OPC maturation into myelinating oligodendrocytes (OLs). These two distinct phases in OL development are defined by transcriptional and morphological changes. How this differentiation program is controlled remains unclear. We used two drugs that stimulate myelin basic protein (MBP) expression (Clobetasol and Gefitinib) alone or combined with epidermal growth factor receptor (EGFR) or Retinoid X Receptor gamma (RXRγ) gene silencing to decode the receptor signaling required for OPC differentiation in myelinating OLs. Electrospun polystyrene (PS) microfibers were used as synthetic axons to study drug efficacy on fiber engagement. We show that EGFR inhibition per se stimulates MBP expression and increases Clobetasol efficacy in OPC differentiation. Consistent with this, Clobetasol and Gefitinib co-treatment, by co-regulating RXRγ, MBP and phosphatidylinositol 4,5-bisphosphate (PIP2) levels, maximizes synthetic axon engagement. Conversely, RXRγ gene silencing reduces the ability of the drugs to promote MBP expression. This work provides a view of how EGFR/ErbB inhibition controls OPC differentiation and indicates the combination of Clobetasol and Gefitinib as a potent remyelination-enhancing treatment.

Seriate cytology vs molecular analysis of peritoneal washing to improve gastric cancer cells detection.

BACKGROUND: Intraperitoneal malignant cells detection in patients with gastric cancer is associated with a significant decrease in overall survival. The overall accuracy of cytological examination of peritoneal lavages, however, is quite low, and intraperitoneal recurrence has been observed even in patients with negative cytology. Immunocytochemistry and molecular techniques have been investigated to improve high-risk patients' identification with variable results. The aim of this study was to compare the performance of different laboratory methods applied to peritoneal washing, to improve the cytological identification of malignant cells. METHODS: We prospectively evaluated 21 patients who underwent surgery and peritoneal lavage for gastric cancer. Among them, 18 had negative cytology and three were positive for malignant cells. For each patient, immunohistochemistry with BerEP4 antibody was performed on seriate sections of cellblock preparation at different levels, using the method reported for sentinel nodes in other types of cancer. Paired frozen quotes of washing fluids were evaluated by qRT-PCR with primer for mRNA of Ceacam5. RESULTS: In 4 of 18 patients with previous negative routine cytology, isolated neoplastic cells in seriate sections of the cellblock inclusion have been found. Results showed to be coherent with molecular analysis for CEA mRNA. CONCLUSION: The sensitivity and specificity of peritoneal washing analyses should be notably improved by immunohistochemistry applied to multilevel cellblock sectioning. The method is less expensive and more widely applicable than molecular analysis, in each laboratory setting. This approach allows detection of minimum peritoneal seeding in patients with gastric cancer.

The long-term follow-up of large-diameter Dacron® vascular grafts in surgical practice: a review.

Synthetic grafts have been widely used in cardiac and vascular surgery since the mid-1970s. Considering the relative lack of randomized clinical trials or systematic analyses in the field of prosthetic large vessel diameter replacement, we reviewed the literature on the long-term performance and surgical management of complications of Dacron® grafts in both thoracic and abdominal aorta reconstruction and in the pediatric population. MedLine, Embase and Cochrane Library databases were searched for meta-analyses, reviews, clinical trials, and case reports pertinent to the study object. Aortic replacement with Dacron® prostheses is widely performed with acceptable outcome and a relatively low rate of graft-related and postimplantation complications, such as rupture, infection and fistulization. However, progressive dilation and mechanical failure of the grafts represent the most worrisome complication in all the districts analyzed. The emerging concept of the mismatch in the biomechanical properties between the prosthetic material and native aorta is thought to be at the root of these complications leading to even more daunting consequences when the ascending aorta is involved. Indeed introduction of a non-compliant prosthesis in place of the native ascending aorta can exert detrimental effects not only at the level of the anastomosis, leading to pseudoaneurysm, but also can influence the optimal performance of the aortic root complex with consequent valve dysfunction and ventricular hypertrophy. Albeit confirming their overall successful performance, this review launches a warning on the current liberal use of non-compliant grafts in aortic position, remarking the need for alternative vascular conduits mimicking the native artery compliance.

Surface functionalization of polyurethane scaffolds mimicking the myocardial microenvironment to support cardiac primitive cells.

Scaffolds populated with human cardiac progenitor cells (CPCs) represent a therapeutic opportunity for heart regeneration after myocardial infarction. In this work, square-grid scaffolds are prepared by melt-extrusion additive manufacturing from a polyurethane (PU), further subjected to plasma treatment for acrylic acid surface grafting/polymerization and finally grafted with laminin-1 (PU-LN1) or gelatin (PU-G) by carbodiimide chemistry. LN1 is a cardiac niche extracellular matrix component and plays a key role in heart formation during embryogenesis, while G is a low-cost cell-adhesion protein, here used as a control functionalizing molecule. X-ray photoelectron spectroscopy analysis shows nitrogen percentage increase after functionalization. O1s and C1s core-level spectra and static contact angle measurements show changes associated with successful functionalization. ELISA assay confirms LN1 surface grafting. PU-G and PU-LN1 scaffolds both improve CPC adhesion, but LN1 functionalization is superior in promoting proliferation, protection from apoptosis and expression of differentiation markers for cardiomyocytes, endothelial and smooth muscle cells. PU-LN1 and PU scaffolds are biodegraded into non-cytotoxic residues. Scaffolds subcutaneously implanted in mice evoke weak inflammation and integrate with the host tissue, evidencing a significant blood vessel density around the scaffolds. PU-LN1 scaffolds show their superiority in driving CPC behavior, evidencing their promising role in myocardial regenerative medicine.

Postbariatric Brachioplasty with Posteromedial Scar: Physical Model, Technical Refinements, and Clinical Outcomes.

BACKGROUND: Brachioplasty is an increasingly performed procedure following massive weight loss. A visible scar is the main hindrance to this surgery. The aims of the study were to develop a physical model to investigate the ideal location of the surgical incision and to present the authors' technical refinements with the posteromedial scar approach. METHODS: Twenty-four postbariatric patients underwent brachioplasty with posteromedial scar placement, concomitant liposuction, fascial plication, and axillary Z-plasty. Skin specimens were tested and a physical model of the arm was set up to investigate the difference in mechanical stress on the posteromedial and medial scars. The validated Patient and Observer Scar Assessment Scale, the Vancouver Scar Scale, and a questionnaire assessing subjective improvements were administered to patients. Preoperative and postoperative photographs were assessed by three independent plastic surgeons. RESULTS: The physical model showed that stress intensity and distribution along the scar were reduced in the posteromedial location, with smaller scar displacement in the loading simulations. Twenty-three patients healed uneventfully. One (4.1 percent) had a 2-cm dehiscence. Mean Patient and Observer Scar Assessment Scale scores were, respectively, 2 ± 0.76 and 2.13 ± 0.64 in the patients' and observers' questionnaires. The mean Vancouver Scar Scale value was 3.5 ± 1.7. Questionnaires assessing the subjective outcomes showed a mean value of 3.45 ± 0.63 of 4. The surgeons' assessment resulted in a score of 4.5 ± 0.4 of 5. CONCLUSIONS: The physical model demonstrated that the posteromedial scar was subjected to lower mechanical stress and displacement. The reported technical refinements allowed pleasant arm recontouring to be achieved with acceptable scarring and a low incidence of complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis.

The possibility of detecting and classifying living cells in a label-free and non-invasive manner holds significant theranostic potential. In this work, Hyperspectral Imaging (HSI) has been successfully applied to the analysis of macrophagic polarization, given its central role in several pathological settings, including the regulation of tumour microenvironment. Human monocyte derived macrophages have been investigated using hyperspectral reflectance confocal microscopy, and hyperspectral datasets have been analysed in terms of M1 vs. M2 polarization by Principal Components Analysis (PCA). Following PCA, Linear Discriminant Analysis has been implemented for semi-automatic classification of macrophagic polarization from HSI data. Our results confirm the possibility to perform single-cell-level in vitro classification of M1 vs. M2 macrophages in a non-invasive and label-free manner with a high accuracy (above 98% for cells deriving from the same donor), supporting the idea of applying the technique to the study of complex interacting cellular systems, such in the case of tumour-immunity in vitro models.

Implantation of a Poly-L-Lactide GCSF-Functionalized Scaffold in a Model of Chronic Myocardial Infarction.

A previously developed poly-L-lactide scaffold releasing granulocyte colony-stimulating factor (PLLA/GCSF) was tested in a rabbit chronic model of myocardial infarction (MI) as a ventricular patch. Control groups were constituted by healthy, chronic MI and nonfunctionalized PLLA scaffold. PLLA-based electrospun scaffold efficiently integrated into a chronic infarcted myocardium. Functionalization of the biopolymer with GCSF led to increased fibroblast-like vimentin-positive cellular colonization and reduced inflammatory cell infiltration within the micrometric fiber mesh in comparison to nonfunctionalized scaffold; PLLA/GCSF polymer induced an angiogenetic process with a statistically significant increase in the number of neovessels compared to the nonfunctionalized scaffold; PLLA/GCSF implanted at the infarcted zone induced a reorganization of the ECM architecture leading to connective tissue deposition and scar remodeling. These findings were coupled with a reduction in end-systolic and end-diastolic volumes, indicating a preventive effect of the scaffold on ventricular dilation, and an improvement in cardiac performance.

Combining Type I Interferons and 5-Aza-2'-Deoxycitidine to Improve Anti-Tumor Response against Melanoma.

Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8+ T cells while inhibiting the homing of immunosuppressive CD11b+ myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects.

Investigating Nonalcoholic Fatty Liver Disease in a Liver-on-a-Chip Microfluidic Device.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide, ranging from simple steatosis to nonalcoholic steatohepatitis, which may progress to cirrhosis, eventually leading to hepatocellular carcinoma (HCC). HCC ranks as the third highest cause of cancer-related death globally, requiring an early diagnosis of NAFLD as a potential risk factor. However, the molecular mechanisms underlying NAFLD are still under investigation. So far, many in vitro studies on NAFLD have been hampered by the limitations of 2D culture systems, in which cells rapidly lose tissue-specific functions. The present liver-on-a-chip approach aims at filling the gap between conventional in vitro models, often scarcely predictive of in vivo conditions, and animal models, potentially biased by their xenogeneic nature. METHODS: HepG2 cells were cultured into a microfluidically perfused device under free fatty acid (FFA) supplementation, namely palmitic and oleic acid, for 24h and 48h. The device mimicked the endothelial-parenchymal interface of a liver sinusoid, allowing the diffusion of nutrients and removal of waste products similar to the hepatic microvasculature. Assessment of intracellular lipid accumulation, cell viability/cytotoxicity and oxidative stress due to the FFA overload, was performed by high-content analysis methodologies using fluorescence-based functional probes. RESULTS: The chip enables gradual and lower intracellular lipid accumulation, higher hepatic cell viability and minimal oxidative stress in microfluidic dynamic vs. 2D static cultures, thus mimicking the chronic condition of steatosis observed in vivo more closely. CONCLUSIONS: Overall, the liver-on-a-chip system provides a suitable culture microenvironment, representing a more reliable model compared to 2D cultures for investigating NAFLD pathogenesis. Hence, our system is amongst the first in vitro models of human NAFLD developed within a microfluidic device in a sinusoid-like fashion, endowing a more permissive tissue-like microenvironment for long-term culture of hepatic cells than conventional 2D static cultures.