Experimental liver models: From cell culture techniques to microfluidic organs-on-chip.

The liver is one of the most studied organs of the human body owing to its central role in xenobiotic and drug metabolism. In recent decades, extensive research has aimed at developing in vitro liver models able to mimic liver functions to study pathophysiological clues in high-throughput and reproducible environments. Two-dimensional (2D) models have been widely used in screening potential toxic compounds but have failed to accurately reproduce the three-dimensionality (3D) of the liver milieu. To overcome these limitations, improved 3D culture techniques have been developed to recapitulate the hepatic native microenvironment. These models focus on reproducing the liver architecture, representing both parenchymal and nonparenchymal cells, as well as cell interactions. More recently, Liver-on-Chip (LoC) models have been developed with the aim of providing physiological fluid flow and thus achieving essential hepatic functions. Given their unprecedented ability to recapitulate critical features of the liver cellular environments, LoC have been extensively adopted in pathophysiological modelling and currently represent a promising tool for tissue engineering and drug screening applications. In this review, we discuss the evolution of experimental liver models, from the ancient 2D hepatocyte models, widely used for liver toxicity screening, to 3D and LoC culture strategies adopted for mirroring a more physiological microenvironment for the study of liver diseases.

Autoradioluminography, a powerful and reliable tool for drug development: Accelera's experience.

A deeper understanding of the pharmacokinetic and pharmacodynamic properties of a drug candidate is a pivotal component of drug discovery and development. Autoradiography is an excellent technique allowing exploiting the advantages of the use of radioisotopes in the drug disscovery field. The introduction of phosphor imaging technology has revolutionized the handling of drug distribution studies providing high-resolution images. Specifically, quantitative whole-body autoradioluminography is employed for preclinical study where the aim is to obtain information about the route of elimination and tissue distribution of a drug candidate. Autoradioluminography is also the technique of choice pursued to deal with all the issue that it is possible to encounter in all stage of drug development (ie, site-specific drug localization and retention, drug-drug interactions, penetration into specific target, specific tissue binding, crossing of brain blood barrier, and placental transfer). The purpose of this review is to give a picture of how autoradiography is employed in our laboratory as a key tool for advances in the assessment of the drug disposition and to validate new experimental models.

Comparative analysis of multilineage properties of mesenchymal stromal cells derived from fetal sources shows an advantage of mesenchymal stromal cells isolated from cord blood in chondrogenic differentiation potential.

BACKGROUND AIMS: Cord blood (CB) and amniotic fluid (AF) could represent new and attractive mesenchymal stromal cell (MSC) sources, but their potential therapeutic applications are still limited by lack of standardized protocols for isolation and differentiation. In particular, chondrogenic differentiation has never been deeply investigated. METHODS: MSCs were obtained from CB and AF samples collected during cesarean sections at term and compared for their biological and differentiation properties, with particular interest in cartilage differentiation, in which quantitative real-time polymerase chain reaction and immunohistochemical analyses were performed to evaluate the expression of type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. RESULTS: We were able to isolate MSCs from 12 of 30 (40%) and 5 of 20 (25%) CB and AF units, respectively. Fluorescence in situ hybridization analysis indicated the fetal origin of isolated MSC strains. Both populations expressed mesenchymal but not endothelial and hematopoietic markers, even though we observed a lower expression of human leukocyte antigen (HLA) I in CB-MSCs. No differences in proliferation rate and cell cycle analysis could be detected. After osteogenic induction, both populations showed matrix mineralization and typical marker expression. Under chondrogenic conditions, pellets derived from CB-MSCs, in contrast with AF-MSCs pellets, were significantly larger, showed cartilage-like morphology and resulted positive for chondrocyte-associated markers, such as type 2 collagen, type 10 collagen, SRY-box9 and aggrecan. CONCLUSIONS: Our results show that CB-MSCs and AF-MSCs collected at term differ from each other in their biological and differentiation properties. In particular, only CB-MSCs showed a clear chondrogenic potential and thus could represent an ideal candidate for cartilage-tissue engineering.

Cholangiocarcinoma-on-a-chip: A human 3D platform for personalised medicine.

Background & Aims: Cholangiocarcinoma (CCA) is a primary liver tumour characterised by a poor prognosis and limited therapeutic options. Available 3D human CCA models fail to faithfully recapitulate the tumour niche. We aimed to develop an innovative patient-specific CCA-on-chip platform. Methods: A CCA tumour microenvironment was recapitulated on a microfluidic three-channel chip using primary CCA cells, cancer-associated fibroblasts (CAFs), endothelial cells, and T cells isolated from CCA specimens (n = 6). CAF and CCA cells were co-cultured in the central channel, flanked by endothelial cells in one lateral channel, recreating a tubular structure. An extensive characterisation of this platform was carried out to investigate its diffusion ability, hydrogel properties, and changes in matrix composition. Cell phenotype and functional properties were assessed. Results: Primary cells seeded on the microfluidic device were shown to reproduce the architectural structure and maintain the original phenotype and functional properties. The tumour niche underwent a deep remodelling in the 3D device, with an increase in hydrogel stiffness and extracellular matrix deposition, mimicking in vivo CCA characteristics. T cells were incorporated into the device to assess its reliability for immune cell interaction studies. Higher T cell migration was observed using cells from patients with highly infiltrated tumours. Finally, the drug trial showed the ability of the device to recapitulate different drug responses based on patient characteristics. Conclusions: We presented a 3D CCA platform that integrates the major non-immune components of the tumour microenvironment and the T cell infiltrate, reflecting the CCA niche. This CCA-on-chip represents a reliable patient-specific 3D platform that will be of help to further elucidate the biological mechanisms involved in CCA and provide an efficient tool for personalised drug testing. Impact and implications: An innovative patient-specific cholangiocarcinoma (CCA)-on-chip platform was successfully developed, integrating the major components of the tumour microenvironment (tumour cells, cancer-associated fibroblasts, endothelial cells, and immune infiltrate) and faithfully mimicking the CCA niche. This CCA-on-chip represents a powerful tool for unravelling disease-associated cellular mechanisms in CCA and provides an efficient tool for personalised drug testing.

Cancer-on-chip: a 3D model for the study of the tumor microenvironment.

The approval of anticancer therapeutic strategies is still slowed down by the lack of models able to faithfully reproduce in vivo cancer physiology. On one hand, the conventional in vitro models fail to recapitulate the organ and tissue structures, the fluid flows, and the mechanical stimuli characterizing the human body compartments. On the other hand, in vivo animal models cannot reproduce the typical human tumor microenvironment, essential to study cancer behavior and progression. This study reviews the cancer-on-chips as one of the most promising tools to model and investigate the tumor microenvironment and metastasis. We also described how cancer-on-chip devices have been developed and implemented to study the most common primary cancers and their metastatic sites. Pros and cons of this technology are then discussed highlighting the future challenges to close the gap between the pre-clinical and clinical studies and accelerate the approval of new anticancer therapies in humans.

Predicting human cardiac QT alterations and pro-arrhythmic effects of compounds with a 3D beating heart-on-chip platform.

Determining the potential cardiotoxicity and pro-arrhythmic effects of drug candidates remains one of the most relevant issues in the drug development pipeline (DDP). New methods enabling to perform more representative preclinical in vitro studies by exploiting induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are under investigation to increase the translational power of the outcomes. Here we present a pharmacological campaign conducted to evaluate the drug-induced QT alterations and arrhythmic events on uHeart, a 3D miniaturized in vitro model of human myocardium encompassing iPSC-CM and dermal fibroblasts embedded in fibrin. uHeart was mechanically trained resulting in synchronously beating cardiac microtissues in 1 week, characterized by a clear field potential (FP) signal that was recorded by means of an integrated electrical system. A drug screening protocol compliant with the new International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines was established and uHeart was employed for testing the effect of 11 compounds acting on single or multiple cardiac ion channels and well-known to elicit QT prolongation or arrhythmic events in clinics. The alterations of uHeart's electrophysiological parameters such as the beating period, the FP duration, the FP amplitude, and the detection of arrhythmic events prior and after drug administration at incremental doses were effectively analyzed through a custom-developed algorithm. Results demonstrated the ability of uHeart to successfully anticipate clinical outcome and to predict the QT prolongation with a sensitivity of 83.3%, a specificity of 100% and an accuracy of 91.6%. Cardiotoxic concentrations of drugs were notably detected in the range of the clinical highest blood drug concentration (Cmax), qualifying uHeart as a fit-to-purpose preclinical tool for cardiotoxicity studies.

Plasma-enhanced protein patterning in a microfluidic compartmentalized platform for multi-organs-on-chip: a liver-tumor model.

A microfluidic technique is presented for micropatterning protein domains and cell cultures within permanently bonded organs-on-chip devices. This method is based on the use of polydimethylsiloxane layers coupled with the plasma ablation technique for selective protein removal. We show how this technique can be employed to generate a multi-organin vitromodel directly within a microscale platform suitable for pharmacokinetic-based drug screening. We miniaturized a liver model based on micropatterned co-cultures in dual-compartment microfluidic devices. The cytotoxic effect of liver-metabolized Tegafur on colon cancer cell line was assessed using two microfluidic devices where microgrooves and valves systems are used to model drug diffusion between culture compartments. The platforms can reproduce the metabolism of Tegafur in the liver, thus killing colon cancer cells. The proposed plasma-enhanced microfluidic protein patterning method thus successfully combines the ability to generate precise cell micropatterning with the intrinsic advantages of microfluidics in cell biology.

Micro-electrode channel guide (µECG) technology: an online method for continuous electrical recording in a human beating heart-on-chip.

Cardiac toxicity still represents a common adverse outcome causing drug attrition and post-marketing withdrawal. The development of relevantin vitromodels resembling the human heart recently opened the path towards a more accurate detection of drug-induced human cardiac toxicity early in the drug development process. Organs-on-chip have been proposed as promising tools to recapitulatein vitrothe key aspects of thein vivocardiac physiology and to provide a means to directly analyze functional readouts. In this scenario, a new device capable of continuous monitoring of electrophysiological signals from functionalin vitrohuman hearts-on-chip is here presented. The development of cardiac microtissues was achieved through a recently published method to control the mechanical environment, while the introduction of a technology consisting in micro-electrode coaxial guides allowed to conduct direct and non-destructive electrophysiology studies. The generated human cardiac microtissues exhibited synchronous spontaneous beating, as demonstrated by multi-point and continuous acquisition of cardiac field potential, and expression of relevant genes encoding for cardiac ion-channels. A proof-of-concept pharmacological validation on three drugs proved the proposed model to potentially be a powerful tool to evaluate functional cardiac toxicity.

Collagenase isoforms for pancreas digestion.

The available information concerning the characteristics and composition of collagenase batches, which are effective in the digestion of human pancreas for islet transplants, is scarce and incomplete. A large inter- and intrabatched variability in activity and efficiency of blend enzymes available for isolation has been observed. The aim of this study was to characterize enzyme blend components. Liberase batches were characterized by SDS-PAGE analyses, microelectrophoresis, and then by MALDI-TOF MS analysis. Three main bands were detected by SDS-PAGE analysis and submitted to MALDI-TOF MS analysis. Two bands were found to correspond to class I (isoform beta and another of 106 kDa) and one to class II (isoform delta) collagenase. These results represent an important step towards a complete characterization of enzymes, with the final aim of identifying key components for a standardized product.

Riboflavin inhibits IL-6 expression and p38 activation in islet cells.

Riboflavin is a water-soluble vitamin that reduces the production of proinflammatory mediators and oxygen radicals. Because islet beta-cells are very sensitive to oxidative stress and to cytokines, we investigated the possible cytoprotective effects of riboflavin on insulinoma NIT-1 cells and on isolated rodent islets. NIT-1 cells and islets cultured in the presence or absence of 10 microM riboflavin were studied at baseline and after exposure to cytokines (TNF-alpha, IL-1beta, INF-gamma). Riboflavin treatment did not affect islet cell viability as assessed by flow cytometry for caspases activation. However, riboflavin prevented the cytokine-induced increase in IL-6 mRNA expression and p38 phosphorylation analyzed by real-time PCR and immunoassay, respectively. In summary, nontoxic doses of riboflavin prevent cytokines-induced p38 phosphorylation and IL-6 upregulation in islet cells. This observation, together with the safety profile of riboflavin in the clinical setting, makes it an appealing agent for islet cytoprotection in islet transplantation protocols.

Characterization of collagenase blend enzymes for human islet transplantation.

BACKGROUND: Efficient islet isolation represents a necessary requirement for successful islet transplantation as a treatment for type 1 diabetes. The choice of collagenase for pancreas digestion is critical for the isolation outcome, and Liberase is the most widely used enzyme, although large intra-batched variability in activity and efficiency has been observed. METHODS: The aim of this study was to characterize Liberase components and their relative role in pancreas digestion. Liberase batches were characterized by microelectrophoresis. RESULTS: By means of microelectrophoresis, we identified three main proteins each with different prevalences between batches. Two proteins were found to correspond to class I (CI) and one to class II (CII) collagenase. In a series of 163 islet isolations, we observed that the CII correlated with islet yield (P<0.001) and digestion time (P<0.001); additionally, CI directly correlated with purity (P=0.028). Finally, when CII and one of the CI isoforms were >50 percentile, 15 of 36 preparations were transplanted, with 27 of 127 transplanted in the other cases (P=0.013). CONCLUSION: These results represent an important step toward the characterization of enzymes, with the final aim of identifying key components for a standardized product.

Islet cell transplantation.

Islet cell transplantation is an attractive alternative therapy to conventional insulin treatment or vascularized whole pancreas transplantation for type 1 diabetic patients. It represents a successful example of somatic cell therapy in humans based on complex procedures for islet isolation from whole pancreas. The islets, that are only 1% of the total pancreas tissue, are isolated by two steps method starting with collagenase digestion that operates a rapid dissociation of the stromal component of the gland, while preserving islet anatomical integrity. After digestion, islets are then separated from exocrine tissue by centrifugation in density gradients. Transplantation consists of a simple injection of few milliliter-purified tissue in the portal vein through a percutaneous trans-hepatic approach performed in local anesthesia. Several studies have now demonstrated that islet transplant can replace pancreatic endocrine function without major side effects and with liver viability preservation in selected patients affected by long-term type 1 diabetes. It can restore endogenous insulin secretion, achieve insulin independence in more than 80% of patients, and recover the metabolism of glucose, protein and lipids. Improved control of glycated HbA1c, reduced risk of recurrent hypoglycemia and of diabetic complications are also seen as important benefits of islet cell transplantation, irrespective of the status of insulin independence. Many protocols are now on going for reduction of immunosuppression therapy in recipients, induction of tolerance, and prolongation of graft function.

Primary biliary cholangitis: a comprehensive overview.

Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by biliary destruction, progressive cholestasis, and potentially liver cirrhosis. Patients develop a well-orchestrated immune reaction, both innate and adaptive, against mitochondrial antigens that specifically targets intrahepatic biliary cells. A puzzling feature of PBC is that the immune attack is predominantly organ specific, although the mitochondrial autoantigens are found in all nucleated cells. The disease results from a combination of genetic and environmental risk factors; however, the exact pathogenesis remains unclear. Serologically, PBC is characterized by presence of antimitochondrial antibodies, which are present in 90-95 % of patients and are often detectable years before clinical signs appear. Like other complex disorders, PBC is heterogeneous in its presentation, symptomatology, disease progression, and response to therapy. A significant number of patients develop end-stage liver disease and eventually require liver transplantation. Recent studies from large international cohorts have better identified prognostic factors, suggesting a change in patient management based on risk stratification. Therapeutic options are changing. In this review we discuss data on the autoimmune responses and treatment of the disease.

Making Sense of Autoantibodies in Cholestatic Liver Diseases.

Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the most common chronic cholestatic liver diseases (CLD) in adults and are associated with immune mechanisms. PBC is considered a model autoimmune disease, and more than 90% of patients present very specific autoantibodies against mitochondrial antigens. Whether PSC should be considered an autoimmune or merely immune-mediated disease is still under debate. This review addresses the clinical relevance of autoantibodies in CLD and their pathogenic mechanisms and illustrates the technology available for appropriate autoantibody detection.

The epigenetics of PBC: The link between genetic susceptibility and environment.

Primary biliary cholangitis (PBC) previously known as primary biliary cirrhosis is an autoimmune disease-associated with progressive cholestasis, the presence of autoreactive T cell and characteristic serological autoantibodies. Genetic and genome-wide association studies (GWAS) have recently shed light on the genetic background of PBC. Besides that some causal nucleotide changes and mechanisms remain largely unknown as suggested for example, by the observation that monozygotic twins have an identical DNA sequence even if presents some phenotypic differences that may be consequences of different exposures to environmental stressors. For this reason, it is believed that epigenetic mechanisms may be involved in PBC pathogenesis, as already demonstrated in many autoimmune diseases and can eventually provide an understanding that has been missed from genetics alone. This review will focus on the most commonly studied epigenetic modifications already demonstrated in PBC; special attention will be paid also to other epigenetic mechanisms so far not demonstrated in PBC patients, but that could increase our understanding in PBC pathogenesis.

Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation.

Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications.

Tissue factor and CCL2/monocyte chemoattractant protein-1 released by human islets affect islet engraftment in type 1 diabetic recipients.

Islet survival in the early posttransplantation period is likely to be influenced by inflammatory events in and around islets. Twenty-seven human islet preparations were transplanted by 24 infusions into 14 patients with brittle type 1 diabetes under the Edmonton protocol. Patients were monitored for their coagulation [cross-linked fibrin degradation products (XDPs)] and liver function test [aspartate and alanine aminotransferase (AST and ALT)] as markers of early posttransplant complications, and these were correlated with in vitro islet number, purification, volume, monocyte-chemoattractant protein-1 (CCL2/MCP-1) and tissue factor (TF) islet release. Consistent with activation of coagulation pathways and hepatic damage, serum XDP values increased early after 11 infusions and transaminase after 13 of 24 infusions. TF and CCL2/MCP-1 were detected in supernatants of 21 and 22 islet preparations, respectively. Serum XDP peak values were correlated with TF/equivalent islets (EI) (r(2)=0.26, P = 0.001) and CCL2/MCP-1/EI (r(2) = 0.42; P < 0.001); serum transaminase areas under the curve in the first week posttransplantation were correlated with CCL2/MCP-1/EI (r(2) = 0.55; P < 0.001 for ALT and r(2) = 0.51; P = 0.001 for AST) and TF/EI (r(2) = 0.31; P = 0.002 for ALT, and r(2) = 0.36; P = 0.002 for AST). These data suggest that reducing the islet proinflammatory state may be a means to reduce the early posttransplant complications and perhaps improve islet engraftment.

Donor and isolation variables associated with human islet monocyte chemoattractant protein-1 release.

Human islets have chemotactic activity toward macrophages mediated by the secretion of monocyte chemoattractant protein-1 (CCL2/MCP-1) that negatively affect clinical outcome in islet after kidney recipients. The aim of the present work was to identify the donor features and the variables involved in the procedures of islet isolation associated with islet CCL2/MCP-1 release in vitro. We used a retrospective approach studying the outcome in 170 islet isolations. The univariate analysis demonstrated that CCL2/MCP-1 release was significantly associated with the surgical team in charge for organ harvesting, the proteins for dilution solution, the type of gradient, the type of enzyme, and the donor noradrenalin treatment. The multivariate analysis confirmed that the surgical team (P = 0.001) and the enzyme (P = 0.001) were independently associated with in vitro CCL2/MCP-1 islet release (r(2) = 17%). Strategies aimed to optimize the procedures of organ harvesting and islet isolation may reduce the pro-inflammatory properties of the preparation and therefore may improve islet engraftment.

Engraftment versus immunosuppression: cost-benefit analysis of immunosuppression after intrahepatic murine islet transplantation.

OBJECTIVE: Immunosuppression (IS) in islet transplantation (Tx) is a double-edged sword: it prevents immunoreaction but has the potential to impair islet engraftment. The aim of this study was to identify in murine animal models the IS platform with the best balance between these two opposite effects. METHODS: To study the impact of IS on islet engraftment diabetic C57BL/6 mice were transplanted with 350 syngeneic islets through the portal vein and treated once-daily with either rapamycin (RAPA; 0.1-0.5-1 mg/kg ip), tacrolimus (FK506; 0.1-0.5-1 mg/kg ip), mycophenolate mofetil (MMF; 60-120-300 mg/kg oral) or vehicle for 14 days. Islet function was evaluated by measuring not-fasting glycemia and by performing an IVGTT on days 15 and 30 post-Tx. RESULTS: RAPA ≥0.5 mg/Kg, FK506 ≥0.5 mg/Kg, and MMF ≥120 mg/kg had detrimental effects on islet engraftment but not on the function of islets already engrafted in the liver. The effect on engraftment was irreversible and persisted even after IS withdrawal. The lower dose of IS that did not affect engraftment was tested for preventing rejection in the full mismatch allogeneic Tx BALB/c to C57BL/6 model. RAPA and/or FK506 were inefficient in preventing rejection, even when anti-IL2R mAb was added to the IS regimen. On the other hand, MMF alone or in association with FK506 significantly prolonged the time to islet rejection. CONCLUSION: IS showed profound dose-dependent deleterious effects on islet cell engraftment. The MMF/FK506 combination proved the best balance with less toxicity at the time of engraftment and more efficacy in controlling graft rejection.

Murine animal models for preclinical islet transplantation: No model fits all (research purposes).

Advances in islet transplantation research have led to remarkable improvements in the outcome in humans with type 1 diabetes. However, pitfalls, mainly linked both to early liver-specific inflammatory events and to pre-existing and transplant-induced auto- and allo-specific adaptive immune responses, still remain. In this scenario research into pancreatic islet transplantation, essential to investigate new strategies to overcome open issues, needs very well-designed preclinical studies to obtain consistent and reliable results and select only promising strategies that may be translated into the clinical practice. This review discusses the main shortcomings of the mouse models currently used in islet transplantation research, outlining the main factors and variables to take into account for the design of new preclinical studies. Since several parameters concerning both the graft (i.e., islets) and the recipient (i.e., diabetic mice) may influence transplant outcome, we recommend considering several critical points in designing future bench-to-bedside islet transplantation research.