Different effects of NK cells and NK-derived soluble factors on cell lines derived from primary or metastatic pancreatic cancers.

Natural killer (NK) cells are cytotoxic lymphoid cells that play a key role in defenses against tumors. However, their function may be severely impaired in patients with pancreatic adenocarcinoma (PA). Indeed, PA cells release soluble factors, thereby generating an immunosuppressive environment that dysregulates NK-cell cytolytic function and favors tumor immune evasion. Here, we analyzed the interactions between NK and PA cells using the PANC-1 and CAPAN-1 cell lines derived from a ductal PA and metastatic lesion, respectively. Metastatic and nonmetastatic cell lines were both able to impair NK cytolytic activity. An analysis of the effect of NK cells and NK-cell-derived exosomes revealed substantial differences between the two cell lines. Thus, NK cells displayed higher cytotoxicity against nonmetastatic PA cells than metastatic PA cells in both 2D cultures and in a 3D extracellular matrix cell system. In addition, NK-derived exosomes could penetrate only PANC-1 spheroids and induce cell killing. Remarkably, when PANC-1 cells were exposed to NK-derived soluble factors, they displayed substantial changes in the expression of genes involved in epithelial-to-mesenchymal transition (EMT) and acquired resistance to NK-mediated cytolysis. These results, together with their correlation with poor clinical outcomes in PA patients, suggest that the induction of resistance to cytolysis upon exposure to NK-derived soluble factors could reflect the occurrence of EMT in tumor cells. Our data indicate that a deeper investigation of the interaction between NK cells and tumor cells may be crucial for immunotherapy, possibly improving the outcome of PA treatment by targeting critical steps of NK-tumor cell crosstalk.

Atomic force microscopy for biomechanical and structural analysis of human dermis: A complementary tool for medical diagnosis and therapy monitoring.

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 µm2 areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun-exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure-the former even more than the latter-are thicker and present a larger D-period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.

Chemical and morphological gradient scaffolds to mimic hierarchically complex tissues: From theoretical modeling to their fabrication.

Porous multiphase scaffolds have been proposed in different tissue engineering applications because of their potential to artificially recreate the heterogeneous structure of hierarchically complex tissues. Recently, graded scaffolds have been also realized, offering a continuum at the interface among different phases for an enhanced structural stability of the scaffold. However, their internal architecture is often obtained empirically and the architectural parameters rarely predetermined. The aim of this work is to offer a theoretical model as tool for the design and fabrication of functional and structural complex graded scaffolds with predicted morphological and chemical features, to overcome the time-consuming trial and error experimental method. This developed mathematical model uses laws of motions, Stokes equations, and viscosity laws to describe the dependence between centrifugation speed and fiber/particles sedimentation velocity over time, which finally affects the fiber packing, and thus the total porosity of the 3D scaffolds. The efficacy of the theoretical model was tested by realizing engineered graded grafts for osteochondral tissue engineering applications. The procedure, based on combined centrifugation and freeze-drying technique, was applied on both polycaprolactone (PCL) and collagen-type-I (COL) to test the versatility of the entire process. A functional gradient was combined to the morphological one by adding hydroxyapatite (HA) powders, to mimic the bone mineral phase. Results show that 3D bioactive morphologically and chemically graded grafts can be properly designed and realized in agreement with the theoretical model. Biotechnol. Bioeng. 2016;113: 2286-2297. © 2016 Wiley Periodicals, Inc.

Guidelines for managing data and processes in bone and cartilage tissue engineering.

BACKGROUND: In the last decades, a wide number of researchers/clinicians involved in tissue engineering field published several works about the possibility to induce a tissue regeneration guided by the use of biomaterials. To this aim, different scaffolds have been proposed, and their effectiveness tested through in vitro and/or in vivo experiments. In this context, integration and meta-analysis approaches are gaining importance for analyses and reuse of data as, for example, those concerning the bone and cartilage biomarkers, the biomolecular factors intervening in cell differentiation and growth, the morphology and the biomechanical performance of a neo-formed tissue, and, in general, the scaffolds' ability to promote tissue regeneration. Therefore standards and ontologies are becoming crucial, to provide a unifying knowledge framework for annotating data and supporting the semantic integration and the unambiguous interpretation of novel experimental results. RESULTS: In this paper a conceptual framework has been designed for bone/cartilage tissue engineering domain, by now completely lacking standardized methods. A set of guidelines has been provided, defining the minimum information set necessary for describing an experimental study involved in bone and cartilage regenerative medicine field. In addition, a Bone/Cartilage Tissue Engineering Ontology (BCTEO) has been developed to provide a representation of the domain's concepts, specifically oriented to cells, and chemical composition, morphology, physical characterization of biomaterials involved in bone/cartilage tissue engineering research. CONCLUSIONS: Considering that tissue engineering is a discipline that traverses different semantic fields and employs many data types, the proposed instruments represent a first attempt to standardize the domain knowledge and can provide a suitable means to integrate data across the field.

A novel scaffold geometry for chondral applications: theoretical model and in vivo validation.

A theoretical model of the 3D scaffold internal architecture has been implemented with the aim to predict the effects of some geometrical parameters on total porosity, Young modulus, buckling resistance and permeability of the graft. This model has been adopted to produce porous poly-caprolacton based grafts for chondral tissue engineering applications, best tuning mechanical and functional features of the scaffolds. Material prototypes were produced with an internal geometry with parallel oriented cylindrical pores of 200 µm of radius (r) and an interpore distance/pores radius (d/r) ratio of 1. The scaffolds have been then extensively characterized; progenitor cells were then used to test their capability to support cartilaginous matrix deposition in an ectopic model. Scaffold prototypes fulfill both the chemical-physical requirements, in terms of Young's modulus and permeability, and the functional needs, such as surface area per volume and total porosity, for an enhanced cellular colonization and matrix deposition. Moreover, the grafts showed interesting chondrogenic potential in vivo, besides offering adequate mechanical performances in vitro, thus becoming a promising candidate for chondral tissues repair. Finally, a very good agreement was found between the prediction of the theoretical model and the experimental data. Many assumption of this theoretical model, hereby applied to cartilage, may be transposed to other tissue engineering applications, such as bone substitutes.

Fluid-Dynamic Culture of Tumour and Immune Cells for More Predictive Infiltration Studies and Immunotherapy Drug Screening.

Immunotherapies represent one of the current most promising challenges in cancer treatment. They are based on the boost of natural immune responses, aimed at cancer eradication. However, the success of immunotherapeutic approaches strictly depends on the interaction between immune cells and cancer cells. Preclinical drug tests currently available are poor in fully predicting the actual safety and efficacy of immunotherapeutic treatments under development. Indeed, conventional 2D cell culture underrepresents the complexity of the tumour microenvironment, while in vivo animal models lack in mimicking the human immune cell responses. In this context, predictability, reliability, and complete immune compatibility still represent challenges to overcome. For this aim, novel 3D, fully humanized in vitro cancer tissue models have been recently optimized by adopting emerging technologies, such as organ-on-chips (OOC) and 3D cancer cell-laden hydrogels. In particular, a novel multi-in vitro organ (MIVO) OOC platform has been recently adopted to culture 3D clinically relevant size cancer tissues under proper physiological culture conditions to investigate anti-cancer treatments and immune-tumour cell crosstalk.The proposed immune-tumour OOC-based model offers a potential tool for accurately modelling human immune-related diseases and effectively assessing immunotherapy efficacy, finally offering promising experimental approaches for personalized medicine.

Shifting the Focus from Dissolution to Permeation: Introducing the Meso-fluidic Chip for Permeability Assessment (MCPA).

In response to the growing ethical and environmental concerns associated with animal testing, numerous in vitro tools of varying complexity and biorelevance have been developed and adopted in pharmaceutical research and development. In this work, we present one of these tools, i.e., the Meso-fluidic Chip for Permeability Assessment (MCPA), for the first time. The MCPA combines an artificial barrier (PermeaPad®) with an organ-on-chip device (MIVO®) and real-time automated concentration measurements, to yield a sustainable, yet effortless method for permeation testing. The system offers three major physiological aspects, i.e., a biomimetic membrane, an optimal membrane interfacial area-to-donor-volume-ratio (A/V) and a physiological flow on the acceptor/basolateral side, which makes the MPCA an ideal candidate for mechanistic studies and excellent in vivo bioavailability predictions. We validated the method with a handful of assorted drug compounds in unstirred and stirred donor conditions, before exploring its applicability as a tool for dissolution/permeation testing on a BCS class III/I drug (pyrazinamide) crystalline adducts and BCS class II/IV (hydrocortisone) amorphous solid dispersions. The results were highly reproducible and clearly displayed the method's potential for evaluating the performance of enabling formulations, and possibly even predicting in vivo performance. We believe that, upon further development, the MCPA will serve as a useful in vitro tool that could push sustainability into pharmaceutics by refining, reducing and replacing animal testing in early-stage drug development.

3D Human Tumor Tissues Cultured in Dynamic Conditions as Alternative In Vitro Disease Models.

The slow knowledge progression about cancer disease and the high drug clinical failure are mainly due to the inadequacy of the simplistic pre-clinical in vitro and in vivo animal tumor models. To overpass these limits, in recent years many 3D matrix-based cell cultures have been proposed as challenging alternatives, since they allow to better recapitulate the in vitro cells-cells and cells-matrix reciprocal interactions in a more physiological context. Among many natural polymers, alginate has been adopted as an extracellular matrix surrogate to mimic the 3D spatial organization. After their expansion, cancer cells are suspended in an alginate solution and dropped within a crosslinking solution enabling gelification. The result is the generation of a 3D hydrogel embedding a single cell suspension: Cells are equally distributed throughout the gel, and they are free to proliferate generating clonal spheroids. Moreover, according to the hydrogel matrix stiffness that can be easily tuned, tumor cells can spread within the 3D structure and migrate outside, where they may become circulating tumor cells and infiltrate secondary tumor sites when these 3D tumor tissues are cultured in a fluid dynamic environment (i.e., organ on chip).

A Human Ovarian Tumor & Liver Organ-on-Chip for Simultaneous and More Predictive Toxo-Efficacy Assays.

In oncology, the poor success rate of clinical trials is becoming increasingly evident due to the weak predictability of preclinical assays, which either do not recapitulate the complexity of human tissues (i.e., in vitro tests) or reveal species-specific outcomes (i.e., animal testing). Therefore, the development of novel approaches is fundamental for better evaluating novel anti-cancer treatments. Here, a multicompartmental organ-on-chip (OOC) platform was adopted to fluidically connect 3D ovarian cancer tissues to hepatic cellular models and resemble the systemic cisplatin administration for contemporarily investigating drug efficacy and hepatotoxic effects in a physiological context. Computational fluid dynamics was performed to impose capillary-like blood flows and predict cisplatin diffusion. After a cisplatin concentration screening using 2D/3D tissue models, cytotoxicity assays were conducted in the multicompartmental OOC and compared with static co-cultures and dynamic single-organ models. A linear decay of SKOV-3 ovarian cancer and HepG2 liver cell viability was observed with increasing cisplatin concentration. Furthermore, 3D ovarian cancer models showed higher drug resistance than the 2D model in static conditions. Most importantly, when compared to clinical therapy, the experimental approach combining 3D culture, fluid-dynamic conditions, and multi-organ connection displayed the most predictive toxicity and efficacy results, demonstrating that OOC-based approaches are reliable 3Rs alternatives in preclinic.

3D Bioprinting as a Powerful Technique for Recreating the Tumor Microenvironment.

In vitro three-dimensional models aim to reduce and replace animal testing and establish new tools for oncology research and the development and testing of new anticancer therapies. Among the various techniques to produce more complex and realistic cancer models is bioprinting, which allows the realization of spatially controlled hydrogel-based scaffolds, easily incorporating different types of cells in order to recreate the crosstalk between cancer and stromal components. Bioprinting exhibits other advantages, such as the production of large constructs, the repeatability and high resolution of the process, as well as the possibility of vascularization of the models through different approaches. Moreover, bioprinting allows the incorporation of multiple biomaterials and the creation of gradient structures to mimic the heterogeneity of the tumor microenvironment. The aim of this review is to report the main strategies and biomaterials used in cancer bioprinting. Moreover, the review discusses several bioprinted models of the most diffused and/or malignant tumors, highlighting the importance of this technique in establishing reliable biomimetic tissues aimed at improving disease biology understanding and high-throughput drug screening.

In vivo lamellar bone formation in fibre coated MgCHA-PCL-composite scaffolds.

Bio-inspired materials with controlled topography have gained increasing interest in regenerative medicine, because of their ability to reproduce the physical features of natural extracellular matrix, thus amplifying certain biological responses both in vitro and in vivo, such as contact guidance and differentiation. However, information on the ability to adapt this high cell potential to 3D scaffolds, effective to be implanted in clinical bone defect, is still missing. Here, we examine the pattern of bone tissue generated within the implant in an ectopic model, seeding bone marrow progenitor cells onto PCL-MgCHA scaffolds. This composite material presented a porous structure with micro/nanostructured surfaces obtained by combining phase inversion/salt leaching and electrospinning techniques. Histological analysis of grafts harvested after 1-2-6 months from implantation highlights an extent of lamellar bone tissue within interconnected pores of fibre coated PCL-MgCHA composites, whereas uncoated scaffolds displayed sparse deposition of bone. Pure PCL scaffolds did not reveal any trace of bone for the overall 6 months of observation. In conclusion, we show that a structural modification in scaffold design is able to enhance bone regeneration possibly mimicking some physiological cues of the natural tissue.

Tumor Microenvironment and Hydrogel-Based 3D Cancer Models for In Vitro Testing Immunotherapies.

In recent years, immunotherapy has emerged as a promising novel therapeutic strategy for cancer treatment. In a relevant percentage of patients, however, clinical benefits are lower than expected, pushing researchers to deeply analyze the immune responses against tumors and find more reliable and efficient tools to predict the individual response to therapy. Novel tissue engineering strategies can be adopted to realize in vitro fully humanized matrix-based models, as a compromise between standard two-dimensional (2D) cell cultures and animal tests, which are costly and hardly usable in personalized medicine. In this review, we describe the main mechanisms allowing cancer cells to escape the immune surveillance, which may play a significant role in the failure of immunotherapies. In particular, we discuss the role of the tumor microenvironment (TME) in the establishment of a milieu that greatly favors cancer malignant progression and impact on the interactions with immune cells. Then, we present an overview of the recent in vitro engineered preclinical three-dimensional (3D) models that have been adopted to resemble the interplays between cancer and immune cells and for testing current therapies and immunotherapeutic approaches. Specifically, we focus on 3D hydrogel-based tools based on different types of polymers, discussing the suitability of each of them in reproducing the TME key features based on their intrinsic or tunable characteristics. Finally, we introduce the possibility to combine the 3D models with technological fluid dynamics platforms, reproducing the dynamic complex interactions between tumor cells and immune effectors migrated in situ via the systemic circulation, pointing out the challenges that still have to be overcome for setting more predictive preclinical assays.

Comparison Between Franz Diffusion Cell and a novel Micro-physiological System for In Vitro Penetration Assay Using Different Skin Models.

In vitro diffusive models are an important tool to screen the penetration ability of active ingredients in various formulations. A reliable assessment of skin penetration enhancing properties, mechanism of action of carrier systems, and an estimation of a bioavailability are essential for transdermal delivery. Given the importance of testing the penetration kinetics of different compounds across the skin barrier, several in vitro models have been developedThe aim of this study was to compare the Franz Diffusion Cell (FDC) with a novel fluid-dynamic platform (MIVO) by evaluating penetration ability of caffeine, a widely used reference substance, and LIP1, a testing molecule having the same molecular weight but a different lipophilicity in the two diffusion chamber systems. A 0.7% caffeine or LIP1 formulation in either water or propylene glycol (PG) containing oleic acid (OA) was topically applied on the Strat-M® membrane or pig ear skin, according to the infinite-dose experimental condition (780 ul/cm2). The profile of the penetration kinetics was determined by quantify the amount of molecule absorbed at different time-points (1, 2, 4, 6, 8 hours), by means of HPLC analysis. Both diffusive systems show a similar trend for caffeine and LIP1 penetration kinetics. The Strat-M® skin model shows a lower barrier function than the pig skin biopsies, whereby the PGOA vehicle exhibits a higher penetration, enhancing the effect for both diffusive chambers and skin surrogates. Most interestingly, MIVO diffusive system better predicts the lipophilic molecules (i.e. LIP1) permeation through highly physiological fluid flows resembled below the skin models.

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model.

The success of immunotherapeutic approaches strictly depends on the immune cells interaction with cancer cells. While conventional in vitro cell cultures under-represent the complexity and dynamic crosstalk of the tumor microenvironment, animal models do not allow deciphering the anti-tumor activity of the human immune system. Therefore, the development of reliable and predictive preclinical models has become crucial for the screening of immune-therapeutic approaches. We here present an organ-on-chip organ on chips (OOC)-based approach for recapitulating the immune cell Natural Killer (NK) migration under physiological fluid flow, infiltration within a 3D tumor matrix, and activation against neuroblastoma cancer cells in a humanized, fluid-dynamic environment. Circulating NK cells actively initiate a spontaneous "extravasation" process toward the physically separated tumor niche, retaining their ability to interact with matrix-embedded tumor cells, and to display a cytotoxic effect (tumor cell apoptosis). Since NK cells infiltration and phenotype is correlated with prognosis and response to immunotherapy, their phenotype is also investigated: most importantly, a clear decrease in CD16-positive NK cells within the migrated and infiltrated population is observed. The proposed immune-tumor OOC-based model represents a promising approach for faithfully recapitulating the human pathology and efficiently employing the immunotherapies testing, eventually in a personalized perspective. An immune-organ on chip to recapitulate the tumor-mediated infiltration of circulating immune cells within 3D tumor model.

An interaction between hepatocyte growth factor and its receptor (c-MET) prolongs the survival of chronic lymphocytic leukemic cells through STAT3 phosphorylation: a potential role of mesenchymal cells in the disease.

BACKGROUND: Chronic lymphocytic leukemia cells are characterized by an apparent longevity in vivo which is lost when they are cultured in vitro. Cellular interactions and factors provided by the microenvironment appear essential to cell survival and may protect leukemic cells from the cytotoxicity of conventional therapies. Understanding the cross-talk between leukemic cells and stroma is of interest for identifying signals supporting disease progression and for developing novel therapeutic strategies. DESIGN AND METHODS: Different cell types, sharing a common mesenchymal origin and representative of various bone marrow components, were used to challenge the viability of leukemic cells in co-cultures and in contact-free culture systems. Using a bioinformatic approach we searched for genes shared by lineages prolonging leukemic cell survival and further analyzed their biological role in signal transduction experiments. RESULTS: Human bone marrow stromal cells, fibroblasts, trabecular bone-derived cells and an osteoblast-like cell line strongly enhanced survival of leukemic cells, while endothelial cells and chondrocytes did not. Gene expression profile analysis indicated two soluble factors, hepatocyte growth factor and CXCL12, as potentially involved. We demonstrated that hepatocyte growth factor and CXCL12 are produced only by mesenchymal lineages that sustain the survival of leukemic cells. Indeed chronic lymphocytic leukemic cells express a functional hepatocyte growth factor receptor (c-MET) and hepatocyte growth factor enhanced the viability of these cells through STAT3 phosphorylation, which was blocked by a c-MET tyrosine kinase inhibitor. The role of hepatocyte growth factor was confirmed by its short interfering RNA-mediated knock-down in mesenchymal cells. CONCLUSIONS: The finding that hepatocyte growth factor prolongs the survival of chronic lymphocytic leukemic cells is novel and we suggest that the interaction between hepatocyte growth factor-producing mesenchymal and neoplastic cells contributes to maintenance of the leukemic clone.

In vitro models replicating the human intestinal epithelium for absorption and metabolism studies: A systematic review.

Absorption, distribution, metabolism and excretion (ADME) studies represent a fundamental step in the early stages of drug discovery. In particular, the absorption of orally administered drugs, which occurs at the intestinal level, has gained attention since poor oral bioavailability often led to failures for new drug approval. In this context, several in vitro preclinical models have been recently developed and optimized to better resemble human physiology in the lab and serve as an animal alternative to accomplish the 3Rs principles. However, numerous models are ineffective in recapitulating the key features of the human small intestine epithelium and lack of prediction potential for drug absorption and metabolism during the preclinical stage. In this review, we provide an overview of in vitro models aimed at mimicking the intestinal barrier for pharmaceutical screening. After briefly describing how the human small intestine works, we present i) conventional 2D synthetic and cell-based systems, ii) 3D models replicating the main features of the intestinal architecture, iii) micro-physiological systems (MPSs) reproducing the dynamic stimuli to which cells are exposed in the native microenvironment. In this review, we will highlight the benefits and drawbacks of the leading intestinal models used for drug absorption and metabolism studies.

High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.

Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.

3D fluid-dynamic ovarian cancer model resembling systemic drug administration for efficacy assay.

Recently, 3D in vitro cancer models have become important alternatives to animal tests for establishing the efficacy of anticancer treatments. In this work, 3D SKOV-3 cell-laden alginate hydrogels were established as ovarian tumor models and cultured within a fluid-dynamic bioreactor (MIVO®) device able to mimic the capillary flow dynamics feeding the tumor. Cisplatin efficacy tests were performed within the device over time and compared with (i) the in vitro culture under static conditions and (ii) a xenograft mouse model with SKOV-3 cells, by monitoring and measuring cell proliferation or tumor regression, respectively, over time. After one week of treatment with 10 µM cisplatin, viability of cells within the 3D hydrogels cultured under static conditions remained above 80%. In contrast, the viability of cells within the 3D hydrogels cultured within dynamic MIVO® decreased by up to 50%, and very few proliferating Ki67-positive cells were observed through immunostaining. Analysis of drug diffusion, confirmed by computational analysis, explained that these results are due to different cisplatin diffusion mechanisms in the two culture conditions. Interestingly, the outcome of the drug efficacy test in the xenograft model was about 44% of tumor regression after 5 weeks, as predicted in a shorter time in the fluid-dynamic in vitro tests carried out in the MIVO® device. These results indicate that the in vivo-like dynamic environment provided by the MIVO® device allows to better model the 3D tumor environment and predict in vivo drug efficacy than a static in vitro model.

A three-dimensional traction/torsion bioreactor system for tissue engineering.

PURPOSE: The aim of this study was to design, develop and validate a simple, compact bioreactor system for tissue engineering. The resulting bioreactor was designed to achieve ease-of-use and low costs for automated cell-culturing procedures onto three-dimensional scaffolds under controlled torsion/traction regimes. METHODS: Highly porous poly-caprolactone-based scaffolds were used as substrates colonized by fibroblast cells (3T3 cell line). Constructs were placed within the cylindrical culture chamber, clumped at the ends and exposed to controlled sequences of torsional stimuli (forward/back-forward sequential cycles of 100 degrees from neutral position at a rate of 600 degrees/min) through a stepper-motor; working settings were defined via PC by an easy user-interface. Cell adhesion, morphology, cytoskeletal fiber orientation and gene expression of extracellular matrix proteins (collagen type I, tenascin C, collagen type III) were evaluated after three days of torsional stimulation in the bioreactor system. RESULTS AND CONCLUSIONS: The 3D bioreactor system was validated in terms of sterility, experimental reproducibility and flexibility. Cells adhered well onto the polymeric scaffolds. Collagen type I, tenascin C and collagen type III gene expression were significantly up-regulated when cells were cultured under torsion in the bioreactor for three days. In conclusion, we have developed a simple, efficient and versatile 3D cell-culture system to engineer ligament grafts. This system can be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system for possible clinical use in the field of regenerative medicine.

3D Perfusable Hydrogel Recapitulating the Cancer Dynamic Environment to in Vitro Investigate Metastatic Colonization.

Metastasis is a dynamic process involving the dissemination of circulating tumor cells (CTCs) through blood flow to distant tissues within the body. Nevertheless, the development of an in vitro platform that dissects the crucial steps of metastatic cascade still remains a challenge. We here developed an in vitro model of extravasation composed of (i) a single channel-based 3D cell laden hydrogel representative of the metastatic site, (ii) a circulation system recapitulating the bloodstream where CTCs can flow. Two polymers (i.e., fibrin and alginate) were tested and compared in terms of mechanical and biochemical proprieties. Computational fluid-dynamic (CFD) simulations were also performed to predict the fluid dynamics within the polymeric matrix and, consequently, the optimal culture conditions. Next, once the platform was validated through perfusion tests by fluidically connecting the hydrogels with the external circuit, highly metastatic breast cancer cells (MDA-MB-231) were injected and exposed to physiological wall shear stress (WSS) conditions (5 Dyn/cm2) to assess their migration toward the hydrogel. Results indicated that CTCs arrested and colonized the polymeric matrix, showing that this platform can be an effective fluidic system to model the first steps occurring during the metastatic cascade as well as a potential tool to in vitro elucidate the contribution of hemodynamics on cancer dissemination to a secondary site.