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Despite advances in the development of in vitro tissue models such as reconstructed human skin, the questions in dermatological research, which can be addressed with these models, are limited. This is mainly due to the lack of key extra-cellular components, which makes dermal compartment an incomplete approach to human ECM in vivo. Today there is a growing awareness of the fact that the ECM has a functional importance as a dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes. We established a human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In our model dermal ECM presents laminin, fibronectin, hyaluronic acid, elastin and collagen arranged and organized as in the human counterpart. As proof of the physiological relevance of such tissue environment we demonstrate that -by using adult human skin cells-it is possible to generate follicle-like structures in vitro resembling what occurs in vivo in the fetal skin. Immunotypization evidences an inward-oriented differentiation of the follicular-like structures through immunopositivity for epithelial stem cell markers such as p63 and K19. Moreover we succeeded in innervating this human skin by inducing rat dorsal root ganglion neurons axon ingrowth and demonstrate the effective functionality of the nerve network. Neurofilaments network infiltrates the entire native dermis ECM until reaching the epidermis, as demonstrated by immunopositivity to neurofilament-M and second harmonic generation imaging. Calcium imaging demonstrates that electrical current travel in the neuronal network proving the its sensing functionality. Our results demonstrate the fundamental role of ECM in morphogenesis, corroborating the awareness of its importance in building up functional in vitro skin model for fundamental research applications.

In this work, we developed an innovative gut-liver-on-chip system useful to predict oral drug administration and first pass metabolism. The two main organs involved in the first pass metabolism are the liver and the intestine. First-pass effects consist mainly in the reduction of bioavailability of drugs and xenobiotics. The prediction of this mechanism is important both for the development of new substances, but also for toxicity testing. For this purpose, we designed a microfluidic device which interconnect 3D human intestinal equivalent (3D-HIE) and HepG2-microtissues, recapitulating the intestinal and hepatic firstpass effect mechanism of ethanol. 3D-HIE were obtained by bottom up approach, using intestinal microtissues moulded into a maturation chamber and HepG2-μTPs were obtained by dynamic cell seeding of Hepg2 and gelatin porous microsphere in a spinner flask bioreactor.

An intestine-on-chip has been developed to study intestinal physiology and pathophysiology as well as intestinal transport absorption and toxicity studies in a controlled and human similar environment. Here, we report that dynamic culture of an intestine-on-chip enhances extracellular matrix (ECM) remodeling of the stroma, basement membrane production and speeds up epithelial differentiation. We developed a three-dimensional human intestinal stromal equivalent composed of human intestinal subepithelial myofibroblasts embedded in their own ECM. Then, we cultured human colon carcinoma-derived cells in both static and dynamic conditions in the opportunely designed microfluidic system until the formation of a well-oriented epithelium. This low cost and handy microfluidic device allows to qualitatively and quantitatively detect epithelial polarization and mucus production as well as monitor barrier function and ECM remodeling after nutraceutical treatment.

Here, we propose an immune-responsive human Microbiota-Intestine axis on-chip as a platform able to reproduce the architecture and vertical topography of the microbiota with a complex extracellular microenvironment consisting of a responsive extra cellular matrix (ECM) and a plethora of immune-modulatory mediators released from different cell populations such as epithelial, stromal, blood and microbial species in homeostatic and inflamed conditions. Firstly, we developed a three-dimensional human intestine model (3D-hI), represented by an instructive and histologically competent ECM and a well-differentiated epithelium with mucus-covered microvilli. Then, we replicated the microenvironmental anaerobic condition of human intestinal lumen by fabricating a custom-made microbiota chamber (MC) on the apical side of the Microbiota-human Intestine on chip (MihI-oC), establishing the physiological oxygen gradient occurring along the thickness of human small intestine from the serosal to the luminal side. The complexity of the intestinal extracellular microenvironment was improved by integrating cells populations that are directly involved in the inflammatory response such as peripheral blood mononuclear cells (PBMCs) and two species of the intestinal commensal microbiota (Lactobacillus rhamnosus and Bifidobacterium longum). We found that lipopolysaccharide (LPS)-induced inflammation elicits microbiota's geographical change and induce Bifidobacterium longum iper-proliferation, highlighting a role of such probiotic in anti-inflammatory process. Moreover, we proved, for the first time, the indirect role of the microbiota on stromal reshaping in immune-responsive MihI-oC in terms of collagen fibers orientation and ECM remodeling, and demonstrated the role of microbiota in alleviating gastrointestinal, immunological and infectious diseases by analyzing the release of key immune-mediators after inflammatory stimulus (reactive oxygen species (ROS), pro- and anti-inflammatory cytokines).

Cystic fibrosis is characterized by lung dysfunction involving mucus hypersecretion, bacterial infections, and inflammatory response. Inflammation triggers pro-fibrotic signals that compromise lung structure and function. At present, several in vitro cystic fibrosis models have been developed to study epithelial dysfunction but none of these focuses on stromal alterations. Here we show a new cystic fibrosis 3D stromal lung model made up of primary fibroblasts embedded in their own extracellular matrix and investigate its morphological and transcriptomic features. Cystic fibrosis fibroblasts showed a high proliferation rate and produced an abundant and chaotic matrix with increased protein content and elastic modulus. More interesting, they had enhanced pro-fibrotic markers and genes involved in epithelial function and inflammatory response. In conclusion, our study reveals that cystic fibrosis fibroblasts maintain in vitro an activated pro-fibrotic state. This abnormality may play in vivo a role in the modulation of epithelial and inflammatory cell behavior and lung remodeling. We argue that the proposed bioengineered model may provide new insights on epithelial/stromal/inflammatory cells crosstalk in cystic fibrosis, paving the way for novel therapeutic strategies.

Here, we proposed an innovative organotypic cervical tumor model able to investigate the bi-directional crosstalk between epithelium and stroma as well as the key disease features of the epithelial-mesenchymal transition (EMT) process in vitro. By using a modular tissue assembling approach, we developed 3D cervical stromal models composed of primary human cervical fibroblasts (HCFs) or cervical cancer-associated fibroblasts (CCAFs) embedded in their own ECM to produce 3D normal cervical-instructed stroma (NCIS) or 3D cervical cancer-instructed stroma (CCIS), respectively. Then, we demonstrate the role of the tumor microenvironment (TME) in potentiating the intrinsic invasive attitude of cervical cancer derived SiHa cells and increasing their early viral gene expression by comparing the SiHa behavior when cultured on NCIS or CCIS (SiHa-NCIS or SiHa-CCIS). We proved the crucial role of the CCAFs and stromal microenvironment in the mesenchymalization of the cancer epithelial cells by analyzing several EMT markers. We further assessed the expression of the epithelial adhesion molecules, matricellular enzymes, non-collagenous proteins as well as ECM remodeling in terms of collagen fibers texture and assembly. This cervical tumor model, closely recapitulating key cervical carcinogenesis features, may provide efficient and relevant support to current approaches characterizing cancer progression and develop new anticancer therapy targeting stroma rather than cancer cells.

Intestine-Liver-on-chip systems can be useful to predict oral drug administration and first-pass metabolism in vitro in order to partly replace the animal model. While organ-on-chip technology can count on sophisticated micro-physiological devices, the engineered organs still remain artificial surrogates of the native counterparts. Here, we used a bottom-up tissue engineering strategy to build-up physiologically functional 3D Human Intestine Model (3D-HIM) as well as 3D Liver-microtissues (HepG2-μTPs) in vitro and designed a microfluidic Intestine-Liver-On-Chip (InLiver-OC) to emulate first-pass mechanism occurring in vivo. Our results highlight the ethanol-induced 3D-HIM hyper-permeability and stromal injury, the intestinal prevention on the liver injury, as well as the synergic contribution of the two 3D tissue models on the release of metabolic enzymes after high amount of ethanol administration.

Transdermal drug delivery represents an appealing alternative to conventional drug administration systems. In fact, due to their high patient compliance, the development of dissolvable and biodegradable polymer microneedles has recently attracted great attention. Although stamp-based procedures guarantee high tip resolution and reproducibility, they have long processing times, low levels of system engineering, are a source of possible contaminants, and thermo-sensitive drugs cannot be used in conjunction with them. In this work, a novel stamp-based microneedle fabrication method is proposed. It provides a rapid room-temperature production of multi-compartmental biodegradable polymeric microneedles for controlled intradermal drug release. Solvent casting was carried out for only a few minutes and produced a short dissolvable tip made of polyvinylpyrrolidone (PVP). The rest of the stamp was then filled with degradable poly(lactide-co-glycolide) (PLGA) microparticles (μPs) quickly compacted with a vapor-assisted plasticization. The outcome was an array of microneedles with tunable release. The ability of the resulting microneedles to indent was assessed using pig cadaver skin. Controlled intradermal delivery was demonstrated by loading both the tip and the body of the microneedles with model therapeutics; POXA1b laccase from Pleurotus ostreatus is a commercial enzyme used for the whitening of skin spots. The action and indentation of the enzyme-loaded microneedle action were assessed in an in vitro skin model and this highlighted their ability to control the kinetic release of the encapsulated compound.

The intestine is a highly heterogeneous hollow organ with biological, mechanical and chemical differences between lumen and wall. A functional human intestine model able to recreate the in vivo dynamic nature as well as the native tissue morphology is demanded for disease research and ​drug discovery. Here, we present a system, which combines an engineered three-dimensional (3D) tubular-shaped intestine model (3D In-tube) with a custom-made microbioreactor to impart the key aspects of the in vivo microenvironment of the human intestine, mimicking the rhythmic peristaltic movement. We adapted a previously established bottom-up tissue engineering approach, to produce the 3D tubular-shaped lamina propria and designed a glass microbioreactor to induce the air–liquid interface ​condition and peristaltic-like motion. Our results demonstrate the production of a villi-like protrusion and a correct spatial differentiation of the intestinal epithelial cells in enterocyte-like as well as mucus-producing-like cells on the lumen side of the 3D In-tube. This dynamic platform offers a proof-of-concept model of the human intestine. Keywords: 3D engineered tubular-shaped intestine model, Air--liquid interface, Peristaltic-like motion, Extracellular matrix, Microbioreactor

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer.

The use of 3D cancer models will have both ethical and economic impact in drug screening and development, to promote the reduction of the animals employed in preclinical studies. Nevertheless, to be effective, such cancer surrogates must preserve the physiological relevance of the in vivo models in order to provide realistic information on drugs’ efficacy. To figure out the role of the architecture and composition of 3D cancer models on their tumor-mimicking capability, here we studied the efficacy of doxorubicin (DOX), a well-known anticancer molecule in two different 3D cancer models: our 3D breast cancer microtissue (3D-μTP) versus the golden standard represented by spheroid model (sph). Both models were obtained by using cancer associated fibroblast (CAF) and breast cancer cells (MCF-7) as cellular component. Unlike spheroid model, 3D-μTP was engineered in order to induce the production of endogenous extracellular matrix by CAF. 3D-μTP have been compared to spheroid in mono- (MCF-7 alone) and co-culture (MCF-7/CAF), after the treatment with DOX in order to study cytotoxicity effect, diffusional transport and expression of proteins related to cancer progression. Compared to the spheroid model, 3D-μTP showed higher diffusion coefficient of DOX and lower cell viability. Also, the expression of some tumoral biomarkers related to cell junctions were different in the two models. Statements of Significance Cancer biology has made progress in unraveling the mechanism of cancer progression, anyway the most of the results are still obtained by 2D cell cultures or animal models, that do not faithfully copycat the tumor microenvironment. The lack of correlation between preclinical models and in vivo organisms negatively influences the clinical efficacy of chemotherapeutic drugs. Consequently, even if a huge amount of new drugs has been developed in the last decades, still people are dying because of cancer. Pharmaceutical companies are interested in 3D tumor model as valid alternative in drug screening in preclinical studies. However, a 3D tumor model that completely mimics tumor heterogeneity is still far to achieve. In our work we compare 3D human breast cancer microtissues and spheroids in terms of response to doxorubicin and drug diffusion. We believe that our results are interesting because they highlight the potential role of the proposed tumor model in the attempts to improve efficacy tests.

Therapeutic approaches based on nanomedicine have garnered great attention in cancer research. In vitro biological models that better mimic in vivo conditions are crucial tools to more accurately predict their therapeutic efficacy in vivo. In this work, a new 3D breast cancer microtissue has been developed to recapitulate the complexity of the tumor microenvironment and to test its efficacy as screening platform for drug delivery systems. The proposed 3D cancer model presents human breast adenocarcinoma cells and cancer-associated fibroblasts embedded in their own ECM, thus showing several features of an in vivo tumor, such as overexpression of metallo-proteinases (MMPs). After demonstrating at molecular and protein level the MMP2 overexpression in such tumor microtissues, we used them to test a recently validated formulation of endogenous MMP2-responsive nanoparticles (NP). The presence of the MMP2-sensitive linker allows doxorubicin release from NP only upon specific enzymatic cleavage of the peptide. The same NP without the MMP-sensitive linker and healthy breast microtissues were also produced to demonstrate NP specificity and selectivity. Cell viability after NP treatment confirmed that controlled drug delivery is achieved only in 3D tumor microtissues suggesting that the validation of therapeutic strategies in such 3D tumor model could predict human response. Statement of Significance A major issue of modern cancer research is the development of accurate and predictive experimental models of human tumors consistent with tumor microenvironment and applicable as screening platforms for novel therapeutic strategies. In this work, we developed and validated a new 3D microtissue model of human breast tumor as a testing platform of anti-cancer drug delivery systems. To this aim, biodegradable nanoparticles responsive to physiological changes specifically occurring in tumor microenvironment were used. Our findings clearly demonstrate that the breast tumor microtissue well recapitulates in vivo physiological features of tumor tissue and elicits a specific response to microenvironmentally-responsive nanoparticles compared to healthy tissue. We believe this study is of particular interest for cancer research and paves the way to exploit tumor microtissues for several testing purposes.

We fabricated three-dimensional microtissues with the aim to replicate in vitro the composition and the functionalities of the tumor microenvironment. By arranging either normal fibroblasts (NF) or cancer-activated fibroblasts (CAF) in two different three dimensional (3D) configurations, two kinds of micromodules were produced: spheroids and microtissues. Spheroids were obtained by means of the traditional cell aggregation technique resulting in a 3D model characterized by high cell density and low amount of extracellular proteins. The microtissues were obtained by culturing cells into porous gelatin microscaffolds. In this latter configuration, cells assembled an intricate network of collagen, fibronectin and hyaluronic acid. We investigated the biophysical properties of both 3D models in terms of cell growth, metabolic activity, texture and composition of the extracellular matrix (via histological analysis and multiphoton imaging) and cell mechanical properties (via Particle Tracking Microrheology). In the spheroid models such biophysical properties remained unchanged regardless to the cell type used. In contrast, normal-microtissues and cancer-activated-microtissues displayed marked differences. CAF-microtissues possessed higher proliferation rate, superior contraction capability, different micro-rheological properties and an extracellular matrix richer in collagen fibronectin and hyaluronic acid. At last, multiphoton investigation revealed differences in the collagen network architecture. Taken together, these results suggested that despite to cell spheroids, microtissues better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the stromal element of the tumor tissues. Statement of Significance This work concerns the engineering of tumor tissue in vitro. Tumor models serve as biological equivalent to study pathologic progression and to screen or validate the drugs efficacy. Tumor tissue is composed by malignant cells surviving in a microenvironment, or stroma. Stroma plays a pivotal role in cancer progression. Current in vitro models, i.e. spheroids, can’t replicate the phenomena related to the tumor stroma remodeling. For this reason, to better replicate the tumor physiology in vitro that include functional and morphological changes, a novel 3D cancer model is proposed.

Despite advances in the development of in vitro tissue models such as reconstructed human skin, the questions in dermatological research, which can be addressed with these models, are limited. This is mainly due to the lack of key extra-cellular components, which makes dermal compartment an incomplete approach to human ECM in vivo. Today there is a growing awareness of the fact that the ECM has a functional importance as a dynamic repository for morphogens, cytokines and growth factors, which in vivo regulate diverse cellular processes. We established a human skin equivalent by means of a tissue engineering process that induces the full morphogenesis of functional dermal and epidermal compartments. In our model dermal ECM presents laminin, fibronectin, hyaluronic acid, elastin and collagen arranged and organized as in the human counterpart. As proof of the physiological relevance of such tissue environment we demonstrate that -by using adult human skin cells-it is possible to generate follicle-like structures in vitro resembling what occurs in vivo in the fetal skin. Immunotypization evidences an inward-oriented differentiation of the follicular-like structures through immunopositivity for epithelial stem cell markers such as p63 and K19. Moreover we succeeded in innervating this human skin by inducing rat dorsal root ganglion neurons axon ingrowth and demonstrate the effective functionality of the nerve network. Neurofilaments network infiltrates the entire native dermis ECM until reaching the epidermis, as demonstrated by immunopositivity to neurofilament-M and second harmonic generation imaging. Calcium imaging demonstrates that electrical current travel in the neuronal network proving the its sensing functionality. Our results demonstrate the fundamental role of ECM in morphogenesis, corroborating the awareness of its importance in building up functional in vitro skin model for fundamental research applications.

Many of the existing three-dimensional (3D) cancer models in vitro fail to represent the entire complex tumor microenvironment composed of cells and extra cellular matrix (ECM) and do not allow a reliable study of the tumoral features and progression. In this paper we reported a strategy to produce 3D in vitro microtissues of pancreatic ductal adenocarcinoma (PDAC) for studying the desmoplastic reaction activated by the stroma–cancer crosstalk. Human PDAC microtissues were obtained by co-culturing pancreatic cancer cells (PT45) and normal or cancer-associated fibroblasts within biodegradable microcarriers in a spinner flask bioreactor. Morphological and histological analyses highlighted that the presence of fibroblasts resulted in the deposition of a stromal matrix rich in collagen leading to the formation of tumor microtissues composed of a heterotypic cell population embedded in their own ECM. We analyzed the modulation of expression of ECM genes and proteins and found that when fibroblasts were co-cultured with PT45, they acquired a myofibroblast phenotype and expressed the desmoplastic reaction markers. This PDAC microtissue, closely recapitulating key PDAC microenvironment characteristics, provides a valuable tool to elucidate the complex stroma–cancer interrelationship and could be used in a future perspective as a testing platform for anticancer drugs in tissue-on-chip technology. Statement of Significance Tumor microenvironment is extremely complex and its organization is due to the interaction between different kind of cells and the extracellular matrix. Tissue engineering could give the answer to the increasing need of 3D culture model that better recapitulate the tumor features at cellular and extracellular level. We aimed in this work at developing a microtissue tumor model by mean of seeding together cancer cells and fibroblasts on gelatin microsphere in order to monitor the crosstalk between the two cell populations and the endogenous extracellular matrix deposition. Results are of particular interest because of the need of heterotypic cancer model that can replicate the complexity of the tumor microenvironment and could be used as drug screening platform.

Decellularized matrices are steadily gaining popularity to study the biology of cells and tissues, as they represent a biomimetic environment in which cells can recapitulate certain behaviours that share similarities with those observed in vivo. Basically, biochemistry, microstructure and mechanics of the decellularized matrices are the most valuable properties that differentiate these culturing systems from conventional bidimensional models. Several procedures to decellularize tissues have been proposed so far, with the common aim to preserve the tissue chemical/physical properties of the original tissue. However, these processes are complex, time-consuming and expensive. In this work, we propose a cost-effective, easy-to-produce decellularized dermal matrix, derived from animal skin. The chemical/physical processes to obtain the matrices proved to not alter matrix structure and did not induce cytotoxicity issues. To test the validity of the decellularized matrices as a model to study the behaviour of tumour cells in vitro, we performed microstructural and mechanical investigations as well as cell proliferation assays. In particular, three different tumour cell lines were used, which proliferated and invaded the matrix with no additional treatments. Decellularized skin scaffold, presented in this work, could be a strong competitor for conventional 3D systems like synthetic porous scaffolds or hydrogels. Copyright © 2016 John Wiley & Sons, Ltd.

The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.

Tissue-on-chip (TOC) systems aim at replicating complex biological dynamics in vitro with the potential either to improve the understanding of human biology or to develop more accurate therapeutic strategies. To replicate faithfully the intricate interrelationships between cells and their surrounding microenvironment, the three-dimensional (3D) tissue model must possess a responsive extracellular matrix (ECM). ECM remodeling plays a pivotal role in guiding cells and tissues functions and such aspect is somewhat denied during in vitro studies. For this purpose, we fabricated a micro-perfusion bioreactor capable to sustain the viability of 3D engineered tissue models recapitulating the process of the native ECM deposition and assembly. Engineered human dermis micro-tissue precursors (HD-mu TP) were used as building blocks to generate a final tissue. HD-mu TP were loaded in the perfusion space of the micro-perfusion bioreactor and, under the superimposition of different fluid dynamic regimes and biochemical stimulation, they synthesized new collagen proteins that were, then, assembled in the perfusion space forming a continuum of cells embedded in their own ECM. The micro-perfusion bioreactor was fabricated to allow the on-line monitoring of the oxygen consumption and the assembly of the newly formed collagen network via real time acquisition of the second harmonic generation (SHG) signal. The possibility to detect the collagen reorganization due to both fluid dynamic and biochemical stimulation, let us to define the optimal perfusion configuration in order to obtain a TOC system based on an endogenous and responsive ECM.

The in vitro fabrication of an endogenous cardiac muscle would have a high impact for both in vitro studies concerning cardiac tissue physiology and pathology, as well as in vivo application to potentially repair infarcted myocardium. To reach this aim, we engineered a new class of cardiac tissue precursor (CTP), specifically conceived in order to promote the synthesis and the assembly of a cardiac extracellular matrix (ECM). The CTPs were obtained by culturing a mixed cardiac cell population, composed of myocyte and non-myocyte cells, into porous gelatin microspheres in a dynamic bioreactor. By engineering the culture conditions, the CTP developed both beating properties and an endogenous immature cardiac ECM. By following a bottom-up approach, a macrotissue was fabricated by molding and packing the engineered tissue precursor in a maturation chamber. During the macrotissue formation, the tissue precursors acted as cardiac tissue depots by promoting the formation of an endogenous and interconnected cardiac network embedding the cells and the microbeads. The myocytes cell fraction pulled on ECM network and induced its compaction against the internal posts represented by the initial porous microbeads. This reciprocal interplay induced ECM consolidation without the use of external biophysical stimuli by leading to the formation of a beating and endogenous macrotissue. We have thus engineered a new class of cardiac micromodules and show its potential for the fabrication of endogenous cardiac tissue models useful for in vitro studies that involve the cardiac tissue remodeling.

In this work, a new model of breast cancer is proposed featuring both epithelial and stromal tissues arranged on a microfluidic chip. The main task of the work is the in vitro replication of the stromal activation during tumor epithelial invasion. The activation of tumor stroma and its morphological/compositional changes play a key role in tumor progression. Despite emerging evidences, to date the activation of tumor stroma in vitro has not been achieved yet. The tumor-on-chip proposed in this work is built in order to replicate the features of its native counterpart: multicellularity (tumor epithelial cell and stromal cell); 3D engineered stroma compartment composed of cell-assembled extracellular matrix (ECM); reliable 3D tumor architecture. During tumor epithelial invasion the stroma displayed an activation process at both cellular and ECM level. Similarly of what repeated in vivo, ECM remodeling is found in terms of hyaluronic acid and fibronectin overexpression in the stroma compartment. Furthermore, the cell-assembled ECM featuring the stromal tissue, allowed on-line monitoring of collagen remodeling during stroma activation process via real time multiphoton microscopy. Also, trafficking of macromolecules within the stromal compartment has been monitored in real time.

Three-dimensional (3D) cell cultures have recently garnered great attention because they promote levels of cells differentiation and tissue organisation not possible in conventional two-dimensional (2D) culture systems. Cancer development is a complex process regulated by interactions between epithelial cells, activated stromal cells, and soluble and insoluble components of the extracellular matrix (ECM). As a consequence, in the field of cancer biology a 3D tumour model that accurately recreates the in vivo tumour phenotype would be a valuable tool for studying tumour biology and would allow better pre-clinical evaluation of anticancer drug candidates. Here, we review the 3D tumour models currently available and the more advanced techniques from the tissue-engineering field used to create a more clinically accurate ex vivo tumour model. Moreover, we highlight the drastic differences in drug responses between 3D and 2D models and give a glance to the emerging multi-organ microdevices that can mimic in vivo tissue-tissue interactions.