Engineering Cell Instructive Microenvironments for In Vitro Replication of Functional Barrier Organs.

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression.

Bioprinting of human dermal microtissues precursors as building blocks for endogenousin vitroconnective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricatein vitrotissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (µTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly ofµTPs into predefined shapes. Compared to cell-aggregated bioink, ourµTPs represent a functional unit where cells are embedded in their specific ECM.µTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days,µTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter.µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel atT> 30 °C), ensuringµTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, untilµTPs fused into a continuous connective tissue.µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

A functional 3D full-thickness model for comprehending the interaction between airway epithelium and connective tissue in cystic fibrosis.

Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodeling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodeling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to fully replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodeling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed gland-like structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy.

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Easy-to-Build and Reusable Microfluidic Device for the Dynamic Culture of Human Bronchial Cystic Fibrosis Epithelia.

Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air-liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.

Capturing the spatial and temporal dynamics of tumor stroma for on-chip optimization of microenvironmental targeting nanomedicine.

Malignant cells grow in a complex microenvironment that plays a key role in cancer progression. The "dynamic reciprocity" existing between cancer cells and their microenvironment is involved in cancer differentiation, proliferation, invasion, metastasis, and drug response. Therefore, understanding the molecular mechanisms underlying the crosstalk between cancer cells and their surrounding tissue (i.e., tumor stroma) and how this interplay affects the disease progression is fundamental to design and validate novel nanotherapeutic approaches. As an important regulator of tumor progression, metastasis and therapy resistance, the extracellular matrix of tumors, the acellular component of the tumor microenvironment, has been identified as very promising target of anticancer treatment, revolutionizing the traditional therapeutic paradigm that sees the neoplastic cells as the preferential objective to fight cancer. To design and to validate such a target therapy, advanced 3D preclinical models are necessary to correctly mimic the complex, dynamic and heterogeneous tumor microenvironment. In addition, the recent advancement in microfluidic technology allows fine-tuning and controlling microenvironmental parameters in tissue-on-chip devices in order to emulate the in vivo conditions. In this review, after a brief description of the origin of tumor microenvironment heterogeneity, some examples of nanomedicine approaches targeting the tumor microenvironment have been reported. Further, how advanced 3D bioengineered tumor models coupled with a microfluidic device can improve the design and testing of anti-cancer nanomedicine targeting the tumor microenvironment has been discussed. We highlight that the presence of a dynamic extracellular matrix, able to capture the spatiotemporal heterogeneity of tumor stroma, is an indispensable requisite for tumor-on-chip model and nanomedicine testing.

Bioengineered Wound Healing Skin Models: The Role of Immune Response and Endogenous ECM to Fully Replicate the Dynamic of Scar Tissue Formation In Vitro.

The healing of deep skin wounds is a complex phenomenon evolving according with a fine spatiotemporal regulation of different biological events (hemostasis, inflammation, proliferation, remodeling). Due to the spontaneous evolution of damaged human dermis toward a fibrotic scar, the treatment of deep wounds still represents a clinical concern. Bioengineered full-thickness skin models may play a crucial role in this direction by providing a deep understanding of the process that leads to the formation of fibrotic scars. This will allow (i) to identify new drugs and targets/biomarkers, (ii) to test new therapeutic approaches, and (iii) to develop more accurate in silico models, with the final aim to guide the closure process toward a scar-free closure and, in a more general sense, (iv) to understand the mechanisms involved in the intrinsic and extrinsic aging of the skin. In this work, the complex dynamic of events underlaying the closure of deep skin wound is presented and the engineered models that aim at replicating such complex phenomenon are reviewed. Despite the complexity of the cellular and extracellular events occurring during the skin wound healing the gold standard assay used to replicate such a process is still represented by planar in vitro models that have been largely used to identify the key factors regulating the involved cellular processes. However, the lack of the main constituents of the extracellular matrix (ECM) makes these over-simplistic 2D models unable to predict the complexity of the closure process. Three-dimensional bioengineered models, which aim at recreating the closure dynamics of the human dermis by using exogenous biomaterials, have been developed to fill such a gap. Although interesting mechanistic effects have been figured out, the effect of the inflammatory response on the ECM remodelling is not replicated yet. We discuss how more faithful wound healing models can be obtained by creating immunocompetent 3D dermis models featuring an endogenous ECM.

Immunoresponsive microbiota-gut-on-chip reproduces barrier dysfunction, stromal reshaping and probiotics translocation under inflammation.

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).

Organ on Chip Technology to Model Cancer Growth and Metastasis.

Organ on chip (OOC) has emerged as a major technological breakthrough and distinct model system revolutionizing biomedical research and drug discovery by recapitulating the crucial structural and functional complexity of human organs in vitro. OOC are rapidly emerging as powerful tools for oncology research. Indeed, Cancer on chip (COC) can ideally reproduce certain key aspects of the tumor microenvironment (TME), such as biochemical gradients and niche factors, dynamic cell-cell and cell-matrix interactions, and complex tissue structures composed of tumor and stromal cells. Here, we review the state of the art in COC models with a focus on the microphysiological systems that host multicellular 3D tissue engineering models and can help elucidate the complex biology of TME and cancer growth and progression. Finally, some examples of microengineered tumor models integrated with multi-organ microdevices to study disease progression in different tissues will be presented.

Geometrical confinement controls cell, ECM and vascular network alignment during the morphogenesis of 3D bioengineered human connective tissues.

Engineered tissues featuring aligned ECM possess superior regenerative capabilities for the healing of damaged aligned tissues. The morphofunctional integration in the host's injury site improves if the aligned ECM elicits the unidirectional growth of vascular network. In this work we used a bottom-up tissue engineering strategy to produce endogenous and highly aligned human connective tissues with the final aim to trigger the unidirectional growth of capillary-like structures. Engineered microtissues, previously developed by our group, were casted in molds featured by different aspect ratio (AR) to obtain final centimeter-sized macrotissues differently shaped. By varying the AR from 1 to 50 we were able to vary the final shape of the macrotissues, from square to wire. We demonstrated that by increasing the AR of the maturation space hosting the microtissues, it was possible to control the alignment of the neo-synthesized ECM. The geometrical confinement conditions at AR = 50, indeed, promoted the unidirectional growth and assembly of the collagen network. The wire-shaped tissues were characterized by parallel arrangement of the collagen fiber bundles, higher persistence length and speed of migrating cells and superior mechanical properties than the square-shaped macrotissues. Interestingly, the aligned collagen fibers elicited the unidirectional growth of capillary-like structures. STATEMENT OF SIGNIFICANCE: Alignment of preexisting extracellular matrices by using mechanical cues modulating cell traction, has been widely described. Here, we show a new method to align de novo synthesized extracellular matrix components in bioengineered connective tissues obtained by means of a bottom-up tissue engineering approach. Building blocks are cast in maturation chambers, having different aspect ratios, in which the in vitro morphogenesis process takes place. High aspect ratio chambers (corresponding to wire-shaped tissues) triggered spontaneous alignment of collagenous network affecting cell polarization, migration and tensile properties of the tissue as well. Aligned ECM provided a contact guidance for the formation of highly polarized capillary-like network suggesting an in vivo possible application to trigger fast angiogenesis and perfusion in damaged aligned tissues.

Modeling the epithelial-mesenchymal transition process in a 3D organotypic cervical neoplasia.

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.

Intrinsic Abnormalities of Cystic Fibrosis Airway Connective Tissue Revealed by an In Vitro 3D Stromal Model.

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.

In Vitro Organotypic Systems to Model Tumor Microenvironment in Human Papillomavirus (HPV)-Related Cancers.

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.

Intestine-Liver Axis On-Chip Reveals the Intestinal Protective Role on Hepatic Damage by Emulating Ethanol First-Pass Metabolism.

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.

Intestine-on-chip device increases ECM remodeling inducing faster epithelial cell differentiation.

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.

Bioengineered Skin Substitutes: the Role of Extracellular Matrix and Vascularization in the Healing of Deep Wounds.

The formation of severe scars still represents the result of the closure process of extended and deep skin wounds. To address this issue, different bioengineered skin substitutes have been developed but a general consensus regarding their effectiveness has not been achieved yet. It will be shown that bioengineered skin substitutes, although representing a valid alternative to autografting, induce skin cells in repairing the wound rather than guiding a regeneration process. Repaired skin differs from regenerated skin, showing high contracture, loss of sensitivity, impaired pigmentation and absence of cutaneous adnexa (i.e., hair follicles and sweat glands). This leads to significant mobility and aesthetic concerns, making the development of more effective bioengineered skin models a current need. The objective of this review is to determine the limitations of either commercially available or investigational bioengineered skin substitutes and how advanced skin tissue engineering strategies can be improved in order to completely restore skin functions after severe wounds.

Non-invasive Production of Multi-Compartmental Biodegradable Polymer Microneedles for Controlled Intradermal Drug Release of Labile Molecules.

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.

Pre-vascularized dermis model for fast and functional anastomosis with host vasculature.

Skin engineering for clinical applications has gained numerous advances, however, most of the available dermis substitutes are exogenous matrices acting for a limited time. Indeed, after implantation these matrices need to be colonized by host cells such as fibroblast and endothelial cells which respectively produce their own extracellular matrix and set a vascular network within the construct. These steps are essential to guarantee implant efficacy, but they may require a long time depending on tissue dimension and lesion severity. Here we show the pre-vascularization process of a dermis equivalent featured by an endogenous matrix produced by human dermal fibroblasts. In this environment, endothelial cells were able to develop mature capillary-like-structures (CLS) as demonstrated by both the inner lumen and the positivity for alpha-SMA, laminin and collagen. The pre-vascularized dermis model (PVD) so obtained had a human matrix populated by fibroblasts as well as a complex capillary network making the construct ready to be implanted. These features make the graft very easy to handle during the surgery. In vivo results showed that 7 days after implantation CLS effectively anastomosed with host vessels. Therefore we argue that the proposed PVD may represent a new class of dermis substitute of strong clinical interest.

A three-dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity.

In this study, we propose the design and fabrication of a liver system on a chip. We first chose the most suitable three-dimensional liver-like model between cell spheroids and microtissue precursors, both based on the use of hepatocellular carcinoma cells (HepG2) to provide proof-of-concept data. Spheroids displayed high cell density but low expression of the typical hepatic biomarkers, whereas microtissue precursors showed stable viability and function over the entire culture time. The two liver-like models were compared in terms of cell viability, function, metabolism, and the P-glycoprotein 1 (P-gp) transport-protein expression with the microtissue precursors showing the best performance. Thus, we cultured them into a microfluidic biochip featured with three parallel channels shaped to mimic the hepatic sinusoids. To assess the detoxification potential of the microtissue-loaded biochip we challenged it with a model molecule (ethanol) at different concentrations and time points. Ethanol cytotoxicity was detected by a noninvasive measurement of cell viability based on cell autofluorescence. As expected, a dose-dependent decrease of albumin and urea secretion was observed in the ethanol-treated samples. We believe that the described totally human-derived platform, suitable for integration into a multiorgan microfluidic system, can provide a consistent innovative platform for drug development and toxicity studies.

Micro-patterned endogenous stroma equivalent induces polarized crypt-villus architecture of human small intestinal epithelium.

The small intestine is the major site for digestion, drug and nutrient absorption, as well as a primary site for many diseases. Current in vitro gut models fail in reproducing the complex intestinal extracellular matrix (ECM) network of the lamina propria and the peculiar architecture of the crypt-villus axis. Here we proposed a novel in vitro human intestine model that mimics the intestinal stromal topography and composition and strictly reproduces the tissue polarity with the crypt-villus architecture. First we developed a 3D human intestinal stromal equivalent (3D-ISE) composed of human intestinal subepithelial myofibroblasts (ISEMFs) embedded in their own extracellular matrix. Then, we seeded human colon carcinoma-derived cells (Caco-2) onto flat or patterned cell-synthetized stromal equivalent structure and cultured them until the formation of a well-oriented epithelium. We demonstrated that the patterned stroma increases the absorbing surface area, the epithelial proliferation rate, and the density of microvilli. In addition it induces changes in the biological functions of the epithelial cells such as enzymes and mucus production, polarization and tightness showing a physiological cell-lineage compartmentalization along the crypt/villi axes with the undifferentiated phenotypes at the base. At last, we reproduced an inflamed intestinal tissue model in which we identified the contribution of the stromal microenvironment by molecular (cytokines release and MMPs production) and immunofluorescence analyses and the effects of the epithelial-stromal cross-talk in the intestinal innate immunity by multiphoton investigation that revealed differences in the collagen network architecture. STATEMENT OF SIGNIFICANCE: The intestinal stroma morphology and composition has a fundamental role in crypt-villus development and appropriate epithelial cell-lineage compartmentalization. On this base, here we develop an engineered organotypic model of human intestine equivalent in which a functional epithelial/ECM crosstalk is recapitulated. Due to its accessible luminal surface it provides a new platform for preclinical studies of mucosal immunology and bowel inflammation as well as the assessment of pharmaco-toxicity studies.