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.

Spatial patterning of PCLµ-scaffolds directs 3D vascularized bio-constructs morphogenesisin vitro.

Modular tissue engineering (mTE) strategies aim to build three-dimensional tissue analoguesin vitroby the sapient combination of cells, micro-scaffolds (µ-scaffs) and bioreactors. The translation of these newly engineered tissues into current clinical approaches is, among other things, dependent on implant-to-host microvasculature integration, a critical issue for cells and tissue survivalin vivo. In this work we reported, for the first time, a computer-aided modular approach suitable to build fully vascularized hybrid (biological/synthetic) constructs (bio-constructs) with micro-metric size scale control of blood vessels growth and orientation. The approach consists of four main steps, starting with the fabrication of polycaprolactoneµ-scaffs by fluidic emulsion technique, which exhibit biomimetic porosity features. In the second step, layers ofµ-scaffs following two different patterns, namely ordered and disordered, were obtained by a soft lithography-based process. Then, the as obtainedµ-scaff patterns were used as template for human dermal fibroblasts and human umbilical vein endothelial cells co-culture, aiming to promote and guide the biosynthesis of collagenous extracellular matrix and the growth of new blood vessels within the mono-layered bio-constructs. Finally, bi-layered bio-constructs were built by the alignment, stacking and fusion of two vascularized mono-layered samples featuring ordered patterns. Our results demonstrated that, if compared to the disordered pattern, the ordered one provided better control over bio-constructs shape and vasculature architecture, while minor effect was observed with respect to cell colonization and new tissue growth. Furthermore, by assembling two mono-layered bio-constructs it was possible to build 1 mm thick fully vascularized viable bio-constructs and to study tissue morphogenesis during 1 week ofin vitroculture. In conclusion, our results highlighted the synergic role ofµ-scaff architectural features and spatial patterning on cells colonization and biosynthesis, and pave the way for the possibility to create in silico designed vasculatures within modularly engineered bio-constructs.

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.

Bioinspired Design of Novel Microscaffolds for Fibroblast Guidance toward In Vitro Tissue Building.

Porous microscaffolds (µ-scaffs) play a crucial role in modular tissue engineering as they control cell functions and guide hierarchical tissue formation toward building new functional tissue analogues. In the present study, we developed a new route to prepare porous polycaprolactone (PCL) µ-scaffs with a bioinspired trabecular structure that supported in vitro adhesion, growth, and biosynthesis of human dermal fibroblasts (HDFs). The method involved the use of poly(ethylene oxide) (PEO) as a biocompatible porogen and a fluidic emulsion/porogen leaching/particle coagulation process to obtain spherical µ-scaffs with controllable diameter and full pore interconnectivity. To achieve this objective, we investigated the effect of PEO concentration and the temperature of the coagulation bath on the µ-scaff architecture, while we modulated the µ-scaff diameter distribution by varying the PCL-PEO amount in the starting solution and changing the flow rate of the continuous phase (QCP). µ-Scaff morphology, pore architecture, and diameter distribution were assessed using scanning electron microscopy (SEM) analysis, microcomputed tomography (microCT), and Image analysis. We reported that the selection of 60 wt % PEO concentration, together with a 4 °C coagulation bath temperature and ultrasound postprocessing, allowed for the design and fabrication of µ-scaff with porosity up to 80% and fully interconnected pores on both the µ-scaff surface and the core. Furthermore, µ-scaff diameter distributions were finely tuned in the 100-600 µm range with the coefficient of variation lower than 5% by selecting the PCL-PEO concentration in the 1-10% w/v range and QCP of either 8 or 18 mL/min. Finally, we investigated the capability of the HDF-seeded PCL µ-scaff to form hybrid (biological/synthetic) tissue in vitro. Cell culture tests demonstrated that PCL µ-scaff enabled HDF adhesion, proliferation, colonization, and collagen biosynthesis within inter- and intraparticle spaces and guided the formation of a large (centimeter-sized) viable tissue construct.

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.

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.

Recapitulating spatiotemporal tumor heterogeneity in vitro through engineered breast cancer microtissues.

Tumor and microenvironmental heterogeneity hinders the study of breast cancer biology and the assessment of therapeutic strategies, being associated with high variability and drug resistance. In this context, it is mandatory to develop three-dimensional breast tumor models able to reproduce this heterogeneity and the dynamic interaction occurring between tumor cells and microenvironment. Here we show a new breast cancer microtissue model (T-µTP) uniquely able to present intra-tumor morphological heterogeneity in a dynamic and responsive endogenous matrix. T-µTP consists of adenocarcinoma cells, endothelial cells and stromal fibroblasts. These three kinds of cells are totally embedded into an endogenous matrix which is rich in collagen and hyaluronic acid and it is directly produced by human fibroblasts. In this highly physiologically relevant environment, tumor cells evolve in different cluster morphologies recapitulating tumor spatiotemporal heterogeneity. Moreover they activate the desmoplastic and vascular reaction with affected collagen content, assembly and organization and the presence of aberrant capillary-like structures (CLS). Thus, T-µTP allows to outline main crucial events involved in breast cancer progression into a single model overcoming the limit of artificial extra cellular matrix surrogates. We strongly believe that T-µTP is a suitable model for the study of breast cancer and for drug screening assays following key parameters of clinical interest. STATEMENT OF SIGNIFICANCE: Tumor and microenvironmental heterogeneity makes very hurdle to find a way to study and treat breast cancer. Here we develop an innovative 3D tumor microtissue model recapitulating in vitro tumor heterogeneity. Tumor microtissues are characterized by the activation of the stromal and vascular reaction too. We underline the importance to mimic different microenvironmental tumor features in the same time and in a single tissue in order to obtain a model of spatiotemporal tumor genesis and progression, suitable for the study of tumor treatment and resistance.

Identification of a novel gene signature of ES cells self-renewal fluctuation through system-wide analysis.

Embryonic Stem cells (ESCs) can be differentiated into ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. In regular culture conditions, ESCs' self-renewal is maintained through molecules that inhibit spontaneous differentiation enabling long-term cellular expansion. This undifferentiating condition is characterized by multiple metastable states that fluctuate between self-renewal and differentiation balance. Here, we aim to characterize the high-pluripotent ESC metastate marked by the expression of Zscan4 through a supervised machine learning framework based on an ensemble of support vector machine (SVM) classifiers. Our study revealed a leukaemia inhibitor factor (Lif) dependent not-canonical pluripotency signature (AF067063, BC061212, Dub1, Eif1a, Gm12794, Gm13871, Gm4340, Gm4850, Tcstv1/3, and Zfp352), that specifically marks Zscan4 ESCs' fluctuation. This novel ESC metastate is enhanced by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK) and Glycogen synthase kinase-3 (Gsk-3) signaling inhibition (2i). Significantly, we reported that the conditional ablation of the novel ESC metastate marked by the expression of Gm12794 is required for ESCs self-renewal maintenance. In conclusion, we extend the comprehension of ESCs biology through the identification of a novel molecular signature associated to pluripotency programming.