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Tissue architecture is a driving force for morphogenetic processes during development as well as for several physiological and regenerative responses. Far from being a passive static environment, tissue architecture is highly dynamic. Hydrogel technology reproduces in vitro geometrical and mechanical constrains that control the three-dimensional self-organization of (3D) organoids and organ-like cultures. This control is restricted to the initial culture conditions and cannot be adapted to the dynamic morphological changes of complex 3D cultures during their developmental trajectory. Here, we developed a method that overcomes this spatiotemporal limit. Using 2P crosslinking approach, high resolution 3D hydrogel structures can be fabricated within pre-existing hydrogel with spatiotemporal (four-dimensional, 4D) control relative to ex-vivo organotypic or organoid culture. This hydrogel-in-hydrogel bioprinting approach enables to continuously instruct the self-organization of the evolving 3D organ-like cultures.

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.

The ability to store effectively excess of electrical energy from peaks of production is key to the development of renewable energies. Power-To-Gas, and specifically Power-To-Methane represents one of the most promising option. This works presents an innovative process layout that integrates Chemical Looping Combustion of solid fuels and a Power-to-Methane system. The core of the proposed layout is a multiple interconnected fluidized bed system (MFB) equipped with a two-stage fuel reactor (t-FR). Performances of the system were evaluated by considering a coal as fuel and CuO supported on zirconia as oxygen carrier. A kinetic scheme comprising both heterogeneous and homogeneous reactions occurring in the MFB was considered. The methanation unit was modelled developing a thermodynamic calculation method based on minimization of the free Gibbs energy. The performance of the system was evaluated by considering that the CO/CO2 stream coming from the t-FR reacts over Ni supported on alumina catalyst with a pure H2 stream generated by an array of electrolysis cells. The number of cells to be stacked in the array was evaluated by considering that a constant H2 production able to convert the whole CO/CO2 stream produced by the CLC process should be attained. The environmental performance of the proposed process was quantified using the Life Cycle Assessment (LCA) methodology. The analysis shows i) that the majority originate from the production and disposal of the oxygen carrier used in the t-FR, and ii) that reusing part of the oxygen produced by the electrolysis cells improves significantly the environmental performance of the proposed process.

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.

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three‐dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models, including nervous system and neuromuscular junctional homing. Since the nervous system plays pivotal roles during skeletal muscle regeneration and in tissue homeostasis, support of reinnervation is a crucial aspect to be considered. However, the effect of decellularized muscles on reinnervation and on neuronal axon growth has been poorly investigated. Here, we characterized residual protein composition of decellularized muscles by mass spectrometry and we show that scaffolds preserve structural proteins of the ECM of both skeletal muscle and peripheral nervous system. To investigate whether decellularized scaffolds could per se attract neural axons, organotypic sections of spinal cord were cultured three dimensionally in vitro, in presence or in absence of decellularized muscles. We found that neural axons extended from the spinal cord are attracted by the decellularized muscles and penetrate inside the scaffolds upon 3D coculture. These results demonstrate that decellularized scaffolds possess intrinsic neurotrophic properties, supporting their potential use for the treatment of clinical cases where extensive functional regeneration of the muscle is required., Decellularized muscles show direct neurotrophic properties. Here, we developed a three‐dimensional in vitro model in which organotypic sections of fetal spinal cord were cultured in presence or in absence of decellularized muscles. Decellularized muscles, which proteomic composition includes both myofibers and peripheral nerve components, sustained axon sprouting and attraction of neurons.

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.

Skeletal muscle is a dynamic tissue in which homeostasis and function are guaranteed by a very defined three-dimensional organization of myofibers in respect to other non-muscular components, including the extracellular matrix and the nervous network. In particular, communication between myofibers and the nervous system is essential for the overall correct development and function of the skeletal muscle. A wide range of chronic, acute and genetic-based human pathologies that lead to the alteration of muscle function are associated with modified preservation of the fine interaction between motor neurons and myofibers at the neuromuscular junction. Recent advancements in the development of in vitro models for human skeletal muscle have shown that three-dimensionality and integration of multiple cell types are both key parameters required to unveil pathophysiological relevant phenotypes. Here, we describe recent achievement reached in skeletal muscle modeling which used biomaterials for the generation of three-dimensional constructs of myotubes integrated with motor neurons.

Background and aim COVID-19 (COronaVIrus Disease 19) is an acute respiratory disease caused by SARS CoV 2 virus. The correlation between SARS-CoV2 infection and comorbidities is complex; patients with multiple comorbidities present often with the most severe symptoms that could potentially lead to death. Patients undergoing hemodialysis are generally frail and immunodeficient. This leads to a greater risk of contracting infectious diseases. In the literature, the estimated incidence of SARS-CoV2 infection is 3.24% in chronic hemodialysis patients. Method Fondazione Policlinico A. Gemelli is a COVID hospital. During the pandemic patients from several dialysis centers converged in our hospital. FPG has two dialysis centers, one for outpatients and one for inpatients. Patients admitted for COVID-19 infection have been treated in three different settings: 1. isolation room within the dialysis center; 2. Bedside; 3. In a COVID-19 dialysis center. We retrospectively collected data of patients treated from March 2020 to January 2021 and analyzed the SARS-CoV2 incidence in our center’s chronic hemodialysis patients. Results 66 hemodialysis patients affected by COVID-19 have been treated in our hospital from March 2020 to January 2021, 60 patients undergoing chronic dialysis and 6 patients diagnosed with acute kidney injury (AKI) stage III non-intensive care unit. Among chronic patients, 64 underwent chronic hemodialysis and 2 patients underwent peritoneal dialysis. Median age was 68.19 (46 males, 20 females), all patients had multiple comorbidities: 37.8% of patients had diabetes mellitus; 72.7% cardiovascular diseases and 16.6% a positive clinical history for cancer. Among the 6 AKI cases, 3 patients regained total kidney function; the other 3 had to continue renal replacement therapy. The mean hospital stay length was 18.5 days with a mean time of COVID-19 infection of 21.23 days. The overall mean Charlson Comorbidty Index was 6.21. Among the 66 treated patients, 43 were diagnosed with COVID-19-related pneumonia, 14 had the infection, no pulmonary involvement, but presented with other complications, and 5 patients resulted positive although asymptomatic. Among the 116 hemodialysis outpatients, only 4 presented with SARS-CoV2 infection, 3 were contacts of a positive family member and 1 resulted positive during a hospital stay for Clostridium Difficile infection. All patients required hospitalization. 14 (21%) patients died. Among the deceased patients, the mean age was 76.90 years (9 males, 3 females), mean Charlson Comorbidity Index was 7.3, mean hospital stay length was 9 days. Among patients who survived the disease the mean age was 76.92 years (34 males, 14 females), mean Charlson Comorbidty Index was 5.87 and mean hospital stay length was 19.47 days. Statistical significance was reached for age (p value 0.005) and Charlson Comorbidty Index (p value 0.39), but not for mean hospital stay length (p value 0.13). All COVID-19 patients were treated with bicarbonate hemodialysis and a Theranova 400 Baxter® filter. This filter was chosen for its efficiency on medium-size molecules removal (between 25 kDa and 60 kDa) that may be associated with inflammation. Bedside treatments were performed using the Genius© Fresenius system. Each treatment lasted 180 minutes, in order to reduce the time of exposure to COVID-19 of medical staff and the risk of virus spread on one hand, but still ensuring an optimal and complication-free treatment to patients. Conclusion Our experience seems to confirm the national data collected so far, both in terms of patients’ outcomes and mortality rate. Our study confirms that age is a risk factor for mortality. How to properly manage chronic hemodialysis patients affected by COVID-19 remains a challenging and burdensome question. However, there is the need of new flexible solutions that guarantee the patients and the medical staff’s safety on one hand and a personalized management on the other.

In questa relazione, sono riportati i risultati ottenuti da prove sperimentali finalizzate alla determinazione del potere calorifico superiore e inferiore di campioni così come forniti dall'azienda Icimendue srl. Le misure del potere calorifico superiore sono state condotte utilizzando la cosiddetta "bomba di Mahler". Il potere calorifico inferiore è stato calcolato scorporando dal potere calorifico superiore il calore di vaporizzazione dell'acqua prodotta durante la combustione. Quest'ultima è stata quantificata attraverso analisi CHN dei campioni di partenza, ipotizzando in essi una presenza trascurabile di acqua - ipotesi questa che è stata verificata utilizzando la titolazione Karl-Fischer.

Fatigue is still present in up to 40–50% of kidney transplant recipients (KTR), the results of studies comparing the prevalence among patients on hemodialysis (HD) and KTR led to conflicting results. Fatigue correlates include inflammation, symptoms of depression, sleep disorders and obesity. Fatigue in KTR leads to significant functional impairment, it is common among KTR poorly adherent to immunosuppressive therapy and is associated with a serious deterioration of quality of life. The following databases were searched for relevant studies up to November 2020: Medline, PubMed, Web of Science and the Cochrane Library. Several studies have compared the prevalence and severity of fatigue between KTR and hemodialysis or healthy patients. They have shown that fatigue determines a significant functional deterioration with less chance of having a paid job and a significant change in quality of life. The aim of the review is to report methods to assess fatigue and its prevalence in KTR patients, compared to HD subjects and define the effects of fatigue on health status and daily life. There is no evidence of studies on the treatment of this symptom in KTR. Efforts to identify and treat fatigue should be a priority to improve the quality of life of KTR.

Italy is the main supplier of leather in Europe, contributing 65% to European production of bovine leather and 15% to the world production. The tanning industry produces wastewater rich in pollutants, especially Cr, which is the chemical species with the strongest environmental impact. The purification process of this wastewater, produces a tanning sludge (TS) with high concentration of Cr(III), generally stored in authorized landfills with high costs. Due the high production of TS, it is necessary to implement sustainable management strategies that aim both to reduce the volume of TS to be disposed of and to produce energy vectors. Therefore, Sludge-to-energy ("StE") technologies are adopted. However, due to the high content of Cr(III) in TS, special attention must be paid to limit/avoid the oxidation of Cr(III) to Cr(VI), extremely harmful. In this context, gasification is particularly suitable for the treatment of TS because, operating in oxygen-deficient conditions, it reduces the risk of oxidation of Cr. In this work, were carried out gasification tests, in a fluidized bed reactor (ID=0.041m and H=1m), of a dried Italian TS, supplied by SSIP. Tests were conducted at 850°C, at a velocity of 0.3 m/s. The volumetric flow rate of fed gas was 350 NL/h, consisting of 85.7% N2 (300 NL/h) and 14.3% air (50 NL/h). The TS (ID

Decellularized skeletal muscle (dSkM) constructs have received much attention in recent years due to the versatility of their applications in vitro. In search of adequate in vitro models of the skeletal muscle tissue, the dSkM offers great advantages in terms of the preservation of native-tissue complexity, including three-dimensional organization, the presence of residual signaling molecules within the construct, and their myogenic and neurotrophic abilities. Here, we attempted to develop a 3D model of neuromuscular tissue. To do so, we repopulated rat dSkM with human primary myogenic cells along with murine fibroblasts and we coupled them with organotypic rat spinal cord samples. Such culture conditions not only maintained multiple cell type viability in a long-term experimental setup, but also resulted in functionally active construct capable of contraction. In addition, we have developed a customized culture system which enabled easy access, imaging, and analysis of in vitro engineered co-cultures. This work demonstrates the ability of dSkM to support the development of a contractile 3D in vitro model of neuromuscular tissue fit for long-term experimental evaluations.

Current multistage CCU technologies using renewable electricity to yield fuels suffer from low energy efficiency and require large capital expenditures. To increase the efficiency of the whole process and reduce its cost, a possible innovative solution is represented by the combined capture and hydrogenation of CO2. The idea is to trap CO2 from a CO2-containing stream (e.g. combustion flue gases) using a dual functioning material (DFM) composed of a highly dispersed supported adsorbent which provides at the same time the CO2 storage function and methanation active element to produce CH4 upon reaction with renewable H2 [1-2]. This process is operated cyclically by alternating phases where the DFM is exposed to the flue gas and CO2 is selectively adsorbed, and phases where H2 is fed and the stored CO2 is hydrogenated to methane [2]. Notably, by eliminating a thermal swing process, the conversion of CO2 to SNG using DFMs constrains the energy input to only renewable sources (in the form of H2), thus allowing the CO2 capture and utilization processes to approach carbon neutrality while integrating more renewable energy into the grid [2]. In a highly innovative scheme aimed at the process intensification, the DFM represents a CO2 carrier that is continuously circulated from a sorption reactor to a methanation reactor and vice versa [3]. Dual interconnected fluidized bed technology appears perfectly suited to perform the chemical looping CO2 Capture and Methanation process since it allows the recirculation of DFM particles between two reactors and ensures their efficient and independent temperature control. Therefore, we set out to experimentally investigate the key features capable to boost the overall performance of DFMs based on Ru and alkali metal (oxides) supported on high surface area aluminas with suitable mechanical and attrition resistance to be used in interconnected fluidized bed reactors. The general scope is to produce synthetic methane via an innovative, renewable energy driven, catalytic looping process, demonstrating effectiveness and reduction of greenhouse gas emissions while also addressing economic, regulatory, environmental and (critical) raw material constraints, as well as socio-economic impact related to the proposed technological pathway.

Peatlands are distinctive wetlands of Patagonia that represent a valuable natural heritage due to its near-pristine conditions and the wide range of the ecosystem services (ES) they provide. The objective of this chapter is to review the state of knowledge about the ES of Patagonian peatlands and the related processes that explain them. Some of these services, such as carbon storage, carbon sink, flood control, and water supply, are outcomes of complex biotic and abiotic interactions that take place in these ecosystems. We identified and classified ES of Patagonian peatlands in nonmonetary terms, following the guidelines of the Common International Classification of Ecosystem Services (CICES) V5.1. Different peatlands may contrast in the quality or essence of respective ES; therefore, the analysis of the ES significance requires information from local studies based on scientific knowledge and field data. We supply evidence obtained from study cases and discuss the accuracy of current estimations of carbon storage in Patagonian peatlands, as well as the peat bogs efficiency on the flood and the erosion control.

Drug screening and disease modelling for skeletal muscle related pathologies would strongly benefit from the integration of myogenic cells derived from human pluripotent stem cells within miniaturized cell culture devices, such as microfluidic platform. Here, we identified the optimal culture conditions that allow direct differentiation of human pluripotent stem cells in myogenic cells within microfluidic devices. Myogenic cells are efficiently derived from both human embryonic (hESC) or induced pluripotent stem cells (hiPSC) in eleven days by combining small molecules and non-integrating modified mRNA (mmRNA) encoding for the master myogenic transcription factor MYOD. Our work opens new perspective for the development of patient-specific platforms in which a one-step myogenic differentiation could be used to generate skeletal muscle on-a-chip.

This study looked at the potential synergy of co-pyrolysis of residual lignocellulosic biomass in the form of olive stone with low-density polyethylene in the absence/ presence of solid acid catalyst in a bench scale continuous bubbling fluidised bed reactor. Despite the catalyst lowering the pyrolytic oil yield, there was significant transition in the class of hydrocarbon derivatives formed with catalytic co-pyrolysis yielding much more deoxygenated hydrocarbons in contrast to the product class from the inert sand bed. .-alumina performed much better improving the H/C molar ratio of bio-oil by ~20% over the inert bed co-pyrolysis experiment, both the .-alumina and HZSM-5 catalyst significantly lowered the O/C molar ratio of bio oil recovered. The product stream from both catalysts was relatively high in polycyclic aromatic hydrocarbons (PAHs)due to the strong acid catalysed reactions which promotes strong aromatisation., Proceedings of the 29th European Biomass Conference and Exhibition, 26-29 April 2021, Online, pp. 817-823

Skeletal muscle is a dynamic tissue in which homeostasis and function are guaranteed by a very defined three-dimensional organization of myofibers in respect to other non-muscular components, including the extracellular matrix and the nervous network. In particular, communication between myofibers and the nervous system is essential for the overall correct development and function of the skeletal muscle. A wide range of chronic, acute and genetic-based human pathologies that lead to the alteration of muscle function are associated with modified preservation of the fine interaction between motor neurons and myofibers at the neuromuscular junction. Recent advancements in the development of in vitro models for human skeletal muscle have shown that three-dimensionality and integration of multiple cell types are both key parameters required to unveil pathophysiological relevant phenotypes. Here, we describe recent achievement reached in skeletal muscle modeling which used biomaterials for the generation of three-dimensional constructs of myotubes integrated with motor neurons.

This work numerically analyzes an innovative process layout considering a torrefaction processes followed by chemical looping combustion of biomass waste, solar hydrogen, and carbon methanation. System performances were evaluated by considering several agro-industrial residues (i.e., sugar beet pulp from sugar production, grape marc from winemaking and olive pits from olive oil production) as fuels, CuO supported on zirconia as oxygen carrier, and Ni supported on alumina as methanation catalyst. The torrefaction pre-treatment was proposed for upgrading the properties, namely heating values, moisture content as well as hydrophobicity, and storability, of the selected biomasses. To this aim, experimental runs were performed at 300 °C and 30 min in a lab-scale fixed bed reactor under an inert atmosphere of nitrogen. The study was complemented with an extensive investigation on fuel properties (i.e., ultimate analysis, proximate analysis, calorific values determination) of both the untreated and the torrefied samples, which provides useful input data for modelling their conversion processes. By considering that only electric energy from renewable sources is used, the capability of the proposed process to be used as an energy storage system was eventually assessed.

The development of sustainable, clean modern energy carriers has swept across the energy sector in line with the goals of moving towards low carbon economies. This has resulted in the increased utilisation of previously neglected solid organic waste resources as alternative carbon feedstocks for fuels and petrochemicals, as evidenced by more research towards valorisation of lignocellulosic and plastic waste resources. Co-pyrolysis has been identified as a potential pathway to benefit from this readily available stream of residual carbon providing a platform to produce valuable products that may fit into the different energy vector streams. Despite a lot of work being done at microscale pyrolysis to determine the synergistical potential of co-feeding, less work has been done on large scale reactors with potential for commercialisation on the impact of co-pyrolysis to improve the quality of pyrolysis oils. In this study, our focus is identifying potential synergy between devolatilization and condensation products that may occur in the presence of an inert and catalytic bed under bench scale fluidised bed conditions. The product distribution from non-catalytic pyrolysis of olive stone and polyethylene was comprised of derivatives indicative of a combination of the individual pyrolysis products i.e., oxygenated biomass derivatives plus olefinic monomers plastic derivatives. This could suggest that condensation synergy as suggested by Gunasee, et al. [1] may be the dominant process, requiring changes in operating procedure to allow for secondary reactions. For the catalytic experiments, the product distribution was shifted due to secondary reactions, however there was no conclusive information to distinguish the type of synergy that may have occurred, i.e., devolatilization/condensation synergy. There is still ongoing work on this project to try to optimise the process parameters towards increasing the interactive devolatilization synergy towards improving the quality of pyrolysis oils.

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.

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 μm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions.