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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In vitro demonstration of intestinal absorption mechanisms of different sugars using 3D organotypic tissues in a fluidic device.

Intestinal permeability is crucial in regulating the bioavailability and, consequently, the biological effects of drugs and compounds. However, systematic and quantitative studies of the absorption of molecules are quite limited due to a lack of reliable experimental models able to mimic human in vivo responses. In this work, we present an in vitro perfused model of the small intestinal barrier using a 3D reconstructed intestinal epithelium integrated into a fluid-dynamic biore­actor (MIVO®) resembling the physiological stimuli of the intestinal environment. This platform was investigated in both healthy and induced pathological conditions by monitoring the absorption of two non-metabolized sugars, lactulose and mannitol, frequently used as indicators of intestinal barrier dysfunctions. In healthy conditions, an in vivo-like plateau of the percentage of absorbed sugars was reached, where mannitol absorption was much greater than lactulose absorption. Moreover, a model of pathologically altered intestinal permeability was generated by depleting extracellular Ca2+, using a calcium-specific chelator. After calcium depletion, the pattern of sugar passage observed under pathological conditions was reversed only in dynamic conditions in the MIVO® chamber, due to the dynamic fluid flow beneath the membrane, but not in static conditions. Therefore, the combination of the MIVO® with the EpiIntestinal™ platform can rep­resent a reliable in vitro model to study the passage of molecules across the healthy or pathological small intestinal barrier by discriminating the two main mechanisms of intestinal absorption.

Cell-Laden Hydrogel as a Clinical-Relevant 3D Model for Analyzing Neuroblastoma Growth, Immunophenotype, and Susceptibility to Therapies.

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody-mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate in vitro models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-γ-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-γ induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring in vivo and not fully appreciated in classical 2D culture conditions. Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way.

"Green-reduced" graphene oxide induces in vitro an enhanced biomimetic mineralization of polycaprolactone electrospun meshes.

A novel green method for graphene oxide (GO) reduction via ascorbic acid has been adopted to realize bio-friendly reduced graphene oxide (RGO)/polycaprolactone (PCL) nanofibrous meshes, as substrates for bone tissue engineering applications. PCL fibrous mats enriched with either RGO or GO (0.25 wt%) were fabricated to recapitulate the fibrillar structure of the bone extracellular matrix (ECM) and the effects of RGO incorporation on the structural proprieties, biomechanics and bioactivity of the nano-composites meshes were evaluated. RGO/PCL fibrous meshes displayed superior mechanical properties (i.e. Young's Modulus and ultimate tensile strength) besides supporting noticeably improved cell adhesion, spreading and proliferation of fibroblasts and osteoblast-like cell lines. Furthermore, RGO-based electrospun substrates enhanced in vitro calcium deposition in the ECM produced by osteoblast-like cells, which was paralleled, in human mesenchymal stem cells grown onto the same substrates, by an increased expression of the osteogenic markers mandatory for mineralization. In this respect, the capability of graphene-based materials to adsorb osteogenic factors cooperates synergically with the rougher surface of RGO/PCL-based materials, evidenced by AFM analysis, to ignite mineralization of the neodeposited matrix and to promote the osteogenic commitment of the cultured cell in the surrounding microenvironment.

A combined low-frequency electromagnetic and fluidic stimulation for a controlled drug release from superparamagnetic calcium phosphate nanoparticles: potential application for cardiovascular diseases.

Alternative drug delivery approaches to treat cardiovascular diseases are currently under intense investigation. In this domain, the possibility to target the heart and tailor the amount of drug dose by using a combination of magnetic nanoparticles (NPs) and electromagnetic devices is a fascinating approach. Here, an electromagnetic device based on Helmholtz coils was generated for the application of low-frequency magnetic stimulations to manage drug release from biocompatible superparamagnetic Fe-hydroxyapatite NPs (FeHAs). Integrated with a fluidic circuit mimicking the flow of the cardiovascular environment, the device was efficient to trigger the release of a model drug (ibuprofen) from FeHAs as a function of the applied frequencies. Furthermore, the biological effects on the cardiac system of the identified electromagnetic exposure were assessed in vitro and in vivo by acute stimulation of isolated adult cardiomyocytes and in an animal model. The cardio-compatibility of FeHAs was also assessed in vitro and in an animal model. No alterations of cardiac electrophysiological properties were observed in both cases, providing the evidence that the combination of low-frequency magnetic stimulations and FeHAs might represent a promising strategy for controlled drug delivery to the failing heart.

A new cell-laden 3D Alginate-Matrigel hydrogel resembles human breast cancer cell malignant morphology, spread and invasion capability observed "in vivo".

Purpose of this study was the development of a 3D material to be used as substrate for breast cancer cell culture. We developed composite gels constituted by different concentrations of Alginate (A) and Matrigel (M) to obtain a structurally stable-in-time and biologically active substrate. Human aggressive breast cancer cells (i.e. MDA-MB-231) were cultured within the gels. Known the link between cell morphology and malignancy, cells were morphologically characterized and their invasiveness correlated through an innovative bioreactor-based invasion assay. A particular type of gel (i.e. 50% Alginate, 50% Matrigel) emerged thanks to a series of significant results: 1. cells exhibited peculiar cytoskeleton shapes and nuclear fragmentation characteristic of their malignancy; 2. cells expressed the formation of the so-called invadopodia, actin-based protrusion of the plasma membrane through which cells anchor to the extracellular matrix; 3. cells were able to migrate through the gels and attach to an engineered membrane mimicking the vascular walls hosted within bioreactor, providing a completely new 3D in vitro model of the very precursor steps of metastasis.

3D Porous Gelatin/PVA Hydrogel as Meniscus Substitute Using Alginate Micro-Particles as Porogens.

One of the current major challenges in orthopedic surgery is the treatment of meniscal lesions. Some of the main issues include mechanical consistency of meniscal implants, besides their fixation methods and integration with the host tissues. To tackle these aspects we realized a micro-porous, gelatin/polyvinyl alcohol (PVA)-based hydrogel to approach the high percentage of water present in the native meniscal tissue, recapitulating its biomechanical features, and, at the same time, realizing a porous implant, permissive to cell infiltration and tissue integration. In particular, we adopted aerodynamically-assisted jetting technology to realize sodium alginate micro-particles with controlled dimensions to be used as porogens. The porous hydrogels were realized through freezing-thawing cycles, followed by alginate particles leaching. Composite hydrogels showed a high porosity (74%) and an open porous structure, while preserving the elasticity behavior (E = 0.25 MPa) and high water content, typical of PVA-based hydrogels. The ex vivo animal model validation proved that the addition of gelatin, combined with the micro-porosity of the hydrogel, enhanced implant integration with the host tissue, allowing penetration of host cells within the construct boundaries. Altogether, these results show that the combined use of a water-insoluble micro-porogen and gelatin, as a bioactive agent, allowed the realization of a porous composite PVA-based hydrogel to be envisaged as a potential meniscal substitute.