Biology subjects

Levato, R.

Publications and source records attributed to Levato, R..

14 recordsLinked to original sources

bioRxiv2026

Spatial control of light-responsive proteins and optogenetics within hydrogels via volumetric bioprinting

Spatiotemporal control over cell fate and behaviour within bioprinted constructs remains a key challenge in tissue engineering. Optogenetics offers versatile potential for non-invasive regulation of biological processes. Yet, its integration within large-scale, cell-laden bioprinted materials is still limited, especially considering spatial constraints of existing light delivery methods. In this study, we introduce a novel approach that repurposes tomographic volumetric bioprinting to enable post-printing stimulation of photosensitive protein-switches and optogenetic circuits in cells deep within hydrogel constructs. By converging different bioprinting approaches, computer vision, context-aware model generation, and synthetic biology and cell engineering, we demonstrated selective activation of a fluorescent, light-responsive protein probe within multi-material centimeter-scale constructs. Moreover, leveraging a multi-wavelength volumetric bioprinter, we further demonstrate this concept by selectively stimulating cells expressing a near-infrared optogenetic system that triggers gene expression and the induction of pancreas-specific transcription factors. The described methods provide platforms for remote, repeatable, and localized control of biological events in volumetric constructs, opening new possibilities for advanced tissue models, and dynamic tuning of cell-mediated protein production in engineered living systems.

bioengineering↗

bioRxiv2026

A Versatile, Biocompatible Additive for Spatiotemporal Volumetric Photografting of Biochemical and Mechanical Cues in Diverse Hydrogel Matrices

Engineering functional tissue constructs requires not only replicating their 3D architecture but also capturing their dynamic biochemical and mechanical environments. While 3D bioprinting technologies enable spatial control over cell and biomaterial deposition, post-fabrication modulation of material properties remains limited. Photografting approaches allow for spatiotemporal functionalization of certain 3D matrices by chemically binding bioactive factors onto spatially determined regions of a material, but current methods often rely on specialized chemistries with narrow material compatibility. Here, we introduce AddGraft, a biocompatible, off-the-shelf additive designed for semi-orthogonal thiol-ene photografting in vinyl-functionalized hydrogels. AddGraft, a heterobifunctional polyethylene glycol, carries an acrylate moiety for network incorporation during photocrosslinking and a norbornene group for post-crosslinking functionalization. AddGraft integrates into the polymer network during gel crosslinking without altering bulk mechanics, enabling precise modification at any time post-fabrication. We demonstrate compatibility with multiple acrylated biomaterial platforms and light-based volumetric photopatterning technology. Photopatterning achieves high spatial resolution and gradient formation in 3D, while grafting of multi-thiolated crosslinkers allows localized stiffening of hydrogels. Encapsulated human mesenchymal stromal cells exhibit high viability and undergo morphological changes in response to the dynamic tuning of their microenvironment. By decoupling structural and functional roles, AddGraft enables on-demand spatial and temporal control over hydrogel properties. This approach expands the biofabrication toolkit for engineering cell-instructive, 4D tissue environments with translational relevance in regenerative medicine.

bioengineering↗

bioRxiv2025

Multi-scale Engineered Vasculature and Hierarchical Porosity via Volumetric Bioprinting-guided Photopolymerization-induced Phase Separation

Vascularization remains a major challenge in hydrogel-based engineered tissues due to the inherent nano-scale porosity of common synthetic and natural biomaterials. Critically, the confinement imposed by the nanoscale network inhibits the outgrowth of blood vessels required for oxygen and nutrient delivery. Despite advancements in biofabrication that enable formation of small channels (0.1-1 mm), achieving vascularization (with capillaries down to 10 {micro}m) throughout cm-scale bioprinted constructs remains a critical bottleneck. Herein, we integrated phase separating cell-interactive gelatin-norbornene hydrogels with volumetric bioprinting to generate architecturally defined centimeter-scale constructs with 0.1-1 mm scale printed channels and interpenetrating micron-scale porosity. Our novel approach allowed us to generate freeform construct designs with light-controllable micron-scale and hierarchical porosity. Importantly, this porosity enabled endothelial cell infiltration and microvessel outgrowth deep into the engineered tissue. Micron-scale vascular structures formed in the pore spaces with feature sizes on the scale of capillaries (<10 {micro}m), crucial to provide oxygen and nutrients to all regions of the hydrogel. The networks remained stable for over 14 days, outperforming classical nanoporous biomaterials. These complex hydrogel-based constructs with engineered multi-scale vascular networks have substantial potential for the generation of actively perfusable advanced tissue models.

bioengineering↗

bioRxiv2025

Hybrid supramolecular-covalent bioresin promotes cell migration and self-assembly in light-based volumetric bioprinted constructs

There is an increasing need for novel biomaterials compatible with advanced biofabrication technologies, which also permit cells to remodel their microenvironment. This remodelling is crucial for maturing tissue constructs into fully functional tissue replacements. Recent progress in supramolecular chemistries has allowed for the production of dynamic biomaterials. Their properties enable bonds to be reversibly broken by cells, facilitating processes requiring morphological changes or migration, crucial for tissue development and homeostasis. Here, we present a one-of-its-kind gelatin-based hybrid covalent/supramolecular biomaterial. We demonstrate the advantage of adding supramolecular-reactive moieties on covalent materials, over covalent bonds alone, in facilitating processes such as cell growth, migration, spreading and organoid proliferation. This is exemplified by enhanced MSC and T cell migration and improved vascular network formation in hybrid hydrogels over covalent-only materials. The combination of supramolecular and covalent bonds further enabled control over photocrosslinking, allowing the use of the material in volumetric bioprinting of complex structures with high shape fidelity. As a proof-of-concept we bioprinted complex breast-like structures from encapsulated normal breast cell lines with a tumor organoid core. We demonstrated that engineered T cells can migrate large distances into the breast tissue, specifically targeting tumor cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/631505v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1e15084org.highwire.dtl.DTLVardef@16d1554org.highwire.dtl.DTLVardef@937d1aorg.highwire.dtl.DTLVardef@fa51f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Basement membrane hydrogels dampen CAR-T cell activation: nanofibrillar cellulose gels as alternative to preserve T cell function in 3D cell cultures.

BackgroundHydrogel-based 3D culture systems are emerging as a valuable tool for preclinical screening of cell-based immunotherapies against solid and hematological malignancies, such as chimeric antigen receptor T (CAR-T) cells. Hydrogels can influence T cell function in a non-desired manner due to their mechanical properties and chemical composition, potentially skewing results in preclinical testing of novel immunotherapeutic compounds. MethodsIn this study, we assess CD4+ T and CAR-T cell activation and proliferation in chemically-undefined matrices (Matrigel and basement membrane extract, BME) and compare them to a synthetic nanofibrillar cellulose (NFC) hydrogel. ResultsRheometric analyses show that NFC is more rigid than Matrigel and BME. Murine CD4+ T cells acquire a regulatory T cell (Treg) phenotype in Matrigel and BME, while this is not observed in NFC. Proliferation and activation of human T cells are higher in NFC than in Matrigel or BME. Similarly, we show that CAR-T cell activation and proliferation is significantly impaired in Matrigel and BME, in contrast to NFC. ConclusionsOur findings highlight the impact of hydrogel choice on (CAR-)T cell behavior, with direct implications for preclinical immunotherapy testing. In contrast to Matrigel and BME, NFC offers a chemically-defined 3D environment where T cell function is preserved. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSIn 3D (preclinical) tumor-killing assays for evaluating engineered T cell cytotoxicity, the surrounding matrix can influence immune cell phenotype and function, potentially skewing T cell activity. Basement membrane hydrogels such as Matrigel and basement membrane extract (BME), widely used as scaffolds for 3D culture, are inherently heterogeneous and contain extracellular matrix components that can influence lymphocyte function. What this study addsHere, we show that (CAR-)T cell function is significantly reduced in Matrigel and BME as compared to standard (2D) culture conditions. In contrast, (CAR-)T cell activity is preserved in synthetic nanofibrillar cellulose (NFC) gels. Importantly, murine T cells spontaneously acquire a Treg phenotype in Matrigel and BME. T cell proliferation and cytokine secretion are >10-fold lower in Matrigel than in NFC. Similarly, CAR-T cell survival and expansion are 10-fold higher in NFC than in Matrigel or BME. How this study might affect research, practice or policyWe report that the intrinsic cytotoxic and proliferative potential of (CAR-)T cells can be underestimated when performing assays in 3D cultures based on Matrigel or BME. As an alternative, we suggest the use of chemically defined synthetic gels, and we show that nanofibrillar cellulose hydrogels are suitable 3D matrices for preserving T cell phenotype and activation.

immunology↗

bioRxiv2024

Novel hepatocyte-like liver organoids recapitulate crucial mature hepatic functions

Accurate liver disease modeling and drug toxicity testing still remain challenging as liver cells in vitro poorly resemble adult hepatocytes, as we previously demonstrated using whole transcriptome and cell identity analysis. To address this, we used our insights into hepatic modeling to develop hepatocyte-like liver organoids (HeLLOs), a novel human organoid model with mature hepatocyte functions superior to existing models. HeLLOs are easily established from (small) healthy or diseased liver tissues and rapidly expanded for an extended period in optimized culture conditions. Transcriptomic and functional analyses revealed that differentiated HeLLOs closely resemble fresh primary human hepatocytes (PHHs) and perform key hepatic functions such as gluconeogenesis, drug metabolism, and bile acid synthesis. We developed a HeLLO-based toxicity assay with higher sensitivity in predicting liver toxicity of known liver-toxic drugs compared to the gold-standard PHHs. By modeling disease-related mechanisms, such as bile acid transport, HeLLOs uncover transport-inhibition toxicity mechanisms of known liver toxic drugs. Single cell sequencing analysis of HeLLOs identified a heterogeneous cluster of cells with cholangiocyte-like and hepatocyte-like cells, overall resembling liver regenerative cells. As such, HeLLOs hold great promise for advancing liver disease modeling and drug testing. To our knowledge, HeLLOs are the best expandable liver model for predicting adverse drug reactions as well as modeling various liver disease mechanisms.

cell biology↗

bioRxiv2024

Multi-material volumetric bioprinting and plug-and-play suspension bath biofabrication via bioresin molecular weight tuning and multiwavelength optics

Volumetric Bioprinting (VBP), enables to rapidly build complex, cell-laden hydrogel constructs for tissue engineering and regenerative medicine. Light-based tomographic manufacturing enables spatial-selective polymerization of a bioresin, resulting in higher throughput and resolution than what achieved using traditional techniques. However, methods for multi-material printing are needed for a broad VBP adoption and applicability. Although converging VBP with extrusion bioprinting in support baths offers a novel, promising solution, further knowledge on the engineering of hydrogels as light-responsive, volumetrically printable baths is needed. Therefore, this study investigates the tuning of gelatin macromers, in particular leveraging the effect of molecular weight and degree of modification, to overcome these challenges, creating a library of materials for VBP and Embedded extrusion Volumetric Printing (EmVP). Bioresins with tunable printability and mechanical properties are produced, and a novel subset of gelatins and GelMA exhibiting stable shear-yielding behavior offers a new, single-component, ready-to-use suspension medium for in-bath printing, which is stable over multiple hours without needing temperature control. As proof-of-concept biological application, bioprinted gels are tested with insulin-producing pancreatic cell lines for 21 days of culture. Leveraging a multi-color printer, complex multi-material and multi-cellular geometries are produced, enhancing the accessibility of volumetric printing for advanced tissue models.

bioengineering↗

bioRxiv2024

A Light Sensitive Hyaluronic Acid-based Hydrogel for 3D Culturing, Differentiation, and Harvesting of Mesenchymal Stromal Cells

Hydrogels derived from natural sources are commonly used for 3D cell culturing due to their favourable interactions with cells. However, these biomaterials (e.g. Matrigel) suffer from variations in composition, limited mechanical tunability, the presence of xenogenic components and difficulties of cell retrieval. Semi-synthetic hydrogels are emerging to address these limitations, making these attractive for drug delivery and tissue engineering. Here, we describe a hydrogel platform based on hyaluronic acid (HA) modified by (1R,8S,9S)-bicycle[6.1.0]non-4-yn-9-ylmethanol (BCN) and a cross-linker composed of light sensitive o-nitrobenzyl moieties and polyethylene glycol (PEG) chains terminating in azides. The two components can undergo strain-promoted azide-alkyne cycloaddition (SPAAC) resulting in rapid gel formation. The stiffness of the hydrogel can be modulated by varying the crosslinker ratio and multi-functionalizing is possible by the incorporation of chemical cues modified by an azide. The fast hydrogelation enables easy encapsulation of cells and harvesting with preservation of viability is possible be short exposure to UV light. Moreover, it is demonstrated that visible light (405 nm) can soften the hydrogel resulting in phenotypical changes of human Mesenchymal Stromal Cells (hMSCs) co-cultured with endothelial colony-forming cells (ECFCs) with high viability. Our findings highlight that the photosensitive HA-based hydrogel provides a versatile and biocompatible platform for cell culture and tissue engineering applications, offering advantages over traditional 3D cell culturing platforms.

biochemistry↗

bioRxiv2024

Tunable bicontinuous macroporous cell culture scaffolds via kinetically controlled phase separation

Three-dimensional (3D) scaffolds enable biological investigations with a more natural cell conformation. However, the porosity of synthetic hydrogels is often limited to the nanometer scale, which confines the movement of 3D encapsulated cells and restricts dynamic cell processes. Precise control of hydrogel porosity across length scales remains a challenge and the development of porous materials that allow cell infiltration, spreading, and migration in a manner more similar to natural ECM environments is desirable. Here, we present a straightforward and reliable method for generating kinetically-controlled macroporous systems using liquid-liquid phase separation between poly(ethylene glycol) (PEG) and dextran. Photopolymerization-induced phase separation resulted in macroporous hydrogels with tunable pore size. Varying light intensity and hydrogel composition controlled polymerization kinetics, time to percolation, and complete gelation, which defined the average pore diameter (O = 1- 300 m) and final gel stiffness of the formed hydrogels. Critically, for biological applications, macroporous hydrogels were prepared from aqueous polymer solutions at physiological pH and temperature using visible light, allowing for direct cell encapsulation. We encapsulated human dermal fibroblasts in a range of macroporous gels with different pore sizes. Porosity improved cell spreading with respect to bulk gels and allowed migration in the porous systems.

bioengineering↗

bioRxiv2023

Sound-based assembly of three-dimensional cellularized and acellularized constructs

Herein we show an accessible technique based on Faraday waves that assist the rapid assembly osteoinductive {beta}-TCP particles as well as human osteoblast pre-assembled in spheroids. The hydrodynamic forces originating at seabed of the assembly chamber can be used to tightly aggregate inorganic and biological entities at packing densities that resemble those of native tissues. Additionally, following a layer-by-layer assembly procedure, centimeter scaled osteoinductive three-dimensional and cellularised constructs have been fabricated. We showed that the intimate connection between biological building blocks is essential in engineering living system able of localized mineral deposition. Our results demonstrate, for the first time, the possibility to obtain three-dimensional cellularised and acellularised anisotropic constructs using Faraday waves.

bioengineering↗

bioRxiv2023

Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion Volumetric Printing of Microgels

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and therefore a bottleneck towards creating physiologically relevant models. Addressing this limitation, we introduced a novel technique, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing to spatially pattern multiple inks/cell types. Light-responsive microgels were developed as permissive microenvironment for cell homing and self-organization, and as bioresins ({micro}Resins) for light-based bioprinting. Tuning the mechanical and optical properties of these gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. {micro}Resins can then be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids within microgels enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP was applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with enhanced functionality, and for developing engineered living systems and (metabolic) disease models.

bioengineering↗

bioRxiv2023

Volumetric Printing across Melt Electrowritten Scaffolds Fabricates Multi-Material Living Constructs with Tunable Architecture and Mechanics

Major challenges in biofabrication revolve around capturing the complex, hierarchical composition of native tissues. However, individual 3D printing techniques have limited capacity to produce composite biomaterials with multi-scale resolution. Volumetric bioprinting recently emerged as a paradigm-shift in biofabrication. This ultra-fast, light-based technique sculpts cell-laden hydrogel bioresins into three-dimensional structures in a layerless fashion, providing unparalleled design freedom over conventional bioprinting. However, it yields prints with low mechanical stability, since soft, cell-friendly hydrogels are used. Herein, for the first time, the possibility to converge volumetric bioprinting with melt electrowriting, which excels at patterning microfibers, is shown for the fabrication of tubular hydrogel-based composites with enhanced mechanical behavior. Despite including non-transparent melt electrowritten scaffolds into the volumetric printing process, high-resolution bioprinted structures were successfully achieved. Tensile, burst and bending mechanical properties of printed tubes were tuned altering the electrowritten mesh design, resulting in complex, multi-material tubular constructs with customizable, anisotropic geometries that better mimic intricate biological tubular structures. As a proof-of-concept, engineered vessel-like structures were obtained by building tri-layered cell-laden vessels, and features (valves, branches, fenestrations) that could be resolved only by synergizing these printing methods. This multi-technology convergence offers a new toolbox for manufacturing hierarchical and mechanically tunable multi-material living structures.

bioengineering↗

bioRxiv2022

Nature's design of the osteochondral unit: microstructural differences between terrestrial and aquatic mammals

During evolution, animals have returned from land to water, adapting with morphological modifications to life in an aquatic environment. We compared the osteochondral units of the humeral head of marine and terrestrial mammals across species spanning a wide range of body weights, focusing on microstructural organization and biomechanical performance. Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of the microarchitecture-function relationship is pivotal for understanding articular biology and, hence, for the development of durable functional regenerative approaches for treatment of joint damage, which are thus far lacking.

evolutionary biology↗

bioRxiv2020

Impact of endotoxins in gelatine hydrogels on chondrogenic differentiation and inflammatory cytokine secretion in vitro

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aiming at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 hours, followed by the assessment of tumour necrosis factor (TNF)- and C-C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with >1000 EU/ml and three with <10 EU/ml), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF- and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

bioengineering↗