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The biocompatibility assessment of biomaterials or the dynamic response of implanted constructs entails inflammatory events primary reflected in cell behavior at the microcirculatory system. Current protocols are based on histopathology which are over 40 years old and require the sacrifice of a huge number of laboratory animal with an unsustainable ethical burden of animal research. Intravital microscopy techniques are actually used to study implantation outcomes in real time. However, no device providing a specific tracking geometry to reposition the field of view of the microscope, for repeated analyses, exists yet. The synthetic photoresist SZ2080 is characterized here, allowing the development and in vivo validation of a miniaturized imaging window, the Microatlas, that, fabricated via two-photon polymerization, is implanted in living chicken embryos and imaged by fluorescence microscopy 3 and 4 days after the implant. The characterization of their elastomechanical and fluorescence properties highlights planar raster spacing as the most important parameter in tuning the mechanical and spectroscopic features of the structures. The quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration and as beacon for 3D repositioning of the microscope field of view and correction of optical aberrations.

The search for new rapid diagnostic tests for malaria is a priority for developing an efficient strategy to fight this endemic disease, which affects more than 3 billion people worldwide. In this study, we characterize systematically an easy-to-operate lab-on-chip, designed for the magnetophoretic capture of malaria-infected red blood cells (RBCs). The method relies on the positive magnetic susceptibility of infected RBCs with respect to blood plasma. A matrix of nickel posts fabricated in a silicon chip placed face down is aimed at attracting infected cells, while healthy cells sediment on a glass slide under the action of gravity. Using a model of infected RBCs, that is, erythrocytes with methemoglobin, we obtained a capture efficiency of about 70% after 10 min in static conditions. By proper agitation, the capture efficiency reached 85% after just 5 min. Sample preparation requires only a 1:10 volume dilution of whole blood, previously treated with heparin, in a phosphate-buffered solution. Nonspecific attraction of untreated RBCs was not observed in the same time interval.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.

Bone tissue is the structural component of the body, which allows locomotion, protects vital internal organs, and provides the maintenance of mineral homeostasis. Several bone-related pathologies generate critical-size bone defects that our organism is not able to heal spontaneously and require a therapeutic action. Conventional therapies span from pharmacological to interventional methodologies, all of them characterized by several drawbacks. To circumvent these effects, tissue engineering and regenerative medicine are innovative and promising approaches that exploit the capability of bone progenitors, especially mesenchymal stem cells, to differentiate into functional bone cells. So far, several materials have been tested in order to guarantee the specific requirements for bone tissue regeneration, ranging from the material biocompatibility to the ideal 3D bone-like architectural structure. In this review, we analyse the state-of-the-art of the most widespread polymeric scaffold materials and their application in in vitro and in vivo models, in order to evaluate their usability in the field of bone tissue engineering. Here, we will present several adopted strategies in scaffold production, from the different combination of materials, to chemical factor inclusion, embedding of cells, and manufacturing technology improvement.

Brand new biomaterials, intended to be used on humans, must undergo in vivo quantification standardized, expensive and unethical procedures mainly based on histopathological analysis, from dissections, as defined by the ISO 10993 normative set. The aim is to prove the biomaterials biocompatibility. There exist no methods based on intravital microscopy able to satisfy the normative quantification requirements both reducing the number of employed animals and related costs. We developed a miniaturized imaging window, the Microatlas, which allows subcutaneous repeated observations in vivo of the foreign body reactions, for example to the implantation of a biomaterial. Confocal and twophoton microscopy inspections at Microatlas implantation sites demonstrated growth of the recipient tissue inside the microgrids both with micro vascularization formation and collagen generation. In conclusion, the Microatlas guided in vivo a quantifiable localized reaction inside its microscaffold, both in terms of cell repopulation, collagen and capillary formation as a probable foreign body reaction.

Nonlinear optical microscopy is a powerful label-free imaging technology, providing biochemical and structural information in living cells and tissues. A possible drawback is photodamage induced by high-power ultrashort laser pulses. Here we present an experimental study on thousands of HeLa cells, to characterize the damage induced by focused femtosecond near-infrared laser pulses as a function of laser power, scanning speed and exposure time, in both wide-field and point-scanning illumination configurations. Our data-driven approach offers an interpretation of the underlying damage mechanisms and provides a predictive model that estimates its probability and extension and a safety limit for the working conditions in nonlinear optical microscopy. In particular, we demonstrate that cells can withstand high temperatures for a short amount of time, while they die if exposed for longer times to mild temperatures. It is thus better to illuminate the samples with high irradiances: thanks to the nonlinear imaging mechanism, much stronger signals will be generated, enabling fast imaging and thus avoiding sample photodamage.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (−30)GFP, to a myogenic transcription factor, MyoD. The (−30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (−30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (−30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Mechanotransduction translates forces into biological responses and regulates cell functionalities. It is implicated in several diseases, including laminopathies which are pathologies associated with mutations in lamins and lamin-associated proteins. These pathologies affect muscle, adipose, bone, nerve, and skin cells and range from muscular dystrophies to accelerated aging. Although the exact mechanisms governing laminopathies and gene expression are still not clear, a strong correlation has been found between cell functionality and nuclear behavior. New theories base on the direct effect of external force on the genome, which is indeed sensitive to the force transduced by the nuclear lamina. Nuclear lamina performs two essential functions in mechanotransduction pathway modulating the nuclear stiffness and governing the chromatin remodeling. Indeed, A-type lamin mutation and deregulation has been found to affect the nuclear response, altering several downstream cellular processes such as mitosis, chromatin organization, DNA replication-transcription, and nuclear structural integrity. In this review, we summarize the recent findings on the molecular composition and architecture of the nuclear lamina, its role in healthy cells and disease regulation. We focus on A-type lamins since this protein family is the most involved in mechanotransduction and laminopathies.

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells' fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.

Mesenchymal stem cells (MSCs) have immune-modulatory and tissue-regenerative properties that make them a suitable and promising tool for cell-based therapy application. Since the bio-chemo-mechanical environment influences MSC fate and behavior, the understanding of the mechanosensors involved in the transduction of mechanical inputs into chemical signals could be pivotal. In this context, the nuclear pore complex is a molecular machinery that is believed to have a key role in force transmission and in nucleocytoplasmic shuttling regulation. To fully understand the nuclear pore complex role and the nucleocytoplasmic transport dynamics, recent advancements in fluorescence microscopy provided the possibility to study passive and facilitated nuclear transports also in mechanically stimulated cell culture conditions. Here, we review the current available methods for the investigation of nucleocytoplasmic shuttling, including photo-perturbation-based approaches, fluorescence correlation spectroscopy, and single-particle tracking techniques. For each method, we analyze the advantages, disadvantages, and technical limitations. Finally, we summarize the recent knowledge on mechanical regulation of nucleocytoplasmic translocation in MSC, the relevant progresses made so far, and the future perspectives in the field.

Peripheral nerve transection is often encountered after trauma and can lead to long-term/permanent loss of sensor/motor functionality. Here, the effect of pure contact interaction of nano/microgrooved substrates on Schwann cells (SCs) is studied in view of their possible use for nerve-repair applications. Elastomeric gratings (GRs; i.e., alternating lines of ridges and grooves) are developed with different lateral periods (1-20 ?m) and depths (0.3-2.5 ?m), leading to two distinct cell-material interaction regimes: contact guidance (grating period < cell body diameter) and boundary guidance (grating period >= cell body diameter). Here, it is shown that boundary guidance leads to the best single-cell polarization, actin organization, and single-cell directional migration. Remarkably, contact guidance is instead more effective in driving collective SC migration and improves functional wound healing. It is also demonstrated that this behavior is linked to the properties of the SC monolayers on different GRs. SCs on large-period GRs are characterized by N-Cadherin downregulation and enhanced single-cell scattering into the wound with respect to SCs on small-period GRs, indicating a less compact monolayer characterized by looser cell-cell junctions in the boundary guidance regime. The present results provide information on the impact of specific sub-micrometer topographical elements on SC functional response, which can be exploited for nerve-regeneration applications.

Graphical abstractDisplay Omitted We engineered micropatterned PDMS membranes to guide Schwann cell migration.Migration occurs along PDMS microgratings for single cells and upon wound healing.We identified topographies that might be exploited for nerve regeneration. We engineered anisotropic microstructured membranes for nerve repair applications by exploiting electron beam lithography on silicon substrates and soft lithography techniques. Our substrates were patterned with polydimethylsiloxane (PDMS) gratings (alternating lines of grooves and ridges) of varying ridge/groove width and depth. These gratings were used as scaffolds to study rat Schwann cell contact interaction for cell migration. We observed that cell motion was affected by the presence of the grating and by its periodicity, while cells on flat substrates showed random spatial migration. Our results allow the identification of specific topographical elements that may be exploited for the production of new devices for enhancing nerve regeneration by promoting Schwann cell invasion, proliferation, and terminal differentiation.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.

By combining spectroscopic measurements under high pressure with molecular dynamics simulations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two Green Fluorescent Protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing ∼100ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning, especially in the case of the sizable 120 cm-1 red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work by Barstow et al [1] on pressure effects on GFP also involved a T203Y mutant. Using cryocooling X-ray crystallography at high pressure they linked the observed, pressure-induced, fluorescence blue shift at low temperature (77 K) to key changes in relative conformation of the chromophore and Y203 phenol ring. At room temperature, however, they observe a red shift at high pressure, analogous to the one in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.[1] Barstow, B., Ando, N., Kim, C. U., & Gruner, S. M. (2008). Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift. Proc Natl Acad Sci USA, 105(36), 13362-13366.

Ex vivo expansion of hematopoietic stem cells has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. Recently, we demonstrated the existence of a circulating myogenic progenitor expressing the CD133 antigen. The relative inability of circulating CD133+ stem cells to reproduce themselves ex vivo imposes substantial limitations on their use for clinical applications in muscular dystrophies. Here we report that the use of cluster-assembled nanostructured titanium dioxide (ns-TiO 2 ) substrates, in combination with cytokine enriched medium, enables high-level expansion of circulating CD133+ stem cells in vitro. Furthermore, we demonstrate that expanded circulating CD133+ stem cells retain their in vitro capacity to differentiate into myogenic cells. The exploitation of cluster-assembled ns-TiO 2 substrates for the expansion of CD133+ stem cells in vitro could therefore make the clinical application of these stem cells for the treatment of muscle diseases practical.

Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. We here present two new molecular rotors characterized by a push-pull hyperconjugated structure and which absorb in the blue-green region of the spectrum. These probes display significant emission and lifetime increase by increasing local microviscosity. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to report on environmental properties, such as viscosity, in the cellular context, i.e. where the fluorescent probe concentration cannot be controlled. Remarkably, we demonstrate that our probes allow for efficient intracellular viscosity measurements by means of the phasor approach to fluorescence lifetime imaging, a fit-free method that overcome the main drawbacks of conventional lifetime imaging (1). More specifically, we show that one probe can be used to monitor membrane organization (i.e. the fractions of liquid ordered and disordered phases) and membrane viscosity, whereas the other reports on the local viscosity in plasma membrane, lysosomes, and mitochondria (2). Notably, measurements were carried out in both physiological and non-physiological conditions, in view of correlating dysfunctional states of the cell with viscosity changes in intracellular organelles.1. Digman, M.A., Caiolfa, V.R., Zamai, M., and Gratton, E. (2008) Biophys J 94, L14-16.2. Battisti, A., Panettieri, S., Abbandonato, G., Jacchetti, E., Cardarelli, F., Signore, G., Beltram, F., and Bizzarri, R. (2013) Anal. Bioanal. Chem. 405, 6223-6233.

Through the interaction with topographical features, endothelial cells tune their ability to populate target substrates, both in vivo and in vitro. Basal textures interfere with the establishment and maturation of focal adhesions (FAs) thus inducing specific cell-polarization patterns and regulating a plethora of cell activities that govern the overall endothelial function. In this study, we analyze the effect of topographical features on FAs in primary human endothelial cells. Reported data demonstrate a functional link between FA dynamics and cell polarization and spreading on structured substrates presenting variable lateral feature size. Our results reveal that gratings with 2 µm lateral periodicity maximize contact guidance. The effect is linked to the dynamical state of FAs. We argue that these results are readily applicable to the rational design of active surfaces at the interface with the blood stream.

The development of biomaterials ensuring proper cell adhesion, polarization, migration and differentiation represents a true enabler for successful tissue-engineering applications. Surface nanostructuring was suggested as a promising method for improving cell-substrate interaction. Here, we study Wharton's Jelly human Mesenchymal Stem Cells (WJ-hMSC) interacting with nanogratings (NGs) having a controlled amount of nanotopographical noise (nTN). Our data demonstrate that unperturbed NGs induce cell polarization, alignment and migration along NG lines. The introduction of nTN dramatically modifies this behavior and leads to a marked loss of cell polarization and directional migration, even at low noise levels. High-resolution focal adhesions (FAs) imaging showed that this behavior is caused by the release of the geometrical vinculum imposed by the NGs to FA shaping and maturation. We argue that highly anisotropic nanopatterned scaffolds can be successfully exploited to drive stem cell migration in regenerative medicine protocols and discuss the impact of scaffold alterations or wear.

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5',5'"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509-14514). P24, but not P18, proved to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca(2+) through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca(2+) influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC(50) of approximately 1 mum. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca(2+)](i) has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146-23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca(2+)](i) to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

Background Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. Results A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. Conclusion The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.