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Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication
Abstract
The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.
... Recently, we demonstrated the fabrication of complex micropatterns of nanostructured zirconia films able to control cell adhesion and their spatial distribution [32,33]. Zirconia nanostructured substrates have been used in a variety of experimental studies [32,34-39] to provide cellular microenvironments with controllable and reproducible biomimetic nanotopographies to unravel their impact on the mechanisms underlying mechanotransduction and to guide and control cellular behaviour [32,33]. ...
... Recently, we demonstrated the fabrication of complex micropatterns of nanostructured zirconia films able to control cell adhesion and their spatial distribution [32,33]. Zirconia nanostructured substrates have been used in a variety of experimental studies [32,34-39] to provide cellular microenvironments with controllable and reproducible biomimetic nanotopographies to unravel their impact on the mechanisms underlying mechanotransduction and to guide and control cellular behaviour [32,33]. Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes. ...
... Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes. For the neuronal cells, it has been shown that they grow and differentiate in a confined manner in these nanostructured zirconia micropatterns [32,33]. For astrocytes, so far the effective confinement of astrocytes in a nanostructured zirconia pattern has also been demonstrated in Ref. [33]. ...
Astrocytes’ organisation affects the functioning and the fine morphology of the brain, both in physiological and pathological contexts. Although many aspects of their role have been characterised, their complex functions remain, to a certain extent, unclear with respect to their contribution to brain cell communication. Here, we studied the effects of nanotopography and microconfinement on primary hippocampal rat astrocytes. For this purpose, we fabricated nanostructured zirconia surfaces as homogenous substrates and as micrometric patterns, the latter produced by a combination of an additive nanofabrication and micropatterning technique. These engineered substrates reproduce both nanotopographical features and microscale geometries that astrocytes encounter in their natural environment, such as basement membrane topography, as well as blood vessels and axonal fibre topology. The impact of restrictive adhesion manifests in the modulation of several cellular properties of single cells (morphological and actin cytoskeletal changes) and the network organisation and functioning. Calcium wave signalling was observed only in astrocytes grown in confined geometries, with an activity enhancement in cells forming elongated agglomerates with dimensions typical of blood vessels or axon fibres. Our results suggest that calcium oscillation and wave propagation are closely related to astrocytic morphology and actin cytoskeleton organisation.
... The EHD printing technique is characterized by straightforward operation without contact or the employment of any molds or photo-masks for the preparation of nanoscale array structures and suspended structures. It is compared with conventional fabrication approaches such as counting lithography [1], laser ablation [2], and nanoimprint lithography [3] as well as mask printing and is considered an advantageous method [4]. EHD printing is an exceedingly efficient additive manufacturing process for the preparation of direct writing nanoscale structures [5]. ...
To increase the printing stability of low-viscosity solutions, an auxiliary method was proposed using a coaxial electrohydrodynamic jet. A high-viscosity solution was employed as the outer layer in the printing process, and it could be removed (dissolved away) after printing the structures. A combination of mechanical and electrical forces was proposed to enhance the consistency, durability, and alignment of the printed versatile structures. The instability of the jet trajectory (which arose from the repulsion between the jet and the base with a residual charge, in addition to the winding effect of the solution) was also reduced using the drag force along the direction of movement. Moreover, the jet velocity, the surface charge, and the influence of various working voltages on the jet speed were simulated. An array of IDT-BT nanostructures measuring about 100 nm was prepared on silicon dioxide (using an inner needle with a diameter of 130 µm) by equating the moving speed (350 mm/s) of the substrate to the speed of the jet. Moreover, the moving speed (350 mm/s) of the substrate was compared exclusively to the speed of the jet. The method proposed throughout this study can provide a reference for enhancing the stability of low-viscosity solutions on substrates for high-efficiency fabrication devices (NEMS/MEMS).
Astrocytes, one of the largest glial cell population in the central nervous system (CNS), play a key function in several events of brain development and function, such as synapse formation and function, control of neurotransmitters release and uptake, production of trophic factors and control of neuronal survival. Initially described as a homogenous population, several evidences have pointed that astrocytes are highly heterogeneous, both morphologically and functionally, within the same region, and across different brain regions. Recent findings suggest that the heterogeneity in the expression profile of proteins involved in astrocyte function may predict the selective vulnerability of brain regions to specific diseases, as well as to the age-related cognitive decline. However, the molecular mechanisms underlying these changes, either in aging as well as in brain disease are scarce. Neuroinflammation, a hallmark of several neurodegenerative diseases and aging, is reported to have a dubious impact on glial activation, as these cells release pro- and anti-inflammatory cytokines and chemokines, anti-oxidants, free radicals, and neurotrophic factors. Despite the emerging evidences supporting that reactive astrocytes have a duality in their phenotype, neurotoxic or neuroprotective properties, depending on the age and stimuli, the underlying mechanisms of their activation, cellular interplays and the impact of regional astrocyte heterogeneity are still a matter of discussion. In this review article, we will summarize recent findings on astrocyte heterogeneity and phenotypes, as well as their likely impact for the brain function during aging and neural diseases. We will focus on the molecules and mechanisms triggered by astrocyte to control synapse formation in different brain regions. Finally, we will discuss new evidences on how the modulation of astrocyte phenotype and function could impact the synaptic deficits and glial dysfunction present in aging and pathological states.
Astrocytes contribute to the formation, function, and plasticity of synapses. Their processes enwrap the neuronal components of the tripartite synapse, and due to this close interaction they are perfectly positioned to modulate neuronal communication. The interaction between astrocytes and synapses is facilitated by cell adhesion molecules and matricellular proteins, which have been implicated in the formation and functioning of tripartite synapses. The importance of such neuron-astrocyte integration at the synapse is underscored by the emerging role of astrocyte dysfunction in synaptic pathologies such as autism and schizophrenia. Here we review astrocyte-expressed cell adhesion molecules and matricellular molecules that play a role in integration of neurons and astrocytes within the tripartite synapse.
Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be “interpreted” by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected.
Micropatterning and manipulation of mammalian and bacterial cells are important in biomedical studies to perform in vitro assays and to evaluate biochemical processes accurately, establishing the basis for implementing biomedical microelectromechanical systems (bioMEMS), point-of-care (POC) devices, or organs-on-chips (OOC), which impact on neurological, oncological, dermatologic, or tissue engineering issues as part of personalizedmedicine. Cell patterning represents a crucial step in fundamental and applied biological studies in vitro, hence today there are a myriad of materials and techniques that allow one to immobilize and manipulate cells, imitating the 3D in vivo milieu. This review focuses on current physical cell patterning, plus chemical and a combination of them both that utilizes different materials and cutting-edge micro-nanofabrication methodologies.
Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly on cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface. Because of the typical piconewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging; sophisticated interdisciplinary methodologies are required. Moreover, an accurate control over the nanotopographical features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographical motifs on the mechanotransductive process. In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs. These probes provided the opportunity to detect nanotopography-specific modulations of the molecular clutch force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the nanotopographical features are responsible for an excessive force loading in single adhesion sites after 20 – 60 s of interaction, causing a drop in the number of adhesion sites. However, by manganese treatment we demonstrated that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics.
In their physiological microenvironment cells interact with the extracellular matrix (ECM); a complex structure build up by intertwined nanometric components (proteins and sugar chains). In the last two decades it became increasingly evident that the topographical complexity of the ECM encountered by cells at the nanoscale decisively influences cell behavior and fate through an intricate signaling pathway called mechanotransduction, i.e., the conversion of microenvironmental biophysical and structural cues into corresponding cellular responses. Classical cell culture devices and experimentation did not sufficiently take into account this aspect, and there is a need in cell biological research for substrates that mimic accurately the ECM nanotopography. This chapter introduces the molecular mechanisms by which cells perceive and “interpret” nanotopographical ECM cues and outlines why cluster-assembled nanostructured materials (produced by supersonic cluster beam deposition) are useful in this context as substrates for mechanobiological studies and bioengineering applications by providing reproducible ECM-like nanotopographical features.
Although many details remain still elusive, it became increasingly evident in recent years that mechanosensing of microenvironmental biophysical cues and subsequent mechanotransduction are strongly involved in the regulation of neuronal cell development and functioning. This review gives an overview about the current understanding of brain and neuronal cell mechanobiology and how it impacts on neurogenesis, neuronal migration, differentiation, and maturation. We will focus particularly on the events in the cell/microenvironment interface and the decisive extracellular matrix (ECM) parameters (i.e. rigidity and nanometric spatial organisation of adhesion sites) that modulate integrin adhesion complex-based mechanosensing and mechanotransductive signalling. It will also be outlined how biomaterial approaches mimicking essential ECM features help to understand these processes and how they can be used to control and guide neuronal cell behaviour by providing appropriate biophysical cues. In addition, principal biophysical methods will be highlighted that have been crucial for the study of neuronal mechanobiology.
Artificially grown neuronal cultures of brain cells have been used for decades in the attempt to reproduce and study in vitro the complexity of brain circuits. It soon became evident that this alone was insufficient, due to the random architecture of these artificial networks. Important groundwork resulted therefore in the development of methods to confine neuronal adhesion at specific locations to match
predefined network topologies and connectivity. Despite this notable progress in neural circuitry engineering, there is still need for micropatterned substrates that recapitulate better biophysical cues of
the neuronal microenvironment, taking into account recent findings of their significance for neuronal differentiation and functioning.
Here we report the development and characterization of a novel approach that; by using supersonic cluster beam deposition of zirconia nanoparticles, allows the patterning of small nanostructured cell-adhesive areas according to predefined geometries onto elsewhere non-adhesive antifouling glass surfaces. As distinguishing features, compared to other micropatterning approaches in this context, the
integrated nanostructured surfaces possess extracellular matrix-like nanotopographies of predetermined roughness; previously shown to be able to promote neuronal differentiation due to their impact on
mechanotransductive processes, and can be used in their original state without any coating requirements.
These micropatterned substrates were validated using: i) a neuron-like PC12 cell line; ii) primary cultures of rat hippocampal neurons. After initial uniform plating, both neuronal cells types were found to converge and adhere specifically to the nanostructured regions. The cell-adhesive areas effectively confined cells, even when these were highly mobile and repeatedly attempted to cross boundaries. Inside these small permissive islands, cells grew and differentiated; in case of the hippocampal neurons up to the formation of mature, functionally active and highly connected synaptic networks. In addition, when spontaneous instances of axon bridging between nearby dots occurred, a functional inter-dot communication between these subgroups of cells was observed.
Nanostructured films obtained by assembling preformed atomic clusters are of strategic importance for a wide variety of applications. The deposition of clusters produced in the gas phase onto a substrate offers the possibility to control and engineer the structural and functional properties of the cluster-assembled films. To date, the microscopic mechanisms underlying the growth and structuring of cluster-assembled films are poorly understood, and, in particular, the transition from the submonolayer to the thin-film regime is experimentally unexplored. Here we report the systematic characterization by atomic force microscopy of the evolution of the structural properties of cluster-assembled films deposited by supersonic cluster beam deposition. As a paradigm of nanostructured systems, we focus our attention on cluster-assembled zirconia films, investigating the influence of the building block dimensions on the growth mechanisms and roughening of the thin films, following the growth process from the early stages of the submonolayer to the thin-film regime. Our results demonstrate that the growth dynamics in the submonolayer regime determines different morphological properties of the cluster-assembled thin film. The evolution of the roughness with the number of deposited clusters reproduces the growth exponent of the ballistic deposition in the 2+1 model from the submonolayer to the thin-film regime.
Astrocytes are neural cells of ectodermal, neuroepithelial origin that provide for homeostasis and defense of the central nervous system (CNS). Astrocytes are highly heterogeneous in morphological appearance; they express a multitude of receptors, channels, and membrane transporters. This complement underlies their remarkable adaptive plasticity that defines the functional maintenance of the CNS in development and aging. Astrocytes are tightly integrated into neural networks and act within the context of neural tissue; astrocytes control homeostasis of the CNS at all levels of organization from molecular to the whole organ.