Francesca Santoro

Interfacing electrogenic cells with 3D nanoelectrodes: position, shape, and size matter.

An in-depth understanding of the interface between cells and nanostructures is one of the key challenges for coupling electrically excitable cells and electronic devices. Recently, various 3D nanostructures have been introduced to stimulate and record electrical signals emanating from inside of the cell. Even though such approaches are highly sensitive and scalable, it remains an open question how cells couple to 3D structures, in particular how the engulfment-like processes of nanostructures work. Here, we present a profound study of the cell interface with two widely used nanostructure types, cylindrical pillars with and without a cap. While basic functionality was shown for these approaches before, a systematic investigation linking experimental data with membrane properties was not presented so far. The combination of electron microscopy investigations with a theoretical membrane deformation model allows us to predict the optimal shape and dimensions of 3D nanostructures for cell-chip coupling.

On Chip Guidance and Recording of Cardiomyocytes with 3D Mushroom-Shaped Electrodes

The quality of the recording and stimulation capabilities of multielectrode arrays (MEAs) substantially depends on the interface properties and the coupling of the cell with the underlying electrode area. The purpose of this work was the investigation of a three-dimensional nanointerface, enabling simultaneous guidance and recording of electrogenic cells (HL-1) by utilizing nanostructures with a mushroom shape on MEAs.

Revealing the Cell–Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy

The interface between cells and nonbiological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influence cellular responses; for example, titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compared to a smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-material interface at the relevant nanometer length scale. Here, we present a method for in situ examination of the cell-to-material interface at any desired location, based on focused ion beam milling and scanning electron microscopy imaging to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary by more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future.

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

Defined Patterns of Neuronal Networks on 3D Thiol-functionalized Microstructures

It is very challenging to study the behavior of neuronal cells in a network due to the multiple connections between the cells. Our idea is then to simplify such a network with a configuration where cells can have just a fixed number of connections in order to create a well-defined and ordered network. Here, we report about guiding primary cortical neurons with three-dimensional gold microspines selectively functionalized with an amino-terminated molecule.

Neurospheres on Patterned PEDOT:PSS Microelectrode Arrays Enhance Electrophysiology Recordings

Microelectrode arrays (MEAs) are a versatile diagnostic tool to study neural networks. Culture of primary neurons on these platforms allows for extracellular recordings of action potentials. Despite many advances made in the technology to improve such recordings, the recording yield on MEAs remains sparse. Here, enhanced recording yield is shown induced by varying cell densities on poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate)‐coated MEAs. It is demonstrated that high cell densities (900 cells mm−2) of primary cortical cells increase the number of recording electrodes by 53.1% ± 11.3%, compared with low cell densities (500 cells mm−2) with 6.3% ± 1.4%. To further improve performance, 3D clusters known as neurospheres are cultured on the MEAs, significantly increasing single unit activity recordings. Extensive spike sorting is performed to analyze the unit activity recording multiple neurons with a single microelectrode. Finally, patterning of polyethylene glycol diacrylate through laser ablation is demonstrated, as a means to more precisely confine neurospheres on top of the electrodes. The possibility of recording single neurons with multiple neighboring electrodes is shown. Overall, a total recording yield of 21.4% is achieved, with more than 90% obtained from electrodes with neurospheres, maximizing the functionality of these planar MEAs as effective tools to study pharmacology‐based effects on neural networks.

Enhanced Cell–Chip Coupling by Rapid Femtosecond Laser Patterning of Soft PEDOT:PSS Biointerfaces

Interfacing soft materials with biological systems holds considerable promise for both biosensors and recording live cells. However, the interface between cells and organic substrates is not well studied, despite its crucial role in the effectiveness of the device. Furthermore, well-known cell adhesion enhancers, such as microgrooves, have not been implemented on these surfaces. Here, we present a nanoscale characterization of the cell-substrate interface for 3D laser-patterned organic electrodes by combining electrochemical impedance spectroscopy (EIS) and scanning electron microscopy/focused ion beam (SEM/FIB). We demonstrate that introducing 3D micropatterned grooves on organic surfaces enhances the cell adhesion of electrogenic cells.

Interfacing Cells with Vertical Nanoscale Devices: Applications and Characterization

Measurements of the intracellular state of mammalian cells often require probes or molecules to breach the tightly regulated cell membrane. Mammalian cells have been shown to grow well on vertical nanoscale structures in vitro, going out of their way to reach and tightly wrap the structures. A great deal of research has taken advantage of this interaction to bring probes close to the interface or deliver molecules with increased efficiency or ease. In turn, techniques have been developed to characterize this interface. Here, we endeavor to survey this research with an emphasis on the interface as driven by cellular mechanisms.

Revealing The Cell-Material Interface With Nanometer Resolution By FIB-SEM

The interface between biological cells and non-biological surfaces profoundly influences cellular activities, chronic tissue responses, and ultimately the success of medical implants. Materials in contact with cells can be plastics, metal, ceramics or other synthetic materials, and their surfaces vary widely in chemical compositions, stiffness, topography and levels of roughness. To understand the molecular mechanism of how cells and tissues respond to different materials, it is of critical importance to directly visualize the cell-material interface at the relevant length scale of nanometers. Conventional ultrastructural analysis by transmission electron microscopy (TEM) often requires substrate removal before microtome sectioning, which is not only challenging for most substrates but also can cause structural distortions of the interface. Here, we present a new method for in situ examination of the cell-to-material interface at any desired cellular location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM). This method involves a thin-layer plastification procedure that preserves adherent cells as well as enhances the contrast of biological specimen. We demonstrate that this unique procedure allows the visualization of cell-to-material interface and intracellular structures with 10nm resolution, compatible with a variety of materials and surface topographies, and capable of volume and multi-directional imaging. We expect that this method will be very useful for studies of cell-to-material interactions and also suitable for in vivo studies such as examining osteoblast adhesion and new bone formation in response to titanium implants.