Small-artery-mimicking multi-layered 3D co-culture in a self-folding porous graphene-based film.

In vitro vessel-mimicking models have been spotlighted as a powerful tool for investigating cellular behaviours in vascular development and diseases. However, it is still challenging to create micro-scale tubular tissues while mimicking the structural features of small arteries. Here, we propose a 3D culture model of small vascular tissue using a self-folding graphene-based porous film. Vascular endothelial cells were encapsulated within the self-folding film to create a cellular construct with a controlled curvature radius ranging from 10 to 100 µm, which is comparable to the size of a human arteriole. Additionally, vascular endothelial cells and smooth muscle cells were separately co-cultured on the inner and outer surfaces of the folded film, respectively. The porous wall worked as a permeable barrier between them, affecting the cell-cell communications like the extracellular layer in the artery wall. Thus, the culture model recapitulates the structural features of a small artery and will help us better understand intercellular communications at the artery wall in physiological and pathological conditions.

Printed Silk Microelectrode Arrays for Electrophysiological Recording and Controlled Drug Delivery.

The use of soft and flexible bioelectronic interfaces can enhance the quality for recording cells' electrical activity by ensuring a continuous and intimate contact with the smooth, curving surfaces found in the physiological environment. This work develops soft microelectrode arrays (MEAs) made of silk fibroin (SF) films for recording interfaces that can also serve as a drug delivery system. Inkjet printing is used as a tool to deposit the substrate, conductive electrode, and insulator, as well as a drug-delivery nanocomposite film. This approach is highly versatile, as shown in the fabrication of carbon microelectrodes, sandwiched between a silk substrate and a silk insulator. The technique permits the development of thin-film devices that can be employed for in vitro extracellular recordings of HL-1 cell action potentials. The tuning of SF by applying an electrical stimulus to produce a permeable layer that can be used in on-demand drug delivery systems is also demonstrated. The multifunctional MEA developed here can pave the way for in vitro drug screening by applying time-resolved and localized chemical stimuli.

Polarity and chirality control of an active fluid by passive nematic defects.

Much like passive materials, active systems can be affected by the presence of imperfections in their microscopic order, called defects, that influence macroscopic properties. This suggests the possibility to steer collective patterns by introducing and controlling defects in an active system. Here we show that a self-assembled, passive nematic is ideally suited to control the pattern formation process of an active fluid. To this end, we force microtubules to glide inside a passive nematic material made from actin filaments. The actin nematic features self-assembled half-integer defects that steer the active microtubules and lead to the formation of macroscopic polar patterns. Moreover, by confining the nematic in circular geometries, chiral loops form. We find that the exact positioning of nematic defects in the passive material deterministically controls the formation and the polarity of the active flow, opening the possibility of efficiently shaping an active material using passive defects.

Filtration-processed biomass nanofiber electrodes for flexible bioelectronics.

An increasing demand for bioelectronics that interface with living systems has driven the development of materials to resolve mismatches between electronic devices and biological tissues. So far, a variety of different polymers have been used as substrates for bioelectronics. Especially, biopolymers have been investigated as next-generation materials for bioelectronics because they possess interesting characteristics such as high biocompatibility, biodegradability, and sustainability. However, their range of applications has been restricted due to the limited compatibility of classical fabrication methods with such biopolymers. Here, we introduce a fabrication process for thin and large-area films of chitosan nanofibers (CSNFs) integrated with conductive materials. To this end, we pattern carbon nanotubes (CNTs), silver nanowires, and poly (3,4-ethylenedioxythiophene):poly (styrenesulfonate) (PEDOT:PSS) by a facile filtration process that uses polyimide masks fabricated via laser ablation. This method yields feedlines of conductive material on nanofiber paper and demonstrates compatibility with conjugated and high-aspect-ratio materials. Furthermore, we fabricate a CNT neural interface electrode by taking advantage of this fabrication process and demonstrate peripheral nerve stimulation to the rapid extensor nerve of a live locust. The presented method might pave the way for future bioelectronic devices based on biopolymer nanofibers.

Lab-on-a-chip based mechanical actuators and sensors for single-cell and organoid culture studies.

All living cells constantly experience and respond to mechanical stresses. The molecular networks that activate in cells in response to mechanical stimuli are yet not well-understood. Our limited knowledge stems partially from the lack of available tools that are capable of exerting controlled mechanical stress to individual cells and at the same time observing their responses at subcellular to molecular resolution. Several tools such as rheology setups, micropipetes, and magnetic tweezers have been used in the past. While allowing to quantify short-time viscoelastic responses, these setups are not suitable for long-term observations of cells and most of them have low throughput. In this Perspective, we discuss lab-on-a-chip platforms that have the potential to overcome these limitations. Our focus is on devices that apply shear, compressive, tensile, and confinement derived stresses to single cells and organoid cultures. We compare different design strategies for these devices and highlight their advantages, drawbacks, and future potential. While the majority of these devices are used for fundamental research, some of them have potential applications in medical diagnostics and these applications are also discussed.

Graphene-based neuron encapsulation with controlled axonal outgrowth.

Neuronal constructs with tuneable 3D geometry can contribute greatly to the construction of brain-like functional tissues for transplantable grafts and robust experimental models. In this study, we propose a self-folding graphene/polymer bilayer film that forms a micro-roll for neuron encapsulation, and highlight the importance of employing pores on the micro-roll to allow neurons to interact with their surroundings. The micro-patterns and varied thicknesses of the bilayer provide control over the 3D geometries of the micro-roll. The pores facilitate the diffusion of reagents, resulting in the adequate loading of probes for imaging and the successful stimulation of the encapsulated neurons. Moreover, the encapsulated neurons inside the micro-roll are functionally integrated into surrounding neuronal networks by extending their axons through the pores. Thus, our method for encapsulating neurons with a porous graphene-laden film allows the construction of precisely shaped neuronal tissues that interact with their surroundings. We believe that the method will open a new avenue for the reconstruction of functional neuronal tissues and is potentially applicable to other self-folding bilayers.

Self-Folded Three-Dimensional Graphene with a Tunable Shape and Conductivity.

Three-dimensional (3D) graphene architectures are of great interest as applications in flexible electronics and biointerfaces. In this study, we demonstrate the facile formation of predetermined 3D polymeric microstructures simply by transferring monolayer graphene. The graphene adheres to the surface of polymeric films via noncovalent π-π stacking bonding and induces a sloped internal strain, leading to the self-rolling of 3D microscale architectures. Micropatterns and varied thicknesses of the 2D films prior to the self-rolling allows for control over the resulting 3D geometries. The strain then present on the hexagonal unit cell of the graphene produces a nonlinear electrical conductivity across the device. The driving force behind the self-folding process arises from the reconfiguration of the molecules within the crystalline materials. We believe that this effective and versatile way of realizing a 3D graphene structure is potentially applicable to alternative 2D layered materials as well as other flexible polymeric templates.

Cell Assembly in Self-foldable Multi-layered Soft Micro-rolls.

Multi-layered thin films with heterogeneous mechanical properties can be spontaneously transformed to realise various three-dimensional (3D) geometries. Here, we describe micro-patterned all-polymer films called micro-rolls that we use for encapsulating, manipulating, and observing adherent cells in vitro. The micro-rolls are formed of twin-layered films consisting of two polymers with different levels of mechanical stiffness; therefore they can be fabricated by using the strain engineering and a self-folding rolling process. By controlling the strain of the films geometrically, we can achieve 3D tubular architectures with controllable diameters. Integration with a batch release of sacrificial hydrogel layers provides a high yield and the biocompatibility of the micro-rolls with any length in the release process without cytotoxicity. Thus, the multiple cells can be wrapped in individual micro-rolls and artificially reconstructed into hollow or fibre-shaped cellular 3D constructs that possess the intrinsic morphologies and functions of living tissues. This system can potentially provide 3D bio-interfaces such as those needed for reconstruction and assembly of functional tissues and implantable tissue grafts.