Real-Time Monitoring of Cellular Cultures with Electrolyte-Gated Carbon Nanotube Transistors.

Cell-based biosensors constitute a fundamental tool in biotechnology, and their relevance has greatly increased in recent years as a result of a surging demand for reduced animal testing and for high-throughput and cost-effective in vitro screening platforms dedicated to environmental and biomedical diagnostics, drug development, and toxicology. In this context, electrochemical/electronic cell-based biosensors represent a promising class of devices that enable long-term and real-time monitoring of cell physiology in a noninvasive and label-free fashion, with a remarkable potential for process automation and parallelization. Common limitations of this class of devices at large include the need for substrate surface modification strategies to ensure cell adhesion and immobilization, limited compatibility with complementary optical cell-probing techniques, and the need for frequency-dependent measurements, which rely on elaborated equivalent electrical circuit models for data analysis and interpretation. We hereby demonstrate the monitoring of cell adhesion and detachment through the time-dependent variations in the quasi-static characteristic current curves of a highly stable electrolyte-gated transistor, based on an optically transparent network of printable polymer-wrapped semiconducting carbon-nanotubes.

High-Aspect-Ratio Semiconducting Polymer Pillars for 3D Cell Cultures.

Hybrid interfaces between living cells and nano/microstructured scaffolds have huge application potential in biotechnology, spanning from regenerative medicine and stem cell therapies to localized drug delivery and from biosensing and tissue engineering to neural computing. However, 3D architectures based on semiconducting polymers, endowed with responsivity to visible light, have never been considered. Here, we apply for the first time a push-coating technique to realize high aspect ratio polymeric pillars, based on polythiophene, showing optimal biocompatibility and allowing for the realization of soft, 3D cell cultures of both primary neurons and cell line models. HEK-293 cells cultured on top of polymer pillars display a remarkable change in the cell morphology and a sizable enhancement of the membrane capacitance due to the cell membrane thinning in correspondence to the pillars' top surface, without negatively affecting cell proliferation. Electrophysiology properties and synapse number of primary neurons are also very well preserved. In perspective, high aspect ratio semiconducting polymer pillars may find interesting applications as soft, photoactive elements for cell activity sensing and modulation.

The Photophysics of Polythiophene Nanoparticles for Biological Applications.

In this work the photophysics of poly(3-hexylthiophene) nanoparticles (NPs) is investigated in the context of their biological applications. The NPs, made as colloidal suspensions in aqueous buffers, present a distinct absorption band in the low-energy region. On the basis of systematic analysis of absorption and transient absorption (TA) spectra taken under different pH conditions, this band is associated with charge-transfer states generated by the polarization of loosely bound polymer chains and originating from complexes formed with electron-withdrawing species. Importantly, the ground-state depletion of these states upon photoexcitation is active even on microsecond timescales, thus suggesting that they act as precursor states for long-living polarons; this could be beneficial for cellular stimulation. Preliminary transient absorption microscopy results for NPs internalized within the cells reveal the presence of long-living species, further substantiating their relevance in biointerfaces.

Photocatalytic Activity of Polymer Nanoparticles Modulates Intracellular Calcium Dynamics and Reactive Oxygen Species in HEK-293 Cells.

Optical modulation of living cells activity by light-absorbing exogenous materials is gaining increasing interest, due to the possibility both to achieve high spatial and temporal resolution with a minimally invasive and reversible technique and to avoid the need of viral transfection with light-sensitive proteins. In this context, conjugated polymers represent ideal candidates for photo-transduction, due to their excellent optoelectronic and biocompatibility properties. In this work, we demonstrate that organic polymer nanoparticles, based on poly(3-hexylthiophene) conjugated polymer, establish a functional interaction with an in vitro cell model (Human Embryonic Kidney cells, HEK-293). They display photocatalytic activity in aqueous environment and, once internalized within the cell cytosol, efficiently generate reactive oxygen species (ROS) upon visible light excitation, without affecting cell viability. Interestingly, light-activated ROS generation deterministically triggers modulation of intracellular calcium ion flux, successfully controlled at the single cell level. In perspective, the capability of polymer NPs to produce ROS and to modulate Ca2+ dynamics by illumination on-demand, at non-toxic levels, may open the path to the study of biological processes with a gene-less approach and unprecedented spatio-temporal resolution, as well as to the development of new biotechnology tools for cell optical modulation.

Tattoo-Paper Transfer as a Versatile Platform for All-Printed Organic Edible Electronics.

The use of natural or bioinspired materials to develop edible electronic devices is a potentially disruptive technology that can boost point-of-care testing. The technology exploits devices that can be safely ingested, along with pills or even food, and operated from within the gastrointestinal tract. Ingestible electronics can potentially target a significant number of biomedical applications, both as therapeutic and diagnostic tool, and this technology may also impact the food industry, by providing ingestible or food-compatible electronic tags that can "smart" track goods and monitor their quality along the distribution chain. Temporary tattoo-paper is hereby proposed as a simple and versatile platform for the integration of electronics onto food and pharmaceutical capsules. In particular, the fabrication of all-printed organic field-effect transistors on untreated commercial tattoo-paper, and their subsequent transfer and operation on edible substrates with a complex nonplanar geometry is demonstrated.

Semiconducting polymers are light nanotransducers in eyeless animals.

Current implant technology uses electrical signals at the electrode-neural interface. This rather invasive approach presents important issues in terms of performance, tolerability, and overall safety of the implants. Inducing light sensitivity in living organisms is an alternative method that provides groundbreaking opportunities in neuroscience. Optogenetics is a spectacular demonstration of this, yet is limited by the viral transfection of exogenous genetic material. We propose a nongenetic approach toward light control of biological functions in living animals. We show that nanoparticles based on poly(3-hexylthiophene) can be internalized in eyeless freshwater polyps and are fully biocompatible. Under light, the nanoparticles modify the light response of the animals, at two different levels: (i) they enhance the contraction events of the animal body, and (ii) they change the transcriptional activation of the opsin3-like gene. This suggests the establishment of a seamless and biomimetic interface between the living organism and the polymer nanoparticles that behave as light nanotransducers, coping with or amplifying the function of primitive photoreceptors.

Poly(3-hexylthiophene) nanoparticles for biophotonics: study of the mutual interaction with living cells.

We report on the mutual interaction between poly(3-hexylthiophene) nanoparticles (P3HT-NPs) and human embryonic kidney (HEK-293) cells. P3HT-NPs, prepared in sterile conditions and efficiently uptaken within the live cells cytosol, show well-ordered morphology, high colloidal stability and excellent biocompatibility. Electrophysiology and calcium imaging experiments demonstrate that physiological functions of live cells are fully preserved in the presence of P3HT-NPs. From a complementary point of view, the photophysical properties of P3HT-NPs are also mainly maintained within the cellular environment, as proven by in situ time-resolved photoluminescence. Interestingly, we detect slight modifications in emission spectra and dynamics, which we ascribe to the contribution from the P3HT-NPs surface, possibly due to conformational changes as the result of the interaction with intracellular proteins or the formation of NPs aggregates. This work demonstrates that P3HT-NPs are excellent candidates for use as light sensitive actuators, due to their remarkable physical properties, optimal biocompatibility and capability of interaction with living cells.

Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

Conjugated polymers for the optical control of the electrical activity of living cells.

The possibility to optically excite the electrical activity of living cells by using exogenous absorbers is gaining more and more interest in the neuroscience and biotechnology community. Conjugated polymers, inherently sensitive to visible light, were recently proposed as candidates to this goal. To date, however, only one polymer type, namely regio-regular poly-3-hexylthiophene, has been tested as the active material. In this work four different conjugated polymers, regarded as prototypes of their category, are investigated as photoactive bio-interfaces. The selected materials have different absorption spectra, morphology, light emission efficiency and charge transport properties. We analyze their key-enabling properties, such as electrochemical stability, surface morphology, wettability, sterilization compatibility, interaction with protein adhesion layers and toxicity, throughout all the necessary steps for the realization of an efficient bio-optical interface. We demonstrate that all considered polymers are characterized by good biocompatibility and cell seeding properties, and can optimally sustain thermal sterilization. Conversely, electrochemical stability and cell photostimulation efficacy can vary a lot among different materials, and should be carefully evaluated case by case. Reported results represent the starting point for the implementation of bio-polymer interfaces sensitive to different colors and, in perspective, for the realization of a three-chromatic artificial visual prosthesis.

Photothermal cellular stimulation in functional bio-polymer interfaces.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications, bearing a huge potential, from basic researches to clinical applications. In particular, light sensitive conjugated polymers can be exploited as a new approach for optical modulation of cellular activity. In this work we focus on light-induced changes in the membrane potential of Human Embryonic Kidney (HEK-293) cells grown on top of a poly(3-hexylthiophene) (P3HT) thin film. On top of a capacitive charging of the polymer interface, we identify and fully characterize two concomitant mechanisms, leading to membrane depolarization and hyperpolarisation, both mediated by a thermal effect. Our results can be usefully exploited in the creation of a new platform for light-controlled cell manipulation, with possible applications in neuroscience and medicine.

Mesenchymal stem cell differentiation on electrochemically modified titanium: an optimized approach for biomedical applications.

PURPOSE: To speed up the osteointegration process, surface-treated titanium has been widely used in dental and orthopedic applications. The present work describes a new silicon-based anodic spark deposition (ASD) treatment and investigates the properties of the surfaces obtained, focusing on their capability to modulate the osteogenic differentiation potential of adult mesenchymal stem cells (MSCs). METHODS: The surfaces examined were obtained from commercially pure grade 2 titanium by a single-step ASD (SUM) eventually followed by a thermal treatment in alkali solution (SUMNa), while acid-etched titanium (AE; NextMaterials s.r.l.) was selected as a control. Their morphology, elemental composition, crystallographic structure of the Ti2O layer, wettability and topography were evaluated by scanning electron microscopy, energy-dispersive X-ray spectroscopy, thin-film X-ray diffraction, contact angle measurements and laser profilometry, respectively. MSCs' response to surface properties was assessed by examining cell morphology and viability by scanning electron microscopy and Alamar Blue assay®, while their osteogenic differentiation potential was investigated by evaluating the levels of the enzyme alkaline phosphatase (ALP) and the degree of calcium accumulation by Alizarin Red-S (AR-S) staining. RESULTS: The proposed ASD treatment has allowed the obtaining of surfaces with round-shaped micrometric pores, enriched in calcium, phosphorus and silicon and significantly more wettable than controls; furthermore, the treatment has been shown to promote MSC proliferation and the degree of in vitro mineralization. CONCLUSIONS: The described ASD treatment may be an effective technique to modify the surface cues of titanium implants, aiming at enhancing the conveying of osteoprogenitor cells and their functional differentiation in bone cells.

A simple method to generate adipose stem cell-derived neurons for screening purposes.

Strategies involved in mesenchymal stem cell (MSC) differentiation toward neuronal cells for screening purposes are characterized by quality and quantity issues. Differentiated cells are often scarce with respect to starting undifferentiated population, and the differentiation process is usually quite long, with high risk of contamination and low yield efficiency. Here, we describe a novel simple method to induce direct differentiation of MSCs into neuronal cells, without neurosphere formation. Differentiated cells are characterized by clear morphological changes, expression of neuronal specific markers, showing functional response to depolarizing stimuli and electrophysiological properties similar to those of developing neurons. The method described here represents a valuable tool for future strategies aimed at personalized screening of therapeutic agents in vitro.