Development and Characterization of PEDOT:PSS/Alginate Soft Microelectrodes for Application in Neuroprosthetics

Reducing the mechanical mismatch between the stiffness of a neural implant and the softness of the neural tissue is still an open challenge in neuroprosthetics. The emergence of conductive hydrogels in the last few years has considerably widened the spectrum of possibilities to tackle this issue. Nevertheless, despite the advancements in this field, further improvements in the fabrication of conductive hydrogel-based electrodes are still required. In this work, we report the fabrication of a conductive hydrogel-based microelectrode array for neural recording using a hybrid material composed of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), and alginate. The mechanical properties of the conductive hydrogel have been investigated using imaging techniques, while the electrode arrays have been electrochemically characterized at each fabrication step, and successfully validated both in vitro and in vivo. The presence of the conductive hydrogel, selectively electrodeposited onto the platinum microelectrodes, allowed achieving superior electrochemical characteristics, leading to a lower electrical noise during recordings. These findings represent an advancement in the design of soft conductive electrodes for neuroprosthetic applications

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Life sciences have a constantly growing need for novel methodological approaches suitable to investigate the intracellular environment with increased temporal and spatial resolutions. Recently, optical near-field probes, such as laser-irradiated pointed metal, uncoated/metal-coated tapered optical fibers, as well as nano-emitters, such as single molecules or nanoparticles, have attracted increasing attention as key components of high-resolution microscopes. In parallel, super-resolution fluorescence microscopy, operating in far-field regime and overcoming the light diffraction limit, has markedly improved imaging resolution. Although both near- and far-field optical approaches drastically improve image resolution, they still represent numerous drawbacks, such as, technical difficulties concerning probe preparation (near-field) or potential damaging through very high light intensities (far-field). The aforementioned shortcomings of high-resolution optical microscopy can be overcome to a great extent by photonic force microscopy (PFM). PFM employs a strongly focused near-infrared (NIR) laser light to hold a dielectric or metallic particle as a local probe. Such an optical trap enables one to ‘cage’ a mesoscopic particle and track its three-dimensional (3D) thermal fluctuations in the surrounding environment, e.g. a viscous liquid. Therefore, besides offering near-field 3D imaging, PFM reports also on other important quantities concerning local mechanical properties, including force and viscosity as well as dynamical properties of the surrounding medium. This additional information is derived from the careful analysis of the jittery motion (so-called Brownian motion) of the trapped single-particle probe that collides with thermally-activated surrounding molecules. In this thesis, we first study in detail the Brownian motion of a single spherical particle that is confined within the harmonic potential of the optical trap. To this end, we optimize the NIR light path and electronic noise floor of our custom-built PFM set-up for detecting and quantifying resonances in the Brownian motion. These resonances arise from the coupling between the hydrodynamic memory and strong strength of the optical trap. Due to the high sensitivity of the short-time dynamics, the size of the particle can be measured by simultaneous fitting of the velocity autocorrelation function and power spectral density of its thermal fluctuations. In order to choose the best-suitable spherical probe for a given experiment in PFM, computational modeling based on a Matlab toolbox is performed. The generalized Lorenz-Mie theory is computed using the T -matrix method for various experimental conditions, including changes in the size and refractive index of the sphere, as well as different laser polarization states. The axial equilibrium position is examined to predict its location compared with the position of the laser focus. Optical forces acting on the sphere are investigated in 3D to highlight, in particular, the limits of the perfectly harmonic potential assumption. These limits are quantified and discussed. Subsequently, we identify different types of inorganic nano/micro-sized particles that can be trapped by the NIR laser of the PFM and used simultaneously as mechanical and near-field light sources. To this end, we take advantage of the visible light emission from single probes confined in the optical trap and excited by the NIR laser light. The emphasis is on particles revealing nonlinear optical properties. In particular, nonlinear optical field enhancement resulting from excitation of surface plasmon resonance (SPR) on trapped gold particles of 200 nm is demonstrated by measuring the emission of that second-harmonic generation (SHG). Furthermore, we show, under optical trapping conditions, SHG emission from single potassium niobate (KNbO3) nano/micro-sized particles. We also report on the multicolor upconversion luminescence (UCL), at the single particle level, for randomly shaped crystals of sodium yttrium fluoride codoped with ytterbium and erbium, NaYF4:Yb,Er (UCC), when they are held in the optical trap. In parallel, 3D Brownian fluctuations of a single trapped particle are quantified. Moreover, luminescence resonance energy transfer (LRET) between a KNbO3 particle or an UCC and molecules of an organic dye (rose bengal) adsorbed on their surface is highlighted and analyzed. Finally, randomly shaped UCCs and hexagonal nanoplates are characterized in detail at the single particle level. The hexagonal β phase in the polycrystalline and monocrystalline crystals is identified, in both randomly and hexagonally shaped particles by electron microscopy. UCL emission from the trapped crystal is quantified in terms of power and found to be dependent on the laser power density, size and shape of the particle as well as its orientation within the trap. Most importantly, the different colors in the UCL spectrum do not vary with the same trend for different orientations of the randomly or hexagonally shaped crystal upon trapping. This suggests the existence of crystalline anisotropy-dependent UCL. Thereby, we should be able to design the best-suited particle probe for LRET experiments with subwavelength resolution

SpikeOnChip ::a custom embedded platform for neuronal activity recording and analysis

In this paper we present SpikeOnChip, a custom embedded platform for neuronal activity recording and online analysis. The SpikeOnChip platform was developed in the context of automated drug testing and toxicology assessments on neural tissue made from human induced pluripotent stem cells. The system was developed with the following goals: to be small, autonomous and low power, to handle micro-electrode arrays with up to 256 electrodes, to reduce the amount of data generated from the recording, to be able to do computation during acquisition, and to be customizable. This led to the choice of a Field Programmable Gate Array System-On-Chip platform. This paper focuses on the embedded system for acquisition and processing with key features being the ability to record electrophysiological signals from multiple electrodes, detect biological activity on all channels online for recording, and do frequency domain spectral energy analysis online on all channels during acquisition. Development methodologies are also presented. The platform is finally illustrated in a concrete experiment with bicuculline being administered to grown human neuronal tissue through microfluidics, resulting in measurable effects in the spike recordings and activity. The presented platform provides a valuable new experimental instrument that can be further extended thanks to the programmable hardware and software

The role of interstitial fluid pressure in cerebral porous biomaterial integration

Recent advances in biomaterials offer new possibilities for brain tissue reconstruction. Biocompatibility, provision of cell adhesion motives and mechanical properties are among the present main design criteria. We here propose a radically new and potentially major element determining biointegration of porous biomaterials: the favorable effect of interstitial fluid pressure (IFP). The force applied by the lymphatic system through the interstitial fluid pressure on biomaterial integration has mostly been neglected so far. We hypothesize it has the potential to force 3D biointegration of porous biomaterials. In this study, we develop a capillary hydrostatic device to apply controlled in vitro interstitial fluid pressure and study its effect during 3D tissue culture. We find that the IFP is a key player in porous biomaterial tissue integration, at physiological IFP levels, surpassing the known effect of cell adhesion motives. Spontaneous electrical activity indicates that the culture conditions are not harmful for the cells. Our work identifies interstitial fluid pressure at physiological negative values as a potential main driver for tissue integration into porous biomaterials. We anticipate that controlling the IFP level could narrow the gap between in vivo and in vitro and therefore decrease the need for animal screening in biomaterial design

Human neural organoids for studying brain cancer and neurodegenerative diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing

Development and characterization of PEDOT:PSS/alginate soft microelectrodes for application in neuroprosthetics

Reducing the mechanical mismatch between the stiffness of a neural implant and the softness of the neural tissue is still an open challenge in neuroprosthetics. The emergence of conductive hydrogels in the last few years has considerably widened the spectrum of possibilities to tackle this issue. Nevertheless, despite the advancements in this field, further improvements in the fabrication of conductive hydrogel-based electrodes are still required. In this work, we report the fabrication of a conductive hydrogel-based microelectrode array for neural recording using a hybrid material composed of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), and alginate. The mechanical properties of the conductive hydrogel have been investigated using imaging techniques, while the electrode arrays have been electrochemically characterized at each fabrication step, and successfully validated both in vitro and in vivo. The presence of the conductive hydrogel, selectively electrodeposited onto the platinum microelectrodes, allowed achieving superior electrochemical characteristics, leading to a lower electrical noise during recordings. These findings represent an advancement in the design of soft conductive electrodes for neuroprosthetic applications

Articular cartilage repair after implantation of hyaline cartilage beads engineered from adult dedifferentiated chondrocytes: cartibeads preclinical efficacy study in a large animal model

Background: Chondrocyte-based cell therapy to repair cartilage has been used for &gt;25 years despite current limitations. This work presents a new treatment option for cartilage lesions. Hypothesis: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. Study design: Controlled laboratory study. Methods: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. Results: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). Conclusion: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. Clinical relevance: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.</p