Defining Biomarkers in Stem Cell-Derived Tissue Constructs for Drug and Disease Screening.

The development of stem cell-derived tissue constructs (SCTCs) for clinical applications, including regenerative medicine, drug and disease screening offers significant hope for detecting and treating intractable disorders. SCTCs display a variety of biomarkers that can be used to understand biological mechanisms, assess drug interactions, and predict disease. Although SCTCs can be derived from patients and share the same genetic make-up, they are nevertheless distinct from human patients in many significant ways, which can undermine the clinical significance of measurements in SCTCs. This study defines biomarkers, how they apply to SCTCs, and clarifies specific ethical issues associated with the use of SCTCs for drug and disease screening.

Limitations in the electrochemical analysis of voltage transients.

Objective. Chronopotentiometric voltage transients (VTs) are used to assess the performance of bionic electrodes. The data obtained from VTs are used to define the safe operating conditions of clinical devices. Various approaches to analysing VTs have been reported, and a number of limitations in the accuracy of the measurements in relation to electrode size have been noted previously.Approach. The impact of electronic hardware and electrode configuration on VTs is discussed.Main results. The slew rate, rise time, sample time, minimum pulse length and waveform averaging characteristics of the electronic hardware, and electrode configuration will impact on VT measurement accuracy. Subsequently, activation and polarisation voltage measurements, and the definition of safe stimulation levels can be affected by the electronic hardware and electrode configuration.Significance. This article has identified some limitations in the previous literature related to the measurement and reporting of VTs and subsequent analysis of access and polarisation voltages. Furthermore, the commonly used Shannon plot used to define safe stimulation protocols does not correct for uncompensated resistance, account for electrode roughness or changes in electrode configuration. The creation of a safe stimulation plot which has been corrected for uncompensated resistance would generate more widely applicable stimulation guidelines for clinical devices used in different anatomical locations such as endovascular neural interfaces.

Electrochemistry in a Two- or Three-Electrode Configuration to Understand Monopolar or Bipolar Configurations of Platinum Bionic Implants.

Electrodes are used in vivo for chemical sensing, electrophysiological recording, and stimulation of tissue. The electrode configuration used in vivo is often optimised for a specific anatomy and biological or clinical outcomes, not electrochemical performance. Electrode materials and geometries are constrained by biostability and biocompatibility issues and may be required to function clinically for decades. We performed benchtop electrochemistry, with changes in reference electrode, smaller counter-electrode sizes, and three- or two-electrode configurations. We detail the effects different electrode configurations have on typical electroanalytical techniques used on implanted electrodes. Changes in reference electrode required correction by application of an offset potential. In a two-electrode configuration with similar working and reference/counter-electrode sizes, the electrochemical response was dictated by the rate-limiting charge transfer step at either electrode. This could invalidate calibration curves, standard analytical methods, and equations, and prevent use of commercial simulation software. We provide methods for determining if an electrode configuration is affecting the in vivo electrochemical response. We recommend sufficient details be provided in experimental sections on electronics, electrode configuration, and their calibration to justify results and discussion. In conclusion, the experimental limitations of performing in vivo electrochemistry may dictate what types of measurements and analyses are possible, such as obtaining relative rather than absolute measurements.

Ethical and regulatory issues of stem cell-derived 3-dimensional organoid and tissue therapy for personalised regenerative medicine.

BACKGROUND: Regenerative medicine has the potential to treat genetic disorders and replace damaged or missing tissue. The use of donor or animal tissue raises many well-known issues, including limited tissue availability, the possibility of rejection and patient infection. Stem cell therapy raised hope of overcoming these issues, but created new risks including tumour formation and limited benefit if the desired target tissue does not form. The recent development of 3-dimensional tissues, including organoids, allows the creation of more complex tissues for personalised regenerative medicine. METHODS: This article details the potential health risks of 3-dimensional organoid and tissue therapy versus dissociated stem cell therapy. The current ethical and regulatory issues surrounding 3-dimensional organoid and tissue therapy are presented with a focus on the highly influential FDA and International Society of Stem Cell Research (ISSCR) guidelines. CONCLUSIONS: The potential use of 3-dimensional organoid and tissue therapy may deliver greater patient benefits than other regenerative medicine approaches, but raises new health and ethical risks. Preclinical testing of these therapies will not mitigate some of their risks; they may only be understood after first-in-human trials. The potential irreversibility and high risk of these therapies affects how clinical trials should be structured, including post-trial care for participants.

CDKL5 deficiency disorder: molecular insights and mechanisms of pathogenicity to fast-track therapeutic development.

CDKL5 deficiency disorder (CDD) is an X-linked brain disorder of young children and is caused by pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene. Individuals with CDD suffer infantile onset, drug-resistant seizures, severe neurodevelopmental impairment and profound lifelong disability. The CDKL5 protein is a kinase that regulates key phosphorylation events vital to the development of the complex neuronal network of the brain. Pathogenic variants identified in patients may either result in loss of CDKL5 catalytic activity or are hypomorphic leading to partial loss of function. Whilst the progressive nature of CDD provides an excellent opportunity for disease intervention, we cannot develop effective therapeutics without in-depth knowledge of CDKL5 function in human neurons. In this mini review, we summarize new findings on the function of CDKL5. These include CDKL5 phosphorylation targets and the consequence of disruptions on signaling pathways in the human brain. This new knowledge of CDKL5 biology may be leveraged to advance targeted drug discovery and rapid development of treatments for CDD. Continued development of effective humanized models will further propel our understanding of CDD biology and may permit the development and testing of therapies that will significantly alter CDD disease trajectory in young children.

Investigating the feasibility and ethical implications of phenotypic screening using stem cell-derived tissue models to detect and manage disease.

Stem-cell-derived tissue models generated from sick people are being used to understand human development and disease, drug development, and drug screening. However, it is possible to detect disease phenotypes before a patient displays symptoms, allowing for their use as a disease screening tool. This raises numerous issues, some of which can be addressed using similar approaches from genetic screenings, while others are unique. One issue is the relationship between disease disposition, biomarker detection, and patient symptoms and how tissue models could be used to define disease. Other issues include decisions of when to screen, what diseases to screen for, and what treatment options should be offered.

Comparison of the In Vitro and In Vivo Electrochemical Performance of Bionic Electrodes.

The electrochemical performance of platinum electrodes was assessed in vitro and in vivo to determine the impact of electrode implantation and the relevance of in vitro testing in predicting in vivo behaviour. A significant change in electrochemical response was seen after electrode polarisation. As a result, initial in vitro measurements were poor predictors of subsequent measurements performed in vitro or in vivo. Charge storage capacity and charge density measurements from initial voltammetric measurements were not correlated with subsequent measurements. Electrode implantation also affected the electrochemical impedance. The typically reported impedance at 1 kHz was a very poor predictor of electrode performance. Lower frequencies were significantly more dependent on electrode properties, while higher frequencies were dependent on solution properties. Stronger correlations in impedance at low frequencies were seen between in vitro and in vivo measurements after electrode activation had occurred. Implanting the electrode increased the resistance of the electrochemical circuit, with bone having a higher resistivity than soft tissue. In contrast, protein fouling and fibrous tissue formation had a minimal impact on electrochemical response. In vivo electrochemical measurements also typically use a quasi-reference electrode, may operate in a 2-electrode system, and suffer from uncompensated resistance. The impact of these experimental conditions on electrochemical performance and the relevance of in vitro electrode assessment is discussed. Recommended in vitro testing protocols for assessing bionic electrodes are presented.

Restoring vision using optogenetics without being blind to the risks.

Retinit is pigmentosa is an incurable degenerative disease that causes loss of light-sensitive cells in the retina and leads to severe vision impairment. The development of optogenetics has created great hype around its potential to treat retinitis pigmentosa by the introduction of light-sensitive proteins into other neural cells in the retina. The first-in-human studies of optogenetic treatment for this disease have recently been reported (NCT02556736 and NCT03326336). The treatment involves irreversible gene therapy and requires access to specially designed goggles to deliver light to the treated eye. These highly innovative and high-profile clinical trials raise numerous ethical issues that must be addressed during the early phases of research and clinical testing to ensure trial participants are treated fairly and can provide appropriate informed consent.

Fibrinogen, collagen, and transferrin adsorption to poly(3,4-ethylenedioxythiophene)-xylorhamno-uronic glycan composite conducting polymer biomaterials for wound healing applications.

We present the conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) doped with an algal-derived glycan extract, Phycotrix™ [xylorhamno-uronic glycan (XRU84)], as an innovative electrically conductive material capable of providing beneficial biological and electrical cues for the promotion of favorable wound healing processes. Increased loading of the algal XRU84 into PEDOT resulted in a reduced surface nanoroughness and interfacial surface area and an increased static water contact angle. PEDOT-XRU84 films demonstrated good electrical stability and charge storage capacity and a reduced impedance relative to the control gold electrode. A quartz crystal microbalance with dissipation monitoring study of protein adsorption (transferrin, fibrinogen, and collagen) showed that collagen adsorption increased significantly with increased XRU84 loading, while transferrin adsorption was significantly reduced. The viscoelastic properties of adsorbed protein, characterized using the ΔD/Δf ratio, showed that for transferrin and fibrinogen, a rigid, dehydrated layer was formed at low XRU84 loadings. Cell studies using human dermal fibroblasts demonstrated excellent cell viability, with fluorescent staining of the cell cytoskeleton illustrating all polymers to present excellent cell adhesion and spreading after 24 h.

Understanding charge transfer on the clinically used conical Utah electrode array: charge storage capacity, electrochemical impedance spectroscopy and effective electrode area.

Objective. The Utah electrode is used for pre/clinical studies on neural recording and stimulation. Anecdotal and empirical reports on their performance have been made, resulting in variable testing methods. An in depth investigation was performed to understand the electrochemical behaviour and charge transfer mechanisms occurring on these clinically important electrodes. The impact of electrode geometry and material on performance was determined.Approach. Platinum and iridium electrodes were assessed by cyclic voltammetry and electrochemical impedance spectroscopy. The effective electrode area was measured by reduction of Ru(NH3)63+.Main results. Pristine Utah electrodes have little to no oxide present and the surface roughness is less than the diffusion length of Ru(NH3)63+during voltammetry, which was ∼30µm. Pristine iridium electrodes pass charge through capacitance and oxide formation. Hydride and anion adsorption occurs on the platinum electrode. Anodic current oxidises both metal surfaces, altering the charge transfer mechanisms at the electrode-solution interface. Charge storage capacity depends on measurement technique and electrode structure, this simplified number ignores more detailed information on charge transfer mechanisms that can be obtained from cyclic voltammetry. Electrode oxidation increases pseudocapacitance, reducing impedance. Charge transfer was non-homogeneous, most likely due to the electrode geometry enhancing charge density at the electrode tip and base. Oxidation of the electrode surface enhanced charge transfer inhomogeneity. The effective electrode area could be measured by reduction of Ru(NH3)63+and calculated with a finite cone geometry.Significance. Increasing electrode pseudocapacitance, demonstrated by metal oxidation, reduces impedance. Increasing electrode capacitance offers a potential route to reducing thermal noise and increasing signal-to-noise ratio of neural recording. The effective electrode area of conical electrodes can be measured. The charge density of the conical electrode was greater than expected compared to a planar disc electrode, indicating modification of electrode geometry can increase an electrodes safe charge injection capacity.in vivoelectrochemical measurements often do not include sufficient details to understand the electrode behaviour. Electrode oxidation most likely accounts for a significant amount of variation in previously published Utah electrode impedance data.

Modeling Emergent Properties in the Brain Using Tissue Models to Investigate Neurodegenerative Disease.

Here we describe emergent properties of the brain and the key challenges associated with modelling them in vitro. Modeling emergent properties of the brain will provide insights into brain function, development, and disease.

Facile Development of a Fiber-Based Electrode for Highly Selective and Sensitive Detection of Dopamine.

A facile one-step method was used to create a selective and sensitive electrode for dopamine (DA) detection based upon a stainless steel (SS) filament substrate and reduced graphene oxide (rGO). The electrode successfully and selectively detects DA in the presence of uric acid and ascorbic acid without the need for a Nafion coating. The proposed electrode is easy to fabricate, low-cost, flexible, and strong. The rGO-SS electrode could also be incorporated into a three-dimensional braided structure enabling DA detection in a two-electrode fiber system. The sensor is an excellent candidate for production of an affordable, robust, and flexible wearable and portable sensor and expands the application of textiles in point of care diagnostic devices.

Using Chronopotentiometry to Better Characterize the Charge Injection Mechanisms of Platinum Electrodes Used in Bionic Devices.

The safe charge injection capacity and charge density of neural stimulating electrodes is based on empirical evidence obtained from stimulating feline cortices. Stimulation induced tissue damage may be caused by electrochemical or biological mechanisms. Separating these mechanisms requires greater understanding of charge transfer at the electrode-tissue interface. Clinical devices typically use a biphasic waveform with controlled current. Therefore, the charge injection mechanism and charge injection capacity of platinum was assessed on a commercial potentiostat by chronopotentiometry (controlled current stimulation). Platinum is a non-ideal electrode, charge injection by chronopotentiometry can be passed via capacitive and Faradaic mechanisms. Electrodes were tested under a variety of conditions to assess the impact on charge injection capacity. The change in electrode potential (charge injection capacity) was affected by applied charge density, pulse length, pulse polarity, electrode size, polishing method, electrolyte composition, and oxygen concentration. The safe charge injection capacity and charge density could be increased by changing the electrode-solution composition and stimulation parameters. However, certain conditions (e.g., acid polished electrodes) allowed the electrode to exceed the water electrolysis potential despite the stimulation protocol being deemed safe according to the Shannon plot. Multiple current pulses led to a shift or ratcheting in electrode potential due to changes in the electrode-solution composition. An accurate measure of safe charge injection capacity and charge density of an implantable electrode can only be obtained from suitable conditions (an appropriately degassed electrolyte and clinically relevant electrode structure). Cyclic voltammetric measurement of charge storage capacity can be performed on implantable electrodes, but will not provide information on electrode stability to multiple chronopotentiometric pulses. In contrast, chronopotentiometry will provide details on electrode stability, but the minimum time resolution of typical commercial potentiostats (ms range) is greater than used in a clinical stimulator (µs range) so that extrapolation to short stimulation pulses is required. Finally, an impedance test is typically used to assess clinical electrode performance. The impedance test is also based on a biphasic chronopotentiometic waveform where the measured potential is used to calculate an impedance value. Here it is shown that the measured potential is a function of many parameters (solution composition, electrode area, and surface composition). Subsequently, impedance test results allow electrode comparison and to indicate electrode failure, but use of Ohm's law to calculate an impedance value is not valid.

Predicting neural recording performance of implantable electrodes.

Recordings of neural activity can be used to aid communication, control prosthetic devices or alleviate disease symptoms. Chronic recordings require a high signal-to-noise ratio that is stable for years. Current cortical devices generally fail within months to years after implantation. Development of novel devices to increase lifetime requires valid testing protocols and a knowledge of the critical parameters controlling electrophysiological performance. Here we present electrochemical and electrophysiological protocols for assessing implantable electrodes. Biological noise from neural recording has significant impact on signal-to-noise ratio. A recently developed surgical approach was utilised to reduce biological noise. This allowed correlation of electrochemical and electrophysiological behaviour. The impedance versus frequency of modified electrodes was non-linear. It was found that impedance at low frequencies was a stronger predictor of electrophysiological performance than the typically reported impedance at 1 kHz. Low frequency impedance is a function of electrode area, and a strong correlation of electrode area with electrophysiological response was also seen. Use of these standardised testing protocols will allow future devices to be compared before transfer to preclinical and clinical trials.

Measuring the effective area and charge density of platinum electrodes for bionic devices.

OBJECTIVE: Neural stimulation is usually performed with fairly large platinum electrodes. Smaller electrodes increase the applied charge density, potentially damaging the electrode. Greater understanding of the charge injection mechanism is required for safe neural stimulation. APPROACH: The charge injection mechanism and charge injection capacity were measured by cyclic voltammetry. Electrodes were cleaned mechanically or by potential cycling in acidic solutions. The effective electrode area was measured by hydrogen adsorption or reduction of [Formula: see text]. MAIN RESULTS: The water window and safe potential window were affected by changes to electrolyte, electrode size, polishing method and oxygen concentration. Capacitance and Faradaic current contribute to the charge injection capacity. Varying voltammetric scan rate (measurement time), electrode size, polishing method, potential window, electrolyte and oxygen concentration affected the charge injection capacity and ratio of oxidation to reduction charge. Hydrogen adsorption in acidic solutions provided an inaccurate effective electrode area. Reduction of a solution phase redox species with a linear or radial diffusion profile could provide an effective electrode area. The charge density (charge injection capacity divided by electrode area) of a platinum electrode is dependent on the charge injection capacity and electrode area measurement technique. By varying cyclic voltammetric conditions, the charge density of platinum ranged from 0.15 to 5.57 mC cm-2. SIGNIFICANCE: The safe potential window, charge injection mechanism, charge injection capacity and charge density of platinum depends on electrolyte, size of the electrode, oxygen concentration and differences in electrode polishing method. The oxidation and reduction charge injection capacities are not equal. Careful control of a platinum electrodes surface may allow larger charge densities and safe use of smaller electrodes. New electrode materials and geometries should be tested in a consistent manner to allow comparison of potential suitability for neural stimulation.