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Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

Implantable cell replacement therapies promise to completely restore the function of neural structures, possibly changing how we currently perceive the onset of neurodegenerative diseases. One of the major clinical hurdles for the routine implementation of stem cell therapies is poor cell retention and survival, demanding the need to better understand these mechanisms while providing precise and scalable approaches to monitor these cell-based therapies in both pre-clinical and clinical scenarios. This poses significant multidisciplinary challenges regarding planning, defining the methodology and requirements, prototyping and different stages of testing. Aiming toward an optogenetic neural stem cell implant controlled by a smart wireless electronic frontend, we show how an iterative development methodology coupled with a modular design philosophy can mitigate some of these challenges. In this study, we present a miniaturized, wireless-controlled, modular multisensor platform with fully interfaced electronics featuring three different modules: an impedance analyzer, a potentiostat and an optical stimulator. We show the application of the platform for electrical impedance spectroscopy-based cell monitoring, optical stimulation to induce dopamine release from optogenetically modified neurons and a potentiostat for cyclic voltammetry and amperometric detection of dopamine release. The multisensor platform is designed to be used as an opto-electric headstage for future in vivo animal experiments.

The pathophysiological progress of Parkinson's disease leads through degeneration of dopaminergic neurons in the substantia nigra to complete cell death and lack of dopamine in the striatum where it modulates motor functions. Transplantation of dopaminergic stem cell-derived neurons is a possible therapy to restore dopamine levels. We have previously presented multifunctional pyrolytic carbon coated leaky optoelectrical fibers (LOEFs) with laser ablated micro-optical windows (µOWs) as carriers for channelrhodopsin-2 modified optogenetically active neurons for light-induced on-demand dopamine release and amperometric real-time detection. To increase the dopamine release by stimulating a larger neuronal population with light, we present here a novel approach to generate µOWs through laser ablation around the entire circumference of optical fibers to obtain Omni-LOEFs. Cyclic voltammetric characterization of the pyrolytic carbon showed that despite the increased number of µOWs, the electrochemical properties were not deteriorated. Finally, we demonstrate that the current recorded during real-time detection of dopamine upon light-induced stimulation of neurons differentiated on Omni-LOEFs is significantly higher compared to recordings from the same number of cells seeded on LOEFs with µOWs only on one side. Moreover, by varying the cell seeding density, we show that the recorded current is proportional to the dimension of the cell population.

Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.

Brain organoids are considered to be a highly promising in vitro model for the study of the human brain and, despite their various shortcomings, have already been used widely in neurobiological studies. Especially for drug screening applications, a highly reproducible protocol with simple tissue culture steps and consistent output, is required. Here we present an engineering approach that addresses several existing shortcomings of brain organoids. By culturing brain organoids with a polycaprolactone scaffold, we were able to modify their shape into a flat morphology. Engineered flat brain organoids (efBOs) possess advantageous diffusion conditions and thus their tissue is better supplied with oxygen and nutrients, preventing the formation of a necrotic tissue core. Moreover, the efBO protocol is highly simplified and allows to customize the organoid size directly from the start. By seeding cells onto 12 by 12 mm scaffolds, the brain organoid size can be significantly increased. In addition, we were able to observe folding reminiscent of gyrification around day 20, which was self-generated by the tissue. To our knowledge, this is the first study that reports intrinsically caused gyrification of neuronal tissue in vitro. We consider our efBO protocol as a next step towards the generation of a stable and reliable human brain model for drug screening applications and spatial patterning experiments.

3D interdigitated pyrolytic carbon microelectrodes (3D IDE) with high complexity and interconnectivity were fabricated taking advantage of suspended interdigitated microstructures. A novel fabrication method for a 3D interdigitated polymer precursor template was developed based on a dual photoresist process including multiple UV exposures at two different wavelengths. The precursor structures were subsequently pyrolyzed at 1100ºC for 1 h in N2 environment to obtain 3D interdigitated carbon microelectrodes. Different 3D electrode designs were fabricated and the electrochemical performance was evaluated by cyclic voltammetry and impedance spectroscopy (EIS). 3D IDE with smaller structural dimensions displayed ultra-microelectrode (UME)-like behavior with a sigmoidal shape of the cyclic voltammograms (CV) and the absence of the linear diffusion regime in EIS. Furthermore, the 3D IDE displayed 2-fold higher peak currents in CV and significantly reduced charge transfer resistance compared to 2D IDE.

A reversible switchable on-demand UV-triggered drug delivery system (DDS) based on interpenetrating polymer networks (IPNs) with silicone as the host polymer and spiropyran (SP)-functionalized guest polymer is designed and demonstrated. The photo-responsive IPNs provide a new triggered drug delivery concept as they exploit the change in intermolecular interactions (work of adhesion) among the drug, matrix, and solvent when the incorporated hydrophobic SP moieties transform into the hydrophilic merocyanine form upon light irradiation without degradation and disruption of the DDS. The change in how the copolymer composition (hydrophilicity and content) and the lipophilicity of the drug (log P) affect the release profile was investigated. A thermodynamic model, based on Hansen solubility parameters, was developed to design and optimize the polymer composition of the IPNs to obtain the most efficient light-triggered drug release and suppression of the premature release. The developed IPNs showed excellent result for dopamine, l-dopa, and prednisone with around 90-95% light-triggered release. The model was applied to study the release behavior of drugs with different log P and to estimate if the light-induced hydrophobic-to-hydrophilic switch can overcome the work of adhesion between polymers and drugs and hence the desorption and release of the drugs. To the best of our knowledge, this is the first time that work of adhesion is used for this aim. Comparing the result obtained from the model and experiment shows that the model is useful for evaluating and estimating the release behavior of specific drugs merocyanine, IPN, DDS, and spiropyran.

A photoresponsive molecular-gated drug delivery system (DDS) based on silicone-hydrogel (poly(HEMA-co-PEGMEA)) interpenetrating polymer networks (IPNs) functionalized with carboxylated spiropyran (SPCOOH) was designed and demonstrated as an on-demand DDS. The triggered-release mechanism relies on controlling the wetting behavior of the surface by light, exploiting different hydrophobicities between the “closed” and “open” isomers of spiropyran as a photoswitchable molecular gate on the surface of IPN (SP-photogated IPN). Light-triggered release of doxycycline (DOX) as a model drug indicated that the spiropyran (SP) molecules provide a hydrophobic layer around the drug carrier and have a good gate-closing efficiency for IPNs with 20–30% hydrogel content. Upon UV light irradiation, SP converts into an open hydrophilic merocyanine state, which triggers the release of DOX. These results were compared with a previously developed SP-bulk modified IPN using the same hydrogel as a control, proving the efficiency of the gated IPN system. The covalent attachment of SPCOOH to the alcohol groups of the hydrogel and the structural change caused by UV light was indicated with FTIR analysis. XPS results also confirm the presence of SP by indicating the atomic percentage of nitrogen with respect to the hydrogel content.

Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.

Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.