Fabrication and Characterization of 3D Printed, 3D Microelectrode Arrays for Interfacing with a Peripheral Nerve-on-a-Chip.

We present a nontraditional fabrication technique for the realization of three-dimensional (3D) microelectrode arrays (MEAs) capable of interfacing with 3D cellular networks in vitro. The technology uses cost-effective makerspace microfabrication techniques to fabricate the 3D MEAs with 3D printed base structures with the metallization of the microtowers and conductive traces being performed by stencil mask evaporation techniques. A biocompatible lamination layer insulates the traces for realization of 3D microtower MEAs (250 µm base diameter, 400 µm height). The process has additionally been extended to realize smaller electrodes (30 µm × 30 µm) at a height of 400 µm atop the 3D microtower using laser micromachining of an additional silicon dioxide (SiO2) insulation layer. A 3D microengineered, nerve-on-a-chip in vitro model for recording and stimulating electrical activity of dorsal root ganglion (DRG) cells has further been integrated with the 3D MEA. We have characterized the 3D electrodes for electrical, chemical, electrochemical, biological, and chip hydration stability performance metrics. A decrease in impedance from 1.8 kΩ to 670 Ω for the microtower electrodes and 55 to 39 kΩ for the 30 µm × 30 µm microelectrodes can be observed for an electrophysiologically relevant frequency of 1 kHz upon platinum electroless plating. Biocompatibility assays on the components of the system resulted in a large range (∼3%-70% live cells), depending on the components. Fourier-transform infrared (FTIR) spectra of the resin material start to reveal possible compositional clues for the resin, and the hydration stability is demonstrated in in-vitro-like conditions for 30 days. The fabricated 3D MEAs are rapidly produced with minimal usage of a cleanroom and are fully functional for electrical interrogation of the 3D organ-on-a-chip models for high-throughput of pharmaceutical screening and toxicity testing of compounds in vitro.

Advances in microfluidic in vitro systems for neurological disease modeling.

Neurological disorders are the leading cause of disability and the second largest cause of death worldwide. Despite significant research efforts, neurology remains one of the most failure-prone areas of drug development. The complexity of the human brain, boundaries to examining the brain directly in vivo, and the significant evolutionary gap between animal models and humans, all serve to hamper translational success. Recent advances in microfluidic in vitro models have provided new opportunities to study human cells with enhanced physiological relevance. The ability to precisely micro-engineer cell-scale architecture, tailoring form and function, has allowed for detailed dissection of cell biology using microphysiological systems (MPS) of varying complexities from single cell systems to "Organ-on-chip" models. Simplified neuronal networks have allowed for unique insights into neuronal transport and neurogenesis, while more complex 3D heterotypic cellular models such as neurovascular unit mimetics and "Organ-on-chip" systems have enabled new understanding of metabolic coupling and blood-brain barrier transport. These systems are now being developed beyond MPS toward disease specific micro-pathophysiological systems, moving from "Organ-on-chip" to "Disease-on-chip." This review gives an outline of current state of the art in microfluidic technologies for neurological disease research, discussing the challenges and limitations while highlighting the benefits and potential of integrating technologies. We provide examples of where such toolsets have enabled novel insights and how these technologies may empower future investigation into neurological diseases.

Modeling chemotherapy-induced peripheral neuropathy using a Nerve-on-a-chip microphysiological system.

Organ-on-a-chip devices that mimic in vivo physiology have the potential to identify effects of chemical and drug exposure in early preclinical stages of drug development while relying less heavily on animal models. We have designed a hydrogel rat nerve-on-a-chip (RNoaC) construct that promotes axon growth analogous to mature nerve anatomy and is the first 3D in vitro model to collect electrophysiological and histomorphic metrics that are used to assess in vivo pathophysiology. Here we culture embryonic rat dorsal root ganglia (DRG) in the construct to demonstrate its potential as a preclinical assay for screening implications of nerve dysfunction in chemotherapy-induced peripheral neuropathy (CIPN). RNoaC constructs containing DRG explants from E15 rat pups were exposed to common chemotherapeutics: bortezomib, oxaliplatin, paclitaxel, or vincristine. After 7 days of treatment, axons were electrically stimulated to collect nerve conduction velocity (NCV) and the peak amplitude (AMP), which are two clinical electrophysiological metrics indicative of healthy or diseased populations. We observed decreased NCV and AMP in a dose-dependent manner across all drugs. At high drug concentrations, NCV and AMP were lower than control values by 10-60%. Histopathological analysis revealed that RNoaC exhibit hallmarks of peripheral neuropathy. IC50 values calculated from dose-response curves indicate significant decrease in function occurs before decrease in viability. Our data suggest electrophysiology recordings collected from our RNoaC platform can closely track subtle pathological changes in nerve function. The ability to collect clinically relevant data from RNoaCs suggests it can be an effective tool for in vitro preclinical screening of peripheral neuropathy.

Engineering a 3D functional human peripheral nerve in vitro using the Nerve-on-a-Chip platform.

Development of "organ-on-a-chip" systems for neuroscience applications are lagging due in part to the structural complexity of the nervous system and limited access of human neuronal & glial cells. In addition, rates for animal models in translating to human success are significantly lower for neurodegenerative diseases. Thus, a preclinical in vitro human cell-based model capable of providing critical clinical metrics such as nerve conduction velocity and histomorphometry are necessary to improve prediction and translation of in vitro data to successful clinical trials. To answer this challenge, we present an in vitro biomimetic model of all-human peripheral nerve tissue capable of showing robust neurite outgrowth (~5 mm), myelination of hNs by primary human Schwann cells (~5%), and evaluation of nerve conduction velocity (0.13-0.28 m/sec), previously unrealized for any human cell-based in vitro system. To the best of our knowledge, this Human Nerve-on-a-chip (HNoaC) system is the first biomimetic microphysiological system of myelinated human peripheral nerve which can be used for evaluating electrophysiological and histological metrics, the gold-standard assessment techniques previously only possible with in vivo studies.

Transdifferentiation of brain-derived neurotrophic factor (BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.

The use of genetically modified mesenchymal stem cells (MSCs) is a rapidly growing area of research targeting delivery of therapeutic factors for neuro-repair. Cells can be programmed to hypersecrete various growth/trophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to promote regenerative neurite outgrowth. In addition to genetic modifications, MSCs can be subjected to transdifferentiation protocols to generate neural cell types to physically and biologically support nerve regeneration. In this study, we have taken a novel approach by combining these two unique strategies and evaluated the impact of transdifferentiating genetically modified MSCs into a Schwann cell-like phenotype. After 8 days in transdifferentiation media, approximately 30-50% of transdifferentiated BDNF-secreting cells immunolabeled for Schwann cell markers such as S100ß, S100, and p75NTR. An enhancement was observed 20 days after inducing transdifferentiation with minimal decreases in expression levels. BDNF production was quantified by ELISA, and its biological activity tested via the PC12-TrkB cell assay. Importantly, the bioactivity of secreted BDNF was verified by the increased neurite outgrowth of PC12-TrkB cells. These findings demonstrate that not only is BDNF actively secreted by the transdifferentiated BDNF-MSCs, but also that it has the capacity to promote neurite sprouting and regeneration. Given the fact that BDNF production remained stable for over 20 days, we believe that these cells have the capacity to produce sustainable, effective, BDNF concentrations over prolonged time periods and should be tested within an in vivo system for future experiments.

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation.

While transplantation of Schwann cells facilitates axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of autologous bone-marrow derived mesenchymal stem cells (MSCs). As MSCs can transdifferentiate to Schwann cell-phenotypes and accelerate nerve regeneration we undertook proteomic evaluation of the cells to uncover the protein contents that affects Schwann cell formulation. Transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. MSC proteins significantly regulated during Schwann cell transdifferentiation included, but were not limited to, GNAI2, MYL9, ACTN4, ACTN1, ACTB, CAV-1, HSPB1, PHB2, TBB4B, CTGF, TGFI1, ARF6, EZR, GELS, VIM, WNT5A, RTN4, EFNB1. These support axonal guidance, myelination, neural development and neural growth and differentiation. The results unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. SIGNIFICANCE STATEMENT: While Schwann cells facilitate axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of bone-marrow derived mesenchymal stem cells (MSCs) transdifferentiated to Schwann cell-phenotypes. In the present study, we undertook the first proteomic evaluation of these transdifferentiated cells to uncover the protein contents that affects Schwann cell formulation. Furthermore, these transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. Our results demonstrate that a number of MSC proteins were significantly regulated following transdifferentiation of the MSCs supporting roles in axonal guidance, myelination, neural development and differentiation. The conclusions of the present work unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. Our study was the first proteomic comparison demonstrating the transdifferentiation of MSCs and these reported results can affect a wide field of stem cell biology, tissue engineering, and proteomics.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we developed and present a method for computationally modeling and measuring oxygen that can easily be implemented in all MPS models. We have applied this method in a liver MPS in which we are then able to control oxygenation in separate devices and demonstrate that zonation-dependent hepatocyte functions in the MPS recapitulate what is known about in vivo liver physiology. We believe that this advance allows a deep experimental investigation on the role of zonation in liver metabolism and disease. In addition, modeling and measuring oxygen tension will be required as investigators migrate from PDMS to plastic and glass devices.

Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes.

In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4×106Pa and pore size of ∼150µm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4×106Pa and pore size of ∼100µm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100ß markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5pg/mL NGF and ∼0.7pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5pg/mL NGF and 0.7pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo. STATEMENT OF SIGNIFICANCE: This manuscript focuses on the effect of microstructure and mechanical properties of gelatin-based 3D conduits on the transdifferentiation of mesenchymal stem cells to Schwann cell-like phenotypes. This work builds on our recently accepted manuscript in Acta Biomaterialia focused on multifunctional 2D films, and focuses on 3D microstructured conduits designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits showed the most favorable environment for MSC transdifferentiation to Schwann-cell like phenotypes, as seen by both immunolabeling as well as secretion of neurotrophic factors. This work demonstrates the importance of controlling the 3D microstructure to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.

Development of multifunctional films for peripheral nerve regeneration.

In this study, a poly(lactic acid) (PLLA) porous film with longitudinal surface micropatterns was fabricated by a dry phase inversion technique to be used as potential conduit material for peripheral nerve regeneration applications. The presence of a nerve growth factor (NGF) gradient on the patterned film surface and protein loaded, surface-eroding, biodegradable, and amphiphilic polyanhydride (PA) microparticles within the film matrix, enabled co-delivery of neurotrophic factors with controlled release properties and enhanced neurite outgrowth from PC12 cells. The protein loading capacity of PA particles was increased up to 80% using the spray drying technique, while the surface loading of NGF reached 300ng/cm2 through ester-amine interactions. The NGF surface gradient provided initial fast release from the film surface and facilitated directional neurite outgrowth along with the longitudinal micropatterns. Furthermore, the variable backbone chemistry and surface eroding nature of protein-loaded PA microparticles within the film matrix ensured protein stability and enabled controlled protein release. This novel co-delivery strategy yielded tunable diffusion coefficients varying between 6×10-14 and 1.67×10-10cm2/min and dissolution constants ranging from 1×10-4 to 1×10-3min-1 with released amounts of ∼100-300ng/mL. This strategy promoted guided neurite extension from PC12 cells of up to 10µm total neurite length per cell in 2days. Overall, this unique strategy can potentially be extended for individually programmed delivery of multiple growth factors through the use of PA microparticle cocktails and can further be investigated for in vivo performance as potential conduit material for peripheral nerve regeneration applications. STATEMENT OF SIGNIFICANCE: This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10µm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

Oriented growth and transdifferentiation of mesenchymal stem cells towards a Schwann cell fate on micropatterned substrates.

While Schwann cells (SCs) have a significant role in peripheral nerve regeneration, their use in treatments has been limited because of lack of a readily available source. To address this issue, this study focused on the effect of guidance cues by employing micropatterned polymeric films to influence the alignment, morphology and transdifferentiation of bone marrow-derived rat mesenchymal stem cells (MSCs) towards a Schwann cell-like fate. Two different types of polymers, biocompatible polystyrene (PS) and biodegradable poly(lactic acid) (PLA) were used to fabricate patterned films. Percentages of transdifferentiated MSCs (tMSCs) immunolabeled with SC markers (α-S100ß and α-p75(NTR)) were found to be similar on patterned versus smooth PS and PLA substrates. However, patterning had a significant effect on the alignment and elongation of the tMSCs. More than 80% of the tMSCs were oriented in the direction of microgrooves (0°-20°), while cells on the smooth substrates were randomly oriented. The aspect ratio [AR, ratio of length (in direction of microgrooves) and breadth (in direction perpendicular to microgrooves)] of the tMSCs on patterned substrates had a value of approximately five, as compared to cells on smooth substrates where the AR was one. Understanding responses to these cues in vitro helps us in understanding the behavior and interaction of the cells with the 3D environment of the scaffolds, facilitating the application of these concepts to designing effective nerve guidance conduits for peripheral nerve regeneration.

Enabling nanomaterial, nanofabrication and cellular technologies for nanoneuromedicines.

Nanoparticulate delivery systems represent an area of particular promise for nanoneuromedicines. They possess significant potential for desperately needed therapies designed to combat a range of disorders associated with aging. As such, the field was selected as the focus for the 2014 meeting of the American Society for Nanomedicine. Regenerative, protective, immune modulatory, anti-microbial and anti-inflammatory products, or imaging agents are readily encapsulated in or conjugated to nanoparticles and as such facilitate the delivery of drug payloads to specific action sites across the blood-brain barrier. Diagnostic imaging serves to precisely monitor disease onset and progression while neural stem cell replacement can regenerate damaged tissue through control of stem cell fates. These, taken together, can improve disease burden and limit systemic toxicities. Such enabling technologies serve to protect the nervous system against a broad range of degenerative, traumatic, metabolic, infectious and immune disorders. From the clinical editor: Nanoneuromedicine is a branch of nanomedicine that specifically looks at the nervous system. In the clinical setting, a fundamental hurdle in nervous system disorders is due to an inherent inability of nerve cells to regenerate after damage. Nanotechnology can offer new approaches to overcome these challenges. This review describes recent developments in nanomedicine delivery systems that would affect stem cell repair and regeneration in the nervous system.

High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.