Fabrication of a Multilayer Implantable Cortical Microelectrode Probe to Improve Recording Potential.

Intracortical neural probes are a key enabling technology for acquiring high fidelity neural signals within the cortex. They are viewed as a crucial component of brain-computer interfaces (BCIs) in order to record electrical activities from neurons within the brain. Smaller, more flexible, polymer-based probes have been investigated for their potential to limit the acute and chronic neural tissue response. Conventional methods of patterning electrodes and connecting traces on a single supporting layer can limit the number of recording sites which can be defined, particularly when designing narrower probes. We present a novel strategy of increasing the number of recording sites without proportionally increasing the size of the probe by using a multilayer fabrication process to vertically layer recording traces on multiple Parylene support layers, allowing more recording traces to be defined on a smaller probe width. Using this approach, we are able to define 16 electrodes on 4 supporting layers (4 electrodes per layer), each with a 30 µm diameter recording window and 5 µm wide connecting trace defined by conventional LWUV lithography, on an 80 µm wide by 9 µm thick microprobe. Prior to in vitro and in vivo validation, the multilayer probes are electrically characterized via impedance spectroscopy and evaluating crosstalk between adjacent layers. Demonstration of acute in vitro recordings in a cerebral organoid model and in vivo recordings in a murine model indicate the probe's capability for single unit recordings. This work demonstrates the ability to fabricate smaller, more compliant neural probes without sacrificing electrode density.

Investigation of red blood cell partitioning in an in vitro microvascular bifurcation.

There is a long history of research examining red blood cell (RBC) partitioning in microvasculature bifurcations. These studies commonly report results describing partitioning that exists as either regular partitioning, which occurs when the RBC flux ratio is greater than the bulk fluid flowrate ratio, or reverse partitioning when the RBC flux ratio is less than or equal to that of the bulk fluid flowrate. This paper presents a study of RBC partitioning in a single bifurcating microchannel with dimensions of 6 to 16 µm, investigating the effects of hematocrit, channel width, daughter channel flowrate ratio, and bifurcation angle. The erythrocyte flux ratio, N*, manifests itself as either regular or reverse partitioning, and time-dependent partitioning is much more dynamic, occurring as both regular and reverse partitioning. We report a significant reduction in the well-known sigmoidal variation of the erythrocyte flux ratio (N*) versus the volumetric flowrate ratio (Q*), partitioning behavior with increasing hematocrit in microchannels when the channel dimensions are comparable with cell size. RBCs "lingering" or jamming at the bifurcation were also observed and quantified in vitro. Results from trajectory analyses suggest that the RBC position in the feeder channel strongly affects both partitioning and lingering frequency of RBCs, with both being significantly reduced when RBCs flow on streamlines near the edge of the channel as opposed to the center of the channel. Furthermore, our experiments suggest that even at low Reynolds number, partitioning is affected by the bifurcation angle by increasing cell-cell interactions. The presented results provide further insight into RBC partitioning as well as perfusion throughout the microvasculature.

Compartmentalized Devices as Tools for Investigation of Human Brain Network Dynamics.

Neuropsychiatric disorders have traditionally been difficult to study due to the complexity of the human brain and limited availability of human tissue. Induced pluripotent stem (iPS) cells provide a promising avenue to further our understanding of human disease mechanisms, but traditional 2D cell cultures can only provide a limited view of the neural circuits. To better model complex brain neurocircuitry, compartmentalized culturing systems and 3D organoids have been developed. Early compartmentalized devices demonstrated how neuronal cell bodies can be isolated both physically and chemically from neurites. Soft lithographic approaches have advanced this approach and offer the tools to construct novel model platforms, enabling circuit-level studies of disease, which can accelerate mechanistic studies and drug candidate screening. In this review, we describe some of the common technologies used to develop such systems and discuss how these lithographic techniques have been used to advance our understanding of neuropsychiatric disease. Finally, we address other in vitro model platforms such as 3D culture systems and organoids and compare these models with compartmentalized models. We ask important questions regarding how we can further harness iPS cells in these engineered culture systems for the development of improved in vitro models. Developmental Dynamics 248:65-77, 2019. © 2018 Wiley Periodicals, Inc.

Coherent Timescales and Mechanical Structure of Multicellular Aggregates.

Multicellular aggregates are an excellent model system to explore the role of tissue biomechanics in specifying multicellular reorganization during embryonic developments and malignant invasion. Tissue-like spheroids, when subjected to a compressive force, are known to exhibit liquid-like behaviors at long timescales (hours), largely because of cell rearrangements that serve to effectively dissipate the applied stress. At short timescales (seconds to minutes), before cell rearrangement, the mechanical behavior is strikingly different. The current work uses shape relaxation to investigate the structural characteristics of aggregates and discovers two coherent timescales: one on the order of seconds, the other tens of seconds. These timescales are universal, conserved across a variety of tested species, and persist despite great differences in other properties such as tissue surface tension and adhesion. A precise mathematical theory is used to correlate the timescales with mechanical properties and reveals that aggregates have a relatively strong envelope and an unusually "soft" interior (weak bulk elastic modulus). This characteristic is peculiar, considering that both layers consist of identical units (cells), but is consistent with the fact that this structure can engender both structural integrity and the flexibility required for remodeling. In addition, tissue surface tension, elastic modulus, and viscosity are proportional to each other. Considering that these tissue-level properties intrinsically derive from cellular-level properties, the proportionalities imply precise coregulation of the latter and in particular of the tension on the cell-medium and cell-cell interfaces.

Microfluidic platforms for the study of neuronal injury in vitro.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury.

Inhibition of glioblastoma dispersal by the MEK inhibitor PD0325901.

BACKGROUND: Dispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization - processes crucial to cell adhesion, substrate attachment, and cell motility - the aim of this study was to determine whether inhibiting this pathway could also impede dispersal. METHODS: A variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods. RESULTS: We show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures. CONCLUSIONS: Inhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery.

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

Modeling the Insertion Mechanics of Flexible Neural Probes Coated with Sacrificial Polymers for Optimizing Probe Design.

Single-unit recording neural probes have significant advantages towards improving signal-to-noise ratio and specificity for signal acquisition in brain-to-computer interface devices. Long-term effectiveness is unfortunately limited by the chronic injury response, which has been linked to the mechanical mismatch between rigid probes and compliant brain tissue. Small, flexible microelectrodes may overcome this limitation, but insertion of these probes without buckling requires supporting elements such as a stiff coating with a biodegradable polymer. For these coated probes, there is a design trade-off between the potential for successful insertion into brain tissue and the degree of trauma generated by the insertion. The objective of this study was to develop and validate a finite element model (FEM) to simulate insertion of coated neural probes of varying dimensions and material properties into brain tissue. Simulations were performed to predict the buckling and insertion forces during insertion of coated probes into a tissue phantom with material properties of brain. The simulations were validated with parallel experimental studies where probes were inserted into agarose tissue phantom, ex vivo chick embryonic brain tissue, and ex vivo rat brain tissue. Experiments were performed with uncoated copper wire and both uncoated and coated SU-8 photoresist and Parylene C probes. Model predictions were found to strongly agree with experimental results (<10% error). The ratio of the predicted buckling force-to-predicted insertion force, where a value greater than one would ideally be expected to result in successful insertion, was plotted against the actual success rate from experiments. A sigmoidal relationship was observed, with a ratio of 1.35 corresponding to equal probability of insertion and failure, and a ratio of 3.5 corresponding to a 100% success rate. This ratio was dubbed the "safety factor", as it indicated the degree to which the coating should be over-designed to ensure successful insertion. Probability color maps were generated to visually compare the influence of design parameters. Statistical metrics derived from the color maps and multi-variable regression analysis confirmed that coating thickness and probe length were the most important features in influencing insertion potential. The model also revealed the effects of manufacturing flaws on insertion potential.

Coating flexible probes with an ultra fast degrading polymer to aid in tissue insertion.

We report a fabrication process for coating neural probes with an ultrafast degrading polymer to create consistent and reproducible devices for neural tissue insertion. The rigid polymer coating acts as a probe insertion aid, but resorbs within hours post-implantation. Despite the feasibility for short term neural recordings from currently available neural prosthetic devices, most of these devices suffer from long term gliosis, which isolates the probes from adjacent neurons, increasing the recording impedance and stimulation threshold. The size and stiffness of implanted probes have been identified as critical factors that lead to this long term gliosis. Smaller, more flexible probes that match the mechanical properties of brain tissue could allow better long term integration by limiting the mechanical disruption of the surrounding tissue during and after probe insertion, while being flexible enough to deform with the tissue during brain movement. However, these small flexible probes inherently lack the mechanical strength to penetrate the brain on their own. In this work, we have developed a micromolding method for coating a non-functional miniaturized SU-8 probe with an ultrafast degrading tyrosine-derived polycarbonate (E5005(2K)). Coated, non-functionalized probes of varying dimensions were reproducibly fabricated with high yields. The polymer erosion/degradation profiles of the probes were characterized in vitro. The probes were also mechanically characterized in ex vivo brain tissue models by measuring buckling and insertion forces during probe insertion. The results demonstrate the ability to produce polymer coated probes of consistent quality for future in vivo use, for example to study the effects of different design parameters that may affect tissue response during long term chronic intra-cortical microelectrode neural recordings.

Scaling relationship and optimization of double-pulse electroporation.

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.

Continuous monitoring of inflammation biomarkers during simulated cardiopulmonary bypass using a microfluidic immunoassay device - a pilot study.

This work demonstrates the use of a continuous online monitoring system for tracking systemic inflammation biomarkers during cardiopulmonary bypass (CPB) procedures. The ability to monitor inflammation biomarkers during CPB will allow surgical teams to actively treat inflammation and reduce harmful effects on postoperative morbidity and mortality, enabling improved patient outcomes. A microfluidic device has been designed which allows automation of the individual processing steps of a microbead immunoassay to allow continuous tracking of antigen concentrations. Preliminary experiments have demonstrated that the results produced by the microimmunoassay are comparable to results produced from a standard enzyme-linked immunosorbent assay (r = 0.98). Additionally, integration of the assay with a simulated CPB circuit has been demonstrated with temporal tracking of C3a concentrations within blood continuously sampled from the circuit. The presented work describes the motivation, design challenges, and preliminary experimental results of this project.

Efficient electrospray deposition of surfaces smaller than the spray plume.

Electrospray deposition (ESD) is a promising technique for depositing micro-/nano-scale droplets and particles with high quality and repeatability. It is particularly attractive for surface coating of costly and delicate biomaterials and bioactive compounds. While high efficiency of ESD has only been successfully demonstrated for spraying surfaces larger than the spray plume, this work extends its utility to smaller surfaces. It is shown that by architecting the local "charge landscape", ESD coatings of surfaces smaller than plume size can be achieved. Efficiency approaching 100% is demonstrated with multiple model materials, including biocompatible polymers, proteins, and bioactive small molecules, on both flat and microneedle array targets. UV-visible spectroscopy and high-performance liquid chromatography measurements validate the high efficiency and quality of the sprayed material. Here, we show how this process is an efficient and more competitive alternative to other conformal coating mechanisms, such as dip coating or inkjet printing, for micro-engineered applications.

Hematocrit skewness along sequential bifurcations within a microfluidic network induces significant changes in downstream red blood cell partitioning.

There has been a wealth of research conducted regarding the partitioning of red blood cells (RBCs) at bifurcations within the microvasculature. In previous studies, partitioning has been characterized as either regular partitioning, in which the higher flow rate daughter channel receives a proportionally larger percentage of RBCs, or reverse partitioning, in which the opposite occurs. While there are many examples of network studies in silico, most in vitro work has been conducted using single bifurcation. When microfluidic networks have been used, the channel dimensions are typically greater than 20 µm, ignoring conditions where RBCs are highly confined. This paper presents a study of RBC partitioning in a network of sequential bifurcations with channel dimensions less than 8 µm in hydraulic diameter. The study investigated the effect of the volumetric flow rate ratio (Q*) at each bifurcation, solution hematocrit, and channel length on the erythrocyte flux ratio (N*), a measure of RBC partitioning. We report significant differences in partitioning between upstream and downstream bifurcations even when the flow rate ratio remains the same. Skewness analysis, a measure of cell distribution across the width of a vessel, strongly suggests that immediately following the first bifurcation most RBCs are skewed toward the inner channel wall, leading to preferential RBC perfusion into one daughter channel at the subsequent bifurcation even at higher downstream flow rate ratios. The skewness of RBC distribution following the first bifurcation can either manifest as enhanced regular partitioning or reverse partitioning at the succeeding branch.

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

Nanoporous membrane-sealed microfluidic devices for improved cell viability.

Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.

A Cerebral Organoid Connectivity Apparatus to Model Neuronal Tract Circuitry.

Mental disorders have high prevalence, but the efficacy of existing therapeutics is limited, in part, because the pathogenic mechanisms remain enigmatic. Current models of neural circuitry include animal models and post-mortem brain tissue, which have allowed enormous progress in understanding the pathophysiology of mental disorders. However, these models limit the ability to assess the functional alterations in short-range and long-range network connectivity between brain regions that are implicated in many mental disorders, e.g., schizophrenia and autism spectrum disorders. This work addresses these limitations by developing an in vitro model of the human brain that models the in vivo cerebral tract environment. In this study, microfabrication and stem cell differentiation techniques were combined to develop an in vitro cerebral tract model that anchors human induced pluripotent stem cell-derived cerebral organoids (COs) and provides a scaffold to promote the formation of a functional connecting neuronal tract. Two designs of a Cerebral Organoid Connectivity Apparatus (COCA) were fabricated using SU-8 photoresist. The first design contains a series of spikes which anchor the CO to the COCA (spiked design), whereas the second design contains flat supporting structures with open holes in a grid pattern to anchor the organoids (grid design); both designs allow effective media exchange. Morphological and functional analyses reveal the expression of key neuronal markers as well as functional activity and signal propagation along cerebral tracts connecting CO pairs. The reported in vitro models enable the investigation of critical neural circuitry involved in neurodevelopmental processes and has the potential to help devise personalized and targeted therapeutic strategies.

Single-cell mechanical analysis and tension quantification via electrodeformation relaxation.

The mechanical behavior and cortical tension of single cells are analyzed using electrodeformation relaxation. Four types of cells, namely, MCF-10A, MCF-7, MDA-MB-231, and GBM, are studied, with pulse durations ranging from 0.01 to 10 s. Mechanical response in the long-pulse regime is characterized by a power-law behavior, consistent with soft glassy rheology resulting from unbinding events within the cortex network. In the subsecond short-pulse regime, a single timescale well describes the process and indicates the naive tensioned (prestressed) state of the cortex with minimal force-induced alteration. A mathematical model is employed and the simple ellipsoidal geometry allows for use of an analytical solution to extract the cortical tension. At the shortest pulse of 0.01 s, tensions for all four cell types are on the order of 10^{-2} N/m.

Novel suction-based in vivo cutaneous DNA transfection platform.

This work reports a suction-based cutaneous delivery method for in vivo DNA transfection. Following intradermal Mantoux injection of plasmid DNA in a rat model, a moderate negative pressure is applied to the injection site, a technique similar to Chinese báguàn and Middle Eastern hijama cupping therapies. Strong GFP expression was demonstrated with pEGFP-N1 plasmids where fluorescence was observed as early as 1 hour after dosing. Modeling indicates a strong correlation between focal strain/stress and expression patterns. The absence of visible and/or histological tissue injury contrasts with current in vivo transfection systems such as electroporation. Specific utility was demonstrated with a synthetic SARS-CoV-2 DNA vaccine, which generated host humoral immune response in rats with notable antibody production. This method enables an easy-to-use, cost-effective, and highly scalable platform for both laboratorial transfection needs and clinical applications for nucleic acid­based therapeutics and vaccines.

Irreversible, direct bonding of nanoporous polymer membranes to PDMS or glass microdevices.

A method for integrating porous polymer membranes such as polycarbonate, polyethersulfone and polyethylene terephthalate to microfluidic devices is described. The use of 3-aminopropyltriethoxysilane as a chemical crosslinking agent was extended to integrate membranes with PDMS and glass microfluidic channels. A strong, irreversible bond between the membranes and microfluidic structure was achieved. The bonding strength in the APTES treated devices was significantly greater than in devices fabricated using either a PDMS "glue" or two-part epoxy bonding method. Evaluation of a filtering microdevice and the pore structure via SEM indicates the APTES conjugation does not significantly alter the membrane transport function and pore morphology.