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.

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.

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.

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.

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.

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.

Self-limiting electrospray deposition on polymer templates.

Electrospray deposition (ESD) applies a high voltage to liquids flowing through narrow capillaries to produce monodisperse generations of droplets down to hundreds of nanometers in diameter, each carrying a small amount of the delivered solute. This deposition method has been combined with insulated stencil masks for fabricating micropatterns by spraying solutions containing nanoparticles, polymers, or biomaterials. To optimize the fabrication process for micro-coatings, a self-limiting electrospray deposition (SLED) method has recently been developed. Here, we combine SLED with a pre-existing patterned polymer film to study SLED's fundamental behavior in a bilayer geometry. SLED has been observed when glassy insulating materials are sprayed onto conductive substrates, where a thickness-limited film forms as charge accumulates and repels the arrival of additional charged droplets. In this study, polystyrene (PS), Parylene C, and SU-8 thin films of varying thickness on silicon are utilized as insulated spraying substrates. Polyvinylpyrrolidone (PVP), a thermoplastic polymer is sprayed below its glass transition temperature (Tg) to investigate the SLED behavior on the pre-deposited insulating films. Furthermore, to examine the effects of in-plane confinement on the spray, a microhole array patterned onto the PS thin film by laser dewetting was sprayed with dyed PVP in the SLED mode. This was then extended to an unmasked electrode array showing that masked SLED and laser dewetting could be used to target microscale regions of conventionally-patterned electronics.

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

The effects of electroporation buffer composition on cell viability and electro-transfection efficiency.

Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m2). The energy of the pulse application primarily dictated cell viability, with Mg2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg2+ also hinders eTE compared to K+-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

Dynamin and reverse-mode sodium calcium exchanger blockade confers neuroprotection from diffuse axonal injury.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

Point-of-Care Fibrinogen Testing in Pregnancy.

Agreement between estimated fibrinogen concentration via thromboelastography and traditional assays is not established in the parturient. We therefore recruited 56 parturients and performed Clauss and functional fibrinogen level (FLEV) tests. Mean difference of measurements was 36.8 mg/dL (95% CI, 21.8-51.9) with a standard deviation of 52.8 mg/dL. Calculated limits of agreement were 140.2 mg/dL (95% CI, 166.3-114.6) and -66.6 mg/dL (95% CI, -40.8 to -92.5), within the maximum allowable difference of 165 mg/dL. We therefore conclude that while most measurements fell within the limits of agreement, more work is needed to clearly define the role of this test in the obstetric population.

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 flow cytometry: The role of microfabrication methodologies, performance and functional specification.

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Evaluating the in vivo glial response to miniaturized parylene cortical probes coated with an ultra-fast degrading polymer to aid insertion.

OBJECTIVE: Despite the feasibility of short-term neural recordings using implantable microelectrodes, attaining reliable, chronic recordings remains a challenge. Most neural recording devices suffer from a long-term tissue response, including gliosis, at the device-tissue interface. It was hypothesized that smaller, more flexible intracortical probes would limit gliosis by providing a better mechanical match with surrounding tissue. APPROACH: This paper describes the in vivo evaluation of flexible parylene microprobes designed to improve the interface with the adjacent neural tissue to limit gliosis and thereby allow for improved recording longevity. The probes were coated with an ultrafast degrading tyrosine-derived polycarbonate (E5005(2K)) polymer that provides temporary mechanical support for device implantation, yet degrades within 2 h post-implantation. A parametric study of probes of varying dimensions and polymer coating thicknesses were implanted in rat brains. The glial tissue response and neuronal loss were assessed from 72 h to 24 weeks post-implantation via immunohistochemistry. MAIN RESULTS: Experimental results suggest that both probe and polymer coating sizes affect the extent of gliosis. When an appropriate sized coating dimension (100 µm × 100 µm) and small probe (30 µm × 5 µm) was implanted, a minimal post-implantation glial response was observed. No discernible gliosis was detected when compared to tissue where a sham control consisting of a solid degradable polymer shuttle of the same dimensions was inserted. A larger polymer coating (200 µm × 200 µm) device induced a more severe glial response at later time points, suggesting that the initial insertion trauma can affect gliosis even when the polymer shuttle degrades rapidly. A larger degree of gliosis was also observed when comparing a larger sized probe (80 µm × 5 µm) to a smaller probe (30 µm × 5 µm) using the same polymer coating size (100 µm × 100 µm). There was no significant neuronal loss around the implantation sites for most device candidates except the group with largest polymer coating and probe sizes. SIGNIFICANCE: These results suggest that: (1) the degree of mechanical trauma at device implantation and mechanical mismatches at the probe-tissue interface affect long term gliosis; (2) smaller, more flexible probes may minimize the glial response to provide improved tissue biocompatibility when used for chronic neural signal recording; and (3) some degree of glial scarring did not significantly affect neuronal distribution around the probe.

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.

The Effect of Adding Subarachnoid Epinephrine to Hyperbaric Bupivacaine and Morphine for Repeat Cesarean Delivery: A Double-Blind Prospective Randomized Control Trial.

BACKGROUND: Spinal anesthesia has become the most common type of anesthetic for cesarean delivery. The major limitation to spinal anesthesia is that the duration of the anesthetic may not be adequate in the event of a prolonged surgery. Some practitioners add epinephrine to hyperbaric bupivacaine to increase the duration, although its effect has not been fully studied. We therefore aimed to evaluate whether adding epinephrine to the spinal medication prolongs the duration of action of the resultant block in women presenting for repeat cesarean delivery. METHODS: Sixty-eight patients were randomized to receive no epinephrine (NE group), epinephrine 100 µg (low-dose [LD] group), or epinephrine 200 µg (high-dose [HD] group) with a standardized spinal mixture (1.5 mL 0.75% hyperbaric bupivacaine with 0.25 mg morphine). Sixty-five patients were included for primary analysis. Our primary outcome was time to intraoperative activation of the epidural catheter or postoperative regression of sensory blockade to T-10 dermatome level as measured by pinprick sensation; motor recovery was a secondary outcome, and graded via a Modified Bromage scale. RESULTS: Block onset time, vital sign changes, and the incidence of hypotension; nausea, and vomiting were similar among groups. Median difference in time to T-10 regression was greatest in the HD group compared to the NE group (median difference [min] [95% confidence interval]: 40 [15-60]; P = .007), followed by the HD group to the LD group (30 [15-45]; P = .007). Comparisons of LD to NE were not significant, but trended to an increase in T-10 regression time (10 [-15 to 30]; P = .76). Median difference in time to knee extension (Bromage 3) was also greatest in the HD group when compared to both the LD and NE group (median difference [min] [95% confidence interval]: 30 [0-60]; P = .034, 60 [0-93]; P = .007). Median difference time to knee extension (min) between the LD and NE group was also significant (37.5 [15-60]; P = .001]. Pain scores during the procedure were higher in the NE group (median [interquartile range] HD: 0 [0-0], LD: 0 [0-0], NE: 0 [0-3]; P = .02) during uterine closure and were otherwise not significantly different from the other groups. CONCLUSIONS: In this single center, prospective, double-blind, randomized control trial, the addition of epinephrine 200 µg to hyperbaric bupivacaine and preservative-free morphine for repeat cesarean delivery prolonged the duration of the sensory blockade. Motor blockade was similarly prolonged and block quality may have been enhanced.