Bioengineering & Translational Medicine

Snake venom composition and its contribution to toxic effects has been heavily researched, but there is a comparative lack of information on venoms fluidic properties and their relationship with fang morphology during the envenomation process. Understanding how venom flows through a fang can shed light on bite site dynamics and potentially explain bite symptoms. In this article we first conduct a broad comparative test of the rheological properties of venom from thirteen snake species, including multiple viperid and elapid snake species, revealing a shear-thinning non-Newtonian flow behavior in all studied species. However, we have not observed strong phylogenetic signal in venom fluidic properties, suggesting that flow properties may vary independently of evolutionary relationships between snake species. Second, we demonstrate that snake venoms fluidic properties can be modeled by other inexpensive, safe, and abundant shear-thinning surrogate fluids. We found that aqueous solutions of bovine serum albumin protein and xanthan gum are useful venom mimics, matching the rheological behavior of venoms from the studied snake species across a range of relevant shear rates. We further evaluated the performance of these snake venom mimics in a simulated venom delivery system, showing good and robust mimetic control of the flow properties as a function of applied pressure. By elucidating the fluidic properties of snake venom and providing a non-toxic, scalable surrogate fluid model to be used in further studies, we provide the biomedical, toxicology, evolutionary biology communities with a tool to study envenomation physics in an inexpensive and safe fashion. We suggest it is possible to design species-specific venom mimics that facilitate research on the biomechanics and fluid dynamics of venom delivery via snake bites, and inform the design of bioinspired puncture and injection devices.

Wound infections are a significant medical challenge, often leading to chronicity or systemic infection. Selective serotonin reuptake inhibitors (SSRIs) have emerged as potential non-antibiotic candidates with demonstrated ability to limit growth and biofilm formation in Gram-negative bacteria, in addition to their pro-healing activity. Here, we compared direct delivery of the SSRI fluoxetine by topical bolus dosing to delivery from an iontophoresis bandage device with an actuator for temporally controlled drug delivery, in a porcine excisional wound model. Device delivery of fluoxetine resulted in a maximum concentration of 12.25 ng fluoxetine per mg tissue, compared to 2.926 ng/mg following bolus dosing, and tissue fluoxetine levels were higher after application using the device than after bolus dosing across the range of doses tested (p=0.0041). The half-life of fluoxetine in the wound tissue was 0.988 {+/-} 0.256 days. Fluoxetine was not detected in the pig plasma, and plasma serotonin levels were not affected by the topical application. Fluoxetine delivery using the device, but not bolus delivery, produced tissue concentrations above the minimum inhibitory concentration (MIC) for some clinically important species of bacteria. The experimental device can effectively deliver topical fluoxetine to the wound, producing higher tissue concentrations of fluoxetine at lower cumulative doses compared to bolus dosing, and with minimal risk of off-target effects. The device may simplify wound treatment by reducing the burden for daily drug application, possibly increasing adherence to a prescribed treatment regimen.

BackgroundNormothermic ex vivo organ perfusion (NEVOP) promises to catalyze organ preservation, therapeutic discovery, and organ-specific disease modeling. Existing technology platforms remain inaccessible for research due to restricted access to commercial organ perfusion devices, high costs of both devices and proprietary consumables, and steep technical learning curves. Additionally, the available technology is not optimized to perfuse smaller organs such as the kidney. MethodsTo overcome these barriers, a custom NEVOP circuit was developed using recycled, repurposed, and low-cost components. Porcine kidneys and autologous blood were used to iteratively optimize circuit design. A porcine kidney autotransplantation protocol was adapted to evaluate in vivo kidney function after ex vivo perfusion. To pilot the flexibility of this system as a multi-organ platform for ex vivo human biology, non-transplantable human donor kidney, spleen, and pancreas specimens were stably perfused using human blood products and analyzed. ResultsAn ultra low-cost NEVOP system engineered to perfuse porcine kidneys and diverse human organs (kidney, pancreas, and spleen) sustained viable organs for up to 24 hours with evidence of both function and viability. Key innovations included a parallel flow resistor to facilitate low-flow perfusion in non-heparinized organs and a containment bag with adjustable magnets to provide vascular stability and recycling of venous overflow. The circuit costs less than 1,500USD to construct, and porcine kidneys perfused for 24 hours on this platform demonstrated healthy in vivo function upon autotransplantation. ConclusionsCustom NEVOP platforms constitute novel and potentially transformative research platforms which use low-cost and readily available materials. Paired with access to non-transplantable research organs from altruistic donors, this model provides a road map for investigators to advance biomedical discovery and human ex vivo biology.

Systemic chemotherapeutics target cancer cells but are also known to impact other cells away from the tumor. Questions remain whether systemic chemotherapy crosses the blood-brain barrier and causes inflammation in the periphery that impacts the central nervous system (CNS) downstream. The meningeal lymphatics are a critical component that drain cerebrospinal fluid from the CNS to the cervical lymph nodes for immunosurveillence. To develop new tools for understanding chemotherapy-mediated effects on the meningeal lymphatics, we present two novel models that examine cellular and tissue level changes. Our in vitro tissue engineered model of a meningeal lymphatic vessel lumen, using a simple tissue culture insert system with both lymphatic endothelial and meningeal cells, examines cell disruption. Our ex vivo model culturing mouse meningeal layers probes structural changes and remodeling, correlating to an explant tissue level. To gain a holistic understanding, we compare our in vitro and ex vivo models to in vivo studies for validation and a three-tier methodology for examining the chemotherapeutic response of the meningeal lymphatics. We have demonstrated that the meningeal lymphatics can be disrupted by systemic chemotherapy but show differential responses to platinum and taxane chemotherapies, emphasizing the need for further study of off-target impacts in the CNS.

BackgroundAccurately controlling drug delivery is crucial for safe anesthesia. Target-controlled infusion (TCI) systems use pharmacokinetic and pharmacodynamic (PK/PD) models to administer intravenous agents to reach target concentrations. However, TCIs operation is restricted to a single-drug and a linear PK/PD model, not accounting for drug interactions. Dose-response interaction (DRI) models quantify such interactions by representing shared effects as a function of agents concentrations. For example, the co-administration of an analgesic and a hypnotic with TCI leads to an uncontrolled synergy. MethodsWe introduce a new administering methodology for multi-drug infusions, interaction target-controlled infusion (iTCI), that combines the PK/PD models of the co-administered drugs and their interactions into a single optimal non-linear dynamic control problem with terminal constraints. ResultsIncorporating DRI and PK/PD models allows novel administration procedures. Simulations of iTCI in different clinical scenarios under propofol and remifentanil co-administrations are presented. These show that: (1) iTCI requires lower administered volumes than TCI to reach simultaneously the same target concentrations. (2) It offers optimal interdependent administrations that address not only concentration targets but also effect targets. (3) iTCI comes with additional constraints on the administration, including controlled titrations along iso-effect conditions (isoboles) or (5) directly limiting plasma concentration levels. (6) Unlike TCI, iTCI can include different exerted effects (ke0) per drug, particularly relevant for opioids. ConclusionThe iTCI is a versatile multi-drug infusion paradigm where effects and interactions play a relevant role - providing better delivery profiles than current TCI while opening the door for using non-linear PK/PD descriptions in anesthesia.

Head and neck cancer (HNC) is the 6th most common malignancy worldwide. 60% of patients present with advanced disease and approximately 50% of patients recur following primary treatment. Chemoradiation remains a standard of care for most patients. However, clinicians lack functional tools to predict which patients will respond to chemoradiation prior to treatment and current models, including organoids and animal model systems, fail to capture either full complexity or patient-to-patient heterogeneity of the individual HNC tumor and microenvironment (TME). Here, we have developed, characterized, and tested a patient-specific microphysiological system (MPS) that reconstructs the HNC TME in a vascularized 3D environment. This MPS was constructed from malignant cells, fibroblasts, and immune cells from a patients surgically resected tumor, seeded within a 3D hydrogel with molded endothelial lumens. Single-cell RNA sequencing confirmed that the MPS preserved 12 transcriptionally distinct cell populations found in matched native tissue. The platform recapitulated tumor hypoxia, with a 12-fold increase in hypoxic marker expression that altered radiation response, consistent with clinical HNC biology. Compartment-resolved imaging revealed distinct treatment dynamics in tumor, stromal, and vascular regions, and individual patients exhibited divergent responses to chemoradiation in spheroid morphology, cell viability, and migration. We found the slope of spheroid area change with treatment tracked with tumor recurrence, suggesting this metric could serve as a functional predictor of therapeutic response.

Since its introduction, vaccination has heavily improved health outcomes. However, implementing vaccination efforts can be challenging, particularly in low and middle-income countries with warmer climates. Microneedle technology has been developed for its simple and relatively painless applications of vaccines. However, no microneedle vaccine has yet been approved by the FDA. A few hurdles must be overcome, including the need to evaluate the safety and biocompatibility of the polymer used to fabricate these microneedles. Additionally, it is important to demonstrate reliable immune responses comparable to or better than those achieved through traditional administration routes. Scalability in manufacturing and the ability to maintain vaccine potency during storage and transportation are also critical factors. In this study, we developed vaccine-loaded dissolvable microneedles that showed preclinical immunogenicity after storage in extreme conditions. We developed our microneedles using the conventional micromolding technique with polyacrylic acid (PAA) polymer, incorporating a novel virus-like particle (VLP) vaccine targeting arboviruses. We performed characterization studies on these microneedles to assess needle sharpness, skin insertion force, and VLP integrity. We also investigated the thermostability of the vaccine after storing the microneedles at elevated temperatures for approximately 140 days. Finally, we evaluated the immunogenicity of this vaccine in mice, comparing transdermal (microneedle) with intramuscular (hypodermic needle) administration. We successfully fabricated and characterized VLP-loaded microneedles that could penetrate the skin and maintain vaccine integrity even after exposure to extreme storage conditions. These microneedles also elicited robust and long-lasting antibody responses similar to those achieved with intramuscular administration.

The hollow fiber membrane bundle is the functional component of artificial lungs, transferring oxygen and carbon dioxide to and from the blood. It is also the primary location of blood clot formation and propagation in these devices. The geometric design of fiber bundles is defined by a narrow range of parameters that determine gas exchange efficiency and blood flow resistance, such as fiber packing density, path length, and frontal area. However, these parameters also affect thrombosis. This study investigated the effect of these parameters on clot formation using 3-D printed flow chambers that mimic the geometry and blood flow patterns of fiber bundles. Hollow fibers were represented by an array of vertical micro-rods (380 micron diameter) arranged with varying packing densities (40, 50, and 60%) and path lengths (2 and 4 cm). Blood was pumped through the device corresponding to three mean blood flow velocities (16, 20, and 25 cm/min). Results showed that (1) clot formation decreases dramatically with decreasing packing density and increasing blood flow velocity, (2) clot formation at the outlet of fiber bundle enhances deposition upstream, and consequently (3) greater path length provides more clot-free fiber surface area for gas exchange than a shorter path length. These results can be used to create less thrombogenic, more efficient artificial lung designs. Translational Impact SentenceFiber bundle parameters, such as decreased packing density, increased blood flow velocity, and a longer path length, can be used to design a less thrombogenic, more efficient artificial lung to extend functionality.

To investigate the potential of an affordable cryotherapy device for accessible treatment of breast cancer, the performance of a novel carbon dioxide-based device was evaluated through both benchtop and in vivo canine models. This novel device was quantitatively compared to a commercial device that utilizes argon gas as the cryogen. The thermal behavior of each device was characterized through calorimetry and by measuring the temperature profiles of iceballs generated in tissue phantoms. A 45-minute treatment from the carbon dioxide device in a tissue phantom produced a 1.67 {+/-} 0.06 cm diameter lethal isotherm that was equivalent to a 7-minute treatment from the commercial argon-based device which produced a 1.53 {+/-} 0.15 cm diameter lethal isotherm. In vivo validation was performed with the carbon dioxide-based device in one spontaneously occurring canine mammary mass with two standard 10-minutes freezes. Following cryotherapy, this mass was surgically resected and analyzed for necrosis margins via histopathology. The histopathology margin of necrosis from the in vivo treatment with the carbon dioxide device at 14 days post cryoablation was 1.57 cm. While carbon dioxide gas has historically been considered an impractical cryogen due to its low working pressure and high boiling point, this study shows that carbon dioxide-based cryotherapy may be equivalent to conventional argon-based cryotherapy in the size of the ablation zone in a standard treatment time. The validation of the carbon dioxide device performed in this study is an important step towards bringing accessible breast cancer treatment to women in low-resource settings.

Individualized and anatomically correct computational models of the brain can be leveraged to improve knowledge of drug dispersal following simulation of drug delivery. Using a patients magnetic resonance image (MRI) scans, we were able to reconstruct the pial surface of the brain of the left hemisphere with strong anatomic accuracy. We then established the major internal features, including the lateral ventricle, a tumor, and drug delivery catheters. These were able to include relevant tissue characteristics such as porosity and permeability in the Multiphysics platform COMSOL to create a platform for brain modeling. To test the performance of this platform, we simulated direct drug infusion in both a healthy patient brain and a diseased patient model, focusing on glioblastoma (GBM). Using this platform, we simulated perturbed convection enhanced delivery of a cancer medication (similar to temozolomide (TMZ) but modeled using methylene blue) to the tumor. Consequently, with our patient derived model, we are able to simulate solute dispersal and fluid flow representative of in vivo conditions.

AO_SCPLOWBSTRACTC_SCPLOWThe placenta is a temporary and complex organ critical for fetal development through its subtle but convoluted harmonization of endocrine, vascular, haemodynamic and exchange adaptations. Yet, due to experimental, technological and ethical constraints, this unique organ remains poorly understood. In silico tools are emerging as a powerful means to overcome these challenges and have the potential to actualize novel breakthroughs. Here, we present an interdisciplinary framework combining in vitro experiments used to develop an elegant and scalable in silico model of oxygen diffusion. We then use in utero imaging of placental perfusion and oxygenation in both control and growth-restricted rodent placentas for validation of our in silico model. Our framework revealed the structure-function relationship in the feto-placental vasculature; oxygen diffusion is impaired in growth-restricted placentas, due to the diminished arborization of growth-restricted feto-placental vasculature and the lack of decelerated flow for adequate oxygen diffusion and exchange. We highlight the mechanisms of impairment in a rat model of growth restriction, underpinned by placental vascular impairment. Our framework reports and validates the prediction of blood flow deceleration impairment in growth restricted placentas with the placentas oxygen transfer capability being significantly impaired, both globally and locally.

Time series clustering is applied to inflammation and neutrophil cell development markers, CD16 and CD10, in sepsis, trauma and surgery patients and a dynamical model with an inflammation function, F, is used to represent their evolution over a two month period. Five patient clusters are identified, characterised and evaluated against medical assessment scores and the literature. A dynamical model for neutrophil and monocyte cell counts and maturity has been constructed based on mass balances and cell kinetics in both blood and bone marrow. Cell proliferation and flow rates, as well as expression of monocyte HLA-DR, depend on concentrations of pro- and anti- inflammatory cytokines, IL6 and IL10, via F. A good fit with the data is obtained for each cluster and the estimated parameters correlate to illness severity. The model is a potential tool for simulation of immunomodulatory therapies.

An important step to fulfill the functionalities of DNA vaccines and therapeutics is transfection in vivo to produce the encoded antigens or therapeutic proteins. A cutaneous suction-based method has demonstrated effectiveness in many animal models and has been successfully applied in human clinical trials, but has not been extended to mouse models, where numerous disease models, transgenic strains, and murine-specific reagents exist. The current work establishes and optimizes methods for cutaneous suction-mediated DNA transfection in mice. By adapting a smaller cup diameter and smaller injection volume, the challenges of skin hyperelasticity and decreased skin thickness can be effectively addressed, and vaccinating mice with the GLS-5310 SARS-CoV-2 DNA vaccine yielded high levels of binding antibody and T cell responses. Additionally, suction following injection of a novel pVAX1-based expression vector yielded systemic levels of a SEAP transgene. Thus, suction-mediated delivery of nucleic acid-based therapies and vaccines can be a valuable tool for the study in pre-clinical mouse models.

The treatment of glioblastoma (GBM) presents significant challenges, with median survival rates remaining low despite standard-of-care therapies. This study expands upon the findings of a novel approach to managing GBM, namely cytostatic hypothermia, through the computational evaluation of a fully implantable system. Our proposed system utilizes a multi-probe array and a novel artificial internal circulation system (AICS) to achieve homogeneous cooling within the brain without overheating any portion of the body. Finite-element modeling was employed to simulate bioheat transfer and fluid dynamics. Our results indicate that the multi-probe array can attain local tissue temperatures within the cytostatic range (20 to 28degC) while minimizing thermal gradients. The use of multiple narrow, thermally conductive probes enhances cooling uniformity with minimal tissue displacement. The revolutionary AICS provides a form of heat management that has not previously been attempted to the best of our knowledge. In this study, it successfully facilitates the transfer of heat from the intracranial region to the skin in the body. Future work will focus on device prototyping and validation through in vitro and in vivo studies in large animal models. These simulations suggest that the proposed intracranial cooling system makes cytostatic hypothermia a practicable approach against GBM. Furthermore, this approach to internal heat management may also open new avenues for treating neurological conditions through local and chronic hypothermia, extending beyond the short-duration (acute) cooling methods currently tested.

Measuring the capacity of microvascular networks in delivering soluble oxygen and nutrients to its organs is essential in health, disease, and surgical interventions. Here, a finite element methodbased oxygen transport program, AngioMT, is designed and validated to predict spatial oxygen distribution and other physiologically relevant transport metrics within both the vascular network and the surrounding tissue. The software processes acquired images of microvascular networks and produces a digital mesh which is used to predict vessel and tissue oxygenation. The image-to-physics translation by AngioMT correlated with results from commercial software, however only AngioMT could provide predictions within the solid tissue in addition to vessel oxygenation. AngioMT predictions were sensitive and positively correlated to spatial heterogeneity and extent of vascularization of 500 different vascular networks formed with variable vasculogenic conditions. The predictions of AngioMT cross-correlate with experimentally-measured oxygen distributions in vivo. The computational power of the software is increased by including calculations of higher order reaction mechanisms, and the program includes defining additional organ and tissue structures for a more physiologically relevant analysis of tissue oxygenation in complex co-cultured systems, or in vivo. AngioMT may serve as a digital performance measuring tool of vascular networks in microcirculation, experimental models of vascularized tissues and organs, and in clinical applications, such as organ transplants.

Precision drug delivery for optimized therapeutic targeting requires knowledge of momentum transport and molecular diffusion of molecules within the patients interstitial tissue, especially for tumor treatment within the brain. Dispersion in the interstitial space is impacted by delivery method, tissue material properties, individual-specific fluid flow, and particle size of the input solute. Knowledge of a drugs dispersion allows for optimizing solute delivery, concentration, and flow rates to maximize drug distribution and biomarker recovery. For delivering drugs, increased knowledge of drug location after delivery can improve therapeutic treatment by optimizing the dosing of healthy and unhealthy tissue. Finite element methods (FEM) tools, such as COMSOL Multiphysics, can simulate molecular distribution inside-individual specific shapes and porous material properties. Furthermore, an additional unmet need is delivery methods that can be adjusted to manipulate diffusion regions through tissue via techniques such as directed flow. This would be especially valuable in targeted drug delivery within tumors to increase the cancerous surface area covered while limiting damage to surrounding tissues. In this project, the directed flow was induced by perfusing the injected solution at an input probe while withdrawing fluid at an output probe, enabling targeted flow through the desired region. FEM computation faithfully replicated these conditions and could be used to determine the effective concentrations perfused over the region of interest. We leveraged COMSOL Multiphysics to perform a computational study simulating convection-enhanced delivery (CED) with an output probe pulling the concentration profile over the region of interest. This simulation system can be applied to therapeutics targeting, vaccine subcutaneous injection, and waste and media diffusion in tissue engineering.

Non-viral approaches to transfection have emerged a viable option for gene transfer. Electro-mechanical transfection involving use of electric fields coupled with high fluid flow rates is a scalable strategy for cell therapy development and manufacturing. Unlike purely electric field-based or mechanical-based delivery methods, the combined effects result in delivery of genetic material at high efficiencies and low toxicity. This study focuses on delivery of reporter mRNA to show electro-mechanical transfection can be used successfully in human T cells. Rapid optimization of delivery to T cells was observed with efficiency over 90% and viability over 80%. Confirmation of optimized electro-mechanical transfection parameters was assessed in multiple use cases including a 50-fold scale up demonstration. Transcriptome and ontology analysis show that delivery, via electro-mechanical transfection, does not result in gene dysregulation. This study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for cell and gene therapy engineering and development. One Sentence SummaryThis study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for development of engineered cellular therapies.

This study investigates the effects of Poloxamer 188 (P188) on neutrophil chemotaxis following burn injury in male and female mice using a microfluidic system. Utilizing male and female CD1 mice, we evaluated neutrophil migration towards two chemoattractants, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) and Leukotriene B4 (LTB4), and FPR1 and BLT1 G protein-coupled receptors after administering P188. Our findings revealed that P188 significantly increased the migration toward LTB4 in both sexes. Additionally, our findings highlight the upregulation of BLT1 and FPR1 markers due to burn injury in both female and male mice in the Burn vs. Sham groups. These results demonstrate the potential of P188 in modulating neutrophil behavior post-burn injury in therapeutic strategies for inflammation management. This microfluidic platform offers a precise and controlled microenvironment for studying neutrophil chemotaxis post-burn injury with and without P188 treatment.

Extremely preterm infants have poor clinical outcomes due to lung immaturity. An artificial placenta could provide extracorporeal gas exchange, allowing normal lung growth outside of the uterus, thus improving outcomes. However, current devices in development use hollow-fiber membrane oxygenators, which have a high rate of bleeding and clotting complications. Here, we present a novel style of oxygenator composed of a stacked array of rigid and flat silicon semi-permeable membranes. Using computational fluid dynamic (CFD) modeling, we demonstrated favorable hemocompatibility properties, including laminar blood flow, low pressure drop, and minimal cumulative shear stress. We then constructed and tested prototype devices on the benchtop and in an extracorporeal pig model. At 20 mL/min of blood flow, the oxygenators exhibited an average oxygen flux of 0.081 {+/-} 0.020 mL (mean {+/-} standard error) and a pressure drop of 2.25 {+/-} 0.25 mmHg. This study demonstrates the feasibility of a building a stacked flat-plate oxygenator with a blood flow path informed by CFD.

Crude mixtures of matrix-degrading enzymes called collagenase Clostridium histolyticum (CCH) have been used to efficiently breakdown tissue for many years. Recently, direct injection of purified CCH has been successfully developed as a treatment for Dupuytrens contracture (DC), a fibrotic disorder of the hand. Given similar histologic and mechanical features between the fibrous bands in DC and cirrhosis, a similar approach may be feasible for the treatment of cirrhosis. Crude and purified CCH were first compared through composition and substrate specificity. The biodistribution of a macromolecule delivered via interstitial infusion in the liver was mapped and quantified with a fluorescent dextran tracer to design a protocol for efficient delivery throughout the liver with minimal off-target exposure. Safety and efficacy of interstitial CCH infusion in the liver was investigated in cirrhotic mice using serum markers of injury and histological analysis of fibrosis. Purified CCH showed high purity and efficient degradation of type I collagen. Tracer experiments showed that interstitial infusion in one lobe of the liver will reach the entire organ in the mouse. A significant amount of the tracer was also found to enter the bloodstream where it is cleared by the kidneys, but was not found to significantly infiltrate other organs. Mild elevation in liver enzymes AST and ALT were observed 1d after infusion of purified CCH, but this was not significantly different than infusion with saline. No elevation in creatinine was observed. Cirrhotic mice infused with purified CCH showed 38% reduction in collagen proportionate area compared to mice infused with saline. These results show interstitial delivery of purified CCH in the cirrhotic liver rapidly decreases collagen content with minimal liver toxicity. This strategy merits further study as a potential treatment for cirrhosis.