Physiological Flow Research Articles

141 Polyvinylpyrrolidone-Coated Catheters Show Fewer Astrocytes and Choroid Plexus Epithelium Adhesion and Better Flow/Pressure Performance Than Other Commercial Catheters in a New Catheter Assay System

INTRODUCTION: The major cause of failure of cerebral spinal fluid (CSF) shunting devices in pediatric patients is proximal catheter occlusion. There are a number of shunt catheters commercially available, but their occlusion properties are unclear. METHODS: There were four catheters tested: 1) antibiotic-impregnated, 2) polyvinylpyrrolidone, 3) barium stripe, and 4) barium impregnated. Catheters were inoculated with cultured astrocytes or choroid cells and incubated in growth media for 7 days prior to testing. Sample catheters were placed into a 3D-printed ventricular replicating system to test occlusion properties. Artificial CSF was pumped through the system to mimic physiologic flow and pressure resistance sensors measured obstruction. Afterward testing, catheters were stained with anti-GFAP or anti-transthyretin to visualize cellular adhesion using a fluorescence microscope. RESULTS: Five catheters of each type were used. Polyvinylpyrrolidone catheters had the highest flow (e.g., least obstruction) in the ventricular replication system (0.12 mL/min) compared to the antibiotic coated (0.10 mL/min), barium stripe (0.02 mL/min) or barium impregnated (0.08 mL/min; p < 0.01). Polyvinylpyrrolidone catheters showed the lowest median cell attachment after cell marker staining (<1 cells per 0.07 mm2) compared to antibiotic coated (4.0 cells per 0.07 mm2), barium stripe (3.5 per 0.07 mm2) or barium impregnated (2.0 per 0.07 mm2). CONCLUSIONS: Polyvinylpyrrolidone catheters were least likely to be occluded by astrocytes or choroid cells during testing in a novel ventricular replicating system due to lower cellular adhesion. Our findings may have clinical significance when choosing proximal ventricular catheters that are least likely to occlude and prevent proximal shunt failure.

Flow dynamic assessment of native mitral valve, mitral valve repair and mitral valve replacement using vector flow mapping intracardiac flow dynamic in mitral valve regurgitation.

The present study aims to assess and describe the intracardiac blood flow dynamic in patients with mitral regurgitation (MR), repaired mitral valves (MV) and mitral valve prostheses using vector flow mapping (VFM). Patients with different MV pathologies and MV disease treatments were analysed. All patients underwent 2D transthoracic echocardiography, and images for flow visualization were acquired in VFM mode in an apical three-chamber view and four-chamber view. Vectors and vortices were qualitatively analyzed. thirty-two (32) patients underwent 2D transthoracic echocardiography (TTE) with VFM analysis. We evaluated intracardiac flow dynamics in 3 healthy subjects, 10 patients with MR (5 degenerative, 5 functional), 4 patients who underwent MV repair, 5 who underwent MV replacement (3 biological, 2 mechanical), 2 surgically implanted transcatheter heart valve (THV), 2 transcatheter edge-to-edge MV repair with MitraClip (TEER), 3 transcatheter MV replacement (TMVR) and 3 transapical off-pump MV repair with NeoChord implantation. Blood flow patterns are significantly altered in patients with MV disease and MV repair compared to control patients. MV repair is superior to replacement in restoring more physiologicalpatterns, while TMVR reproducesan intraventricular flowcloser to normal than surgical MVR and TEER. Intracardiac flow patterns can be clearly defined using VFM. Restoration of a physiological blood flow pattern inside the LV directly depends on the procedure used to address MV disease.

Interstitial fluid flow contributes to prostate cancer invasion and migration to bone; study conducted using a novel horizontal flow bioreactor

Prostate cancer bone metastasis is the leading cause of cancer-related mortality in men in the United States, causing severe damage to skeletal tissue. The treatment of advanced-stage prostate cancer is always challenging due to limited drug treatment options, resulting in low survival rates. There is a scarcity of knowledge regarding the mechanisms associated with the effects of biomechanical cues by the interstitial fluid flow on prostate cancer cell growth and migration. We have designed a novel bioreactor system to demonstrate the impact of interstitial fluid flow on the migration of prostate cancer cells to the bone during extravasation. First, we demonstrated that a high flow rate induces apoptosis in PC3 cells via TGF-β1 mediated signaling; thus, physiological flow rate conditions are optimum for cell growth. Next, to understand the role of interstitial fluid flow in prostate cancer migration, we evaluated the migration rate of cells under static and dynamic conditions in the presence or absence of bone. We report that CXCR4 levels were not significantly changed under static and dynamic conditions, indicating that CXCR4 activation in PC3 cells is not influenced by flow conditions but by the bone, where CXCR4 levels were upregulated. The bone-upregulated CXCR4 levels led to increased MMP-9 levels resulting in a high migration rate in the presence of bone. In addition, upregulated levels of α v β 3 integrins under fluid flow conditions contributed to an overall increase in the migration rate of PC3 cells. Overall, this study demonstrates the potential role of interstitial fluid flow in prostate cancer invasion. Understanding the critical role of interstitial fluid flow in promoting prostate cancer cell progression will enhance current therapies for advanced-stage prostate cancer and provide improved treatment options for patients.

In-vitro modeling of intravenous drug precipitation by the optical spatial precipitation analyzer (OSPREY)

Intravenous (IV) administration of poorly water-soluble small molecule therapeutics can lead to precipitation during mixing with blood. This can limit characterization of pharmacological and safety endpoints in preclinical models. Most often, tests of kinetic and thermodynamic solubility are used to optimize the formulation for solubility prior to infusion in animals, but these do not capture the dynamic precipitation processes that take place during in-vivo administration. To better capture the fluid dynamic processes that occur during IV administration, we developed the Optical Spatial PREcipitation analYzer (OSPREY) as a method to quantify the amount and size of compound precipitates in whole blood using a flow-through system that mimics IV administration. Here, we describe the OSPREY device and its underlying imaging processing methods. We then validate the ability to accurately segment particles according to their size using monodisperse suspensions of microspheres (diameter 50 to 425 µm). Next, we use a tool compound, ABT-737, to study the effects of compound concentration, vessel flow rate, compound infusion rate and vessel diameter on precipitation. Finally, we use the physiological diameter and flow rate of rat femoral vein and dog saphenous vein to demonstrate the potential of OSPREY to model in-vivo precipitation in a controlled, dynamic in-vitro assay.

MarioHeart: Novel In-Vitro Flow Model for Testing Heart Valve Prostheses and Anticoagulant Therapies.

Mechanical heart valve (MHV) prostheses present a risk of thromboembolic complications despite antithrombotic therapy. Further steps in the development of more hemocompatible MHVs and new anticoagulants are impeded due to the lack of adequate in-vitro models. With the development of a novel in-vitro model (MarioHeart), a pulsatile flow similar to the arterial circulation is emulated. The MarioHeart design owns unique features as 1) a single MHV within a torus with low surface/volume ratio, 2) a closed loop system, and 3) a dedicated external control system driving the oscillating rotational motion of the torus. For verification purposes, a blood analog fluid seeded with particles was used to assess fluid velocity and flow rate using a speckle tracking method on high-speed video recordings of the rotating model. The flow rate resembled the physiological flow rate in the aortic root, in both shape and amplitude. Additional in-vitro runs with porcine blood showed thrombi on the MHV associated with the suture ring, which is similar to the in-vivo situation. MarioHeart is a simple design which induces well-defined fluid dynamics resulting in physiologically nonturbulent flow without stasis of the blood. MarioHeart seems suitable for testing the thrombogenicity of MHVs and the potential of new anticoagulants.

Peristaltic Transport of Bingham Fluid through Nonuniform Channel with Convective Conditions and Lorentz Forces

In this study, the impacts of changing the liquid characteristics of the Bingham fluid are highlighted under MHD peristaltic transport. Conditions for convective and porous boundary conditions are taken into consideration. By using proper similarity transformations, the governing equations become dimensionless. For temperature, velocity, and streamlines, the series solution is found. The MATLAB 2019b programming offers a graphical depiction of relevant metrics on physiological flow parameters. The findings demonstrate that lowering the velocity and temperature profiles occurs when the magnetic parameter is increased. Furthermore, the effect of changing viscosity increases the trapped bolus size by a small amount. Once again, the results of the present investigation have significance for our knowledge of the complex rheological mechanisms governing blood flow via small arteries.

Abstract Number ‐ 151: Impact of Stent‐Retriever Tip Design on Distal Embolization during Mechanical Thrombectomy

Introduction Mechanical thrombectomy (MT) is a widely performed procedure for acute ischemic stroke (AIS) due to large vessel occlusion. Repeated number of passes, clot fragmentation, and distal embolization during MT lead to worse clinical outcomes. We aim to evaluate the impact of different stent‐retriever (SR) distal tip designs on distal emboli generation during MT. Methods Fragment‐prone clot analogs (diameter = 3.53±0.14mm; length = 6.74±0.61mm) were used to create proximal middle cerebral artery (MCA‐M1) occlusions in an in vitro neurovascular model featuring a complete circle of Willis. The anatomical replica was connected to a flow loop with circulating saline at physiological flow rate and temperature; 100‐µm filters were placed at the neurovascular outflow points of the model to collect generated distal emboli. After initial embolization, experiments were randomized into one of the three treatment arms based on SR tip design: open‐end (Open‐SR: Solitaire 6.0×40mm), closed‐end (Closed‐SR: Embotrap II 5.0×33mm), and filter‐end (Filter‐SR: NeVa NET 5.5×37mm). A balloon guide catheter was inflated at the internal carotid artery C1 level immediately after SR deployment, and the SR was pulled out under continuous proximal pump aspiration. A total of 90 cases were performed (30 cases/treatment arm). A single attempt was performed per case and after each pass, distal emboli collected in the outflow filters were analyzed by an image processing algorithm. Successful first pass recanalization (FPR) was confirmed if no residual clot was observed at the initial location or in a distal branch of the model. Primary study endpoints were: FPR rate (%FPR), the size of the largest embolus (largest‐E), the total emboli count (total‐E), the total count of emboli larger than 1mm (total>1mm‐E), and the total area of the filter covered by emboli (area‐E). Results FPR was achieved in 57.8% of cases (52/90): Filter‐SR achieved a non‐significantly higher %FPR (70%) than closed‐SR (50%) and open‐SR (53.3%) (p = 0.244). In comparison to open‐ and closed‐SR, filter‐SR significantly reduced the largest‐E (open‐SR = 1.66±0.68mm vs. closed‐SR = 1.77±0.90mm vs. filter‐SR = 1.22±0.77mm; p = 0.013) as well as the total>1mm‐E (open‐SR = 2.27±2.33 vs. closed‐SR = 3.97±5.68 vs. filter‐SR = 0.93±1.28; p = 0.002), and the area‐E (open‐SR = 18.22±14.47mm2 vs. closed‐SR = 23.98±22.39mm2 vs. filter‐SR = 10.14±8.81mm2; p = 0.013). The differences between distal tip designs were not clearly evidenced in the total‐E (open‐SR = 22.7±10.75 vs. closed‐SR = 23.13±13.16 vs. filter‐SR = 20.63±12.77; p = 0.464). No significant differences were found between open‐ and closed‐SR (p>0.05 in all metrics). Conclusions When facing fragment‐prone clots with low SR engagement, the filter‐SR significantly reduces the number of large clot fragments (>1mm), the size of the largest embolus, and the overall surface area of clot fragments that embolize distally during an MT procedure.

The value of arterial spin labelling perfusion MRI in brain age prediction.

Current structural MRI-based brain age estimates and their difference from chronological age-the brain age gap (BAG)-are limited to late-stage pathological brain-tissue changes. The addition of physiological MRI features may detect early-stage pathological brain alterations and improve brain age prediction. This study investigated the optimal combination of structural and physiological arterial spin labelling (ASL) image features and algorithms. Healthy participants (n = 341, age 59.7 ± 14.8 years) were scanned at baseline and after 1.7 ± 0.5 years follow-up (n = 248, mean age 62.4 ± 13.3 years). From 3 T MRI, structural (T1w and FLAIR) volumetric ROI and physiological (ASL) cerebral blood flow (CBF) and spatial coefficient of variation ROI features were constructed. Multiple combinations of features and machine learning algorithms were evaluated using the Mean Absolute Error (MAE). From the best model, longitudinal BAG repeatability and feature importance were assessed. The ElasticNetCV algorithm using T1w + FLAIR+ASL performed best (MAE = 5.0 ± 0.3 years), and better compared with using T1w + FLAIR (MAE = 6.0 ± 0.4 years, p < .01). The three most important features were, in descending order, GM CBF, GM/ICV, and WM CBF. Average baseline and follow-up BAGs were similar (-1.5 ± 6.3 and - 1.1 ± 6.4 years respectively, ICC = 0.85, 95% CI: 0.8-0.9, p = .16). The addition of ASL features to structural brain age, combined with the ElasticNetCV algorithm, improved brain age prediction the most, and performed best in a cross-sectional and repeatability comparison. These findings encourage future studies to explore the value of ASL in brain age in various pathologies.

Portal Vein Recanalization-Transjugular Intrahepatic Portosystemic Shunt (PVR-TIPS) Facilitates Liver Transplantation in Cirrhotic Patients with Occlusive Portal Vein Thrombosis.

Portal vein thrombosis (PVT) is a heterogeneous condition with multiple possible etiologies and to varying degrees has historically limited candidacy for liver transplant (LT) in the cirrhotic patient population due to resultant difficulties in constructing a robust portal vein anastomosis. While intraoperative approaches to managing PVT are well-described, methods which approximate normal portal physiology are not always feasible depending on the extent of PVT, and other nonphysiologic techniques are linked with substantial morbidity and poor long-term outcomes. Portal vein recanalization-transjugular intrahepatic portosystemic shunt (PVR-TIPS) creation is an efficacious method of restoring physiologic portal flow in cirrhotic patients prior to LT allowing for end-to-end PV anastomosis, and is the product of decades-long institutional expertise in TIPS/LT and the support of a multidisciplinary liver tumor board. To follow is a review of the pertinent pathophysiology of PVT in cirrhosis, the rationale leading to the development and subsequent evolution of the PVR-TIPS procedure, technical lessons learned, and a summary of outcomes to date.

The resignations among nurses in the Veneto healthcare institutions. A retrospective study

The phenomenon of large-scale resignations is complex, heterogeneous and cannot be attributed solely to the pandemic crisis, during which many people reconsidered the role of work in their lives. The health system was particularly exposed to the shocks caused by the pandemic. To describe the turnover and analyse the resignations of nurses in the NHS hospitals and districts of the Veneto Region. The hospitals were categorised into 4 types: Hub and Spoke of level 1 and 2. The positions of nurses with permanent contracts between 1 January 2016 and 31 December 2022 were analysed, considering active, nurses on duty for at least one day. The data were extracted from the human resource management database of the Region. Unexpected resignations were considered those occurring before the retirement date (59 years for women and 60 for men). Negative and overall turnover rates were calculated. The risk of unexpected resignations was increased for nurses not resident in Veneto, of male gender and employed at a Hub hospital. The flight from the NHS is in addition to the physiological flow of retirements, due to increase in the coming years. It is necessary to: act on the retention capacity and attractiveness of the profession; implement organisational models based on task sharing and shifting; implement digital tools; favour flexibility and mobility to improve the balance between work and personal life; efficiently integrate professionals quali-fied abroad.

Direct 0D-3D coupling of a lattice Boltzmann methodology for fluid-structure aortic flow simulations.

This work introduces a numerical approach and implementation for the direct coupling of arbitrary complex ordinary differential equation- (ODE-)governed zero-dimensional (0D) boundary conditions to three-dimensional (3D) lattice Boltzmann-based fluid-structure systems for hemodynamics studies. In particular, a most complex configuration is treated by considering a dynamic left ventricle- (LV-)elastance heart model which is governed by (and applied as) a nonlinear, non-stationary hybrid ODE-Dirichlet system. Other ODE-based boundary conditions, such as lumped parameter Windkessel models for truncated vasculature, are also considered. Performance studies of the complete 0D-3D solver, including its treatment of the lattice Boltzmann fluid equations and elastodynamics equations as well as their interactions, is conducted through a variety of benchmark and convergence studies that demonstrate the ability of the coupled 0D-3D methodology in generating physiological pressure and flow waveforms-ultimately enabling the exploration of various physical and physiological parameters for hemodynamics studies of the coupled LV-arterial system. The methods proposed in this paper can be easily applied to other ODE-based boundary conditions as well as to other fluid problems that are modeled by 3D lattice Boltzmann equations and that require direct coupling of dynamic 0D boundary conditions.

Biological Role of Folic Acid in Pregnancy and Possible Therapeutic Application for the Prevention of Preeclampsia.

The rationale and importance of folic acid supplementation during pregnancy for fetal congenital defect prevention are accepted worldwide. Moreover, a sufficient plasma concentration of folates can reduce the incidence of spontaneous abortions, and support the normal expansion of placental blood vessels, ensuring physiological placental blood flow, thus promoting appropriate fetal growth and development. Furthermore, there is emerging evidence that long-term supplementation with folic acid can effectively prevent preeclampsia. Preeclampsia is unique to the human species in complications during pregnancy, which contributes to maternal and perinatal mortality worldwide. In the pathogenesis of preeclampsia abnormal placental invasion, the excess of antiangiogenic factors and maternal-placental syndrome play a key role. Increased blood levels of homocysteine during pregnancy are associated with the risk of preeclampsia. Moreover, hyperhomocysteinemia has been proposed to be an independent risk factor for preeclampsia. Folate supplementation helps to decrease elevated levels of homocysteine; thus, the role of folic acid supplementation in pregnancy is even more important. Multiple reports suggest that folate administration decreases the level of serum homocysteine and, therefore, reduce the risk and severity of preeclampsia. However, the association between folic acid supplementation and the decreased risk of preeclampsia has been investigated with controversial conclusions. Currently, the optimal dose of folic acid that is effective for preeclampsia prevention remains uncertain. In this review, we aim to summarize the accumulated knowledge on the role of folic acid in the pathogenesis of preeclampsia, and the possible impact of folate supplementation on the decreased risk of preeclampsia.

Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonization strategies of uropathogenic Escherichia coli under shear stress.

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic Escherichia coli (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work in vivo revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.

Maternal Smoking in the First Trimester and its Consequence on the Early Placenta

Smoking during pregnancy increases the risk of adverse pregnancy outcomes, such as stillbirth and fetal growth restriction. This suggests impaired placental function and restricted nutrient and oxygen supply. Studies investigating placental tissue at the end of pregnancy have revealed increased DNA damage as a potential underlying cause, which is driven by various toxic smoke ingredients and oxidative stress induced by reactive oxygen species (ROS). However, in the first trimester, the placenta develops and differentiates, and many pregnancy pathologies associated with reduced placental function originate here. Therefore, we determined DNA damage in a cohort of first-trimester placental samples of verified smokers and nonsmokers. In fact, we observed an 80% increase in DNA breaks (P < .001) and shortened telomeres by 5.8% (P = .04) in placentas exposed to maternal smoking. Surprisingly, there was a decrease in ROS-mediated DNA damage, ie, 8-oxo-guanidine modifications, in placentas of the smoking group (−41%; P = .021), which paralleled the reduced expression of base excision DNA repair machinery, which restores oxidative DNA damage. Moreover, we observed that the increase in placental oxidant defense machinery expression, which usually occurs at the end of the first trimester in a healthy pregnancy as a result of the full onset of uteroplacental blood flow, was absent in the smoking group. Therefore, in early pregnancy, maternal smoking causes placental DNA damage, contributing to placental malfunction and increased risk of stillbirth and fetal growth restriction in pregnant women. Additionally, reduced ROS-mediated DNA damage along with no increase in antioxidant enzymes suggests a delay in the establishment of physiological uteroplacental blood flow at the end of the first trimester, which may further add to a disturbed placental development and function as a result of smoking in pregnancy.

Mechanical Assist Device-Assisted Percutaneous Coronary Intervention: The Use of Impella Versus Extracorporeal Membrane Oxygenation as an Emerging Frontier in Revascularization in Cardiogenic Shock.

The extracorporeal membrane oxygenation (ECMO) procedure aids in the provision of prolonged cardiopulmonary support, whereas the Impella device (Abiomed, Danvers, MA) is a ventricular assist device that maintains circulation by pumping blood into the aorta from the left ventricle. Blood is circulated in parallel with the heart by Impella. It draws blood straight into the aorta from the left ventricle, hence preserving the physiological flow. ECMO bypasses the left atrium and the left ventricle, and the end consequence is a non-physiological flow. In this article, we conducted a detailed analysis of various publications in the literatureand examined various modalities pertaining to the use ofECMO and Impella for cardiogenic shocks, such as efficacy, clinical outcomes, cost-effectiveness, device-related complications, and limitations. The Impella completely unloads the left ventricle, thereby significantly reducing the effort of the heart. Comparatively, ECMO only stabilizes a patient with cardiogenic shock for a short stretch of time and does not lessen the efforts of the left ventricle ("unload" it). In the acute setting, both devices reduced left ventricular end-diastolic pressure and provided adequate hemodynamic support. By comparing patients on Impella to those receiving ECMO, it was found that patients on Impella were associated with better clinical results, quicker recovery, limited complications, and reduced healthcare costs; however, there is a lack of conclusive studies performed demonstrating the reduction in long-term mortality rates. Considering the effectiveness of given modalities and taking into account the various studies described in the literature, Impella has reported better clinical outcomes although more clinical trials are needed for establishing the effectiveness of these interventional approaches in revascularization in cardiogenic shock.

In Vitro Morphogenesis and Differentiation of Human Intestinal Epithelium in a Gut-on-a-Chip.

The establishment of a three-dimensional (3D) epithelial structure and cytodifferentiation in vitro is necessary to recapitulate in vivo-relevant structure and function of the human intestine. Here, we describe an experimental protocol to build an organomimetic gut-on-a-chip microdevice that allows inducing 3D morphogenesis of human intestinal epithelium using Caco-2 cells or intestinal organoid cells. Under physiological flow and physical motions, intestinal epithelium spontaneously recreates 3D epithelial morphology in a gut-on-a-chip that offers enhanced mucus production, epithelial barrier, and longitudinal host-microbe co-culture. This protocol may provide implementable strategies to advance traditional in vitro static cultures, human microbiome studies, and pharmacological testing.

Dispersion of Heterogeneous Medium in Pulsatile Blood Flow and Absolute Pulsatile Flow Velocity Quantification.

Heterogeneous medium enhanced angiogr- ams are key diagnostic tools in clinical practice; the associated hemodynamic information is crucial for diagnosing cardiovascular diseases. However, the dynamics of such medium in physiological blood flow are poorly understood. Herein, we report a previously unnoticed dispersion pattern, which is a universal phenomenon, of a medium in pulsatile blood flow. We present a physical theory for studying the dispersion of a steadily injected heterogeneous medium into a thin tubular blood vessel in which the blood flow is pulsatile. In a thin tubular blood vessel, we demonstrate that variations of concentration associated with the heterogeneous medium obey a one-dimensional advection diffusion equation, and the diffusion has limited effect whenever a short vascular segment is considered. A distinct feature of the distribution of the medium in the axial distance-time plane is a "dilation-retraction" pattern. The time evolution signals at different axial positions exhibit distinct concentration waveforms. A numerical scheme is proposed for exploiting this information to estimate the pulsatile velocity. Artificial data are adopted to validate the scheme. Real X-ray angiography is also analyzed to support our theory and method. The theory is applicable whenever imaging protocols involve a heterogeneous medium in pulsatile flow.

Continuously perfusable, customisable, and matrix-free vasculature on a chip platform.

Creating vascularised cellular environments in vitro is a current challenge in tissue engineering and a bottleneck towards developing functional stem cell-derived microtissues for regenerative medicine and basic investigations. Here we have developed a new workflow to manufacture vasculature on chip (VoC) systems efficiently, quickly, and inexpensively. We have employed 3D printing for fast-prototyping of bespoke VoC and coupled them with a refined organotypic culture system (OVAA) to grow patent capillaries in vitro using tissue-specific endothelial and stromal cells. Furthermore, we have designed and implemented a pocket-size flow driver to establish physiologic perfusive flow throughout our VoC-OVAA with minimal medium use and waste. Using our platform, we have created vascularised microtissues and perfused them at physiologic flow rates for extended time (>2 weeks) observing flow-dependent vascular remodelling. Overall, we present for the first time a scalable and customisable system to grow vascularised and perfusable microtissues, a key initial step to grow mature and functional tissues in vitro. We envision that this technology will empower fast prototyping and validation of increasingly biomimetic in vitro systems, including interconnected multi-tissue systems.

Central lymphatic surgery

In recent years, microsurgical reconstruction of the lymphatic system has opened new frontiers in the treatment of central lymphatic lesions. Central lymphatic lesions can be congenital or acquired. While the latter can result from any surgery or trauma in the area of the thoracic duct, congenital lymphatic lesions can show a plethora of manifestations, ranging from singular thoracic duct abnormalities to complex multifocal malformations. Regardless of the anatomical location of the thoracic duct lesion, these conditions cause recurrent chylous effusions and downstream lymphatic congestion and are associated with increased mortality due to the permanent loss of protein and fluid. In case of disruption of the lymphatic flow, microsurgical reconstructive surgery is indicated to treat downstream congestion leading to bronchitis plastica, protein-loosing enteropathy, chylothorax, and chylascites. Thoracic duct-vein anastomoses can reconstruct the physiological lymphatic flow.

VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry.

Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite's pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.