Perfusion Flow Rate Research Articles

Neural Network-based Estimation of Microbubbles Generated in Cardiopulmonary Bypass Circuit: A Clinical Application Study.

The cardiopulmonary bypass system used in cardiac surgery can generate microbubbles (MBs) that may cause complications, such as neurocognitive dysfunction, when delivered into the blood vessel. Estimating the number of MBs generated, thus, is necessary to enable the surgeons to deal with it. To this end, we previously proposed a neural network-based model for estimating the number of MBs from four factors measurable from the cardiopulmonary bypass system: suction flow rate, venous reservoir level, blood viscosity, and perfusion flow rate. However, the model has not been adapted to the data collected from actual surgery cases. In this study, the accuracy of MBs estimated by the proposed model was examined in four clinical cases. The results showed that the coefficient of determination between estimated MBs and the measured MBs throughout the surgeries was R2=0.558 (p<0.001). We found that the surgical treatments, such as administration of drugs, fluids and blood transfusions, increased the number of measured MBs. The coefficient of determination increased to R2= 0.8762 (p<0.001) by excluding the duration of these treatments. This result indicates that the model can estimate the number of MBs with high accuracy under the clinical environment.

Development and Characterization of a Nonelectronic Versatile Oxygenating Perfusion System for Tissue Preservation.

Oxygenated machine perfusion of human organs has been shown to improve both preservation quality and time duration when compared to the current gold standard: static cold storage. However, existing machine perfusion devices designed for preservation and transportation of transplantable organs are too complicated and organ-specific to merit use as a solution for all organs. This work presents a novel, portable, and nonelectronic device potentially capable of delivering oxygenated machine perfusion to a variety of organs. An innovative pneumatic circuit system regulates a compressed oxygen source that cyclically inflates and deflates silicone tubes, which function as both theoxygenator and perfusion pump. Different combinations of silicone tubes in single or parallel configurations, with lengths ranging from 1.5 to 15.2 m, were evaluated at varying oxygen pressures from 27.6 to 110.3 kPa. The silicone tubes in parallel configurations produced higher peak perfusion pressures (70% increase), mean flow rates (102% increase), and oxygenation rates (268% increase) than the single silicone tubes that had equivalent total lengths. While pumping against a vascular resistance element that mimicked a kidney, the device achieved perfusion pressures (8.4-131.6mmHg), flow rates (2.0-40.2mLmin-1), and oxygenation rates (up to 388μmolmin-1) that are consistent with values used in previous kidney preservation studies. The nonelectronic device achieved those perfusion parameters using 4.4Lmin-1 of oxygen to operate. These results demonstrate that oxygenated machine perfusion can be successfully achieved without any electronic components.

Coronary Flow Assessment Using 3-Dimensional Ultrafast Ultrasound Localization Microscopy

BackgroundDirect assessment of the coronary microcirculation has long been hampered by the limited spatial and temporal resolutions of cardiac imaging modalities. ObjectivesThe purpose of this study was to demonstrate 3-dimensional (3D) coronary ultrasound localization microscopy (CorULM) of the whole heart beyond the acoustic diffraction limit (<20 μm resolution) at ultrafast frame rate (>1000 images/s). MethodsCorULM was performed in isolated beating rat hearts (N = 6) with ultrasound contrast agents (Sonovue, Bracco), using an ultrasonic matrix transducer connected to a high channel–count ultrafast electronics. We assessed the 3D coronary microvascular anatomy, flow velocity, and flow rate of beating hearts under normal conditions, during vasodilator adenosine infusion, and during coronary occlusion. The coronary vasculature was compared with micro-computed tomography performed on the fixed heart. In vivo transthoracic CorULM was eventually assessed on anaesthetized rats (N = 3). ResultsCorULM enables the 3D visualization of the coronary vasculature in beating hearts at a scale down to microvascular structures (<20 μm resolution). Absolute flow velocity estimates range from 10 mm/s in tiny arterioles up to more than 300 mm/s in large arteries. Fitting to a power law, the flow rate–radius relationship provides an exponent of 2.61 (r2 = 0.96; P < 0.001), which is consistent with theoretical predictions and experimental validations of scaling laws in vascular trees. A 2-fold increase of the microvascular coronary flow rate is found in response to adenosine, which is in good agreement with the overall perfusion flow rate measured in the aorta (control measurement) that increased from 8.80 ± 1.03 mL/min to 16.54 ± 2.35 mL/min (P < 0.001). The feasibility of CorULM was demonstrated in vivo for N = 3 rats. ConclusionsCorULM provides unprecedented insights into the anatomy and function of coronary arteries at the microvasculature level in beating hearts. This new technology is highly translational and has the potential to become a major tool for the clinical investigation of the coronary microcirculation.

A microfluidic chip carrier including temperature control and perfusion system for long-term cell imaging

Microfluidic devices are widely used for biomedical applications but there is still a lack of affordable, reliable and user-friendly systems for transferring microfluidic chips from an incubator to a microscope while maintaining physiological conditions when performing microscopy. The presented carrier represents a cost-effective option for sustaining environmental conditions of microfluidic chips in combination with minimizing the device manipulation required for reagent injection, media exchange or sample collection. The carrier, which has the outer dimension of a standard well plate size, contains an integrated perfusion system that can recirculate the media using piezo pumps, operated in either continuous or intermittent modes (50–1000 µl/min). Furthermore, a film resistive heater made from 37 µm-thick copper wires, including temperature feedback control, was used to maintain the microfluidic chip temperature at 37 °C when outside the incubator. The heater characterisation showed a uniform temperature distribution along the chip channel for perfusion flow rates up to 10 µl/min. To demonstrate the feasibility of our platform for long term cell culture monitoring, mouse brain endothelial cells (bEnd.3) were repeatedly monitored for a period of 10 days, demonstrating a system with both the versatility and the potential for long imaging in microphysiological system cell cultures.

Characterizing and Tuning Perfusion Parameters Within an Innovative, Versatile Oxygenating Perfusion System.

The advantages of oxygenated perfusion are continuing to be demonstrated by many groups focused on improving the efficacy of tissue preservation for transplant, bioreactors for studies of basic tissue physiology, and closed-loop resuscitation. This work presents a novel and portable device that supplies oxygenated and pulsatile perfusion, both of which are regulated by a single pump-oxygenator component comprised of silicone tubes that are cyclically inflated/deflated with compressed oxygen. In this study, pump variables (oxygen supply pressure and length of a silicone tube) were evaluated against hydraulicelements that mimicked the vascular resistance of kidneys, livers, and hearts. The perfusion pressures, flow rates, and oxygenation rates produced by the device were characterized for all configurations of pump variables, and the pulse rates were tuned to improve performance. The device supplied perfusion pressures ranging from 3.5 to 109 mmHg, flow rates ranging from 1.4 to 71.8 mL min-1, and oxygenation rates up to 316.6 µmol min-1. From those results, it was determined that the device was capable of achieving perfusion parameters used in previous kidney, liver, and heart preservation studies. Ultimately, this research demonstrated the efficacy of a novel device that is designed to supply oxygenated perfusion across a range of applications.

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

O67: PERFUSATE GLUCOSE REFLECTS TISSUE GLYCOGENATION DURING LIVER PERFUSION

Abstract Introduction Normothermic machine perfusion (NMP) is a method of organ preservation that aims to replicate the physiological environment, achieved by perfusing the livers with a blood-based perfusate at physiological inflow pressures and temperature. NMP also permits viability assessment through evaluation of the perfusate flow rates through the portal vein and hepatic artery. In addition to this, biochemical assessment and perfusate gas analysis can be performed to provide insights into the metabolic activity of the liver. Method Discarded human liver grafts (n=6), were perfused for 24 hours. Core biopsies and perfusate samples were taken from each liver at 5 distinct time intervals over 24 hours. Core biopsies were fixed and stained with periodic acid-Schiff and analysed with Leica software to provide a quantitative estimate of the hepatocellular glycogen content. Result Hepatic glycogen concentration rose during the first hour, followed by a steady decline thereafter until the end of perfusion. Contrary to our initial hypothesis that glucose concentration within the circuit would show an inverse relationship to glycogen stores in the liver cells, we found that glucose concentration closely followed the same trend. Conclusion Change in hepatocyte glycogen content provides an important insight into the synthetic function of a liver destined for transplant. Our research suggests that glucose concentration can be used as a surrogate marker for the synthetic function of a liver on NMP and provides valuable information on the glycogen-synthesising capability of the hepatocytes. In future, this could potentially aid the decision-making process with regards to liver graft transplant viability. Take-home message Perfusate glucose concentration could provide an insight into the viability of liver transplants

Reduced Flow during Ex-Situ Heart Perfusion Provides Superior Function Preservation and Less Edema Formation

PurposeEx-situ heart perfusion (ESHP) allows continuous functional and metabolic resuscitation of donor hearts before transplantation. Evidence indicates that hearts that are perfused under physiologic conditions lose the normal vasomotor tone and fail in a few hours. We aim to evaluate the reduced coronary flow during ex situ heart perfusion on cardiac function and coronary vascular resistance.MethodsPorcine hearts (n=6 each group) were perfused in working mode for 6 hours either by constant diastolic pressure perfusion (40 mmHg) or constant coronary blood flow rate perfusion (500mL/min). Cardiac functional parameters, coronary vascular resistance (CVR) and metabolism were monitored continuously. Myocardial injury parameter of cardiac troponin I and lipid peroxidation marker oxidized low-density lipoprotein were evaluated in the perfusate. These parameters were compared between groups at different time.ResultsCardiac function was better preserved in the flow control group at 5 hours (T5) of perfusion, indicated by higher stroke work and cardiac index (p<0.05, Figure AC). After perfusion the flow control group showed less edema than the pressure control group (p<0.001, Figure B). Both groups have a significant decrease of CVR, with relative higher in the flow control group (p<0.01, Figure D). cTnI was lower in the flow control group starting at T3, indicating less myocardial injury (p<0.05, Figure E). oxLDL increased in both groups, indicating lipid peroxidation formation; the level in the flow control group was significantly lower in comparison to the pressure control group (p<0.01, Figure F), suggesting less oxidative stress in this group.ConclusionReduced coronary flow during ex-situ heart perfusion decreased edema formation, showed less myocardial damage and oxidative stress which may contribute to better cardiac function preservation. The relative higher coronary vascular resistance induced by reduced coronary flow perfusion may help to maintain the cardiac function. Ex-situ heart perfusion (ESHP) allows continuous functional and metabolic resuscitation of donor hearts before transplantation. Evidence indicates that hearts that are perfused under physiologic conditions lose the normal vasomotor tone and fail in a few hours. We aim to evaluate the reduced coronary flow during ex situ heart perfusion on cardiac function and coronary vascular resistance. Porcine hearts (n=6 each group) were perfused in working mode for 6 hours either by constant diastolic pressure perfusion (40 mmHg) or constant coronary blood flow rate perfusion (500mL/min). Cardiac functional parameters, coronary vascular resistance (CVR) and metabolism were monitored continuously. Myocardial injury parameter of cardiac troponin I and lipid peroxidation marker oxidized low-density lipoprotein were evaluated in the perfusate. These parameters were compared between groups at different time. Cardiac function was better preserved in the flow control group at 5 hours (T5) of perfusion, indicated by higher stroke work and cardiac index (p<0.05, Figure AC). After perfusion the flow control group showed less edema than the pressure control group (p<0.001, Figure B). Both groups have a significant decrease of CVR, with relative higher in the flow control group (p<0.01, Figure D). cTnI was lower in the flow control group starting at T3, indicating less myocardial injury (p<0.05, Figure E). oxLDL increased in both groups, indicating lipid peroxidation formation; the level in the flow control group was significantly lower in comparison to the pressure control group (p<0.01, Figure F), suggesting less oxidative stress in this group. Reduced coronary flow during ex-situ heart perfusion decreased edema formation, showed less myocardial damage and oxidative stress which may contribute to better cardiac function preservation. The relative higher coronary vascular resistance induced by reduced coronary flow perfusion may help to maintain the cardiac function.

Aortic arch aneurysm repair – Unsteady hemodynamics and perfusion at different heart rates

The aortic arch aneurysm is a complex disease that requires branching of one or more aortic arch vessels and can be fatal if left untreated. In this in vitro study, we examine the effect of the treatment approach on the unsteady hemodynamics and blood perfusion to the upper vessel's in models of an aortic arch aneurysm, and of the three common repair approaches: open-chest surgical repair, chimney, and hybrid approach. A particle image velocimetry method was used to quantify the unsteady hemodynamics in the four models simulated in a mock circulatory loop, to evaluate unsteady hemodynamic parameters and measure perfusion to the brain and the upper body.According to the findings, in terms of perfusion to the brain and upper body, the surgery model has the highest flow rate comparing to the other models in most heart-rate conditions. It also shows oscillatory parameters in the upper vessels which in normal arteries are correlated with a better arterial function. Between the two endovascular procedures, the hybrid model exhibits slightly better hemodynamic characteristics than the chimney model, with lower shear stresses and more oscillatory flow and WSS in the upper vessels. The hybrid model had lower perfusion flow rates to upper vessels during rest conditions (90BPM). However, unlike the other models, perfusion in the hybrid model increased with heart rate, thus at 135 BPM, it results in flow rate to upper vessels similar to that of the chimney model. The results of this study may shed light on future endograft' design and placement techniques.

3D ultrasound coronarography: first proof of concept study on isolated beating rat hearts

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Bettencourt Foundation Background/Introduction We demonstrated recently that Ultrafast ultrasound Doppler imaging can image the intramyocardial coronary circulation in beating hearts of large animals and patients [1]. Yet, ultrasound spatial resolution remains limited by wave physics and coronaries smaller than ∼100 µm could not be imaged. Ultrasound Localization Microscopy (ULM) [2] was recently introduced to tackle this issue and exploit the micrometric localization of microbubble contrast agents at ultrafast frame rate in order to image blood flows in micrometer vessels. Purpose The objective of this work was to demonstrate that 3D ultrafast ultrasound with contrast agents can provide the full 3D mapping of the coronary microcirculation with quantitative flow velocity on a beating rat heart. Methods Acquisitions were performed on ex vivo rat hearts (n = 5) with retrograde perfusion (Langendorff model). A flow of a Krebs–Henseleit solution mixed with a diluted microbubbles solution (0.22%) was perfused at controlled pressure into the coronary arteries (between 5 and 15 mL/min). We used a 32 × 32 elements, 8-MHz matrix-array ultrasound transducer connected to a 1024-channel programmable ultrasound scanner. An ultrafast Doppler imaging sequence consisting of 9 plane waves was transmitted at a PRF of 20 kHz during 270 ms and repeated 40 times. After beamforming and SVD clutter filtering, the microbubbles were localized and tracked in 3D. Flow velocity were mapped at baseline and after infusion of Adenosine (10e-5 µMol) at constant coronary perfusion pressure (120 mm Hg). Eventually, the hearts were fixed using formaldehyde perfusion and imaged by µCT after injection of radio opaque agent. Results We successfully imaged the coronary blood flows of entire rat hearts. It revealed the entire vasculature from large main coronaries arteries (cross section up to 1 mm) to small arterioles (smaller than 40 µm). Coronary flow velocities ranged from [1 – 50] cm/s depending on the arteries diameter. Velocity estimates were validated in vitro in tubes of Ø0.58mm and were in good agreement with theoretical values of a Poiseuille’s flow (relative ratio of 10% for maximum velocities). After Adenosine infusion, perfusion flow rates increased 102% ± 50% (p &amp;lt; 0.05) on average. Eventually, anatomy revealed by 3D ultrasound coronarography was in accordance with the anatomy revealed by the µCT. Conclusion(s) We demonstrated the feasibility of 3D ultrasound coronarography on isolated beating rat hearts. This technique has the potential to become a novel imaging tool to investigate the coronary micro-circulation and quantify non-invasively the Coronary Flow Reserve (CFR). Abstract Figure. Ultrasound coronarography

Original Method for Evaluating Endothelial Vasomotor Function of Isolated Vessel in Experiment.

We describe a method for experimental assessment of endothelial dysfunction in an isolated using extracorporeal machine perfusion and vasoactive substrates agents (norepinephrine and acetylcholine). Quantitative assessment of endothelial vasomotor dysfunction was proposed, based on registration of changes in volumetric perfusate flow rate through the isolated vessel. Functional activity of the isolated aorta endothelium and total content of stable NO degradation products in blood plasma were studied in outbred white male rats after subtotal liver resection. Impaired vasodilation ability of the aorta and increased content of NO degradation products in blood plasma observed after surgery indicate the development of vasomotor endothelial dysfunction associated with oxidative stress, cholemia, and endotoxemia.

Neural network-based modeling of the number of microbubbles generated with four circulation factors in cardiopulmonary bypass

The need for the estimation of the number of microbubbles (MBs) in cardiopulmonary bypass surgery has been recognized among surgeons to avoid postoperative neurological complications. MBs that exceed the diameter of human capillaries may cause endothelial disruption as well as microvascular obstructions that block posterior capillary blood flow. In this paper, we analyzed the relationship between the number of microbubbles generated and four circulation factors, i.e., intraoperative suction flow rate, venous reservoir level, continuous blood viscosity and perfusion flow rate in cardiopulmonary bypass, and proposed a neural-networked model to estimate the number of microbubbles with the factors. Model parameters were determined in a machine-learning manner using experimental data with bovine blood as the perfusate. The estimation accuracy of the model, assessed by tenfold cross-validation, demonstrated that the number of MBs can be estimated with a determinant coefficient R2 = 0.9328 (p < 0.001). A significant increase in the residual error was found when each of four factors was excluded from the contributory variables. The study demonstrated the importance of four circulation factors in the prediction of the number of MBs and its capacity to eliminate potential postsurgical complication risks.

Evaluation of the suitability of a donor heart for transplantation after various asystole periods in experiment

The lack of donor hearts for transplantation in patients with end-stage chronic heart failure makes us look for new ways to increase the pool of donors. One of the possible ways to increase the donor base may be the use of asystolic donors, i. e. patients with in-hospital cardiac arrest or patients with cardiac arrest after brain death. The aim of the study was to examine the morphofunctional state of the heart after various periods of normothermic asystole in an experiment on rats. Materials and methods . Experiments were performed on female Wistar rats. After cervical dislocation and registration of respiratory arrest, the body temperature of the rat was maintained above at 36.5±0.5 °C for 10, 15 and 20 minutes, depending on the group. After asystole period, the heart was connected to the Langendorff apparatus to provide perfusion for 90 minutes. During perfusion of the heart, the pulse pressure inside the left ventricle (LVPP) and the volumetric flow rate of coronary perfusion (CP) were recorded every 15 minutes. After the end of the experiment, the volume of necrosis was calculated. Results . In the group with 20-minute ischemia, there was no recovery of ventricular contractions, the volume of necrosis was 34,9±11,7 %. In the group with 15-minute ischemia, the volume of necrosis was 19,5±7,6 %. In the group with 10-minute normothermic ischemia, the volume of necrosis was 13,1±5,1 %. Conclusions . 15 minutes of normothermic cardiac arrest in situ causes irreversible damage to the heart of the animal, which does not allow us to consider it as a potential donor heart. However, this volume of damage to the myocardium and microvasculature, together with the restoration of the contractile activity of the heart, makes this group the main one for the continuation of such experiments using various methods of heart reconditioning.

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation.

Normothermic ex vivo liver perfusion (NEVLP) is a promising strategy to increase the donor pool in liver transplantation. Small animal models are essential to further investigate questions regarding organ preservation and reconditioning by NEVLP. A dual vessel small animal NEVLP (dNEVLP) model was developed using metamizole as a vasodilator and compared to conventional portovenous single vessel NEVLP (sNEVLP). Livers of male Wistar rats were perfused with erythrocyte-supplemented culture medium for six hours by either dNEVLP via hepatic artery and portal vein or portovenous sNEVLP. dNEVLP was performed either with or without metamizole treatment. Perfusion pressure and flow rates were constantly monitored. Transaminase levels were determined in the perfusate at the start and after three and six hours of perfusion. Bile secretion was monitored and bile LDH and GGT levels were measured hourly. Histopathological analysis was performed using liver and bile duct tissue samples after perfusion. Hepatic artery pressure was significantly lower in dNEVLP with metamizole administration. Compared to sNEVLP, dNEVLP with metamizole treatment showed higher bile production, lower levels of transaminases during and after perfusion as well as significantly lower necrosis in liver and bile duct tissue. Biochemical markers of bile duct injury showed the same trend. Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion. The administration of metamizole effectively ameliorates arterial vasospasm allowing for six hours of dNEVLP, with superior outcome compared to sNEVLP.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Effect of cardiopulmonary bypass on the development of acute kidney injury after open-heart surgery

THE AIM. Assessment of factors associated with cardiopulmonary bypass (CPB) in acute renal dysfunction in patients in the early postoperative period after cardiac surgery. PATIENTS AND METHODS. Monocentric observational study in patients (n = 97) who underwent elective open-heart cardiac surgery (coronary artery bypass grafting -50.44 %, aortic valve prosthetics – 31.04 %, mitral valve prosthetics – 12.61 %) using cardiopulmonary bypass. Inclusion criteria: the study included patients not younger than 18 years old, undergoing cardiac surgery with CPB lasting up to 95 minutes (coronary bypass surgery, valve replacement), without signs of end-stage renal disease. Using nonparametric correlation analysis, we evaluated the effect on the development of acute kidney injury (AKI) of the following extracorporeal circulation factors: duration of CPB, aortic cross-clamp, mean arterial pressure (MAP), cardiac index (CI), perfusion flow rate (PFR), transport, consumption, and oxygen extraction variables. The diagnosis of AKI was made on the basis of the KDIGO classification, the studied parameters were recorded initially (before the operation), 15 minutes after the start of general anesthesia, 30 minutes after the start of cardiopulmonary bypass and 15 minutes after the end of general anesthesia. RESULTS. The frequency of AKI in 24 hours after surgery was 56.3 % (58 cases): including stage 1 in 37 (35.9 %), stage 2 in 17 (16.5 %) , stage 3 – in 4 (3.9 %) patients. In the 48th hour of the postoperative period, signs of AKI regressed and were presented in only 26 people (25.2 %), including the stage 1 in 18 (17.5 %), the stage 2 – in 5 (4.8 %), stage 3 – in 3 (2.9 %). Among the risk factors for AKI in cardiac surgery with CPB, the main effect of the anemia was revealed, especially a decrease in hemoglobin levels of less than 90 g / l and hematocrit of less than 25 %. CONCLUSION. Hemodilution below the "threshold" values of hemoglobin and hematocrit during the CPB provoke acute kidney injury in patients undergoing open-heart surgery.

P0115EFFECT OF CARDIOPULMONARY BYPASS ON THE DEVELOPMENT OF ACUTE KIDNEY INJURY AFTER OPEN-HEARD SURGERY

Abstract Background and Aims Assessment of factors associated with cardiopulmonary bypass (CPB) in acute renal dysfunction in patients in the early postoperative period after cardiac surgery. Method Monocentric observational study in patients (n = 97) who underwent elective open-heart cardiac surgery (coronary artery bypass grafting -50.44%, aortic valve prosthetics - 31.04%, mitral valve prosthetics - 12.61%) using cardiopulmonary bypass. Inclusion criteria: the study included patients not younger than 18 years old, undergoing cardiac surgery with CPB lasting up to 95 minutes (coronary bypass surgery, valve replacement), without signs of end-stage chronic kidney disease (CKD). Using nonparametric correlation analysis, we evaluated the effect on the development of acute kidney injury (AKI) of the following extracorporeal circulation factors: duration of CPB, aortic cross-clamp, mean arterial pressure (MAP), cardiac index (CI), perfusion flow rate (PFR), transport, consumption and oxygen extraction variables. The diagnosis of AKI was made on the basis of the KDIGO classification, the studied parameters were recorded initially (before the operation), 15 minutes after the start of general anesthesia, 30 minutes after the start of cardiopulmonary bypass and 15 minutes after the end of general anesthesia. Results The frequency of AKI in 24 hours after surgery was 56.3% (58 cases): including stage 1 in 37 (35.9%), stage 2 in 17 (16.5%), stage 3 - in 4 (3.9%) patients. In the 48th hour of the postoperative period signs of AKI regressed, and were presented in only 26 people (25.2%), including the stage 1 in 18 (17.5%), the stage 2 - in 5 (4.8%), stage 3 - in 3 (2.9%). Among the risk factors for AKI in cardiac surgery with CPB, the main effect of of the anemia was revealed, especially a decrease in hemoglobin levels of less than 90 g / l and hematocrit of less than 25%. Conclusion Hemodilution below the "threshold" values of hemoglobin and hematocrit during the CPB provoke acute kidney injury in patients undergoing open-heart surgery.

Fluid flow-induced cell stimulation in bone tissue engineering changes due to interstitial tissue formation in vitro.

In tissue engineering experiments in vitro, bioreactors have been used for applying wall shear stress (WSS) on cells to regulate cellular activities. To determine the loading conditions within bioreactors and to design tissue engineering products, in silico models are used. Previous in silico studies in bone tissue engineering (BTE) focused on quantifying the WSS on cells and the influence on appositional tissue growth. However, many BTE experiments also show interstitial tissue formation (i.e., tissue infiltrated in the pores rather than growing on the struts ‐ appositional growth), which has not been considered in previous in silico studies. We hereby used a multiscale fluid‐solid interaction model to quantify the WSS and mechanical strain on cells with interstitial tissue formation, taken from a reported BTE experiment. The WSS showed a high variation among different interstitial tissue morphologies. This is different to the situation under appositional tissue growth. It is found that a 35% filling of the pores results (by mineralised bone tissue) when the average WSS increases from 1.530 (day 0) to 5.735 mPa (day 28). Furthermore, the mechanical strain on cells caused by the fluid flow was extremely low (at the level of 10−14‐10−15), comparing to the threshold in a previous mechanobiological theory of osteogenesis (eg, 10−2). The output from this study offers a significant insight of the WSS changes during interstitial tissue growth under a constant perfusion flow rate in a BTE experiment. It has paved the way for optimising the local micro‐fluidic environment for interstitial tissue mineralisation.

Automated dendritic cell generation in a closed system

Background & Aim Dendritic cells (DC) are an attractive vehicle for therapeutic manipulation and are used in antigen-pulsed autologous DC therapies, DC-stimulated autologous T cell therapies, and a variety of in vitro cellular immunology assays. Given the low abundance of DC in blood, they are typically generated ex vivo from monocytes or stem cell precursors in a manual culture process. The standard multiwell plate or flask culture used for DC generation requires several solution exchanges including cytokine refresh and wash steps. When DCs must be generated in a patient-specific manner, the multistep nature of the protocol presents significant logistical challenges for consistent results. To this end, the Corning MicroDEN® system relies upon medium perfusion to supply fresh cytokines throughout the duration of monocyte-derived DC generation. This study demonstrates performance of the MicroDEN system for immature DC (iDC) generation from a source of enriched monocytes. Methods, Results & Conclusion Monocytes were isolated from whole blood via Ficoll separation and CD14 magnetic bead purification. Monocyte differentiation to iDCs was achieved by a 6 day culture with CellGenix DC medium containing IL4 (350 U/mL;CellGenix), GM-CSF (350 U/mL;CellGenix), and 1% penicillin-streptomycin in both 6-well tissue culture-treated (TCT) multiwell plates (Corning) and the MicroDEN system. Flow cytometry data indicated that the iDCs from MicroDEN and 6-well plates were phenotypically comparable with regard to expression of CD209, CD80, CD83, CD86 and CD14. Allogeneic T cell proliferation assays and an antigen-specific assay demonstrated that iDCs generated via MicroDEN are functionally competent and can successfully generate a T cell response. A DOE-based approach was employed to characterize the relationship between perfusion flow rate, monocyte seeding density, and yield of dendritic cells. DCs generated by MicroDEN are functionally equivalent to DCs generated by the standard well plate culture. The larger surface area of MicroDEN cartridge allows generation of a higher number of dendritic cells in single run when compared to the standard 6-well plate, thereby reducing time and labor involved in harvesting cells from multiple plates. Higher perfusion flow rates enable the processing of larger numbers of seeded monocytes, upto a seeding density level of 1.3 million monocytes/cm2. MicroDEN is an effective tool to generate dendritic cells for applications ranging from cell-based assays to cell-based immunotherapies.

Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.