Perfusion System Research Articles

Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients.

Simple SummaryDiabetic nephropathy is one of the most frequent complications of diabetes, resulting from diffuse damage to different kidney cells. The identification of subjects at risk is mandatory to prevent its development and provide appropriate therapies reducing the unmanageable evolution towards end-stage kidney disease. The aim of this work was to identify urinary-derived extracellular vesicles (EVs) miRNA cargo to be used as biomarker of kidney damage in diabetic patients. The miRNA profile was then correlated with the molecular mechanism associated with the glomerular and tubular damage using a diabetic-like model. In patients, miR145 and miR126 in urinary EVs increased together with albuminuria. MiR145 and miR126 increased in parallel in EVs from renal epithelial cells undergoing transition to a fibrotic mesenchymal phenotype. These data unveiled a role for miR126 and miR145 as the biomarkers of damage progression and proteinuria development in diabetic nephropathy. Diabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney’s physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-β1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.

Save the Leg: Utilization of Distal Perfusion Catheter With Impella CP® May Prevent Morbidity of Limb.

Leg ischemia is a potential complication of percutaneous left ventricular assist device (Impella CP®) placement. To avoid leg ischemia in at-risk patients, a distal perfusion catheter (DPC) should be placed. In utilizing a passive distal perfusion system from the contralateral femoral artery, we optimized blood flow to the distal limb mitigating leg ischemia. A 65-year-old female with dilated cardiomyopathy complicated by hemodynamic instability was placed on an Impella CP via the right femoral artery. A DPC was placed to the right distal femoral artery and connected to the wire re-access port of the Impella CP. Despite this, the leg became ischemic shortly after admission to the ICU. A contralateral femoral arterial line was placed in standard fashion, and it was connected to the DPC while the wire re-access port was capped. Shortly after placement of the new DPC system, the right lower extremity distal pulses returned, and distal leg ischemia was resolved. Another patient, a 67-year-old male with acute myocardial infarction, was placed on an Impella CP via the left femoral artery for cardiogenic shock. His hemodynamics continued to deteriorate, requiring initiation of veno-arterial extracorporeal membrane oxygenation (VA ECMO) via the right femoral artery and vein with associated DPC placement. Shortly after the initiation of VA ECMO, the Impella CP-related extremity (left leg) became ischemic. A left femoral DPC was placed and connected to the side port of the right femoral arterial cannula. After initiation of the additional DPC system, the left leg ischemia resolved. Distal leg ischemia with Impella CP is not a rare event. Utilization of a DPC to Impella CP may decrease the morbidity of limb malperfusion.

The administration of valproic acid exerts a protective effect by reducing the infarct size and the triggering of ventricular fibrillation in an experimental model of chronic myocardial infarction

Abstract Introduction In myocardial infarction there are complications associated with adverse cardiac remodeling that occurs during the fibrotic process, producing an arrhythmogenic substrate that favors the triggering of ventricular arrhythmias such as ventricular fibrillation (VF). These complications also contribute to infarct expansion and mortality. Purpose To analyze, in a model of chronic infarction (5 weeks) in rabbits, the effects of valproic acid (VA), an inhibitor of histone deacetylase enzymes, on the inducibility of ventricular fibrillation and infarct size. Methods 47 New Zealand rabbits were assigned to sham (n=15), control (n=22) and VA-treated (n=10) groups. After midsternal thoracotomy and pericardiotomy, the circumflex coronary artery was occluded (1 hour), followed by reperfusion (control and treated groups). 500 mg/kg of VA were administered every 24 hours for 5 weeks. Then, the animals were sacrificed, their hearts excised and placed in a Langendorff perfusion system. We used a multielectrode recording plate (256 electrodes) and ventricular stimulation electrodes in the left ventricular epicardium. The inducibility of VF was analyzed using ventricular extrastimulus tests (VEST) with two basic stimulation cycle lengths (250 and 150 ms) and up to the three extrastimuli in each cycle. VEST was applied in four peri-infarct stimulation zones. After the electrophysiological study, the circumflex coronary artery was occluded, and the aorta was perfused with thioflavin-S to determine the area at risk (AR). We made sections of the left ventricle (LV) which were incubated in a triphenyltetrazolium chloride solution to determine the infarct size. The images obtained were digitized and manually quantified. The quantification was performed in 3 sections, located below the ligature of the coronary occlusion. Unpaired Student's t-test or chi-square test were used when appropriate (p<0.05). Results Infarct size decreased in the VA-treated group compared to the control group, when expressed as a percentage of AR (40.3±5.3% vs. 69.8±20.4%, respectively) and when expressed as percentage of LV (19.3±7.8% vs. 38.6±16.4% respectively). The myocardium of the sham group did not exhibit areas of necrosis. Regarding VF inducibility, the treated group presented lower arrhythmia inducibility, regardless of the number of extrastimuli and pacing cycle length, triggering the arrhythmia in 40% of cases compared to 59% in the control group (p=0.011). Likewise, more extrastimuli were needed in VA-treated animals to trigger VF than controls (2 (50%) and 3 (50%) extrastimuli vs. 1 (15%), 2 (46%) and 3 (38%) extrastimuli needed, respectively; χ2=16.526; p<0.005). Conclusion VA did exhibit a protective effect, as shown by its ability to limit the extension of the infarct size and to reduce the inducibility of ventricular fibrillation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Carlos III Health Institute and Institute of Health Research (INCLIVA)

Optimization of an adenovirus-vectored zoster vaccine production process with chemically defined medium and a perfusion system.

ObjectivesCells grown in chemically defined medium are sensitive to shear force, potentially resulting in decreased cell growth. We optimized the perfusion process for HEK293 cell-based recombinant adenovirus-vectored zoster vaccine (Ad-HER) production with chemically defined medium.MethodsWe first studied the pseudo-continuous strategies in shake flasks as a mimic of the bioreactor equipped with perfusion systems. Using design of experiment (DoE) in shake flasks, we obtained the regression models between Ad-HER titer/virus input–output ratio and three production process parameters: time of infection (TOI), multiplicity of infection (MOI), and virus production pH (pH). We then confirmed the effect of Pluronic F68 (PF-68) at 3.0 g/L on HEK293 cell growth and Ad-HER production in shake flasks and a 2 L benchtop bioreactor.ResultsThe optimized process was scale-up to a 2 L benchtop bioreactor with the PATFP perfusion system, which yielded cell density of 7.4 × 106 cells/mL and Ad-HER titer of 9.8 × 109 IFU/mL at 2 dpi, comparable to the bioreactor with a ATF2 system.ConclusionThis optimization strategy could be used to develop a robust process with stable cell culture performance and adenovirus titer. Increasing PF-68 concentration in chemically defined medium could protect cells from shear stress generated by perfusion system.

THE TELL-TALE HEART. MACHINE PERFUSION IN HEART TRANSPLANTATION

Hear Transplantation (HTx) is the current best surgical treatment for patients affected by end-stage heart failure. However, the improvement of medical and interventional therapies has led to an increasingly old and high-risk population of HTx candidates. Moreover, the use of “extended” donor criteria to face the shortage of donors could increase the risk of worse outcomes after HTx. In this setting, donor organ preservation strategy could significantly affect HTx results. The cold storage (CS) represents the standard practice worldwide for donor graft preservation. However, prolonged ischemic time is known to be an independent risk factor for mortality after HTx. Machine perfusion (MP) systems continuously perfuse the donor heart, reducing ischemic time. Also, since MP permits to evaluate marginal donor organs, they could represent a safe and effective technique to expand the available donor pool. Despite the increasing number of donor hearts preserved with MP, whether MP could be considered superior to traditional CS still represents a matter of debate. The aim of this paper is to summarize and critically assess the available clinical data on MP employed for HTx.

In Vivo Study of Monoamine Oxidases Using Multisite Intracerebral Microdialysis.

The activity of monoamine oxidases (MAOs) in the brain is often associated with neurodegenerative diseases. The study of MAOs in vivo or ex vivo is generally performed using MAO inhibitors and rarely using substrates. We present a pharmacological approach using intracerebral microdialysis to study the activity of MAO in the striatum and the prefrontal cortex of rats. It consists of applying ascending concentrations of 3-methoxytyramine (3-MT) as a substrate via the probes and measuring the indirect product homovanillic acid generated by MAO activity. We present herein the methodologies comprising our in-house stereotaxic procedures in rats, the microdialysis perfusion system and the substrate application, and the neurochemical analysis of the samples.

Perfusable Vessel-on-a-Chip for Antiangiogenic Drug Screening with Coaxial Bioprinting.

Vessel-on-a-chips, which can be used to study microscale fluid dynamics, tissue-level biological molecules delivery and intercellular communication under favorable three-dimensional (3D) extracellular matrix microenvironment, are increasingly gaining traction. However, not many of them can allow for long-term perfusion and easy observation of angiogenesis process. Since angiogenesis is necessary for the expansion of tumor, antiangiogenic drugs play a significant role in cancer treatment. In this study, we established an innovative and reliable antiangiogenic drug screening chip that was highly modularly integrated for long-term perfusion (up to 10 days depending on the hydrogel formula) and real-time monitoring. To maintain an unobstructed flow of cell-laden tubes for subsequent perfusion culture on the premise of excellent bioactivities, a polycaprolactone stent inspired by coronary artery stents was introduced to hold up the tubular lumen from the inside, while the perfusion chip was also elaborately designed to allow for convenient observation. After 3 days of perfusion screening, distinct differences in human umbilical vein endothelial cell sprouting were observed for a gradient of concentrations of bevacizumab, which pointed to the effectiveness and reliability of the drug screening perfusion system. Overall, a perfusion system for antiangiogenic drug screening was developed, which can not only conduct drug evaluation, but also be potentially useful in other vessel-mimicking scenarios in the area of tissue engineering, drug screening, pharmacokinetics, and regenerative medicine.

Retinal organoids and microfluidic chip-based approaches to explore the retinitis pigmentosa with USH2A mutations.

Retinitis pigmentosa (RP) is a leading cause of vision impairment and blindness worldwide, with limited medical treatment options. USH2A mutations are one of the most common causes of non-syndromic RP. In this study, we developed retinal organoids (ROs) and retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs) of RP patient to establish a sustainable in vitro RP disease model. RT-qPCR, western blot, and immunofluorescent staining assessments showed that USH2A mutations induced apoptosis of iPSCs and ROs, and deficiency of the extracellular matrix (ECM) components. Transcriptomics and proteomics findings suggested that abnormal ECM-receptor interactions could result in apoptosis of ROs with USH2A mutations via the PI3K-Akt pathway. To optimize the culture conditions of ROs, we fabricated a microfluidic chip to co-culture the ROs with RPE cells. Our results showed that this perfusion system could efficiently improve the survival rate of ROs. Further, ECM components such as laminin and collagen IV of ROs in the RP group were upregulated compared with those maintained in static culture. These findings illustrate the potential of microfluidic chip combined with ROs technology in disease modelling for RP.

Low levels of nitric oxide promotes heme maturation into several hemeproteins and is also therapeutic

Nitric oxide (NO) is a signal molecule and plays a critical role in the regulation of vascular tone, displays anti-platelet and anti-inflammatory properties. While our earlier and current studies found that low NO doses trigger a rapid heme insertion into immature heme-free soluble guanylyl cyclase β subunit (apo-sGCβ), resulting in a mature sGC-αβ heterodimer, more recent evidence suggests that low NO doses can also trigger heme-maturation of hemoglobin and myoglobin. This low NO phenomena was not only limited to sGC and the globins, but was also found to occur in all three nitric oxide synthases (iNOS, nNOS and eNOS) and Myeloperoxidase (MPO). Interestingly high NO doses were inhibitory to heme-insertion for these hemeproteins, suggesting that NO has a dose-dependent dual effect as it can act both ways to induce or inhibit heme-maturation of key hemeproteins. While low NO stimulated heme-insertion of globins required the presence of the NO-sGC-cGMP signal pathway, iNOS heme-maturation also required the presence of an active sGC. These effects of low NO were significantly diminished in the tissues of double (n/eNOS−/−) and triple (n/i/eNOS−/−) NOS knock out mice where lung sGC was found be heme-free and the myoglobin or hemoglobin from the heart/lungs were found be low in heme, suggesting that loss of endogenous NO globally impacts the whole animal and that this impact of low NO is both essential and physiologically relevant for hemeprotein maturation. Effects of low NO were also found to be protective against ischemia reperfusion injury on an ex vivo lung perfusion (EVLP) system prior to lung transplant, which further suggests that low NO levels are also therapeutic.

Impact of various periods of perfusion-pause and reperfusion on the severity of myocardial injury in the langenodorff model.

To explore impact of various periods of ischemia and reperfusion on the severity of myocardial injury. Langendorff model of isolated cardiac perfusion system was established in 56 rat hearts. They were randomly assigned into four groups with four different ischemia (perfusion-pause) time and reperfusion time. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were measured and the size of myocardial infarction was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The levels of AST, ALT, LDH, and CK-MB in the heart tissues and perfusate were lowest in the group I (shortest time of perfusion-pause and reperfusion) followed by the groups II, III, and IV (longest time of perfusion-pause and reperfusion) (p < 0.05). The myocardial infarction size was smallest in the group I (6.63 ± 0.47) followed by group II (15.12 ± 1.03), group III (20.32 ± 2.18), and group IV (32.29 ± 5.42) (p < 0.05). Two-way ANOVA analysis revealed that period of perfusion-pause and reperfusion independently and significantly affected the levels of AST and ALT in both heart tissues and perfusate (p < 0.001). The interaction of pausing period and reperfusion significantly affected the level of AST (p = 0.046) and CK-MB (p = 0.001) in the perfusate. In addition, perfusion-pause period significantly affected levels of LDH and CK-MB only in the perfusate (p < 0.001). Neither perfusate nor heart tissue LDH level was significantly affected by the interaction of perfusion-pause and reperfusion period (p > 0.05). The severity of myocardial injury in the Langendorff model was affected by the period of perfusion-pause and reperfusion. The longer period of perfusion-pause followed by the longer the period of reperfusion, the severe myocardial injury was found.

P11.11: A New Designed Machine Perfusion System to Mimic the Cardiac Cycle’s Heart Function for a Better Hepatic Microcirculation Perfusion

Background: Machine perfusion is a relatively new concept developed for protecting marginal liver transplantation. However, so far, limited studies have reported on liver transplantation donated after cardiac death (DCD). Objective: To establish and verify a novel machine perfusion concept to eliminate these risk factors and recover DCD livers. Methods: To explore a novel hypothermic oxygenated perfusion (HOPE) system, two pumps and an elastic water sac were integrated into the perfusion system to mimic the cardiac cycle’s heart function. Compared to the other two traditional systems (HOPE S1 and S2), the novel HOPE system (HOPE S3) was verified in rats by no injured livers (perfused with methylene blue diluted by UW-G) or DCD livers subjected to 60 min in situ warm ischemia, without application of heparin. Liver perfusion outcomes were compared using macroscopy, microscopy, molecular tests, and orthotopic liver transplantation (OLT). Results: DCD livers, subjected to HOPE systems’ perfusion, disclosed reduced injury and improved survival compared to static cold storage after warm ischemia of 60 min (DCD+SCS). The post-transplantation survival rates of 4 weeks were 0%, 20 and 30% in DCD+SCS, DCD+SCS+HOPE S1, and DCD+SCS+HOPE S2 group, respectively. In contrast, HOPE S3 protected from hepatocyte and non-parenchymal cell injury and led to 60% (6/10) animal survival after 60 min of warm donor ischemia (DCD+SCS+HOPE S3). These data were further demonstrated by monitoring the hepatic sinusoid microcirculation, function morphological, and molecular changes of preserved livers. Conclusion: The newly designed novel machine perfusion system achieved complete and homogeneous liver perfusion and is regarded as a useful tool for resuscitating DCD liver grafts with severe warm ischemic injuries. Key words：Liver transplantation; Hypothermic oxyenated perfusion; Donation after cardiac death; Organ recovery; Perservayion manner; Ischemic reperfusion injury; Static cold storage; Hepatic sinusoidal; Hepatic microcirculation; Inferior vena cava. National Natural Science Foundation of China (Grant No. 81970548). Medical Science Advancement Program (Youth Scholars) of Wuhan University (Grant No. TFZZ2018035). Science and Technology Innovation Cultivation Fund of Zhongnan Hospital of Wuhan University(No.znpy2019041).

313.16: Glycocalyx Damage Marker Syndecan-1 Correlates With Early Allograft Dysfunction During Hypothermic Liver Machine Perfusion

Introduction: The endothelial glycocalyx is mainly consisting of Syndecan-1 (Sdc-1) which is released into human serum upon glycocalyx degradation. Glycocalyx alterations are connected to different aspects of organ damage and have been recently investigated in the context of liver transplantation. Due to shortage of donor organs, new preservation methods as hypothermic oxygenated machine perfusion (HOPE) have been introduced to enable transplantation of organs with a higher risk profile. For safe expansion of the donor pool, reliable organ assessment markers for evaluation during machine perfusion are missing. We aimed to measure glycocalyx damage during hypothermic liver perfusion. Methods: HOPE was performed with the Organ Assist® perfusion system on 40 livers, prior to organ transplantation. Samples were collected during (perfusate at 0 and 60 min) and after HOPE (effluent). Sdc-1 concentration in samples was measured by ELISA as indicator for glycocalyx destruction. We compared clinical parameters with Sdc-1 levels in patients regarding the development of early allograft dysfunction (EAD) using Mann-Whitney U test, Pearson correlation and receiver operating characteristics (ROC). Results: The 13 patients which developed EAD, showed an elevation in Sdc-1 concentration compared to non-EAD patients during HOPE at 0 min: [598 (±526) vs. 276 (±150) ng/ml; p=0.076] and 60 min: [1099 (±739) vs. 521 (±382) ng/ml; p=0.016], as well as afterwards in the effluent: [2074 (±1273) vs. 443 (±226) ng/ml; p=0.001 (n=15, 4 with EAD)]. Concentration of Sdc-1 during HOPE correlated with EAD at 0 min: (R=0.433, p=0.006), at 60 min: (R=0.471, p=0.003) and in the effluent: (R=0.769, p<0.001, n=15, 4 with EAD). Furthermore, an association between EAD and Sdc-1 concentrations was indicated by ROCs: during HOPE at 60 minutes: (AUC=0.704 and p=0.018) and in the effluent: (AUC=1 and p=0.004 (n=15, 4 with EAD)). No association of graft survival with Sdc-1 (p=0.339) was detected in cox regression analysis however, reduced graft survival (log-rank=0.009) of EAD patients compared to those without EAD could be shown. Conclusion: We found that measuring glycocalyx degradation during HOPE could indicate transplantation outcome regarding EAD. Consequently, we argue that Sdc-1 could be a useful biomarker for organ assessment during HOPE.

313.5: Meeting Packed Red Blood Cell Requirement for the Transmedics Organ Care System (OCS) Liver Transplantation Trial (PROTECT Trial) At Henry Ford Hospital

Introduction: In January 2016, HFHS Transplant Institute began the PROSPECT trial, an interventional trial studying normothermic preservation of the donated liver before transplant using the Liver Organ Care System (OCSTM). The Henry Ford Hospital Transfusion Service (TS) provided the required packed red blood cells (PRBC). Background: The TransMedics (OCSTM) liver trial was an international, randomized trial studying the intervention of a portable liver system to preserve donor livers for transplant. The system utilizes an oxygenated blood-based perfusate mixed with nutrient solution in normothermic perfusion for organ preservation. The TS provided PRBC needed for the study, final PRBC used dictated by the condition and size of the retrieved liver. TS study goal was to secure proper transfusion needs for liver perfusion while also adhering to blood inventory management. Methods: Organ procurement candidates for the PROTECT trial were selected based on pre-determined conditions and consented according to that protocol. Once the patient was consented and the liver transplantation was scheduled, the PROTECT trial team notified the TS,and 5 units of group O Positive, leukoreduced PRBC less than 10 days old were selected, and placed inside a temperature-controlled packaging container along with a remote temperature monitoring device. The container was then transported with the procurement team to the harvesting location. Once the insulated container was returned to the TS, the temperature-monitoring device information was uploaded to verify that the returned PRBC have been stored within their proper temperature limits. Results: The TS support for the PROTECT trial at HFHS occurred from January 2016 until September 2021. The table below shows the activity related to this trial. - Issued Utilized Returned Wasted PRBC units 240 132 104 4 In summary, 29/48 study events used 4 units; 3/48 study events used 5 units; on 15/48 events the organ harvesting was cancelled; all units returned had an acceptable temperature range and returned to the available inventory. For one study event, 4 PRBC were spiked in preparation for use in the perfusate prior to the event being cancelled. These PRBC were discarded and deemed wasted. Conclusions: During the PROTECT trial no PRBCs were wasted due to inappropriate storage conditions. The joint use of a temperature-controlled container and remote temperature monitoring device allowed for the appropriate return of unused PRBCs to the TS blood inventory, thus avoiding any wastage. Only 3/48 events used 5 units, the\us issuing 4 PRBC to meet the OCS™ support may suffice, further decreasing the risk for wastage of any PRBC units. After receiving FDA in September 2021, alternatives to maintain the required PRBC temperature should be addressed to minimize wastage at this time of limited blood availability for the support of the TransMedics Organ Care System (OCSTM) for liver organ perfusion.

Normothermic liver machine perfusion as a dynamic platform for regenerative purposes: What does the future have in store for us?

Liver transplantation has become an immense success; nevertheless, far more recipients are registered on waiting lists than there are available donor livers for transplantation. High-risk, extended criteria donor livers are increasingly used to reduce the discrepancy between organ demand and supply. Especially for high-risk livers, dynamic preservation using machine perfusion can decrease post-transplantation complications and may increase donor liver utilisation by improving graft quality and enabling viability testing before transplantation. To further increase the availability of donor livers suitable for transplantation, new strategies are required that make it possible to use organs that are initially too damaged to be transplanted. With the current progress in experimental liver transplantation research, (long-term) normothermic machine perfusion may be used in the future as a dynamic platform for regenerative medicine approaches, enabling repair and regeneration of injured donor livers. Currently explored therapeutics such as defatting cocktails, RNA interference, senolytics, and stem cell therapy may assist in the repair and/or regeneration of injured livers before transplantation. This review will provide a forecast of the future utility of normothermic machine perfusion in decreasing the imbalance between donor liver demand and supply by enabling the repair and regeneration of damaged donor livers.

Effects of cGMP/Akt/GSK-3β signaling pathway on atrial natriuretic peptide secretion in rabbits with rapid atrial pacing.

Atrial natriuretic peptide (ANP) plays a pivotal role in the regulation of the cardiovascular system. The ANP level increases during atrial fibrillation (AF), suggesting that AF may provoke ANP secretion, but its potential mechanism is still unclear. In the present study, the potential mechanisms of rapid atrial pacing (RAP) regulating ANP secretion was explored. Rabbits were subjected to burst RAP, ANP secretion increased whereas cyclic guanosine monophosphate (cGMP) concentrations decreased during RAP. The p-Akt and p-GSK-3β levels decreased in atrial tissues. Natriuretic peptide receptor A (NPR-A) protein and particulate guanylate cyclase (PGC) activity were detected. The sensitivity of NPR-A to ANP decreased, leading to the decrease of PGC activity. Also, the isolated atrial perfusion system were made in the rabbit model, cGMP was shown to inhibit ANP secretion, and the Akt inhibitor LY294002 (LY) and GSK-3β inhibitor SB216763 (SB) attenuated the inhibitory effects of cGMP on ANP secretion and enhanced the inhibitory effects of cGMP on atrial dynamics. In conclusion, NPR-A interacts with ANP to regulate PGC expression, and influence the expression of cGMP during RAP, which involves in the Akt/GSK-3β signaling pathway. From the aforementioned points we conclude that cGMP regulates ANP secretion by the Akt/GSK-3β signaling pathway during atrial pacing.

Microfluidic-Assisted Human Cancer Cells Culturing Platform for Space Biology Applications.

In the paper, the lab-on-chip platform applicable for the long-term cultivation of human cancer cells, as a solution meeting the demands of the CubeSat biological missions, is presented. For the first time, the selected cancer cell lines—UM-UC-3 and RT 112 were cultured on-chip for up to 50 days. The investigation was carried out in stationary conditions (without medium microflow) in ambient temperature and utilizing the microflow perfusion system in the incubation chamber assuring typical cultivation atmosphere (37 °C). All the experiments were performed to imitate the conditions that are provided before the biological mission starts (waiting for the rocket launch) and when the actual experiment is initialized on a CubeSat board in space microgravity. The results of the tests showed appropriate performance of the lab-on-chip platform, especially in the context of material and technological biocompatibility. Cultured cells were characterized by adequate morphology—high attachment rate and visible signs of proliferation in each of the experimental stage. These results are a good basis for further tests of the lab-on-chip platform in both terrestrial and space conditions. At the end of the manuscript, the authors provide some considerations regarding a potential 3-Unit CubeSat biological mission launched with Virgin Orbit company. The lab-on-chip platform was modelled to fit a 2-Unit autonomous laboratory payload.

Electric catfish hearts are not intrinsically immune to electric shocks.

High voltage electric shocks cause life threatening cardiac injuries such as sudden cardiac standstill or severe myocardial injury. Here, we analysed the physiology of the heart of the strongly electric catfish (Malapterurus beninensis) that stuns prey with high-voltage shocks but is immune to its own, as well as external, high-voltage shocks. Neither a detailed analysis of the electrocardiogram nor the structure of the heart indicated a specialized cardiac conduction system. Using a suitable perfusion system, we discovered that, despite its immunity in vivo, the explanted heart of electric catfish can readily be activated by external electrical currents and is equally sensitive to electric shock-induced arrhythmias as similar-sized goldfish hearts. The surprise thus is that the electric catfish has a vulnerable heart that requires to be protected by highly efficient but presently unknown means.

Determining the role of von Willebrand factor in thrombotic post transplantation complications using ex vivo lung perfusion system

Background and Aims : A major complication of lung transplantation is thrombosis, which may lead to allograft failure. Increased production of procoagulant molecules, such as von Willebrand factor (VWF), is a significant risk factor for thrombus formation. VWF is a pro-coagulant glycoprotein expressed only in endothelial cells and megakaryocytes, which mediates adhesion of platelets to the endothelium/sub-endothelium. External stimuli, including hypoxia can upregulate VWF. Since during organ-transplantation donor organs are under hypoxic conditions, we explored whether this may lead to alterations in VWF expression, and whether ex vivo lung perfusion (EVLP) as an innovative method for organ preservation prior transplantation can moderate the effect of VWF upregulation and its thrombogenic consequences.

Large Area Microfluidic Bioreactor for Production of Recombinant Protein.

To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm2 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.8 mL. The LM bioreactor was connected to a syringe pump system for culture media perfusion, and the cells’ culture was monitored by photomicrograph imaging. CHO-ahIFN-α2b adherent cell line expressing the anti-hIFN-2b recombinant scFv-Fc monoclonal antibody (mAb) for the treatment of systemic lupus erythematosus were cultured on the LM bioreactor. Cell culture and mAb production in the LM bioreactor could be sustained for 18 days. Moreover, the anti-hIFN-2b produced in the LM bioreactor showed higher affinity and neutralizing antiproliferative activity compared to those mAbs produced in the control condition. We demonstrate for the first-time, a large area microfluidic bioreactor for mammalian cell culture that enables a controlled microenvironment suitable for the development of high-quality biologics with potential for therapeutic use.

Generation of an Adequate Perfusion Network within Dense Collagen Hydrogels Using Thermoplastic Polymers as Sacrificial Matrix to Promote Cell Viability.

Dense collagen hydrogels are promising biomaterials for several tissue-engineering applications. They exhibit high mechanical properties, similar to physiological extracellular matrices, and do not shrink under cellular activity. However, they suffer from several drawbacks, such as weak nutrient and O2 diffusion, impacting cell survival. Here, we report a novel strategy to create a perfusion system within dense and thick collagen hydrogels to promote cell viability. The 3D printing of a thermoplastic filament (high-impact polystyrene, HIPS) with a three-wave shape is used to produce an appropriate sacrificial matrix. The HIPS thermoplastic polymer allows for good shape fidelity of the filament and does not collapse under the mechanical load of the collagen solution. After the collagen gels around the filament and dissolves, a channel is generated, allowing for adequate and rapid hydrogel perfusion. The dissolution process does not alter the collagen hydrogel’s physical or chemical properties, and the perfusion is associated with an increased fibroblast survival. Here, we report the novel utilization of thermoplastics to generate a perfusion network within biomimetic collagen hydrogels.