Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

Efficacy of Pulsatile Flow Perfusion in Adult Cardiac Surgery: Hemodynamic Energy and Vascular Reactivity

Background: The role of pulsatile (PP) versus non-pulsatile (NP) flow during a cardiopulmonary bypass (CPB) is still debated. This study’s aim was to analyze hemodynamic effects, endothelial reactivity and erythrocytes response during a CPB with PP or NP. Methods: Fifty-two patients undergoing an aortic valve replacement were prospectively randomized for surgery with either PP or NP flow. Pulsatility was evaluated in terms of energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE). Systemic (SVRi) and pulmonary (PVRi) vascular resistances, endothelial markers levels and erythrocyte nitric-oxide synthase (eNOS) activity were collected at different perioperative time-points. Results: In the PP group, the resultant EEP was 7.3% higher than the mean arterial pressure (MAP), which corresponded to 5150 ± 2291 ergs/cm3 of SHE. In the NP group, the EEP and MAP were equal; no SHE was produced. The PP group showed lower SVRi during clamp-time (p = 0.06) and lower PVRi after protamine administration and during first postoperative hours (p = 0.02). Lower SVRi required a higher dosage of norepinephrine in the PP group (p = 0.02). Erythrocyte eNOS activity results were higher in the PP patients (p = 0.04). Renal function was better preserved in the PP group (p = 0.001), whereas other perioperative variables were comparable between the groups. Conclusions: A PP flow during a CPB results in significantly lower SVRi, PVRi and increased eNOS production. The clinical impact of increased perioperative vasopressor requirements in the PP group deserves further evaluation.

Pilot Study of Blood Perfusion Changes at PC4 and Its Surrounding Points Induced by Acupuncture and Moxibustion

Acupuncture and moxibustion are widely used in clinical practice; however, the differences between their mechanisms are unclear. In the present study, the response of blood perfusion resulting from acupuncture or moxibustion at Ximen (PC4) and its surrounding points was explored. Using the wavelet method, the differences in the frequency interval of blood flux were observed. Furthermore, the correlations between these points were analyzed. The results suggested that moxibustion could significantly improve blood flow perfusion at PC4 compared to acupuncture; however, there was no significant difference around PC4. The response of blood flux at PC4 to different stimulations was related to the frequency V (0.4–1.6 Hz) component. However, a difference in response at other points was not observed. Correlation analysis showed that both acupuncture and moxibustion could cause a decline in the correlation of blood flux signals at these recorded points, but there was no significant difference between these techniques. The results suggested that, at least in the forearm, the acupuncture or moxibustion only influenced the level of blood perfusion locally.

Efficacy of Regional Chemotherapy Approach in Peritoneal Metastatic Gastric Cancer

Peritoneal spread is frequent in gastric cancer (GC) and a palliative condition. After failure to systemic chemotherapy (sCTx) remaining therapeutic options are very limited. We evaluated the feasibility and efficacy of locoregional chemotherapy (RegCTx) in peritoneal metastatic GC. In total, 38 (23 male and 15 female) patients with peritoneal metastatic GC after failure of previous sCTx and unresectable disease were enrolled in this study. Using the hypoxic abdominal stop-flow perfusion, upper abdominal perfusion and intraarterial infusion technique in total 114 cycles with Cisplatin, Adriamycin and Mitomycin C were applied. No significant procedure related toxicity was noticed- especially no Grade 3 or 4 toxicity occurred. With the RegCTx approach a median overall survival of 17.4 months was achieved. Patients who had undergone previously resection of the GC the median overall survival was even better with 23.5 months. RegCTx is a promising, safe and efficient approach in diffuse metastatic GC. The evaluation of RegCTx in the setting of multimodal treatment approach at less advanced stages is also warranted.

Universal single-board computer based control unit for biomedical test benches

Continuous adaptation of international standards for medical devices requires recurrent modification of test benches. A universal control unit using an open software environment is presented to simplify the maintenance of various test benches. Our developed control unit is equipped with a Raspberry Pi 4, with standard communication interfaces and application-specific electronic assemblies. The software is based on Node-RED, a browser-based editor. A measuring setup was adapted for a flow perfusion system. Our control unit simplifies the handling of the flow perfusion system by controlling a hydraulic pump and all required valves. A software programmed PID algorithm adapts the speed of the pump to adjust the pressure automatically. Actuators like proportional pinch valves are handled to control volumetric flow and pressure within the circulation. Consequently, the user directly observes changes inside the system. The measured data are stored and are available for documentation.

Parallelizable Microfluidic Platform to Model and Assess In Vitro Cellular Barriers: Technology and Application to Study the Interaction of 3D Tumor Spheroids with Cellular Barriers

Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.

Radial Flow Perfusion Enables Real-Time Profiling of Cellular Metabolism at Low Oxygen Levels with Hyperpolarized 13C NMR Spectroscopy

In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.

High fibrinogen γ′ levels in patient plasma increase clot formation at arterial and venous shear

Fibrinogen γ' accounts for 3% to 40% of plasma fibrinogen. Earlier studies indicated that fibrinogen γ' forms altered fibrin clots under static conditions, whereas clinically, altered plasma γ' levels are associated with arterial and venous thrombosis. However, the effects of static vs flow conditions on the role of γ′ throughout the pathophysiological range is unknown. This study explores the effects of γ' levels on clot formation and structure in static and flow conditions. Coagulation of plasma samples with low (n = 41; 3%), normal (n = 45; 10%), or high (n = 33; 30%) γ′ levels were compared with that of purified fibrinogen mixtures with increasing ratios of γ′ (3%, 10%, 30%). Clots were analyzed by confocal microscopy, permeation, turbidity, and lysis techniques. In a novel 2-step flow-perfusion model, fibrinogen-deficient plasma repleted with increasing ratios of γ′ (3%, 10%, 30%) or plasmas with low (n = 5, 3%) or high (n = 5, 30%) γ′ were flowed over preformed platelet aggregates at arterial (500 s−1) and venous (150 s−1) shear rates. Increasing γ′ percentages within the pathophysiological range (3%-30%) did not result in any change in clot-formation rates; however, it led to significantly higher clot density, thinner fibers, and slower lysis in static conditions. Under flow at arterial shear, high γ′ (30%) led to faster (+44.1%-75.3%) and increased (+104%-123%) fibrin deposition, with clots exhibiting a larger volume (+253%-655%) and height (+130%-146%). These trends were magnified at venous shear. Overall, our findings demonstrate the significant impact of pathophysiological fibrinogen γ′ levels on clot structure and provide new flow-dependent mechanisms to explain how γ′ increases thrombosis risk.