Pulsatile Flow Perfusion Research Articles

Determining stemness and exosome production from nutrient-dependent cells: Influence of the molecular weight cut-off in hollow fiber membrane systems

This study aimed to optimize parameters for cultivating adipose-derived stem cells (ADSCs) in hollow fiber membrane systems. Enhanced ADSC cultivation was achieved in this study by utilizing a higher cell seeding rate (10 mL/min) and fibronectin coating (8 μg/cm²) on the membrane. A pulsatile flow perfusion (30 min on / 3 h off) for 24 h, followed by continuous flow perfusion at a lower rate (<20 mL/min), was employed. Notably, a two-day medium change maintained CD73 expression (79.26 %) compared to scenarios without changes (1.92 %) over eight days. Using a membrane cut-off of 20 kDa and <0.1 μm, a 1.4- and 2.8-fold increase in proliferated cells was observed; however, CD73 expression decreased by 10.07 and 33.63 %, respectively. The membrane with a pore size of <0.1 μm exhibited 1.4-fold higher exosome particle numbers. In conclusion, nutrient supplementation emerged as crucial for optimal ADSC cultivation in hollow fiber systems.

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.

Effect of pulsatile flow perfusion on decellularization

BackgroundDecellularized animal organs have been used as scaffolds for tissue engineering. To make a properly functioning scaffolds, the extracellular matrix (ECM) components must be preserved after decellularization. Because pulsatile flow is known to be beneficial for tissue perfusion, pulsatile perfusion of a detergent might decrease the exposure time of the tissues to the detergent used for decellularization. Using Energy Equivalent Pressure (EEP) as a pulsatility parameter, the effect of pulsatile flow in decellularization process is studied.ResultsTwelve rat hearts were decellularization with 1% sodium dodecyl sulfate (SDS) solution for 2 h. They are divided into two groups, one with pulsatile perfusion (n = 6), the other with non-pulsatile perfusion (n = 6) of SDS. The initial mean perfusion pressures were same in both group. The result indicated that the EEP and the perfusion flow were increased significantly in the pulsatile group compared to the non-pulsatile group. Photographs taken during the decellularization showed more profound decellularization in the pulsatile group. The residual DNA content in the scaffolds was significantly lower in the pulsatile group. However, the level of ECM components, collagen and GAG showed no significant differences between the groups.ConclusionsDecellularization is more efficient in pulsatile flow than in non-pulsatile flow but still preserves the ECM molecules.

Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd.

Alteration of inflammatory response by shock wave therapy leads to reduced calcification of decellularized aortic xenografts in mice†.

Tissue-engineered xenografts represent a promising treatment option in heart valve disease. However, inflammatory response leading to graft failure and incomplete in vitro repopulation with recipient cells remain challenging. Shock waves (SWs) were shown to modulate inflammation and to enhance re-epithelialization. We therefore aimed to investigate whether SWs could serve as a feasible adjunct to tissue engineering. Porcine aortic pieces were decellularized using sodium deoxycholate and sodium dodecylsulphate and implanted subcutaneously into C57BL/6 mice (n = 6 per group). The treatment (shock wave therapy, SWT) group received SWs (0.1 mJ/mm(2), 500 impulses, 5 Hz) for modulation of inflammatory response directly after implantation; control animals remained untreated (CTR). Grafts were harvested 72 h and 3 weeks after implantation and analysed for inflammatory cytokines, macrophage infiltration and polarization, osteoclastic activity and calcification. Transmission electron microscopy (TEM) was performed. Endothelial cells (ECs) were treated with SWs and analysed for macrophage regulatory cytokines. In an ex vivo experimental set-up, decellularized porcine aortic valve conduits were reseeded with ECs with and without SWT (0.1 mJ/mm(2), 300 impulses, 3 Hz), fibroblasts as well as peripheral blood mononuclear cells (all human) and tested in a pulsatile flow perfusion system for cell coverage. Treated ECs showed an increase of macrophage migration inhibitory factor and macrophage inflammatory protein 1β, whereas CD40 ligand and complement component C5/C5a were decreased. Subcutaneously implanted grafts showed increased mRNA levels of tumour necrosis factor α and interleukin 6 in the treatment group. Enhanced repopulation with recipient cells could be observed after SWT. Augmented macrophage infiltration and increased polarization towards M2 macrophages was observed in treated animals. Enhanced recruitment of osteoclastic cells in proximity to calcified tissue was found after SWT. Consequently, SWT resulted in decreased areas of calcification in treated animals. The reseeding experiment revealed that fibroblasts showed the best coverage compared with other cell types. Moreover, SW-treated ECs exhibited enhanced repopulation compared with untreated controls. SWs reduce the calcification of subcutaneously implanted decellularized xenografts via the modulation of the acute macrophage-mediated inflammatory response and improves the in vitro repopulation of decellularized grafts. It may therefore serve as a feasible adjunct to heart valve tissue engineering.

Effects of elevated perfusion pressure and pulsatile flow on human saphenous veins isolated from diabetic and non-diabetic patients

This study was carried out to determine high pressure and pulsatile flow perfusion effects on human saphenous vein (HSV) segments obtained from diabetic and non-diabetic patients. The veins were perfused with oxygenated Krebs solution for 3 h, with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 or 300 × 250 mmHg. After perfusion, veins were studied by light microscopy; nitric oxide synthase (NOS) isoforms, CD34 and nitrotyrosine immunohistochemistry and tissue nitrite/nitrate (NO(x)) and malondialdehyde (MDA) quantification. Light microscopy revealed endothelial denuding areas in all HSV segments subjected to 300 × 250 mmHg perfusion pressure, but the luminal area was similar. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between groups. The endothelial nitric oxide synthase (eNOS) isoform immunostaining decreased significantly in diabetic patients' veins independent of the perfusion pressure levels. The inducible NOS (iNOS), neuronal NOS (nNOS) and nitrotyrosine immunostaining were similar. Significant CD34 differences were observed between the diabetic 300 × 250 mmHg perfusion pressure group and the non-diabetic control group. Tissue nitrite/nitrate and MDA were not different among groups. Pulsatile flow and elevated pressures for 3 h caused morphological changes and decreased the eNOS expression in the diabetic patients' veins.

A positron emission tomography approach to visualize flow perfusion in hollow-fiber membrane bioreactors

Despite the success of hollow-fiber membrane bioreactors in tissue engineering, few evaluations of steady- and pulsatile-flow perfusion through these bioreactors have been made. Such evaluations are vital to the optimization of bioreactor culture conditions. In this study, positron emission tomography (PET) was proposed and used to visualize steady- and pulsatile-flow perfusion in hollow-fiber membrane bioreactors for tissue-engineering applications. PET is a noninvasive method that allows measuring the spatial distribution of a radioactive tracer by detecting its activity within porous scaffolds. A radioactive tracer, 18-fluoro-deoxy-glucose ((18)FDG), was injected into a fluid circuit having a hollow-fiber membrane bioreactor with gel-devoid or gel-filled extracapillary space. Dynamic PET scans of the inlet section were acquired and followed by volumetric PET scans of the whole bioreactor. Results were used to reconstruct dynamic and volumetric two- and three-dimensional images. Pulsatile inlet flow improved the uniformity of perfusion flow within the bioreactor in comparison to the steady inlet flow. Pulsatile flow also reduced the accumulation of radioactive tracer for both gel-devoid and gel-filled bioreactors compared to the steady flow. The stability of the radioactive tracer for both conditions was evaluated. The potential of the PET approach was demonstrated by the quantification of the imaging results for steady- and pulsatile-flow perfusions that can be used for the development of bioreactors for tissue-engineering applications.

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real-time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48-h culture period. Cells were uniformly dispersed within the 14.40 mm x 17.46 mm x 6.35 mm chamber. A larger fraction of the cells suspended in 6.35-mm thick gels and cultured in a traditional CO(2) incubator were found to be round and dead [corrected]. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel.

Changes of opening angle in hypertensive and hypotensive arteries in 3-day organ culture

To study the effect of pressure changes on the opening angle of arteries in organ culture, tubular segments of porcine common carotid arteries were cultured with pulsatile flow perfusion under hypertensive (150±20 mmHg), normotensive (100±20 mmHg), or hypotensive (30±10 mmHg) pressure while maintaining the arteris at a physiological wall shear stress of ∼15 dyn/cm 2 for up to 3 days. Arteries were then cut into short ring segments by sections perpendicular to the axis and then cut open radially to observe the opening angle in aerated phosphate buffered saline solution (37 °C). Norepinephrine (NE, 10 μM), carbacol (CCh, 100 μM), and sodium nitroprusside (SNP, 10 μM) were added after the radial cut at 30, 20, and 30 min intervals, the opening angles were measured, respectively. Results show that hypertensive arteries developed a significantly larger opening angle than normotensive and hypotensive arteries, associated with a significant increase in cell proliferation. In addition, with smooth muscle contraction activated by NE, the opening angle decreases significantly in hypertensive arteries but has little change in hypotensive and normotensive arteries, indicating an enhancement of smooth muscle contraction on the lumen side of the hypertensive arterial wall. In comparison, hypotensive pressure has little effect on arterial opening angle and cell proliferation.

体外循環における拍動流潅流と定常流潅流の比較検討

今回我々は,より生理的であると考える拍動流灌流法を施行し,大動脈遮断中における拍動流灌流法の末梢への影響をパルスオキシメータおよび温度計を用い,定常流灌流法と比較検討した.その結果,拍動流灌流法を施行することにより,末梢への脈圧がパルスオキシメータにより確認できた.平均収縮期血圧および平均拡張期血圧において,定常流灌流法よりも拍動流灌流法が低い値を示した.食道温と末梢温の温度差は拍動流灌流法で低い値を示した.末梢血管抵抗は,拍動流灌流法が低い値を示した.これらは拍動流灌流による脈圧が生体の圧受容体に関与した結果,末梢血管抵抗を減少させ,血液流量を増加させたと考えられた.

Microembolism during cardiopulmonary bypass: a comparison of bubble oxygenator with arterial line filter and membrane oxygenator alone

Microemboli can be detected in the cerebral circulation during cardiopulmonary bypass with transcranial Doppler. They are likely to be responsible for the microvascular occlusions that have been seen in the retina and they may contribute to neuropsychological damage that has been demonstrated after cardiac surgery. Twenty patients undergoing routine coronary artery bypass graft surgery were allocated to either a Harvey 1700 bubble oxygenatorwith a Pall 40μ arterial line filter or a Harvey 4000 membrane oxygenator with no arterial line filter. All cases received pulsatile flow perfusion with core cooling to 28°C. A standard surgical technique was used with distal anastomoses performed during cold cardioplegic arrest and proximal anastomoses performed with coronary perfusion during systemic rewarming. Microembolic events were measured by the counting of flow disturbances over the pattern of pulsatile flow as detected by Doppler. Median bypass time was 69 minutes (range 45-95) for the bubbler group and 79 minutes (range 50-115) for the membrane group. The microembolic event count was normalized to the number of events per 15 minute period of bypass. The median number of microembolic events per 15 minute period of bypass was 28 (range 6-54) for the bubble group and 21 (range 10-65) for the membrane group. There were no significant differences between the lengths of bypass between the two groups (Mann-Whitney p = 0.202) or the number of microemboli recorded per 15 minute period of bypass (Mann-Whitney p = 0.224). We conclude that the previously demonstrated excess generation of gaseous microemboli from bubble oxygenators over membrane oxygenators is negated by the use of a 40μ arterial line filter with the bubble oxygenator.

Perfusion cooling by pulsatile flow.

Organ temperature changes and the temperature gradient between organs with cooling and rewarming were studied in rabbits using pulsatile flow perfusion. The temperature gradient between organs was within 3 degrees C. At the initial stage of cooling and rewarming, organ temperatures changed rapidly. During circulatory arrest, organ temperatures rose gradually. Brain temperature changes were similar to other organs.

Physiologic role of pulsatile flow perfusion in extracorporeal circulation

Physiologic role of pulsatile flow perfusion in extracorporeal circulation