perfusion system
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Micromachines ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 131
Author(s):  
Nicholas J. Ginga ◽  
Raleigh Slyman ◽  
Ge-Ah Kim ◽  
Eric Parigoris ◽  
Sha Huang ◽  
...  

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.


Author(s):  
Said H. Audi ◽  
Swetha Ganesh ◽  
Pardis Taheri ◽  
Xiao Zhang ◽  
Ranjan K. Dash ◽  
...  

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia, and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm, and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone-induced and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.


2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Eriselda Keshi ◽  
Peter Tang ◽  
Marie Weinhart ◽  
Hannah Everwien ◽  
Simon Moosburner ◽  
...  

Abstract Background Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daisuke Ishii ◽  
Naoto Matsuno ◽  
Mikako Gochi ◽  
Hiroyoshi Iwata ◽  
Tatsuya Shonaka ◽  
...  

AbstractThis study examined the efficacy of end-ischemic hypothermic oxygenated machine perfusion preservation (HOPE) using an originally developed machine perfusion system for split-liver transplantation. Porcine split-liver grafts were created via 75% liver resection after 10 min of warm ischemia. In Group 1, grafts were preserved by simple cold storage (CS) for 8 h (CS group; n = 4). In Group 2, grafts were preserved by simple CS for 6 h and end-ischemic HOPE for 2 h (HOPE group; n = 5). All grafts were evaluated using an isolated ex vivo reperfusion model with autologous blood for 2 h. Biochemical markers (aspartate aminotransferase and lactate dehydrogenase levels) were significantly better immediately after reperfusion in the HOPE group than in the CS group. Furthermore, the HOPE group had a better histological score. The levels of inflammatory cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1β, and interleukin-10) were significantly lower after reperfusion in the HOPE group. Therefore, we concluded that end-ischemic HOPE for split-liver transplantation can aid in recovering the graft function and reducing ischemia–reperfusion injury. HOPE, using our originally developed machine perfusion system, is safe and can improve graft function while attenuating liver injury due to preservation.


HardwareX ◽  
2021 ◽  
pp. e00245
Author(s):  
Federico Cantoni ◽  
Gabriel Werr ◽  
Laurent Barbe ◽  
Ana Maria Porras ◽  
Maria Tenje

Author(s):  
Haneen Abusharkh ◽  
Terreill Robertson ◽  
Juana Mendenhall ◽  
Bulent Gozen ◽  
Edwin Tingstad ◽  
...  

The present study is focused on designing an easy-to-use novel perfusion system for articular cartilage (AC) tissue engineering and using it to elucidate the mechanism by which interstitial shear upregulates matrix synthesis by articular chondrocytes (AChs). Porous chitosan-agarose (CHAG) scaffolds were synthesized, freeze-dried, and compared to bulk agarose (AG) scaffolds. Both scaffold types were seeded with osteoarthritic human AChs and cultured in a novel perfusion system for one week with a shear-inducing medium flow velocity of 0.33 mm/s corresponding to an average surficial shear of 0.4 mPa and a CHAG interstitial shear of 40 mPa. While there were no statistical differences in cell viability for perfusion vs. static cultures for either scaffold type, CHAG scaffold cultures exhibited 3.3-fold higher (p<0.005) cell viability compared to AG scaffold cultures. Effects of combined superficial and interstitial perfusion for CHAG showed 150- and 45-fold (p<0.0001) increases in total collagen (COL) and 13- and 2.2-fold (p<0.001) increases in glycosaminoglycans (GAGs) over AG’s scaffold non-perfusion and perfusion cultures, respectively, and a 1.5-fold and 3.6-fold (p<0.005) increase over non-perfusion CHAG cultures. Contrasting CHAG perfusion and static cultures, chondrogenic gene comparisons showed a 3.5-fold increase in collagen type II/type I (COL2A1/COL1A1) mRNA ratio (p<0.05), and a 1.3-fold increase in aggrecan mRNA. Observed effects are suggested to be the result of inhibiting the inflammatory NF-κB signal transduction pathway as confirmed by a further study that indicated a reduction by 3.2-fold (p<0.05) upon exposure to perfusion. Our results demonstrate that the presence of pores plays a critical role in improving cell viability and that interstitial flow caused by medium perfusion through the porous scaffolds enhances the expression of chondrogenic genes and ECM components through the downregulation of NF-κB1.


2021 ◽  
Vol 28 (6) ◽  
Author(s):  
J. B. Montgomery ◽  
M. Klein ◽  
J. R. Boire ◽  
C. Beck ◽  
D. Häusermann ◽  
...  

Laminitis is an extremely painful and debilitating condition of horses that can affect their athletic ability and even quality of life. The current gold standard for assessment of laminar tissue is histology, which is the only modality that enables detailed visualization of the lamina. Histology requires dissection of the hoof and therefore can only represent one specific time point. The superior spatial and contrast resolution of synchrotron computed tomography (sCT), when compared with readily available imaging modalities, such as radiographs and conventional CT, provides an opportunity for detailed studies of the lamina without the need for hoof dissection and histological assessment. If the resolution of histology can be matched or even approached, dynamic events, such as laminar blood flow, could also be studied on the microscopic tissue level. To investigate this possible application of sCT further, two objectives are presented: (i) to develop a protocol for sCT of an equine digit using cadaver limbs and (ii) to apply the imaging protocol established during (i) for sCT imaging of the vasculature within the foot using an ex vivo perfusion system to deliver the vascular contrast. The hypotheses were that sCT would allow sufficient resolution for detailed visualization to the level of the secondary lamellae and associated capillaries within the equine digit. Synchrotron CT enabled good visualization of the primary lamellae (average length 3.6 mm) and the ex vivo perfusion system was able to deliver vascular contrast agent to the vessels of the lamina. The individual secondary lamellae (average length 0.142 mm) could not be seen in detail, although differentiation between primary and secondary lamellae was achieved. This approaches, but does not yet reach, the current gold standard, histology, for assessment of the lamellae; however, with further refinement of this imaging technique, improved resolution may be accomplished in future studies.


2021 ◽  
pp. 152-161
Author(s):  
Eliel Eduardo Montijo-Valenzuela ◽  
Elvis Osiel Covarrubias-Burgos ◽  
Darío Soto-Patrón ◽  
Esthela Fernanda Torres-Amavizca

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