scholarly journals Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1880
Author(s):  
Hristo Zlatev ◽  
Charlotte von Horn ◽  
Thomas Minor
Keyword(s):  
Cold Storage ◽  
Negative Impact ◽  
Ex Vivo ◽  
Coupling Efficiency ◽  
Sodium Reabsorption ◽  
Enzyme Release ◽  
Machine Perfusion ◽  
Early Functional Outcome ◽  
Perfusion Period

Background: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. Methods: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 °C (controlled oxygenated rewarming—COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. Results: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 °C was effectuated in the same controlled manner. Conclusion: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

scholarly journals Generation of vascular chimerism within donor organs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shahar Cohen ◽  
Shirly Partouche ◽  
Michael Gurevich ◽  
Vladimir Tennak ◽  
Vadym Mezhybovsky ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Patient Specific ◽  
Machine Perfusion ◽  
Organ Perfusion ◽  
Perfusion Process ◽  
Hind Limbs ◽  
Porcine Organs ◽  
Immunological Barrier

AbstractWhole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

scholarly journals Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Frank Elbers ◽  
Claudia Woite ◽  
Valentina Antoni ◽  
Sara Stein ◽  
Hiroshi Funakoshi ◽  
...  
Keyword(s):  
Essential Amino Acid ◽  
Negative Impact ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Effector Functions ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Oxygen Conditions

Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxiain vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO andex vivomurine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occurin vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

1998 ◽  
Vol 7 (4) ◽  
pp. 345-355 ◽  
Author(s):  
Edgardo Elvio Guibert ◽  
María Gabriela Mediavilla ◽  
María Eugenia Mamprin ◽  
Joaquín Valentín Rodríguez
Keyword(s):  
Cold Storage ◽  
Trypan Blue ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Enzyme Release ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Hypothermic Storage

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96h-4°C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37°C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

Normothermic Ex Vivo Kidney Perfusion Improves Early DCD Graft Function Compared With Hypothermic Machine Perfusion and Static Cold Storage

2020 ◽  
Vol 104 (5) ◽  
pp. 947-955 ◽  
Author(s):  
Peter Urbanellis ◽  
Matyas Hamar ◽  
J. Moritz Kaths ◽  
Dagmar Kollmann ◽  
Ivan Linares ◽  
...  
Keyword(s):  
Cold Storage ◽  
Ex Vivo ◽  
Graft Function ◽  
Machine Perfusion ◽  
Hypothermic Machine Perfusion ◽  
Kidney Perfusion

scholarly journals A simple ex vivo model of human renal allograft preservation using the gonadal vein

2019 ◽  
Vol 101 (8) ◽  
pp. 609-616
Author(s):  
WP Ries ◽  
Y Marie ◽  
K Patel ◽  
C Turnbull ◽  
TB Smith ◽  
...  
Keyword(s):  
Cold Storage ◽  
Organ Preservation ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Human Model ◽  
Machine Perfusion ◽  
Flow Conditions ◽  
Ex Vivo Perfusion ◽  
Gonadal Vein ◽  
Hypothermic Machine Perfusion

Introduction Hypothermic machine perfusion, an organ preservation modality, involves flow of chilled preservation fluid through an allograft’s vasculature. This study describes a simple, reproducible, human model that allows for interrogation of flow effects during ex vivo organ perfusion. Materials and methods Gonadal veins from deceased human renal allografts were subjected to either static cold storage or hypothermic machine perfusion for up to 24 hours. Caspase-3, Krüppel-like factor 2 expression and electron microscopic analysis were compared between ‘flow’ and ‘no-flow’ conditions, with living donor gonadal vein sections serving as negative controls. Results The increase in caspase-3 expression was less pronounced for hypothermic machine-perfused veins compared with static cold storage (median-fold increase 1.2 vs 2.3; P < 0.05). Transmission electron microscopy provided ultrastructural corroboration of endothelial cell apoptosis in static cold storage conditions. For static cold storage preserved veins, Krüppel-like factor 2 expression diminished in a time-dependent manner between baseline and 12 hours (P < 0.05) but was abrogated and reversed by hypothermic machine perfusion (P < 0.05). Conclusions Our methodology is a simple, reproducible and successful model of ex vivo perfusion in the context of human organ preservation. To demonstrate the model’s utility, we establish that two widely used markers of endothelial health (caspase-3 and Krüppel-like factor 2) differ between the flow and no-flow conditions of the two predominant kidney preservation modalities. These findings suggest that ex vivo perfusion may mediate the induction of a biochemically favourable endothelial niche which may contribute tohypothermic machine perfusion’s association with improved renal transplantation outcomes.

scholarly journals Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Katarzyna Drela ◽  
Luiza Stanaszek ◽  
Adam Nowakowski ◽  
Zuzanna Kuczynska ◽  
Barbara Lukomska
Keyword(s):  
Stem Cell ◽  
Negative Impact ◽  
Ex Vivo ◽  
Biological Properties ◽  
Therapeutic Effects ◽  
Functional Changes ◽  
Cell Propagation ◽  
Ex Vivo Culture

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

Negative impact of prolonged cold storage time before machine perfusion preservation in donation after circulatory death kidney transplantation

2016 ◽  
Vol 29 (10) ◽  
pp. 1117-1125 ◽  
Author(s):  
Siegfredo Paloyo ◽  
Junichiro Sageshima ◽  
Jeffrey J. Gaynor ◽  
Linda Chen ◽  
Gaetano Ciancio ◽  
...  
Keyword(s):  
Kidney Transplantation ◽  
Cold Storage ◽  
Storage Time ◽  
Negative Impact ◽  
Machine Perfusion ◽  
Donation After Circulatory Death ◽  
Circulatory Death

scholarly journals Development of ex vivo normothermic perfusion as an innovative method to assess pancreases after preservation

2021 ◽  
Vol 2 (4) ◽  
Author(s):  
Ann Ogbemudia
Keyword(s):  
Cold Storage ◽  
Ex Vivo ◽  
Gas Analysis ◽  
Machine Perfusion ◽  
Assessment Strategy ◽  
Hypothermic Machine Perfusion ◽  
Normothermic Perfusion ◽  
Pancreas Preservation ◽  
Normothermic Machine Perfusion ◽  
Lactate Levels

Ann Ogbemudia, Julien Branchereau (Joint first authors), Gabriella Hakim, Fungai Dengu, FaysalEl-Gilani, John Mulvey, Kaithlyn Rozenberg, Thomas Prudhomme, Letizia Lo Faro, James Hunter,Paul Johnson, Rutger Ploeg and Peter Friend   Objective Static cold storage (SCS) is the standard method for pancreas preservation but does not facilitate objective organ assessment prior to transplantation. Normothermic machine perfusion (NMP) has been used to test other abdominal and thoracic organs’ function and viability in transplantation settings. Our aim was to develop a NMP protocol specific for pancreases and then investigate its potential as an organ assessment strategy. Method 8 porcine pancreases were procured in conditions replicating donation after circulatory death with warm ischaemia time of 25 minutes. After 3 hours of static cold storage (SCS) the pancreases were divided into 3 experimental groups 1) the feasibility group (n=2) that underwent 2.5 hours of NMP 2) the SCS group (n = 2) that underwent an additional 6 hours of SCS prior to assessment on NMP for an hour and 3) the Oxygenated Hypothermic Machine Perfusion (oxyHMP) group (n = 4) that underwent 6 hours of oxyHMP followed by 1-hour assessment on NMP. The NMP protocol used autologous, leucodepleted blood delivered at a mean arterial pressure of 40mmHg with a temperature of 37oC. At timed intervals during NMP, perfusate samples were collected for gas analysis and perfusion parameters were recorded. Results The feasibility group was used to develop the NMP protocol and demonstrated stable perfusion parameters throughout NMP. Compared to the SCS group the oxyHMP group demonstrated better average perfusion characteristics with lower resistances, higher flow rates, lower mean lactate levels and physiological pH. The oxyHMP group maintained normal macroscopic appearances during NMP. At the end of NMP the SCS group had an average 32% weight increase compared to the oxyHMP group that were found to have a 17% weight reduction. Conclusion Normothermic machine perfusion of whole pancreases is feasible after cold preservation and potentially useful as an assessment strategy. Furthermore, it demonstrated that oxygenated HMP may be beneficial for pancreas preservation compared to SCS.

scholarly journals A Concise Review of Current In Vitro Chemical and Cell-Based Antioxidant Assay Methods

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4865
Author(s):  
Ifeanyi D. Nwachukwu ◽  
Roghayeh Amini Sarteshnizi ◽  
Chibuike C. Udenigwe ◽  
Rotimi E. Aluko
Keyword(s):  
Free Radicals ◽  
Negative Impact ◽  
Ex Vivo ◽  
Scientific Information ◽  
Antioxidant Properties ◽  
Antioxidant Assays ◽  
Antioxidant Agents ◽  
Synthetic Molecules ◽  
Negative Side Effects

Antioxidants remain interesting molecules of choice for suppression of the toxic effects of free radicals in foods and human systems. The current practice involves the use of mainly synthetic molecules as potent antioxidant agents. However, due to the potential negative impact on human health, there is an intensive effort within the research community to develop natural alternatives with similar antioxidant efficacy but without the negative side effects of synthetic molecules. Still, the successful development of new molecules depends on the use of reliable chemical or cell culture assays to screen antioxidant properties. Chemical antioxidant assays include the determination of scavenging ability against free radicals such as DPPH, superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, and nitric oxide. Other antioxidant tests include the ability of compounds to bind and sequester prooxidant metal cations, reduce ferric iron, and attenuate the rate of lipid oxidation. Ex vivo tests utilize cell cultures to confirm entry of the molecules into cells and the ability to quench synthetic intracellular free radicals or to stimulate the increased biosynthesis of endogenous antioxidants. In order to assist researchers in their choice of antioxidant evaluation methods, this review presents background scientific information on some of the most commonly used antioxidant assays with a comparative discussion of the relevance of published literature data to food science and human nutrition applications.

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