531 results on '"Uygun, Korkut"'
Search Results
202. Injury of Peribiliary Glands of Donor Liver Bile Ducts Plays a Pivotal Role in the Development of Non-anastomotic Biliary Strictures after Liver Transplantation
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Karimian, Negin, Op Den Dries, Sanna, Weeder, Pepijn D., Westerkamp, Andrie, Bomfati, Fernanda, Wiersema-Buist, Janneke, Bruinsma, Bote G., Gouw, Annette S., Markmann, James F., Lisman, Ton, Yeh, Heidi, Uygun, Korkut, Paulo N. Martins, Porte, Robert J., Groningen Institute for Organ Transplantation (GIOT), and Vascular Ageing Programme (VAP)
203. Evolving utilization of donation after circulatory death livers in liver transplantation: The day of DCD has come.
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Haque, Omar, Yuan, Qing, Uygun, Korkut, and Markmann, James F.
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LIVER transplantation , *BRAIN death , *TRANSPLANTATION of organs, tissues, etc. , *MYOCARDIAL reperfusion , *HOMOGRAFTS - Abstract
Compared to donation after brain death (DBD), livers procured for transplantation from donation after circulatory death (DCD) donors experience more ischemia‐reperfusion injury and higher rates of ischemic cholangiopathy due to the period of warm ischemic time (WIT) following withdrawal of life support. As a result, utilization of DCD livers for liver transplant (LT) has generally been limited to short WITs and younger aged donor grafts, causing many recovered DCD organs to be discarded without consideration for transplant. This study assesses how DCD liver utilization and outcomes have changed over time, using OPTN data from adult, first‐time, deceased donor, whole‐organ LTs between January 1995 and December 2019. Results show that increased clinical experience with DCD LT has translated into increased use of livers from DCD donors, shorter ischemic times, shorter lengths of hospitalization after transplant, and lower rates of retransplantation. The data also reveal that over the past decade, the rate of increase in DCD LTs conducted in the United States has outpaced that of DBD. Together, these trends signal an opportunity for the field of liver transplantation to mitigate the organ shortage by capitalizing on DCD liver allografts that are currently not being utilized. [ABSTRACT FROM AUTHOR]
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- 2021
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204. The Autonomization Principle in Vascularized Flaps: An Alternative Strategy for Composite Tissue Scaffold In Vivo Revascularization.
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Berkane, Yanis, Kostyra, David M., Chrelias, Theodoros, Randolph, Mark A., Lellouch, Alexandre G., Cetrulo Jr., Curtis L., Uygun, Korkut, Uygun, Basak E., Bertheuil, Nicolas, and Duisit, Jérôme
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TISSUE scaffolds , *PLASTIC surgeons , *SURGICAL flaps , *COMPLEX matrices , *EXTRACELLULAR matrix , *BIOENGINEERING , *PERFORATOR flaps (Surgery) - Abstract
Autonomization is a physiological process allowing a flap to develop neo-vascularization from the reconstructed wound bed. This phenomenon has been used since the early application of flap surgeries but still remains poorly understood. Reconstructive strategies have greatly evolved since, and fasciocutaneous flaps have progressively replaced muscle-based reconstructions, ensuring better functional outcomes with great reliability. However, plastic surgeons still encounter challenges in complex cases where conventional flap reconstruction reaches its limitations. Furthermore, emerging bioengineering applications, such as decellularized scaffolds allowing a complex extracellular matrix to be repopulated with autologous cells, also face the complexity of revascularization. The objective of this article is to gather evidence of autonomization phenomena. A systematic review of flap autonomization is then performed to document the minimum delay allowing this process. Finally, past and potential applications in bio- and tissue-engineering approaches are discussed, highlighting the potential for in vivo revascularization of acellular scaffolds. [ABSTRACT FROM AUTHOR]
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- 2023
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205. Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study.
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Berkane, Yanis, Lellouch, Alexandre G., Goudot, Guillaume, Shamlou, Austin, Filz von Reiterdank, Irina, Goutard, Marion, Tawa, Pierre, Girard, Paul, Bertheuil, Nicolas, Uygun, Basak E., Randolph, Mark A., Duisit, Jérôme, Cetrulo Jr., Curtis L., and Uygun, Korkut
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FREE flaps , *VASCULAR resistance , *PERFUSION , *PLASTIC surgery , *TRANSPLANTATION of organs, tissues, etc. , *ARTIFICIAL blood circulation , *MACHINERY - Abstract
Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques. [ABSTRACT FROM AUTHOR]
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- 2023
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206. Zebrafish as a high throughput model for organ preservation and transplantation research.
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Da Silveira Cavalcante, Luciana, Lopera Higuita, Manuela, González‐Rosa, Juan Manuel, Marques, Beatriz, To, Samantha, Pendexter, Casie A., Cronin, Stephanie E. J., Gopinathan, Kaustav, de Vries, Reinier J., Ellett, Felix, Uygun, Korkut, Langenau, David M., Toner, Mehmet, and Tessier, Shannon N.
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Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co‐cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high‐throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation‐specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at −10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation. [ABSTRACT FROM AUTHOR]
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- 2023
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207. Metabolic profiling during ex vivo machine perfusion of the human liver.
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Bruinsma, Bote G., Sridharan, Gautham V., Weeder, Pepijn D., Avruch, James H., Saeidi, Nima, Özer, Sinan, Geerts, Sharon, Porte, Robert J., Heger, Michal, van Gulik, Thomas M., Martins, Paulo N., Markmann, James F., Yeh, Heidi, and Uygun, Korkut
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- 2016
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208. Response Regarding: Continuous Versus Pulsatile Flow in 24-h Vascularized Composite Allograft Machine Perfusion in Swine: A Pilot Study.
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Tawa, Pierre, Goutard, Marion, Andrews, Alec R., de Vries, Reinier J., Rosales, Ivy A., Yeh, Heidi, Uygun, Basak, Randolph, Mark A., Lellouch, Alexandre G., Uygun, Korkut, and Cetrulo, Curtis L.
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PULSATILE flow , *HOMOGRAFTS , *SWINE , *PERFUSION , *PILOT projects - Published
- 2023
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209. Gluteal Propeller Perforator Flaps: A Paradigm Shift in Abdominoperineal Amputation Reconstruction.
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Chrelias, Theodoros, Berkane, Yanis, Rousson, Etienne, Uygun, Korkut, Meunier, Bernard, Kartheuser, Alex, Watier, Eric, Duisit, Jérôme, and Bertheuil, Nicolas
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PERFORATOR flaps (Surgery) , *PATIENT compliance , *AMPUTATION , *PROPELLERS , *OPERATIVE surgery , *DISEASE relapse , *SURGICAL wound dehiscence - Abstract
Abdominoperineal amputation (AAP) is a gold standard procedure treating advanced abdominal and pelvic cancers. The defect resulting from this extensive surgery must be reconstructed to avoid complications, such as infection, dehiscence, delayed healing, or even death. Several approaches can be chosen depending on the patient. Muscle-based reconstructions are a reliable solution but are responsible for additional morbidity for these fragile patients. We present and discuss our experience in AAP reconstruction using gluteal-artery-based propeller perforator flaps (G-PPF) in a case series. Between January 2017 and March 2021, 20 patients received G-PPF reconstruction in two centers. Either superior gluteal artery (SGAP)- or inferior artery (IGAP)-based perforator flaps were performed depending on the best configuration. Preoperative, intraoperative, and postoperative data were collected. A total of 23 G-PPF were performed—12 SGAP and 11 IGAP flaps. Final defect coverage was achieved in 100% of cases. Eleven patients experienced at least one complication (55%), amongst whom six patients (30%) had delayed healing, and three patients (15%) had at least one flap complication. One patient underwent a new surgery at 4 months for a perineal abscess under the flap, and three patients died from disease recurrence. Gluteal-artery-based propeller perforator flaps are an effective and modern surgical procedure for AAP reconstruction. Their mechanic properties, in addition to their low morbidity, make them an optimal technique for this purpose; however, technical skills are needed, and closer surveillance with patient compliance is critical to ensure success. G-PPF should be widely used in specialized centers and considered a modern alternative to muscle-based reconstructions. [ABSTRACT FROM AUTHOR]
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- 2023
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210. Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.
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Goutard, Marion, de Vries, Reinier J., Tawa, Pierre, Pendexter, Casie A., Rosales, Ivy A., Tessier, Shannon N., Burlage, Laura C., Lantieri, Laurent, Randolph, Mark A., Lellouch, Alexandre G., Cetrulo Jr, Curtis L., and Uygun, Korkut
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COLD storage , *PERFUSION , *PRESERVATION of organs, tissues, etc. , *LABORATORY rats , *VASCULAR resistance , *KIDNEY transplantation - Abstract
Background For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. Methods Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. Results Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). Conclusion To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation. [ABSTRACT FROM AUTHOR]
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- 2023
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211. Continuous versus Pulsatile Flow in 24-Hour Vascularized Composite Allograft Machine Perfusion in Swine: A Pilot Study.
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Tawa, Pierre, Goutard, Marion, Andrews, Alec R., de Vries, Reinier J., Rosales, Ivy A., Yeh, Heidi, Uygun, Basak, Randolph, Mark A., Lellouch, Alexandre G., Uygun, Korkut, and Cetrulo, Curtis L.
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PULSATILE flow , *VASCULAR resistance , *PERFUSION , *YORKSHIRE swine , *SWINE - Abstract
Multiple perfusion systems have been investigated on vascularized composite allografts, with various temperatures and different preservation solutions, most using continuous flow (CF). However, physiological flow is pulsatile and provides better outcomes in kidney and lung ex vivo perfusions. The objective of this pilot study is to compare pulsatile flow (PF) with CF in our 24-h subnormothermic machine perfusion protocol for swine hindlimbs. Partial hindlimbs were harvested from Yorkshire pigs and perfused with a modified Steen solution at 21°C for 24 h either with CF (n = 3) or with pulsatile flow (PF) at 60 beats/min (n = 3). Perfusion parameters, endothelial markers, and muscle biopsies were assessed at different timepoints. Overall, lactate levels were significantly lower in the PF group (P = 0.001). Glucose uptake and potassium concentration were similar in both groups throughout perfusion. Total nitric oxide levels were significantly higher in the PF group throughout perfusion (P = 0.032). Nitric oxide/endothelin-1 ratio also tends to be higher in the PF group, reflecting a potentially better vasoconductivity with PF, although not reaching statistical significance (P = 0.095). Arterial resistances were higher in the PF group (P < 0.001). Histological assessment did not show significant difference in muscular injury between the two groups. Weight increased quicker in the CF group but reached similar values with the PF after 24 h. This pilot study suggests that PF may provide superior preservation of vascularized composite allografts when perfused for 24 h at subnormothermic temperatures, with potential improvement in endothelial function and decreased ischemic injury. [ABSTRACT FROM AUTHOR]
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- 2023
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212. Development of a rat forelimb vascularized composite allograft (VCA) perfusion protocol.
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Pendexter, Casie A., Haque, Omar, Mojoudi, Mohammadreza, Maggipinto, Sarah, Goutard, Marion, Baicu, Simona, Lellouch, Alexandre G., Markmann, James F., Brandacher, Gerald, Yeh, Heidi, Tessier, Shannon N., Cetrulo, Curtis, and Uygun, Korkut
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FORELIMB , *HINDLIMB , *HOMOGRAFTS , *RATS - Abstract
Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies. [ABSTRACT FROM AUTHOR]
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- 2023
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213. Addressing the Donor Liver Shortage with EX VIVO Machine Perfusion
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Izamis, Maria-Louisa, a. Berendsen, Tim, Uygun, Korkut, and L. Yarmush, Martin
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Despite a critical shortage of viable donor livers for transplantation, only a fraction of the available organs are used. Donor organ defects, which in the majority of cases are caused by extensive exposure to ischemia, cannot be reversed by static cold storage, the current gold standard of organ preservation. In this review, the role of machine perfusion (MP) in the recovery of non-transplantable ischemic donor organs is discussed. Though still in the experimental phase, various models of MP have consistently demonstrated that ischemic donor organs can be recovered to a transplantable state through continuous perfusion. MP can also provide dynamic quantitative assessments of the extent of ischemia, in addition to predicting the likelihood of organ recovery. Continued endeavors to translate MP into clinical use and eventually incorporate it into routine donor organ care will have a significant impact on the quality and availability of transplantable donor organs.
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- 2012
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214. Continuous oxygen monitoring to enhance ex-vivo organ machine perfusion and reconstructive surgery.
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Berkane, Yanis, Cascales, Juan Pedro, Roussakis, Emmanuel, Lellouch, Alexandre G., Slade, Julian, Bertheuil, Nicolas, Randolph, Mark A., Cetrulo, Curtis L., Evans, Conor L., and Uygun, Korkut
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PERFUSION , *PLASTIC surgery , *VASCULAR resistance , *TECHNOLOGICAL innovations , *OXYGEN detectors , *TISSUE viability - Abstract
Continuous oxygenation monitoring of machine-perfused organs or transposed autologous tissue is not currently implemented in clinical practice. Oxygenation is a critical parameter that could be used to verify tissue viability and guide corrective interventions, such as perfusion machine parameters or surgical revision. This work presents an innovative technology based on oxygen-sensitive, phosphorescent metalloporphyrin allowing continuous and non-invasive oxygen monitoring of ex-vivo perfused vascularized fasciocutaneous flaps. The method comprises a small, low-energy optical transcutaneous oxygen sensor applied on the flap's skin paddle as well as oxygen sensing devices placed into the tubing. An intermittent perfusion setting was designed to study the response time and accuracy of this technology over a total of 54 perfusion cycles. We further evaluated correlation between the continuous oxygen measurements and gold-standard perfusion viability metrics such as vascular resistance, with good agreement suggesting potential to monitor graft viability at high frequency, opening the possibility to employ feedback control algorithms in the future. This proof-of-concept study opens a range of research and clinical applications in reconstructive surgery and transplantation at a time when perfusion machines undergo rapid clinical adoption with potential to improve outcomes across a variety of surgical procedures and dramatically increase access to transplant medicine. [ABSTRACT FROM AUTHOR]
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- 2024
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215. Specific patterns of vital sign fluctuations predict infection and enable sepsis diagnosis in pediatric burn patients.
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Sadeq, Farzin, Poster, Jonah, Chu, Chris, Weber, Joan, Lydon, Martha, Begis, Maggie Dylewski, Sheridan, Robert Leo, and Uygun, Korkut
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BURN patients , *CHILD patients , *VITAL signs , *BODY surface area , *BURN care units , *SEPSIS - Abstract
Early recognition of the clinical signs of bloodstream infection in pediatric burn patients is key to improving survival rates in the burn unit. The objective of this study was to propose a simple scoring criteria that used readily available temperature, heart rate (HR) and mean arterial pressure (MAP) data to accurately predict bloodstream infection in pediatric burn patients. A retrospective chart review included 100 patients admitted to the pediatric burn unit for >20% total body surface area (TBSA) burn injuries. Each patient had multiple blood culture tests, and each test was treated as a separate and independent "infection event" for analysis. The time at each blood culture draw was time 0 for that event, and temperature, HR and MAP data was collected for 24 hours after the blood culture was drawn. "Infection events" included in this study had at least six complete sets of temperature, HR and MAP data entries. Median temperature, HR and MAP, as well as mean fever spikes, HR spikes and MAP dips, were compared between infection group (positive blood cultures) and control group (negative blood cultures). These vital sign fluctuations were evaluated individually and as a combination of all three as timely predictors of bloodstream infection. In addition, we tested the prediction of Gram-negative bacteria versus Gram-positive or fungi present in blood cultures. Patients in the infection group had significantly higher median temperatures (p<0.001), mean fever spikes (p<0.001) and mean HR spikes (p<0.001), compared to the control group. Using the combination scoring criteria to predict bloodstream infection, the strongest predictive values in the 24-hour timeframe had high sensitivity (93%) and specificity (81%). The predictive test metric based on vital sign spikes predicted Gram-negative bacteria, but with limited sensitivity (57%) and specificity (44%). A simple scoring criteria using a combination of fever spikes, HR spikes and MAP dips predicted bloodstream infection in pediatric burn patients, and can be feasibly implemented in routine clinical care. There is also potential to use the predictive metric to detect a few select organisms based on vital signs, however further work is necessary to enhance accuracy to levels that would allow consideration for clinical use. [ABSTRACT FROM AUTHOR]
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- 2022
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216. Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts.
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Burlage, Laura C., Lellouch, Alexandre G., Taveau, Corentin B., Tratnig-Frankl, Philipp, Pendexter, Casie A., Randolph, Mark A., Porte, Robert J., Lantieri, Laurent A., Tessier, Shannon N., Cetrulo, Curtis L., and Uygun, Korkut
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OXYGEN carriers , *HOMOGRAFTS , *PERFUSION , *COLD storage , *FACIAL transplantation - Abstract
• Six hours of ex vivo subnormothermic machine perfusion (SNMP) of rodent vascularized composite allografts (VCA) is feasible using acellular oxygen carrier HBOC-201 • SNMP results in superior tissue preservation compared to conventional static cold storage (SCS) preservation • Successful transplantation of VCA grafts preserved with 6 h of SNMP is feasible and 30-d • survival rates are comparable to SCS preserved graft Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS. [ABSTRACT FROM AUTHOR]
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- 2022
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217. Activation of autophagy during normothermic machine perfusion of discarded livers is associated with improved hepatocellular function.
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Ohman, Anders, Raigani, Siavash, Santiago, John C., Heaney, Megan G., Boylan, Joan M., Parry, Nicola, Carrol, Cailah, Baptista, Sofia G., Uygun, Korkut, Gruppuso, Philip A., Sanders, Jennifer A., and Heidi Yeh
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AUTOPHAGY , *LIVER , *PRINCIPAL components analysis , *PERFUSION , *PROTEIN analysis , *HEPATOCELLULAR carcinoma - Abstract
Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function. [ABSTRACT FROM AUTHOR]
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- 2022
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218. High-throughput single cell arrays as a novel tool in biopreservation
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Roach, Kenneth L., King, Kevin R., Uygun, Korkut, Hand, Steven C., Kohane, Isaac S., Yarmush, Martin L., and Toner, Mehmet
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MICROFABRICATION , *MANUFACTURING processes , *CYTOLOGY , *BIOLOGY - Abstract
Abstract: Microwell array cytometry is a novel high-throughput experimental technique that makes it possible to correlate pre-stress cell phenotypes and post-stress outcomes with single cell resolution. Because the cells are seeded in a high density grid of cell-sized microwells, thousands of individual cells can be tracked and imaged through manipulations as extreme as freezing or drying. Unlike flow cytometry, measurements can be made at multiple time points for the same set of cells. Unlike conventional image cytometry, image analysis is greatly simplified by arranging the cells in a spatially defined pattern and physically separating them from one another. To demonstrate the utility of microwell array cytometry in the field of biopreservation, we have used it to investigate the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes. Even with optimized cryopreservation protocols, the stress of freezing almost always leads to dysfunction or death in part of the cell population. To a large extent, cell fate is dominated by the stochastic nature of ice crystal nucleation, membrane rupture, and other biophysical processes, but natural variation in the initial cell population almost certainly plays an important and under-studied role. Understanding why some cells in a population are more likely to survive preservation will be invaluable for the development of new approaches to improve preservation yields. For this paper, primary hepatocytes were seeded in microwell array devices, imaged using the mitochondrial dyes Rh123 or JC-1, cryopreserved for up to a week, rapidly thawed, and checked for viability after a short recovery period. Cells with a high mitochondrial membrane potential before freezing were significantly less likely to survive the freezing process, though the difference in short term viability was fairly small. The results demonstrate that intrinsic cell factors do play an important role in cryopreservation survival, even in the short term where extrinsic biophysical factors would be expected to dominate. We believe that microwell array cytometry will be an important tool for a wide range of studies in biopreservation and stress biology. [Copyright &y& Elsevier]
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- 2009
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219. The effect of blood cells retained in rat livers during static cold storage on viability outcomes during normothermic machine perfusion.
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Haque, Omar, Pendexter, Casie A., Wilks, Benjamin T., Hafiz, Ehab O. A., Markmann, James F., Uygun, Korkut, Yeh, Heidi, and Tessier, Shannon N.
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COLD storage , *BLOOD cells , *PERFUSION , *LIVER , *RATS , *HISTOLOGY - Abstract
In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush. [ABSTRACT FROM AUTHOR]
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- 2021
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220. Non-invasive quantification of the mitochondrial redox state in livers during machine perfusion.
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de Vries, Reinier J., Cronin, Stephanie E. J., Romfh, Padraic, Pendexter, Casie A., Jain, Rohil, Wilks, Benjamin T., Raigani, Siavash, van Gulik, Thomas M., Chen, Peili, Yeh, Heidi, Uygun, Korkut, and Tessier, Shannon N.
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REPERFUSION injury , *RESONANCE Raman spectroscopy , *MITOCHONDRIA , *OXIDATION-reduction reaction , *LIVER transplantation - Abstract
Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI. [ABSTRACT FROM AUTHOR]
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- 2021
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221. In Vivo Activity of Genetically Modified Cells Preseeded in Rat Vascularized Composite Allografts.
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Taveau, Corentin B., Lellouch, Alexandre G., Chin, Ling-Yee, Mamane, Olivia, Tratnig-Frankl, Philipp, Lantieri, Laurent A., Randolph, Mark A., Uygun, Korkut, Cetrulo, Curtis L., and Parekkadan, Biju
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HOMOGRAFTS , *PLASTIC surgery , *FACIAL transplantation , *RATS , *CELL populations - Abstract
• We bioengineered rat fibroblasts to secrete a biomarker: Gaussia Luciferase. • Machine perfusion was used to engraft these genetically modified cells in rat limbs. • After transplantation we could track the biomarker secretion for 4 days. • It is the first step toward engraftment of biosensor cells before transplantation. • Vascularized Composite Allotransplantation would benefit from in situ cell therapy. Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model. Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P =.02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs—an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes. [Display omitted] [ABSTRACT FROM AUTHOR]
- Published
- 2021
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222. Liver donor age affects hepatocyte function through age-dependent changes in decellularized liver matrix.
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Acun, Aylin, Oganesyan, Ruben, Uygun, Korkut, Yeh, Heidi, Yarmush, Martin L., and Uygun, Basak E.
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EXTRACELLULAR matrix , *BIOENGINEERING , *LIVER transplantation , *CELLULAR signal transduction , *LIVER cells - Abstract
The only treatment available for end stage liver diseases is orthotopic liver transplantation. Although there is a big donor scarcity, many donor livers are discarded as they do not qualify for transplantation. Alternatively, decellularization of discarded livers can potentially render them transplantable upon recellularization and functional testing. The success of this approach will heavily depend on the quality of decellularized scaffolds which might show variability due to factors including age. Here we assessed the age-dependent differences in liver extracellular matrix (ECM) using rat and human livers. We show that the liver matrix has higher collagen and glycosaminoglycan content and a lower growth factor content with age. Importantly, these changes lead to deterioration in primary hepatocyte function potentially due to ECM stiffening and integrin-dependent signal transduction. Overall, we show that ECM changes with age and these changes significantly affect cell function thus donor age should be considered as an important factor for bioengineering liver substitutes. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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223. Cell release during perfusion reflects cold ischemic injury in rat livers.
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de Vries, Reinier J., Pendexter, Casie A., Cronin, Stephanie E. J., Marques, Beatriz, Hafiz, Ehab O. A., Muzikansky, Alona, van Gulik, Thomas M., Markmann, James F., Stott, Shannon L., Yeh, Heidi, Toner, Mehmet, Uygun, Korkut, and Tessier, Shannon N.
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BIOMARKERS , *TRANSPLANTATION of organs, tissues, etc. , *LIVER cells , *ISCHEMIA , *LABORATORY rats - Abstract
The global shortage of donor organs has made it crucial to deeply understand and better predict donor liver viability. However, biomarkers that effectively assess viability of marginal grafts for organ transplantation are currently lacking. Here, we showed that hepatocytes, sinusoidal endothelial, stellate, and liver-specific immune cells were released into perfusates from Lewis rat livers as a result of cold ischemia and machine perfusion. Perfusate comparison analysis of fresh livers and cold ischemic livers showed that the released cell profiles were significantly altered by the duration of cold ischemia. Our findings show for the first time that parenchymal cells are released from organs under non-proliferative pathological conditions, correlating with the degree of ischemic injury. Thus, perfusate cell profiles could serve as potential biomarkers of graft viability and indicators of specific injury mechanisms during organ handling and transplantation. Further, parenchymal cell release may have applications in other pathological conditions beyond organ transplantation. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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224. Supercooling extends preservation time of human livers.
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de Vries, Reinier J., Tessier, Shannon N., Banik, Peony D., Nagpal, Sonal, Cronin, Stephanie E. J., Ozer, Sinan, Hafiz, Ehab O. A., van Gulik, Thomas M., Yarmush, Martin L., Markmann, James F., Toner, Mehmet, Yeh, Heidi, and Uygun, Korkut
- Abstract
The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at –6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at –4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood. Preservation of human livers at subzero temperatures with ice-free supercooling extends ex vivo organ life. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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225. Resonance Raman spectroscopy detects leaky mitochondrion in rat livers with ischemia-reperfusion injury.
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Nguyen, Khanh T., Ozgur, O. Sila, Romfh, Padraic, Jain, Rohil, Ajenu, Emmanuella, Pugeda, Tyler, Vakhshoori, Daryoosh, Uygun, Korkut, and Tessier, Shannon N.
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RESONANCE Raman spectroscopy , *REPERFUSION injury , *MITOCHONDRIA , *LIVER injuries , *RATS , *REPERFUSION - Published
- 2023
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226. 24-hour supercooling preservation of vascularized composite allografts in rats: A proof-of-concept study.
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Von Reiterdank, Irina Filz, Tawa, Pierre, Goutard, Marion, Pendexter, Casie, Rosales, Ivy, Lellouch, Alexandre, Cetrulo, Curtis, and Uygun, Korkut
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SUPERCOOLING , *HOMOGRAFTS - Published
- 2022
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227. Tools for heart preservation research.
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Higuita, Manuela Lopera, Da Silveira Cavalcante, Luciana, González Rosa, Juan Manuel, Pendexter, Casie, Gopinathan, Kaustav, To, Samantha, De Vries, Reinier, Ellett, Felix, Uygun, Korkut, Langenau, David M., Toner, Mehmet, and Tessier, Shannon N.
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CARDIAC research , *DIGITAL preservation - Published
- 2022
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228. Gene expression correlation analysis of livers undergoing normothermic machine perfusion identifies interactions between donor demographics and liver function.
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Santiago, John C., Raigani, Siavash, Ohman, Anders, Uygun, Korkut, Yeh, Heidi, and Sanders, Jennifer
- Abstract
R596 --> 526.18 --> Background: Liver transplantation is currently the only cure for end‐stage liver disease, but the shortage of donor livers severely limits the effectiveness of transplantation. This shortage is made worse by challenges in preserving and assessing livers pre‐transplant that can result in discarding of functional livers. Normothermic machine perfusion (NMP) is a method of liver preservation that aims to provide physiological conditions while allowing real‐time assessment of transplant viability criteria. The aim of this study was to understand the effect of machine perfusion on the transcriptome and to correlate changes in gene expression with functional liver metrics. Methods: Ten discarded livers (5 steatotic, 5 non‐steatotic), rejected by local transplant centers, were obtained from donation after circulatory death (DCD). The livers underwent 12 hours of NMP with oxygenated red blood cells. Serial tissue and plasma samples were collected at various times during perfusion. Plasma lactate samples were used to categorize liver as meeting viability criteria (viable) or not (nonviable) following NMP. Transcriptome sequencing was performed on serial biopsies taken at 0, 3, and 6hr during perfusion. Weighted gene co‐expression network analysis (WGCNA) was performed to correlate each module with demographics and functional metrics. Pathway and transcription factor analysis were performed on significant modules of interest. Results: Expression data at 0, 3 and 6 hr of perfusion was used to construct the weighted co‐expression network. Gene co‐expression modules were then correlated with various clinical demographics and functional data collected during NMP. To evaluate the functional association of genes in the modules that significantly correlated with arterial lactate levels, we performed Gene Ontology enrichment analysis. By using lactate clearance as an indicator of transplant viability we were able to identify co‐expression gene sets enriched for biological processes including drug metabolism, immune response, autophagy, vascular development, nutrient metabolism, and gene expression. Conclusions: Current donor liver assessment models are limited by traditionally accessible parameters and can often lead to discarding potentially viable organs. NMP provides a novel data collection platform that could be used to improve traditional assessment models. By performing WGCNA, we were able to capitalize on an extensive set of metrics to identify interactions between the diverse donor demographics, the functional data collected during NMP and changes in gene expression. This may ultimately allow us to identify novel biomarkers to improve our current methods of donor liver assessment and assist the development of targeted therapeutics for more successful preservation and utilization of diverse donor livers. [ABSTRACT FROM AUTHOR]
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- 2022
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229. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.
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Uygun, Basak E., Soto-Gutierrez, Alejandro, Yagi, Hiroshi, Izamis, Maria-Louisa, Guzzardi, Maria A., Shulman, Carley, Milwid, Jack, Kobayashi, Naoya, Tilles, Arno, Berthiaume, Francois, Hertl, Martin, Nahmias, Yaakov, Yarmush, Martin L., and Uygun, Korkut
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LIVER transplantation , *LIVER failure , *TISSUE engineering , *REGENERATIVE medicine , *REGENERATION (Biology) , *BIOMEDICAL engineering - Abstract
Orthotopic liver transplantation is the only available treatment for severe liver failure, but it is currently limited by organ shortage. One technical challenge that has thus far limited the development of a tissue-engineered liver graft is oxygen and nutrient transport. Here we demonstrate a novel approach to generate transplantable liver grafts using decellularized liver matrix. The decellularization process preserves the structural and functional characteristics of the native microvascular network, allowing efficient recellularization of the liver matrix with adult hepatocytes and subsequent perfusion for in vitro culture. The recellularized graft supports liver-specific function including albumin secretion, urea synthesis and cytochrome P450 expression at comparable levels to normal liver in vitro. The recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage. These results provide a proof of principle for the generation of a transplantable liver graft as a potential treatment for liver disease. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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230. Oxygenated machine perfusion of donor livers and limbs: Studies on endothelial activation and function
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Burlage, Laura, Porte, Robert, Lisman, Ton, and Uygun, Korkut
- Abstract
Donor organ viability is closely linked to transplant success. In an attempt to overcome the ongoing organ shortage, the criteria for organ donation are progressively being extended. These suboptimal grafts are, however, more prone to preservation and reperfusion related injury and are associated with in inferior transplant outcomes. Oxygenated machine perfusion is a new method of organ preservation whereby an organ is ‘mechanically flushed’ outside the human body with an oxygenated fluid. Oxygenated machine perfusion is gaining increasing attention as an alternative method of organ preservation, as it can be used to improve and assess organ viability of marginal donor organs prior to transplantation. Moreover, the technique of machine perfusion also enables the development of more extreme methods of organ preservation such as cryopreservation (preservation below 0 degrees Celsius) and biobanking within a new areas of transplant surgery, such as vascularized tissue transplantation. This ultimately leads to more and better quality organs and vascularized tissue grafts. In this thesis I investigate the effects of oxygenated machine perfusion on both donor livers (part A) and limbs (part B) in more detail. The results of this thesis contribute to a better understanding of the effect of new methods of organ preservation on the quality of the organ to be transplanted.
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- 2019
231. Gradual Rewarming Preservation of Liver and Kidney Grafts
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Paria Mahboub, de Leij, Lou, and Uygun, Korkut
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Kidney ,medicine.medical_specialty ,medicine.anatomical_structure ,business.industry ,Liver and kidney ,A hemoglobin ,medicine ,Urology ,business ,Perfusion - Abstract
The aim of this thesis was to study the role of gradual rewarming on enhancing organ quality in organs with low quality such as ECD and especially DCD during the organ preservation phase and before implantation. This thesis also addresses an improvement in gradual rewarming protocol by adding a hemoglobin-based oxygen carrier (HBOC) to the perfusion solution in the preclinical kidney and liver rodent models.
- Published
- 2019
232. Transcriptomic Analysis during Normothermic Machine Perfusion Reveals Similar Responses in Steatotic and Lean Discarded Human Livers.
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Sanders, Jennifer, Ohman, Anders, Raigani, Siavash, Santiago, John, Heaney, Megan, Carroll, Cailah, Boylan, Joan, Uygun, Korkut, and Yeh, Heidi
- Abstract
R4157 --> Chronic liver disease is the 12th leading cause of death in the U.S., with end‐stage liver disease alone accounting for 60,000 deaths in the U.S. each year.The only available treatment for liver failure is orthotopic liver transplantation.However, the severe shortage of viable donor organs results in less than 30% of patients on the wait list receiving transplants.The morbidity and mortality associated with liver disease could be dramatically reduced by expanding the donor organ pool by rendering marginal donor livers, especially steatotic grafts, transplantable.Steatotic livers are more susceptible to ischemia‐reperfusion (I/R) injury, the major complication leading to graft dysfunction.Normothermic machine perfusion (NMP) combined with adjunct therapies may be able to rehabilitate discarded steatotic livers, thereby increasing the supply of organs for transplant.The goal of this study was to develop an understanding of the transcriptomic changes associated with NMP in steatotic compared to lean livers. Twelve discarded livers (6 lean, 6 steatotic), rejected by local transplant centers, were obtained and underwent 12 hours of NMP with oxygenated blood. Serial tissue and plasma samples were collected. Clinical viability criteria were used to determine if livers would have been transplantable (viable) or not (nonviable). Transcriptome sequencing was performed on biopsies taken at 0 (pre‐), 3, and 6hr of perfusion. Comparative analysis between transcriptomes of individual conditions was performed using R. Ingenuity Pathway Analysis (IPA) was performed on significant genes to identify canonical pathways and upstream regulators. Three of the six livers from each group met viability criteria for transplant. IPA comparing viable livers at 0 and 3hr of perfusion demonstrated robust activation of innate immune responses, such as Toll‐like receptor, IL‐10, and IL‐6 canonical pathways (P<10E‐7) in both lean and steatotic livers, compared to nonviable livers. However, many of the same canonical pathways identified after 3 hours of perfusion in viable livers were not significant in the nonviable livers until 6hr of perfusion in both lean and steatotic livers, suggesting a delayed transcriptomic response in nonviable livers of both groups. There was significant overlap in upstream regulators identified in both viable and nonviable livers. However, these shared regulators demonstrated remarkably divergent downstream target activation.This is the first study to demonstrate activation of regenerative signaling in discarded lean and steatotic human livers during NMP. Comparison of gene expression profiles between lean and steatotic viable livers were similar as were the transcriptomic profiles of the nonviable livers from both groups.These data suggest that the response to NMP is similar in lean and steatotic liver.Our analysis provides numerous potential therapeutic targets for improving function and transplantability of nonviable lean and steatotic livers during ex vivo machine perfusion. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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233. Extending preservation duration of hearts and livers with partial freezing.
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Da Silveira Cavalcante, Luciana, Pendexter, Casie A., Cronin, Stephanie E.J., de Vries, Reinier J., Ellett, Felix, Marques, Beatriz, Gopinathan, Kaustav, González-Rosa, Juan Manuel, Langenau, David M., Uygun, Korkut, Toner, Mehmet, and Tessier, Shannon N.
- Subjects
- *
FREEZING , *LIVER , *HEART , *CORONARY circulation , *TRANSPLANTATION of organs, tissues, etc. - Published
- 2020
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234. Optimized Partial Freezing Protocol Enables 10-Day Storage of Rat Livers.
- Author
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Ozgur OS, Taggart MS, Mojoudi M, Pendexter C, Kharga A, Yeh H, Toner M, Longchamp A, Tessier SN, and Uygun K
- Abstract
Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15°C, enabling 5 days storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations including increased concentration of propylene glycol to reduce ice recrystallization and maintained osmotic balance through an increase in bovine serum albumin, all while minimizing sheer stress during cryoprotectant unloading with an acclimation period. These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched cold stored livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation and thereby revolutionizing transplant medicine., Competing Interests: Conflict of interests Some authors declare competing interests. Drs. Uygun, Tessier, Yeh and Toner have patent applications relevant to this study. Drs. Uygun, Tessier and Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies. All the remaining authors declare no conflict of interest.
- Published
- 2024
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235. Two-day Static Cold Preservation of α1,3-Galactosyltransferase Knockout Kidney Grafts Before Simulated Xenotransplantation.
- Author
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Mojoudi M, Taggart M, Karadagi A, Hassan M, Tomosugi T, Tomofuji K, Agius T, Lyon A, Nakamura T, Taveras C, Ozgur OS, Kharga A, Matheson R, Riella LV, Kimura S, Yeh H, Markmann JF, Kawai T, Uygun K, and Longchamp A
- Subjects
- Animals, Swine, Humans, Animals, Genetically Modified, Perfusion methods, Heterografts, Cryopreservation methods, Gene Knockout Techniques methods, Mice, Transplantation, Heterologous methods, Kidney Transplantation methods, Galactosyltransferases genetics, Galactosyltransferases deficiency, Organ Preservation methods, Kidney
- Abstract
Transplantation remains the preferred treatment for end-stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off-site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3-galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub-normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA-sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub-normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2024
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236. Gradual rewarming with a hemoglobin-based oxygen carrier improves viability of donation after circulatory death in rat livers.
- Author
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Mahboub P, Aburawi M, Ozgur OS, Pendexter C, Cronin S, Lin FM, Jain R, Karabacak MN, Karimian N, Tessier SN, Markmann JF, Yeh H, and Uygun K
- Abstract
Background: Donation after circulatory death (DCD) grafts are vital for increasing available donor organs. Gradual rewarming during machine perfusion has proven effective in mitigating reperfusion injury and enhancing graft quality. Limited data exist on artificial oxygen carriers as an effective solution to meet the increasing metabolic demand with temperature changes. The aim of the present study was to assess the efficacy and safety of utilizing a hemoglobin-based oxygen carrier (HBOC) during the gradual rewarming of DCD rat livers., Methods: Liver grafts were procured after 30 min of warm ischemia. The effect of 90 min of oxygenated rewarming perfusion from ice cold temperatures (4 °C) to 37 °C with HBOC after cold storage was evaluated and the results were compared with cold storage alone. Reperfusion at 37 °C was performed to assess the post-preservation recovery., Results: Gradual rewarming with HBOC significantly enhanced recovery, demonstrated by markedly lower lactate levels and reduced vascular resistance compared to cold-stored liver grafts. Increased bile production in the HBOC group was noted, indicating improved liver function and bile synthesis capacity. Histological examination showed reduced cellular damage and better tissue preservation in the HBOC-treated livers compared to those subjected to cold storage alone., Conclusion: This study suggests the safety of using HBOC during rewarming perfusion of rat livers as no harmful effect was detected. Furthermore, the viability assessment indicated improvement in graft function., Competing Interests: KU is inventor on pending patents relevant to this study, has a financial interest in Sylvatica Biotech, a company focused on developing organ preservation technology, whose interests are managed by the MGH and Mass General Brigham in accordance with their conflict of interest policies. ST and KU have several patent applications disclosures on extended organ preservation that may be relevant to this study. The HOBC-201 used in this study was provided by HBO2 Therapeutics LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Mahboub, Aburawi, Ozgur, Pendexter, Cronin, Lin, Jain, Karabacak, Karimian, Tessier, Markmann, Yeh and Uygun.)
- Published
- 2024
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237. Polyethylene glycol and caspase inhibitor emricasan alleviate cold injury in primary rat hepatocytes.
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Chen H, Ellis BW, Dinicu AT, Mojoudi M, Wilks BT, Tessier SN, Toner M, Uygun K, and Uygun BE
- Abstract
Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation., Competing Interests: Declaration of competing interest Some authors declare competing interests. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have patent applications relevant to this study. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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238. Enhancing Vascularized Composite Allograft Supercooling Preservation: A Multifaceted Approach with CPA Optimization, Thermal Tracking, and Stepwise Loading Techniques.
- Author
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von Reiterdank IF, Dinicu AT, Cetrulo CL Jr, Coert JH, Mink van der Molen AB, and Uygun K
- Abstract
Vascularized composite allografts (VCAs) present unique challenges in transplant medicine, owing to their complex structure and vulnerability to ischemic injury. Innovative preservation techniques are crucial for extending the viability of these grafts, from procurement to transplantation. This study addresses these challenges by integrating cryoprotectant agent (CPA) optimization, advanced thermal tracking, and stepwise CPA loading strategies within an ex vivo rodent model. CPA optimization focused on various combinations, identifying those that effectively suppress ice nucleation while mitigating cytotoxicity. Thermal dynamics were monitored using invasive thermocouples and non-invasive FLIR imaging, yielding detailed temperature profiles crucial for managing warm ischemia time and optimizing cooling rates. The efficacy of stepwise CPA loading versus conventional flush protocols demonstrated that stepwise (un)loading significantly improved arterial resistance and weight change outcomes. In summary, this study presents comprehensive advancements in VCA preservation strategies, combining CPA optimization, precise thermal monitoring, and stepwise loading techniques. These findings hold potential implications for refining transplantation protocols and improving graft viability in VCA transplantation., Competing Interests: COMPETING INTEREST STATEMENT I.F.R., C.C. and K.U. have patent applications relevant to this field. K.U. has financial interests in and serves on the Scientific Advisory Board for Sylvatica Biotech Inc., a private company developing high subzero organ preservation technologies. Competing interests for Massachusetts General Hospital investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies. A.D., J.C. and A.M.M. have no competing interests.
- Published
- 2024
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239. UW Supplementation with AP39 Improves Liver Viability Following Static Cold Storage.
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McLean ST, Holkup S, Tchir A, Mojoudi M, Hassan M, Taveras C, Ozge SO, James FM, Yeh H, Uygun K, and Longchamp A
- Abstract
Static cold storage of donor livers at 4°C incompletely arrests metabolism, ultimately leading to decreases in ATP levels, oxidative stress, cell death, and organ failure. Hydrogen Sulfide (H
2 S) is an endogenously produced gas, previously demonstrated to reduce oxidative stress, reduce ATP depletion, and protect from ischemia and reperfusion injury. H2 S is difficult to administer due to its rapid release curve, resulting in cellular death at high concentrations. AP39, a mitochondrially targeted, slow-release H2 S donor, has been shown to reduce ischemia-reperfusion injury in hearts and kidneys. Thus, we investigated whether the addition of AP39 during 3-day static cold storage can improve liver graft viability. At the end of storage, livers underwent six hours of acellular normothermic machine perfusion, a model of transplantation. During simulated transplantation, livers stored with AP39 showed reduced resistance, reduced cellular damage (ALT and AST), and reduced apoptosis. Additionally, bile production and glucose, as well as energy charge were improved by the addition of AP39. These results indicate that AP39 supplementation improves liver viability during static cold storage., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Korkut Uygun reports financial support was provided by Massachusetts General Hospital. Korkut Uygun reports a relationship with National Institutes of Health that includes: funding grants. Some authors declare competing interests. Drs. Uygun and Yeh have patent applications relevant to this study. Dr. Uygun, has a financial interest in and serves on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.- Published
- 2024
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240. Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.
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Charlès L, Filz von Reiterdank I, Lancia HH, Shamlou AA, Berkane Y, Rosales I, Mink van der Molen AB, Coert JH, Cetrulo CL Jr, Lellouch AG, and Uygun K
- Abstract
Background: Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT., Methods: Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d., Results: Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d., Conclusions: Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2024
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241. Effect of Donor Age and Liver Steatosis on Potential of Decellularized Liver Matrices to be used as a Platform for iPSC-Hepatocyte Culture.
- Author
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Acun A, Fan L, Oganesyan R, Uygun KM, Yeh H, Yarmush ML, and Uygun BE
- Subjects
- Humans, Adult, Tissue Donors, Middle Aged, Cells, Cultured, Age Factors, Tissue Scaffolds chemistry, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology, Fatty Liver metabolism, Fatty Liver pathology, Extracellular Matrix metabolism, Hepatocytes metabolism, Hepatocytes cytology, Liver metabolism, Liver pathology, Cell Differentiation
- Abstract
Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450 enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization., (© 2024 Wiley‐VCH GmbH.)
- Published
- 2024
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242. Anti-apoptotic treatment of warm ischemic male rat livers in machine perfusion improves symptoms of ischemia-reperfusion injury.
- Author
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Mojoudi M, Taggart MS, Kharga A, Chen H, Dinicu AT, Wilks BT, Markmann JF, Toner M, Tessier SN, Yeh H, and Uygun K
- Abstract
Background: Liver donation after cardiac death (DCD) makes up a small percentage of the organs used in transplantation and poses a higher risk of graft loss compared to donation after brain death (DBD); this is a result of ischemia reperfusion for which the exact injury mechanisms are currently not fully understood. However, reperfusion injury has been shown to lead to necrosis as well as apoptosis through oxidative stress and mitochondrial dysfunction. In this work, we propose that use of the pro-survival, anti-apoptotic CEPT cocktail in post-ischemia normothermic machine perfusion (NMP) may improve recovery in rat livers subjected to extended durations of warm ischemia., Materials and Methods: Livers procured from male Lewis rats were subjected to 90 min of warm ischemia, followed by 6 h of NMP where they were treated either with the survival-enhancing anti-apoptotic cocktail (CEPT), the vehicle (DMSO) or the base media with no additives., Results: The CEPT-treated group exhibited lower expression of hepatic injury biomarkers, and improvement in a range of hepatocellular symptoms associated with the hepatic parenchyma, biliary epithelium and the sinusoidal endothelium, including recovery of bile secretion and lowered vascular resistance., Conclusions: This study's findings suggest apoptosis plays a more significant role in ischemia-reperfusion injury than previously understood, and provide useful insight for further investigation of the specific underlying mechanisms and development of novel treatment methods., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Korkut Uygun reports financial support was provided by 10.13039/100005294Massachusetts General Hospital. Korkut Uygun reports a relationship with 10.13039/100000002National Institutes of Health that includes: funding grants. Some authors declare competing interests. Drs. Uygun, Tessier, Yeh and Toner have patent applications relevant to this study. Drs. Uygun, Tessier and Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)
- Published
- 2024
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243. Acellular Perfusate is an Adequate Alternative to Packed Red Blood Cells During Normothermic Human Kidney Perfusion.
- Author
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Longchamp A, Fontan FM, Aburawi MM, Eymard C, Karimian N, Detelich D, Pendexter C, Cronin S, Agius T, Nagpal S, Banik PD, Tessier SN, Ozer S, Delmonico FL, Uygun K, Yeh H, and Markmann JF
- Abstract
Background: Brief normothermic machine perfusion is increasingly used to assess and recondition grafts before transplant. During normothermic machine perfusion, metabolic activity is typically maintained using red blood cell (RBC)-based solutions. However, the utilization of RBCs creates important logistical constraints. This study explored the feasibility of human kidney normothermic perfusion using William's E-based perfusate with no additional oxygen carrier., Methods: Sixteen human kidneys declined for transplant were perfused with a perfusion solution containing packed RBCs or William's E medium only for 6 h using a pressure-controlled system. The temperature was set at 37 °C. Renal artery resistance, oxygen extraction, metabolic activity, energy metabolism, and histological features were evaluated., Results: Baseline donor demographics were similar in both groups. Throughout perfusion, kidneys perfused with William's E exhibited improved renal flow ( P = 0.041) but similar arterial resistance. Lactic acid levels remained higher in kidneys perfused with RBCs during the first 3 h of perfusion but were similar thereafter ( P = 0.95 at 6 h). Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding oxygen consumption ( P = 0.41) and reconstitution of ATP tissue concentration ( P = 0.55). Similarly, nicotinamide adenine dinucleotide levels were preserved during perfusion. There was no evidence of histological damage caused by either perfusate., Conclusions: In human kidneys, William's E medium provides a logistically convenient, off-the-shelf alternative to packed RBCs for up to 6 h of normothermic machine perfusion., Competing Interests: K.U. and H.Y. have provisional patent applications relevant to this study. K.U. serves on the Scientific Advisory Board for Sylvatica Biotech Inc, a company focused on developing high subzero organ preservation technology. All competing interests are managed by the Mass General Brigham in accordance with their conflict of interest policies., (Copyright © 2024 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)
- Published
- 2024
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244. A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds.
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Berkane Y, Jain R, Ajenu EO, Shamlou AA, Nguyen K, McCarthy M, Uygun BE, Lellouch AG, Cetrulo CL Jr, Uygun K, Randolph MA, and Tessier SN
- Subjects
- Swine, Animals, Humans, Swine, Miniature, Bandages, Disease Models, Animal, Wound Healing physiology, Skin
- Abstract
Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the "shadow zone" remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.
- Published
- 2024
- Full Text
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245. Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21.
- Author
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Agius T, Emsley R, Lyon A, MacArthur MR, Kiesworo K, Faivre A, Stavart L, Lambelet M, Legouis D, de Seigneux S, Golshayan D, Lazeyras F, Yeh H, Markmann JF, Uygun K, Ocampo A, Mitchell SJ, Allagnat F, Déglise S, and Longchamp A
- Subjects
- Animals, Female, Humans, Male, Mice, Dietary Carbohydrates metabolism, Dietary Proteins metabolism, Liver surgery, Liver metabolism, Mice, Inbred C57BL, Diet, Carbohydrate Loading, Fibroblast Growth Factors metabolism, Reperfusion Injury metabolism, Surgical Procedures, Operative
- Abstract
Dietary restriction promotes resistance to surgical stress in multiple organisms. Counterintuitively, current medical protocols recommend short-term carbohydrate-rich drinks (carbohydrate loading) prior to surgery, part of a multimodal perioperative care pathway designed to enhance surgical recovery. Despite widespread clinical use, preclinical and mechanistic studies on carbohydrate loading in surgical contexts are lacking. Here we demonstrate in ad libitum-fed mice that liquid carbohydrate loading for one week drives reductions in solid food intake, while nearly doubling total caloric intake. Similarly, in humans, simple carbohydrate intake is inversely correlated with dietary protein intake. Carbohydrate loading-induced protein dilution increases expression of hepatic fibroblast growth factor 21 (FGF21) independent of caloric intake, resulting in protection in two models of surgical stress: renal and hepatic ischemia-reperfusion injury. The protection is consistent across male, female, and aged mice. In vivo, amino acid add-back or genetic FGF21 deletion blocks carbohydrate loading-mediated protection from ischemia-reperfusion injury. Finally, carbohydrate loading induction of FGF21 is associated with the induction of the canonical integrated stress response (ATF3/4, NF-kB), and oxidative metabolism (PPARγ). Together, these data support carbohydrate loading drinks prior to surgery and reveal an essential role of protein dilution via FGF21., (© 2024. The Author(s).)
- Published
- 2024
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246. Role of Machine Perfusion in Liver Transplantation.
- Author
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Longchamp A, Nakamura T, Uygun K, and Markmann JF
- Subjects
- Humans, Aged, Organ Preservation adverse effects, Perfusion adverse effects, Liver, Liver Transplantation adverse effects, Reperfusion Injury etiology, Reperfusion Injury prevention & control
- Abstract
Given the current severe shortage of available livers for transplantation, there is an urgent need to maximize the utilization of donor organs. One of the strategies to increase the number of available livers for transplantation is to improve organ utilization through the use of elderly, overweight, or organs donated after circulatory death. However, the utilization of these "marginal" organs was associated with an increased risk of early allograft dysfunction, primary nonfunction, ischemic biliary complications, or even re-transplantation. Ischemia-reperfusion injury is a key mechanism in the pathogenesis of these complications., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2024
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247. Sub-Zero Non-Freezing of Vascularized Composite Allografts Preservation in Rodents.
- Author
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von Reiterdank IF, Tawa P, Berkane Y, de Clermont-Tonnerre E, Dinicu A, Pendexter C, Goutard M, Lellouch AG, van der Molen ABM, Coert JH, Cetrulo CL Jr, and Uygun K
- Abstract
Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4-6 hours of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by cryoprotective agent (CPA) cocktail developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation., Competing Interests: COMPETING INTEREST STATEMENT Y.B., A.G.L., C.L.C. and K.U. have patent applications relevant to this field. K.U. has financial interests in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a private company developing high subzero organ preservation technologies. Competing interests for Massachusetts General Hospital investigators are managed by Mass General in accordance with their conflict-of-interest policies. I.F.R., P.T., E.C.T., A.T.D., C.P., M.G., A.M.M. and J.H.C. have no competing interests.
- Published
- 2023
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248. Polyethylene Glycol and Caspase Inhibitor Emricasan Alleviates Cold Injury in Primary Rat Hepatocytes.
- Author
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Chen H, Ellis BW, Dinicu AT, Mojoudi M, Wilks BT, Tessier SN, Toner M, Uygun K, and Uygun BE
- Abstract
Current methods of storing explanted donor livers at 4°C in University of Wisconsin (UW) solution result in loss of graft function and ultimately leads to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4°C, we investigated the effects of lowering the storage temperature to -4°C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5% PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation., Competing Interests: Additional Information Some authors declare competing interests. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have patent applications relevant to this study. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies.
- Published
- 2023
- Full Text
- View/download PDF
249. Sodium Hydrosulfide Treatment During Porcine Kidney Ex Vivo Perfusion and Transplantation.
- Author
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Agius T, Songeon J, Lyon A, Longchamp J, Ruttimann R, Allagnat F, Déglise S, Corpataux JM, Golshayan D, Buhler L, Meier R, Yeh H, Markmann JF, Uygun K, Toso C, Klauser A, Lazeyras F, and Longchamp A
- Abstract
Background: In rodents, hydrogen sulfide (H
2 S) reduces ischemia-reperfusion injury and improves renal graft function after transplantation. Here, we hypothesized that the benefits of H2 S are conserved in pigs, a more clinically relevant model., Methods: Adult porcine kidneys retrieved immediately or after 60 min of warm ischemia (WI) were exposed to 100 µM sodium hydrosulfide (NaHS) (1) during the hypothermic ex vivo perfusion only, (2) during WI only, and (3) during both WI and ex vivo perfusion. Kidney perfusion was evaluated with dynamic contrast-enhanced MRI. MRI spectroscopy was further employed to assess energy metabolites including ATP. Renal biopsies were collected at various time points for histopathological analysis., Results: Perfusion for 4 h pig kidneys with Belzer MPS UW + NaHS resulted in similar renal perfusion and ATP levels than perfusion with UW alone. Similarly, no difference was observed when NaHS was administered in the renal artery before ischemia. After autotransplantation, no improvement in histologic lesions or cortical/medullary kidney perfusion was observed upon H2 S administration. In addition, AMP and ATP levels were identical in both groups., Conclusions: In conclusion, treatment of porcine kidney grafts using NaHS did not result in a significant reduction of ischemia-reperfusion injury or improvement of kidney metabolism. Future studies will need to define the benefits of H2 S in human, possibly using other molecules as H2 S donors., (Copyright © 2023 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)- Published
- 2023
- Full Text
- View/download PDF
250. Anti-apoptotic treatment of warm ischemic male rat livers in machine perfusion improves symptoms of ischemia-reperfusion injury.
- Author
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Mojoudi M, Taggart MS, Kharga A, Chen H, Dinicu AT, Wilks BT, Markmann JF, Toner M, Tessier SN, Yeh H, and Uygun K
- Abstract
Liver donation after cardiac death (DCD) makes up a small percentage of the donor pool and poses a higher risk of graft loss compared to donation after brain death (DBD); this is a result of ischemia reperfusion for which the exact injury mechanisms are currently not fully understood. However, reperfusion injury has been shown to lead to necrosis as well as apoptosis at the cellular level. In this work, we propose that use of the pro-survival, anti-apoptotic CEPT cocktail in post-ischemia normothermic machine perfusion (NMP) may improve recovery in rat livers subjected to extended durations of warm ischemia. Livers procured from male lewis rats were subjected to 90 minutes of warm ischemia, followed by 6 hours of NMP where they were treated with the survival-enhancing anti-apoptotic cocktail (CEPT), the vehicle (DMSO) or the base media with no additives. The CEPT-treated group exhibited lower expression of hepatic injury biomarkers, and improvement in a range of hepatocellular functions associated with the hepatic parenchyma, biliary epithelium and especially the sinusoidal endothelium. This study's findings provide useful insight for further investigation of the extent of apoptotic contribution to ischemia reperfusion injury (IRI)., Competing Interests: Additional Information Some authors declare competing interests. Drs. Uygun, Tessier, Yeh and Toner have patent applications relevant to this study. Drs. Uygun, Tessier and Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies. The remaining others have no competing interests to report.
- Published
- 2023
- Full Text
- View/download PDF
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