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Articles by Maria-Louisa Izamis in JoVE
JoVE Bioengineering
Tüm karaciğerleri Decellularization ve Recellularization
Perfüzyon decellularization organ ekstraselüler matriks kompozisyonu ve mikromimarisini korur tüm karaciğer iskeleleri üretmek için yeni bir tekniktir. Burada, hepatositler ile perfüzyon decellularization ve sonraki repopulation kullanarak tüm organı iskeleleri hazırlama yöntemi açıklanmıştır. Fonksiyonel ve transplante karaciğer greft tekniği kullanılarak oluşturulabilir.
Other articles by Maria-Louisa Izamis on PubMed
A Model for Normothermic Preservation of the Rat Liver
Current techniques for the preservation of donor livers typically rely on cold temperatures (approximately 0-4 degrees C) to slow down metabolic processes. Recently, normothermic extracorporeal liver perfusion (NELP) has regained interest as a potentially promising approach for long-term liver preservation. Unlike cold-storage techniques, NELP attempts to maintain the liver in a near physiological environment, thus enabling normal metabolic and tissue repair processes to take place, which may help in the recovery of ischemically damaged and fatty donor livers, both of which represent significant untapped sources of donor livers. However, NELP is technically more complex than cold-storage techniques, and the lack of standardized small animal models limits its development. Here we describe a rat NELP system that is simple and cost-effective to run. We show that rat livers that underwent NELP for 6 h could be routinely transplanted into syngeneic recipient rats with excellent 1-month survival. During perfusion, the release of cytosolic enzymes, bile and urea production, and oxygen uptake rate could be readily monitored, thus providing a comprehensive picture of hepatic function before transplantation. This system will help in the optimization of NELP in several ways, such as for the improvement of perfusion conditions and the development of quantitative metabolic criteria for hepatic viability.
Recovery of Warm Ischemic Rat Liver Grafts by Normothermic Extracorporeal Perfusion
Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.
Effects of Burn Injury on Markers of Hypermetabolism in Rats
The basic metrics of hypermetabolism have not been thoroughly characterized in rat burn injury models. We examined three models expected to differ in sensitivity to burn injury to identify that which group(s) exhibited the most clinically relevant metabolic response. Six and 12 weeks old male CD (6 week mCD and 12 week mCD) rats, and 12 weeks old female Fischer (12 week fFI) rats received a 20% total body surface area burn, followed by saline resuscitation. Activity, core body temperature, heart rate (via implanted telemetry devices), body weight, food and water intake, and fecal output were measured daily for 1 week before and after burn. Rats lost weight initially postburn but resumed weight gain by 1 week, except for 12 week mCD rats. Core body temperature increased above normal 2 days postburn and returned to baseline by 1 week. Food intake, normalized to body weight, remained unchanged postburn for 12 week mCD rats, but decreased in 6 week mCD rats and increased in 12 week fFI rats. Heart rate in the 12 week mCD and 12 week fFI rats remained at 10 to 15% above baseline, whereas, in 6 week mCD, heart rates returned to baseline after 4 days. Activity levels were unchanged for 12 week fFI and 6 week mCD rats postburn, but decreased for 12 week mCD rats. Postburn hypermetabolism was most significant and sustained in 12 week mCD rats, of least consequence and brief in 6 week mCD rats, and intermediate in 12 week fFI rats. The disparate responses indicate that the choice of animal model should be carefully considered in hypermetabolism studies.
Organ Reengineering Through Development of a Transplantable Recellularized Liver Graft Using Decellularized Liver Matrix
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.
In Situ Metabolic Flux Analysis to Quantify the Liver Metabolic Response to Experimental Burn Injury
Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses. Biotechnol. Bioeng. © 2010 Wiley Periodicals, Inc.
In Situ Metabolic Flux Analysis to Quantify the Liver Metabolic Response to Experimental Burn Injury
Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses.
Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation
BACKGROUND: Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C. METHODS: Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls. RESULTS: Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values. CONCLUSIONS: MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.
A Metabolic Index of Ischemic Injury for Perfusion-recovery of Cadaveric Rat Livers
With over 110,000 patients waiting for organ transplantation, the current crisis in organ transplantation is based on a lack of donors after brain-death (DBD). A very large alternative pool of donor organs that remain untapped are the donors after cardiac death (DCD), recovered after cardiac activity has ceased and therefore sustained some ischemic injury. Machine perfusion has been proposed as a novel modality of organ preservation and treatment to render such cadaveric organs, and in particular livers, transplantable. Two key issues that remain unaddressed are how to assess whether a DCD liver is damaged beyond repair, and whether machine perfusion has rendered an injured organ sufficiently viable for transplantation. In this work, we present a metabolic analysis of the transient responses of cadaveric rat livers during normothermic machine perfusion (NMP), and develop an index of ischemia that enables evaluation of the organ ischemic injury level. Further, we perform a discriminant analysis to construct a classification algorithm with >0.98 specificity to identify whether a given perfused liver is ischemic or fresh, in effect a precursor for an index of transplantability and a basis for the use of statistical process control measures for automated feedback control of treatment of ischemic injury in DCD livers. The analyses yield an index based on squared prediction error (SPE) as log(SPE) >1.35 indicating ischemia. The differences between metabolic functions of fresh and ischemic livers during perfusion are outlined and the metabolites that varied significantly for ischemic livers are identified as ornithine, arginine, albumin and tyrosine.
