functional-human-liver-preservation-recovery-means-subnormothermic

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Massachusetts General Hospital, Harvard Medical School

A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation.

Bacterial contamination during cold organ preservation occurs without major complications. However, with organ preservation steering toward (sub)normothermic temperatures, bacterial contamination may be detrimental with limited evidence to support the choice of antibiotic.

Antibiotic prophylaxis in (sub)normothermic organ preservation: in vitro efficacy and toxicity of cephalosporins.

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability.  

Determination and extension of the limits to static cold storage using subnormothermic machine perfusion.

The peribiliary glands of large bile ducts have been identified as a niche of progenitor cells that contribute to regeneration of biliary epithelium after injury. We aimed to determine whether injury to the peribiliary glands of donor livers is a risk factor for development of non-anastomotic biliary strictures (NAS) after liver transplantation.

Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures.

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.

Supercooling enables long-term transplantation survival following 4 days of liver preservation.

Organ culture systems are instrumental as experimental whole-organ models of physiology and disease, as well as preservation modalities facilitating organ replacement therapies such as transplantation. Nevertheless, a coordinated system of machine perfusion components and integrated regulatory control has yet to be fully developed to achieve long-term maintenance of organ function ex vivo. Here we outline current strategies for organ culture, or organomatics, and how these systems can be regulated by means of computational algorithms, or organometrics, to achieve the organ culture platforms anticipated in modern-day biomedicine.

Organomatics and organometrics: Novel platforms for long-term whole-organ culture.

Irreversible electroporation (IRE) is an emerging, minimally invasive technique for solid tumors ablation, under clinical investigation for cancer therapy. IRE affects only the cell membrane, killing cells while preserving the extracellular matrix structure. Current reports indicate tumors recurrence rate after IRE averaging 31% of the cases, of which 10% are local recurrences. The mechanisms for these recurrences are not known and new explanations for incomplete cell death are needed. Using finite elements method for electric field distribution, we show that presence of vascular structures with blood leads to the redistribution of electric fields leading to the areas with more than 60% reduced electric field strength in proximity to large blood vessels and clustered vessel structures. In an in vivo rat model of liver IRE ablation, we show that cells located in the proximity of larger vessel structures and in proximity of clustered vessel structures appear less affected by IRE ablation than cells in the tissue parenchyma or in the proximity of small, more isolated vessels. These findings suggest a role for "electric field sinks" in local tumors recurrences after IRE and emphasize the importance of the precise mapping of the targeted organ structure and conductivity for planning of electroporation procedures.

Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by "electric field sinks".

The current standard for liver preservation involves cooling of the organ on ice (0-4 °C). Although it is successful for shorter durations, this method of preservation does not allow long-term storage of the liver. The gradual loss of hepatic viability during preservation puts pressure on organ sharing and allocation, may limit the use of suboptimal grafts and necessitates rushed transplantation to achieve desirable post-transplantation outcomes. In an attempt to improve and prolong liver viability during storage, alternative preservation methods are under investigation. For instance, ex vivo machine perfusion systems aim to sustain and even improve viability by supporting hepatic function at warm temperatures, rather than simply slowing down deterioration by cooling. Here we describe a novel subzero preservation technique that combines ex vivo machine perfusion with cryoprotectants to facilitate long-term supercooled preservation. The technique improves the preservation of rat livers to prolong storage times as much as threefold, which is validated by successful long-term recipient survival after orthotopic transplantation. This protocol describes how to load rat livers with cryoprotectants to prevent both intracellular and extracellular ice formation and to protect against hypothermic injury. Cryoprotectants are loaded ex vivo using subnormothermic machine perfusion (SNMP), after which livers can be cooled to -6 °C without freezing and kept viable for up to 96 h. Cooling to a supercooled state is controlled, followed by 3 h of SNMP recovery and orthotopic liver transplantation.

Supercooling preservation and transplantation of the rat liver.

Bariatric surgery is the only definitive solution currently available for the present obesity pandemic. These operations typically involve reconfiguration of gastrointestinal tract anatomy and impose profound metabolic and physiological benefits, such as substantially reducing body weight and ameliorating type II diabetes. Therefore, animal models of these surgeries offer unique and exciting opportunities to delineate the underlying mechanisms that contribute to the resolution of obesity and diabetes. Here we describe a standardized procedure for mouse and rat models of Roux-en-Y gastric bypass (80-90 min operative time) and sleeve gastrectomy (30-45 min operative time), which, to a high degree, resembles operations in humans. We also provide detailed protocols for both pre- and postoperative techniques that ensure a high success rate in the operations. These protocols provide the opportunity to mechanistically investigate the systemic effects of the surgical interventions, such as regulation of body weight, glucose homeostasis and gut microbiome.

Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice.

Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range.

Article	Year
A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation. Transplantation research\| PubMed ID: 23369351	2012
Antibiotic prophylaxis in (sub)normothermic organ preservation: in vitro efficacy and toxicity of cephalosporins. Transplantation\| PubMed ID: 23411744	2013
Supercooling as a viable non-freezing cell preservation method of rat hepatocytes. PloS one\| PubMed ID: 23874947	2013
Determination and extension of the limits to static cold storage using subnormothermic machine perfusion. The International journal of artificial organs\| PubMed ID: 24338652	2013
Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. Journal of hepatology\| PubMed ID: 24560661	2014
Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nature medicine\| PubMed ID: 24973919	2014
Organomatics and organometrics: Novel platforms for long-term whole-organ culture. Technology\| PubMed ID: 25035864	2014
Tissue heterogeneity in structure and conductivity contribute to cell survival during irreversible electroporation ablation by "electric field sinks". Scientific reports\| PubMed ID: 25684630	2015
Supercooling preservation and transplantation of the rat liver. Nature protocols\| PubMed ID: 25692985	2015
Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice. Nature protocols\| PubMed ID: 25719268	2015
Warm Ischemic Injury Is Reflected in the Release of Injury Markers during Cold Preservation of the Human Liver. PloS one\| PubMed ID: 25822248	2015

Bote G. Bruinsma

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