Orthotopic liver transplantation is the only therapeutic option for end-stage liver disease, but it is limited by the scarcity of suitable grafts. Macrosteatotic livers exhibit elevated triglyceride (TG) levels in the form of large lipid droplets (LDs), elevated reactive oxygen species (ROS) and reduced adenosine triphosphate (ATP) levels in the liver’s hepatocytes. These abnormalities increase sensitivity of hepatocytes to ischemia and reperfusion (I/R) stress during transplantation and to increased hepatocyte death and graft failure following transplantation. Macrosteatosis reversal several weeks prior to live-donor liver transplantation reduces graft I/R sensitivity and enables successful transplantation. However, to apply this concept to livers from deceased donors, the defatting process must be accelerated using ex-vivo perfusion with defatting agents. To explore macrosteatosis defatting phenomena and to identify potent defatting agents prior to whole liver studies, an in-vitro system simulating macrosteatosis was developed. Primary rat hepatocyte cultures incubated for several days with elevated free fatty acid (FFA) levels exhibited characteristics of clinical macrosteatosis based on LD morphology, elevated TG levels and elevated sensitivity to hypoxia and reoxygenation (H/R) induced stress, simulating I/R. This novel system was used to explore the ability of selected defatting cocktails to reverse macrosteatosis and reduce H/R sensitivity as measured by hepatocyte viability and function. We found that pretreatment of macrosteatotic cultures with an L-carnitine supplemented defatting cocktail under hyperoxic conditions for 48h prior to H/R induction led to a ~82% reduction in the number of macrosteatotic LDs and to a ~57% reduction in intrahepatic TG by promoting bete-oxidation of FFAs. Furthermore, this treatment reduced ROS stress by ~32%, elevated the ATP levels to that of lean controls and fully abolished H/R associated hepatic death. Treated cultures maintained ~83% viability and exhibited superior functionality compared to untreated macrosteatotic cultures as assessed by urea secretion and bile canalicular transport 48h post H/R stress. The developed system is suitable for exploring additional defatting cocktails that may reverse macrosteatosis and elevated H/R sensitivity. The developed in-vitro defatting routine can now be scaled-up for whole organ perfusion systems to recover macrosteatotic livers in order to mitigate the persistent shortage of suitable livers for transplantation.
Subject (authority = RUETD)
Topic
Biomedical Engineering
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_5358
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
viii, 108 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = statement of responsibility)
by Nir Israel Nativ
Subject (authority = ETD-LCSH)
Topic
Liver--Diseases
Subject (authority = ETD-LCSH)
Topic
Liver cells--Viability
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
Rutgers University. Graduate School - New Brunswick
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License
Name
Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.