Primary Rat Hepatocyte Spheroids as a Model of MetALD
Location
LSU Health Sciences Center - New Orleans
Event Website
https://www.medschool.lsuhsc.edu/genetics/2023_medical_student_research_poster_symposium.aspx
Presentation Date
23-10-2023 8:29 AM
Description
Objective: Metabolic and alcohol associated steatotic liver disease (MetALD) is a newly established diagnosis that considers both metabolic and alcohol associated factors contributing to liver injury. The objective of this study was to use a three-dimensional cell culture technique to model metabolic and inflammatory manifestations of MetALD in primary rat hepatocyte spheroids treated with high fats and sugars (HFS) plus ethanol (E) after chronic control- or alcohol-diet feeding. Methods: Primary rat hepatocytes were isolated from Fischer 344 rats that were given either a Lieber-DeCarli control (n=1) or alcohol liquid diet (n=1) for 15-weeks. Hepatocytes were isolated by canulating the hepatic portal vein, perfusing the liver with a chelating buffer, and then digesting the liver with a collagenase IV solution. The liver was then resected and mechanically digested. Through a series of centrifugation steps and a 45% percoll isolation, viable rat hepatocytes were placed in ultra-low attachment plates to facilitate hepatocyte aggregation & spheroid formation. Five days later, the spheroids were changed to a serum free media, and treated with control (C; vehicle (1% BSA) + 5.5 mM glucose), ethanol (E; 50 mM ethanol), or high fat and sugar (HFS; 5:5:1 palmitic acid:oleic acid:BSA (400mM FAs), 1:1 glucose/fructose (11 mM sugars) media in combination (EHFS) or separately. After five days of incubation, supernatants from individual spheroids were collected. Using a sandwich ELISA assay, the concentration of IL-6 and C-reactive protein (CRP) in the supernatants from these spheroids were determined (n=3 technical replicates, n=2 spheroid supernatants from each group). The ATP and triglyceride (TG) contents of individual spheroids (n=6 each group) were also quantified using spheroid lysis bioluminescent assays. Results: Spheroids incubated with the various conditions from both control- and alcohol-fed rats showed similar viability as indicated by ATP content relative to control media at endpoint (p=0.479). TG accumulation was significantly increased by in vitro HFS and EHFS 3.3-fold in control- and 5.5- and 5.3-fold, respectively, in alcohol-fed rat spheroids compared to control media treated spheroids (p
Recommended Citation
Davis, Brice A., "Primary Rat Hepatocyte Spheroids as a Model of MetALD" (2023). Medical Student Research Poster Symposium. 33.
https://digitalscholar.lsuhsc.edu/sommrd/2023MSRD/Posters/33
Primary Rat Hepatocyte Spheroids as a Model of MetALD
LSU Health Sciences Center - New Orleans
Objective: Metabolic and alcohol associated steatotic liver disease (MetALD) is a newly established diagnosis that considers both metabolic and alcohol associated factors contributing to liver injury. The objective of this study was to use a three-dimensional cell culture technique to model metabolic and inflammatory manifestations of MetALD in primary rat hepatocyte spheroids treated with high fats and sugars (HFS) plus ethanol (E) after chronic control- or alcohol-diet feeding. Methods: Primary rat hepatocytes were isolated from Fischer 344 rats that were given either a Lieber-DeCarli control (n=1) or alcohol liquid diet (n=1) for 15-weeks. Hepatocytes were isolated by canulating the hepatic portal vein, perfusing the liver with a chelating buffer, and then digesting the liver with a collagenase IV solution. The liver was then resected and mechanically digested. Through a series of centrifugation steps and a 45% percoll isolation, viable rat hepatocytes were placed in ultra-low attachment plates to facilitate hepatocyte aggregation & spheroid formation. Five days later, the spheroids were changed to a serum free media, and treated with control (C; vehicle (1% BSA) + 5.5 mM glucose), ethanol (E; 50 mM ethanol), or high fat and sugar (HFS; 5:5:1 palmitic acid:oleic acid:BSA (400mM FAs), 1:1 glucose/fructose (11 mM sugars) media in combination (EHFS) or separately. After five days of incubation, supernatants from individual spheroids were collected. Using a sandwich ELISA assay, the concentration of IL-6 and C-reactive protein (CRP) in the supernatants from these spheroids were determined (n=3 technical replicates, n=2 spheroid supernatants from each group). The ATP and triglyceride (TG) contents of individual spheroids (n=6 each group) were also quantified using spheroid lysis bioluminescent assays. Results: Spheroids incubated with the various conditions from both control- and alcohol-fed rats showed similar viability as indicated by ATP content relative to control media at endpoint (p=0.479). TG accumulation was significantly increased by in vitro HFS and EHFS 3.3-fold in control- and 5.5- and 5.3-fold, respectively, in alcohol-fed rat spheroids compared to control media treated spheroids (p
https://digitalscholar.lsuhsc.edu/sommrd/2023MSRD/Posters/33
Comments
Mentor: Dr. Liz Simon LSUHSC, Department of Physiology