Rapid and sensitive assessment of human hepatocyte functions.
Transplantation of human hepatocytes (HTx) has gained recognition as a bridge to, or an alternative to, orthotopic liver transplantation for patients with acute liver failure or genetic defects in liver function. Although the quality of the hepatocytes used for cell transplantation is critical, no consensus exists on protocols to assess the function of hepatocytes prior to HTx. Application of this cell therapy in clinical practice could be aided by fast and reliable assays to evaluate the functional competence of isolated hepatocytes prior to clinical transplantation. Traditional assays for measuring metabolic functions in primary hepatocytes frequently involve highly technical equipment, time-consuming methods, and large numbers of cells. We describe a novel approach for the rapid assessment of the metabolic capabilities of human hepatocytes. This report details simple procedures to evaluate 11 endpoints from cells isolated from human liver that can be performed by a single operator within approximately 2 h of isolation. Longer term cultured hepatocytes were also analyzed to determine if the results from the 2-h tests were predictive of long-term hepatic function. The assays simultaneously measure five cytochrome P450 activities, one phase II activity, plating efficiency, and ammonia metabolism in addition to viability and cell yield. The assays require fewer than 20 million cells and can be completed using commonly available and inexpensive laboratory equipment. The protocol details methods that can be used in a time frame that would allow analysis of hepatic functions in freshly isolated hepatocytes prior to their use for clinical transplantation.
Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden.
Roberto
R
Gramignoli
Veysel
V
Tahan
Kenneth
K
Dorko
Raman
R
Venkataramanan
Ira J
IJ
Fox
Ewa C S
EC
Ellis
Massoud
M
Vosough
Stephen C
SC
Strom
D000293:Adolescent; D000328:Adult; D000368:Aged; D000641:Ammonia / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D020169:Caspases / Q000378:metabolism; D002469:Cell Separation; D002470:Cell Survival; D002648:Child; D002675:Child, Preschool; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007223:Infant; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D055815:Young Adult
D000641:Ammonia; D003577:Cytochrome P-450 Enzyme System; D020169:Caspases