title: Optimization of conditions for clinical human hepatocyte infusion.
abstract: Cytotoxicity and apoptosis are common problems in the isolation and storage of human hepatocytes. In vitro environments of hepatocytes during cell infusion may be critical to reducing cellular damage and enhancing cell viability. We examined the effects of donor liver histology (40-50% steatosis vs. normal), incubation time, temperature, and three solutions for infusion on banked primary human hepatocytes, by studying: trypan blue exclusion, AST release, LDH release, MTT assay, detection of DNA ladder, and a hepatocyte proliferation assay. In addition, the microstructure functions of the endoplasmic reticulum and mitochondria of the intact hepatocytes were determined by measuring correlates of UGT 1A1 and cytochrome P-450 3A (CYP3A4) activity. In general, hepatocyte viability decreased significantly within 60 min after thawing. Cells suspended in 5% dextrose lactated Ringers solution (D5LR) maintained greater cell viability. Hepatocytes from normal liver donors showed less AST and LDH enzyme leak in comparison with cells from fatty liver donors. Mild hypothermic temperature (32 degrees C) inhibited cellular damage that otherwise significantly increased at 60 min. Hepatocytes did not proliferate until 12 h from thaw, regardless of supernatant or conditions of suspension. CYP3A4 activity and a marker for UGT 1A1 activity in hepatocytes from normal donor livers were higher than those from steatotic donor livers. These findings suggest that hepatocytes suspended for infusion after isolation from normal liver donors have normal biological functions and less cellular damage/necrosis in contrast with those isolated from fatty liver donors. These damages are inhibited significantly by maintaining hepatocytes at a mild hypothermic temperature (32 degrees C). D5LR alone maintained the best cell viability for up to 60 min. Media of D5LR + adenosine and HMM were able to partially inhibit hepatocyte apoptosis in hepatocytes from steatotic livers.
authors.affiliation: Department of Surgery, Virginia Commonwealth University Medical Center, Richmond, VA 23298, USA. rafisher@hsc.vcu.edu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fisher
authors.forename: Dawen
authors.initials: D
authors.lastname: Bu
authors.forename: Melissa
authors.initials: M
authors.lastname: Thompson
authors.forename: Luke
authors.initials: L
authors.lastname: Wolfe
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Ritter
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000293:Adolescent; D017209:Apoptosis / Q000187:drug effects; D001219:Aspartate Aminotransferases / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods*; D002648:Child; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D005234:Fatty Liver / Q000473:pathology; D005260:Female; D015870:Gene Expression / Q000235:genetics; D014453:Glucuronosyltransferase / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D000099148:UGT1A1 Enzyme
chemical_list: D000241:Adenosine; D001219:Aspartate Aminotransferases; D016895:Culture Media, Serum-Free; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase; D007770:L-Lactate Dehydrogenase; D000099148:UGT1A1 Enzyme; C104464:CYP3A protein, human; C510163:CYP3A4 protein, human