Optimization of rat hepatocyte culture in citrated human plasma.
BACKGROUND
Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma.


METHODS
Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated.


RESULTS
Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml).


CONCLUSIONS
Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.
Washizu
J
J
Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Berthiaume
F
F
Chan
C
C
Tompkins
R G
RG
Toner
M
M
Yarmush
M L
ML
D000418:Albumins / Q000378:metabolism; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002951:Citrates / Q000494:pharmacology*; D003470:Culture Media / Q000494:pharmacology*; D005260:Female; D005934:Glucagon / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D006854:Hydrocortisone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D010949:Plasma; D051381:Rats; D011917:Rats, Inbred Lew; D012016:Reference Values; D014508:Urea / Q000378:metabolism
D000418:Albumins; D000596:Amino Acids; D002951:Citrates; D003470:Culture Media; D007328:Insulin; D014508:Urea; D005934:Glucagon; D006854:Hydrocortisone