Three techniques for measuring oxygen consumption rate (OCR) of cultured cells relevant to the development of bioartificial liver devices are reported. In an oxystat apparatus, HepG2 cells immobilised on Cytodex 3 microcarriers at a concentration of 10(6) cells ml-1 had a mean OCR of 0.7 nmol s-1/10(6) cells. The OCR decreased with increasing cell density, a characteristic previously reported for other cell lines. Rat hepatocytes immobilised on single collagen layers in a flow cell and challenged with ammonia had a mean OCR of 0.59 nmol s-1/10(6) cells. A novel two-compartment oxystat system was used to determine the OCR of rat hepatocytes during the attachment phase. OCR declined from 1.0 nmol s-1/10(6) immediately after seeding to 0.7 nmol s-1/10(6) cells at nine hours. The low OCR for HepG2 reflects loss of certain oxygen dependent metabolic pathways. The OCR measured for rat hepatocytes during and post-attachment are significantly higher than those reported elsewhere and have major implications for the development of bioartificial liver devices.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

Supply ofO2 is a crucial consideration in the development of bioartificial liver assist ( B A L ) devices for the treatment of patients with acute liver failure. However, few device designs address this problem. Currently, BAL are used seeded with e i t h e r h u m a n h e p a t o m a c e l l s [ l ] , or p r i m a r y h e p a t o c y t e s [ 2 ] a s t h e s o u r c e of h e p a t i c metabolising capacity. We have investigated the sensitivity of function in both hepatoma cells and primary hepatocytes to different o2 environments. Dawley rats by collagenase perfusion of the livers and seeded ( 5x106 c e l l s ) onto collagen-coated (0.25pg/cma) lOOmm diameter Petri dishes in 5ml of modified Earle's medium containing 5% foetal calf serum (FCS). They were cultured for 24h before use i n e x p e r i m e n t s . H e p 6 2 c e l l s w e r e s e e d e d (5x104/cm') on 100 mm diameter Petri dishes in 5ml of Williams' E medium with 10% FCS.They were used at confluence 7 days later. concentration in the liquid phase the dishes were mounted on a rocking platform (f25O at 2 cycles/min) contained within an incubation chamber (37f0.5'C). Cells were exposed to 5 , 1 2 , 2 0 , 2 8 and 35% 02, with 5% C02 and the balance N2. After this time viability was assessed by lactate d e h y d r o g e n a s e ( L D H ) leakage into t h e m e d i u m a s d e s c r i b e d b e f o r e [3].T h e parameters of cell function measured were the total cytochromes P450 and P420 content, the cytochrome P450 dependent e t h o x y r e s o r u f i n 0 -d e a l k y l a t i o n ( E R O D ) , a n d incorporation of H3-leucine into protein [ 3,4]. Table 1 shows that the function of Hep 6 2 cells was relatively insensitive to the O2 environment, whereas both cytochromes P450 and P420 (Table 2 ) , and EROD activity (Table 1) were better maintained at the lower O2 concentration in primary cultures. L o s s o f P 4 5 0 c o n t e n t c a n n o t be e x p l a i n e d by degradation t o P420, degradation to apoprotein is occurring particularly at 35% 02. Protein synthesis increased in primary hepatocytes at 35% 02, and this may reflect repair of oxidative damage. Viability was not altered by the O2 environment (LDH data not shown). O2 t e n s i o n is an important modulator of t h e regio-specific expression of xenobiotic metabolism in the liver [5,6].Albumin secretion of primary rat hepatocytes increases at 4% O2 !5], and t h e expression of P450 dependent metabolism, and its control by hormones, is also modulated by the O2 environment [ 6 J . The oxygen environment of cells in the BAL will influence the retention of function especially if primary cells are being used.Hepatoma cells seem relatively insensitive to O2 concentrations, perhaps because they are less metabolically active [ 7 ] and therefore require leas O2 than primary hepatocytes. Hepatocytes were isolated from male Sprague-T o e n s u r e u n i f o r m t e m p e r a t u r e a n d o x y g e nExposure time was 24h. Table 1. Protein svnthesis and EROD activitv in Hep G2 cells and 48h tximarv cultures ...