The effect of oxygen concentration on the function of human Hep G2 hepatoma cells and primary cultures of rat hepatocytes

Abstract

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 ...