The influence of medium composition on the maintenance of cytochrome P-450, glutathione content and...

Journal of Hepatology 1995; 231 605-612 Printed in Denmark AN rights reserved

Copyright 0 Journal of Heporology I995

Journal of Hepatology

ISSN 0168-8278

The influence of medium composition on the maintenance of cytqchrome P-450, glutathione content and urea synthesis: a comparison of rat and

sheep primary hepatocyte cultures

Patricia Watts’, Mark D. Smith’, Ian Edwards*, Victor Zammit3, Vivienne Brown* and Helen Grant’

‘Bioengineering Unit, University of Strathclyde, Wolfson Centre, Glasgow, ‘Biotechnology Department, Bldg 353, HarweN Laboratories, Oxford, and 3Hannah Research Institute, Ayr, UK

Rat and sheep primary hepatocytes have been cultured in four different medium formulations: Willi- ams’ E, Cheek, Medium 199 and Mod&d Earle’s. The total cytochrome P450 content, intracellular concentration of reduced glutathione, rate of urea synthesis and total protein content of cultures of cells from both species in each medium have been determined. Modified Earle’s and Chee’s medium proved to be the most favourable formulations for the culture of rat hepatocytes. After 48 h, cells cultured in Modified Earle’s had significantly more cytochrome P450 and a significantly greater rate of urea synthesis than cells in any other medium. After 6 days in culture the difference in cytochrome P450 levels between rat hepatocytes in Chee’s medium and those in Mod&d Earle’s medium was abrogated. The difference in the rate of urea synthesis between rat hepatocytes cultured in each of these two media was shown to be more de-

T” USE of hepatocytes in vitro to provide detoxi-

fication in an artificial biological liver support system depends on the maintenance of hepatocyte function in culture. However, in common with most differentiated mammalian cells, the function of liver cells deteriorates in culture. De-differentiation in primary cultures of liver cells occurs particularly rapidly, with many key aspects of function being lost within 48 h (1,2). To provide effective liver support for patients in acute liver failure, it will be necessary to optimise culture conditions that prolong retention of specific liver functions in the hepatocytes.

One of the first considerations is which culture me-

Received I December 1994; revised 24 February: accepted 5 April 1995

Correspondence: Patricia Watts, Bioengineering Unit, University of Strathclyde, Wolfson Centre, 106 Rottenrow, Glasgow G4 ONW, UK.

pendent on the medium in which the cells were maintained during the period of urea synthesis measurement than on the medium in which the cells had been previously cultured. Sheep hepatocytes cultured in Chee’s medium ruptured and died within 24 h. Apart from this, sheep cells were less sensitive to changes in medium formulation than were rat hepatocytes. The initial plating efficiency was lower in sheep cells. Total cytochrome P450 content was the most discriminatory of the four parameters for evaluating the status of rat hepatocyte cultures. However, urea synthesis may be the most useful parameter for assessment of hepatocyte function in hybrid liver devices such as bioartificial liver support systems where access to the cells during operation of the device is restricted.

Key words: Culture medium; Liver cell culture; Liver function; Species differences. 0 Journal of Hepatology.

dium to choose, and there are numerous options available as described in the catalogues of companies such as Gibco BRL and Flow Laboratories. In this paper we have examined the stability of hepatic functions in four different media: Modified Earle’s Medium (3,4), Williams’ E Medium, Medium 199(M 199) and Chee’s Medium (5). They were chosen for the following rea- sons: Williams’ E is one of the most widely used media for the culture of liver cells; Modified Earle’s has been shown to be one of the most efficient media for cytochrome P450 maintenance (1,3); Chee’s is a new serum- free medium in which hepatocytes have been shown to respond to phenobarbitone induction of cytochrome P450 (5), a characteristic which has previously been rapidly lost in cultured hepatocytes (6); and Ml99 has previously been used in bioreactors for hybrid liver support systems (7,8). A second consideration is the choice of species. Rats were chosen because of the vast

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literature available. The number of cells required for a liver device is estimated to be 1O’O cells (equivalent to 20 rat livers); thus an animal with a larger liver was required. Sheep livers were chosen to supply the necessary numbers of cells for a bioreactor.

We have measured three parameters as indicators of retained liver function: (i) the content of cytochrome P450, (ii) the ability to convert ammonia to urea, and (iii) the intracellular reduced glutathione (GSH) content. The toxicity and carcinogenicity of many chemi- cals depends upon activation and detoxification by the liver microsomal cytochrome P450 monooxygenase system and the conjugating enzymes. Conjugating with GSH plays an important role in detoxification of elec- trophilic xenobiotics, and this tripeptide is also in- volved in the termination of free radical reactions which may otherwise cause oxidative damage to cell macromolecules. GSH takes part in metabolic functions that protect the cell membrane including thiol transfer reactions, whilst GSH peroxidase prevents lipid peroxidation that can lead to hepatocellular lysis (9, 10). The conversion of ammonia to urea is a vital liver function, in which the ammonia produced through amino acid deamination is detoxified via the urea cycle. This pathway is relatively stable in culture in comparison to cytochrome P450 activity (11). In liver failure, plasma ammonia concentrations can rapidly reach toxic levels. During treatment of a pa- tient with a bioartificial liver support system, ammonia clearance is a convenient parameter of liver function that could. be monitored, via discrete plasma sampling, to give an indication of device performance.

Materials and Methods Preparation and culture of rat hepatocytes Hepatocytes were isolated from male Sprague-Dawley rats (190-220 g) by collagenase (Type IV collagenase from Clostridium histolyticum, Sigma Chemical Co.) perfusion (12). The viability of the hepatocytes determined by Trypan blue exclusion was 80-95%. One- hundred-millimetre tissue culture dishes (Falcon) coat- ed with type 1 collagen (1.5 mg/dish) isolated from rat tail tendons as described by Elsdale & Bard (13) were seeded with 5X106 cells in 10 ml of medium. The cultures were incubated in a 5%COz/air atmosphere at 37°C. For 48-h cultures the medium was changed at 24 h. Six-day cultures had additional medium changes on day 3 and day 5.

Preparation and culture of sheep hepatocytes Sheep hepatocytes were prepared from 6-month-old wethers by collagenase (Sigma Type 1) perfusion of the caudate lobe of the liver, as described previously (14).

Briefly, the caudate lobe was immediately cleared of blood upon removal by perfusion with Ca*+-free buffer containing 140 mM NaCl, 6.7 mM KCI, 10 mM Hepes, 2.5 mM glucose and 0.5 mM EDTA, pH 7.4. This was followed by a recirculating collagenase perfusion with buffer containing 30 mg of collagenase/lOO ml, 2.5 mg of trypsin inhibitor/ml, 140 mM NaCl, 6.7 mM KCl, 30 mM-Hepes, 2.5 mM glucose and 5 mM CaC12. Digestion was continued for various periods (30-70 min), depending on the size of the lobe. The cell yields were in the range of 1.34.5X lo8 cells per perfusion. The determination of viability (87-98%) and the preparation of cultures were the same as for rat hepatocyte cultures. Sheep hepatocytes were cultured for 24 h. Medium was changed on the cultures 6 h before harvesting.

Media tested Four media were tested: Modified Earle’s Medium (3, 4), M199, Williams’ E Medium, and Chee’s Medium (5). All media were suplied by Gibco BRL, Paisley. Chee’s is a serum-free formulation, the other media were supplemented with 5% (v/v) foetal calf serum (Seralab). Penicillin (100 U/ml), streptomycin (100 pg/ ml) and fungizone (2.5 ,&ml) were added to all cultures.

Analytical methods Measurements of cytochrome P450 were carried out by a modified method of Omura & Sato (15). Fresh cells (5 X 106/ml) were homogenised in 0.1 M sodium phosphate buffer, pH 7.6, containing 1 mM dithiotreitol, 1 mM ethylene glycol-bis(beta-amino-ethyl ether) N,N,N’N’-tetra-acetic acid (EGTA), 20% (v/v) gly- cerol and 0.02% (v/v) Nonidet P-40 (Sigma Chemical Co.) (P450 buffer) using a motor-driven teflon-glass homogeniser and stored at -70°C. Cultures of hepatocytes were rinsed with phosphate-buffered saline, pH 7.4 , (PBS) to dislodge dead cells and cellular debris. The cells were scraped with a rubber policeman in 2 ml P450 buffer, then homogenised and stored at -70°C. Carbon monoxide-treated homogenates were compared to CO-treated and sodium-dithionite-reduced homogenates with a Shimadzu UV-2 10 1 PC two-chan- nel spectrophotometer, using 1 ml quartz cuvettes.

The concentration of GSH was measured as described by Hissin & Hilf (16). Freshly isolated cells were suspended in 10% (w/v) trichloroacetic acid (TCA) at a concentration of lo6 viable cells per 500 ,ul TCA and stored at -70°C. Prior to measurement, the cell/TCA suspension was centrifuged to pellet the cellular debris at 13 000 rpm for 1 min. Hepatocyte culture plates were rinsed with PBS and 2 ml lO%TCA was added to each

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Effect of medium on hepatocyte cultures

plate. After 15 min at room temperature the acidic GSH extracts were stored at -70°C until analysis.

The rate of urea synthesis from ammonia was determined with the Sigma Urea Kit No. 535-B. Freshly isolated cells (5x lo6110 ml) were incubated, in rotating 50- ml round-bottomed flasks under an atmosphere of 95% 02/5%C02, in each of the media with 10 mM am- monium chloride. Hepatocytes were cultured as monolayers in each of the media for 24 h (sheep), 48 h (rat) or 6 days (rat). After this time the appropriate fresh medium containing 10 mM NH&l was added to the monolayers on the Petri dishes. The rate of urea synthesis was measured over 2 h in both freshly prepared and cultured cells. Additional measurements were taken from cultures supplemented with 10 mM glucose, 2 mM lactate, and 5 mM ornithine during the assay period (17).

Plating efficiency was determined by comparing the amount of protein present in the incubated cultures to the amount in freshly seeded cultures. The amount of cellular protein present was measured by the method of Lowry and co-workers (18) with bovine serum albu- min as a standard. Protein values were corrected for the collagen coating on the dishes. Freshly isolated cells were suspended in 0.5 M NaOH (106/ml). Cul- tured cells were rinsed with PBS and 4 ml 0.5 M NaOH was added to each plate for 30 min before samples were stored at -70°C until analysis.

Statistical analyses

Data were analysed statistically using one-way analysis of variance and Fisher’s pair-wise comparisons. The four media were compared pairwise for each species. All data are reported as mean+SEM with nZ4.

Results Plating efJiciency

Rat hepatocytes. The plating efficiencies, defined as the protein content as a percentage of the initial value for the number of fresh cells seeded, for the cells cultured for 48 h in Modified Earle’s, Chee’s and Ml99 media were 62%, 76% and 66%, respectively; while in cells cultured in Williams’ E medium the plating efficiency was significantly lower (pcO.05) at 39% (Fig. 1). After 6 days’ incubation the amount of protein remaining in cultures in the Modified Earle’s medium was reduced to only 42% of the original, whereas the protein in the cell cultures in Chee’s medium had decreased only slightly to 71% of the original (Fig. 2). Sheep hepatocytes. After 24 h, plating efficiencies of 42%, 45%, and 44% were attained by cells cultured in Modified Earle’s, Williams’ E, and M199, respectively; a significantly lower ($0.05) plating efficiency of 10% was achieved in Chee’s medium (Fig. 3)

200

160

Value 120

60

40

0 GSH Protein Urea p450

Parameter

Fig. I. Rat hepatocyte function after 48 h culture in Modi- fied Earle’s, Chee’s, Ml99 and Williams E media. All values represent the means?SEM of at least 4 experiments (*=signi$cant at 95% confidence level). Cytochrome P450 is expressed as pmobmg protein; GSH as nmoUmg protein; urea synthesis as nmoUh per mg protein and protein content as the % of the initial value for the number of fresh cells seeded.

p450 GSH Protein

Parameter

Urea

Fig. 2. Rat hepatocyte function after 6 days culture in Modified Earle S and Cheek media. All values represent the meanskSEA4 of at least 4 experiments. The units are the same as in Fig. I.

Concentration of GSH

Rat hepatocytes. Freshly isolated cells had an average concentration of intracellular GSH of 13.9k2.5 nmoll mg protein. The amounts of GSH present in the cells cultured for 48 h in Williams’ E and Ml99 media were

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P450 GSH Protein

Parameter

Urea

Fig. 3. Sheep hepatocyte function after 24 h culture in each medium. All the values represent the means?SEM of at least 4 experiments. The units are the same as in Fig. 1.

similar to fresh cell values, 14.821.4 and 15.6k2.5 nmol/mg protein, respectively (106% and 112% of fresh cell values). In comparison, the amounts in the cells cultured in Modified Earle’s and Chee’s media were nearly two-fold greater, 26.6k5.1 and 24.322.2 nmol/ mg, respectively, (191% and 175% of fresh cell values). Although these differences are marked they are not statistically significant, p=O. 153. These data are shown in Fig. 1. After 6 days culture in Modified Earle’s medium and Chee’s medium, the GSH levels had returned to levels found in freshly isolated cells, 11.75 1 and 16.424.1 nmollmg, respectively, (84% and 118% of fresh cell values) (Fig. 2).

Sheep hepatocytes. Freshly isolated sheep hepatocytes had a concentration of GSH of 13.2+2 nmol/mg protein. The concentrations after 24 h in culture were not significantly different from fresh cell values in Modified Earle’s, Williams’ E or M199, 1422.6, 19.321.3, and 19.821.7, respectively, (106%, 146% and 150% of fresh cell values). No measurable amount of GSH was detected in cells cultured in Chee’s (Fig. 3).

Cytochrome P450 content

Rat hepatocytes. Freshly isolated cells contained 2162 16 pmol/mg protein of cytochrome P450. After 48 h in culture the cells in Modified Earle’s medium, Chee’s, M199, and Williams’ E contained 80.628, 66.025, 49.3+7 and 30.62 13 pmol/mg, respectively, (37%, 31%, 23% and 14% of fresh cell values). Hepato- cytes cultured in Modified Earle’s medium retained significantly more (~~0.005) cytochrome P450 than cells

cultured in any of the other three media. After 6 days in culture there was no difference in the cytochrome P450 levels of cells cultured in either Modified Earle’s medium or Chee’s medium; the respective values were 5 126 and 492 18 pmol/mg protein (24% and 23% of fresh cell values). The 6-day cultures in Modified Earle’s medium and Chee’s medium retained as much P450 as did the 48-h cultures in Williams’ E or Ml99 medium. These data are shown in Fig. 1 and 2.

Sheep hepatocytes. Freshly isolated sheep hepatocytes contained 198.6224 pmol/mg protein of cytochrome P450. The levels of cytochrome P450 in cells cultured in Modified Earle’s, Williams’ E and Ml99 did not differ significantly from each other: 117.Ok19.5, 112.6214.4, 106.829.4, respectively, (59%, 57%, and 54% of fresh values). No cytochrome P450 was found in cells cultured in Chee’s (Fig. 3).

Urea synthesis

Rat hepatocytes. Freshly isolated rat hepatocytes were assayed in Modified Earle’s synthesised urea at the rate of 1712 18 nmol/h per mg protein. The ability of cultured hepatocytes to convert ammonia into urea was significantly greater (~~0.001) for cells cultured for 48 h in Modified Earle’s (1842 19 nmolh per mg protein) than in cells cultured in Williams’ E, Ml99 or Chee’s (84?24, 84+8, 3126, respectively) (Fig. 1). Further- more, the rate of urea synthesis of cells cultured in Chee’s medium was significantly less than in cells cultured in Williams’ E or Ml99 (~~0.001). After 6 days, the rate of urea synthesis in cells cultured in Modified Earle’s medium had diminished to approximately 40% of that recorded at 48 h, while cells cultured in Chee’s medium maintained the level of urea synthesis found at 48 h (Fig. 2). However, when cells cultured in Modified Earle’s medium were subsequently assayed for urea synthesis in Chee’s medium, and likewise when cells cultured in Chee’s medium for 48 h were assayed in Modified Earle’s medium, the rate of urea synthesis was found to be more dependent upon the assay medium (~~0.0001) than on the culture medium (p= 0.014) (Fig. 4). The addition of supplements to each medium, as described in Methods, resulted in the rate of urea synthesis being independent of the type of culture medium used (Fig. 5).

Sheep hepatocytes. Freshly isolated sheep hepatocytes assayed in Modified Earle’s medium synthesised urea at the rate of 13622 nmol/h per mg protein. In 24-h cultures, cells cultured in Modified Earle’s con- verted significantly more (~K0.05) ammonia to urea than those cultured in the other three media (Williams’ E, 51? 18; M199, 70?7; Chee’s, 0). It should be noted

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nmol/h per mg protein that Chee’s medium did not support retention of urea synthesis in cultures (Fig. 3).

120 •j ChW’S

[7 Mod. Earles

100 r-l

60

Cheek Mod. Earles

Medium

Fig. 4 The effect of assay medium on urea sythesis in 48 h cultures of rat hepatocytes. AN values represent the meankSEA4 of at least 4 experiments. Urea synthesis is shown as nmol/h per mg protein. Filled bars represent values from cells cultured in Chee ‘s medium while empty bars indi- cate values from cells cultured in Modtfied Earle’s medium. The cells were then assayed for urea synthesis in either Chee ‘s medium or Modtf?ed Earle S medium as indicated on the X-axis.

nmol/h per mg protein

250,

200-

150-

loo-

50 -

0-r WmE Ml99 Chee’s Mod.Esrle’s

Medium

Fig. 5. Effect of addition of 10 mM glucose, 2 mM lactate and 5 mM ornithine to each medium on the rate of urea synthesis in rat hepatocytes. Units are expressed as nmobh per mg pro tein. All values represent mean 2 SEA4 of at least 4 experiments.

Hepatocyte morphology

Rat cells cultured in Modified Earle’s or Chee’s media had more canaliculae present and were more flattened at 48 h than cells cultured in either Williams’ E or M199. At 6 days the cells cultured in Chee’s medium looked healthier, were less granular and contained more canaliculae than those cultured in Modified Earle’s (Fig. 6).

Sheep cells were different from rat hepatocytes in terms of both morphology and behaviour in culture. They looked smaller, rounder and took longer to flat- ten and attach after seeding. In contrast to rat hepatocytes, sheep hepatoytes could not survive in Chee’s medium; they formed blebs, ruptured and died within 24 h.

Discussion It has been shown here that the choice of medium for successful culture of primary hepatocytes depends upon the species of hepatocyte origin, and to some ex- tent upon the differentiated function that is being investigated. The phenotype of rat hepatocytes was more sensitive than that of sheep to the different media, with the exception of Chee’s medium which was unique in that the sheep cells in this medium ruptured and died within 24 h of culture. The inability of Chee’s medium to support sheep hepatocyte cultures was not abrogated by the addition of 5% FCS (Watts, unpublished data). Rat hepatocytes cultured in the four media showed significant differences in plating efficiency, cytochrome P450 content, and rate of urea synthesis, but not in intracellular concentration of GSH. No- tably, Williams’ E, which is widely used for rat hepatocyte culture, performed rather badly in our tests. Sheep hepatocytes cultured in William’s E, Ml99 and Modi- fied Earle’s media showed a significant difference only in the rate of urea synthesis.

Although GSH concentration per se, does not represent a specific hepatocyte function, the presence of GSH in the 6-day rat hepatocyte cultures in Modified Earle’s and Chee’s media does present evidence of maintained differentiation. In most cells GSH is synthesised from three substrates: L-glutamate, L-cysteine and L-glycine; however, hepatocytes are one of only a few cell types able to utilise L-methionine for GSH synthesis in place of L-cysteine via the cystathionine pathway (19). However, the activity of the cystathionine pathway is rapidly lost in de-differentiated liver cells (20-23). In the Modified Earle’s and Chee’s media there is no L-cysteine or L-cystine; therefore hepato-

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Fig. 6. Hepatocyte morphology: a) sheep, 24 h in Williams’ E; b) rat, 24 h in Modi- fied Earle ‘s; c) rat, 48 h in Chee’s medium d) rat, 6-day cultures in ModiJied Earle’s and e) rat, 6-day cultures in Chee’s medium (magniJication a-c: x2.50, d-e:

cytes cultured in these media can synthesise GSH only through the cystathionine pathway using L-methionine. GSH levels in rat hepatocytes are well maintained in these two media for at least 6 days in culture, indi- cating that these cells have retained this differentiated function.

The hepatic microsomal cytochrome P450 system plays an important role in the detoxification function of hepatocytes. In rat hepatocytes, P450 levels were significantly higher at 48 h in cells cultured with Modified Earle’s medium than in those cultured with any of the other media. This may be explained by increased production and/or reduced degradation of P450 in Modi- fied Earle’s medium. This is the only medium which contains 5-amino-laevulinic acid (ALA) which stimu- lates P450 production in cultured hepatocytes (24). In addition, it has been shown that the presence of L- cysteine and L-cystine in media results in the loss of P450 due to enhanced degradation (3). Modified Earle’s contains neither L-cysteine nor L-cystine. The two media in which the loss of P450 was greatest, Wil- liams’ E and M199, contain both L-cysteine and L-

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cystine. After 6 days in culture the cells in Modified Earle’s and Chee’s media contained less P450 than at 48 h, but these 6-day levels were comparable with the 48-h levels in cells cultured in Williams’ E and Ml99 media. Sheep hepatocytes, in contrast, show no significant difference in cytochrome P450 content in the three media which supported growth. Recent evidence suggests that the loss of total P450 in cultured hepatocytes is concomitant with a reduction in mRNAs cod- ing for P450 isoenzymes (25,26), and that substantial amounts of this loss may even occur during the collagenase perfusion before culture has been initiated. If this is the case, then the protection offered by L-cysteine-free media will be less important.

The liver is the major site of ammonia detoxification, and one of the main features of this metabolism is the conversion of ammonia to urea. A measure of the function of hepatocytes can be made by determin- ing the rate of synthesis of urea from ammonia, and this is a convenient parameter that could be used for monitoring the function of hybrid artificial liver devices. Of the four media tested, the rate of urea syn-


thesis was significantly greater in Modified Earle’s, while cells cultured in Chee’s medium gave the slowest rate of urea synthesis. This emergence of urea synthesis as the most discriminatory parameter of liver function in the study was unexpected because the expression in culture of the enzymes of urea synthesis has been found to be more stable than those of the cytochrome P450 system (11). However, the medium used during the urea analysis proved to be more significant than the medium in which the cells had been cultured prior to the assay. Furthermore, no significant differences in the rate of urea synthesis were found between cells cultured in the different media when glucose, lactate and ornithine were added to each. It appears that all four media under investigation support expression of the urea enzymes in culture and that the rate of urea synthesis is mainly a function of the composition of the assay medium, rather than of the differentiated status of the hepatocytes at this time point.

We conclude that Chee’s medium does not support sheep hepatocytes and that Williams’ E and Ml99 are the least hospitable of the media tested in which to culture rat hepatocytes. Modified Earle’s supported the P450 levels of rat hepatocytes better than any of the other media in the first 48 h of culture, but this difference was reduced to parity with the Chee’s medium over 6 days. It is of particular importance to note that Chee’s is a serum-free formulation and thus a com- pletely defined medium. Serum-free conditions have previously been demonstrated to increase the activity of cytochrome P450 in cultured rat hepatocytes (27). The same authors demonstrated that GSH content was not affected by the absence of serum. In addition, MacDonald et al. (23) have also reported improved expression of bilirubin UDP-glucuronosyltransferase activity, but not ghttathione-S-transferase activities, in immortalised rat hepatocytes cultured in serum-free conditions. Clearly these serum-free conditions do not improve retention of all liver specific functions in hepatocytes.

We have shown that there are significant differences in retained hepatocyte functions in rat and sheep cell cultures in the four media investigated, and that there are startling species differences. In addition to the ob- servation that Chee’s medium, one of the best for rat hepatocytes, did not support sheep hepatocytes, we have demonstrated that rat hepatocytes retained function significantly better in Modified Earle’s, while sheep hepatocyte function was maintained equally well in Williams’ E and M199. We therefore recommend careful choice of media for artificial liver bioreactor cultures dependent on the source of the cells used.

Acknowledgements This work was supported by Science and Engineering Research Council Link Scheme Grant No. GR- H25522.

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