Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation

Research in VeterinaryScience1997, 62, 233-237

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Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation N. YAMANAKA, H. KITANI, O. MIKAMI, Y. NAKAJIMA, K. MIURA, National Institute of Animal Health, 3-1-1

Kannon-dai, Tsukuba, Ibaraki 305, Japan

SUMMARY

The morphological and biochemical characterisation of adult chicken hepatocytes in a serum-freeculture are described. When cultured in positively charged plastic dishes, chicken hepatocytes formed a monolayer cell sheet. The monolayer morphologyof these chicken hepatocytes was quite distinct from the spheroid shape of rat hepatocytes cultured under similar conditions. Electron microscopy showed that the cytoplasmicorganelles of chicken hepatocytes were well preserved in vitro. Two-dimensionalgel electrophoresis showed that the chicken hepatocytes secreted liver-specific proteins. Several enzymes of ghicose-6-phosphatase, cytochrome P-450 or glutathione S-transferase, involved in metabolic and biotransformationpathways in the liver, were retained in the chicken hepatocytes in a serum free condition. These findings suggest that the primary culture of adult chicken hepatocytes with a serum-free culture system could be useful to study the hepatic metabolicpathway in the chicken and its responseto various chemicals.

PRIMARY cultured hepatocytes have been used to investigate hepatic metabolism and biotransformation. Unlike hepatocyte cell fines derived from hepatoma, primary cultured hepatocytes provide simple systems for experiments, while maintaining various liver-specific functions. The procedure for primary cultures was originally developed for rat hepatocytes (Bissell et ai 1973), although it has also been used for other mammalian species (Miyazaki et al 1981, Urlich et al 1990, Chesn6 et al 1993). Although the metabolic functions of the poultry liver differ vastly from those in mammals very few reports have dealt with chicken primary hepatocytes after hatching (Fraslin et al 1992). Chicken embryo hepatocytes have also been applied because they are easy to isolate and to culture. However, the metabolic functions of these hepatocytes differ from those of adult hepatocytes (Plant et al 1983). Serum contains a number of bioactive substances unidentified in both quality and quantity, eg, growth factors, hormones and cytokines. These substances are normally essential to support cell growth, but they can also interfere with biochemical assessments by affecting cell functions. Therefore, serum free culture systems have become important for in vitro investigation using cultured hepatocytes. To investigate biochemical and toxicological functions of adult chicken hepatocytes, we developed a serum free culture system, characterised their metabolic functions and the stages of biotransformation of the hepatocytes and compared them with the rat system.

MATERIALS AND METHODS

Tissue culture procedures Male white Leghorn chickens, one to two months old and approximately 1 kg in bodyweight, were euthanised by bleeding under anesthesia with sodium pentobarbital. The abdominal cavities was opened immediately, the mesenteric veins were catheterised in situ (Kuhlenschmidt et al 1982), and the right atriums were incised. Seglen's (1973) method of sequential perfusion was car0034-5288/97/030233 + 05 $18.00/0

ried out with several modifications. The liver was perfused briefly at first with a Ca2+-free Hanks solution supplemented with 500 ~M EGTA and 10 mM Hepes, and secondarily with a 0-03 per cent collagenase (cell dispersion grade, WAKO Pure Chemical Industries Ltd., Japan) in Hanks solution containing Ca 2+. The perfused liver was then removed from each chicken, cut into pieces and immersed in minimal essential medium. The dispersed cells were filtered through a nylon mesh to remove cell debris. The filtrate was then centrifuged three times at 50 x g for 1 minute to isolate parenchymal hepatocytes. These hepatocytes were seeded onto plastic dishes with a positively charged surface (Primaria, Becton Deckinson and Co., USA) at a density of 106 cells ml -t in a total volume of 4 ml. A modified serum free culture medium (Koide et al 1990) was employed: William's E medium, containing 300 mg -I litre -1 glucose, 10.9 M insulin, 10-9 M dexamethasone, 5 klg approtinin, trace elements (0.1 jaM CuSO4-5H20 , 3 nM H2SeO 3, 50 pM ZnSO4-7H20), and antibiotics (100 U m1-1 penicillin, 100 mg m1-1 streptomycin, 250 ~tU m1-1 amphotericin B). The cells were incubated at 37°C under a humidified atmosphere of 5 per cent CO 2 in air. To remove unattached cells the medium was changed four hours after initiating incubation. The medium was then renewed every two days during the culture period, and 24 hours before cell harvest. To compare species difference in hepatocyte culture, rat primary hepatocytes were prepared from male Fischer 344 rats, 200 grams of bodyweight. The procedure was the same as for the chicken hepatocytes, except for adding 50 ng m1-1 epidermal growth factor (EGF, human recombinant, WAKO Pure Chemical Industries Ltd., Japan).

Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was carried out by the method of Manabe et al (1983). The first dimension involved electrofocusing (PI 4-5-10), while the second © 1997 W. B. Saunders CompanyLtd

234

N. Yamanaka, H. KitanL O. Mikami, Y. Nakajima, K. Miura

dimension used native polyacrylamide gel electrophoresis (PAGE) in a 4-17 per cent concentration gradient gel. The secondary slab gel was stained using a silver staining kit (WAKO Pure Chemical Industries Ltd., Japan).

Enzyme-linked immunosorbent assay The chicken albumin content in the culture medium was determined using a sandwich method of the enzyme-linked immunosorbent assay (ELISA) by the modified method of Oikawa et ai (1995) as follows. Chicken albumin (Cappel Research Products, USA) or diluted culture medium were coated onto a flat-bottom polystyrene 96 well plastic microplate (Flow Laboratories, Inc. USA) overnight at 4°C. After blocking with 3 per cent skimmed milk in PBS(-), they were sequentially reacted for two hours at 37°C with anti-chicken albumin rabbit IgG (Cappel Research Products, USA) as the first antibody and then alkaline phosphatase-conjugated anti-rabbit IgG goat serum (Tago Products, USA) as the second antibody. Finally, the albumin content in the medium was detected as a 405 nm absorption using 1 mg m1-1 of p-nitrophenyl phosphate (WAKO Pure Chemical Industry Ltd., Japan) in a 1 M diethylamine (WAKO Pure Chemical Industry Ltd., Japan) buffer (pH 9.8) as substrate and 0.75 M NaOH as the reaction terminator.

Biochemical analysis

cytochrome P-450 contents were method of Omura and Sato (1964).

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Electron microscopy Primary cultured chicken hepatocytes were fixed in 1 per cent glutaraldehyde-4 per cent formaldehyde phosphate buffer (pH 7.4). Then they were postfixed in 1 per cent osmium tetroxide phosphate buffer, and were processed routinely for embedding in epoxy resin. Ultra-thin sections were stained with uranyl acetate and lead citrate, and they were examined with a transmission electron microscope OEM-IOOCX)

RESULTS

Light microscopy Using the serum-free system, the isolated chicken hepatocytes had adhered to the positively charged plastic dish and began to develop four hours after seeding. A monolayer sheet was formed after three days. This sheet monolayer was retained in culture for at least eight days (Fig la). In contrast, the rat hepatocytes formed multicellular spheroids (Fig lb) within a week under the same primary culture condition as the chicken hepatocytes, except for supplementation of EGF (Koide et al 1990). Addition of EGF into chicken hepatocytes culture had no effect on forming multicellular spheroids (data not shown),

To examine protein synthesis and enzyme activity of cultured hepatocytes, the cells were harvested using a rubber policeman after washing with PBs(-). The cells were re-washed with PBS(-) and then they were homogenised by sonication. Glucosc 6-phosphatase (G6Pase) activity was assayed by the method of Gierow & Jergil (1982). Glutathione S-transferase (GST) activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (Warholm et al 1985). The

Fig 2 shows the ultrastmcture of primary cultured chicken hepatocytes. The nucleus, mitochondria, and rough endoplasmic reticulums were well preserved. Microvillus-like structures were formed on the cell surface adjacent to the neighbouring hepatocytes, after two days of culture. These structures were similar to the structures found in vivo.

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Ultrastructure of primary cultured hepatocytes

FIG 1: Phase contrast photomicrographs of chicken and rat hepatocytes in primary culture. (a) Chicken hepatocytes spread as a sheet monolayer on a positively charged plastic dish under a serum-free condition at four days in culture. (b) Rat hepatocytes formed multicellular spheroids under similar culture conditions as in (a), except for adding EGF at nine days in culture. Magnification x200

In vitro chicken hepatocytes

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the arrows indicate. Thus, the cultured monolayer of hepatocytes were shown to synthesise and secrete albumin and other serum proteins.

Metabolic functions The amount of albumin, a major protein secreted by hepatocytes, was monitored throughout the experiments by a sandwich ELISAmethod. Fig 4 illustrates the time course of albumin secretion into the culture. Albumin levels gradually decreased over the first three days and then maintained the levels from 0-3 to 0-7 lag m1-1 throughout the observation period. Fig 5 shows G6Pase activity. G6Pase is one of the key enzymes in gluconeogenesis. The activity of this enzyme normally reflects carbohydrate metabolic functions of the liver. This activity was maintained constantly up to eight days.

Biotransformation functions Fig 6 shows the changes in the activity of xenobioticmetabolising enzymes. The content of cytochrome P-450 (Fig 6a), the first phase enzyme, gradually increased and reached its highest levels at eight days. Fig 6b shows the activity of GST, the second phase enzyme. The activity was decreased at two days of the culture period, but the activity maintained levels of approximately 50 mU for the following six days of culture.

FIG 2: Electron microscopy of chicken primary hepatocytes after two days in serum-free culture. Various cell organelles are well preserved and microvillus-like structures (arrow) are formed. Bar=500 nm

Two-dimensional electrophoresis Fig 3 shows two-dimensional electrophoresis profiles of the media of the cultured monolayer of chicken hepatocytes and chicken serum. Secretion proteins were more clearly observed by studying undenatured protein profiles by electrofucusing and native PAGE rather than by a SDS-PAGE polypeptide profile. The electrophoresis profile of the culture medium (Fig 3a) greatly resembled that of the chicken serum (Fig 3b) as (a)

DISCUSSION Primary chicken hepatocytes cultured by our method maintained liver-specific functions including protein secretion, glyconeogenesis, and biotransformation, as suggested from two-dimensi0nal electrophoresis, enzyme activities of G6Pase and GST, P-450 content and albumin secretion. The (b)

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FIG 3: Two-dimensional gel electrophoresis of chicken serum and conditioned medium of chicken hepatocytes. (a) Five-thousand-fold diluted normal chicken serum. (b) Ten-fold concentrated conditioned medium of chick primary cultured hepatocytes at two days after isolation and culture. Isoelectric point (PI) and apparant molecular weight of proteins are shown. Arrows show the proteins with the same F'l and apparent molecular weight

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well-Preserved cell organelles ultrastructurely substantiated the maintenance of hepatic functions in these cells. Under the serum-free condition, the chicken hepatocytes displayed monolayer morphology on the positively charged surface culture dish. The morphology was quite distinct from rat hepatocytes, which produced multicellular spheroids, under the same culture condition. The difference may be attributed to the diversity in cytoskeleton protein and/or extracellular matrix structures. Rat multicellular spheroids maintained liver-specific functions, such as albumin production, more readily than the rat monolayer culture (Koide et al 1989). By another method, rat hepatocytes can form small spheroids through recognition and binding properties of asialo-glycoprotein receptor on the cytoplasmic membrane (Kobayashi and Akaike 1986). Although chicken hepatocytes could also form small spheroids by this receptor-ligand interaction, they did not secrete much more albumin than the monolayer culture (Yamanaka et al, unpublished data). The patterns of enzyme expressions also varied between the two species. The amount of cellular cytochrome P-450 appeared to increase throughout the culture period in chicken primary hepatocytes, and the level was equal to or higher than those in vivo (Kobayashi et al 1993), while in rat primary hepatocytes, the P-450 level decreased in the early stages (Utesch et al 1991). However, to confirm whether various P-450 isoforms are widely expressed should be determined in cultured chicken hepatocytes. In contrast to P-450, the activity of GST among chicken primary hepatocytes rapidly decreased to one quarter of that in the rat (Gebhardt et al 1990). In the rat system, GST activity in vitro is lower than in vivo, but it is induced by sodium phenobarbital and 3-methylcholanthrene by supplementation in the culture medium (Gebhardt et al 1990). It may be that chicken hepatocytes in vitro retain the ability of drug metabolism by GST. Serum supplementation inhibits the survival of primary cultured hepatocytes by inducing the strong growth of contaminated fibroblasts. Elimination of this problem is the major reason why a serum-free culture is needed. In a culture method of chicken primary hepatocytes previously described by Fraslin et al (1992), supplementation of serum in the culture is needed to adhere the cells onto the culture dish as a first step. Cytokines contained in the serum may immediately induce various signal transductions such as an acute phase protein synthesis in the hepatocytes. The serum

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proteins may also disturb the estimation of proteins secreted by hepatocytes. Thus, our completely serum-free primary culture system is more suitable to study hepatic functions of metabolism and biotransformation. ACKNOWLEDGEMENTS The authors thank Dr. W. K. Sung from the Center for Reproductive Sciences, International Institute for the Study of Human Reproduction, Columbia University, for reading the manuscript and for his valuable comments.

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