In Vitro Cell. Dev. Biol.~Animal 42:255 262, September and October 2006 �9 2006 Society for In Vitro Biology 1071-2690/06 $18.00+0.00

ISOLATION AND PRIMARY CULTURE OF NECTURUS MACULOSUS (AMPHIBIA: URODELA) HEPATOCYTES

PETRA-MAJA PRELOV~EK, 1 UR~KA BATISTA, AND BORIS BULOG

Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia (P. M. P., B. B.), Institute of Biophysics, Faculty of Medicine, University of Ljubljaaa, Lipiceva 2, 1000 Ljubljana, Slovenia (U. B.)

(Received 28 March 2006; accepted 26 April 2006)

SUMMARY

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochenlical analyses. A typical perfusion yielded 1.7 • 10 ~ viable hepatocytes per gram body weight with an average viability of 86 + 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 ~ C in Leibovitz L-15 medium supplemented with 5% FCS. The uhrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autopha- gosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

Key words: liver; isolated hepatocytes; uhrastructure; bioassay model system; tyrosine aminotransferase induction.

INTRODUCTION

Primary cultures of hepatocytes are a well-established system for the study of many aspects of hepatic biology. Furthermore, isolated hepatoeytes are used for pharmacological and toxicological studies, as therapeutic tools (Ben7 and Edwards, 2000), and are a useful model for comparative interspecies studies. Among the poikilo- therlns, fish received the most attention. Many articles have been published about the isolation and cultivation of fish hepatocytes (for reviews see Segner, 1998; Braunbeck and Segner, 2000). Little work, however, has been directed toward isolation and characterization of amphibian hepatoeytes in primary culture.

Chiakulas et al. (1965) described the behavior of urodele liver cells in vitro after explantation of the liver tissue. This was before Berry and Friend (1969) first described the isolation of rat hepa- tocytes by collagenase perfusion. Later; isolated amphibian hepa- tocytes, mostly anuran, have been used in several studies of am- phibian hepatic functions (Stanchfield and Yager, 1978, 1979, 1980; Janssens et al., 1986; Ade et al., 1995) and in environmental toxicology (Kloas et al., 1999; Rouhani Rankouhi et al., 2005). In the majority of these studies short-term cell suspensions or primary

1 To whom con'espondence should be addl~ssed at Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia, EU. E-mail: maja.prelovsek@bf.uni-lj.si

cultures were used without further investigation of how different culture conditions (e.g., culture medium, temperature of incubation, and serum concentration) may have affected the viability of the ceils. Surprisingly, there are no systematic investigations on the presmvation of arnphibian hepatocyte structure in primary culture.

There is a serious concern about the worldwide decline of ant- phibian populations. Since environmental pollution is one of the factors responsible for the disappearance of amphibians, cultured amphibian hepatoeytes could serve as a model in the field of en- vironmental toxicology. In order to evaluate their suitability for physiological and ecotoxicological studies, hepatoeytes were isolat- ed from the common mudpuppy (Necturus maculosus, Amphibia: Urodela). N. maculosus was chosen because of its large body size and commercial accessibility. Due to the large body size, perfusion is easier to perform, and a large number of ceils are isolated. The purpose of the present study was to establish a primm~ culture of N. maculosus hepatocytes and to examine the influence of different culture conditions on hepatocyte viability. We also described he- patocyte ultrastructural and functional characteristics during cul- turing. The functional integrity of hepatocytes was estimated by tyrosine aminotransferase induction with hydrocortisone.

MATERIALS AND METHODS

Animals. Common mudpuppies (N. maculosns) of 100-200 g body weight were supplied by the Carolina Biological Supply Company (Burlington, NC).

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2 5 6 PRELOV~EK ET AL.

The animals were kept in aquaria at 8--10 ~ C. They were fed with trout fry (Oneorhynchus mykiss) or Gammarus sp. once a week.

Chemicals. Antibiotics, culture media, and insulin were obtained from In- vitrogen (Paisley, U.K.), hydroeortisone and other biochemieals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Buffers. The following buffers were used for isolation of hepatocytes. (A) Buffer 1; 110 mM NaC1, 5.40 mM KC1, 0.34 lnM Na2HPO4, 0.44 mM KH2PO4, 0.05 mM NaHCO3, 15 mM HEPES, 5 mM ethylenediamine-tetra- acetic acid (EDTA), 2500 U/100 ml heparin, 100 U/ml penicillin, 100 I-~g/ ml streptomycin. (B) Buffer 2; 110 n~/ NaC1, 5.40 mM KC1, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.05 mM NaHCQ, 0.80 mM MgSOr 1.86 mM CaClz.2H20 , 15 n~/HEPES, 100 U/ml penicillin, 100 txg/ml strep- tomycin, 25 mg/ml collagenase. All media were autoclaved. CaC12-2H20, hep- arin, antibiotics, and collagenase were added after sterilization.

Hepatocyte isolation. The animals were anesthetized with a 0.03% solution of MS-222 (ethyl 3-aminobenzoate methane sulfonic acid salt). They were weighed, superficially disinfected with 70% ethanol, and transferred to a sterile dissection tray. Hepatocytes were isolated by a two-step collagenase perfusion (Berry and Friend, 1969) using a modified procedure described for Xenopus laevis (Ade et at., 1995). The body cavity was opened and the liver was cannulated with a 0.5-mm needle fixed to the portal vein. If the gall bladder was full of bile, the bile was removed by aspiration into a sterile syringe to expose the portal vein. The bulbus arteriosus was cut to allow free blood and buffer release. The liver was cleared of blood with Ca2+-and Mg a+- free buffer 1 (pH 7.5, 4 ~ C, peffusion rate 1.5 ml/min). A tulal 30-40 ml of buffer 1 was used. After 20-30 min, perfusion was switched to buffer 2 (pH 7.5, 4 ~ C, perfusion rate 1.5 ml/min) containing 0.25 mg/ml collagenase. Perfusion was stopped when the liver tissue became soft. The liver was re- moved from the animal, transferred to cold Leibovitz L-15 medium (L-15) supplemented with penicillin (100 U/ml) and streptomycin (100 Ixg/ml), and cut into small pieces. The resulting cell suspension was repeatedly pipetted and filtered through a sieve into a sterile beaker to remove cell aggregates and connective tissue remnants. Dispersed cells were transferred to several 15-ml sterile centrifuge tubes and washed by settling. After 5 minutes at 8 ~ C hepatocytes sedimented and the cell pellet was resuspended in fresh L- 15 supplemented with antibiotics twice more. Cold L-15 media was added to bring the volume to 15 ml. The final cell pellet was resuspended in a final culture medium. Cell density was determined in a Neubauer hemocytometer (Brand, Wertheim, Germany). Cell viability was estimated by exclusion of 0.4% try'pan blue.

Culture conditions. The hepatocyte suspensions (5 ml) were seeded into 25-era 2 gas-tight tissue culture flasks (Techno Plastic Products AG, Trasa- dingen, Switzerland). Cell density was adjusted to 0.9 • 10 '~ to 1.1 • 1@ cells/ml. Cells were incubated at 8 ~ or 20 ~ C without additional OJCO~ gas- sing for up to 14 d. Two different media were used: Eagle's Minimum Es- sential Medium with Hanks' salts (EMEM), and L-15 containing 15 mM HEPES. All media were supplemented with penicillin (100 U/ml), strepto- mycin (100 txg/ml), and 5% or 10% FCS. The media were changed every second day.

Cell viability. Viability was determined by cell ability to exclude trypan blue. Cells were sampled immediately after isolation and after 1, 2, 3, 4, 6, 8, 10, and 14 d of culture.

Cell morphology. Structural characteristics of hepatocytes immediately af- ter isolation and after 1, 2, 3, 4, 6, 8, 10, and 14 d of culture were examined by light and transmission electron microscopy. Culture medium was carefully removed by aspiration and cells were fixed in the culture flasks by adding ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). After rinsing in cacodylate buffer, cells were transferred to centrifuge tubes and postfixed for 30 rain at 4 ~ C with 1% osmium ferrocyanide (1:1 mixture of 2% aqueous osmium tetroxide and 3% K4[Fe(CN)6]) and once more rinsed in 0.1 M cacodylate buffer. Cells sedimented by settling after each step, and the supernatants were removed by aspiration. After rinsing, cells were sus- pended in 2% liquid agar. After solidification, small pieces of agar-cell mix- ture were cut, dehydrated in a graded series of ethanol solutions, aod em- bedded in Spurr's medium (SPI Supplies, West Chester PA). Ultrathin sec- tions were stained with 3% uranyl acetate and 4% lead citrate. Observations were carried out using a Phillips CM 100 transmission electron microscope and documented by a Gatan Bioscan Camera using Digital Mierograph soft- ware (Gatan, Pleasanton, CA). The same software was used for all measure- ments.

For fixation of control liver in vivo, a small piece of liver tissue was re- moved before perfusion and processed as described above.

Glycogen content. Cells were stored at - 8 0 ~ C. After thawing, the cells were homogenized in 1 ml of 100 mM potassium phosphate buffer, pH 7.3, and centrifuged (1800 • g, 10 min, 4 ~ C). Aliquots of the supernatant (140- 500 Ixl) were analysed for glycogen content using anthrone reagent (Seifter et al., 1950). Glycogen content was expressed in milligrams per milligrams of protein.

Protein content. Protein content was measured in the supernatant using BCA Protein Assay reagents (Pierce, Rockford, IL). A standard curve was generated using bovine serum albumin.

Tyrosine aminotransferase inducibility. Tyrosine aminotransferase (TAT; EC 2.6.1.5) was induced with hydrocortisone. Cultures to be analysed were fed with L-15 containing 5% FCS, insulin (10 p~g/ml), and hydrocortisone (10 _5 M in ethanol) or the control medium, L-15 containing an equal amount of ethanol, 5% FCS, and insulin (10 Ixg/ml). After 16 h exposure, the cells were collected and stored at - 8 0 ~ C. Upon thawing, the cells were homog- enized in 1 ml of 100 mM potassium phosphate buffer, pH 7.3, and centri- fuged (30,000 • g, 30 rain, 0-4 ~ C). The supernatant was then analyzed for TAT activity as described by Diamondstone (1966). The incubation mixture contained 2.6 ml of 19.2 txmol L-tyrosine, 0.1 ml of 1.2 mM pyridoxal-5'- phosphate (both dissolved in 0.2 M potassium phosphate buffer, pH 7.3), 0.2 ml of 60 mM diethyldithioearbamic acid (dissolved in water), and 0.2 ml of the superuatant. Reaction was initiated by the addition of 0.1 ml of 0.3 M c~-ketoglutarate in 0.2 M potassium phosphate buffer, pH 7.3, run for 30 min at 25 ~ C, and stopped by 0.2 ml 10 N NaOH. The absorbance at 331 nm was measured after 30 min at room temperature against a zero-time con- trol prepared by adding the NaOH just before a-ketoglutarate. The TAT ac- tivity was expressed in milliunts (nmol ofp-hydroxyphenylpyruvate performed per minute) per gram of protein. Protein content was measured in the su- pernatant as described above.

RESULTS

Uhrastructure of N. macu losus hepatocytes in vivo. The liver of

N. maculosus consis ts of an unlobula ted mass of hepatocytes, ar-

ranged in plates, which are mostly two cells thick. Beside hepato-

cytes, hepat ic p igment cells, cells of granulocytopoietic t issue, en-

dothelial cells, and stellate (Ito) cells are present . Intercel lular bile

canaliculi lie between two to four adjacent hepatocytes. Hepatocytes

have distinct polarity. The apical portion of the cell with numerous

microvilli faces the bile canaliculi , at the lateral surfaces tight junc-

tions and dict iosomes connect neighboring cells, while the basal

pole faces the endothel ial l ining of the s inusoids.

Hepatocytes are relatively large (average size 46 by 30 Ixm) and

contain one nuc leus (Fig. 1A). The nuc leus is centrally or slightly

basal ly located, is oval or lightly ovoid in shape, and contains het-

erochromatin in several isolated pa tches present benea th the nu-

clear envelope and scattered over the nuc leoplasm (Fig. 1A). The

main cytoplasmic inclusions are rough and smooth endoplasmic re-

t iculum (RER and SER), mitochondria, peroxisomes, lysosomes,

Golgi complexes, glycogen, and lipid droplets. Lipid droplets are

mostly located at the basal and lateral portion of the cell (Fig. 1A);

they vary between the cells in shape and electron density. The nu-

merous mitochondria are oval, ovoid, or rod shaped; their matrix is

med ium electron dense and contains few lamel lar cristae (Fig. 1/3).

The amount of RER and SER varies among individuals. RER is

present as individual cis ternae or arranged in parallel s tacks around

the nucleus , at the apical portion of the cell and parallel to the

lateral p l a sma lemma (Fig. 1A and B). SER is only poorly developed;

it is present as an irregular network of tubular or vesicular profiles

among the glycogen (Fig. 1B). Glycogen rosettes are d ispersed

throughout the cytoplasm, with some aggregations also present (Fig.

1A and B). The Golgi complex, numerous vesicles, lysosomes, and

autophagic vacuoles of varying size and content are present between

the nuc leus and bile canal iculus (not shown).

NECTURUS HEPATOCYTE PRIMARY CULTURE 257

100

80

60 > ,

..Q 40

20

FIG. 2.

- - L - 1 5

- - - EMEM

0 2 4 6 8 10 12 14

Culture durat ion (days)

Viability of isolated N. rnaculosus hepatocytes at 8 ~ C in different culture media (Leibovitz L-15 medium [L-15] and Eagle's Minimum Essential Medium [EMEM]) supplemented with 5% fetal calf serum (means + SE; N = 4).

1 oo

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FIG. 3.

-~- 0% FCS

5% FCS . . . . . . 10% FCS

2 4 6 8 10 12 14

Culture duration (days)

Viability of isolated N. maculosus hepatocytes at 8 ~ C in Leibovitz L-15 medium supplemented with different concentrations of fetal calf serum (FCS); (means -+ SE; N = 3).

FIG. 1. Transmission electron micrographs of a N. maculosus hepatocyte in vivo. (A) Large nucleus (N), abundant glycogen deposits (G), lipid droplets (L), and rough endoplasmic reticulum (RER) are characteristic features. The arrow indicates bile canaliculus. (B) Perinuclear part of the cytoplasm with mitochondria (M), RER, SER (bent arrow), and glycogen deposits (--->).

Isolation. The cell suspension obtained after two-step collagenase peffusion contained hepatocytes and variable nmnbers of damaged cells, nonparenchimal cells, cell aggregates, and subcellular debris. After washing by settling, the final suspension of hepatocytes with an average viability of 86 -4- 5% (rain. 81%, max. 93%) was ob- tained. In 2000 randomly counted cells from five different isola- tions, hepatocytes represented 92% of the isolated cells, with the remaining 8% consisting mainly of hepatic pigment ceils; erythro- cytes and small, probably endothelial and granulocytopoietic cells

were only very rarely observed. The yield of isolated hepatocytes varied between preparations. On average 1.7 • 105 viable hepato- eytes per gram of body weight were obtained from six different isolations (rain. 1.3 X 105, max. 2.3 X 1@). Viability in fi'eshly isolated suspensions and cell yield per gram of body weight were the highest when 0.15-0.25 ml/g body weight of collagenase-con- raining buffer was used for peffusion.

Cell adhesion was re17 weak. Coating flasks with collagen or fibronectin and increasing the concentration of FCS in the medium did not improve attachment. Since most hepatocytes remained in suspension, their characteristics are described.

Necturus maculosus hepatocytes in vitro. The two tested culture media showed similar viability. A slightly higher viability was ob- served in L-15 medium at the beginning of the culture period (Fig. 2). The addition of FCS to the incubation medium improved the viability of hepatocytes during the first days in culture (Fig. 3). Furthermore, whereas isolated hepatocytes incubated in serum-con- taining medium usually formed aggregates, hepatocyte aggregation in serum-free medium was weak. Viability was temperature depen- dent. The effect of temperature on hepatocyte viability is shown in Fig. 4. Cells maintained at 20 ~ C showed a rapid decline in viability after day 4 and fell to 0% after 14 d. In addition, cells incubated at 20 ~ C developed blebbing of the plasma membrane and formed

258 PRELOV~EK ET AL.

100

80

60

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0 t i - - i

0 4 6 8 10

Culture duration (days)

• J_ ' ~

\ \ \

\ \

�9 8 o c \ \

_ ~ _ 2 0 o c \ \ \

\

2 12 14

FIG. 4. Viability of isolated N. maculosus hepatocytes at different incu- bation temperatures in Leibovitz L-15 medium supplemented with 5% fetal calf serum (ineans _+ SE; N = 3).

smaller aggregates compared to cells incubated at lower tempera- tures.

For further uhrastructural and biochemical analyses, cells were incubated at 8 ~ C in L-15 medium supplemented with 5% FCS. The viability under those conditions was 86 -+ 5% at the beginning of the mdture period and dropped to 78 -+ 13% after 14 d (Fig. 2). Dead cells were removed during the medium change and the total number of viable hepatocytes declined during culturing. On average (8.1 -4- 0.5) • 104 viable cells/inl were seeded after isolation. The number of viable cells dropped to (5.6 -+ 0.4) • 104 cells/ml after 4 d. After 14 d only (2.2 + 0.4) X 104 viable cells/ml were present.

Hepatocytes in the freshly isolated suspension were present as individual cells; occasionally small groups of two to three cells, which had not completely dissociated, were observed. After isola- tion hepatocytes lost their typical polygonal configuration and as- sumed a spherical shape (Fig. 5A). At uhrastructural level, hepa- tocyte polarity, evident by microvilli-lined apical plasmalemma, a Golgi complex, and numerous small spherical vesicles (presumably lysosomes) in the apical cytoplasin, were retained in most cells (Fig. 5A). Peroxisomes, mitochondria, and the arrangement of RER did not differ obviously from hepatocytes in vivo (Fig. 5B). Compared to hepatocytes in vivo, the amount of heterochromatin increased, and the amount of hepatoeyte glycogen stores decreased.

Since the viability of isolated hepatocytes was best at 8 ~ C in L-15 medium supplemented with 5% FCS, the following description is of hepatocytes cultured under those conditions. Within 24 h in primary culture hepatocytes formed aggregates of a few (2-10) vi- able cells. With extension of the culture period, aggregates in- creased in size (Fig. 6). The majority of hepatocytes retained struc- tural characteristics similar to in vivo up to 4 d (Figs. 7 and 8). Loss of cellular polarity, fragmentation of RER, formation of new autophagosomes, and exhaustion of cellular glycogen deposits were observed with increased time in culture (Fig. 9). With extension of the culture period the number of structurally changed cells in- creased.

Biochemical analyses confirmed a significant reduction of gly- cogen content during the first 3 d in culture. Hepatocytes were able to partially restore their glycogen levels between days 3 and 4, and maintained a steady level of glycogen reserves up to day 8 (Fig. 10).

FIG. 5. Transmission electron inicrographs of a N. maculosus hepatocyte immediately after isolation. (A) Hepatocyte polarity is well preserved. The arrow indicates the apical part of the cell, where numerous microvilli, pre- viously lining bile canaliculi, are visible. Nucleus (N) with condensed chro- matin; lipid droplets (L) in basal and lateral part of the cell. (B) Perinuelear part of the cell with rough endoplasmic reticulmn (RER), lipid droplets (L), and mitochondria (M). Glycogen deposits (G) are reduced compared to the cells in vivo.

Functional integrity of hepatocytes was estimated by TAT induc- tion with 10 tam hydrocortisone. The highest induction was mea- sured after the first (175% of the control value) and second day (159% of the control value), and dropped to 110% of the control value by day 10 (Fig. 11). The control value of TAT activity during the culture period ranged between 35.5 and 21.5 mU/g protein.

NECTURUSHEPATOCYTE PRIMARY CULTURE 259

Fro. 6. Aggregate of N. maculosus hepatoeytes alter 4 d in primm~y cul- ture. Light micrograph.

DISCUSSION

The isolation of N. maculosus hepatocytes described in this paper is a modification of the well-established two-step perfusion method. Few attempts to isolate and culture amphibian hepatocytes have been made. Ade et. al. (1995) achieved a viability of over 90% in X. laevis, Stanchfield and Yager (1978) reported a viability around 96% in Rana catesbeiana, and the viability in N. maculosus he- patocytes was 86 + 5% (this study). The isolation procedure de- scribed in this study gave an average yield of 1.7 • 10 s viable hepatocytes per gram of body weight (rain. 1.3 • l0 s, max. 2.3 • lOS). Cell yield was lower than in fish isolations (Klaunig et al., 1985; Braunbeck and Segner, 2000) and could be at least partly explained by the very large size of N. maculosus hepatocytes. It is also known that cell yields from fish liver perfusion vary with spe- cies, strain, sex, nutritional stratus, and age of the donor fish (Braunbeck and Segner, 2000). Some of these factors (e.g., nutri- tional status and sex) probably also contribute to variability in cell yields in N. maculosus.

Addition of FCS to the culture medium improved N. maculosus

hepatocyte viability in primary culture. There are no reports about the effect of FCS on hepatocyte viability in vitro in other amphib- ians. In the case of rainbow trout (O. mykiss) hepatocytes in vitro, serum supplementation both improved (Klaunig et al., 1985) and reduced (Braunbeck and Storch, 1992) survival. Serum in primary

FIG. 7. Transmission electron micrographs of a N. maculosus hepatocyte 1 d after isolation. (A) Cell is electron lucent due to the low amount of glycogen present. Other uhrastructural features are similar to the cells in vivo. Large euchromatic nuclens (N); numerous lipid droplets (L). (B) Peri- nuclear part of the cell with well-preselved rough endoplasmic retieulum (RER) and mitoehondria (M).

culture of mammalian hepatocytes improves cell attachment, sur- vival, and morphology (LeCluyse et al., 1996), but in general, fac- tors in serum do not aid maintenance of differentiated function (Ber- ry et al., 1991). The viability of IV. maculosus hepatoeytes in serum- free medium declined during the first 2 d, and then partially re- covered by day 4. N. rnaculosus hepatoeytes retained higher viability in serum-containing medium; FCS also promoted hepato- eytes aggregation. It seems that ceils needed longer time to recover

260 PRELOV~EK ET AL.

FIG. 8. Transmission electron micrographs of N. maculosus hepatocytes 4 d after isolation. (A) Cell is electron lucent due to the huge reduction in glycogen content. Nucleus (N); lipid droplets (L); parallel stacks of rough endoplasmic reticulum in perinuclear position (--~). (B) Perinuclear part of the cell with rough endoplasmic reticulum (RER).

from the isolation stress in serum-free medium. It is known that cell-cell interactions in vitro contribute to the survival of mam- malian and fish hepatocytes (Berry et al., 1991; Segner, 1998). It is likely that the formation of aggregates contributed to quicker recovery and higher viability of N. maculosus hepatocytes in serum- containing medium. Serum may also contribute protease inhibitor(s) that aid cell survival (Berry et al., 1991). Despite beneficial effects it should be considered that mammalian sera, due to their specific lipid composition, may evoke adverse cellular changes in cultured

FIG. 9. Transmission electron microgr~phs of a N. maculosus hepatocyte 14 d after isolation. (A) Majority of hepatocytes are ultrastructurally changed compared to the cells in vivo. Nucleus (N); lipid droplets (L). (B) Avrmagement of rough endoplasmic reticulum is changed. It is in part still mTanged in parallel stacks (RER), but fragmentation of RER (--~) was also observed. (C) Autophagosome containing mitochondria (M), RER, and lipid droplet (L).

fish cells such as altered membrane composition or lipid accumu- lation (Haschemeyer and Mathews, t983; Hayashi and Ooshiro, 1986). No such studies have been performed in amphibian hepa- tocytes in vitro.

While anuran hepatocytes attached to commercially available culture dishes (Stanchfield and Yage~, 1978, 1979, 1980; Ade et al., 1995; Kloas et al., 1999; Rouhani Rankouhi et al., 2005), N. macuIosus hepatocytes did not. Coating flasks with collagen or fi-

NECTURUS HEPATOCYTE PRIMARY CULTURE 261

v

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Culture duration (days)

FIG. 10. Changes in the amount of cellular glycogen during primary cul- ture of N. maculosus hepatocytes. The amount of glycogen is expressed as the percentage of the initial value measured in hepatocytes immediately after isolation (means +- SE; N = 4).

FIG. 11. Time course of lyrosine amino/ransferase induction in primary culture of N. maculosus hepatocytes. The induction is presented as a per- centage of the control value, which is 100%. Results are expressed as the mean from two separate experiments performed in duplicate.

bronectin and increasing the concentration of FCS in the medium did not improve attachment. Anaehment of fish hepatocytes to cul- ture surfaces has also been a problem. Generally it is held that monolayer cultures of fish hepatocytes last signifcantly longer than hepatocytes held in suspension (Braunbeck and Segner, 2000). The morphological and functional characteristics of N. maculosus he- patocytes in primary culture would perhaps improve in monolayer culture. On the other hand, N. maculosus hepatoeytes in suspension euhure showed a high affinity for each other, and formation of large aggregates in suspension could be a useful nmdel for studying cell- to-cell interactions in vitro.

Despite the high and relatively stable viability during all 14 d in primary euhure, uhrastmetural and functional characteristics changed during the culture period. The ultrastrueture of viable he- patoeytes in vitro was similar to hepatocyte ultrastrueture in vivo up to 4 d. With extension of the culture period the number of struc- turally changed cells increased. Changes in uhrastructural charac- teristics were not accompanied by a drastic increase of necrotic cells. For comparison, O. rnykiss hepatocytes in vitro retain ultra- structural characteristics similar to hepatocytes in vivo up to 5 d. From day 5 in culture, progressive degradation of cellular ultra-

structure takes place, together with an increase of necrotic ceils (Braunbeck and Storch, 1992).

Concomitant analyses of TAT induction also demonstrated the highest induction during the first 2 d in primary culture of N. ma- culosus hepatocytes. Induction of TAT is a commonly used indicator of hepatocyte differentiation and is widely used in studies of mam- malian cell cultures (Gurr and Potter, 1980; Malan-Shibley and Iype, 1981; Ikeda et at., 1998); it was also used with fish (Lipsky et at., 1986; R'~bergh et al., 1995) and amphibian (Stanchfield and Yager, 1978) hepatocytes in primary cuhure. The level of TAT in- duction in hepatocytes isolated from N. maculosus is comparable to that measured in some other poikilotherms. TAT inductions in he- patocytes isolated from R. catasbeiana were 190% on day 2, and 160% on day 6 (Stanchfield and Yagm, 1978). In primary cuhure of O. mykiss hepatocytes the level of TAT induction ranged from 118% to 200% (Lipsky et al., 1986; R~bergh et al., 1995). The inducibility of TAT in hepatocytes isolated from poikilotherms is of a lower magnitude than that observed in rat hepatocytes in which a 400% to 600% induction has been observed (Kreamer et at., 1986; Kido et al., 1987; Juillerant et at., 1997). It is unknown whether this is due to basic physiological differences between the organisms. It should also be considered that hepatocytes isolated from poikilotherms are incubated at lower temperatures (N. macu- losus at 8 ~ C, O. mykiss at 15 ~ C, R. catasbeiana at 25 ~ C). It appears that TAT induction in poikilotherms is a less sensitive indicator of differentiation compared to mammals, and other more sensitive in- dicators should be established for routine screening of differentia- tion in poikilotherms, since this was also suggested for fish hepa- tocytes in primary culture (R~bergh et al., 1995).

Chemical analysis and electron microscopy of N. maculosus he- patocytes in culture demonstrated huge glycogen loss during the culture period. Hepatocytic glycogen reduction may be regarded as a response to stress due to isolation and has also been reported in 0. mykiss hepatocytes in vitro (Braunbeck and Segner, 2000). N. maculosus hepatocytes in vitro were, however, capable of glycogen synthesis, as was evident by partial restoration of glycogen stores between days 3 and 4 in culture. For X. laevis hepatocytes in vitro, Ade et al, (1995) reported a variable glycogen content during the first d in primary culture, and a significant reduction after 48 and 72 h in EMEM and L-15 medium. Only the addition of a high amount of glucose (3400 rag/l) to the medium elevated the glycogen level and compensated for glycogen loss. Studies in rats also found hepatocytes in suspension to be in negative glycogen balance up to 20 mM glucose (Agius et at., 2000).

Ultrastructural and biochemical analyses of N. maculosus hepa- tocytes in primary culture indicate the need for further improvement of culture conditions. Nevertheless, hepatocytes cultured under the described conditions up to 4 d in primary culture could be rec- ommended for physiological and ecotoxicological studies.

ACKNOWLEDGMENTS

The help, motivation, and cooperation t}'om Dr. L. Bizjak Mall, T. Herzog, and K. Zde~ar are gratefully acknowledged. This research was supported by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia, grant 3311-02-8317436.

REFERENCES

Ade, T.; Segner, H.; Hanke, Vr Hormonal response of primary hepatocytes of the clawed mad, Xenopus laev2s. Exp. Clin. Endocrinol. 103:21-27; 1995.

262 PRELOVgEK ET AL.

Agius, L.; Alam, N.; Aiston, S. Short-term regulation by insulin of glucose metabolism in isolated and cultured hepatocytes. In: BetTy, M. N.; Edwards, A. M., ed. The hepatocyte review. Dordrecht: Klnwer Ac- ademic. Publishers; 2000:317-341. M. N.; Edwards, A. M. The hepatocyte review. Dord,~cht: Kluwer Academic Publishers; 2000.

M. N.; Edwards, A. M.; Barritt, G. J. Isolated hepatocytes preparation, properties and applications. In: Burdon, R. H.; van Knippenberg, P. H., ed. Laboratory techniques in biochemistry and molecular biology. Vol. 21. Amsterdam: Elsevier; 1991:237-354.

Ben'y, M. N.; Friend, D. S. High yield preparation of isolated rat liver pa- renchymal cells. J. Cell Biol. 43:506-520; 1969.

Braunbeck, T.; Segner, H. Isolation and cultivation of teleost hepatocytes. In: Berry, M. N.; Edwards, A. M., ed. The hepatocyte review. Dordrecht: Kluwer Academic Publishers; 2000:49 71.

Braunbeck, T.; Storch, V. Senescence of hepatocytes isolated from rainbow trout (Oncorhy~whus mykiss) in primary culture. Protoplasma 170:138-159; 1992.

Chiakulas, J. J.; Seheving, L. E.; %ai, T. H. The behavior of urodele liver cells in vitro. Acta Hepato-Splenol. 12:257 268; 1965.

Diamondstone, T. I. Assay for tyrosine transaminase activity by conversion of p-hydroxyphenylpyruvate to p-hydroxybenzaldehyde. Anal. Bio- chem. 16:395-401; 1966.

Gum J. A.; Potter, V. R. Independent induction of tyrosine aminotransferase activity by dexamethasone and glucagons in isolated rat liver paren- chymal cells in suspension and in monolayer culture in serum-free media. Exp. Cell Res. 126:237 248; 1980.

Hasehemeyei, A. E. V.; Mathews, R. W. Temperature dependency of protein synthesis in isolated hepatocytes of Antarctic fish. Physiol. Zool. 56:77~7; 1983.

Hayashi, S.; Ooshiro, Z. Primary culture of the eel hepatocytes in the serum- free medium. Bull. Jap. Soc. Sei. Fish 52:1641-1651; 1986.

Ikeda, T.; Sawada, N.; Satoh, M.; Mori, M. Induction of tyrosine aminotrans- ferase of primary cultured rat hepatocytes depends on the organiza- tion of mierotubules. J. Cell. Physiol. 175:41-49; 1998.

Janssens, R A.; Kleineke, J.; Caine, A. G. Calcium-independent stimulation of glycogenolysis by arginine vasotoein and eateholamines in liver of the axolotl (Ambys~oma mexicanum) in vitro. J. Endocrinol. 109:75-84; 1986.

Juillerant, M.; Marceau, N.; Coeytaux, S.; Sierra, E; Kolodziejczyk, E.; Gui- goz, Y. Expression of organ-specific structures and functions in long- term cultures of aggregates from adult rat liver cells. Toxicol. in Vitro 11:57~59; 1997.

BetTy,

BeiTy,

Kido, H.; Fukusen, N.; Katunuma, N. Epidermal growth factor as a new regulator of induction of tyrosine aminotrasferase and tryptophan ox- ygenase by glucocorticoids. FEBS Lett. 223:223-226; 1987.

Klaunig, J.E.; Ruch, R.J.; Goldblatt, P.J. Trout hepatocyte culture: isolation and primary culture. In Vitro Cell. Dev. Biol. 21A:221-228; 1985.

Kloas, W.; Lutz, I.; Einspanier, R. Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of enviromnental ehmnicals in v#ro and in vivo. Sci. Total Environ. 225:59-68; 1999.

Kreamer, B. L.; Staecker, J. L.; Sawada, N.; Sattler, G. L.; Hsia, M. T. S.; Pitot, H. C. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated *'at hepatocyte prepara- tions. In Vitro Cell. Dev. Biol. 22A:201-211; 1986.

LeCluyse, E. L.; Bullock, P. L.; Parkinson, A. Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv. Drug Deliv. Rev. 22:133-186; 1996.

Lipsky, M. M.; Sheridan, T. R.; Bennett, R. 04 May, E. B. Comparison of trout hepatocytes on different substrates. In Vitro Cell. Dev. Biol. 22A:360-362; 1986.

Malan-Shibley, L.; Iype P. The influence of culture conditions on cell mor- phology and tyrosine aminotransferase levels in rat liver epithelial cell lines. Exp. Cell Res. 131:363-371; 1981.

Rgtbergh, C. M.; Kane, A. S.; Reimschuessel, R.; Lipsky, M. M. Viability and induction of tyrosine aminotransferase in rainbow trout bepatocytes cultured on laminin and polylysine in a serum-free medium. Methods Cell Sci. 17:207-215; 1995.

Rouhani Rankouhi, T; Sanderson, J. T.; van Holsteijn, L; van Kooten, P.; Bosveld, A. T. C.; van der Berg, M. Effects of environmental and natural estrogens on vitellogenin production in hepatocytes of the brown frog (Rana temporaria). Aquat. Toxicol. 71:97-101; 2005.

Segner, H. Isolation and primary culture of teleost hepatocytes. Comp. Bio- chem. Physiol. 120A:71-81; 1998.

Seifter, S.; Dayton, S.; Novic, B.; Muntwyler, E. The estimation of glycogen with anthrone reagent. Arch. Biochem. 25:191-200; 1950.

Stanchfield, J. E.; Yager, J. D. An estrogen responsive primary amphibian liver cell culture system. Exp. Cell Res. 116:239-252; 1978.

Stanchfield, J. E.; Yager, J. D. Insulin effects on protein synthesis and se- cretion in primary cuhures of amphibian hepatoeytes. J. Celt. Physiol. 100:279-290; 1979.

Stanchfield, J. E.; Yager, J. D. Primary induction of vitellogenin synthesis in monolayer cultures of amphibian hepatoeytes. J. Cell Biol. 84:468-475; 1980.