JOURNAL OF CELLULAR PHYSIOLOGY 147:495-504 (1991)

Amino Acid-Rich Medium (Leibovitz L-15) Enhances and Prolongs Proliferation of Primary Cultured Rat Hepatocytes in the Absence of Serum TOSHlHlRO MITAKA, GERALD 1. SATTLER, AND HENRY C. PITOT* McArdle laboratory for Cancer Research, Department of Oncology, Medical School, University of Wisconsin, Madison, Wisconsin 53706 Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO, and 10 ngiml EGF in a 5% C0,/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEMiFl2 medium in that there were four peaks of 'H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x l o 5 and 6.0 x l o 5 cells per 35-mm dish. Even at a density of 9.0 x 10' cellsidish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleatewith a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEMiFl2 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-I5 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.

Adult liver parenchymal cells have the potential t o proliferate in vivo and in vitro under various conditions (Bucher, 1987). The most dramatic example is the regeneration of liver after a two-thirds partial hepatectomy (Higgins and Anderson, 1931). In partially hepatectomized rats, both the hormonal and the nutritional conditions in the liver are very important for the replication of hepatocytes. In order to study the factors that may stimulate the proliferation of hepatocytes in vitro, man investigators are using primary monolayer cultures ofyrat hepatocytes in serum-free medium. More than 20 hormonal and growth factors have been shown to stimulate the DNA synthesis of primary cultured rat he atocytes (McGowan, 1986; Michalopoulos, 1990).In ad ition, various culture conditions have been shown to modulate the response of hepatocytes to these growth stimuli, e.g., cell density (Nakamura et al., 1983a; Tomomura et al., 1987) and the extracellular matrix used as substratum (Enat et al., 1984; Sawada et al., 1986). Attention has also been given to those components of the medium, such as amino acids, vitamins, and inorganics, that may effect cell replication. In early studies with serum-free medium, a high concentration of pyruvate (Hasegawa and Koga, 1981; McGowan and Bucher, 1983) and lactate (McGowan and Bucher,

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0 1991 WILEY-LISS, INC.

1983) was reported to stimulate the replicative DNA synthesis of rat hepatocytes with or without EGF. That proline is required for DNA synthesis of hepatocytes was also confirmed by several studies (Hase awa et al., 1982; Nakamura et al., 1984; Houck and hfichalopoulos, 1985). Furthermore, a low Ca2+ ion concentration in the medium was recommended for the induction of multiple cell divisions in primary cultured hepatocytes (Hasegawa et al., 1982; Eckl et al., 1987). In addition, we have recently demonstrated that a high concentration of nicotinamide in serum-free medium can induce hepatocytes to progress through multiple cell divisions (Mitaka et al.. 1991). Leibovitz L-15 medium was originally designed for virus production in cell lines cultured in a 100% air incubator (Leibovitz, 1963). In our laboratory, L-15 medium in a 100% air incubator has proven to be quite effective for the maintenance of differentiated Received November 16: 1990; accepted March 8, 1991. "To whom reprint requestskorrespondence should be addressed. T. Mitaka is now at the Cancer Research Institute, Dept. of Pathology, Sapporo Medical College, Chuo-Ku, S-1, W-17, Sapporo 060, Japan.

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MITAKA ET AL.

functions of adult hepatocytes in culture (Michalopoulos et al., 1976; Sirica et al., 1979; Sawada et al., 1987; Staecker et al., 1988), but unsatisfactory in sup orting cell division (Sawada et al., 1987). In the stulies described herein, however, we show that primary cultured adult rat hepatocytes can go through multiple cell divisions in a modified L-15 medium when it contains sodium bicarbonate in a 5% coz/95% air atmosphere. MATERIALS AND METHODS Isolation and culture of rat hepatocytes Male Sprague-Dawley rats (Madison, WI), weighing 200-250 g, were used to isolate hepatocytes by the two-step liver perfusion method of Seglen (1976) with some modification (Mitaka et al., 1991). The isolated cells were purified by Percoll isodensity centrifugation (Kreamer et al., 1986) and their viabilities examined by the trypan blue exclusion test. Hepatocyte preparations exhibiting > 95% viability were used in these experiments. The cells were suspended in L-15 medium with 0.2%bovine serum albumin (BSA; Sigma Chemical, St. Louis), 20 mM HEPES (Research Organics, Cleveland), 5 pgiml (0.08 pM) insulin, 5 pg/ml (0.66 pM) transferrin, 5 ng/ml (0.01 pM) sodium selenite (ITS, Collaborative Research, Lexington, MA), M dexamethasone (Elkins-Sinn, Cherry Hill, NJ), 30 pgiml (0.26 mM) proline, and penicillin-streptomycin (Sigma).The cells were plated in 35mm culture dishes or six-well plates (Corning, Corning, NY), which were coated with 30 pg each of type I collagen (Vitrogen, Collagen Corp., Palo Alto, CA), and the cultures were incubated in an 100% air incubator at 37°C. After 2-3 h, the cultures were changed to either L-15 medium supplemented with 0.2% BSA, 20 mM HEPES, 5 pgiml (0.66 pM) transferrin (Collaborative Research), 1 mg/ml (5.56 mM) galactose, 30 pgiml (0.26 mM) proline, 0.2 pgiml (0.8 pM) CuS04.5H20, 0.5 p /ml (2.2 FM) FeS04.4H20,0.05 Fgiml(O.33 pM) I# nS04, 0.75 Fgiml (2.6 pM) ZnS04.7Hz0,2 ngiml(O.01 pM)NazSe03, 0.5 pgi’ml (0.08 pM) insulin, lop7 M dexamethasone, 20 mM NaHC03, 10 ng/ml EGF and antibiotics, or to DMEMiFl2 (1:l) medium supplemented with 0.2% BSA, 20 mM HEPES, 5 pg/ml transferrin, 1 mgiml galactose, 3 0 ~ g i mproline, l 5 ngiml NazSe03, 0.5 giml insulin, 10- M dexamethasone, 6 mM NaHCdL,, 10 n iml EGF, and antibiotics. The molarity of the supp emented L-15 medium containing the additions indicated above was increased by about 46 mM over that of the unsupplemented L-15. The cultures were then placed in a humidified incubator with a 5% coz/95% air atmosphere at 37°C. These media were replaced every other day. Measurement of DNA content and number of cells To determine changes in the numbers of hepatocytes in situ, the same fields of cultured hepatocytes, identified by needle marks, were photographed each day on a Nikon Diaphot hase-contrast microscope. Three fields per dish at ran om and two dishes per experiment were photographed for 6 days. The number of cells in the photomicrographs were counted. For the measurement of total DNA per culture dish, the cells were rinsed with

B

B

cold, phosphate-buffered saline (PBS) on ice, and then 0.5 ml of DNA buffer (0.1 M NaC1, 0.01 M Tris-HC1, 0.01 M EDTA, pH 7.0) was added. The cells were then scraped from the dish and collected quantitatively. The measurement of DNA was done by the method of Brunk et al. (1979), with calf thymus DNA (Sigma) as a control. Measurement of DNA synthesis and autoradiography For the measurement of DNA synthesis, cultures in six-well plates were treated with 1.2 pCi of 3H-thymidine (DuPont, Boston; 81.9 Ciimmol) for 2 h or 24 h before harvest. The incorporation of 3H-thymidine into DNA was measured as previously described (Mitaka et al., 1991). For autoradiography, cultures in 35mm dishes were treated with 3H-thymidine for 24 h, and the cells were fixed in cold absolute ethanol after three rinses with cold PBS. The dishes were covered with emulsion (Eastman Kodak, Rochester, NY) for 1 week, and the autoradiographs then were developed and counterstained with hematoxylin and eosin. Labeling index To determine labeling indices, immunocytochemical staining for BrdU (Sigma) was used. Fifty pM BrdU was added to the medium for 24 h, and each dish was washed with cold PBS twice. Then, cultures were fixed with cold 70% ethanol. Immunocytochemistry for BrdU was carried out with mouse anti-BrdU antibody (DAKO Co., Santa Barbara, CA) as primary antibody, followed by the ABC method (Vectastain ABC Kit, Vector Laboratory, Burlingame, CA). Labeling indices were calculated by counting the number of stained cells within a 0.5mm-square area of a lenticle in the eyepiece of a Nikon Diaphot phase-contrast microscope (X200 objective).Ten fields per dish (except where mentioned otherwise) were observed, and two or three dishes per experiment were examined. For more precise studies, 100 areas of two dishes (unevenly seeded) were observed at random, and more than 100 areas from the separately seeded dishes at different densities were chosen. More than 1,000 nuclei were counted for each index. Double labeling with BrdU and 3H-thymidine To determine the percentage of hepatocytes traversing more than one round of DNA replication, both immunocytochemical staining for BrdU and 3H-thymidine incorporation into DNA were done as described previously (Mitaka et al., 1991). Combined staining for albumin with 3H-thymidine To show that proliferating hepatocytes in these cultures produced albumin, we carried out both immunocytochemical staining for albumin and 3H-thymidine incorporation into DNA. Twenty-four h before harvest, 2.4 pCi of 3H-thymidinewas added to the medium. The cells were fixed with cold absolute ethanol at 120 h after plating. Immunocytochemical staining was done on the fixed cells with rabbit antirat albumin antibody (United States Biochemical, Cleveland) as a primary

PROLIFERATION OF RAT HEPATOCYTES IN L-15 MEDIUM

antibody, followed by the ABC method. Rabbit serum was used as a negative control. After immunocytochemistry, the dishes were covered with emulsion for 2 days. The autoradiographs were then developed. Western blotting for albumin For examination of the secretion of albumin into the culture medium, western blot analysis was carried out; 0.1 pl of the culture medium was loaded onto and run in a discontinuous 10% polyacrylamide gel containing SDS (Laemmli, 1970) and electroblotted to nitrocellulose (Bowen et al., 1980).Blots were probed as described by Towbin et al. (1979) with antibody directed against rat albumin. The antibody did not cross-react with BSA. The blot was developed by the ABC method with 3,3'-diaminobenzidine as the substrate. Addition of amino acids to DMEM/Fl2 medium Concentrated solutions of essential amino acids (EAA) ( X 50) and nonessential amino acids ( x 100) were purchased from Sigma Chemical. Both concentrated solutions were made according to the DMEM formula. As a test of the effect of these essential amino acids, 1 ml ( ~ 1 . 5 )2, ml ( ~ 2 . 0 )and , 4 ml (X3.0) of the concentrated EAA solution were added into each 100 ml of the modified DMEMlFl2 medium. For nonessential amino acids, 1 ml (~2.01,2 ml ( ~ 3 . 0 1and , 3 ml (X4.0) of the concentrated non-EAA solution were added into each 100 ml of the medium. These media were replaced at 2 h and 48 h after plating; 1.2 FCi of ,H-thymidine was added to the culture media 24 h prior to analysis.

RESULTS Although L-15 medium was originally designed for the culture of viruses in cell lines under a non-bicarbonate buffer system (Leibovitz, 19631, it is now also commonly used in the primar culture of rat hepatocytes for the maintenance o f t eir differentiated functions. The choice of this medium is, in part, due to the high level of amino acids in L-15 compared with other commercially available media. In our laboratory L-15 medium has been used for the maintenance of differentiated hepatic functions (Michalopoulos et al., 1976; Sirica et al., 1979; Sawada et al., 1987; Staecker et al., 19881, but we have not been able to increase DNA synthesis nor mitoses in this medium in a 100% air incubator (Sawada et al., 1987). Recently, we found that primary cultured rat hepatocytes could not only increase their DNA synthesis but also go through cell division in our modified L-15 medium when NaHC03 was added and the cultures were placed in a 5% C02/95% air incubator (Mitaka et al., unpublished observations). As shown in Figure 1, the effect of the dose of NaHC0, on the DNA synthesis of primary cultured hepatocytes indicated that an increase of NaHCO, concentration in the medium gradually enhanced DNA synthesis and that 20 mM NaHCO, was the optimal concentration. Up to 48 h after plating, little incorporation of 3H-thymidine into DNA was seen, but the DNA synthesis of the cells suddenly increased during the next 24 h. This high incorporation continued up to 96 h after platin Even without EGF, a relatively high incorporation of %H-thymidine was seen in the hepato-

H

497

60000

4

I

Control

24-48

48-72

Hours after

72-96

Plating

Fig. 1. Dose response of the effect of sodium bicarbonate on DNA synthesis of primary cultured rat hepatocytes in modified L-15 medium treated with 10 ng/ml EGF. The intervals designate the hours after the initial plating in culture medium containing 1.2 FCi of ,H-thymidine. Control means L-15 medium supplemented with 20 mM NaHCO, but without EGF. Bars indicate ,H-thymidine incorporation of cells treated with various concentrations of sodium bicarbonate. T's above the bars indicate standard deviations of triplicate dishes.

x

c tes during the period 72-96 h after plating. During

t is period without EGF, we found few mitoses in the

cultures. This pattern of DNA synthesis of the hepatocytes cultured in modified L-15 medium was much different from that of the DNA synthesis in cells in DMEMiFl2 medium, which had been previously used in our laboratory (Sawada et al., 1986; Tomomura et al., 1987; Staecker et al., 1988) and by others (Bottenstein and Sato, 1979). We then compared the time course of DNA synthesis in hepatocytes cultured in modified L-15 medium to that of hepatocytes cultured in modified DMEM/F12 (Fig. 2). In order to make the culture conditions more comparable, an additional 6 mM NaHC03 was added to the modified DMEMiF12 medium to bring it up to the 20 mM NaHC0, concentration of our modified L-15 medium. Compared with the cells in modified DMEMIF12 medium, the first peak of DNA synthesis in the cells cultured in modified L-15 medium was delayed until 60 h after plating, but then much more DNA synthesis was observed. The acute increase of the DNA synthesis in hepatocytes after 48 h of culture indicated that many cells have synchronously entered the S-phase of the cell cycle. Furthermore, a second, third, and fourth peak of DNA synthesis occurred at 82 h, 96 h, and 108 h, respectively. This result sug ests that many hepatocytes may have entered a secon round of the cell cycle and that the cycle interval of these cells was about 24 h. This pattern of DNA synthesis is very similar to that of primary hepatocytes cultured in modified DMEMiFl2 medium with added nicotinamide (Mitaka et al., 1991). When primary adult rat hepatocytes are cultured in modified L-15 medium under the stated conditions,

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MITAKA ET AL. 20000

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2 0

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60

80

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100

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Fig. 2. The time course of DNA synthesis in primary cultured rat hepatocytes in modified L-15 medium and modified DMEMiFl2 medium. EGF (10 ngiml) was added a t 2-3 h after plating. Cultures in six-well plates were treated with 1.2 pCi of 3H-thymidinc for 2 h before harvest, The values are means of triplicates. Small capped lines indicate standard deviations. The ordinate indicates the level of 3H-thymidine incorporation ( d p d 2 hipg DNA).

their DNA synthesis is not affected by the concentration of Ca2+ ion (data not shown). The morphology of the cells cultured in modified L-15 medium is shown in Figure 3. Within 48 h after plating, a small percentage of the hepatocytes attached to the dish were dead or degenerating, with many vacuoles apparent (Fig. 3). At day 3 many mitoses were observed in hepatocytes cultured in modified L-15 medium, and even at 120 h many cells were seen to have divided. From day 3 on, dividing hepatocytes became increasingly smaller than resting cells. These mononucleate cells, which had a smaller cytoplasmic mass than most hepatocytes, occurred in small clusters, iving a colonylike appearance as seen in Figure 3. he number of hepatocyte cultures in modified L-15 medium suddenly increased from day 3 and reached about 155% of the initial value at day 5, although some of the cells had died during the 48 h just after plating (Fig. 4A). Cell proliferation was observed at cell densities of both 1.5 x lo5 and 6.0 x lo5 cellsidish. After day 6 in culture the loss of cells increased significantly. The total DNA of cells grown in modified L-15 medium increased from day 3 and reached 165% of the initial value at day 5 even though some of the cells that were attached to the dish had died by day 2 (Fig. 4B). In contrast, in modified DMEMiF12 medium, the DNA content of the cells reached a peak at day 3, but they rapidly decreased after day 4. In media without EGF, the DNA content of the hepatocytes decreased with time in culture (Fig. 4B). In order to identify these proliferating cells as hepatocytes, albumin staining was carried out (Fig. 5). All cells in the culture stained positive for albumin, even the cells labeled with 'H-thymidine. Albumin secretion

4

by the cells into the culture medium was followed by the use of western blot analysis (data not shown). For the first 5 days cultured hepatocytes were producing albumin. These results strongly suggest that proliferating cells in this culture condition were derived from hepatocytes. At day 5, no a-fetoprotein was detected by immunocytochemistry or by western blot analysis in either culture medium or cell homogenate (data not shown). Labeling indices of cells cultured in modified L-15 and DMEMiF12 media at different densities are shown in Table 1. In modified L-15 medium, less than 10% of the cells were labeled within 48 h regardless of the plating density. In the sequential periods of BrdU treatment up to 120 h after plating, at cell densities of 0.75 x lo5 to 6.0 x lo5 cellsidish more than 60% of the cells were labeled. However, at a cell density of 9.0 X lo5 cellsidish, DNA synthesis was inhibited. In contrast, in modified DMEMiFl2 medium, the labeling indices of the cells were affected by cell densities higher than 3.0 x lo5 cellsidish in the period of 24-48 h. Even in the period of 48-72 h, DNA synthesis of the cells was inhibited by the higher densities of cells. We also examined small areas of dishes that had different densities of cells in different areas of the culture dish. The percentage of the labeled cells per 0.25 mm2 was measured and plotted in Figure 6. In modified L-15 medium, 60-80% of the hepatocytes were labeled in the period of 48-72 h until the number of the cells per 0.25 mm2 reached about 175. Between 175 and 200 cells/ unit area, the labeling index suddenly decreased and then gradually ta ered off. This pattern of the labeling index was also s own with 3H-thymidine instead of BrdU. We could find no difference between experiments in which various densities of cells were plated on separate dishes and those in which cells were unevenly seeded on each dish. In contrast, in modified DMEMi F12 medium, 50-60% of the cells were labeled at a cell density of 100 cellsiunit area. However, the labeling index gradually decreased between 100 and 200 cells/ unit area, and less than 5% of the cells were labeled at cell densities over 200 cellsiunit area. Table 2 shows the mitotic indices of the cells cultured in modified L-15 medium; 2 4 % of the cells went into the M-phase of the cell cycle in the periods of 69-72 h and 93-96 h regardless of the number of' cells plated. To study what percentage of the cells went through a second round of the cell cycle, BrdU-immunocytochemistry and autoradiography were combined on the same cells. In Figure 7, darkened nuclei show the incorporation of BrdU into DNA, and dots over the nuclei indicate the incorporation of 3H-thymidine. Some nuclei exhibited many dots on darkened nuclei, indicating that these cells had entered the second round of the cell cycle. As shown in Table 3, about 20% of the hepatocytes replicate their DNA twice within 96 h of culture. Since the amino acid concentrations of L-15 medium are much higher than those of DMEMiFl2 medium, we added various amounts of amino acid concentrates to our modified DMEMiF12 medium and compared their effects on DNA synthesis in our cultured hepatocytes to that in our modified L-15 medium. As shown in Figure 8, during the first 24 h (24-48 h of culture), the DNA synthesis of the cells in x3 EAA medium was

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PROLIFERATION OF RAT HEPATOCYTES IN L-15 MEDIUM

499

Fig. 3. Photographs, all within the same field of cells in modified L-15 medium with 20 mM NaHCO, and 10 ngiml EGF. A. Day 1 (24 h after plating); B. day 2; C. day 3; D. day 4; E. day 5; F. day 6. All photographs are the same magnification ( x 140). Arrows show the colony-like features of mononucleate cells.

inhibited. During the second 24 h (48-72 h), the cells in X 3 EAA medium incorporated much more 3H-thymidine into their DNA than the cells in the control medium, although the inco oration of the cells in L-15 medium was much higher t an that in other media. In the period 72-96 h, the DNA synthesis of the cells in x3 EAA medium was much higher than that in the control medium, and the value was near to that in L-15

TI

medium. In contrast, the addition of nonessential amino acids except proline did not affect the DNA synthesis of the cells. DISCUSSION The distinctive finding of this study is that the addition of NaHC03 to a supplemented L-15 medium incubated in a 5% C02/95%air atmosphere resulted in

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MITAKA ET AL.

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Fig. 4. The percentage change of the cell number of primary cultured rat hepatocytes (A) in modified L-15 medium with 20 mM NaHCO, and 10 ng/ml EGF and (B) in modified L-15 and modified DMEMIF12 medium with or without 10 ngiml EGF. In A, 1.5 x lo5 and 6.0 x lo5 cellsI35mm dish were plated. The values show the average of two experiments. Three fields per dish and two dishes per experiment were observed. In B, both media contain 20 mM NaHCO,. 3.0 x lo5 cells/35mm dish were plated. The values are the mean of seven independent experiments (L-15 + EGF) and two independent experiments (DMEMIF12 + EGF and L-15). The content ofDNA on day 1was 15.9 2.7 pg per dish (mean +- standard deviation).

*

Fig. 5. Immunocytochemistry for rat albumin in primary hepatocytes cultured in modified L-15medium combined with autoradiography at 120 h after plating. 3H-Thymidine (2.4 pCi) was added to the culture medium at 96 h. Immunocytochemistry for albumin was done first, and then the dishes were subjected to autoradiography. Nearly all cells exhibited albumin staining in their cytoplasm. Arrows show the small mononucleate cells with 3H-thymidine incorporation into their DNA ( X 170).

a more than 10-fold stimulus of DNA synthesis in primary cultures of hepatocytes as compared to those maintained in air only in the absence of bicarbonate. In young adult rats, when two-thirds of the liver is removed, the remnant cells undergo tissuewide replication; and the original mass of the tissue is restored

within 1 week. Clearly, if 100% of the hepatocytes replicate during the first cycle, 50% of the cells replicating twice would restore the total initial number of cells (McGowan, 1986). Thus in vivo, many cells may replicate two or more times to complete a cycle of regenerative restoration. However, the focus of most

501

PROLIFERATION OF RAT HEPATOCYTES IN L-15 MEDIUM TABLE 1. Labeling indices of primary rat hepatocytes cultured in the medium of L-15 or DMEM/F12 supplemented with 20 mM NaHC03 and 10 ng/ml EGF

Number of cells/dish (cells/cm?

Media employed

24-48 h

L15 DMEM/F12 L15

75,000 (9,400) 150,000 (18,800) 300,000 (37,500) 600,000 (75,000) 900,000 (112.500)

5.12 N.D.3 5.0 34.1 3.9 5.7 3.1 26.4 1.6 8.7 4.4 f 1.2 0.6 0.5 0.2

+ + +

DMEM/F12 L15 DMEM/F12 L15 DMEM/F12 L15 DMEM/F12

Labelinz indices Wd Periods of BrdU treatment' 48-72 h 72-96 h 57.5 N.D. 66.0 4B4 57.0 f 2.7 64.1 7.0 56.2 5.9 70.7 c! 7.4 26.4 6.1 43.4 9.1 f 2.1

* + * *

96-120 h

60.8

32.6 N.D. 47.7 f 5.7

N.D. 57.5 f 6.1 N.D. 54.8 zt 9.2 N.D. 38.6 10.0

N.D.

N.D. 69.1 f 4.7

*

N.D.

47.9 N.D. 38.6 5 7.2 N.D. 16.4 N.D.

'The dishes were treated with 50 fiM bromodeoxyuridine (BrdU) for the 24-h intervals noted following plating at 0 time. 'The number is the average of two separate experiments. JNot determined. 4These numbers are the mean standard deviation of four or five separate experiments.

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150

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200

225

250

275

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Fig. 6. Scatter plots of the relationship between the labeling index and cell density. Labeling indices were measured as described in Methods. Fifty KM BrdU was added to the culture medium 48-72 h after plating. At 72 h cells were fixed with 70% ethanol. Experiment L15 (1):The isolated hepatocytes (6 x lo5 cellsidish) were plated on 35mm dishes unevenly. Experiment L15 (2): The isolated hepatocytes were seeded on 35mm dishes at the densities of 3 x lo5, 6 x lo5, and 9 x lo5 cellsidish as evenly as possible. More than 30 areas were observed in each dish and more than 100 areas were totally measured and plotted. DMEMiF12: Labeling index of the cells in modified DMEMiFl2 medium, Experiments L15 (l), L15 (2) and DMEM/F12 were independent experiments, but the results were reproduced several times.

TABLE 2. Mitotic indices of primary rat hepatocytes cultured in modified L15 supplemented with 10 ng/ml EGF

Number of cells Cells/p35/dish (cells/cm9 75,000 150,000 300,000 600,000 ~,~

(9,400) (18,800) (37,500) (75,000) (112,500)

24-48 h O2 O2

0.032 0.l2 02

investigators studying proliferating hepatocytes in vitro has been on the initial aspects of the proliferation (Richman et al., 1976; McGowan et al., 1981; Francavilla et al., 1987; LaBrecqua et al., 1987; Gohda et al., 1988; Brenner et al., 1989; Kan et al., 1989; Michalopoulos et al., 1984; Nakamura et al., 1986; Mead and Fausto, 1989). There has been very little investigation of what nutrients themselves in the culture medium can modulate the DNA synthesis of primary cultured rat hepatocytes. Amino acids may be articularly important as liver cell nutrients because t ey can serve as protein precursors, as energy substrates (Se len and Solheim, 1978), and as natural inhibitors o hepatic protein degradation (Schworer and Mortimore, 1979). Seglen et al. (1983) reported that the inclusion of higher concentrations of amino acids and insulin resulted in a stimulation of protein synthesis as well as an inhibition of protein degradation. By using their medium (SM-1)supplemented with insulin and glucocorticoid hormone, they could maintain hepatocytes on a collagen substratum in a protein-free medium without any detectable protein loss for at least a week. However, they could not induce the cells to proliferate. It is of interest that a protein-free diet or protein deprivation for 3 days followed by an amino acid meal stimulated the DNA synthesis of the liver in an intact rat (Short et al., 1974; Bucher et al., 1978). The results of our experiments may be an in vitro analogue of this phenomenon. The isolation of the hepatocytes and their adjustment to a cultural environment may correspond to protein deprivation, and the amino acid-rich medium (L-15) can be likened to an amino acid meal. Since a certain amount of essential amino acids is contained in every commercial medium, many investigators have been more concerned with the nonessential amino acids, which some media do not contain. Thus the proline requirement for active hepatocyte DNA synthesis was found by several researchers (Hasegawa et al., 1982; Nakamura et al., 1984; Houck and Michalopoulos, 1985). However, McGowan and Bucher (1983) reported that a high concentration of alanine and glutamine inhibited the DNA synthesis of primary cultured rat hepatocytes. Although without proline, an

Periods of BrdU treatment1 48-72 h 72-96 h 96-120 h 4.g2 5.6 l.Z3 3.9 1.3 4.6 f 0.6 5.72

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1.@

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1.@ 0.22

'Thedishes weretreatedwith 20rM bromcdeoxyuridine(BrdU)for24hasinTable1 and 0.2 pg/dish Colcemid for 3 h before harvest. 'The number is the average of two separate experiments. ,'These numbers are the mean standard deviation of four separate experiments.

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MITAKA ET AL.

Fig. 7. Double scoring of cell nuclei by (1) BrdU by immunocytochemistry and (2) 3H-thymidine incorporation into DNA by autoradiography. 3H-thymidine (0.6 $3)was added at 48 h after plating, and 50 mM BrdU was added at 72 h. The cells were fixed with cold 70% ethanol at 96 h. Details are described in Methods. Darkened nuclei are those stained with BrdU, and dots over the darkened nuclei show a second round of the cell cycle (Arrows) (X170).

TABLE 3. Double scoring of BrdU and Wthymidine in rat hepatocytes cultured in L-15 medium supplemented with 20 mM NaHC03 and 10 ng/ml EGF Periods of treatment First 48-72 h

Second 72-96 h

BrdIJ' "H-thymidine

3H-thymidinez BrdU

First period 79.4 71.0

*+ 0.8 1.53

Percentage of stained cells Second period 27.3 k 3.2 32.9 2.6

*

Both periods 22.4 16.0

+ 1.5 + 4.4

'50 p M of Bromodeoxyuridine (BrdU) was added to the culture medium for the 24 h interval shown after plating a t 0 time.

'0.6 pCi of 3H-thymidine was added to the culture medium for 24 h. 3The number is the mean standard deviation of triplicate dishes.

*

additional 1 mM of one or more of several amino acids was reported to have no effect on DNA synthesis, in the presence of proline, tyrosine, and phenylalanine slightly increased the 3H-thymidine incorporation (Houck and Michalopoulos, 1985). As shown in the present study, the addition of all essential amino acids can increase the DNA synthesis of hepatocytes, although the increase was delayed (72-96 h) and DNA synthesis was inhibited until 48 h after plating. These results suggest that the balance of individual amino acid concentrations as well as the high concentrations of amino acids are requirements for the multiple replication of primary cultured rat hepatocytes. Hasegawa et al. (1982)and Eckl et al. (1987)reported that primary cultured rat hepatocytes required low Ca2+ ion concentration to complete mitoses, whereas McGowan et al. (1981) and Vintermyr and Doskeland (1989)observed high rates of mitoses with normal Ca2' ion (0.1-1.25 mM). In the present studies, 0.2-3 mM Caz+in our modified L-15 medium had no effect on the

DNA synthesis of the primary cultured rat hepatocytes, confirming the results of the latter investigators. A low cell density of cultured hepatocytes has also been reported to be required for cell division. Nakamura et al. (198313) demonstrated that the response of hepatocytes to the stimulatory activity of insulin and EGF decreases as the cell density is increased, with no stimulation above a density of 5 x lo4 cellsicm'. The importance of low plating density was also suggested by Michalopoulos et al. (1982) in studies with rat serum. However, in the present study, with our modified L-15 medium, even when more than 7.5 x lo4 cells/cm2were plated on the dish, a high labeling index and mitotic index were observed. A more precise study (Fig. 7) revealed that the DNA synthesis of primary cultured rat hepatocytes was suddenly inhibited when the density of the cells became more than 200 cellsi0.25 mm', which number corresponded to 6.4 x lo5 cellsi35mm dish. In contrast, in the modified DMEMIF12 medium, the inhibition of the DNA synthesis started at a density of less than 3 x lo4 cells/cm2. These results suggest

503

PROLIFERATION OF RAT HEPATOCYTES IN L-15 MEDIUM 80000

A

B

H Control ffl x1.5 60000

ffl

Control x2.0

x3.0

x2.0 x3.0

El x4.0

h

40000

20000

0 24-48

48-72

Hours after

24-48

72-96

Essential Amino Acids Control X 1.5 mM mM Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine

2.8 1.3

0.85 0.16

1.9

0.4

0.95 0.4 1.0 1.5 5.0 0.1 1.65 1.7

0.45 0.5 0.11 0.21 0.45 0.04 0.3 0.45

72-96

Plating

Non-Essential Amino Acids

L-15 DMEM/F12 x 2.0 mM mM 1.21 0.27 0.6 0.65 0.7 0.16 0.31 0.65 0.065 0.4 0.65

48-72

Hours after

Plating

1.57 0.38 0.8 0.85 0.9 0.21 0.41 0.85 0.09 0.5 0.85

X 3.0

Control X 2.0 mM mM

mM 2.29 0.6 1.2 1.25 1.3 0.31 0.61 1.25 0.14 0.7 1.25

Alanine Asparagine Aspartic acid Cysteine Cystine Glutamic acid Glutamine Serine Praline

5.0 1.9

-

1.0

-

2.0 1.9 0.26

0.05 0.057

-

0.15 0.13

2.5 0.25 0.26

L-15 DMEM/F12 X 3.0 mM mM

X 4.0

0.25 0.257 0.2 0.15 0.13 0.2 2.5 0.45 0.26

0.35 0.357 0.3 0.15 0.13 0.3 2.5 0.55 0.3

0.15 0.157 0.1 0.15 0.13

0.1 2.5 0.35 0.26

mM

Fig. 8. The effect of amino acid concentration in modified DMEMiFl2 medium on the DNA synthesis of primary cultured rat hepatocytes. Ten ngiml EGF was added to the medium at 2 h after plating. The intervals designate the hours in culture medium containing 1.2 KCi of "-thymidine from the time of plating. T represents the standard deviation values. Bars show 3H-thymidine incorporation of the cells (dpmi24 hikg DNA). A. The effect of essential amino acids. B. The effect of nonessential amino acids.

that the factors that induce the inhibition of replicative DNA synthesis of cultured hepatocytes may exist more in the culture medium than in the cell density, although a very high cell density does inhibit the proliferation of the cells. One of these limiting factors in the medium may be the amino acid concentration since there is a large difference between the concentration of amino acids in L-15 and DMEMIF12 media. Our results showed that essential amino acids play an important role in the DNA synthesis of primary cultured hepatocytes. Although nonessential amino acids are also important, except for proline they did not have a direct action on DNA synthesis. Furthermore, the early inhibition of DNA synthesis observed in the eriod of 24-48 h after plating may be due to the hig concentration of methionine, threonine, and tryptophan (Mitaka et al., unpublished observations). These data further support the importance of the balance among amino acids, as Ham (1974)has previously pointed out. Thus nutrients in the culture medium, especially the amino acids, can modulate the proliferation of primary cultured hepatocytes. Further experiments are neces-

K

sary to reveal which amino acids can regulate the proliferation of the cells and whether these phenomena can be observed using other growth factors instead of EGF. ACKNOWLEDGMENTS The authors thank Jane A. Weeks for her expert technical assistance. We also thank Dr. Ilse L. Riegel for her editorial advice and Mrs. Mary J o Markham for expert typing of the manuscript. The investigations described in this study were supported in part by grants from the National Cancer Institute (CA-07175, CA22484, and CA-45700).

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