[CANCER RESEARCH 42, 3858-3863. September 1982]0008-5472/82/0042-0000$02.00

Growth of Human Hepatoma Cell Lines with Differentiated Functions in

Chemically Defined Medium

Hidekazu Nakabayashi, Kazuhisa Taketa, Keiko Miyano, Takashi Yamane, and Jiro Sato1

Division of Pathology, Cancer Institute [H. N.. K. M., J. S.I, and Department of Orthopedics ¡T.Y.I, Okayama University School of Medicine, 2-5-1 Shikata-cho,Okayama (700), and Health Research Center, Kagawa University, 1-1 Saiwai-cho, Takamatsu (760) [K. T.], Japan

ABSTRACT

A human hepatoma cell line, HuH-7, which was established

from a hepatocellular carcinoma, was found to replicate continuously in a chemically defined medium when the medium wassupplemented with Na2SeO3. The cells grew better in thismedium than in serum-containing medium without any adaptation period. Other established human hepatoma and hepa-toblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, andhuH-4, also grew in the defined medium. Although HLEC-1

cells failed to proliferate continuously with Na2SeO3 alone, theygrew if a cell-free conditioned medium from HuH-7 cells wasadded to the medium. These cell lines, except the HLEC-1 cell

line, produced the following human plasma proteins amongthose examined: albumin, prealbumin, c^-antitrypsin, cerulo-plasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, /?-lipoprotein, a2-macroglobulin, /S2-microglobulin, transferrin,Complement Components 3 and 4, and «i-fetoprotein. Besideplasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anti-carcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, andhuH-4 medium contained hepatitis B surface antigen. Theseproteins were detected during periods of serial cultivation over9 months under the above culture conditions. The hepatomacell lines grown in the fully defined synthetic medium mayprovide a new approach for investigating the growth and metabolism of human hepatoma cells in vitro.

INTRODUCTION

Several efforts have been made during the past few years toestablish cell lines with differentiated functions of their originalcells in tissues. Among established human hepatoma cell lines,those with differentiated functions of the liver, such as HuH-6(10), PLC/PRF/5 (2), Hep 3B (1 ), huH-1 (1 5), and huH-4 (1 5),

are now available. However, the growth of these cell linesrequires the addition of serum and other undefined substancesto the medium. The availability of hepatoma cell lines that couldbe propagated in a culture medium containing exclusivelydefined components would permit us to study systematically invitro the effect of various agents on the growth and metabolismof hepatoma cells.

A human hepatoma cell line, HuH-7, was found to replicate

continuously over 9 months in a chemically defined syntheticmedium when the medium was supplemented with selenium asNa2SeO3. HuH-7 produced a number of plasma proteins andliver-specific enzymes under the above culture conditions. Inthe present paper, we report the growth and some biologicalcharacteristics of HuH-7 cells in comparison with those of other

cultured human hepatoma and hepatoblastoma cell lines, whichwe similarly attempted to grow in the chemically defined medium.

MATERIALS AND METHODS

Cell Lines and Culture Medium. The human hepatoma cell lineHuH-7 was established as follows. A hepatoma tissue removed atsurgical operation from a 57-year-old Japanese male with well-differentiated hepatocellular carcinoma (serum ai-fetoprotein, 16.0 jig/ml;serum CEA,2 1.6 ng/ml; and HBsAg, negative) was minced and culti

vated in RPMI 1640 supplemented with 20% heat-inactivated bovine

serum and 0.4% LAH. An epithelial cell colony isolated from primaryculture on Day 28 was designated as HuH-7. Injection of HuH-7 cells

s.c. into nude mice produced tumors, and a cell line reestablished inculture from the tumor tissue was designated as H7R. The establishedHuH-7 and H7R and other human hepatoma and hepatoblastoma celllines used in the present study, HLEC-1 (715 total culture days, 66passages) (11), HuH-6 cl-5 (1096 total culture days, 58 passages)

(10), PLC/PRF/5 (422 days of culture in our laboratory, 99 passagesin total) (2), huH-1 (1980 total culture days) (15), and huH-4 (543 total

culture days) (15), were cultivated for indicated periods of time in agrowth medium of RPMI 1640 supplemented with Na2SeO3, unlessotherwise indicated, for the study of growth and plasma protein production.

Growth Experiments. Numerical estimation of growth was performed in 24-well multiplates (Falcon Plastics, Los Angeles, Calif.).

After the medium was removed in late logarithmic phase, the cells wererinsed once with PBS, incubated in PBS with 0.01% EDTA for 10 to15 min at 37°, and trypsinized with a solution of 0.1% trypsin and0.005% EDTA for 15 min at 37°. The detached cells were dispersed

into single cells by gentle pipeting and collected by centrifugation at1200 rpm for 5 min. The cell pellet was suspended in the growthmedium and used for subculture or growth experiments. Viable cellcount was determined by erythrosin B exclusion in a hemocytometer,and appropriate dilutions of viable cells were made. The cells werethen seeded in wells each with 0.5 ml growth medium, and the mediumwas renewed every 2 days. The number of cells was counted bytreatment with trypsin-EDTA using a Coulter Counter at the indicated

intervals after seeding. Results represent the mean of 3 counts eachon triplicate wells.

Detection of Human Plasma Proteins. Human plasma proteins weredemonstrated by immunoelectrophoresis against anti-whole human

serum protein sera and confirmed by double immunodiffusion withindividual antibodies. CEA and HBsAg were detected by enzyme im-

munoassay methods. Four to 5 spent media each after incubation ofcells for 48 hr in RPMI 1640 supplemented with Na2SeO3 were combined and concentrated for analysis of plasma proteins. The 500-fold

' To whom requests for reprints should be addressed.

Received February 22. 1982; accepted June 9. 1982.

2 The abbreviations used are: CEA, carcinoembryonic antigen; HBsAg. hepa

titis B surface antigen; RPMI 1640, Roswell Park Memorial Institute Medium1640; LAH, lactalbumin hydrolysate; PBS, phosphate-buffered saline [KCI, 0.2g/liter; KH2PO„,0.2 g/liter; Na2HPOa •12H2O, 2.89 g/liter; NaCI, 8.0 g/liter (pH7.2)]; G6Pase, glucose-6-phosphatase; FDPase, fructose-1,6-diphosphatase;EGF. epidermal growth factor; FGF, fibroblast growth factor; CFCM, cell-freeconditioned medium.

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Human Hepatoma Cells in Defined Medium

concentrated medium in a collodion bag (Sartorius GmbH, Gottingen,Germany) was dialyzed against PBS. Antibodies against whole humanserum rabbit sera were prepared in this laboratory. Antisera againstalbumin, prealbumin, a,-antitrypsin, ceruloplasmin, fibrinogen, hapto-globin, hemopexin, /J-lipoprotein, a2-macroglobulin, /f2-microglobulin,transferrin, Complement Components 3 and 4, and ai-fetoprotein were

purchased from Medical and Biological Laboratories, Ltd., Nagoya,Japan. Anti-fibronectin was obtained from Cappel Laboratories, Coch-

ranville, Pa.Other Methods. Periodic acid-Schiff staining was performed ac

cording to the method of Lillie (16) with preparations pretreated withdiastase as controls. The homogenates prepared in 0.154 M KCI-4 mw

EDTA, pH 7.5, were analyzed for G6Pase (EC 3.1.3.9) and 25,000 xg 60-min supernatants for low- and high-Km hexokinase (EC 2.7.1.2),glucose-6-phosphate dehydrogenase (EC 1.1.1.49), FDPase (EC

3.1.3.11), pyruvate kinase (EC 2.7.1.40), and phosphofructokinase(EC 2.7.1.11) activities as described earlier (31, 32). Isoenzymes ofhexokinase and pyruvate kinase were separated by electrophoresis oncellulose acetate membrane (Cellogel; Chemetron, Milan, Italy) andquantitated by densitometry (31, 32). Enzyme activities analyzed at37° were expressed as units/g protein (homogenate protein for

G6Pase and supernatant protein for other enzymes). Cytogenetic studywas performed at 357 days (19 passages) in culture as describedpreviously (22). Tumorigenicity was determined by s.c. injection of 3.6or 8 x 106 cells into athymic BALB/c nude mice (nu/nu).

Chemicals. The reagents used in these experiments werepurchased from the following: RPMI 1640, Nissui Pharmaceutical Co.,Tokyo, Japan; insulin, hydrocortisone, glucagon, and triiodothyronine,Sigma Chemical Co., St. Louis, Mo.; EGF and FGF, CollaborativeResearch Co., Waltham, Mass.; and Na2SeO3, Wako Pure ChemicalIndustries, Ltd., Osaka, Japan.

RESULTS

Characterization of HuH-7 Cells. On the 198th day (10passages) in culture with serum, the growth medium for HuH-7 cells was changed to serum-free RPMI 1640 supplementedwith 0.4% LAH. The cells in the serum-free medium grew at amore rapid rate than did those in serum-containing medium.The population-doubling times of HuH-7 cells at the 266th day(15 passages) in serum-containing and serum-free medium

were 126.5 and 78.9 hr, respectively. The growth rate beganto rise in about 340 days, and the population-doubling time

decreased to 35.8 hr at the 352nd day (19 passages) inculture. At this time, they seemed to acquire the capacity togrow indefinitely and therefore were considered to be established as a cell line. The morphology of HuH-7 cells differed

depending on the presence or absence of serum in the medium.The cells cultured in serum-containing medium grew in multi-

layered islands, which often piled up, and the peripheral cellssurrounding the island appeared to be flattened (Fig. 1a). Inthe absence of serum, the cells exhibited a typical epithelialfeature with pavement-like cell arrangement (Fig. 10) andformed a confluent monolayer. The periodic acid-Schiff-stainedcells had strongly positive, large cytoplasmic granules, whichwere mostly digested by treatment with 0.1 % diastase solution.The pattern of carbohydrate-metabolizing enzymes in the cul

tured cells was characterized by the resemblance to theoriginal tumor enzyme pattern (Table 1). Small activities ofliver-specific enzymes, G6Pase and FDPase, were present,and higher high-Km hexokinase activities in the absence of

glucokinase were observed, probably due to the presence ofa relatively high level of hexokinase II. Inhibition kinetics ofFDPase by AMP showed a normal liver-type pattern with a K¡

of 0.04 rriM and an n value of 2.23 in the Hill plot. These resultssupport the author's previous contention that the loosely linked

and fixed gene expression best describes the altered pheno-

type in hepatoma cells (30, 32). Cytogenetic studies revealedthat the range in chromosome number lay between 50 and 59with some marker chromosomes. When HuH-7 cells were in

jected s.c. into athymic nude mice, they induced tumors (4 of7 mice tested) which resembled histologically the original hepatoma tissue of the patient (Fig. 2).

Growth Characteristics of HuH-7 Cells. Because HuH-7cells grew even better in the serum-free medium supplementedwith LAH, a further attempt was made to eliminate LAH fromthe medium. Since selenium was suggested by McKeehan efal. (18) to be essential for clonal growth of human fibroblasts,LAH, which was presumed to contain selenium, was replacedwith different concentrations of Na2SeO3 (Chart 1). At concentrations of Na2SeO3 ranging from 1 x 10~8 to 3 x 10~7 M the

maximum growth was obtained. Thus, a Na2SeO3 concentration of 3 x 10~8 M was chosen for the subsequent experiments.

The effect of simultaneous removal of serum and LAH on thegrowth of HuH-7 cells maintained in RPMI 1640 containing

20% bovine serum and 0.4% LAH was studied. In addition to

Table 1Activities of carbohydrate-metabolizing enzymes in HuH-7 cells and in original

tissue of hepatoma and tumor-bearing liver tissue

Enzyme activities (units/g protein)

EnzymesG6PaseGlucose-6-phosphate

dehydrogenaseLow-Kmhexokinase1IIIIIHigh-Km

hexokinaseIV(glucokinase)PhosphofructokinaseFDPasePyruvate

kinaseLPyruvatekinase M2HuH-7a2.6275.817.63.713.9013.9o0141.85.7e04102Liver14.485.720.616.104.52.60116.532.8109.01538Hepatoma5.4110.537.824.413.4Q11.7fa0105.59.502859

a HuH-7 cells growing for 1 month in RPMI 1640 supplemented with Na2SeO3

at the 436th day (27 passages) of culture.6 The presence of high-Km hexokinase in the absence of glucokinase is

probably due to the increase in high-Km hexokinase II.c K, with 5'-AMP was 0.04 mw, and n value in the Hilll plot was 2.23.

7

6

! 5

t .ÃŽ2

O IO'9 IO'8 IO'7 IO'6

N»2S*03concentration (M)

Chart 1. Dose-response curves of HuH-7 cell growth to added NazSeO3 inchemically defined medium. Cells grown in RPMI 1640 supplemented with 0.4%LAH (352 total culture days, 19 passages) were deprived of LAH by replacingthe medium with LAH-free medium 2 days before harvest, harvested by treatmentwith trypsin-EDTA, and plated at a density of 5 x 104 viable cells/well (2.1-sq

cm surface area) with 0.5 ml medium. After 2 days, the medium was changed toRPMI 1640 with varying concentrations of Na2SeO3. The number of cells wascounted at 10th day after seeding. Bars, S.D.

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H. Nakabayashi et al.

Na2SeO3, several hormones and growth factors were tested inthis system; the results are presented in Chart 2. The cellsgrew better in serum-free medium without any adaptation pe

riod if the medium was supplemented with LAH or Na2SeO3.When Na2SeO3 was absent from the serum- and LAH-free

medium, cell death occurred within 4 to 6 days, although theinitial growth was not impaired. With this chemically definedmedium, insulin, glucagon, triiodothyronine, hydrocortisone,EGF, and FGF had no effect on the growth when they wereadded individually (data not shown) or in combination at 10~8to 10~4 g/ml for insulin, glucagon, and hydrocortisone and at10~11 to 1CT7 g/ml for triiodothyronine, EGF, and FGF, cov

ering the physiological ranges of the hormones. Growth curvesof HuH-7 cells in the defined medium supplemented withNa2SeO3 and tested at different inoculum size with or withoutCFCM for the first 2 days are shown in Chart 3. Cells failed toattach to the surface of the dish when they were plated at lowcell densities in the absence of CFCM. Therefore, the cellswere routinely plated into CFCM, and the medium was replacedwith fresh medium after 2 days of culture. Once cells attachedto the dish, they grew continuously. The population-doublingtime of HuH-7 cells in the chemically defined medium was 43.7

hr. After omitting serum and LAH from the culture medium atthe 406th day (23 passages) in culture, HuH-7 cells had been

growing over 9 months and through 20 passages in the chemically defined medium.

Growth of Established Human Hepatoma Cell Lines inChemically Defined Medium. Other established human hepa-toma and hepatoblastoma cell lines grown in serum-containingmedia were transferred to the defined synthetic medium whichwas developed for HuH-7 cells, and their growth was alsostudied. The cells examined, except HLEC-1 cells, continued

to proliferate in the defined medium supplemented withNa2SeO3 without an adaptation period (Chart 4a). AlthoughNa2SeO3 alone failed to sustain the proliferation of HLEC-1cells, a CFCM from HuH-7, when added to the synthetic me

dium, allowed the cell to grow (Chart 40). Thus, the cultured

10_,1ìT

Co5K-SnD

8 days~M '6days•-'F;ri±î-±ÕL•îiÉ«TÕ1

A B C D E F

Chart 2. Number of cells after 8 days (logarithmic phase) and 16 days(stationary phase) of culture in medium with or without additives. The HuH-7 cellsmaintained in RPMI 1640 supplemented with 20% bovine serum and 0.4% LAH(374 total culture days. 20 passages) were harvested by trypsin-EDTA treatmentand plated at a density of 5 x 10" cells. After 2 days, the cultures were changed

by the indicated experimental media. A, RPMI 1640 plus 20% bovine serum; B,RPMI 1640 plus 20% bovine serum plus 0.4% LAH; C, RPMI 1640 plus 0.4%LAH; D, RPMI 1640 alone; E, RPMI 1640 plus 3 x 10"" M Na2SeO¡;F, RPMI1640 plus 3 X 10" M Na2SeO3 plus insulin (3 fig/ml) plus triiodothyronine (2

ng/ml) plus hydrocortisone (6.8 (ig/ml) plus glucagon (30 ng/ml) plus EGF (10ng/ml) plus FGF (10 ng/ml). Bars, S.D.

100 p

30

Õ 10

° 3

0.3

0.10 2 16i. 6 8 10 12 14

Days after plating

Chart 3. Growth curves of HuH-7 cells in chemically defined medium. Cellsgrowing for 1 month in RPMI 1640 supplemented with NajSeO:, (442 total culturedays, 28 passages) were harvested by trypsin-EDTA treatment, plated at densities of 1 x 10" ( ) and 5 x 10" ( ) cells/well with (O) or without (•)

50% CFCM and incubated for 2 days; then, the medium was replaced by a freshmedium without CFCM. The supernatant fluid collected from semiconfluentpopulations of HuH-7 cells was used as CFCM The fluid was centrifuged at 1500rpm for 10 min and filtered through 0.3-/im Millipore filters (Millipore Corp.,Bedford, Mass.). S.D.s for cell counts were generally less than 10% when theywere expressed as variation coefficients.

10

Õ I

S-0.5

b 3

0.3

O.t0 2 4 6 8 10 12 U 16

Days after plating

Chart 4. Growth curves of established human hepatoma and hepatoblastomacell lines in chemically defined medium, a, cells grown in serum-containing mediawere deprived of serum by replacing RPMI 1640 supplemented with Na2SeO3 2days before harvest in order to eliminate the effect of serum carried over to thenew medium. Cells were harvested by trypsin-EDTA treatment, plated at a densityof 5 x 10* or 1 x 105 cells/well with 50% CFCM of each cell line, and incubated

for 2 days; then, the medium was replaced by fresh medium without CFCM. •,H7RO41 days after reculturing. 14 passages); A, HuH-6cl-5 (1096 total culturedays, 58 passages); D, PLC/PRF/5 (422 days of culture in our laboratory. 99passages); A, huH-1 (1980 total culture days); •huH-4 (543 total culture days),b, similarly prepared cell suspension of HLEC-1 (715 total culture days, 66passages) plated at a density of 3 x 10" cells/well and cultured with (O) or

without (x) CFCM of HuH-7 cells. CFCM was prepared as described in Chart 3.S.D.s for cell counts were generally less than 10% when they were expressed asvariation coefficients.

hepatoma cells seemed to produce some hormones or growthfactors required for the replication of HLEC-1 cells in theserum-free medium. The growth rates of these establishedhepatoma cell lines in the serum-free medium were usuallyequal to those obtained with serum-supplemented media. Thelag phase observed for HuH-6 cl-5 was also present in the

medium which contained serum.

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Human Hepatoma Cells in Defined Medium

Table 2

Plasma protein analyzed in spent media of human hepatoma and hepatoblastoma cell lines which were grown in a chemically defined synthetic medium

Cells ALB PRE AAT CP FIB FN HP HX BLP AMG BMG TF C3 C4 AFP CEA HBsAg

HuH-7 (D, 2; P, 10)HuH-7 (D, 27; P, 26)HuH-7 (D, 277; P, 44)H7R(D, 39; P, 18)HLEC-1 (D, 2; P, 68)HuH-6cl-5(D, 155; P, 73)huH-1 (D, 97)huH-4 (D, 77)PLC/PRF/5 (D, 98; P, 106)

NO ND

ALB. albumin; PRE, prealbumin; AAT a.-antitrypsin; CP, ceruloplasmin; FIB, fibrinogen; FN, fibronectin; HP, haptoglobulin; HX, hemopexin; BLP. /8-lipoprotein;AMG, az-macroglobulin; BMG, ft-microglobulin; TF. transferrin; C3 and C4, Complement Components 3 and 4; AFP, t>,-fetoprotein; D, days in the chemically definedmedium before collection of spent medium; P, number of passages in culture; ND, not done.

" HuH-7 cells grown in RPMI 1640 supplemented with 20% bovine serum and 0.4% LAH and kept for 2 days in the absence of the added serum.c Intensities of precipitin lines were scored as follows; +, clearly visible; -, not present; ±, partial cross-reaction to albumin. Cutoff levels obtained by the

immunodiffusion technique were approximately 10 pg/ml. CEA and HBsAg were detected by enzyme immunoassay and the cutoff levels were 1 and 10 ng/ml.respectively.

Plasma Protein Production in Chemically Defined Medium.Plasma proteins produced by HuH-7 and other cell lines wereanalyzed on concentrated spent media by immunoelectropho-resis and immunodiffusion. The plasma proteins studied arelisted in Table 2. All the cells tested, except HLEC-1 cells,

produced plasma proteins in the chemically defined mediumfor more than 9 months. Each cell line produced proteinsdifferent with respect to number and amount; e.g., the level ofalbumin attained in 9 ml medium with 1 x 107 cells of HuH-7

in 2 days was approximately 1/500 the level of albumin inserum. Beside plasma proteins, anti-CEA-reactive protein(s)were found in the medium of HuH-7, HuH-6 cl-5, huH-1, andPLC/PRF/5, and HBsAg was found in the medium of huH-1,huH-4, and PLC/PRF/5 as reported (13, 15, 17). Similar

plasma protein patterns were obtained when cells were grownin RPMI 1640 supplemented with serum and LAH and kept for2 days in the absence of the added serum.

DISCUSSION

In the present studies, it was possible to eliminate serum andLAH from culture medium for the growth of hepatoma cells byadding a trace element, selenium, to a chemically definedmedium. The trace element selenium has been routinely addedto the serum-free medium for a variety of established cell lines(4-6, 19, 21, 28) with stimulated growth after its discovery by

McKeehan ef al. (18). Thompson ef al. (34) and Niwa ef al.(24, 25) were able to grow the rat hepatoma cell lines, HTCand H35, respectively, in chemically defined media withoutselenium. Under these conditions, both cell lines required anadaptation period or multistep selection for cell growth. It isimportant to note that no adaptation period was required forthe growth of the present hepatoma cell lines after replacingserum or LAH with Na2SeO3. The results suggest that neitherselection of cells capable of growing in chemically definedmedium nor extensive adaptation to the growth condition isrequired.

The hepatoma cells studied in this experiment replicatedcontinuously and produced plasma proteins, except for HLEC-1, in chemically defined medium without any added hormonesand growth factors. This suggested that the hepatoma cellcultures may be utilized for the production of a set of attachment and growth factors which stimulated their own growth.The failure of HLEC-1 to produce plasma proteins is in accord

with the fact that this cell line does not grow in the defined

synthetic medium, probably because the cell line does notsynthesize some factors, such as transferrin (3-6, 14, 19, 21,27, 28), fibronectin (4-6, 27); ai-protein (4, 5), or fetuin (6,

19), which are needed for its own growth or adhesion to thesubstratum. The assumption may be validated by the fact thata variety of hormones and growth factors, such as multiplication-stimulating activity (6, 7, 29), nerve growth factor (26), orunidentified factors (3, 6, 8, 9, 12, 23), were found in theconditioned medium of a number of cell lines. The identificationof such growth regulators in the hepatoma cells may facilitatethe analysis of growth-promoting and growth-inhibiting factors

of hepatoma cells.The hepatoma cells grown in fully defined synthetic medium

retained differentiated functions of liver cells in vivo. Besidesplasma proteins, 2 liver-specific enzymes, G6Pase andFDPase, were present in HuH-7 in the analysis of carbohydrate-metabolizing enzymes. The maintenance of differ

entiated functions in these cell lines derived from hepatomas incontrast with the loss of differentiated functions in normalhepatocytes in culture (20, 32, 35) indicated that the geneexpression in hepatoma cells is rather stable or fixed under theculture condition. Thus, the enzyme pattern in HuH-7 resem

bled that of the original hepatoma tissue. Accordingly, thehepatoma cell lines grown in the defined medium offer a goodmodel not only for the investigation of oncogenesis but also forthe elucidation of regulatory mechanisms of gene expression.

ACKNOWLEDGMENTS

We thank Drs. N. Huh and T. Utakoji, Department of Cell Biology, CancerInstitute, Tokyo, for their gift of the huH-1 and huH-4 cell lines and T. Yabe for

her excellent technical assistance.

REFERENCES

1. Aden. D. P., Fogel, A.. Plotkin, S.. Damjanov, I., and Kowles, B. B. Controlledsynthesis of HBsAg in a differentiated human liver carcinoma-derived cellline. Nature (Lond.), 282: 615-616, 1979.

2. Alexander. J. J.. Bey. E. M., Geddes, E. W., and Lecatsas, G. Establishmentof a continuously growing cell line from primary carcinoma of the liver. S.Afr. Med. J., 50: 2124-2128, 1976.

3. Allegra. J. C., and Lippman, M. E. Growth of a human breast cancer cellline in serum-free hormone-supplemented medium. Cancer Res., 38:3823-3829, 1978.

4. Barnes, D., and Sato, G. Growth of a human mammary tumour cell line ina serum-free medium. Nature (Lond.), 287: 388-389, 1979.

5. Barnes. D.. and Sato, G. Methods for growth of cultured cells in serum-freemedium. Anal. Biochem., )02: 255-270, 1980.

6. Bottenstein, J., Hayashi. I., Hutchings. S., Masui, H., Mather, J.. McClure,D. B., Ohasa. S., Rizzino, A., Sato, G., Serrerò, G., Wolfe, R., and Wu, R.

SEPTEMBER 1982 3861

on July 14, 2018. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

H. Nakabayashi et al.

The growth of cells in serum-free hormone-supplemented media. MethodsEnzymol., 58 94-109, 1979.

7. Bourne, H. R., and Rozengurt, E. An 18,000 molecular weight polypeptideinduces early events and stimulates DNA synthesis in cultured cells. Proc.Nati. Acad. Sei. U. S. A., 73: 4555-4559, 1976.

8. De Larco, J. E., and Todaro, G. J. A human fibrosarcoma cell line producingmultiplication stimulating activity (MSA)-related peptides. Nature (Lond.),272. 356-358, 1978.

9. De Larco, J. E., and Todaro, G. J. Growth factors from murine sarcomavirus-transformed cells. Proc. Nati. Acad. Sei. U. S. A., 75. 4001-4005,1978.

10. Doi, I. Establishment of a cell line and its clonal sublines from a patient withhepatoblastoma. Gann, 67. 1-10, 1976.

11. Doi, I., Namba, M.. and Sato, J. Establishment and some biological characteristics of human hepatoma cell lines. Gann, 66. 385-392, 1975.

12. Gajdusek, C., DiCorleto, P., Ross. R., and Schwartz, S. M. An endothelialcell-derived growth factor. J. Cell Biol., 85. 467-472. 1980.

13. Gerber, M. A., Gar'inkel, E., Hirschman, S. Z., Thung, S. N., and Panagiota-

tos, T. Immune and enzyme histochemical studies of a human hepatocellularcarcinoma cell line producing hepatitis B surface antigen. J. Immunol., 726:1085-1089, 1981.

14. Hayashi, I., and Sato. G. H. Replacement of serum by hormones permitsgrowth of cells in a defined medium. Nature (Lond.). 259. 132-1 34, 1976.

15. Huh, N.. and Utakoji, T. Production of HBs-antigen by two new humanhepatoma cell lines and its enhancement by dexamethasone. Gann, 72:178-179, 1981.

16. Lillie. R. D. Reticulum staining with Schiff reagent after oxidation by acidifiedsodium periodate. J. Lab. Clin. Med., 32: 910-912. 1947.

17. Macnab, G. M., Alexander, J. J., Lecatsas, G., Bey, E. M., and Urbanowicz,J. M. Hepatitis B surface antigen produced by a human hepatoma cell line.Br. J. Cancer, 34: 509-515, 1976.

18. McKeehan, W. L.. Hamilton, W. G., and Ham, R. G. Selenium is an essentialtrace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Nati.Acad. Sei. U. S. A.. 73. 2023-2027, 1976.

19. Medina. D., and Oborn, C. J. Growth of preneoplastic mammary epithelialcells in serum-free medium. Cancer Res.. 40: 3982-3987. 1980.

20. Miyazaki. M. Primary culture of adult rat liver cells. II. Cytological andbiochemical properties of primary cultured cells. Acta Med. Okayama. 32.11-22. 1978.

21. Murakami. H., and Masui, H. Hormonal control of human colon carcinomacell growth in serum-free medium. Proc. Nati. Acad. Sei. U. S. A., 77.3464-3468. 1980.

22. Nakabayashi. H. Spontaneous neoplastic transformation in vitro of a diploidclone of rat liver cells under conditions of large inoculum size of cells at thetransfer. Gann, 72: 377-387, 1981.

23. Nissley, S. P.. Short, P. A., Rechler, M. M., Podskalny, J. M., and Coon,H. G. Proliferation of Buffalo rat liver cells in serum-free medium doesnot depend upon multiplication-stimulating activity (MSA). Cell, 11:441-446, 1977.

24. Niwa. A., Yamamoto, K., Sorimachi, K., and Yasumura, Y. Continuousculture of Reuber hepatoma cells in serum-free arginine-, glutamine- andtyrosine-deprived chemically defined medium. In Vitro (Rockville), 76:987-993, 1980.

25. Niwa, A., Yamamoto, K., and Yasumura. Y. Establishment of a rat hepatomacell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium. J. Cell Physiol., 98. 177-184, 1979.

26. Oger, J., Arnason, B. G. M., Pantazis, N., Lehrich, J., and Young, M.Synthesis of nerve growth factor by L and 3T3 cells in culture. Proc. Nati.Acad. Sei. U. S. A., 71: 1554-1558, 1974.

27. Orly, J.. and Sato, G. Fibronectin mediated cytokinesis and growth offollicular cells in serum-free medium. Cell, 17: 295-305, 1979.

28. Simms, E., Gazdar, A. F., Abrams, P. G.. and Minna. J. D. Growth of humansmall cell (oat cell) carcinoma of the lung in serum-free growth factor-supplemented medium. Cancer Res., 40: 4356-4363, 1980.

29. Smith, G. L., and Temin, H. M. Purified multiplication-stimulating activityfrom rat liver cell conditioned medium: comparison of biological activitieswith calf serum, insulin, and somatomedin. J. Cell Physiol., 84: 181-192,1974.

30. Taketa. K., Shimamura, J., Kobayashi, M.. Nagashima. H., and Okajima, K.Onco-fetal isoenzymes in human hepatomas and preneoplastic livers. In: F.G. Lehmann (ed.), Carcino-embryonic proteins, Vol. 1, pp. 381-389. Amsterdam: Elsevier/North-Holland BiomédicalPress, 1979.

31. Taketa, K., Shimamura, J., Takesue, A., Tanaka. A., and Kosaka, K. Undif-ferentiated pattern of key carbohydrate-metabolizing enzymes in injuredliver. II. Human viral hepatitis and cirrhosis of the liver. Enzyme, 27:200-210, 1976.

32. Taketa, K., Watanabe, A., Shimamura, J., Takesue, A., Ueda, M., Masuji,H., and Sato, J. Undifferentiated patterns of key glucolytic and gluconeo-genic enzymes in epithelial cell lines derived from rat liver. Acta Med.Okayama, 29: 151-159, 1975.

33. Taketa, K., Ueda, M., and Watanabe, A. A case of minimal deviationhepatoma in man with elevated liver-type pyruvate kinase isozyme. Gann,68: 29-35, 1977.

34. Thompson, E. B., Anderson, C. U., and Lippman, M. E. Serum-free growthof HTC cells containing glucocorticoid- and insulin-inducible tyrosine ami-notransferase and cytoplasmic glucocorticoid receptors. J. Cell Physiol.,86: 403-412, 1975.

35. Tokiwa, T., Nakabayashi, H., Miyazaki. M., and Sato, J. Isolation andcharacterization of diploid clones from adult and newborn rat liver cell lines.In Vitro (Rockville), 75: 393-400, 1979.

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1982;42:3858-3863. Cancer Res   Hidekazu Nakabayashi, Kazuhisa Taketa, Keiko Miyano, et al.   Functions in Chemically Defined MediumGrowth of Human Hepatoma Cell Lines with Differentiated

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