Induction of mitosis in primary cultures of adult rat hepatocytes under serum-free conditions

Vol. 104, No. 1, 1982 January 15, 1982

BIOCHEMICAL

INDUCTION OF MITOSIS Kaoru Department

Received

IN PRIMARY CULTURES OF ADULT RAT HEPATOCYTES UNDER SERUM-FREE CONDITIONS

Hasegawa,

Kazuto

of Physiology, Mibu,

November

RESEARCH COMMUNICATIONS Pages 259-265

AND BIOPHYSICAL

Watanabe

and Mutuyosi

Koga

Dokkyo University School Tochigi 321-02, Japan

of Medicine,

30, 1981

When adult rat hepatocytes were cultured in a serum-free, hormone-supplemented medium containing l&i of Ca2+,DNA synthes is was induced in about 50% of hepatocytes. Howwer, mitosis could not be observed since hepatocytes became aggregated and detached from culture flasks after 3 days. On the other band, when Ca2+ concentration was reduced to below 0.4 mM, not only survival time of hepatocytes was prolonged but cell divisions were observed in morphologically identified hepatocytes. About 50% of hepatocytes synthesized DNA These results indicate that low and divided under the latter conditions. concentration of Ca2+ in the culture medium permit post DNA synthetic hepatocytes to complete cell division. INTRODUCTION Adult

rat

hepatocytes

and functions few days,

at

levels

similar

and therefore,

in hepatocyte cell

in primary

cultured

(l-3).

that

marked

study

of serum

have developed

methods

defined

in culture

medium

hepatocytes

under

vate

agents adult

inducing A lag

rat

for

DNA synthesis period

was close

to that

of hepatocytes degeneration

replacement (5).

rise

observed

cell

years,

been observed

of hepatocytes

after

we have previousrat Sato

hepatectomy in culture

owing

hepatocytes and co-workers

hormones

and chemically tried

to culti-

and succeeded

and other

in these

mechanisms

show little

we have also

conditions

the first

of regulatory

in adult

of hormones

partial

during

however,

of serum with Recently,

characteristics

division,

In recent

serum-free

have not

Abbreviations used: acid, FD= fibrinogen

cells,

in DNA synthesis after

study

was induced

(4).

by combination

of initial

specific --in vivo

the

to induce

DNA synthesis

in the presence

for

Such cultured

As an initial

ly reported

exhibit

to the hepatocytes

have been used

metabolism

division.

culture

chemicals

cultured (6).

in (6,7).

hepatocytes

However,

mitosis

to the progressive

3 days.

HEPES= N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic digests, EDTA= ethylenediamine-tetraacetic

acid.

0006-291X/82/010259-07$01.00/0 259

Copyright 0 1982 b.vAcademtc Press, Inc. All rights of reproducrton m any form reserved.

Vol. 104, No. 1, 1982 During adult

the course rat

reduced

induction

of the

hepatocytes to below

In this

report

BIOCHEMICAL

could

to improve

undergo

culture

mitosis

RESEARCH COMMUNICATIONS conditions,

we found

when Ca2+ concentration

that

was

0.4 mM.

communication, of mitosis

microscopical

study

AND BIOPHYSICAL

we report

in adult

rat

observations MATERIALS

the

culture

hepatocytes of the

conditions in primary

process

necessary culture,

of hepatocyte

for

the

and also divisions.

AND METHODS

Male Wistar rats weighing 200 Isolation of Hepatocytes and Primary Cultures. to 300 g were used for experiments. Hepatocytes (parenchymal liver cells) were isolated aseptically using a modification of the previously reported methods (4,8) which was firstly described by Haward --et al. (9). Liver was perfused vein with 02-saturated warm (37'C) perfusion --in situ via the portal buffer [in mg/l; NaCl (7,000), KC1 (400), NaHC03 (420), Na-pyruvate (550), HEPES (40 mM, pH 7.4)] at a flow rate of 20 ml/ min for 5 to 6 min. The perfusate was then changed to 30 ml of 0.05% of collagenase buffer (perfusion buffer plus 5 mM of Ca2+). After perfusion, the liver was removed and minced finely with scissors. Minced liver was suspended in 30 ml of collagenase solution and incubated at 37'C for 10 to 15 min with shaking. The digests were filtered through double layers of stainless mesh (167 and 67 jna pore size The cells in the filtrate were sedimented at 10 g for 3 min. The cell ). pellet was washed thrice by suspending in glucose-free, pyruvate (5 &)-supThe cells were finally plemented Hanks' solution followed by centrifugation. resuspended in an appropriate volume of the culture medium. At the expence of cell yield (1 to 2 x 108 cells from 5 to 7 g of liver), the resulting cell suspension consist almost of hepatocytes (98% or more). For hepatocytes to anchorage to culture flask, 0.12 A280 unit of FD (4,6) was supplemented to 5 ml of culture medium. FD was prepared as follows. To 0.5% bovine fibrinogen buffer, Urokina& (E.C.3.4.21.31, Green Cross (Sigma, Type I) in perfusion Japan) was added (100 U/ml) and incubated at 37“C for 60 min. At the Corp., end of incubation, Trasylol@ (Sayer, 200 I.U./ml) was added to stop the reaction. Other procedures for primary culture of hepatocytes were essentially the same as reported previously (4,6) and briefly described in the legend of Fig. 2. Culture Medium. Koga's Medium L (KL) was used for primary culture of adult rat hepatocytes. Composition of KL was as follows (in mg/l). NaCl (7,400), KC1 (250), CaC12.2H20 (ZO), MgS04.7H20 (150), NaH2P04.2H20 (150), MnC12.4H20 (0.05), ZnS04.7H20 (0.75), FeS04. 7H20 (0.5), H2Se03 (0.002), CuSO4.5H20 (0.2) NaHC03 (420), L-alanine (ZOO), L-asparagine.H20 (40), L-aspartic acid (lo), L-cysteine HCl H20 (80), L-cystine HCl (40), L-glutamine (700). L-glutamic acid (60), L-histidine.HClaH20 (40), L-isoleucine (50), L-leucine (go), Llysine.HCl (150), L-methionine (30), L-ornithine.HCl (loo), L-phenylalanine (40), L-proline (120), L-serine (60), L-threonine (go), L-tryptophan (45), L-tyrosine (60), L-valine (75), glutathione (reduced, ZO), ascorbic acid (60), biotin (O.Ol), Ca-pantothenate (O-5), choline chloride (lo), cyanocobalamine (O.l), folic acid (l.O), inositol (lo), menadione (O.Ol), nicotinamide (0.5), pyridoxine.HCl (0.5), riboflavin (O.l), thiamine.HCl (l.O), erotic acid (O.l), lipoic acid (0.04), Na-linoleate (0.5), DL-carnitinesHCl (3), DL-mwalonic acid lactone (Z), adenine-HCl (12), thymidine (0.15), galactose (ZOO), Na-pyruvate (4,400), phenol red (5), kanamycin (30), bovine serum albumin (Fraction V, Reheis Chemical Co., 500), transferrin (human, Sigma, 5) Trasylol@ (2,000 II), and HEPES (20 mM, pH 7.4). In the present experiments, Ca2+ concentration was adjusted to levels between 0 and 2 mM by addition of features of KL are: 1) Glucose was CaC12*2H20 to Ca2+-free KL. The special omitted. Instead, a high concentration of Na-pyruvate was supplemented. An 260

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

unusually high concentration of Na-pyruvate to enhance DNA synthesis (7). 2) Considerable amino acids were supplemented, while arginine inhibitor Trasylol was supplemented. This cultivation (6). Although albumin (Fraction sary for the induction of DNA synthesis and ed survival of hepatocytes under serum-free

RESEARCH COMMUNICATIONS

is required to replace serum and amounts of all non-essential was omitted. 3) A protease is also required for serum-free V) and transferrin are not necesmitosis, these components prolon8conditions.

RESULTS AND DISCUSSION About

80% of plated

and formed

a monolayer

plating.

ml4 of Ca2+. detached

Several the

1B shows

flask

distributed

cultured

and one is in

homogenously

situated

at the center

difference

of DNA synthesis 1, about

Reduced

between

was higher other

among hepatocytes

outlined

shape

hepatocytes

(Fig.

this

idea.

50 h in

Fig.

This

cell of Ca2+.

Several

rhombic

Fig.

As shown

area

in the

of them

area

Although

of a

marked

lB,

C, degree

autoradiograph were

are is

is composed

and binucleated)

(Fig. labeled

with

plating.

than

0.4 mM, hepatocytes

morphological

changes.

loose would

under

Observations hepatocytes

loose by the

actually 261

for

closely

was below

and each hepatocyte be easier

When

contacted

when Ca2+ concentration

division

three granules

1A and Fig.

induced

It

figures

rhombic

made by chromosomes. between

were

Several

figures,

and non-granular

1

remained.

0.1 mM Ca2+ medium,

also

was very

containing

1C shows magnified

In metaphase

same.

hand,

lB,C,D).

to complete

by low concentration

for

26 and 50 h after

On the

one another.

seen.

(mononucleated

Ca2+ concentration

Ca2+ concentration

75 h.

was observed

50% of hepatocytes

[3H]thymidine

at 0.1 mM of Ca2+ for are

1A

hepatocytes debris

cytoplasm.

the

Fig.

cell

plate

was almost

almost

the medium

except

4 h

one day after

consists

was taken,

cells

in the cytoplasm of the

75 h in

photograph

telophase.

body and an equatorial

morphological

support

cultured

within

non-hepatocytes.

for

of the

(encircled)

hepatocytes

in metaphase

contact

and none

flask

morphology

the monolayer

cultured the

culture

epithelial

contaminating

after

hepatocytes

to the

1 show that

without

hours

hepatocytes

of dividing

spindle

in Fig.

of hepatocytes

from

dividing

attached

pavement-like

of hepatocytes

shows aggregates

are

with

Photographs

exclusively

Fig.

hepatocytes

cell-to-cell time entered

0.4 mM

showed

post-DNA

simple

synthetic contact

lapse mitotic

to

caused

cinemicrography phase

even

1D

Vol. 104, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Photomicrographs of adult rat hepatocytes cultured in a serum-free Fig. 1. medium containing 1mM (A) and O.lmM (B,C,D) of Ca2+. (A) Phase contrast photomicrograph of hepatocytes which formed aggregates at 75 h in high Ca2+ medium. Arrows indicate aggregates of hepatocytes that axhibits blebiog and (B) Phase contrast photomicrograph of hepatohalooning of cell membrane. cytes cultured for 75 h in low Ca2+ medium. Dividing hepatocytes are indicated by circles. (C) Phase contrast photanicrograph showing 4 dividing Arrows indicate three metaphase and one telophase hepatocytes. hepatccytes. Fifty hours after plating. (D) Autoradiograph of Gieasa stained hepatocytes labeled with [jH]thymidine between 26 and 50 h. The arrow indicate a telophase hepatocyte. in high

Ca 2+ (In&l)

mitotic

figures

microscope, ited

resulted loose

were

since

extensive

medium when observed almost

ruffling

blebing

in detachment cell-to-cell

of cell

was relatively

division

but

membrane

the

On the

in

from

low Ca 2+ medium and not

only

of Ca2+. Concerning

the hepatocyte

degeneration

reported

that

death

flask.

twice

other

hand,

toxins

under

of cell underwent

of cultures

Ca2+ medium,

exhib-

1A) which

mM), ruffling

of that

in high

contrast

(Fig.

50 % of hepatocytes

by membrane-active 262

(0.1

phase

such

and hepatocytes

of cells

was prolonged

cell

was active

membrane

time

(1Oj

under

of the cell

quiet

survival

when observed

However,

and balooning

contact

membrane

unnoticed

by cinemicrography.

it

in primary

with

cell 1 mM

has been cultures

BIOCHEMICAL

Vol. 104, No. 1, 1982 of adult

rat

hepatocytes

was 3.6 mM, while Therefore,

observed

cell

intracellular In the

Ca2+ medium

in high

lapse

cinemicrography, during into

hepatocyte

both

synthesized

Third,

divided DNA prior

hepatocytes

fused,

of division

accompanied

Fig.

and third or polyploid

and reached

gradually terns

(not similar

to cell

number

in the number

began

a peak at 4-day

DNA and protein

shown).

of the

experiment.

since

rats

content

suggest

over

the

one did

not.

many cells

are

l-day

since

2B,C)

increase

in cell

that

two types

(12).

(Fig.

Maximal

number.

values

latter

3- to 4-day),

Up to 50% of increase These

experiments.

cell

the

to increase

(usually

two daughter

The former

noteworthy,

of intact

by autoradio-

divided,

hepatocyte.

one

hepatocytes;

as observed

while

types

Second,

daughter

hepatocyte

are

different

hepatocytes.

division

increase,

liver

of

one mononucleated

two mononucleated

of division

cell

to that

35% in this other

types

three

First,

daughter

one binucleated cell

the

by accumulation

observed

culture.

one mononucleated

forming

2A shows that

2-day,

into

from

(11). we have

primary

of Ca2+ in the

hepatocytes

may be caused

two mononucleated

soon after

binucleated

which

in the medium

in the absence

of Ca*+ protects

Ca*+

binucleated

The second

was observed

(damage)

divided

nuclei

death

low concentration

division

hepatocyte

gram.

cell

RESEARCH COMMUNICATIONS

when Ca2+ concentration

degeneration

time

of hepatocyte

was prominent

little

medium.

AND BIOPHYSICAL

all

then

at

decreased

changed

in pat-

in cell

number

number

almost

level

was

was observed

hepatocytes

in

which

synthesized DNA (SO%, see Fig. 1D) might have divided. stimulated It has been reported that low Ca*+ concentration

growth

of human

epidermal

(15).

Our

keratinocytes

results

which

consistent

partial tion

that

to the mitosis

cell

T51B rat normal

level

in both

cases.

in primary

epithelium

by low Ca*+ concentration

to our results,

inhibited

and in cultured

of hepatocytes

established

Contrary

Ca2+ deprivation

and enter

bladder

of hepatocytes

reports.

of Ca2+ concentration

requirement in the

these

hepatectomy

DNA synthesis

and urinary

show proliferation

with

have reported

(13,14)

culture

line. 263

Whitfield

DNA synthesis liver

cells.

enabled It differs

--et al.

in the

is unclear from

(16)

liver

However, the cells

are

after

restora-

to initiate

why Ca2+ that

--in vivo

and

Vol. 104, No. 1, 1982

“‘r

Fig.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A

2.

Time

courses

of

cell

Days

after

number

(A),

Rating

DNA content

(B)

ad

protein

(C) during primary culture of adult rat hepatccytes. About 1.5 x lo6 viable hepatocytes in 5 ml of culture medium (KL) were plated in a glass culture flask (40 cm*) and incubated at 37'C under air in a closed system. Culture period was divided into Pl (plating to 4 h), P2 (4 to 22 h) and P3 (22 h -). Medium was changed at 4, 22 h and at 24-h interval thereafter. Only during the initial 4 h, FD was added. Glucagon (50 rig/ml) arkl insulin (50 rig/ml) were supplemented after 4 h. Hepatocytes were harvested by EDTA For determination -trypsin and cell number was counted with a hemocytoaeter. of DNA and protein content, hepatocytes were lysed with 1 N NaOH and the lysate was assayed by the method of Burton (21) and Lowry -7 et al. (22), respectively. Each point shows the average of duplicate determinations in one and expressed as per cent of typical experiment of five similar experiments, values obtained at l-day. content

Low concentration non-essential

[FD (4,6), ration),

hormones

pyruvate

(7,17)

in primary culture

ed by other (18),

CLeibovitz) Waymouth's However, with

these

amino

(insulin is

as other

acids,

especially

for

culture

the

under

in original

Dulbecco

and Vogt's

modification

medium (3), Ham's F-12 medium MB 752/l

mitosis

(17)]

was not

was observed

forms

successful

(2),

which

minimum medium

Wiiliams'

mediumE

the

induction

above media

serum-free

have been adopt-

of Eagle's

for

when each of the

In our

[Eagle's

of Na-

proliferation

conditions. media

in prepa-

concentration

of hepatocyte

available

medium

(manuscript

and high

serum-free

or modified

in the culture

proline

induction

the use of commercially

groups

factors

and glucagon)(4,6,7),

essential

monolayer system,

medium

of Ca2+ as well

essential (19), (2U),

L-15 and

of mitosis. was supplemented

factors.

ACKi?OWLEDGi%ENT: We thank Professors H. Terayama and M. Yamada of University of Tokyo and Drs. J. Enami and H. Ohtake in our laboratory for reading the manuscript. Supported in part by grants from the Ministry of Education, Science and Culture of Japan. REFERENCES

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Bissell, D. M., Hammaker, L. E., and Meyer, U, A. (1973) 59, 722-734. Bonney, R. J., Becker, J. E., Walker, P. R., and Potter, In Vitro 9, 399-413. 264

J. Cell

Biol.

V. R. (1974)

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BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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