Vol.
No.
184,
April
1, 1992
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
15, 1992
RECEPTOR-MEDIATED CULTURED
FORMATION
ADULT
OF
RAT HEPATOCYTES
Seishiro Kazukiyo
'Kanagawa
Academy
of
2Faculty
of
Agriculture,
3Faculty
of
Bioscience
February
and
and
OF
and
Toshihiro
POLYSTYRENE
Akaike3
Sakado,
Kawasaki-shi
Chikusa-ku,
University,
PRIMARY
Takei'.
Technology,
Nagoya
AGGREGATES
LACTOSE-SUBSTITUTED
Yuka
Kobayashi'
Science
28,
ON
Tobe',
Biotechnology,
Midori-ku,
Received
MULTILAYER
225-230
Nagoya-shi
Tokyo
Yokohama-shi
Institute
227,
of
213.
Japan
464,
Japan
Technology,
Japan
1992
We used a lactose-substituted polystyrene, poly-i\l-p-vinylbenzyl-DSummary: lactonamide (PVLA). as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLAcoated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates. 0 1992 ACxkrnlC Press, 1°C.
A primary system
to
culture
artificial
A
on
various
hepatocyte
improved
by
using
laminin
to
whose
have
and
the
the
the
receptor-mediated hepatocytes in
hepatocyte
(6)
(7,8)
surfaces
recognition
following
to of
that
and
binding
adult
were
coated
PVLA
coated-dishes
1) by
primary
reported
that
functions (1,2),
were
fibronectin
substrata. rat with
a
We previously
2)
pointed
resemblance the
residues (9).
attached
lactose-substituted
from
B-D-galactose
hepatocytes
hepatocytes
showed
asialoglycoproteins
respects.
highly in
been
collagen
a model a hybrid
express
cellular
(PVLA).
attachment
to
has
their as
as
construct
hepatocytes It
of
to
made
substrata.
as
papers
clearance in
been
such
used
and
of
expression matrices
previous
extensively
proliferation
surfaces and
been functions
poly-N-p-vinylbenzyl-D-lactonamide
that
signals
liver
attempts
proteoglycans in
dishes
out
by
of
extracellular (5),
the
polystyrene,
solid
have in
and/or
attachment
We reported well
lot
functions
cultures
(3,4),
hepatocytes
metabolisms
liver.
differentiated
the
of
investigate
blood were
Calcium
to stream
specific ion
0006-29lX'92 225
was
$1.50
Copwigizr 0 1992 6~ Academic Press, fnc. All rights of reproduction in any form reserved.
Vol.
184,
No.
required
1, 1992
essentially
cytes
was
(10).
inhibited
addition,
dishes
(12).
It
based
on
cell
the
Thus,
PVLA
clustered
be
regarded
Recently, were
found
the to
form
stimulation
formation insulin. level
determined
by
collagen
and In
between during
cells
of
multilayer
in
the
factor
aggregates
differentiated
and
uptake
to
PVLA-coated
of
PVLA
than
as
well
dishes
receptors
was on
substratum
on
anchored
on
and the
PVLA
the (13).
a lower in
through
(14.15).
The
of
long-term
EGF
and
survivability,
ability
those
substratum
substratum
insulin
concentrations
expressed
functions
[3H]-thymidine
dishes
attached
on
In
asialoglycoprotein.
(ECF)
depended
we
study,
asialoglycoprotein formation
attempted
to
receptors of
multilayer
clarify on
of
proliferation
monolayer
cultures
on
the
the cell
role
surface
of
the
and
PVLA
interaction substratum
aggregates.
MATERIALS Preparation ml aliquot polystyrene solution Dulbecco's
of
aggregates
growth
B (11). asialoceruloplasmin-
PVLA-coated
residues model
hepato-
fibronectin.
this
the
on
hepatocytes
aggregates
on
asialoglycoprotein
a synthetic
epidermal
into
cytochalasin
attachment
spherical-shaped
multilayer The
higher
cultured
between
as
by cultured
hepatocyte
COMMUNICATIONS
asialoglycoproteins
not
B-D-galactose
stable of
of
those
the
of
hepatocytes
interaction
and may
uptake but
of in
that
specific
BIOPHYSICALRESEARCH
colchicine,
retained
assumed
surface
The
morphology
was
was
3)
by
spherical
coated
the
BIOCHEMICALAND
PVLA was of culture dish. of an aqueous PVLA solution dish (Falcon 1008) at room was decanted and the surface phosphate buffer solution.
AND
METHODS
synthesized (0.01%) was temperature. of dish was
as reported (16,17). placed into 35-mm After IO min. rinsed three times
An 1 diameter the PVLA with a
Preparation and primary culture of hepatocytes. Adult rat hepatocytes were prepared from 5-week old female Sprague-Dawley rats (150-200 g) by the method a slight modification. The basal medium was William's of Seglen (18) with 100 U/ml streptomycin and 1 ug/ml medium E containing 100 U/ml penicillin, The hormone-supplemented medium was made by supplementing EGF fungizone. insulin (Sigma Chemical Co., US) and dexamethasome (Takara Shuzo, Japan), (Si ma Chemical Co.) to the basal medium at final concentration of 50 rig/ml, IO- !I M. and 10-g M, respectively. The isolated hepatocytes were finally cells/ml in the basal medium. suspended at a concentration of 5 x 105 Aliquots (2.0 ml) of the cell suspension were placed into PVLA-coated dishes and incubated for 4 h in a humidified atmosphere of 5% CO2 and 95% air at After incubation, the medium and dead hepatocytes were removed and 37°C. placed with the hormone-supplemented medium containing cytochalasin B (Sigma colchicine (Sigma Chemical Co.), PVLA molecules, aprotinin Chemical Co.), and fetal calf serum (GIBCO, US) at various concentra(Sigma Chemical Co.), tions, and then cultured for 48 h. Treatment incubated hepatocytes containing
with for
allo A lectin. 4 h in the basal were removed and concentrations various
Hepatocytes seeded to PVLA-coated dishes were the medium and dead medium. After incubation, placed with the hormone-supplemented medium of allo A lectin (Allomyrina dichotoma
226
Vol.
184,
No.
BIOCHEMICAL
1, 1992
AND
Japan) which lectin, COSMO BIO Co., then incubated for 20 min at 37°C residues of PVLA substratum, and then
BIOPHYSICALRESEARCH
can specifically in order to cultured for
COMMUNICATIONS
bind B-D-galactose, block exposed B-D-galactose 48 h.
MorphologiAnchored were gently diameter of particle-size
Morphological observation and measurement of aggregate diameter. cal observation was carried out using a phase-contrast microscope. multilayer aggregates of hepatocytes cultured on PVLA substratum The average removed from the dish with a silicon rubber scraper. measured with electronic one thousand of the aggregates was analyzer (MULTISIZER II, Coulter Electronic Ltd., UK).
RESULTS When were not
freshly
cultured
on
spread
then
pm within
formed 48
substratum
the
h of
and
substratum
formed
was
continuous
hepatocytes
laminin.
cultured
a
in
the
medium,
shape
to
The
morphological
on
contrast on
to
adhesive
hormone-supplemented
that
of of
proteins.
the
of as
cells
cells
80-100 anchored
collagen,
cultured
on
flattened the
excluded
Fiq. 1. Phase-contrast microscopic features of adult rat hepatocytes on PVLA substratum for 48 h. (A) In the basal medium. (B) In the supplemented medium (50 rig/ml of EGF). (C) In the hormone-supplemented excluded calcium ion. The scale bar represents 100 urn.
227
other,
of
such
did
hormone-
each
formation
On medium
cells
diameter
the
cells,
the
with
proteins
features
the In
assemble
the
adhesive
of
IA).
average
In contrast,
1B). observed
with
single
most
(Fig.
gradually
not
striking
monolayer
spherical
basal
aggregates
(Fig. was
the
migrated
multilayer
culture
hepatocytes,
spherical
cells
anchored
DISCUSSION
rat in
their
aggregates
fibronectin
adult
retained
medium,
multilayer
PVLA
PVLA
and
supplemented and
isolated
AND
and
other
and hand,
calcium
cultured hormonemedium
ion,
Vol.
184, No. 1, 1992
z t
120
s
100
F b 0
80
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
120
iiE
40
.! 0
20
B
600 i-
1
~ 1 o-7
10-G
PVLA
and
of
inhibitors
such
average cytochalasin hand,
average is
mediated
by
the
B
aggregates
of
receptor-mediated into
formation
the
the
same
as
formation
1C).
aggregates
on
the
of
actin
uptake
scarcely
as
by
the
It
was
of
by
which
was
but
not
colchicine,
may of
On decreased
formation
uptake
the by
28).
hepatocytes
substratum
the
2A,
influenced
(Fig.
inhibited
PVLA
Fig.
remarkably
into
the
using
functions.
was
that
well
in
functions
was
multilayer
examined
shown
filament
aggregates
on
as
was
As was
suggested
cultured
of
substratum
microtubule
receptors
interaction of
culture
surface, block multilayer culture
multilayer
the
asialoglycoproteins
interaction
(Fig.
multilayer
be
related
to
asialoglycoproteins
hepatocytes. The
the
was
colchicine.
of
of
This
hepatocytes
form
on
of
of
(11).
to
PVLA
inhibitor
inhibitor
uptake
o-5
I (M)
aggregates
ion
aggregates
asialoglycoprotein
cytochalasin
calcium
B and
an
10.7 concentration
multilayer
on
diameter an
10-B
(10).
multilayer is
the
that
by
of
which
reported
of
cultured
B which
colchicine
stable
essentially
cytochalasin
diameter
control Colchicine
cytoskeletons
hepatocytes as
form
ion
of
0
colchicine on the formation of on PVLA substratum. The cells medium for 48 h. (A) The culture of cytochalasin B. (B) The culture of colchicine. The values of mean + multilayer aggregates.
hepatocytes
influence
aggregates
20
B and hepatocytes
requirement into
The
not
calcium The
asialoglycoproteins
other
could
required
substratum.
40
(M)
Fia. 2. Effects of cytochalasin multilayer aggregates of adult rat were cultured in the hormone-supplemented medium contained various concentrations medium contained various concentrations SD were calculated from one thousand
Hepatocytes
60
10-5
B concentration
slightly
80
~
control Cytochalasin
spread
100
between
multilayer medium
and exposed
by
aggregates in
order
treating
to
medium.
was As
was block
shown
residues
Fig.
examined
dishes of
effectively in
and
PVLA by
adding
asialoglycoprotein
PVLA-coated
D-D-galactose aggregates
hepatocytes
PVLA
3A,
the
228
allo-A
PVLA
added diameter
The PVLA
into
on
the
in
order
cell to
formation
molecules of
the
molecules
lectin
substratum. by
average
during
receptors
with
inhibited
substratum
the
of in
aggregates
the
Vol.
BIOCHEMICAL
184, No. 1, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
40: 20
L
0 PVLA
concentration
0
(Kg/ml)
2
Allo
4
A lectin
6
6
10
concentration
(Kg/ml)
Effects of PVLA molecules and allo A lectin on the formation of Fig. 3. The cells multilayer aggregates of adult rat hepatocytes on PVLA substratum. (A) The culture were cultured in the hormone-supplemented medium for 48 h. (6) PVLA-coated medium contained various concentrations of PVLA molecules. dishes were used after the treatment with various concentrations of allo A lectin solutions as described in Materials and Methods. The values of mean ? SD were calculated from one thousand multilayer aggregates.
decreased
with
increasing
concentration
of
PVLA
of
In
cultures
culture.
inhibited
by
lectin
(Fig.
receptor
that
the
by
blocking
the
role
in
floating
spheroids
protease
inhibitor, of
with
of
be
to
multilayer ug/ml
of
aprotinin,
5% of
calf
serum
were
The
formation
scarcely
influenced
by
aprotinin
A PVLA to
suggested
substratum
was
inhibited
surface,
substratum.
PVLA
allo with
results
cell
and It
substratum
h
also
specifically
is
by
likely
plays
in
the
5% of
of
24
by
a
The
adding
major
which inhibitors.
whether
the serum.
calf
on
calf
serum, 86
multilayer calf
229
f
28
of
The
94
aggregates serum.
These
0.12
on
PVLA
results
of and
a
diameter alone,
ug/ml urn
is
multilayer
average
+ 23
of
substratum
medium
with urn,
PVLA
formation
some
formation
aprotinin
formed
and
on
protease
hormone-supplemented
urn.
and
cultured
several
aggregates
examined
*
hepatocytes (spheroids).
inhibited
aprotinin
with 88
of
compete
bind
48
was
ug/ml can
the
containing
multilayer
formed
and
rat
aggregates
serum
We
0.12
PVLA
PVLA
and
adult
completely
by
aggregates
respectively.
that
compare
influenced
formation
in
aggregates.
calf
spheroids. was
the
can
on
hepatocytes
spheroidal
and
on
of
hepatocytes
These
receptors residues
of
2.5
substratum.
Higher
molecules.
medium
A lectin
aggregates
(19-21)
can
than
culture
PVLA
multilayer
floating
interest
these
aggregates
of
between of
reported
formed
allo
&D-galactose
formation
surfaces
and
multilayer
interaction
was
the
asialoglycoprotein
exposed
the It
is
of
of of
that
residues
formation
blocking
It
binding,
A lectin,
more
in
PVLA
spreading
allo
with
molecules
added
about
with
treatment
D-D-galactose
of
brought
treated
PVLA
for
exposed
molecules
the
36).
substratum
concentration
with
aprotinin
91
substratum suggested
+ 21
urn, was that
Vol.
184, No. 1, 1992
anchored
multilayer
formed
by In
rat
a different
form
stable to
be
aggregates. are
in
attached
on
PVLA
floating
the
studies
using the
in
substratum on
aggregates trigger
reveal
cultured
the
receptors
to
to
hepatocytes
described
multilayer able
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from
observations
Further progress
of
mechanism
summary,
asialoglycoprotein
found
cell
aggregates
hepatocytes
between to
BIOCHEMICAL
on
study
cell
anchored
mechanism
of
suggested the
were
on
and
substratum. formation
culture receptor-mediated
that
specific
surface
receptor-mediated hepatocyte
substratum
spheroids. this
required the
PVLA
system
adult
interaction PVLA
substratum
EGF of
was
also
multilayer
described regulation
here in
the
behaviors.
REFERENCES
:: 3. 4. 5. 6.
7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
Michal opoulos, G., and Pitot, H.C. (1975) Exp. Cell Res. 94, 70-78. Rubin, K ., Hook, M., Obrink, B., and Timpl, R. (1981) Cell 24. 463-470. Marceau, N., Noel, M., and Deschenes, J. (1982) in vitro 18, l-11. Johansson, S., and Hook, M. (1984) J. Cell Biol. 98, 810-817. Timpl, R., Johansson, S., Delden, V.V., Cberbaumer, I., and Hook, M. (1983) J. Biol. Chem. 258, 8922-8927. Spray, D.C., Fujita, M., Saez, J.C., Choi, H., Watanabe, T., Hertzberg, E., Rosenberg, L.C., and Reid, L.M. (1987) J. Cell Biol. 105. 541-551. Kobayashi, A., Akaike, T., Kobayashi, K., and Sumitomo, H. (1986) Makromol. Chem., Rapid Commun. 7, 645-650. Kobayashi, K., Sumitomo, H., Kobayashi, A., and Akaike, T. (1988) J. Macromol. Sci. Chem. A25, 655-667. Ashwell, G., and Harford, J. (1982) Ann. Rev. Biochem. 51, 531-554. Pricer, W.E. Jr., and Ashwell, G. (1971) J. Biol. Chem. 246, 4825-4833. Kolset, S.O., Tolleshauf, H., and Berg, T. (1979) Exp. Cell Res. 122, 159-167. Hook, M., Rubin, K., Oldberg, A., Obrink, B., and Vaheri, A. (1977) Biochem. Biophys. Res. Commun. 29. 726-733. Akaike, T., Kobayashi, A., Kobayashi, K., and Sumitomo, H. (1989) J. Bioactive Compatible Polym. 4, 51-55. Tobe, S., Takei, Y., Maeda, A., Yagawa, A., Kobayashi, K., and Akakike, T. (1990) Artif. Organs 14 (Supple 2). 262-264. Tobe, S., Takei, Y., Kobayashi, K., and Akakike, T. (1990) Commun. Applied Cell Biol. 8, 16-22. Kobayashi, K., Sumitomo, H., and Ina, Y. (1983) Polym. J. 15, 667-671. Y. (1985) Polym. J. 17, 567-575. Kobayashi, K.. Sumitomo, H., and Ina, Seglen.P.0. (1976) In Methods in Cell Biology (D.M. Prescott, eds) vol. 13. pp. 29-83. Academic Press, New York. Koide, N., Sakaguchi, K., Koide. Y., Asano, K., Kawaguchi, M., Matsushima, H ., Takenami, T., Shinji, T., Mori, M.. and Tsuji, T. (1990) Exp. Cell Res. 186, 227-235. Zhong-Tong, J., Bernard, O., and Alvarez, F. (1990) Exp. Cell Res. 189. 87-92. Sakai, Y., and Suzuki, M. (1991) In Animal Cell Culture and Production of Biologicals (R. Sasaki and K. Ikura, eds) pp. 127-134, Kluwer Academic Press, Dordrecht Boston London.
230
No.
184,
April
1, 1992
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS Pages
15, 1992
RECEPTOR-MEDIATED CULTURED
FORMATION
ADULT
OF
RAT HEPATOCYTES
Seishiro Kazukiyo
'Kanagawa
Academy
of
2Faculty
of
Agriculture,
3Faculty
of
Bioscience
February
and
and
OF
and
Toshihiro
POLYSTYRENE
Akaike3
Sakado,
Kawasaki-shi
Chikusa-ku,
University,
PRIMARY
Takei'.
Technology,
Nagoya
AGGREGATES
LACTOSE-SUBSTITUTED
Yuka
Kobayashi'
Science
28,
ON
Tobe',
Biotechnology,
Midori-ku,
Received
MULTILAYER
225-230
Nagoya-shi
Tokyo
Yokohama-shi
Institute
227,
of
213.
Japan
464,
Japan
Technology,
Japan
1992
We used a lactose-substituted polystyrene, poly-i\l-p-vinylbenzyl-DSummary: lactonamide (PVLA). as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLAcoated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates. 0 1992 ACxkrnlC Press, 1°C.
A primary system
to
culture
artificial
A
on
various
hepatocyte
improved
by
using
laminin
to
whose
have
and
the
the
the
receptor-mediated hepatocytes in
hepatocyte
(6)
(7,8)
surfaces
recognition
following
to of
that
and
binding
adult
were
coated
PVLA
coated-dishes
1) by
primary
reported
that
functions (1,2),
were
fibronectin
substrata. rat with
a
We previously
2)
pointed
resemblance the
residues (9).
attached
lactose-substituted
from
B-D-galactose
hepatocytes
hepatocytes
showed
asialoglycoproteins
respects.
highly in
been
collagen
a model a hybrid
express
cellular
(PVLA).
attachment
to
has
their as
as
construct
hepatocytes It
of
to
made
substrata.
as
papers
clearance in
been
such
used
and
of
expression matrices
previous
extensively
proliferation
surfaces and
been functions
poly-N-p-vinylbenzyl-D-lactonamide
that
signals
liver
attempts
proteoglycans in
dishes
out
by
of
extracellular (5),
the
polystyrene,
solid
have in
and/or
attachment
We reported well
lot
functions
cultures
(3,4),
hepatocytes
metabolisms
liver.
differentiated
the
of
investigate
blood were
Calcium
to stream
specific ion
0006-29lX'92 225
was
$1.50
Copwigizr 0 1992 6~ Academic Press, fnc. All rights of reproduction in any form reserved.
Vol.
184,
No.
required
1, 1992
essentially
cytes
was
(10).
inhibited
addition,
dishes
(12).
It
based
on
cell
the
Thus,
PVLA
clustered
be
regarded
Recently, were
found
the to
form
stimulation
formation insulin. level
determined
by
collagen
and In
between during
cells
of
multilayer
in
the
factor
aggregates
differentiated
and
uptake
to
PVLA-coated
of
PVLA
than
as
well
dishes
receptors
was on
substratum
on
anchored
on
and the
PVLA
the (13).
a lower in
through
(14.15).
The
of
long-term
EGF
and
survivability,
ability
those
substratum
substratum
insulin
concentrations
expressed
functions
[3H]-thymidine
dishes
attached
on
In
asialoglycoprotein.
(ECF)
depended
we
study,
asialoglycoprotein formation
attempted
to
receptors of
multilayer
clarify on
of
proliferation
monolayer
cultures
on
the
the cell
role
surface
of
the
and
PVLA
interaction substratum
aggregates.
MATERIALS Preparation ml aliquot polystyrene solution Dulbecco's
of
aggregates
growth
B (11). asialoceruloplasmin-
PVLA-coated
residues model
hepato-
fibronectin.
this
the
on
hepatocytes
aggregates
on
asialoglycoprotein
a synthetic
epidermal
into
cytochalasin
attachment
spherical-shaped
multilayer The
higher
cultured
between
as
by cultured
hepatocyte
COMMUNICATIONS
asialoglycoproteins
not
B-D-galactose
stable of
of
those
the
of
hepatocytes
interaction
and may
uptake but
of in
that
specific
BIOPHYSICALRESEARCH
colchicine,
retained
assumed
surface
The
morphology
was
was
3)
by
spherical
coated
the
BIOCHEMICALAND
PVLA was of culture dish. of an aqueous PVLA solution dish (Falcon 1008) at room was decanted and the surface phosphate buffer solution.
AND
METHODS
synthesized (0.01%) was temperature. of dish was
as reported (16,17). placed into 35-mm After IO min. rinsed three times
An 1 diameter the PVLA with a
Preparation and primary culture of hepatocytes. Adult rat hepatocytes were prepared from 5-week old female Sprague-Dawley rats (150-200 g) by the method a slight modification. The basal medium was William's of Seglen (18) with 100 U/ml streptomycin and 1 ug/ml medium E containing 100 U/ml penicillin, The hormone-supplemented medium was made by supplementing EGF fungizone. insulin (Sigma Chemical Co., US) and dexamethasome (Takara Shuzo, Japan), (Si ma Chemical Co.) to the basal medium at final concentration of 50 rig/ml, IO- !I M. and 10-g M, respectively. The isolated hepatocytes were finally cells/ml in the basal medium. suspended at a concentration of 5 x 105 Aliquots (2.0 ml) of the cell suspension were placed into PVLA-coated dishes and incubated for 4 h in a humidified atmosphere of 5% CO2 and 95% air at After incubation, the medium and dead hepatocytes were removed and 37°C. placed with the hormone-supplemented medium containing cytochalasin B (Sigma colchicine (Sigma Chemical Co.), PVLA molecules, aprotinin Chemical Co.), and fetal calf serum (GIBCO, US) at various concentra(Sigma Chemical Co.), tions, and then cultured for 48 h. Treatment incubated hepatocytes containing
with for
allo A lectin. 4 h in the basal were removed and concentrations various
Hepatocytes seeded to PVLA-coated dishes were the medium and dead medium. After incubation, placed with the hormone-supplemented medium of allo A lectin (Allomyrina dichotoma
226
Vol.
184,
No.
BIOCHEMICAL
1, 1992
AND
Japan) which lectin, COSMO BIO Co., then incubated for 20 min at 37°C residues of PVLA substratum, and then
BIOPHYSICALRESEARCH
can specifically in order to cultured for
COMMUNICATIONS
bind B-D-galactose, block exposed B-D-galactose 48 h.
MorphologiAnchored were gently diameter of particle-size
Morphological observation and measurement of aggregate diameter. cal observation was carried out using a phase-contrast microscope. multilayer aggregates of hepatocytes cultured on PVLA substratum The average removed from the dish with a silicon rubber scraper. measured with electronic one thousand of the aggregates was analyzer (MULTISIZER II, Coulter Electronic Ltd., UK).
RESULTS When were not
freshly
cultured
on
spread
then
pm within
formed 48
substratum
the
h of
and
substratum
formed
was
continuous
hepatocytes
laminin.
cultured
a
in
the
medium,
shape
to
The
morphological
on
contrast on
to
adhesive
hormone-supplemented
that
of of
proteins.
the
of as
cells
cells
80-100 anchored
collagen,
cultured
on
flattened the
excluded
Fiq. 1. Phase-contrast microscopic features of adult rat hepatocytes on PVLA substratum for 48 h. (A) In the basal medium. (B) In the supplemented medium (50 rig/ml of EGF). (C) In the hormone-supplemented excluded calcium ion. The scale bar represents 100 urn.
227
other,
of
such
did
hormone-
each
formation
On medium
cells
diameter
the
cells,
the
with
proteins
features
the In
assemble
the
adhesive
of
IA).
average
In contrast,
1B). observed
with
single
most
(Fig.
gradually
not
striking
monolayer
spherical
basal
aggregates
(Fig. was
the
migrated
multilayer
culture
hepatocytes,
spherical
cells
anchored
DISCUSSION
rat in
their
aggregates
fibronectin
adult
retained
medium,
multilayer
PVLA
PVLA
and
supplemented and
isolated
AND
and
other
and hand,
calcium
cultured hormonemedium
ion,
Vol.
184, No. 1, 1992
z t
120
s
100
F b 0
80
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
120
iiE
40
.! 0
20
B
600 i-
1
~ 1 o-7
10-G
PVLA
and
of
inhibitors
such
average cytochalasin hand,
average is
mediated
by
the
B
aggregates
of
receptor-mediated into
formation
the
the
same
as
formation
1C).
aggregates
on
the
of
actin
uptake
scarcely
as
by
the
It
was
of
by
which
was
but
not
colchicine,
may of
On decreased
formation
uptake
the by
28).
hepatocytes
substratum
the
2A,
influenced
(Fig.
inhibited
PVLA
Fig.
remarkably
into
the
using
functions.
was
that
well
in
functions
was
multilayer
examined
shown
filament
aggregates
on
as
was
As was
suggested
cultured
of
substratum
microtubule
receptors
interaction of
culture
surface, block multilayer culture
multilayer
the
asialoglycoproteins
interaction
(Fig.
multilayer
be
related
to
asialoglycoproteins
hepatocytes. The
the
was
colchicine.
of
of
This
hepatocytes
form
on
of
of
(11).
to
PVLA
inhibitor
inhibitor
uptake
o-5
I (M)
aggregates
ion
aggregates
asialoglycoprotein
cytochalasin
calcium
B and
an
10.7 concentration
multilayer
on
diameter an
10-B
(10).
multilayer is
the
that
by
of
which
reported
of
cultured
B which
colchicine
stable
essentially
cytochalasin
diameter
control Colchicine
cytoskeletons
hepatocytes as
form
ion
of
0
colchicine on the formation of on PVLA substratum. The cells medium for 48 h. (A) The culture of cytochalasin B. (B) The culture of colchicine. The values of mean + multilayer aggregates.
hepatocytes
influence
aggregates
20
B and hepatocytes
requirement into
The
not
calcium The
asialoglycoproteins
other
could
required
substratum.
40
(M)
Fia. 2. Effects of cytochalasin multilayer aggregates of adult rat were cultured in the hormone-supplemented medium contained various concentrations medium contained various concentrations SD were calculated from one thousand
Hepatocytes
60
10-5
B concentration
slightly
80
~
control Cytochalasin
spread
100
between
multilayer medium
and exposed
by
aggregates in
order
treating
to
medium.
was As
was block
shown
residues
Fig.
examined
dishes of
effectively in
and
PVLA by
adding
asialoglycoprotein
PVLA-coated
D-D-galactose aggregates
hepatocytes
PVLA
3A,
the
228
allo-A
PVLA
added diameter
The PVLA
into
on
the
in
order
cell to
formation
molecules of
the
molecules
lectin
substratum. by
average
during
receptors
with
inhibited
substratum
the
of in
aggregates
the
Vol.
BIOCHEMICAL
184, No. 1, 1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60
40: 20
L
0 PVLA
concentration
0
(Kg/ml)
2
Allo
4
A lectin
6
6
10
concentration
(Kg/ml)
Effects of PVLA molecules and allo A lectin on the formation of Fig. 3. The cells multilayer aggregates of adult rat hepatocytes on PVLA substratum. (A) The culture were cultured in the hormone-supplemented medium for 48 h. (6) PVLA-coated medium contained various concentrations of PVLA molecules. dishes were used after the treatment with various concentrations of allo A lectin solutions as described in Materials and Methods. The values of mean ? SD were calculated from one thousand multilayer aggregates.
decreased
with
increasing
concentration
of
PVLA
of
In
cultures
culture.
inhibited
by
lectin
(Fig.
receptor
that
the
by
blocking
the
role
in
floating
spheroids
protease
inhibitor, of
with
of
be
to
multilayer ug/ml
of
aprotinin,
5% of
calf
serum
were
The
formation
scarcely
influenced
by
aprotinin
A PVLA to
suggested
substratum
was
inhibited
surface,
substratum.
PVLA
allo with
results
cell
and It
substratum
h
also
specifically
is
by
likely
plays
in
the
5% of
of
24
by
a
The
adding
major
which inhibitors.
whether
the serum.
calf
on
calf
serum, 86
multilayer calf
229
f
28
of
The
94
aggregates serum.
These
0.12
on
PVLA
results
of and
a
diameter alone,
ug/ml urn
is
multilayer
average
+ 23
of
substratum
medium
with urn,
PVLA
formation
some
formation
aprotinin
formed
and
on
protease
hormone-supplemented
urn.
and
cultured
several
aggregates
examined
*
hepatocytes (spheroids).
inhibited
aprotinin
with 88
of
compete
bind
48
was
ug/ml can
the
containing
multilayer
formed
and
rat
aggregates
serum
We
0.12
PVLA
PVLA
and
adult
completely
by
aggregates
respectively.
that
compare
influenced
formation
in
aggregates.
calf
spheroids. was
the
can
on
hepatocytes
spheroidal
and
on
of
hepatocytes
These
receptors residues
of
2.5
substratum.
Higher
molecules.
medium
A lectin
aggregates
(19-21)
can
than
culture
PVLA
multilayer
floating
interest
these
aggregates
of
between of
reported
formed
allo
&D-galactose
formation
surfaces
and
multilayer
interaction
was
the
asialoglycoprotein
exposed
the It
is
of
of of
that
residues
formation
blocking
It
binding,
A lectin,
more
in
PVLA
spreading
allo
with
molecules
added
about
with
treatment
D-D-galactose
of
brought
treated
PVLA
for
exposed
molecules
the
36).
substratum
concentration
with
aprotinin
91
substratum suggested
+ 21
urn, was that
Vol.
184, No. 1, 1992
anchored
multilayer
formed
by In
rat
a different
form
stable to
be
aggregates. are
in
attached
on
PVLA
floating
the
studies
using the
in
substratum on
aggregates trigger
reveal
cultured
the
receptors
to
to
hepatocytes
described
multilayer able
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from
observations
Further progress
of
mechanism
summary,
asialoglycoprotein
found
cell
aggregates
hepatocytes
between to
BIOCHEMICAL
on
study
cell
anchored
mechanism
of
suggested the
were
on
and
substratum. formation
culture receptor-mediated
that
specific
surface
receptor-mediated hepatocyte
substratum
spheroids. this
required the
PVLA
system
adult
interaction PVLA
substratum
EGF of
was
also
multilayer
described regulation
here in
the
behaviors.
REFERENCES
:: 3. 4. 5. 6.
7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21.
Michal opoulos, G., and Pitot, H.C. (1975) Exp. Cell Res. 94, 70-78. Rubin, K ., Hook, M., Obrink, B., and Timpl, R. (1981) Cell 24. 463-470. Marceau, N., Noel, M., and Deschenes, J. (1982) in vitro 18, l-11. Johansson, S., and Hook, M. (1984) J. Cell Biol. 98, 810-817. Timpl, R., Johansson, S., Delden, V.V., Cberbaumer, I., and Hook, M. (1983) J. Biol. Chem. 258, 8922-8927. Spray, D.C., Fujita, M., Saez, J.C., Choi, H., Watanabe, T., Hertzberg, E., Rosenberg, L.C., and Reid, L.M. (1987) J. Cell Biol. 105. 541-551. Kobayashi, A., Akaike, T., Kobayashi, K., and Sumitomo, H. (1986) Makromol. Chem., Rapid Commun. 7, 645-650. Kobayashi, K., Sumitomo, H., Kobayashi, A., and Akaike, T. (1988) J. Macromol. Sci. Chem. A25, 655-667. Ashwell, G., and Harford, J. (1982) Ann. Rev. Biochem. 51, 531-554. Pricer, W.E. Jr., and Ashwell, G. (1971) J. Biol. Chem. 246, 4825-4833. Kolset, S.O., Tolleshauf, H., and Berg, T. (1979) Exp. Cell Res. 122, 159-167. Hook, M., Rubin, K., Oldberg, A., Obrink, B., and Vaheri, A. (1977) Biochem. Biophys. Res. Commun. 29. 726-733. Akaike, T., Kobayashi, A., Kobayashi, K., and Sumitomo, H. (1989) J. Bioactive Compatible Polym. 4, 51-55. Tobe, S., Takei, Y., Maeda, A., Yagawa, A., Kobayashi, K., and Akakike, T. (1990) Artif. Organs 14 (Supple 2). 262-264. Tobe, S., Takei, Y., Kobayashi, K., and Akakike, T. (1990) Commun. Applied Cell Biol. 8, 16-22. Kobayashi, K., Sumitomo, H., and Ina, Y. (1983) Polym. J. 15, 667-671. Y. (1985) Polym. J. 17, 567-575. Kobayashi, K.. Sumitomo, H., and Ina, Seglen.P.0. (1976) In Methods in Cell Biology (D.M. Prescott, eds) vol. 13. pp. 29-83. Academic Press, New York. Koide, N., Sakaguchi, K., Koide. Y., Asano, K., Kawaguchi, M., Matsushima, H ., Takenami, T., Shinji, T., Mori, M.. and Tsuji, T. (1990) Exp. Cell Res. 186, 227-235. Zhong-Tong, J., Bernard, O., and Alvarez, F. (1990) Exp. Cell Res. 189. 87-92. Sakai, Y., and Suzuki, M. (1991) In Animal Cell Culture and Production of Biologicals (R. Sasaki and K. Ikura, eds) pp. 127-134, Kluwer Academic Press, Dordrecht Boston London.
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