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BIOTEC 00595
Summary
Introduction
Electrid field effects on cells have beenstudied since the 18th century and applied
to cell engineering in the 197Os, exemplified by electrically induced cell fusion
(Zimmermann, 1982; Berg et al., 1984; Kurischiko and Berg, 1986) and electro-pola-
tion (Neumann, 1984; Karube et al., 1985). Most of the research regarding electrical
effects on cells have been done with cells suspended between two electrodes, anodic
and cathodic ones. The level of electric field varies from large pulses in the order of
500 V cm-’ to a small potential of 100 mV cm-’ (Grosse et al., 1988; Berg, 1987).
On the other hand, intensive research has long been made on the interaction of
cells and the surface charge of cell-supporting material as weli as on ionic con-
centration of cell culture medium. Investigation of biomedical material has sug-
gested that weak electrostatic field effect should be important for cell adsorption
and cell proliferation (Okano et al., 1981). The modulation of ionic balance across a
cell membrane might induce the expression of untapped cellular functions for
various types of cells. These facts indicate that if the electrode was used as a cell
supporting material, the interactions between cells and material surface could be
electrochemically controlled. Our current results of the electrical effects on mam-
malian cells attached to the surface of an electrode suggest that the structure of
plasma membrane and cytoskeleton are appreciably modulated by electrical stimu-
lation (Yaoita et al., 1988a, 1989). Furthermore, HeLa cells cultured on the
electrode surface are drastically inhibited in cell proliferation with a morphological
change (Yaoita et al., 1988b, 1990).
Controlled cell proliferation has become attractive in cell engineering because
mammalian cells are becoming one of the major materials used to produce func-
tional proteins. Cell cycle and cell proliferation are mostly controlled using chemical
factors represented by growth factors. In this research electrically controlled cell
proliferation has been demonstrated with MKN45 cells. The MKN45 cell strained
from human stomach cancer tissue in 1975 (Motoyama et al., 1979) specifically
produces CEA (carcinoembryonic antigen, Gold and Freedman, 1965a,b; Benchi-
mol et al., 1989). MKN45 cells were cultured on the surface of an electrode and
cellular behavior was observed under controlled potential.
C. E.
Cell
A7
\
Pt-coated plate
Culture dish
Fig. 1. Schematic illustration of experimental set-up for electrically controlled cell culture. MKN45 cells
were cultured on the surface of a platinum-coaled electrode which was installed in a culture dish. To the
cover of culture dish, a working electrode (Pt), counter electrode (Pl) and reference electrode (Ag/AgCl)
were fixed.
was poured into the culture dish in which the 3-electrode system was installed. After
the cells were attached to the surface of an electrode, electrode potential was
immediately controlled against the reference electrode by a potentiostat (Type
HAB-151 of Hokuto Denko). The culture dish was placed in a 5% CO1 37°C
incubator.
MKN45 cells were cultured for more than 100 h on the surface of an electrode
whose potential was controlled. Morphological observation and counting of cell
population were performed once a day with an inverted microscope which was
connected to a TV monitor and videotape. During the observation of cell mor-
phology, potential application was stopped. Potential-controlled culture started
again after counting of the cell population. The pictures of cells were photographed
and videotaped if necessary. The cell population was measured at 10 fields of
microscope (one field is 3 X 10m3 cm’) for each plate and averaged.
in a saline solution containing 100 PM ANS at 37°C and washed with PBS. The
In,O, electrode was placed in a cuvette for fluorescent measurement with a
platinum counter electrode and an Ag/AgCl reference electrode. The polarization
of the ANS fluorescence in MKN45 cell membrane was detected with a fluorescence
spectrophotometer (Jasco, FP-550A) under potential application, (Yaoita et al.,
1988a,b). A polarizer and an analyzer were mounted at the excitation site and at the
emission site of a cuvette, respectively. The fluorescent intensity of polarized
emission was measured, following which the fluorescent intensity was measured
after the analyzer was rotated 90 O. The degree of polarization was calculated by the
formula of P = (I,, - I,)/( I,, + I,). Furthermore the fluorescent anisotropy was
calculated as r = 2P/(3 - P) (Miyake et al.. 1984; Kashiwayagi and Kurihara,
1985). I,, stands for the parallel component of the fluorescent intensity to the plane
of polarized excitation beam, and I, for the perpendicular component. Increase of
fluorescent anisotropy may correspond to the loss of membrane fluidity (Tao, 1969;
Lentz. 1989).
Results
MKN45 cells were cultured on the surface of an electrode for more than 100 h at
several potentials and the cell morphology and proliferation of MKN45 cells were
observed. The electrical effect of resting potential was discussed at first. Fig. 2
shows the picture of MKN45 cells cultured both on a platinum-coated plate and on
a normal plastic plate. There were no prominent differences in cell morphology, and
cell attachment to the platinum-coated plate seemed better than that to the normal
plate. The proliferation profiles of both are shown in Fig. 3. With regard to cell
proliferation, cells grew on a platinum coated plate as well as on a normal plate.
These results indicated that platinum did not induce any harmful effects on cell
morphology and proliferation. However, when cells were cultured at the potential
above 0.4 V vs. Ag/AgCl, the rate of cell proliferation was markedly decreased with
a morphological change as shown in Figs. 4 and 5. The outline of each cell became
indistinctive and some cells became round in shape. The proliferation was stopped
when the potential was applied for 120 h. This indicates that the electrical effect of
the electrode surface induces the inhibition of cell proliferation at positive potentials
vs. Ag/AgCl. The potential dependency of cell proliferation is also shown in Fig. 6.
The cells whose proliferation was stopped were stained by trypan blue to confirm if
they were alive. As a result, more than 90% of cells cultured at 0.4 V vs Ag/AgCl
were not stained after 100 h culture (Table 1). These results suggest that cell
proliferation is electrically inhibited but the cell membrane was not fatally affected
by a breakdown of plasma membrane.
It was decided to begin proliferation again for the cells whose proliferation was
stopped at 0.4 V vs. Ag/AgCl. The applied potential was shifted from 0.4 V vs.
Ag/AgCl to 0.1 V vs. Ag/AgCl. As a result, cells started to proliferate again as
shown in Fig. 7. It should be noted that cell proliferation is restored by shifting
Fig 2. Morphology of MKN45 cells cultured on a platinum-coated plate (a) and on a tissue culture pl late
(b).
apI plied potential and the cells whose proliferation was halted still had the ability to
di\ ride.
The state of plasma membrane fluidity was studied by measuring fluorescr :nt
134
l‘!me I h
Fig. 3. Proliferation of MKN45 cells cultured on a tissue culture plate (0) and platinum-coated plate
electrode at resting potential (0).
anisotropy with a probe which was incorporated in the plasma membrane to clarify
the mechanism of electrical effect on plasma membrane structure. The time course
of fluorescent anisotropy change is shown in Fig. 8. The fluorescent anisotropy did
Fig. 4. Morphological change of MKN45 cells on a platinum-coated plate electrode controlled at 0.4 V
vs. Ag/AgCI for 120 h.
135
0 20 40 60 80 100 120
Time I h
Fig. 5. Proliferation halting of MKN45 cells by potential application. MKN45 cells were cultured on the
surface of a platinum-coaled plate electrode at 0.4 V vs. Ag/AgCI (0) and al resting potential (0). The
population of cells were counted through a inverted microscope.
O-0
\ o--o-o
Fig. 6. Dependency of cell proliferation on applied potential. MKN45 cells were cultured on a potential
controlled electrode for 4 d. Final cell numbers on the eleclrode were counted.
TABLE 1 ,
Trypan blue stain of MKN45 cells on a platinum-coated plate electrode after 120 h culture at 0.4 V, 0.5
V vs. Ag/AgCI
Potential
V vs. Ag/AgCl 0.4 0.5
Trypan blue stained
cells (%I) 7.0 15.9
136
I I
30 - o/o-o- 1
0
20 - / I
0
IO - O/O /
/ 0
A-0 4 l/
C,L&o-.-.-e/.’
0- I 1 I I I
0 50 100 150 200 250
Trme I h
Fig. 7. Controlled proliferation rate of MKN45 cells on a platinum-coated plate electrode. MKN45 cells
were cultured at 0.4 V vs. Ag/AgCI for 100 h, then (I) the applied potential was shifted to 0.1 V vs.
Ag/AgCI (0). Open circle shows the proliferation profiles at resting potential.
not change at the resting potential. However, when the potential of 0.4 V vs.
Ag/AgCl was applied to the electrode, it was increased. It is known that the
increase of fluorescent anisotropy reflects the decrease of membrane fluidity. The
120
II0
100
90
0 20 40 60 80
Time I h
Fig. 8. Time course of fluorescent anisotropy change of ANS in MKN45 cell membrane. MKN45 cells
were cultured on a surface of electrode and the fluorescent anisotropy under potential application was
measured by taking ANS as a fluorescent probe. The applied potentials were at 0.5 V (A), 0.4 V (A), 0.3 V
(0) vs. Ag/AgCl and resting potential (0). A r is the change in anisotropy, i.e., r% = lOO(r/r,,). where r,,
is the anisotropy at the beginning of potential application.
137
Discussion
Platinum compounds like cis platin are known to bind DNA and inhibit the
activity of DNA polymerase. No appreciably harmful effect on cell proliferation
and morphology, however, was observed when cells were cultured on a platinum-
coated plate. As regards the cell attachment, cells attached more strongly to the
surface of a platinum plate as compared with a normal plastic plate. It should be
noted that a platinum-coated plate can be used as a culture bed without any
harmful effects.
Electrical effects on cell proliferation were observed in the potential range in
which no electrolysis of water occurred. With an increasing applied potential, the
rate of cell proliferation was sharply depressed and some cells became round in
shape. It is indicated that there should be a critical potential which completely
inhibits cell proliferation. Proliferation of MKN45 cell is completely inhibited at 0.4
V vs. Ag/AgCl. The reversibility of the electrically induced halting of cell prolifera-
tion has been confirmed. When the applied potential was shifted from 0.4 V vs.
Ag/AgCl to 0.1 V vs. Ag/AgCl, cells started proliferating again, although there was
a time lag. This fact demonstrates that the electrical halting of cell proliferation is
not an irreversible process. It was concluded that our new cell culture method could
control cell proliferation reversibly.
The investigation of cell membrane structure clarified that the fluorescent ani-
sotropy started to increase at the critical potential of cell proliferation. The
fluorescent anisotropy is estimated from mobility of a fluorescent probe incorpo-
rated into the plasma membrane and reflects the state of membrane fluidity. The
increase of fluorescent anisotropy may be attributed to the decrease of membrane
fluidity. Plasma membrane structure is seemingly modulated to become rigid at
positive potentials. Although the mechanism of electrical effects on membrane
fluidity was not clear, one of the possible interpretations concerns the modulation of
the interaction between membrane charge and electrode. The positive electrode
potential supposedly made cells strongly adherent because the surface of plasma
membrane might be negatively polarized to compensate for the surface charge of the
electrode. It could reflect the decrease in membrane fluidity. Electrically induced
change in plasma membrane structure must cause the cytoskeleton and cell metabo-
lism to fluctuate. The changing of cell morphology should be due to the alternation
of the cytoskeleton. Concerning cell metabolism, the details should be clarified in
further investigations.
It is concluded that the surface charge of the electrode causes plasma membranes
to reduce fluidity with resulting inhibition of cell proliferation.
13s
In the potential range below 0 V vs. Ag/AgCl, most of cells desorbed from the
surface of an electrode. The intrinsically negative charge of the plasma membrane
might be repulsed against the surface charge of the electrode in the potential range.
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