Note: Descriptions are shown in the official language in which they were submitted.
1341049
The present invention relates generally to an improved cell
culture dish, for example a petri dish, and more specifically to
culture dishes for culturing adherent normal cell monolayer
cultures and to transparent thin film electrodes for use in
conjunction with such dishes.
The term "pet:ri dish" as used herein refers to that
"shape/function", familiarly known to those skilled in the art
of cell culture as a petri dish. The term "pepetri dish" is used
herein to refer to a planar electrode petri-type dish according
to the invention.
The expressions "cell/s", "culture/s", and "cell cultures"
as used herein include tho~~e operations starting from the process
of "plating", and up to and including the stage known as
"confluency" ; i . a . f:rom a :starting point of one cell to the point
at which the culture surface is entirely covered by a monolayer
of cells and substantially no further cell division occurs in
normal cells.
Diverse biological responses to electric fields, both
applied and endogenous, continue to motivate experimental
searches for mechanisms of electromagnetic interactions with
~~ells. Jaffe, L.F. ((1979) Control of development by ionic
~~urrents. In Membrane Transduction Mechanisms. R.A. Cone and
~J.E. Downing editors. Raven, N.Y. 199-231) has shown that cell
development is affected by an electric field, while Borgens,
:~.B., J.W. Vanable, ~Jnr., and L.F. Jaffe ((1977) Bioelectricity
,end Regeneration. I. Initiation of frog limb) describe the
effect of electric fields on limb regeneration. Many other basic
~~ellular functions, including motility and receptor regulation
,ire also modulated by applied external electric fields. In
;addition, cell membrane permeabilization and fusion have been
;effected by applied fielda (see Zimmerman, U. , and J. Vienken
(1982) Electric fie7.d-induced cell-to-cell fusion. J. Membr.
Biol. 67:165-182; Te;~sie, J., V.P. Knutson, T.Y. Tsong, and M.D.
:bane (1982) Electric pulse-induced fusion of 3T3 cells in
~T 1
~ 3410 ~
2
monolayer culture. Science (Wash.D.C.). 216:537-538; and
Potter, H., L. Wie:r, and P. Leder (1984) Enhancer-dependent
expression of human :K immu:noglobulin genes introduced into mouse
pre-B lymphocytes by elect:roporation).
Local perturbation o:E plasma membrane potentials provides
a hypothetical mechanism for the interaction of applied electric
fields with cells.
A large percentage of interest in mammalian cell lines lies
in the group known as adhesion-dependent types. Most primary
fibroblasts proliferate when attached to glass or plastic, but
do not grow in suspension culture. Cells do not adhere well to
metallic surfaces. Studies into "anchorage dependence", a term
that described the inabi'.Lity of normal cells to grow unless
attached to a substr;~tum, have shown that cells do not enter the
S-phase (i.e. the portion of the cell cycle when DNA is
'undergoing replication) unless attached to an appropriate
substratum. While significant work has been done towards
'understanding the int=eraction between cells and applied electric
fields, this has ~~een virtually restricted to single cell
suspensions, and is i~herefore of very limited application in the
.study of the far more: comp7Lex interplay between applied electric
fields and cells in monolayer tissue culture and in the S-phase
~~f growth.
Thus, Canadian Patent No. 1,208,146 (Wong) describes a
method of transferring genes into cells which comprises
;subjecting a mixture of the genes and the cells to an electric
:Field.
U.S. Patent N~~. 4,561,961 (Hofmann) (see Figure 3),
discloses an electrofusion apparatus wherein a sandwiched chamber
~~ontaining the electrodes may be placed under a microscope, while
c3erman Offer. 3,321,239 (Zimmermann et al) describes an
~~lectrofusion cell of very simple structure.
U.S. Patent No.. 4,695,547 (Hilliard et al) relates to a
multi-cell cuvette including a ring-shaped
,.,..
.,
....
~ 341 04 9
electrode that is received from above within the cell and wherein
the electrode conf:Lguration does not interfere with visual
observation with an inverted microscope during the procedure.
Finally, U.S. Patent No. 4,071,430 (Liebert) supports the
proposition that electrophoretic devices having thin-film
electrodes deposited. on a transparent non-conducting substrate,
such as glass, are known in the electrophoresis apparatus art.
It is an object of the present invention to provide a
structure and methc>d for the growth and study of monolayer
adherent cell cultures so that changes occurring during the
stages of the life cycle of a cell, especially during the S-
phase; the effect of an applied electrical field upon the cell;
and the cell's interaction with a contact electrode surface may
be studied optically with i~he growth surface either electrically
neutral or in an electrically ionized state. Accordingly, one
aspect of the inveni~ion provides a cell culture device, which
comprises: a planar substrate, an electrically conductive coating
thereon, and electrode means in contact with said coating for
.applying an electric potential or electrical ionizing source to
said coating to establish an electrical field above said coated
substrate.
In one embodiment the invention provides an apparatus for
subjecting adherent cell cultures to electrical fields while in
situ on an electrode surface, the apparatus comprising a
substrate; an electrical:Ly conductive coating thereon affording
.an upper surface ccmducive to cell adhesion and growth; and
electrode means in contact with the coating, for applying an
electric potential or electrical ionizing source to the coating
vo establish an electrical field above the coated substrate.
In particular, t:he invention provides a cell culture device,
~Nhich comprises a transparent planar substrate, an electrically
conductive, optically transparent coating thereon, and electrode
means in contact with the coating for applying an electric
~~otential or electrical ionizing source to the coating to
establish an electrical field above the coated substrate.
1 3 41 04 9
3a
In a further em);~odiment, the invention provides an apparatus
for subjecting adherent cell cultures to substantially uniform
electrical fields while i» situ on an electrode surface, which
comprises a planar substrate; an electrically conductive coating
thereon, the coating affording an upper surface conducive to cell
adhesion and growth; a di~;tribution electrode formed of a layer
of material of greater conductivity than said coating,
interjacent the planar substrate and the coating; means for
keeping cells immersed in a nutrient medium while adherent to the
electrode surface; and electrode means in contact with the
coating or the distribution electrode.
A further aspect of tine invention provides an apparatus for
subjecting adherent cell cultures to electrical fields while in
situ on an electrode surface, which comprises a transparent
:planar substrate; an electrically conductive, optically
transparent coating thereon, the coating affording an upper
surface conducive to cell adhesion and growth forming a first
electrode; a counter'-electrode in close proximity to the first
electrode and adapted to create an electrical field substantially
:perpendicular to the first electrode; means for keeping cells
~~ultured on the first elecarode immersed in a fluid, the fluid
disposed interjacent the first electrode and the counter-
electrode; and electrode means in contact with the first
=lectrode and the counter-Electrode, the electrode means adapted
to create an electrical potential between the first electrode and
'the counter-electrode.
The invention also provides a method of culturing cells
~~omprising the steps of: culturing the cells on a substrate
~~oated with an electrically conductive layer affording an upper
surface conducive to cell adhesion and growth; and subjecting
~auch cells to an ionizing electric field or electrical potential
~Nhile in situ on the substrate during monolayer adherent cell
1 34? ~4 9
3b
culture by applying an elE~ctrical potential to the layer.
Hitherto, a restriction to researchers in this area has been
the fact that experiments involving adherent cells and electric
fields have been esse~ntial7Ly constrained to non-replicative phase
periods in the cell': life" In addition, in conventional devices
the lines of force generated by an electric field are propagated
in a side-to-side fashion across the cells being grown.
Petri dishes hive bec=_n in existence since before the turn
of the century and the ability to create transparent conducting
thin films was first: noticed by Baedeker in 1907, but remained
a scientific curiosity until the Second World War, when they were
used to deice aircrai_t windows . A wide variety of materials may
be used for the thin film and a wide variety of techniques used to
~ 341 04 9
4
<~pply them, such as are disclosed in Jarzebski, Z.M. , Preparation
~~nd Physical Properties of: Transparent Conducting Oxide Films,
Institute of Solid State Physics, Zabrze, Poland (1982); Vossen,
~J.L., Transparent Conducting Films, RCA Corporation David Sarnoff
research Center, Princeton, N.J.; and Haacke, G., Transparent
~~onducting Coatings, Ann. Rev. Mater. Sci., 1977.7:73-93.
As indicated above, the invention contemplates the
,application onto the upper surface and side of a substrate which
forms the transparent floor of a dish, of a layer of optically
transparent, electrically conductive material that is amenable
to cell adhesion. The substrate is optically and chemically and
electrically neutral, and may be formed, for instance, of glass
and certain plastics. The applied surface coating may be
circumferentially attached to an annular wire or strip of metal
which passes through a wall of the tubular enclosure forming the
walls of the dish, so as t.o provide an electrical connection at
the outer face of the wal7_ .
The coating it~~elf m<~y be varied in application such that
a number of differing iso-electric potentials may be created,
such as with a flat thin film in cross-section, or, as preferred,
a layer that is thin at t;he annular outside, and increases in
depth at a controlled rate' to the center of the dish, such that
a relatively uniform iso-electric potential may be manifested
over the entire surface o:E the floor of the dish.
Another aspect of the invention provides a method of
culturing cells, which comprises placing cells to be cultured on
a transparent planar substrate coated with an optically
transparent, electrically conducting layer, subjecting such cells
to an ionizing electric field or electrical potential while in
situ on said substrate during monolayer adherent cell culture by
applying an electrical potential to said layer and, if desired,
optically studying the cells through the coated substrate during
charging of the coai~ed substrate and cell growth.
1 341 04 9
Thus, the invention also affords a method of subjecting
monolayer adherent cells to a planar projected ionizing electric
field or electric discharge while they are being cultivated,
thereby removing the need to transfer the cells to an electrode
5 chamber and thereby removing the disruption of their growth with
either chemical or mechanical methods, such as the application
of a proteolytic enzyme ox~ scraping off the cells from the dish
with a rubber policeman.
The electrical potential may be applied continuously or
intermittently and may be as high as about 2000V. The electrical
potential may be applied at least partially during the S-phase
of the cell cycle.
Cells to be cultured may be eucaryotic (e.g., plant or
mammalian) or procaryotic.
It will be apparent to the skilled observer that systems
hitherto available, do not allow, nor have they provided for the
possibility of, subjE'Cting monolayer adherent cells to an applied
planar electric field, in the petri dish in which they have been
grown. This represents an important advantage of the system
provided by the present invention.
Embodiments of the invention will now be described, by way
of example, with reference to the accompanying drawings, in
which:
Figure 1 is a perspective view, partly from above, of a
:planar electrode cel7_ cultivation dish according to an embodiment
~~f the present invention;
Figure 2 is a cutaway isometric view of the dish of Figure
1 on an enlarged scale, showing the inter-relationship of the
~~omponent parts of the die~h;
Figure 3 is a cross-section of the dish shown in Figures 1
,end 2, taken along line III-III of Figure 2;
Figure 4a show~~ diagrammatically a conventional substrate
aurface with charged cells adherent thereon;
Figure 4b shows diagrammatically the shape of the electric
Force field emanating f=rom a conventional charged plate
electrode;
1 341 04 9
Figure 4c: shows the shape of the electric force
field using a thin film conductor in accordance with an
embn_diment of t-he invention;
Figure 5 is a cross-section of part of a planar
electrode cell cultivation dish according to another
embodiment of i~he invention
Figure 6 is a plan view of an embodiment of
apparatus for u.~~e with the planar electrode cell
cultivation d.i~:hes;
Figure 6b is a cross-section of part of the
apparatus of Figure ~, taken along line R-B of Figure 6;
and
Fig~_a_rP 6c is a cross-section of part of the
apparatus of Figure .6, taken along line r-C of Figure 6.
Referring now to Figures 1 to 3, a planar
electrode cell Ctllt:ivation dish is shown which comprises a
substrate 1 in the form of a circular disc of optically
transparent glass, for example rorning's Pyrex* brand,
having polished edges, disposed in a tubular glass or
plastic enclosure 6.
A r_.ondu.c-t ive surface coating 2, preferably
having a thickness of from 0.1 to 5 microns, is deposited
over the ,tipper surface -and the edges of substrate 1 , and
extends over these aurfaces as a.n uninterrupted coating.
A distribution elP~:~trnde 4 in the form of a thin annular
metal strip enclose=. the edge of the substrate 1, on the
outside of the stir face coating 2. The distribution
electrode 4 is formed, for instance, of a cond,_ictive
material such us copper, tin, platinum, silver or an alloy
containing one or more thereof.
Positioned between the s,_irface coating 2 and the
distribution e:~ectrode 4, is a wettable metallic coating 3
that intimatel~T contacts and wets both the outer surface
of the surface coating 2 and the inner surface of the
distribution electrode 4, so as to minimize the contact
resistance to ~elec~rrical flow which might otherwise occta.r
as a res,_~lt of s~.irfacP or dimensional impPrfer_.tinns in
either or both the substrate 1 and the distribution
1341049
electrode 4. The rnetallic coating 3 is preferably formed
of a conducti~~e alloy which is liquid at room temperature,
for example a gallium indium alloy and particularly a Gain
90:10 alloy.
The distribution electrode 4 completely
encircles the substrate 1, and overlaps upon itself a
sufficient distance to allow bonding together of the two
ends thereof.
Attached to one point on the distribution
electrode 4 and exi:ending radially from the outer surface
thereof is a lead-i.n wire 5 (see Figure 3), for example a
platinum or copper wire, that passes cleanly through one
wall of the tabular enclosure 6, and terminates in contact
with an outer electrical contact '1 disposed in or on the
outer surface of the enclosure 6.
The outer electrical contact Z is a metal strip,
for example, formed of platinum, preferably mounted flush
into the outer surface of tubular enclosure 6, so as to
allow the e;Kternal application of electricity to be
propagated entirely around the outer circumference of the
substrate 1 b~~ means of the annular distribution electrode
4 and thence ;long the plane of the coating 2 on the upper
surface of the substrate 1.
The tubular enclosure 6, circumferentially
encloses the substrate 1, the surface coating 2, the
metallic coating 3, and the distribution electrode 4. The
enclosure 6 ins provided with spaced annular flanges 6a and
6b which extend radially inwards over the upper and lower
surfaces of the substrate 1 a sufficient distance so as to
preclude the leakage of fluid. The enclosure 6 also
extends perpendicularly upwards from the plane of the
upper surface of the substrate 1, i.e. in the manner of a
tube, so as to create a well or chamber of sufficient
depth to allow the cultivation of cells on the surface of
the surface coating 2.
The exact method of deposition used to form the
surface coating 2 will depend upon parameters such as the
particular materials employed, the desired thickness of
? 34~ 04 9
8
the coating, the substrate/coating interface shape, the
availability of equipment, economic factors associated with each
of the methods, et:c. ~~ome suitable techniques include R.F.
sputtering, D.C. reactive sputtering, thermal evaporation,
electron beam evaporation, dipping and curing. Those skilled in
the art will be aware of other suitable methods or will be able
to ascertain them using no more than routine experimentation.
Those skilled in the art will also be aware of variations of the
above techniques, such as electric field ion depletion of the
substrate so as to enhance the conductivity of the coating.
Since conductive than films tend to suffer from high in-
the-plane resistance, an alternate method of fabricating the
distribution electrode is to deposit a transparent, thin layer
of preferentially a noble metal, e.g. platinum or gold, of
approximately 50 to 200, e.g. 100, Angstroms thickness onto the
base substrate followed bit a thin layer having a thickness of 0.1
to 5 microns of suitable material for cell adhesion, preferably
tin oxide. This ~~rrangE~ment overcomes the high in-the-plane
resistance of the c:oatinc~ and in turn allows the generation of
a uniform planar isoelect:ric potential.
The thickness of the coating will depend on the material
employed and the de~,iderat:a of the intended application. Thicker
coatings have better conductivity but poorer light transmission
properties, and vice versa. Generally, for transparent
applications, the c~~ating will be formed with a thickness in the
range of 0.1 to 5 microns, and , where transparency is not of
great importance, such as in bio-reactors, the coating may be of
any convenient thickness.
The exact material/materials used for the coating/coatings
will depend upon such parameters as transparency, resistivity,
chemical stability, mechanical stability, biological, inertness,
cost, preferred methods of application, etc. However, a
preferred material :Eor foaming the coating 2 is tin oxide (Sn02) .
Other materials su~.table as transparent thin film conductors and
1 ~'~41 04 9
8a
which may be employe~3 for forming the coating 2 include tin oxide
doped with either fluorine or antimony, indium oxide, indium
oxide doped with tin (ITO) ,, cadmium oxide, cadmium stannate, zinc
oxide, zinc cadmium sulfii~e, and
1341049
titanium nitride (TiN). Material currently showing promise for
use as transparent electrodes and which may also be contemplated
for forming the coating 2 are: rubidium silver iodide (RbAg4I5),
dieuropium trioxide, lanthanum hexaboride, rhenium trioxide, and
divanadium pentaboride.
In addition to being non-cytotoxic and capable of supplying
a surface suitable f:or cellular adhesion, the coating material
must also have the added properties of withstanding attack by
acidic and basic organic solutions, nondegradation by
autoclaving, and rel~~tively resistant to mechanical degradation.
Although the me~chani:~m is not yet fully understood, early
observations appear i:.o ind:icate that the pyrolytically deposited
Sn02 surfaces may give rise to mitogenesis enhancing properties
over that of current:Ly used surfaces . Whether this is due to the
mechanical surface properties induced by pyrolytic deposition,
a chemical effect of: tin :ions nearby, the conductive nature of
the Sn02 film, or a combination between these properties, is not
.at this time discern<~ble. It is felt that the surface properties
(i.e. smoothness, et~~.) along with the conductive nature are the
essentially interactive components and that materials other than
that of Sn02 will show the same effect, although to differing
extents.
Figure 4a shows diagrammatically the conventional method of
subjecting adherent cells to an electric field. Cells 8 grow
~~ahile attached to a substrate 9 and a circular electrode 10 is
placed on either side of the cell. Figure 4b shows the shape of
'the electric force field emanating from a charged plate electrode
11.
Lines of force a.re shown in short dashes and the proximity of
lines to each other indicates the relative intensity of force. Lines
~~f current flow are shown with longer dashed lines. It can be seen
'that the lines of force and of current flow are propagated in a side-
~to-side fashion, across the cells. This will inevitably lead to
r
l0 1341049
an electrical interaction between the cells. The
difficulty in establishing lines of force perpendicularly
through the ~~ells is overcome by the use of the thin
transparent cc~nduct_'ive coating of the invention.
Figure 4c: shows the configuration of lines of
force and potential current flow from a thin film
conductor 12 <~ccording to an embodiment of the invention.
It can be seen that: the lines of force are perpendicular
to the dire ction of cell spread, creating the highest
equipotential point near to the upper surface of the cell.
It will be apparent that this shape can be of definite
use, for example, if one wanted to "charge sweep" the
upper surface of the cell of proteins on the surface, or
of those occurring :fn the outer membrane surface.
Figure 5 shows another embodiment of the device
of the invention which replaces the metallic coating 3 and
the distribution electrode 4, lead-in wire 5, and external
electrical contact ? of Figure 3, with a deposition of a
metal film :L3, e.g. copper. Electrical contact is
permitted by a~n opening 14 in the side wall of the tubular
enclosure 6, which allows contact with an external
electrode that: passes through the opening. However, this
technique rec;uires; relatively sophisticated materials
engineering and handling, and more expensive manufacturing
techniques.
The subsi:rate may also be frusto-conical in
shape, i.e. the outer edge surface on which the film 13 is
deposited malt slope downwardly, outwardly, so that the
upper edge of the substrate has a bevelled surface. This
permits simpl.ificaition of the process of depositing the
metal film 13 which can then be accomplished in a single
step.
Figure 6 shows a view from above of a style of
receptacle for use with the pepetri dish of Figure 5. The
pepetri-dish :L5 with an electrode indent 16, slides over
bevel-edged circumferential supports 22 held in place by a
supporting body 21, whereupon a gold-plated spring
electrode 1?, connected to an electrical input jack 19 by
1341049
11
~~ connecting wire 18, completes electrical contact with the
indented electrode 1.. (see Figure 5) while a retaining spring 20
prevents the pepetri dish from slipping out of electrical
<:ontact. The dish 1!~ is provided with a cover 23.
The electrical jack 7_9 can be connected to an electrical
lionizing source of p:refere:nce, depending on the requirements of
each experiment . It can be' seen that the receptacle provides an
Efficient and convenient method for charging the pepetri-dish
while allowing virtually t:otal freedom for optical examination
of a culture in the dish.
Given the current stage of the art in molecular manipulation
~~f plastic-forming materials, it will be apparent that there
=xists the distinct ;possibility that the coated glass materials
could be dispensed with in favour of a transparent conducting
:plastic, conforming to the other constraints applied, such as
adhesivity, non-toxicity, etc. While replacement of the
conductive coating on a glass substrate with a plastic type
material would remove the need for the coating, it still would
represent the use of a conductive substrate material for growing
monolayer cells while treating them with applied electrical
fields of a simiar nature..
It will be apparent that many further uses for the novel
device of the invention will be readily obvious to those skilled
in the theories and procedures of molecular and cell biology, and
especially to those akilled in the art of application of electric
fields to cells, such as c=_lectroporation.
To those skillE~d in the art it will be apparent that this
device can be used to subject cells to a contact planar
electrical field, and thereby optically to study the cells while
under an ionized condition.
It will be further apparent that an ancillary effect of
the unique architecture of the electrode allows the
propagation of an electric force field substantially
perpendicular to the direction of cell spread, a condition
1 341 (l4 9
12
not hitherto achievable in relation to cell culture and
having a direct effect on reducing the potential fusion of
the cells.
To one skilled in the art of electroporation,
especially in the art of electro-transfection, it will be
seen that this: device offers a new and radically different
and potentially more efficient system to employ for the
purpose of e~lectroporation, and with which to study
optically the process of electroporation. Thus, an upper
electrode made of the same materials as the pepetri
coating may be disposed opposite the pepetri dish with the
conductive sup~face facing the upper surface of the lower
pepetri-dish and the material to be transfected arid the
cells to be porated placed between the two plate
electrodes and dii'ferent electrical charges applied to
each plate.
To i:hose skilled in the art of electrofusion of
cells, it will be readily apparent that slight
modification: to the device, such as the growing of
another monol<<yer of cells on another pepetri surface, and
bringing the 'two of them into contact such that the upper
cell surface of one is in contact with the upper cell
surface of t:he other and an electrical charge applied,
offers a new and radically different way with which to
subject the cells ~:o a condition to promote cell fusion
and with which to tie able to study optically the process
of cell fusion.
It will also be apparent that the study of cells
and micro-orc~anism~s not requiring to be adherent to a
surface, will also benefit from the ability to expose them
to a uniforrn electric field while under optically
observable conditions, such as generated by the invention.
Finally, to those skilled in the art of cell culture, it
will be apparent that the new geometry and other features
of the device also have application in the field of
bioreactors.
