CA1341049C - Optically transparent cell cultivation dish with planar electrode - Google Patents
Optically transparent cell cultivation dish with planar electrodeInfo
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- CA1341049C CA1341049C CA 568588 CA568588A CA1341049C CA 1341049 C CA1341049 C CA 1341049C CA 568588 CA568588 CA 568588 CA 568588 A CA568588 A CA 568588A CA 1341049 C CA1341049 C CA 1341049C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/06—Plates; Walls; Drawers; Multilayer plates
- C12M25/08—Plates; Walls; Drawers; Multilayer plates electrically charged
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Abstract
A method and device are disclosed for culturing of adherent cell monolayer cultures on a planar electrode. The device generally comprises cell culture dish, for example a petri-type dish, having a planar bottom, with an electrically conductive, optically transparent coating amenable to cell adhesion on the upper surface of said planar bottom. An annular electrode surrounds and contacts the coated bottom and is connected to a source of electrical power. The method involves use of the device for culturing the cells under the influence of an electric field and/or during establishment of an electrical potential.
Description
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 ~
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 ~
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.
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 <~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.
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;
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 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 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 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 ~~ 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 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.
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.
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 <~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.
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;
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 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 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 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 ~~ 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 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.
Claims (52)
1. Apparatus for subjecting adherent cell cultures to electrical fields while in situ on an electrode surface, said apparatus comprising:
a substrate;
an electrically conductive coating thereon affording an upper surface conducive to cell adhesion and growth; 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.
a substrate;
an electrically conductive coating thereon affording an upper surface conducive to cell adhesion and growth; 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.
2. Apparatus for subjecting adherent cell cultures to electrical fields while in situ on an electrode surface, said apparatus comprising:
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon ,affording an upper surfaces conducive to cell adhesion and growth;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface; and electrode means in contact with and enclosing perimeter of said coating, for applying an electric potential or electrical ionizing source to said coating to establish an electrical field above said coated substrate.
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon ,affording an upper surfaces conducive to cell adhesion and growth;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface; and electrode means in contact with and enclosing perimeter of said coating, for applying an electric potential or electrical ionizing source to said coating to establish an electrical field above said coated substrate.
3. Apparatus for subjecting adherent cell cultures to electrical fields while in situ on an electrode surface, said apparatus comprising:
an electrically conductive material affording an upper surface conducive to cell adhesion and growth thereon forming an electrode;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface;
means for applying an electric potential or electrical ionizing source to perimeter of said electrically conductive material;
wherein said electrically conductive material is selected from the group consisting of (a) tin oxide, (b) a mixture of tin and indium oxides, and (c) titanium nitride.
an electrically conductive material affording an upper surface conducive to cell adhesion and growth thereon forming an electrode;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface;
means for applying an electric potential or electrical ionizing source to perimeter of said electrically conductive material;
wherein said electrically conductive material is selected from the group consisting of (a) tin oxide, (b) a mixture of tin and indium oxides, and (c) titanium nitride.
4. Apparatus for subjecting adherent cell cultures to substantially uniform electrical fields while in situ on an electrode surface, said apparatus comprising:
a planar substrate;
an electrically conductive coating affording an upper surface conducive to cell adhesion and growth;
a distribution electrode formed of a layer of material of greater conductivity than said coating, interjacent said planar substrate and said coating;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface; and electrode means in contact with said coating or said distribution electrode.
a planar substrate;
an electrically conductive coating affording an upper surface conducive to cell adhesion and growth;
a distribution electrode formed of a layer of material of greater conductivity than said coating, interjacent said planar substrate and said coating;
means for keeping cells immersed in a nutrient medium while adherent to said electrode surface; and electrode means in contact with said coating or said distribution electrode.
5. Apparatus for subjecting adherent cell cultures to substantially uniform electrical fields while in situ on an electrode surface, said apparatus comprising:
a transparent planar substrate;
a transparent electrically conductive coating affording an upper surface conducive to cell adhesion and growth;
a distribution electrode formed of a transparent layer of material greater conductivity than said coating, interjacent said substrate and said coating;
means for keeping cells immersed in a nutrient medium while adherent to said coating; and electrode means in contact with said coating or said distribution electrode.
a transparent planar substrate;
a transparent electrically conductive coating affording an upper surface conducive to cell adhesion and growth;
a distribution electrode formed of a transparent layer of material greater conductivity than said coating, interjacent said substrate and said coating;
means for keeping cells immersed in a nutrient medium while adherent to said coating; and electrode means in contact with said coating or said distribution electrode.
6. An apparatus pursuant to claim 2, 3 or 5, wherein the transparent planar substrate comprises the bottom of a petri dish.
7. An apparatus pursuant to claim 2, 3 or 5, wherein the substrate comprises the bottom of a petri dish and the electrode means passes through a side wall of the petri dish.
8. An apparatus pursuant to claim 2, 3 or 5, wherein said substrate comprises the bottom of a petri dish and the side wall of the petri dish is provided with an aperture, through which the electrode means is connected to a source of electric potential or an electrical ionizing source.
9. An apparatus pursuant to claim 1 or 2, wherein the conductive coating is formed of tin oxide.
10. An apparatus pursuant to claim 4 or 5, wherein the conductive coating is formed of tin oxide.
11. An apparatus pursuant to claim 1 or 2, wherein the conductive coating is formed of tin oxide and indium oxide.
12. An apparatus pursuant to claim 4 or 5, wherein the conductive coating is formed of tin oxide and indium oxide.
13. An apparatus pursuant to claim 4 or 5, wherein the conductive coating is formed of indium oxide.
14. An apparatus pursuant to claim 1, 2 or 3, wherein the electrically conductive coating is selected from the group consisting of (a) tin oxides doped with fluorine or antimony, (b) indium oxide doped with cadmium oxide, (c) cadmium stannate, (d) zinc oxide, and (e) zinc cadmium sulfite.
15. An apparatus pursuant to claim 4 or 5, wherein the electrically conductive coating is selected from the group consisting of (a) tin oxides doped with fluorine or antimony, (b) indium oxide doped with cadmium oxide, (c) cadmium stannate, (d) zinc oxide, and (e) zinc cadmium sulfite.
16. An apparatus pursuant to claim 1, 2 or 3, wherein the conductive coating is formed of rubidium silver iodide, dieuropium trioxide, lanthanum hexaboride, rhenium trioxide or divanadium pentaboride.
17. An apparatus pursuant to claim 4 or 5, wherein the conductive coating is formed of rubidium silver iodide, dieuropium trioxide, lanthanum hexaboride, rhenium trioxide or divanadium pentaboride.
18. An apparatus pursuant to claim 1, 2 or 3, wherein the substrate is a material capable of supporting a conductive coating affording an upper surface conducive to cell adhesion and growth.
19. A culture device for culturing adherent cell monolayer cultures, said device comprising:
a cell culture device having a planar bottom surrounded by a wall for retaining culture media;
said planar bottom forming a substrate;
an electrically conductive, optically transparent coating affording an upper surface conducive to cell adhesion and growth completely covering the top and edge surfaces of said substrate;
an electrode surrounding and in contact with the edge of said planar bottom; and electrical contact means adapted to connect said electrode through said wall with a source of electric potential or an electrical ionizing source.
a cell culture device having a planar bottom surrounded by a wall for retaining culture media;
said planar bottom forming a substrate;
an electrically conductive, optically transparent coating affording an upper surface conducive to cell adhesion and growth completely covering the top and edge surfaces of said substrate;
an electrode surrounding and in contact with the edge of said planar bottom; and electrical contact means adapted to connect said electrode through said wall with a source of electric potential or an electrical ionizing source.
20. An apparatus pursuant to claim 19, wherein a thin, transparent layer of metal is disposed interjacent said substrate and said transparent coating thereon and wherein said metal layer has a thickness in the range of 50 to 200 Angstroms.
21. An apparatus pursuant to claim 19, wherein said device wall is provided with a pair of spaced flanges between which the peripheral portion of said planar bottom is gripped.
22. An apparatus pursuant to claim 2, 4 or 19, wherein the thickness of the conductive coating is in the range of 0.1 to 5 microns or greater.
23. An apparatus pursuant to claim 4 or 5, wherein the coating is comprised of two or more layers of different semi-conducting materials, and wherein the top-most layer in contact with the cells is of a semi-conductor material conducive to cell adhesion and growth, and wherein the lower layer or layers are composed of semi-conducting materials having greater conductivity than said top-most layer.
24. An adherent cell culture device, said device comprising:
an electrically conductive surface formed of a conductive elastic material affording an upper surface conducive to cell adhesion and growth; and electrode means in contact with said surface for applying an electric potential to said surface to establish an electric field emanating from the upper surface of said surface, thereby permitting charging of the electrode means during cell growth.
an electrically conductive surface formed of a conductive elastic material affording an upper surface conducive to cell adhesion and growth; and electrode means in contact with said surface for applying an electric potential to said surface to establish an electric field emanating from the upper surface of said surface, thereby permitting charging of the electrode means during cell growth.
25. An adherent cell culture device comprising:
an optically transparent, electrically conductive planar surface formed of a transparent, conductive plastic material and affording an upper surface conducive to cell adhesion and growth;
and electrode means in contact with said surface for applying an electric potential to said surface to establish an electric field emanating from the upper surface of said surface, thereby permitting optical study of cell growth during charging of the electrode means.
an optically transparent, electrically conductive planar surface formed of a transparent, conductive plastic material and affording an upper surface conducive to cell adhesion and growth;
and electrode means in contact with said surface for applying an electric potential to said surface to establish an electric field emanating from the upper surface of said surface, thereby permitting optical study of cell growth during charging of the electrode means.
26. An apparatus for the exposure of cells in suspension to electrical fields, said apparatus comprising:
a planar substrate;
an electrically conductive coating affording an upper surface conducive to cell adhesion and growth thereon;
means of retaining fluid over said surface; and electrode means in contact with said coating for applying an electric potential or electrical ionizing source to said coating.
a planar substrate;
an electrically conductive coating affording an upper surface conducive to cell adhesion and growth thereon;
means of retaining fluid over said surface; and electrode means in contact with said coating for applying an electric potential or electrical ionizing source to said coating.
27. An apparatus pursuant to claim 26, wherein disposed interjacent said substrate and said coating is a layer of material of greater conductivity than said coating.
28. Apparatus for subjecting adherent cell cultures to electrical fields while in situ on an electrode surface, said apparatus comprising:
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to cell adhesion and growth and forming a first electrode;
a counter-electrode in close proximity to said first electrode and adapted to create an electrical field substantially perpendicular to said first electrode;
means for keeping cells cultured on said first electrode immersed in a fluid, said fluid disposed interjacent said first electrode and said counter-electrode; and electrode means in contact with said first electrode and said counter-electrode, said electrode means adapted to create an electrical potential between said first electrode and said counter-electrode.
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to cell adhesion and growth and forming a first electrode;
a counter-electrode in close proximity to said first electrode and adapted to create an electrical field substantially perpendicular to said first electrode;
means for keeping cells cultured on said first electrode immersed in a fluid, said fluid disposed interjacent said first electrode and said counter-electrode; and electrode means in contact with said first electrode and said counter-electrode, said electrode means adapted to create an electrical potential between said first electrode and said counter-electrode.
29. Apparatus for subjecting cells to electrical fields, said apparatus comprising:
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to call adhesion and growth and forming a first electrode;
a counter-electrode in close proximity to said first electrode and adapted to create an electrical field substantially perpendicular to said first electrode;
means for keeping a suspension of cells in a fluid on said first electrode, said fluid disposed interjacent said first electrode and said counter-electrode; and electrode means in contact with said first electrode and said counter-electrode, said electrode means adapted to create an electrical potential between said first electrode and said counter-electrode.
a transparent planar substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to call adhesion and growth and forming a first electrode;
a counter-electrode in close proximity to said first electrode and adapted to create an electrical field substantially perpendicular to said first electrode;
means for keeping a suspension of cells in a fluid on said first electrode, said fluid disposed interjacent said first electrode and said counter-electrode; and electrode means in contact with said first electrode and said counter-electrode, said electrode means adapted to create an electrical potential between said first electrode and said counter-electrode.
30. An apparatus pursuant to claim 28 or 29, wherein a distribution electrode is disposed interjacent said first electrode and said substrate, said distribution electrode composed of a material having greater conductivity than said first electrode.
31. An apparatus pursuant to claim 28 or 29, wherein said counter-electrode comprises:
a substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to cell adhesion and growth and forming a first electrode; and means for supporting said counter-electrode in close proximity to said first electrode.
a substrate;
an electrically conductive, optically transparent coating thereon, said coating affording an upper surface conducive to cell adhesion and growth and forming a first electrode; and means for supporting said counter-electrode in close proximity to said first electrode.
32. An apparatus pursuant to claim 28 or 29, wherein said counter-electrode comprises:
a substrate;
an electrically conductive, optically transparent coating affording an upper surface conducive to cell adhesion and growth thereon forming a counter-electrode;
a distribution electrode disposed interjacent said counter-electrode and said substrate, said distribution electrode composed of a material having greater conductivity than said counter-electrode;
and means for supporting said counter-electrode in close proximity to said first electrode.
a substrate;
an electrically conductive, optically transparent coating affording an upper surface conducive to cell adhesion and growth thereon forming a counter-electrode;
a distribution electrode disposed interjacent said counter-electrode and said substrate, said distribution electrode composed of a material having greater conductivity than said counter-electrode;
and means for supporting said counter-electrode in close proximity to said first electrode.
33. An apparatus pursuant to claim 28 or 29, wherein said counter-electrode comprises a transparent, metallic film disposed on a transparent substrate.
34. A method of culturing cells, said method comprising the steps of:
culturing the cells on a substrate coated with an electrically conductive layer affording an upper surface conducive to cell adhesion and growth; and 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.
culturing the cells on a substrate coated with an electrically conductive layer affording an upper surface conducive to cell adhesion and growth; and 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.
35. A method of culturing cells, said method comprising the steps of:
culturing the cells on a transparent planar substrate coated with an optically transparent, electrically conductive layer affording an upper surface conducive to cell adhesion and growth;
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.
culturing the cells on a transparent planar substrate coated with an optically transparent, electrically conductive layer affording an upper surface conducive to cell adhesion and growth;
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.
36. A method of culturing cells, said method comprising the steps of:
culturing cells on a surface formed of a conductive plastic affording an upper surface conducive to cell adhesion and growth;
subjecting such cells to an ionizing electric field or electrical potential while in situ on said surface during monolayer adherent cell culture by applying an electrical potential to said plastic.
culturing cells on a surface formed of a conductive plastic affording an upper surface conducive to cell adhesion and growth;
subjecting such cells to an ionizing electric field or electrical potential while in situ on said surface during monolayer adherent cell culture by applying an electrical potential to said plastic.
37. A method of culturing cells, said method comprising the steps of:
culturing cells on an optically transparent planar surface formed of a transparent, conductive plastic affording an upper surface conducive to cell adhesion and growth; and subjecting such cells to an ionizing electric field or electrical potential while in situ on said surface during monolayer adherent cell culture by applying an electrical potential to said plastic.
culturing cells on an optically transparent planar surface formed of a transparent, conductive plastic affording an upper surface conducive to cell adhesion and growth; and subjecting such cells to an ionizing electric field or electrical potential while in situ on said surface during monolayer adherent cell culture by applying an electrical potential to said plastic.
38. A method of electroporating adherent cell cultures, said method comprising they steps of:
culturing the cells to be porated on a first electrode, said first electrode providing a surface conducive to cell adhesion and growth;
providing a counter-electrode in close proximity to said first electrode; and subjecting the cells to an electrical field of sufficient intensity to induce poration, said electrical field created by applying an electrical potential to said first electrode and said counter-electrode.
culturing the cells to be porated on a first electrode, said first electrode providing a surface conducive to cell adhesion and growth;
providing a counter-electrode in close proximity to said first electrode; and subjecting the cells to an electrical field of sufficient intensity to induce poration, said electrical field created by applying an electrical potential to said first electrode and said counter-electrode.
39. A method of electroporating cells, said method comprising the steps of:
suspending cells in an electroporation fluid;
disposing said electroporation fluid on a first electrode, said first electrode providing a surface conducive to cell adhesion and growth;
providing a counter-electrode in close proximity to said first electrode; and subjecting the electroporation fluid to an electrical field of sufficient intensity to induce poration of cells suspended therein, said electrical field created by applying an electrical potential to said first electrode and said counter-electrode.
suspending cells in an electroporation fluid;
disposing said electroporation fluid on a first electrode, said first electrode providing a surface conducive to cell adhesion and growth;
providing a counter-electrode in close proximity to said first electrode; and subjecting the electroporation fluid to an electrical field of sufficient intensity to induce poration of cells suspended therein, said electrical field created by applying an electrical potential to said first electrode and said counter-electrode.
40. A method pursuant to claim 34 or 35, wherein the applied electrical potential is 2000 volts or less.
41. A method pursuant to claim 36 or 37, wherein the applied electrical potential is 2000 volts or less.
42. A method pursuant to claim 34 or 35, wherein the electrical potential is applied at least partially during the S-phase of the cell cycle.
43. A method pursuant to claim 36, 37 or 38, wherein the electrical potential is applied at least partially during the S-phase of the cell cycle.
44. A method pursuant to claim 34 or 35, wherein said electrical potential is applied intermittently or continuously during cell culture on said electrically conductive layer.
45. A method pursuant to claim 36 or 37, wherein said electrical potential is applied intermittently or continuously during cell culture on said surface.
46. A method pursuant to claim 34, 35 or 36, wherein the cells are cultured in synchrony.
47. A method pursuant to claim 37 or 38, wherein the cells are cultured in synchrony.
48. A method pursuant to claim 34, 35 or 36, wherein said cell culture comprises eucaryotic cells or procaryotic cells.
49. A method pursuant to claim 37, 38 or 39, wherein said cell culture comprises eucaryotic cells or procaryotic cells.
50. In a cell culture device for effecting electroporation, which includes a container to receive and permit growth of selected yells in a growth medium therefor and means to achieve an electric field in said container, the improvement comprising:
an optically transparent, electrically conductive planar substrate forming at least one wall of said container and selected from an electrical conducting polymeric material and an optically transparent non-conductive material coated with an optically transparent electrically conductive material.
an optically transparent, electrically conductive planar substrate forming at least one wall of said container and selected from an electrical conducting polymeric material and an optically transparent non-conductive material coated with an optically transparent electrically conductive material.
51. A method for culturing cells which comprises:
providing a container having an optically transparent electrically conductive planar substrate forming at least one wall thereof upon which cells to be cultured adhere and means to achieve an electric field in said container;
placing said cells and a growth medium therefor in said container and forming an adherent layer of said cells on said substrate, and subjecting said cells to said electric field.
providing a container having an optically transparent electrically conductive planar substrate forming at least one wall thereof upon which cells to be cultured adhere and means to achieve an electric field in said container;
placing said cells and a growth medium therefor in said container and forming an adherent layer of said cells on said substrate, and subjecting said cells to said electric field.
52. A method pursuant to claim 48 or 49, wherein the eucaryotic cells are plant cells or mammalian cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 568588 CA1341049C (en) | 1988-06-03 | 1988-06-03 | Optically transparent cell cultivation dish with planar electrode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 568588 CA1341049C (en) | 1988-06-03 | 1988-06-03 | Optically transparent cell cultivation dish with planar electrode |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1341049C true CA1341049C (en) | 2000-07-11 |
Family
ID=33315116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 568588 Expired - Fee Related CA1341049C (en) | 1988-06-03 | 1988-06-03 | Optically transparent cell cultivation dish with planar electrode |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1341049C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016436A (en) * | 2019-04-26 | 2019-07-16 | 北京康源嘉成生物科技有限公司 | A kind of electrical environment biological culture system |
| CN113528343A (en) * | 2021-07-19 | 2021-10-22 | 中国科学院重庆绿色智能技术研究院 | Adherent cell culture device for terahertz wave irradiation |
| WO2022109303A1 (en) * | 2020-11-20 | 2022-05-27 | Syngenta Crop Protection Ag | Plant cell chromosome doubling by application of electromagnetic field |
-
1988
- 1988-06-03 CA CA 568588 patent/CA1341049C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016436A (en) * | 2019-04-26 | 2019-07-16 | 北京康源嘉成生物科技有限公司 | A kind of electrical environment biological culture system |
| WO2022109303A1 (en) * | 2020-11-20 | 2022-05-27 | Syngenta Crop Protection Ag | Plant cell chromosome doubling by application of electromagnetic field |
| CN113528343A (en) * | 2021-07-19 | 2021-10-22 | 中国科学院重庆绿色智能技术研究院 | Adherent cell culture device for terahertz wave irradiation |
| CN113528343B (en) * | 2021-07-19 | 2022-08-02 | 中国科学院重庆绿色智能技术研究院 | Adherent cell culture device for terahertz wave irradiation |
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