CA2025022A1 - Device for in situ electroporation of adherent cells - Google Patents

Abstract

ABSTRACT

A method and device are disclosed for culturing of adherent cell monolayer cultures on an electrode surface and subjecting the cells to an electrical field.
The device may generally comprises cell culture dish, for example a petri-type dish, having a bottom, with an electrically conductive, optically transparent coating amenable to cell adhesion on the upper surface of the bottom. A metal electrode may contact the underside of the coating and is connected to a source of electrical power. The method involves use of the device for culturing the cells under the intermittent or continuous influence of an electric field, or during the establishment of an electrical field or potential.
Further, a method and device are disclosed for the sensing of cell poration, and the use of this information to automate the process of electroporation. Further yet, a device and method are disclosed for use in assisting in the time-correlated mapping of the sequence of serial and parallel gene replication.

Description

2~'~5~22 METHOD AND ~EVICE FOR CELL
CULTIVATION ON ELECTRODES
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of my application Serial No. 489,976 filed March 7, 1990.
BACKGROUND OF THE INVENTION

The present invention relates generally to devices and methods for the culturing of cells on electrode surfaces, and more specifically to culture dishes for the culturing of adherent normal cell monolayer cultures wherein they may be subjected to an electric field or discha~ge and to the use of transparent thin film electrodes conducive to cell adhesion for use in conjunction with such applications.
The term "petri dish" as used herein refers to that "shape/function", familiarly known to those skilled in the art o~ cell culture as a petri dish. The term "pepetri dish" is used herein to refer to a planar el-ctrode petri-type dish according to one embodiment o~
the invention. The terms "fluid", "media" and "medium" as as used herein re~er to such materials as may be used ~or the culturing and or suspension Or cell cultures.
Similarly, the terms llelQctroporation fl~ud" or ''electroporation medium" refer to those groups of materials and solutions that may be used in the process of electroporation.
The expressions "cell/s", "culture/s", and "cell culture/s" as used herein include those operations starting from the process of "plating", and up to and including the stage known as "confluency"; i.e. from a 3S startlng 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. This range is meant to include known cell culturing techniques, such as synchronous culturing, and those that make up the wide range that would be used 2~2~2~

in micro-biology, neuro-biology, pharmacology and other related fields of endeavour.
The term "exogenous materials" as used herein refers to macromolecules such as DNA, RNA, proteins, plasmids, and other such materials that may be of interest for introduction into a living cell.
Adherent cells/cultures as used herein refers to those types of cells that are anchorage-dependent for growth.
Diverse biological responses to electric fields, both applied and endogenous, continue to motivate experimental searches for mechanisms of electromagnetic interactions with cells. Jaffe, L.F. (1979) Control of development by ionic currents. In Membrane ~ransduction ~echanisms. R.A. Cone and J.E. Downing editors. Raven, N.Y. 199-231, has shown that cell development is effected by an electric field, while Borgens, R.B., J.W. Vanable, Jnr., and L.F. Jaffe (1977) Bioelectricity and Regeneration. I. Initiation of frog limb, describe the effect of electric fields on cell regeneration. Many other basic cellular functions, including motility and receptor regulation are also modulated by applied external electric fields. In addition, cell membrane permeabilization and ~usion have been effected by applied fields (see Zimmerman, U., and J. Vienken (1982) Electric field-induced cell-to-cell ~usion. J. Membr. Biol.
67:165-182; Tessie, J., V.P. Knutson, T.Y. Tsong, and M.D. Lane (1982) Electric pulse-induced fusion of 3t3 cells in monolayer culture. Science (Wash.D.C.). 216:537-538; and Potter, H., L. Wier, and P. Leder (1984) Enhancer-dependent expression of human K immunoglobulin genes introduced i~to mouse pre-B lymphocytes by electroporation).
Local perturbation of plasma membrane potentials provides a hypothetical mechanism for the interaction of applied electric fields with cells.
Thus, Canadian Patent No. 1,208,146 (Wong) describes a method of transferring genes into cells which : :, ',:. ''~',', "~

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comprises subjecting a mixture of the genes and the cells suspended in a liquid medium, and subjected to an electric field and electric discharge. U.S. Patent No.
4,663,292 (Wong) discloses a method of transferring genes into cells and fusing cells, which comprises subjecting suspensions of genes and cells and cells to an electric discharge.
U.S. Patent No. 4,695,547 (Hilliard et al) relates to a multi-welled tray that contains a suspension o~ cells and the foreign molecule, and wherein a ring-shaped metallic electrode configuration that does not interfere with visual observation by inverted microscope during the procedure is received from above within the well. U.S. Patent 4,764,473 (Matschke et al) discloses a device for the electroporation and electrofusion of cells using a double helical metallic electrode configuratio~
and i9 for the application of electric fields to cells in suspension.
U.S. Patent No. 4,561,961 (Hofmann, see Figure ~0 3,) discloses an electrofusion apparatus wherein a sandwlched chamber containing the metallic electrodes may be placed in the microscope, while German O~en.

3,321,239 ~zimmermann et al) describes an electro~uslon cell of very simple structure.
A large percentage o~ interest in mammalian cell lines lies in the group known as adhesion-dependent types. Most primary fibroblasts prolirerate when attached to glass or plastic, but do not grow while in suspension. Adhesion dependent cells do not adhere to metallic surfaces. Studies into "anchorage dependance", a term that describes the inability o~ normal cells to grow unless attached to a substratum, have shown that cells do not enter the S-phase ~i.e. the reproduction cycle of cell growth when DNA i9 undergoing replication) 3S unless attached to an appropriate substratum. While signi~icant work has been done towards understanding the interaction between cells and applied electric ~ields, this has been virtually restricted to single cells in 4 23~5~2~

suspension, and is therefore of very limited application in the study of the far more complex interplay between applied electric fields and cells in monolayer culture tissue, and virtually no work has been able to be done on cells while in the S-phase of growth.
Certain procedures such as electroporation and electrofusion require subjecting cell samples to electric fields. Treating the cells entails trypsinizing the cells, rinsing, suspending them in an appropriate fluid carrying the foreign molecule, in most cases chilling the resulting suspension, subjecting the cells to the electric field, rinsing, and then re-plating them.
Current cell electroporation equipment requires the significant expenditure of relatively high voltage and current, to the point that cooling is often a concern. This is due to the relative inefficiencies in mutually dependent system parameters, such as the number of cells per unit volume of electrolytic media; the conductivity and composition of the electrolytic media;
the voltage being applied; the time period of pulse, the location of the cell relative to the electrode; and the area of membrane surface being presented in relation to the electrodes (spherical) are all factors which require attention when using suspensions and does not account for any change incurred ln the cell or to the membrane as a result of being put into suspension. The exogenous material (DNA/RNA code, etc.) - that it would be of interest to have expressed as a measurable cell function upon successful integration is open to cyto-enzymatic attack until integrated or metabolized. Normal cell function is recovered after when the electrical inducing poration is no longer present, but progression of normal cell functions such as replication can not occur until surricient adhesion is regained to induce a growth signal. A significant period of time may elapse between the poration process and potential integration and has an affect on efficiency. Depending upon the cell condition after transmembrane inductance of exogenous material and 2 ~ 2 ~

the type of material introduced, transient, stable or no expression may be expected. Due to the adverse conditions imposed in the process to date, a large percentage of cells die, some give a transient expression and fewer still exhibit a permanent stable expression.
Because of this large number of cell deaths, a shift in the base population occurs since a first level of selection (survivors vs non-survivors) has been made which further complicates matters.
It is the purpose of this invention to overcome many of the above stated drawbacks as currently known in the art and to significantly advance the state of the art.

SUMMARY OF THE INVENTION
The object of the present invention is to provide means and methods wherein monolayer adherent cells may be cultured on electrode surfaces.
It is another object of the present invention to a O provide a structure and method for the growth and study of monolayer adherent cell cultures on while on electrode surfaces and while being sub~ected to electric fields, th- ob~ect being that changes that occur during the various stages of the life cycle of a cell, and especially of~ those that occur the S-phase of a cell's life; the ef~ects of applied electrical fields upon growing cells; and cell interactions with contact electrode surfaces, may all be studied optically with the growth surface either electrically neutral or in an 30 electrically ionized state. -~
It is another object of the invention to allow an in vitro culture of aells to be equally affected by an el-ctrical discharge.
It is another ob~ect of the present invention to 3S be able to automatically determine the required intensity of electrical discharge to induce poration.
It is another ob~ect of the invention to be able to sub;ect the cells to an electric discharge intense ., .:,..

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enough to induce poration, and not cause damage to the cells by heat or other secondary effects.
Accordingly, one aspect of the invention provides cell culturing devices, which comprise:
substrates, electrically conductive coatings conducive to cell adhesion and growth thereon, and electrode means in contact with said coatings for applying an electric potentials or electrical ionizing sources to said coatings to establish an electrical field above said coated substrates.
A further aspect of the invention provides culture devices, which comprise transparent substrates, electrically conductive, optically transparent coatings conducive to cell adhesion and growth thereon, and electrode means in contact with the coatings for applying electric potentials or electrical ionizing sources to the coatings to establish an electrical field above the coated substrates.
A further aspect of the invention provides an electro-culturing petri dish, comprising a transparent planar substrate, an electrically conductive, optically transparent coating conducive to cell adhesion and growth thereon, and electrode means ln contact with the coating for applying an electric potential or electrical ionizing source to the coating to establish an electrical field above the coated substrate. A further aspect of the invent$on provides an electrode, which may be transparent, disposed above the cell culture creating an electrode chamber wherein the cells may be subjected to an electrical discharge.
A further aspect provides detector electrodes disposed on the opposed electrodes in the chamber so as to be able to detect the real-time effect of the electrical discharge on the cells and the electrode 3S c~amber fluid.
A yet further embodiment provides microprocessor means to control the time and intensity of electric discharge so as to induce poration and uses the detection ~2~22 of the poration to terminate the process.
Hitherto, a restriction to researchers in the area of applying electrical fields to cells, and to adherent monolayer cells in particular, has been the fact that experiments involving adherent cells and electric fields have been essentially constrained to non-replicative phases in cell's lives because cells are usually treated while in suspension. Those devices that ~ay be used while the cells are not in suspension, suffer from the fact that the lines of force generated by an electric field are propagated in a side-to-side fashion across the cells being grown.
Petri dishes have been 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 aircraft windows. A wide variety of materials may be used for the thin film and a wide variety of techniques used to apply them,'such as are disclosed in Jarzebski, Z.M., Preparation and Physical Properties of 'rransparent Conducting Oxide Fllms, Institute of Solid State Physics, Zabrze, Poland (1982); Vossen, J.L., ~ 'rransparent Conducting Films, RCA Corporation David Sarnoff Research as Center, P.rinceton, N.J.; and Haacke, G., Transparent Conducting Coatings! Ann. Rev. Mater.'Sci., 1977.7:73-93.

As indicated above, one aspect of 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.
'rhe substrate is optically, chemically and electrically neutral, and may be formed, for instance, of glass or 3S certain plastics. 'rhe applied surface coating may be clrcum~erentially attached to an annular wire, thin-film layer of a metal deposited between the substrate and the coating, or strip of metal which passes through a wall of ;, .~ ~' ,..' ''' ,~

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the tubular enclosure forming the walls of the dish, so as to provide an electrical connection at the outer face of the wall. -The coating itself may 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, a layer that is thin at the 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 of the floor of the dish. An alterant method is to dispose a layer of a thin-film conductor, which may be transparent, having greater conductivity than the cell adherent coating, between the coating and substrate.
Another aspect of the invention provides a method of culturing cells on electrode surfaces, which comprises culturing the cells on a substrate coated with an electrically conducting layer conducive to cell adhesion and growth, and subjecting such cells to an ionizing electric field or electrical potential while ln on the substrate during the monolayer adherent cell culturing by applying an electrical potential to the layer. If desired, this method may be carried out on a device comprised o~ a transparent substrate and transparent cell adherent electrode coating means 80 that one may optically study the cells, their behaviour, and their response to various intensities of electrical fields through the coated substrate during charging of, and while the coated substrate is charged.
Thus, the invention affords a method and means of subjecting monolayer adherent cells to a planar pro~ected ionizing electric field or electric discharge while they are being cultivated, thereby removing the need to transfer the cells to an electrode chamber and thereby removing the disruption of their normalized growing phase with either chemical or mechanical methods, such as the application of a proteolytic enzyme or scraping off the cells from the culturing dish with a 2~2~22 rubber policeman.
The electrical potential may be applied continuously or intermittently and may be as low as a few volts or as high as about 2000V depending upon the desired range and may be constant or variable in nature.
The electrical potential may be applied at least partially during the S-phase of a synchronized cultures' life for instance.
The device offers an exceptional and hitherto nonexisting means of sub;ecting adherent cell cultures to electric fields, but is not limited in use to only that type of cell culture. Cells to be cultured may be, for instance, eucaryotic, procaryotic, plant or mammalian cells.
It will be apparent to the skilled observer that systems hitherto available, do not allow, nor have they provided ~or the possibility o~, sub~ecting monolayer adherent cell cultures to an applied electric field and to an applied planar-projected electric field while ~a 91~, in the petri dish in which they have been cultured.
This represents an important advantase of the system provided by the present invention over the prior art.
~ Other ~oatures, advantages, ob~ects and ; embodlments o~ the invention will be readily apparent to 2S those ~killed in the art from the ~ollowing description of a pre~erred embodiment taken in con~unction with the appended ¢laims, and accompanying drawings. ;~
DESCRIPTION OF ~HE DRAWINGS ;~ ~;

Figure 1 is a perspective view, partly from above, of a planar electrode cell cultivation dish according to an embodiment o~ the present invention;
Figure 2 i9 a cutaway isometric view of the dish of Figure 1 on an enlarged 9cale, showing the inter- ;
relationship o~ the component parts o~ the dish;
Figure 2a i9 a cutaway isometric view of a dish of an alternate embodiment showing a distribution electrode layer formed under the coating of the instant 1~ ~

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invention.
Figure 3 is a partial cross-section of the dish .
shown in Figures 1 and 2, taken along line III-III of Figure 2; . .
5Figure 3a is a partial cross-section of the dish shown in Figure 2a, taken along line III-III of Figure 2a;
Figure 4a shows diagrammatically a conventional , substrate surface with charged cells adherent thereon;
10Figure 4b shows diagrammatically the shape of the ..
electric force field emanating from a conventional ~ .
charged plate electrode;
Figure 4c shows the shape of the electric force field using a thin film conductor in accordance with an 15 embodiment of the invention; ;
Figure 5 is a partial cross-section o~ part of a electrode cell cultivation dish according to another :. .
embodiment of the invention;
Figure 5a is a partial cross-section of part of a planar electrode cell cultivation dish according to another embodiment of the invention; ..
Figure 5b shows an alternate embodiment of Figure 2a:
Figure 5c is a partial cross-section of part of a 2S planar electrode cell cultivation dish according to the embodiment of Figure 5b;
Figure 6 is a cutaway isometric view of another embodiment of the invention showing the relationship --between a counter electrode and a pepetri dish; .
Figure 6a is a partial cross-section of the emboiment shown in Figure 6;
~ Figure 7 shows a cross-section through an electrode surface of the invention an~ showing an lonization sensing electrode;
Figure 7a shows a crosss-section through an ionization sensing electrical contact point, according to one embodiment of the invention;
Figure 7b shows a partial section of Figure 6 2~5~22 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 ~orm the surface coating 2 will depend upon parameters such as the particular materials employed, the desired thickness of the coating, the substrate/coating interface shape, the availability of equipment, economic factors associated with each of the methods, etc. Some 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 suitabl.e 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 and semi-conductive thin films tend to suffer from high in-the-plane resistance, an alternate method is shown in Figure 2a where there is ' deposlted a transparent, thin film 24 o~ metal, preferentially one o~ the noble metal family, e.g.
platinum or gold, of approximately S0 to 200, e.g. 100, 2S Angstroms thickness onto the base substrate 1 forming a radial distribution electrode upon which a thin layer of coating 2 having a thickness of 0.1 to 5 microns of suitable material for cell adhesion, preferably of tin and/or indium oxides, is deposited. This arrangement gives rise to an overall reduction in resistance. In a further embodiment,~the preferably noble metal may be replaced by a further sami-conductor having better conductlvity properties, but unusable as a cell cultivation surface for reasons such as toxicity, electro-chemical stability, etc. This second semi-conductor would, as the noble metal film, have deposited on its' surface, semi-conductor materials conducive to cell growth such as tin oxide or tin oxide with indium, .: .
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2~2~22 etc. If a transparent thin-film layer of metal, or a layer of a more conductive transparent thin-film semi-conductor is applied firstly to the substrate, the coating may be in the thinner range stated above.

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In yet a further alternative embodiment, where transparency is not required, the conductive coating conducive to cell adhesion and growth may be directly applied onto a metal wherein the metal would serve as the means to reduce the in-the-plane resistance by conducting the electrical potential to the entire sub-surface of the coating at one time, much as with the transparent thin-film noble metal layer.
The thickness of the coating will depend on the materlal employed and the desiderata of the intended application. Thicker coatings have better conductivity but poorer light transmission properties, and vice versa.
Generally, for transparent applications, the coating will be ~ormed with a thickness in the range of O.1 to 5 mlcron~, and , where transparency is not of great importance, such as in bio-reactor~ and associated applic~tions, the coating may be of any convenient thi¢knes~.
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, preferred materials for forming the coating 2 are tin oxide (SnO2) and indium oxide and various combinations of the two, (the ITO
fam~ly) and various combinations of the two doped with other materials. Other materials suitable as transparent thin film conductors and which may be employed ~or forming the coating 2 or layer 2a include tin oxide doped with either fluorine or antimony and indium oxide doped cadmium oxide, cadmium stannate, zinc oxide, zinc cadmium sulfite, and titanium nitride (TiN).

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Materials currently showing promise for use as transparent electrodes and which may also be contemplated for forming the coating 2 or layer 2a 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 for cellular adhesion and growth, the coating material must also have the added properties of withstanding attack by acidic and basic organic solutions, nondegradation by autoclaving, and be relatively resistant to mechanical degradation, and resist if not withstand electro-chemical attack in application.
Although the mechanism is not yet fully understood, early observations appear to indicate that the pyrolytically deposited SnO2 surfaces may give rise to mitogenesis enhancing properties over that of currently 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 Sno2 film, or a combination between these properties, is not at this time discernable. It is felt that the surface properties as ~i.e. ~moothness, etc.) along with the inert conductive nature are the essentlally interactlve components and that materials other than that of SnO2 will show the same effect, although to differing extent. Other as yet unrecognized effects may also be found.
Figure 4a shows diagrammatically the conventional method of subjecting adherent cells to an electric field~ Cells 8 grow while 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 elQctrode 11.
Lines of force are shown in short dashes and the proximity of lines to each other indicates the relative '.,' '.

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intensity of force. Lines of 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 may lead to an electrical interaction between the cells, especially if the cell membranes are touching. The difficulty in establishing lines of force perpendicularly through the cells is overcome by the use of the thin transparent conductive coating o~ the invention.
Figure 4c shows the configuration of lines of force and potential current flow from a thin film conductor 12 according to an embodiment of the invention.
It can be seen that the lines of force are perpendicular to the direction 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 de~inite use, ~or example, if one wanted to "charge sweep" the upper sur~ace of the cell of proteins on the surface, or o~ those occurring in the outer membrane sur~ace.
Figures 5, 5a, and 5b show other embodiments o~
the device o~ the invention which replace the metallic coating 3 and the distribution electrode 4, lead-in wire S, and external electrical contact 7 o~ Figures 3 and 3a, as with a deposition of a metal film 13, e.g. gold, silver, platinum, copper. Electrical contact is permitted by an aperture 14 in the side wall o~ the tubular enclosure 6, which allows contact with an external power electrode that passes through the opening. Figure 5 shows the metallic deposition electrode 13 formed in a ring encircling the vertical circumference and extending onto the upper sur~ace o~ the coating 2 The ring electrode 13 may encircle only the vertical circum~erence.
Figure 5a shows the radial distribution electrode 3S 24 below the cell adhesion conducive coating 2 in an alternate embodiment of Figure 2a. The radial distribution electrode may be deposited over the face o~
the substrate 1 and the vertical perimeter ~irst, and the ~3~ 2 cell adhesion conducive coating 2 deposited afterwards, with a window to the metallic layer 24 being left open through the coating 2. Alternatively, only the upper surface of the radial distribution electrode need be coated with - the cell adhesion conducive coating 2.
Figures 5b and 5c show a further alternate e~bodiment where the metal film forming the radial distribution electrode 24, is deposited on the upper face of the substrate and has extending down the face of the vertical perimeter a strip forming a contact point 34 for externally applied power via aperture 35.
The substrate may also be frusto-conical in shape, i.e. the outer edge surface on ~hich the film 13 is deposited may slope downwardly, outwardly, so that the upper edge of the substrate has a bevelled surface. This permits simplification of the process of depositing the metal film 13 which can then be accomplished in a single step. As may be appreciated, there are many ways and geometries available and conceivable in which to combine the coating, the distribution layer, the distribution electrode and the lead-in electrode.
Figures 8, 8a and 8b show a view from above of a ~tyle of r-ceptacle for use with the pepetri dish of Flgures 5 and 5a. The pepetri-dish 15 with an external power electrode aperture 16, slides over bevel-edged circumferential supports 22 held in place by a supporting body 21, whereupon a gold-plated spring electrode 17, connected to an electrical input jack 19 by a connecting wire 18, completes electrical contact with the indented electrode 13 (see Figures 5 and 5a) while a retaining spring 20 prevents the pepetri dish from slipping out of electrical contact. 'rhe dish 15 is provided with a cover 23.
'rhe electrical ~ack 19 can be connected to an electrical ionizing source of preference, depending on the re~uirements of each experiment. It can be seen that the receptacle provides an efficient and convenient method for charging the pepetri-dish while allowing .:' .; , ~ ~ .
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virtually total freedom for optical examination of a culture in the dish. It is recognized that the shape of the retaining means and contact means may be varied in several ways, but have the basic element of the ability to supply an ~electrical connection to the pepetri dish, and many ways of doing this may be readily envisioned.

Given the current state of the arts in molecular manipulation of plastic-~orming materials, it will be apparent that there exists the distinct possibility that coated substrate materials could be dispensed with in favour of a conducting or transparent conducting plastic, conforming to the other constraints applied, such as adhesivity, non-toxicity, etc. While replacement of the conductive coa~ing on a substrate with a plastic type material would remove the need for the coating, it still would represent the use of a conductive material for growing monolayer cells while treating them with applied electrical fields of a similar nature.
2~
Using the pepetri as a starting point, a device for electroporation purposes may be fabricated wherein the cells are not sub~ect to suspension insult and which present~ a large area of cell membrane per cell, and 2S wherein all cells may be presented equi-distantly from, and at an extremely close distances to a counter-olectrode, which is preferably planar. In addition, in some cells the nuclear bulge will present a raised area of membrane as a portion of the upper membrane, depending upon the percentage of normal confluency of the culture, among other things.
, Thus, an upper electrode made of the same (if desired) transparent, conducting, non-cyto-toxic material as the pepetri coating may be disposed opposite the pepetri dish with the monolayer of cells to be porated, with the conductive surface facing the upper surfaca of the lower pepetri-dish, and the material to be transfected ~ . . . . . ~ .... , ,., .. - ..... .

~ J~ Jo2 suspended in an appropriate medium placed between the two planar electrodes and different electrical charges applied to each of the electrodes. Alternatively, the material to be transfected may be first electrostatically (or similar method) drawn onto a carrier surface which is then placed onto the upper (counter) electrode. The counter electrode and material carrier may then be disposed at a very close distance, such as lmm or less from the upper sur~ace of the cells in the pepetri-dish, and appropriate electrical charges applied. ~he distance that the opposing electrodes may be placed relative to each other is an obvious advantage of the instant invention over any prior art, in this manner. Further targeting of the material to be transfected may be achieved by t:he use of appropriate electroporation media in which a large majority, if not all of the otherwise ionizable material i9 removed, leaving the material to be transfected as the only ionizable material. With this approach, matsrial such as DNA and RNA and materials 20 having only a very small charge may be made the current ;
carrier, further increasing its uptake into the cell. ~;
It is recognized that this new geometry will simplify if not remove the current significant problem of determining at exactly what field intensity individual cell lines react to (for poration purposes and others), and the calculation of what field intensity a cell i9 .
actually being sub~ected to.
Figures 6 and 6a show an embodiment of the above device. , The lower (cell growth) electrode 30 base material 31 may be plastic or glass and having disposed over the upper surface and down the outer circumference a thin-film layer of metal or ~;emi-conductor material forming the radial distribution electrode (RDE) 32 and wherein the metal may either of the noble type when exposure to the electrolytic trans~er media (ETM) or poration media PM is preferred, or of another metal when it is expected to be overlaid with the electrically conductive cell adhesion ;
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conducive coating (CACC) 33 or transparent CACC (TCACC) 33. The total coating thickness of the radial distribution electrode and cell adhesion conducive coating for transparent purposes such as optical viewing should not be so great as to substantially interfere with light passage. When non-view apparatuses are required, the coatings may be of any thickness, excepting those that would fall below minimum function parameters.
The radial distribution electrode 24 extending across the face of the disc and onto the disc edge, terminates in a built up layer of metal in the form of a square or rectangle that acts as an electrical contact 34 for the exterior power electrode 37 and aligns with an aperture 35 in the pepetri fluid retaining wall 36 allowing contact with the exterior power electrode 37.
This type of contact arrangement is shown in Figures 3 and 5. Alternatively, the radial distribution electrode may extend substantially the total diameter and leave the faee onto the edge as a strip which ends in an electrical eontaet point 34 as shown in Figures 5a, 5b and 5c.
Extending radially inward on both the lower and upper edge of the disc, are gripping flanges formed in such manner so as to provide a water and moisture proof bond with the disc. ~he inner flange 38, thicker than the outer flange 39, serves as the counter electrode support means and depth gage. The counter eleetrode support means 38 does not neeessarily have to keep the eounter eleetrode 40 and lower electrode 30 planar to each other, although this may be preferred. The vertical inner face and upper surface of the flange 38 may be made of, or coated in a material non-conducive to cell adhesion so as to prevent eell growth up the face and over the upper surface of the flange. Depending upon style Or eounter electrode used, the counter electrode support means 38 may also include around its inner diameter and upper surface, surface tension relief openings 41 to facilitate removal of the counter eleetrode 40. Surface tension relief openings 41 may be 2~5~22 in the form of grooves or notches or similar and extend completely around either the outer edge of the counter electrode 40 or the inner edge of the counter electrode support means 38. These notches aid in breaking the surface tension of the fluid when the counter electrode i9 removed form the lower electrode.

The counter (upper) electrode 40 may be of the radial distribution type, having disposed upon it either a transparent or non-transparent cell adhesion canducive c~ating as the electrode. Alternatively, the counter electrode may be formed only of a thin-film of preferentially noble metal. In another alternative, the substrate and coating may be dispensed with and a solid metallic electrode used.
According to the first embodiment, the outer dlameter of the substrate disc 42 forming the counter electrode 40 and the support tube counter electrode support tube 43 is sufficiently less than the inner diameter of the upper fluid retaining walls 36 of the lower electrode 30 so as to allow the counter electrode 40 to be inserted into the pepetri dish and allow the counter electrode surfacs tension relie~ openings 41 to rest on the upper face of the counter electrode support means 38; The radial distribution electrode 45 film if employed, is of a diameter approximately equal to the inner circumference of the counter electrode support means 38 while the coating of electrically conductive cell adhesion conducive coating 46 disposed on it has a nominally larger diameter than the radial distribution electrode 45 or inner diameter of the counter electrode support means 38 so as to prevent direct contact of radial distribution electrode with the electroporation media. The radial distribution electrode 45 film extends out from under the cell adhesion conducive coating 46 at one point, over the edge of the base material 42 and continues up the outside of the counter electrode support tube 43 in the form of a strip 48 which terminates in an 2~2~: O?J2 electrical contact point 47. The radial distribution electrode connecting strip 48 is insulated by means of insulating strip 49 formed of a thin film strip coating of sio2 or equivalent material, which terminates next to s the electrical contact point 47, so that electrical contact with the radial distribution electrode 45 occurs in an insulated fashion from contact with the electroporation media, which may by displacement extend up the annular opening between the counter electrode support tube 43 and the pepetri fluid retaining wall 36.
Mounted externally at the top of the counter electrode support tube 43 is the counter electrode support tube flange S0 which is an L-shaped toroid with the base of the L inwardly and upwardly directed. The counter electrode support tube flange 50 is bonded onto the exterior of the counter electrode support tube 43 and nominally overlaps the insulating strip 49, and has provided an external power electrode aperture S1, situated over the electrical contact point 47, to receive the external power electrode 52.
The counter electrode support tube flange 50 and the top of the pepetri dish wall 36 may have interlocking registration mar~s 90 that when assembled the counter electrode external power electrode aperture 51 and the lower eleqtrode external power electrode aperture 35 are vertically disposed from each other. These may take the form of a downward pro~ecting V from the counter electrode flange and a complimentary V groove in the top of the pepetri dish wall 36.
The distance that the lower electrode 30 and counter electrode 40 are separated, along with other factors such as media composition, cell type, will doterminQ the voltage requirements, and subsequently, ths current requirements for poration to occur. In the 3S preferred embodiment the electrodes are displaced 1 mm or less from each other, but need not be as close as this.
Given potential process refinements they may be disposed within a few cell diameters of each other. In the preferred embodiment, the voltage will be 25 volts or less to create sufficient potential for poration. -Thus, the instant invention permits a method of subjecting ~n ~ adherent cells to electrical fields of sufficient intensity to induce electroporation of n situ adherent cells while said cells are adhered to an eleetrode surface, which comprises the steps of eultivating adherent cells in a culture media on an electrically conductive surfaee which is characterized by affording an upper surface eondueive to cell adhesion and growth; replacing said culture media with a liquid media appropriate for electroporation of cells; supporting a eounter-electrode at a close- distance from said surface having said cells adherent thereon so that said counter-lS electrode is in contact with said electroporation media; ;
applying a source electric potential of su~ficient ~ ;
intensity to effect electroporation to said surface and said counter electrode; removing said counter-electrode and replacing said electroporation media with said eulture media. This represents a tremendous saving in labour, cost and in the risk of contamination over, and affords results hitherto unaehievable by, the prior art.

With a readily eontrolled planar-pro~eeted eleetrie field sueh as made possible by the instant invention a deviee with appropriate sensing means, Such as thin-film eleetrodes disposed onto the cell adhesion eondueive eoating and isolated from the coating so as to be only in eonductive eontact with the media, and eonneeted to an eleetrical field intensity measuring deviee appropriately interfaced to a micro-proeessor, ~
that one has an "intelligent" electroporatiQn device --~i.e. a deviee capable of applying ever increasing eleetrical potential until the eells have porated, and eapable of sensing at what field intensity the eells have porated)~ as well as a device for reeording hitherto unavailable information eoneerning the poration potential required for various eell lines and the effects of .:~

-~ 2~5~2 ;. .:. `

various media compositions on the types and sizes of porations that may occur.
A yet further embodiment of the device allows the hitherto unachievable process of time-targeted S-phase introduction of exogenous material such as DNA, RNA, and proteins via electro-transfection into synchronized cell cultures which would allow researchers to determine the start time of a particular section of code replication, which is complex information to deduce because replication is a multi-point parallel process rather than a serial process. To allow the material introduced into the cell to be incorporated into the DNA
of the target cell for a permanent expression, it is advantageous to know when the target site will replicate.
Incorporation prior to replication obtains the greatest probability that the daughter cells will permanently express the new code, whereas incorporation after target site replication may result in only a transient expression. Since the applied voltages are very small, and no lnsult incurred by the cells, a large percentage of eells survive the eleetroporation.
$he deviee assists in the mapping process by allowlng time eorrelated introductlon of exogenous material into synchronized eells. Since virtually no as cell death .oecurs, and cells do not leave the S-phase, extremely high expression fregueneies beeome possible.
~y using time eorrelation and serum stimulated synehronized cells, resulting expression effieiencies of integrated ~aterial may be directly correlated to whether or not transfection occurred prior to a given site's replication period. ~y using batch trans~ection of ~everal different marXers simultaneously, and stepping through the S-phase in discrete timed steps in a sueeession of related experiments, information as to what sites are undergoing replication at what time becomes available. Some of the markers making up a given batch may prevent or enhance the expression of other markers in the given batch. Since cell populations remain . ~. -202~0~

essentially intact and no base population shift occurs,very high experiment-to-experiment correlations can be made, further increasing the available data. The entire procedure may be effected and recorded by microprocessor means, and is down-loadable to a system processor for recordal purposes by means of appropriate software.
Very high rates of expression should be obtainable, barring loses due to replication correction undertaken by the cell. Information received as a result can be used to build a map of replication sites in relation to time and thusly be used to determine the replication sequence for a given DNA strand.
A further embodiment allQws a plurality of precisely timed electroporations of synchronized cultures to be effected sequentially by a microprocessor and appropriate software operating as a control unit. Since there is very little if any shift in the base population of the synchronized cells, the experiments will have a high degree of correlation with each other, further increasing the amount of information that may be deduced.
The control unit may have provided a program to correlate the marker, the time into S-phase of introduction and resulting expression or non-expression into a map of the sites undergoing replication at any given time in the S-phase.
Thus, the instant invention may be used in amQthod o~ mapping DNA replication sequences by time-correlated in situ electroporetic introduction of exogenous material into synchronized adherent cell cultures on an electrode surface while said cells are in their S-phase, which comprises the steps of: preparing, culturing and serum stimulating adherent cells cultures in one or more lower units of davices for subjecting adherent in ~ cell cultures to an electrical field while said calls are adherent to an electrode surface, said one or more lower electrode units comprised of an electrically conductive coating characterized by affording an upper surface conducive to cell adhesion and 2 ~ 2 ~

growth, and having a sensing electrode disposed on an insulating layer disposed on said coating and electrode means in contact with said coating, inputting serum-stimulation time and start of S-phase into control unit, said control unit comprising means to detect electroporation fluid ionization, means to disariminate the transmembrane flow of ions from said cells into said electroporation ~luid when said cells porate and generate a corresponding modulating signal, means for said modulating signal to control a source of external electrical potential, and software to control the procedure; preparing an electroporation media, said electroporation media consisting of a solution osmotically balanced for cell survival and ionizable components and having in suspension one or more types of code, proteins or other materials desired to be entered into the cells, replacing culture media with said electroporation media, inserting into each of said one or more lower units of said device, upper upper units comprising counter-electrodes at a close distance from ~ald coating having said cells adherent thereon, wherein said counter electrodes have a sensing electrode disposed on an insulating layer disposed on their sur~ace and electrode means in contact with said counter-electrode;
ensuring said electroporation fluid is in contact with said cells, said coating, said counter-electrode, and said sensing electrodes in each of said devices;
connecting the sensing electrodes of each of the one or more devices to means for detecting poration of said cells in said one or more devices; connecting said lower electrode unit and said counter-electrode unit of each o said. one or more devices to their respective external lectrical terminalst inputting into the control unit the electroporatlon sequence for each of one or ~ore said devices, the delay period into S-phase of the first poration, and the delay period between each subseguent poration; enabling the control unit, replacing the electroporation fluid with culture media; correlating the 2~2~22 - -:-expression vs non-expression results and inputting resulting data along with the type of code, protein or other material into the control unit data-mapping program. The device may also give an indication when the process has been completed.
Referring now to Figures 7, 7a and 7b, ionization potential sensing means 70 may be placed on the lower electrode 30 and counter electrode 40 by laying down on the T/CACC 2 or 46 coated radial distribution electrodes 24 or 45, an insulating thin film strip 71 of SiO2 or similar material, extending from a contact source on the upper-outer face of the substrate 31 or 42 and over the outer circumference and thence outward onto the electrode field area of the lower or counter electrode surface 2 or 46 in the form of a strip. Over laying this strip 71 of insulating material is the sensing electrode 70 formed of thin film deposition noble metal or other material. The ionization potential electrode 70 extends in a thin strip centered on the insulating thin film strip 71 and is of a width narrower than the strip 71 so as to preclude contact with the T/CACC 2 or 46, and terminating in a rectangular or other shaped external sensing electrode contact area 72.
Shown in Figure 6 i9 the lower electrode sensing means contact 72a. and not shown is counter electrode sensing means contact 72b. In the case of the counter electrode, the ionization sensing electrode strip 70 extends over the outer circumference and up the face of the counter electrode support tube 43, and terminates in a rectangular or other shaped external sensing eleotrode contact area 72b (not shown). Apertures are provided for the external sensing electrodes for access the contact areas 72a and 72b in the same fashion as for the external power electrodes discussed earlier.
3S The sensing electrode contact means and the power electrode contact means may be radially displaced from each other,such as 90 degrees apart, so that no errors, such as attempting to sense from the power 202~02~

electrode (coating 2 or 46), or applying external power to the sensing electrodes can occur.
When the externally applied electric field climbs in intensity, electrophoretic disassociation of the electroporatlon media takes place as motile ions are drawn to their corresponding electrodes and an electrical charge will be stored as an electrophoretically imbalanced solution. The ionization sensing electrodes, coupled to a high impedance circuit as part of a tuned tank, or other circuit capable of being used for this purpose, serve to detect the extent of electrophoretic disassociation of the solution at any given instant. As the current carriers native to the electroporation media dri~t to their respective electrodes, the conductivity of the solution decays (resistance increases) resulting in the drifting of the tuned circuit from a given starting ~requency. This measurement corresponds to the electroporation media disassociation only, until such time as poration of the cell membrane occurs. The cell membranes prevent the ionizable components of the cytoplasm from dri~ting externally to the membrane until su¢h time as poration occurs. At such time, a momentary ~urge o~ non-native current carriers becomes available to the electroporation media. This momentary surge causes a ~ ;
momentary rapid shift in the electrophoretic disassociation curve of the ETM, causing a momentary deflection in the frequency o~ the tuned circuit and is converted into a control signal that is used to control the external power source to the lower and counter 30 electrodes. ~ -Referring now to Figure 10, the ionization ; ;
sensing electrodes 72a and 72b, placed so as to be ;~ ~;
vertlcally disposed ~rom each other and in electrical contact by means o~ the electroporation media 75 3S ~repre~ented here as a variable resistor in series with a leaky capacitor), when coupled with an electrically isolated high impedance detector means 76 may be used to determine the degree of polarization o~ the . . . .. ~, ' 2~2~022 electroporation media caused by the electric strain.
Detector means 76 may be comprised of a differential amplifier circuit or tuned tank circuit. In an alternative embodiment, detector means 76 may comprise a conductivity meter.
The instant invention thus may be used in a method of detecting electroporation of ~n situ adherent cells while said cells are adherent to an electrode surface, which comprises the steps of cultivating adhehent cells in culture media on an electrically conductive surface characterized by affording an upper surface conducive to cell adhesion and growth and having a first or second sensing electrode disposed on an insulating means disposed on said surface; replacing said culturing media with a media appropriate for electroporation; disposing a counter-electrode, having a flrs~t or second sensing electrode disposed on an insulating means disposed on said counter-electrode, at a close distance from said surface having said cells adherent thereon so that said electroporation media is in contact with said cells, said surface, said counter-eleotrode, and said ~irst and second sensing electrodes, said electroporation media characterized by being in osmotic balance with said cells and containing motile lons; connecting said sensing electrodes to means for detecting polarization o~ said media upon the application o~ an electrical potential to -said surface and said counter-electrode; connecting means for discriminating the transmem~rane flow of ions from said cells when said cells porate to said polarization detection means, connecting said surface and said counter electrode to a source of electrical potential; applying an electrical potential to -~aid surface and said counter-electrode to porate said cells; recording said detection o~ said poration of said cells; removing said counter-electrode and replacing said electroporation media with said culturing media.
The output signal from the detector stage 76 may --~ 2~2~02~

be converted from an analog signal to digital via A/D 77.
Connected to the lower and counter electrodes of the electroporation chamber is a source of external electrical power EP controlled by switch 78 which may be transistorized. These units may form the basis of an electroporation module 79.
Several electroporation modules 79 may be connected to a versatile interface adapter 80 which would allow microprocessor 81 to have fast access to the output of each module to the hardware addressing approach which is significantly faster than other techniques.
Microprocessor 81 may be used to calibrate or set the detector circuit 76 operational parameters. The microprocessor may also control via output signals a bank of addressable switches (not shown), with the electrical power being delivered individually to each electroporation module 79. With this arrangement, switch 78 would be replaced by an equivalent unit in the above mentioned bank of switches.
The microprocessor program 82 may be controlled either by operator keyboard 83 input, or external mlcroprocessor 84, or by a combination of both.
Additionally, the program 82 may retrieve and store information concerning the conductivity, field intensity at time of poration, cell type, time into S-phase among, other data the operator may wish to have available.
Several electroporation modules 79 may be linked to the microprocQssor~s, providing very accurate time control over a large number of related porations.
The use, design and applications of microprocessors for gathering information and process controlling is well known in the art of electronics, and it is beyond the scope of this specification to outline all ~easible methods of designing such circuitry. ~o those s~illed in the art of microprocessor applications, such criteria are a merely a matter of design. The same is true of the program required to conduct the process.
The software is highly dependent on the hardware ..

--` 2~50~2 features. The process as outlined should provide a person skilled in the art of electronics and programming sufficient direction as to the features pertinent to the design of appropriate hardware and software.
The ~evice would allow accurate analysis of media composition changes on time/ voltage/ integration ~requency. The device may be programmed to calculate duty-cycle parameters in an automated process where different cell lines exposed in different media would be used primarily.
In this embodiment, the device may be used in a method of controlling an electrical field being used to induce poration of in situ adherent cells while said cells are adherent to an electrode surface, which comprises the steps of: culturing adherent cells in a culturing media on an electrode surface forming part of a means for subjecting adherent cells to an electrical field wherein said means for subjecting adherent cells to an electrical field also has means for detecting the electrically induced ionization of an electroporation media; connecting means for discriminating a trans-membrane flow of ions from said cells when said cells porate to said means for detecting the electrically induced ionization of an electroporation media forming 2S part of said means for sub~ecting adherent cells to an electrical field; connecting means for converting said discriminated transmembrane flow of ions into a modulating signal; replacing said culturing media with an electroporation media characterized by being in osmotic balance with said cells and containing motile ions;
connecting means for applying said electrical field to means of sub~ecting s,aid cells said electrical potential under control of said modulating signal; enabling said di~criminating means to apply said electrical potential to said device for sub~ecting said adherent cells to said electrical field; replacing said electroporation media with said culturing media.

` -` 2~2~022 In an alternative technique, instead of being placed in suspension in the electroporation media, the material to be transfected may be first electro-statically (or similar method) drawn onto a carrier surface which is placed onto the upper (counter) electrode. The carrier surface may be of materials like PAG or similar filter and membrane materials. The counter electrode and material carrier may then be disposed at a very close distance, such as lmm or less from the upper surface of the cells in the pepetri-dish, and appropriate electrical charges applied. The distance that the opposing electrodes may be placed relative to each other is an obvious advantage of the instant invention over any prior art, in this manner. Further targeting of the material to be transfected may be achieved by the use of an appropriate electroporation media in which a large ma~ority, if not all o~ the otherwise ionizable material is removed, leaving the material to be transfected as the ~ ionizable material. With this approach, material uch as DNA and RNA and materials having only a very small charge may be made the current carrier, ~urther increasing its upta~e into the cell. It is recognized that this new geometry will simplify if not remove the current signi~icant problem of determining at exactly what ~ield intensity individual cell lines react to (for poration purposes and others), and the calculation of what field intensity a cell is actually being subjected to and what effect media composition plays in the process .
~ A look-up table may be generated so that for a given cell type, and ~or a given exogenous material, requiring a giv~n media, the apparatus would need only a program selection number relating to cell type, a media type, and a target molecule (extra-cytoplasmic material such as DNA, RNA, etc.) re~erence name or number, the device can set the optimum conditions ~or each poration, rather than having the experimenter having to continuously reset the intensity/time functions for "`"~
- ~ .

2~a2~

change in cell line, cell type, media, specialized media commitments, etc as is the current practice. Since the device measures the conditions required to induce poration, and detects when poration occurs, substantial reductions in current mediated cell death will be realised since only enough energy to induce poration is introduced into the system.
Microprocessor controlled bursts of short duty-cycle ionization may be employed so as to intermittently drsg the targeted molecule without incurring damage to the coating. In addition, the microprocessor may have control over a wave-form generator allowing electrical power of many varying waveforms to be applied.
By use of low ionization potential electrotransfection media, such as the earlier mentioned poration media, higher electrical field intensities may be used if desired. Removal of the not required ions such as Cl will reduce electrode damage at occurs at higher voltages and currents. Power levels sufficiently high enough to damage the T/CACC should not be required because of the distance of the opposed electrodes. Since electrically induced cell membrane perturbations are the bAsls of pores opening in the cell and research shows that this poratlon occurs virtually instantly when the trans-membrane potential passes the 0.7 to l.l V region, voltage fields of sufficient intensity to induce poration should be achievable in the 20 volt and less region.
This is of course highly dependent on the CESM height and m~dia being used. Some variance may be found due to the cell type, depth or thic~ness, targeted molecule (net charge, size, shape).
The above procedures may be advantageously effected by the chilling of the cell cultures prior to the application o~ electrical energy. This also holds true for chllling during the procedure. While the chilling is not required to prevent over-heating of the cells and poration media, studies have shown that the pores produced, begin to "heal" or close almost as soon 2 ~ 2 2 ~ ~

as the pore inducing field is removed. Chilling the cells results in a somewhat greater time required for the membrane materials to flow back into their original position. The desirability of chilling, will however, need to be assessed for different applications.
Figure 9 shows the relationship of an assembled lower and upper electrode forming an electroporation device and inserted into a handling device. The pepetri ~lower electrode 30) and counter electrode 40 may be inserted into a handling device 89 used to both transport the electroporation apparatus and act as an electrical interface jack connecting the lower electrode 30 and counter electrode 40 and the external ionizing source leads 37 and 52 together. Lower electrode electrical contact 34 is made accessible to ionizing source electrode 37 via means of aperture 35. Counter electrode eleatrical contact 47 is made accessible to ionizing source electrode 52. Both contact electrodes 37a and S2a may be in the form of a spring, and when the electroporation device is inserted into the handling device 89, make conductive contact with their appropriate lectrical contact. The arms of the handling device are separated at their outer ends by a distance nominally larger than the outside diameter of the pepetri, and then converge slightly to a distance somewhat less than the diameter of the pepetri. The longer arm of the two has disposed at its outer end, and directed inwardly, a retain~ent spring (element 20 of Figure 8), which serves to lock the pepetri and counter electrode into the ~handling device and bring electrical contacts 37a and 52a into contact with electrical contacts 34 and 47.
Electrical contacts for the sensing electrodes may be built in the same fashion as the power electrodes and match up with their appropriate apertures at the ~unction 3~ of the two arms o~ the handling device. Further, there may be provided in both arms at appropriate locations, compression springs 9o serving to keep the counter electrode 40 and lower electrode 30 in close contact. The ,':, .".' ''. '' ,'`.`'.....
.~ . . ,- , r ~ 9 i 2 ~ 2 ~
. .
. .

handling device may be so designed so as to allow its use on optical microscope stages when it is of interest to visually inspect the cells and/or process for film recording.
Althouh the aspects of the present in~ention have been fully described by way of example with reference to the accompanying drawings, it should be noted that many of the ebodiments disclosed may be interchanged in part or in total with other embodiments outlined, and as a result numerous variations in design become possible, and as such, these various changes, modifications and other embodiments will be apparent to those skilled in the art.
Therefore, unless such changes and modifications otherwise depart from the scope of the present invention, they should be construed as being included herein.
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 cell microbiology, and especially to those skilled in the art of application of electric fields to cells, such as for use in electro-fusion.
To those skilled in the art o~ electrofusion of cells, it will be readily apparent that slight modifications to the device, such as the culturing of another monolayer o~ a different cell line on another ~odified 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 the other and an electrical charge applied, offers a new and radically different way with which to subject substantial numbers of the cells to a condition to promote cell fusion and with which to be abl~a to study optically the process of cell fusion. In this case the electrodes would serve to deliver either AC fields and/or DC ~ields.
It will also be apparent that the study of cells, and the study of micro-organisms such as bacteria and viruses not requiring to be adherent to a surface, will also benefit from the ability to expose them to a 2 ~ 2 2 .... ..

uniform electric field while under optically observable conditions, such as generated by the instant invention.
Finally, to those skilled in the art of cell culture, it will be apparent that the new geometry and other features of the instant invention also have application in the field of bio-reactors and assoeiated devices and that eleetrically eonduetive eoatings eonducive to cell adhesion and growth have direct and wide spread potential applieations in the field of bio-sensors and bio-sensory apparatuses.
. It will also be apparent that the culture surface may be fabricated in the form of a checkerboard, in which, using current ion beam etching and similar techniques such as used in the fabrication of very large scale integrated eireuits, that individual "checkerboard squares" may be fabrieated of alternate areas of eonduetive and non-eonductive material wherein eaeh eonduetive square may be individually addressable, sueh as by eurrent eomputer means, and have one or more nerve eells or brain eells attaehed thereto and adressed thereby, and will have application in such areas as nerve eell growth and communication studies, bio-sensors, bio-implants and similar.
A ~urther modi~ication of the instant invention contemplates the disposition of an electrically insulating layer of, for instance sio2 type materials onto the upper surface of a pepetri dish (the coating 2 now beeomes optional), a similar disposition of sio2 type material (those materials applicable to thin-film techniques and that have the required properties to be conducive to eell adhesion and growth) onto an opposing eounter-eleetrode where,in a radial distribution eleetrode is formed on the substrate, and the application of eleetrieal potQntials to the opposing eleetrodes so that 3S the eells eultured on the lower (or upper) sio2 surfaee are sub;eet to intense eleetrostatie fields, (beeoming unit di-polar eapaeitors in an ionizable solution comprising one half of two capacitors) suffieient to ~ '~ ''~ ''' ,. . -~: - .. - .: ~ . , , - , -~` 2~2~2~

induce poration of the cell membrane. A further alternate embodiment of this device is the automation of the device by using thin-film electrode strips disposed onto the SiO2 coatings, serving as media ionization sensing electrodes so as to be able to sense the permeabilization of the cells by means of sensing the outrush of ions from with-in the cell membrane. The momentary current pulse occurring, although small, can serve as means to sense the poration of the cells, (earlier alternate embodiment) and under appropriate micro-processor control, electro-static fields can be applied and controlled until such time as poration occurs, and all significant data recorded, Devices of the nature above can serve to automate the process of electroporation so that prior experimentation to determine the required electrical potential needed for poration of a given cell line may be dispensed with. Further, the device will provide measurable information as to the effect of media composition on required field intensities for electroporation when current is not applied.
The method of use is almost identical of that to the above disclosed devices, with the exception that the electroporation fluid would require a somewhat higher number of motile ions and the electrical potential required to induce poration may be substantially higher and for a longer period of time.

The foregoing material serves to demonstrate the tremendous untapped potential of the novel instant invention for use in the fields of microbiology, cell microbiology, genetic engineering, bio-reactors and bio-sensory apparatuses.
Those skilled in the art of microbiology or eleotronics will see that many various combinations and embodiments may be foreseen using the nature of the adhesion conducive properties of some semi-electrode materials, and as such, this disclosure should be construed as referring to these alternatives as well.

Claims (36)

1. Apparatus for subjecting in situ adherent cells to an electrical field while said cells are adhered to an electrode surface, said apparatus comprising:
an electrically conductive surface conducive to cell adhesion and growth;
means for maintaining cells submerged in an appropriate culture medium while adherent to said surface; and means for applying an electric potential or electrical ionizing source to said surface.

2. Apparatus for subjecting in situ adherent cells to an electrical field of sufficient intensity to induce electroporation of in situ adherent cells while said cells are adhered to an electrode surface, said apparatus comprising:
an electrically conductive surface conducive to cell adhesion and growth;
means for maintaining cells submerged in an appropriate medium while adherent to said surface;
a counter-electrode:
means for supporting said counter electrode in contact with said medium and at a close distance from said surface; and means for applying an electric potential or electrical ionizing source to said surface and said counter-electrode.

3. Apparatus for detecting electroporation of in situ adherent cells while said cells are adherent to an electrode surface, said apparatus comprising:
an electrically conductive surface conducive to cell adhesion and growth;
means for maintaining cells submerged in an appropriate medium while adherent to said surface;
a counter-electrode supported in close proximity to said surface;
first and second sensing electrodes disposed on but insulated from said surface and said counter-electrode;
means for insulating said first or second sensing electrode from direct physical contact with said surface or said counter electrode;
means for retaining a fluid in contact with the cells, said surface, said counter-electrode, and said first and second sensing electrodes;
means connected to said sensing electrodes for detecting polarization of the fluid upon the application of an electrical potential to said surface and said counter-electrode;
means connected to said polarization detection means for discriminating the transmembrane flow of ions from the cells when they porate; and means for applying an electrical potential to said surface and said counter-electrode.

4. Apparatus for controlling an electrical field being used to induce poration of in situ adherent cells while said cells are adherent to an electrode surface, said apparatus comprising:
a chamber means wherein in situ adherent cells may be subjected to an electrical field of sufficient intensity to cause poration of cells while the cells are adherent to an electrode surface; said chamber characterized by having at least one internal face being an electrically conductive surface conducive to cell adhesion and growth;
means for applying an increasing electrical potential to said chamber means;
means for detecting a transmembrane flow of ions when cells porate;
means for converting said detected transmembrane flow of ions into a modulating signal; and means for applying said electrical potential under control of said modulating signal to said chamber means.

5. Apparatus for the transfection of exogenous material into is in situ adherent cells by means of electroporation while said cells are adherent to an electrode surface, said apparatus comprising:
an electrically conductive surface conducive to cell adhesion and growth;
fluid retaining means for maintaining cells submerged in an appropriate medium while adherent to said surface;
a counter-electrode, means for supporting said counter electrode in close proximity to said surface having cells adherent thereon;
means for applying an electric potential or electrical ionizing source to said surface and said counter electrode;
means for detecting the poration of cells by means of sensing electrodes and converting said detected poration into a modulating signal;
means for retaining a fluid in contact with cells, said surface, said counter-electrode, and said sensing electrodes; and means for controlling by said modulating signal, said electrical potential or electrical ionizing source.

6. Apparatus for mapping DNA replication sequences by time-correlated electroporetic introduction of exogenous material into synchronized in situ adherent cell cultures while said cells are in their S-phase and adherent to an electrode surface, said apparatus comprising:
one or more electroporation units, said units characterized by having at least one electrically conductive surface conducive to cell adhesion and growth, fluid retaining means, a counter-electrode, means for supporting said counter electrode in close proximity to said surface, together providing a chamber wherein a fluid may be in contact with cells, said surface, said counter-electrode, and said sensing electrodes;
means for detecting poration of cells in said one or more units by means of said sensing electrodes and converting said detected poration of said cells in said one or more units into a modulating signal;
means for controlling by said modulating signal said electrical potential or electrical ionizing source;
means for recording the time each of said one or more units cells are serum-stimulated to synchrony;
means for detecting the elapse of a predetermined time inputted by an operator; and means for applying an electric potential or electrical ionizing source to each of said one or more units when said predetermined time has elapsed.

7. Apparatus for subjecting cells to an electrical field, said apparatus comprising:
an electrically conductive surface conducive to cell adhesion and growth;
fluid retaining means for maintaining cells in an appropriate medium while on said surface; and means for applying an electric potential or electrical ionizing source to said surface.

8. Apparatus for electrofusing in situ adherent cells while said cells are adherent to an electrode surface.

9. Apparatus according to claim 3, 4, 5 or 6, wherein disposed on the electrically conductive surface are means for electrically isolating the electrodes from the electroporation medium, said means comprising:
a thin film layer of insulating material, wherein said insulating material is characterized by affording a surface conducive to cell growth and adhesion;
and further characterized by having a high dielectric value and being of sufficient thickness so as to prevent dielectric breakdown across it when subjected to electric fields of sufficient intensity so as to cause the poration of cells adherent thereon.

10. Method of subjecting in situ adherent cells to an electrical field while said cells are adherent to an electrode surface, said apparatus comprising the steps of:
cultivating adherent cells on an electrically conductive surface conducive to cell adhesion and growth, applying an electric potential or electrical ionizing source to said surface to establish an electrical field emanating from said surface.

11. Method of subjecting in situ adherent cells to an electrical field of sufficient intensity to induce electroporation of in situ adherent cells while said cells are adhered to an electrode surface, which comprises the steps of: cultivating adherent cells in a culture medium on an electrically conductive surface which is conducive to cell adhesion and growth;
replacing the culture medium with a liquid medium appropriate for electroporation of cells;
providing a counter-electrode in close proximity to said surface having cells adherent thereon so that said counter-electrode is in contact with the electroporation medium;
applying a source electric potential of sufficient intensity to said surface and said counter electrode to effect electroporation; and removing said counter-electrode and replacing the electroporation medium with culture medium.

12. Method of detecting electroporation of in situ adherent cells while said cells are adherent to an electrode surface, which comprises the steps of:
cultivating adherent cells in culture medium on an electrically conductive surface conducive to cell adhesion and growth and having a first sensing electrode disposed on an insulating means disposed on said surface;
replacing said culturing medium with a medium appropriate for electroporation;
providing a counter-electrode, having a second sensing electrode disposed on an insulating means disposed on said counter-electrode, in close proximity to said surface having cells adherent thereon so that the electroporation medium is in contact with cells, said surface, said counter-electrode, and said first and second sensing electrodes;
connecting said sensing electrodes to means for detecting polarization of the medium upon the application of an electrical potential to said surface and said counter-electrode;
connecting means for discriminating the transmembrane flow of ions from cells when the cells porate to said polarization detection means;
connecting said surface and said counter electrode to a source of electrical potential;
applying an electrical potential to said surface and said counter-electrode to porate the cells;
recording said detection of said poration of the cells;
removing said counter-electrode and replacing the electroporation medium with culturing medium.

13. Method of controlling an electrical field being used to induce poration of in situ adherent cells while said cells are adherent to an electrode surface, which comprises the steps of: providing an electrode surface conducive to cell adhesion and growth which forms part of a device for subjecting adherent cells to an electrical field, wherein said device for subjecting adherent cells to an electrical field also has means provided for detecting the electrically induced polarization of an electroporation medium;
connecting means for discriminating a transmembrane flow of ions from cells when the cells porate to said means for detecting the electrically induced polarization of an electro-poration medium used in said device for subjecting adherent cells to an electrical field;
connecting means for converting said discriminated transmembrane flow of ions into a modulating signal;
replacing the culturing medium with an electroporation medium; connecting means for applying an electrical field to means of subjecting said cells said electrical potential under control of said modulating signal;
enabling said discriminating means to apply said electrical potential to said device for subjecting said adherent cells to an electrical field; and replacing the electroporation medium with culturing medium.

14. Method of transfecting exogenous material into in situ adherent cells by means of electroporation while said cells are adherent to an electrode surface, which comprises the steps of:
providing an electrode surface conducive to cell adhesion and growth which forms part of a device for subjecting adherent cells to an electrical field, wherein said device for subjecting adherent cells to an electrical field also has means provided for detecting the electrically induced polarization of an electroporation medium;
connecting means for discriminating a transmembrane flow of ions from cells when the cells porate to said means for detecting the electrically induced polarization of an electro-poration medium used in said device for subjecting adherent cells to an electrical field;
connecting means for converting said discriminated transmembrane flow of ions into a modulating signal;
replacing the culturing medium with an electroporation medium having in suspension code, proteins or other materials desired to be entered into the cells;
connecting means for applying an electrical field to device for subjecting cells said electrical potential under control of said modulating signal;
enabling said discriminating means to apply said electrical potential to said device for subjecting adherent cells to an electrical field; and replacing the electroporation medium with culturing medium.

15. Method of mapping DNA replication sequences by time-correlated in situ electroporetic introduction of exogenous material into synchronized adherent cell cultures on an electrode surface while said cells are in their S-phase, which comprises the steps of:
preparing, culturing and serum stimulating adherent cell cultures in one or more lower units of devices for subjecting adherent in situ cell cultures to an electrical field while the cells are adherent to an electrode surface, said one or more lower electrode units comprised of an electrically conductive surface conducive to cell adhesion and growth, and having a sensing electrode disposed on an insulating layer disposed on said coating and electrode means in contact with said surface;
inputting serum-stimulation time and start of S-phase into the control unit, said control unit comprising means to detect electroporation fluid ionization, means to discriminate the transmembrane flow of ions from said cells into said electroporation fluid when cells porate and means to generate a corresponding modulating signal, means for said modulating signal to control a source of external electrical potential, a micro-processor, and control software;
preparing electroporation fluid, the electroporation fluid consisting of a solution having in suspension one or more types of code, proteins or other materials desired to be entered into the cells;
replacing the culture medium with the electroporation medium;
inserting into each of said one or more lower units of said device, upper units comprising counter-electrodes in close proximity to said surface having said cells adherent thereon, wherein said counter electrodes have a sensing electrode disposed on an insulating layer disposed on their surface and electrode means in contact with said counter-electrode;
ensuring that the electroporation fluid is in contact with the cells, said surface, said counter-electrode, and said sensing electrodes in each of said devices;
connecting the sensing electrodes of each of the one or more devices to means for detecting poration of cells in said one or more devices;
connecting said lower electrode unit and said counter-electrode unit of each of said one or more devices to their respective external electrical terminal;
inputting into the control unit the electroporation sequence for each of one or more said devices, the delay period into S-phase of the first poration, and the delay period between each subsequent poration;
enabling the control unit;
replacing the electroporation fluid with culture medium;
correlating the expression vs non-expression results and inputting resulting data along with the type of code, protein or other material into the control unit data-mapping program.

16. Method for electrofusing in situ adherent cells while said cells are adherent to an electrode surface.

17. Method for electrostatically inducing poration in in situ adherent cell cultures while said cells are adherent to a di-electric or insulating surface disposed on an electrode.

18. Apparatus as claimed in claim 1, 2, 3, 4, or 5 wherein the electrically conductive surface affording a surface conducive to cell adhesion and growth is comprised of a semi-conductor material.

19. Apparatus as claimed in claim 6, 7, or 8 wherein the electrically conductive surface affording a surface conducive to cell adhesion and growth is comprised of a semi-conductor material.

20. Apparatus as claimed in claim 18, wherein the semiconductor material is selected from the group consisting of tin oxide, tin oxide and indium oxide, indium oxide.

21. Apparatus as claimed in claim 19, wherein the semiconductor material is selected from the group consisting of tin oxide, tin oxide and indium oxide, indium oxide.

22. Apparatus as claimed in claim 18, wherein the conductive coating is selected from the group consisting of stannic oxide doped with fluorine or antimony, indium oxide doped with cadmium oxide, cadmium stannate, zinc oxide, zinc cadmium sulfite or titanium nitride.

23. Apparatus as claimed in claim 19, wherein the conductive coating is selected from the group consisting of stannic oxide doped with fluorine or antimony, indium oxide doped with cadmium oxide, cadmium stannate, zinc oxide, zinc cadmium sulfite or titanium nitride.

24. Apparatus as claimed in claim 18, wherein the conductive coating is selected from the group consisting of rubidium silver iodide, dieuropium trioxide, lanthanum hexaboride, rhenium trioxide or divanadium pentaboride.

25. Apparatus as claimed in claim 19, wherein the conductive coating is selected from the group consisting of rubidium silver iodide, dieuropium trioxide, lanthanum hexaboride, rhenium trioxide or divanadium pentaboride.

26. Apparatus as claimed in claim 18, wherein the electrically conductive coating is deposited on one or more layers of material more electrically conductive than said electrically conductive coating.

27. Apparatus as claimed in claim 19, wherein the electrically conductive coating is deposited on one or more layers of material more electrically conductive than said electrically conductive coating.

28. Apparatus as claimed in claim 26, wherein the electrically conductive layer or layers are transparent to an extent sufficient to permit optical observation of cells adherent thereon.

29. Apparatus as claimed in claim 27, wherein the electrically conductive layer or layers are transparent to an extent sufficient to permit optical observation of cells adherent thereon.

30. Apparatus as claimed in claim 3, 4, 5, 6, or 7, wherein said polarization detecting means is selected from the group consisting of differential amplifier, tunable tank circuit or conductivity meter.

31. Apparatus as claimed in claim 9, wherein said insulating material is disposed on a counter-electrode surface.

32. Method pursuant to claim 14 wherein some of the codes, proteins or other materials desired to be entered into the cell DNA effects the expression of some of the other codes, proteins or other materials entered simultaneously, if integrated or expressed.

33. Apparatus as claimed in claim 1, 2, 3, 4 or 5, wherein the electrically conductive surface affording a surface conducive to cell adhesion and growth is comprised of conductive plastic.

34. Apparatus as claimed in claim 6, 7 or 8, wherein the electrically conductive surface affording a surface conducive to cell adhesion and growth is comprised of conductive plastic.

35. A method pursuant to claim 11, 12, 13, 14, or 15, wherein the cells are chilled prior to being subjected to electrical fields inducing electroporation.

36. A method pursuant to claim 16, or 17, wherein the cells are chilled prior to being subjected to electrical fields inducing electroporation.