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Methods and devices for the electrically tight adhesion of a cell to a surface

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Publication number
WO2002059597A1
WO2002059597A1 PCT/IB2001/000095 IB0100095W WO2002059597A1 WO 2002059597 A1 WO2002059597 A1 WO 2002059597A1 IB 0100095 W IB0100095 W IB 0100095W WO 2002059597 A1 WO2002059597 A1 WO 2002059597A1
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WO
Grant status
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Patent type
Prior art keywords
cell
carrier
layer
surface
membrane
Prior art date
Application number
PCT/IB2001/000095
Other languages
French (fr)
Inventor
Christian Schmidt
Original Assignee
Cytion Sa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/48707Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728Investigating individual cells, e.g. by patch clamp, voltage clamp

Abstract

An apparatus is disclosed for the electrically tight adhesion of a cell to the surface of a carrier despite the presence of obstacles to form an electrically sealed patch of the cell membrane over an aperture in the carrier, thereby allowing accurate measurement of electrical properties of the cell and cell membrane. The carrier includes a compressible surface layer having hydrophobic and cell attracting properties. The hydrophobic nature of the layer insulates against current leakage through water and ions between the cell and the carrier, the cell attracting properties aid in adhering the cell to the carrier over the obstacles between the cell and the carrier which sink into the compressible layer. The layer can be formed of various materials such as a lipid bilayer, a silicone based oil or aminosilanes. Seal formation is further aided by the reduction of cell stiffness including changes in the osmolarity of the cell solution or a reduction in the stiffness of the cell cytoskeleton which allows the cell membrane to more closely adhere over the obstacles and onto the surface of the carrier.

Description

METHODS AND DEVICES FOR THE ELECTRICALLY TIGHT ADHESION OF A

CELL TO A SURFACE

FIELD OF THE INVENTION The present invention relates to a method and apparatus for manipulating and testing biological membranes, more particularly to the electrically tight adhesion of cells for the electrical testing and measurement of the cell membranes.

BACKGROUND OF THE INVENTION The screening of potential pharmaceutical agents such as ion channel agonists/antagonists, hormones and cytokines often requires measurement of a cell's electrical properties over time and after exposure of the cell to the pharmaceutical agents. One way to measure the electrical properties of a cell and its membrane is to conduct patch clamp recordings, an example of which is disclosed in B. Sakmann and E. Neher, Editors, Single-Channel Recording (1st edition, 1983). Patch clamp recording involves placing a fluid filled glass-micropipette onto a cell or liposomal membrane or to place cells or liposomes on a hard planar surface (typically constructed of SiO2 or PYREX) containing a small liquid filled hole. The diameter of the hole is typically less than 5 μm and smaller than the diameter of the cell which is to be measured. The portion of the cell's membrane that covers the hole is a "patch" that is electrically isolated, or sealed, with a resistance on the order of 1 GΩ or greater as disclosed in the above referenced book by Neher and Sakmann as well as in the PCT publication WO 99/31503. The electrical properties of the patch of cell membrane can then be subjected to various electrical measurements. The carrier surface on which the cell rests must be free of nano and micro scale obstacles to allow for close adhesion of the cell membrane that is necessary to form seal resistances higher than about 1 GΩ ("Gigaseal"). Resistances lower than 1 GΩ cause significant electrical noise and are generally less effective for the accurate measurement of the electrical properties of the cell and its membrane. These obstacles include proteins, lipids, and intracellular organelles, which are hard to avoid in cell suspensions. The obstacles interfere with the formation of the seal by acting like a spacer and preventing the cell membrane from coming into intimate contact with the carrier surface. One exception to the problem caused by the presence of obstacles is the relatively easy formation of a seal that occurs during the testing and measurement of liposomes. Liposomal membranes have a high flexibility that allows a large part of the membrane to attach tightly to the carrier surface and form a Gigaseal even in the presence of obstacles. Seal formation of liposomal membranes without cleaning or other precautions has been shown to achieve resistances of up to 200 GΩ. See, Schmidt et al., 39 Angew. Chem. Int. Ed. 3137 (2000). However, the formation of Gigaseals on most cells with membranes stiff er than those of a liposome is generally still a difficult task.

It would be desirable to have a method and apparatus for the formation of electrically sealed patches that can be applied to most cells, especially those cells having membrane characteristics that differ from liposomal membranes, and that is still effective in the presence of debris and other obstacles on the carrier and in the cell membrane.

SUMMARY OF THE INVENTION The present invention relates to a carrier with a modified surface that facilitates the use of patch clamp techniques in pharmaceutical screening of biological cells of a wide variety despite the presence of obstacles in the cell suspension, including debris and factors (e.g., proteins and lipids) attached to the cell membrane. Reducing sensitivity to obstacles advantageously reduces the normally high constraints placed on the cleanliness of the cell suspension and on the types of cells that can be measured. The seal resistance of the patch and the patch clamp technique are further improved by combining the modified carrier surface with a method of modifying the mechanical properties of the cell and cell membrane to promote improved cell adhesion to the carrier. An apparatus for recording the electrical properties of a membranous object includes a carrier of the invention for supporting the membranous object. The carrier includes a compressible and insulated layer attached to a diaphragm. The compressibility of the layer allows the layer to absorb obstacles trapped between the membranous object and the layer. Absorption of the obstacles allows an electrically tight seal to form between the layer and the membranous object despite the presence of the obstacles. The carrier further comprises an aperture surrounded by the layer and providing access to a patch of the membranous object adhered to the layer. The patch is the portion of the cell membrane that covers the aperture and is electrically sealed at its periphery. The seal at the patch periphery electrically isolates the patch and allows electrical measurements to be taken through patch.

The layer includes a hydrophilic layer on top of a hydrophobic layer. The hydrophilic layer attracts and binds the membranous object while the hydrophobic layer insulates against currents between the membranous object and the carrier. Each layer can be formed by a number of methods using different materials. In one aspect, aminosilanes that allow an interaction between the amino-group and cell membrane are attached to the diaphragm using covalent bonds. In another aspect, the layer can be formed of amphiphilic molecules bound to the diaphragm and functionalized to bind specific cells. The layer can also be formed of long alkyl-chain silanes and amphiphilic molecules to promote cell adhesion. Other materials include the use of a lipid bilayer bound to a Si3N4/SiO2 diaphragm or thin oil films that have been functionalized at the surface, e.g. by physisorption of amphiphilic molecules or exposure to a plasma or glow discharge.

In another embodiment, electrically tight adhesion of a cell to the modified carrier, or even rigid carrier surfaces, can be aided or accomplished via a reduction in the rigidity of the cell membrane. The rigidity of the cell can be reduced through the temporary or permanent modification of the cytoskeleton, membrane fluidity or osmotic cell pressure.

Thus, the invention provides, among other things, devices and methods for improving the analysis of electrical properties of cell membranes by reducing sensitivity to debris and the stiffness of the cell membrane. BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein: Figure 1 is a schematic side view of a first embodiment of the apparatus for the electrically tight adhesion of a cell to a surface despite the presence of obstacles and other debris;

Figure 2 is a schematic side view of a carrier prepared with an insulated layer for cushioning objects for use in the apparatus shown in Figure 1;

Figure 3 is a schematic side view of a rigid cell spaced from a carrier due to the presence of obstacles between the cell and the carrier;

Figure 4 is a schematic side view of the cell in Figure 3 treated to increase its flexibility and adhere closely to the carrier; and Figure 5 is a graph of current versus time data collected using the apparatus depicted in Figure 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout.

As shown in Figure 1, the present invention includes an apparatus 10 for the electrically tight adhesion of a small membranous object, such as a biological cell and portions thereof, to form an electrically sealed membrane patch even in the presence of various debris, thereby allowing sensitive electrical measurements to be performed on the membranous object. The apparatus 10 includes a soft and compressible insulated carrier 11 with an aperture 16 connecting a fluid filled cell compartment 17 to a fluid filled reference compartment 18. An electrically sealed patch 20 is formed for electrical testing when a cell 15 in the cell compartment 17 sediments (or is otherwise urged) to adhere to the carrier 11 over the aperture 16. The seal is electrically tight even in the presence of obstacles 23 because the compressibility of the carrier 11 allows the obstacles to be absorbed (i.e., to sink) into the carrier surface. Current is passed through the patch 20 by a first electrode 21 located in the cell compartment 17 and a second electrode 22 located in the reference compartment 18. In the case of pharmaceutical screening assays, various stimuli can be introduced into either, or both, compartments and the resultant change in the electrical properties of the patch 20 can be measured and recorded.

Figure 2 is a schematic depiction of a side cross-sectional view of the carrier 11. The carrier 11 includes a diaphragm 12, a hydrophobic cushioning layer 13 and a functionalized layer 14 that mediates cell adhesion. The layers 13 and 14 are shown covering the entire surface of the diaphragm 12, but alternatively may cover only a small portion or zone of the diaphragm 12. The carrier 11 could also have several of these zones for the study of several cells and/or other membranous objects.

The characteristics of the layers 13 and 14 are that they are compatible with and attachable to the diaphragm 12, they exhibit charges or other anchoring points for cells to the cell compartment 17, and a certain fluidity or softness (compressibility) that allows obstacles 23 to be pushed into the layers. The combined thickness of layers 13 and 14 can be as in a range of 1 nm to 5 mm, but typically will be in a range of 3-20 nm. However, the production processes for a thickness between 4nm and 5 μm are generally less complicated.

In one embodiment, the hydrophobic layer 13 is attached or self-assembled onto the diaphragm 12 using functionalized alkanes that bind with their functional group to the diaphragm surface. The hydrophobic nature of the layer 13 excludes water and ions thereby preventing any kind of current flow inside the layer. Even in a case where the layer 13 is not perfect, such as when coverage of the diaphragm is less than 100%, lateral currents are still greatly inhibited.

The functional layer 14 is attached on top of the hydrophobic cushioning layer and preferably shows a hydrophilic surface to the recording buffer or cell suspension of the cell compartment 17. The layers 13 and 14 can be constructed of amphiphilic molecules such as lipids or surfactant molecules and may be functionalized to promote specific cell-carrier interactions. It is also possible to use molecules as the layers 13 and 14 that show a functional group on their ends pointing into the cell compartment 17 and that allow directly the interaction and attachment of cells. For instance, long-chain aminosilanes such as (17-aminoheρtadecyl)trimethoxysilane as disclosed in Harder et al., 13 Langmuir 445 (1997) can be covalently bound to a glass surface diaphragm while allowing on the other terminus the direct attachment of cells by a charge interaction between the amino-group and cell surface charge, which is usually negative- Long alkyl chain silanes use covalent bonds to attach to a diaphragm 12 having a SiO or PYREX surface. Amphiphilic molecules are then added that self-assemble on the hydrophobic alkyl-chain layer and provide, by means of the hydrophilic end, for a charge interaction with cells. The hydrophilic end can also be functionalized to allow a molecule-specific interaction with cells.

For instance, a monolayer of octadecyltricholrosilane (OTS) is self-assembled onto the SiO2 surface of the diaphragm 12 by immersion of the freshly cleaned diaphragm 12 surface into 1 mM OTS solution in bicyclohexyl for a 24 hour period. The OTS layer is stable allowing the carrier 11 to be kept for days under clean conditions without the danger of surface degradation or of excessive oxidation. Long-term oxidation may degrade surface properties more easily when molecules are attached to the surface that are already charged, that is if an entirely coated (i.e. with layer 13 and 14) diaphragm is stored. For instance, when using bilayers containing l-palmitoyl-2-oleoyl- sn-glycero-3-[phospho-rac-(l-glycerol)] (POPG) or silanes terminated with an amine group (aminosilanes). However, even under such conditions oxidation and consequently surface degradation can be largely prevented if appropriate storage conditions are chosen, for instance by storing the carrier 11 in Argon. The OTS layer can be modified directly before the cell adhesion, e.g., by addition of an aqueous solution containing sodium bis- (2-ethyl-l-hexanol) sulphosuccinate (AOT), sodium dodecyl sulfate (SDS) or didodecyldimethylammonium bromide (DODAB) in the range of the critical micellar concentration (CMC). After washing the solution away a surfactant layer adjacent to the OTS layer remains that promotes cell adhesion. When washing the surfactants away, it may be necessary to prevent a total removal of all free surfactant molecules so that a certain concentration in solution remains to avoid inducing desorption of the physisorped upper layer. In the case of AOT, at least cmc/100 should remain as described in Fragneto et al., 178 Journal of Colloid and Interface Science 531 (1996). In another embodiment, the layers 13 and 14 are preferably a lipid bilayer attached to a silicon oxide surface (the diaphragm 12). The lipid film is created by detergent removal of a solution of solubilized lipids or lipid-like compounds in contact with the diaphragm 12. The lipid bilayer can interact with both the oxide surface and the cells by electrostatic or hydrophobic hydrophilic interaction. As mentioned above, the hydrophobic nature of the cushioning layer 13 excludes water and ions, which could carry current laterally between the cell and the diaphragm 12. The layer 13 is also able to spatially buffer small obstacles 23 so as to allow electrically tight cell adhesion.

In still another embodiment, a thin oil, grease, or oil-like film is disposed on the diaphragm 12 surface to form layers 13 and 14. These thin films usually have a thickness that varies up to several hundred μm. Well suited to forming layers 13 and 14 are sufficiently flexible silicon oils or plastics typically having a low dielectric constant (ε). For instance, polydimethylsiloxane (PDMS), one brand of which is called SYLGARD, has a low dielectric constant (ε = 2.4). Oil films can be added to the carrier by evaporation or aerosol sputtering (spraying) or removal of solvent of an oil-solvent solution that is in contact with the diaphragm 12' s surface. Oil films can also be polymerized to improve physical and chemical properties. An example is the polymerization of SYLGARD. The degree of polymerization can be chosen to still allow small obstacles to be pushed into the layer.

The oil and oil-like layers are advantageously very hydrophobic and long-term stable, and can be activated or prepared with an outer surface compatible to cell adhesion. Examples for such a preparation is the self-assembly of surfactants as explained above. A direct activation of the hydrophobic layer is achieved by high-energy treatments, such as a plasma or glow-discharge exposure. One suitable plasma is an argon plasma that hydrophilizes the surface of the oil film thereby making it adhesive to cells.

Surface activation by an argon plasma has the advantage over an oxygen plasma of activating the surface without the danger of forming a glass-like layer on the top of the oil film. For the plasma process it is usually required to use oils that do not evaporate in the plasma chamber. After the plasma process, molecules mediating contact to cell membranes (e.g., poly-L-lysine) can be physisorped or covalently bound to the activated surface. Oil films can also be modified analogous to the modification of hydrophobic silane coatings as described above, such as by the self-assembly of amphiphilic molecules (e.g., surfactants and lipids).

Other methods could also be used to construct the layers 13 and 14 so as to have the desired properties necessary for the apparatus 10 to create tight electrical seals. For instance, vesicles could be spread on a hydrophilic carrier surface. Layers 13 and 14 could also be formed using plasma enhanced chemical vapor deposition of hydrophobic/hydrophilic molecules. Detergent removal of a solution containing solubilized amphiphilic molecules, such as lipids in contact with a hydrophobic or hydrophilic diaphragm 12 surface (which may already be pretreated with OTS), is another possibility. It is also feasible to position a large, flattened vesicle at the spot on the carrier 11 chosen for cell adhesion and adhere the cell 15 to the flattened vesicle. To increase the stability of the adsorbed layer, a cross-linking of the adsorbed molecules can be induced for some specific molecules (e.g., lipids with unsaturated fatty acid tails). The adhesion of specific cell types can be selectively enabled by using layers that posses cell specific binding sites, including, but not limited to, cell adhesion molecules, antibodies, lectins, receptor ligands and analogues, such as integrin and laminin and parts thereof. The layer containing the desired specificity could also be added just prior to cell adhesion. A very strong binding of cells to the carrier 11 is achieved by covalent attachment of cells to the layer 14. Because under such conditions the interaction carrier - cell is extremely strong, debris between carrier surface and the cell can be pushed into the cell membrane, i.e. the cell membrane will be bend inward at these points, if the carrier surface does not absorb these obstacles. Consequently, the cushioning layer 13 can be chosen very thin. The cushioning layer can even be omitted, if under these conditions the cell membrane allows for a sufficient cushioning or embedding itself. Examples for such a covalent binding is a SiO2 diaphragm 12 that is silanized with Aminosilanes, which in turn are bound to linker molecules by immersion in glutaraldehyde solution. The aldehyde group of the covalently bound glutaraldehyde binds cells covalently. Another example is the coupling of gamma-Maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) to the amino-group of aminosilanes bound to the diaphragm 12. This is typically done at pH 7.5 (6.5-8.5). After a short incubation in a solution containing e.g. 1% mercaptoethanol, cells are covalently attached to the GMBS layer in a solution buffered at pH 6.8 (6.5-7.0).

The layers 13 and 14 may be applied to diaphragm 12 materials such as SiO2, Si3N , PYREX and various plastics and is not limited to any particular geometry or size of diaphragm. The creation of the final insulation layers 13 and 14 can be done an instant before the adhesion of the cell 15 takes place. Over such a short time period, the part of the layers 13 and 14 interacting with the cell 15 is not prone to degradation or mechanical disruption. Diaphragm 12 surfaces may be patterned so as to allow cell contact at specific sites. Multiple specific site contact is important for a carrier 11 with multiple recording apertures. For multiple recording apertures, the diaphragm 12 surface may be patterned before addition of the insulation layers 13 and 14 (e.g., partition in hydrophobic/hydrophilic areas), selective addition of the insulation layers (e.g., addition by ink-jet technology) or after addition by selective activation and modification of the layer (e.g., coverage of parts of the insulation layer during plasma or UV-light activation).

The obstacles 23 can include any common microscopic debris found in suspension and includes such things as proteins, lipids, small organelles and bacteria. Insulation layers 13 and 14 may also be adapted to conditions of the cell suspension in the cell compartment 17 and the obstacles 23 contained therein, i.e., to the cleanliness of the solution containing the cells, as well as the cells themselves. To provide enough flexibility to cushion larger debris particles, such as membrane fragments, DNA, large sugar molecules, cellular organelles (e.g., mitochondria), the thickness of layers 13 and 14 can be varied accordingly. The thickness is preferably increased (or decreased) by the enlargement of the hydrophobic layer 13. The hydrophobic layer could be enlarged by the deposition of hydrophobic molecules on an already hydrophobic layer, e.g. by detergent removal. It is also feasible to stack several layers 13 and 14 on each other. For instance, using bi-functional molecules having a hydrophobic core region, the surface of the last deposited layer can be activated to bind the next layer of molecules. This process can be repeated several times.

The cell compartment 17 is filled with a fluid medium suitable for the biological sample of cell 15 that is being tested and measured. The reference compartment 18 is filled with a suitable recording fluid, which is electrically conductive and provides electrical access to the sealed membrane patch 20 or, by perforation of the patch, to the entire cell 15 without contact with the fluid in the cell compartment 17. Current passage through the patch 20 (or the cell 15) is then the current path of least resistance due to the insulating characteristics of the hydrophobic layer 13 and the electrically tight contact between the rest of the cell membrane and the carrier 11. Thus, current flowing from the second electrode 22 passes through the conducting fluid and the patch 20 (or the cell 15) before entering the fluid in the cell compartment 17 on the way to the first electrode 21. Conversely, current generated by the first electrode 21 passes through the fluid in cell compartment 17 and the cell 15 (or patch 20) before entering the conducting fluid in the reference compartment 18 on the way to the second electrode 22. The current direction is determined by the polarity of the voltage applied between the electrodes 21 and 22.

The electrodes 21 and 22 are immersed in the sample compartments 17 and 18, but could also be attached to the carrier 11 surface, such as in proximity to the aperture 16, or even in the aperture itself. The electrodes 21 and 22 are attached, respectively, to a pair of electrical leads (or wires) 24, which are in turn connected to a desired electrical testing device or devices (not shown) for electrical measurement, data collection and data storage. In another embodiment, the second electrode 22 could be placed directly in the aperture 16 on the reference side of the carrier 11, thereby eliminating the need for the reference compartment 18. Once the cell 15 has been electrically sealed, a range of measurements can be performed on the cell or its components. For example, the apparatus 10 could be used in voltage clamping, voltage sensing and impedance spectroscopy. The electrically tight adhesion allows measurements of very small currents because the apparatus 10 has a high signal to noise ratio. The currents typically are in the range of 1 pA to 100 nA and current leakage through the seal for accurate measurements should be at least one order of magnitude smaller.

A range of membranous objects (in addition to biological cells) can be measured using the apparatus 10, including, but not limited to, artificial simulations of biological cells, liposomes or portions thereof such as portions of cell membranes, vesicles, protoplasts, cell organelles (such as mitochondria and chloroplasts), and lipid bilayers. The voltage applied by the electrodes 21 and 22 can also be used to analyze channels protein, and other artifacts, embedded in the membranes.

In another embodiment, electrically tight adhesion of a cell to the modified carrier 11, or even rigid carrier surfaces, can be aided or accomplished via a reduction in the rigidity of the cell membrane. Without being wed to any particular theory, the relatively easy Gigaseal formation with liposomal membranes can be explained by their high flexibility, which allows a large part of the membrane to attach tightly to the carrier surface. Thus, making the cell membrane and cell mechanical properties resemble those of a liposomal bilayer membrane should promote a tight electrical seal. Changes in the cell's mechanical properties can be accomplished through the temporary or permanent modification of the cytoskeleton, membrane fluidity or osmotic cell pressure.

Figure 3 depicts a round, relatively rigid cell 115 perched on a pair of obstacles 123 resting on the surface of a rigid carrier 111 having a cell attracting layer 114. The obstacles 123 prevent the cell 115' s membrane from adhering tightly to the cell-attracting layer 114 of the carrier's surface. The osmolarity outside the cell 115 can be increased from 300 - 350 milliosmoles kg to 400 - 600 milliosmoles/kg (or more) while the cell is already attached to the rigid carrier 111. An osmolarity increase leads to a temporary reduction of cell rigidity that promotes attachment of membrane parts normally spaced too far away from the carrier 111 to adhere to its cell-attracting layer 114. As shown in Figure 4, the temporary or permanent reduction of cell rigidity causes the obstacles 123 becoming embedded into the cell membrane with the membrane conforming to the shape of the obstacles and adhering to the surface of the cell attracting layer 114 of the carrier 111.

To reduce membrane stiffness permanently, cells can be incubated, prior to adhesion, using substances interfering with the cells cytoskeleton, such as by the addition of colchicin or cytochalasin to the suspension. Some other substances suited are phalloidin, iota toxin of Clostridium perfringens and taxol. For instance, human embryonic kidney (HEK 293) cells incubated with 0.5 mM colchicin for 120 minutes show a significantly reduced membrane stiffness. Seal resistances obtained with 106 HEK 293 cells suspended in 1 ml, washed twice in 12 ml Ringer Solution (including 1 mM CaCl2) by centrifugation, on SiO2 carrier surfaces covered by poly-L-lysine physisorped by 12 hour immersion of the carrier in 0.01 %-0.1 % Poly-L-Lysine solution (Sigma Diagn, Inc., Stock #P8920) were between 192 MΩ and 11 GΩ. Note these measurements were performed with an aperture size of 2 μm and without the hydrophobic cushioning layer 13.

Membrane adhesion can also be improved by augmentation of the membrane fluidity. Substances can be used that incorporate into the membrane, such as surfactants, or that stimulate metabolic processes leading to membrane destabilization, such as by stimulation of lipases or the addition of spermidine, or increased production of specific membrane components, such as certain lipids or membrane proteins.

Figure 5 is a graphical depiction of data from a voltage-clamp recording performed on the carrier 11 with the Poly-L-Lysine coated diaphragm and an aperture diameter of 1.5 μm, but otherwise the same as the embodiment shown in Figure 1. The Poly-L-Lysine is physisorped onto the oxygen plasma activated (20 min) SiO2 carrier surface. A buffer of 137 mM NaCl, 5.6 mM KCl, 10 mM HEPES, ImM CaCl2 with a pH = 7.35 is added in two 5 μl volumes to the cell compartment 17 and reference compartment 18. The Ag/AgCl electrodes 21 and 22 are inserted into their respective compartments 17 and 18.

A giant lipid vesicle, more specifically a POPG vesicle as described in Schmidt et al., 39 Angew. Chem. Int. Ed. 3137 (2000), is added to the cell compartment 17 and positioned on the aperture 16. Application of a -60 mV voltage by the first (recording) electrode 21 moves the POPG vesicle to the aperture. Other methods of positioning could be used such as dielectrophoresis with an alternating current, or the application of under-pressure on the reference compartment 18 side. After obtaining a seal with one giant liposome, vesicles are completely removed from the cell compartment 17 by extensive washing leaving a lipid bilayer, which comprises the hydrophobic cushioning layer 13 and charged hydrophilic layer 14. The lipid membrane patch 20 covering the aperture 16 is removed resulting in a current increase 26.

Shortly thereafter, 200 nl of HEK 293 with a density of 107 cells/ml is added to the cell compartment 17 resulting in a drop in current magnitude 27. The step-like reduction of the current 27 is interpreted as positioning of the cells on the aperture 16. After cell positioning, CaCl2 is added to the cell compartment 17 at a concentration of 40 mM, which results in a near-complete current reduction 28 with a seal resistance measured at 10 GΩ. As described above, the Gigaseal results because the hydrophobic layer 13 prevents lateral currents (i.e., currents parallel to the surface of the carrier 11), currents underneath the cell 15 or another membranous object attached to the carrier. In particular the mechanical flexibility of the layers 13 and 14 prevents leak currents even in the case of small particles (and other obstacles 23) in solution or attached to the cell membrane that would otherwise act as a spacer forming a cleft between the carrier surface and the cell 15.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

THAT WHICH IS CLAIMED:
1. An apparatus for recording the electrical properties of a membranous object, said apparatus comprising a carrier for supporting the membranous object thereon, said carrier having an attracting layer and an insulating layer attached to a diaphragm, the attracting layer attracting the membranous object, the insulating layer insulating against current flow between the membranous object and the carrier and said layers being sufficiently compressible to absorb obstacles trapped between the membranous object and the carrier to ensure electrically tight contact between the carrier and the membranous object despite the presence of obstacles trapped there between.
2. The apparatus of claim 1, wherein said attracting layer has an electrical charge attractive to the membranous object.
3. The apparatus of claim 1 or 2, wherein the carrier further comprises an aperture surrounded by the layers and providing access to a patch of the membranous object covering the aperture, said patch electrically sealed by the electrically tight contact between the carrier and the membranous object.
4. The apparatus of claim 3, further including an electrode positioned on each side of the patch for applying voltages and measuring currents there through.
5. The apparatus of anyone of the preceding claims, wherein the layers have a combined thickness of between 1 nm and 5 mm.
6. The apparatus of claim 5, wherein the layers have a combined thickness of between 4 nm and 10 μm.
7. The apparatus of anyone of the preceding claims, wherein said layers are comprised of a self-assembled and compressible amphiphilic molecule layer.
8. The apparatus of anyone of the preceding claims, wherein said layers are comprised of a long-chain aminosilane layer that allows direct attachment of the membranous object.
9. The apparatus of anyone of claims 1 to 7, wherein said layers are comprised of a compressible lipid bilayer formed by a deposition of vesicles or detergent removal of a solution solubilized lipid.
10. The apparatus of anyone of the preceding claims, wherein said insulating layer is comprised of an oil or oil-based substance.
11. The apparatus of claim 10, wherein said insulating layer is comprised of a silicon oil or silicon oil-based substance, including polymerized silicones.
12. A method for promoting electrically tight adhesion of membranous objects to a carrier, comprising: applying a compressible insulating layer to the carrier; positioning the membranous object on the carrier; and absorbing small obstacles in the flexible insulating layer thereby facilitating the formation of an electrically tight seal between the membranous object and the carrier despite the presence of obstacles.
13. A method for promoting electrically tight adhesion of membranous objects to a carrier, comprising: positioning the membranous object on the carrier; increasing the osmolarity outside of the membranous object to within a range of 400 to 600 milliosmoles/kg to decrease the osmotic pressure within the membranous object and reducing the rigidity of the membranous object; and adhering the membranous object to the carrier by conforming to any obstacles there between and forming an electrically tight seal.
14. A method for promoting electrically tight adhesion of membranous objects to a carrier, comprising: positioning the membranous object on the carrier; reducing the stiffness of the membranous object using substances modifying the cytoskeleton of the cell; and adhering the membranous object to the carrier by conforming to any obstacles trapped in between the carrier and the membranous object.
15. A method as in claim 14, using colchicin, cytochalasin, phalloidin, iota toxin and taxol to modify the cytoskeleton of the cell adhering to the carrier.
PCT/IB2001/000095 2001-01-26 2001-01-26 Methods and devices for the electrically tight adhesion of a cell to a surface WO2002059597A1 (en)

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