CA2589114A1 - Sample transfer system - Google Patents

Abstract

The present invention relates to a sample transfer system that is suitable for electrophysiological measurements on voltage-clamped cells, patches of membrane, tissue, embryo, small living organisms or inorganic samples, that can be exposed to different test compound solutions, wherein said test compound solutions can be retrieved after the measurements. The invention also provides for a method to transfer a cell or biological sample, e.g. from one aqueous solution bath to another, while protecting it from being exposed to mechanical forces at the air-liquid interface.

Description

Sample transfer system Field of the invention The present invention relates to a sample transfer system which, for example, may be used for moving a cell or a group of cells between microwells.
Microwells can be on a commecrcally available 'microplate' or can be custom manufactured. A group of cells may for examplc also include tissues, organs, parasites or other small living organisms. The sample transfer system can also be used to transfer solid substrates or biosensors between different solutions. The sample transfer system may also be used for cell-fusion, cytoplasmic or nuclear transfer from cell to cell.

State of the art Current protocols for testing compounds on ion channels include, low throughput assays with a high information content such as patch claniping. This technique is slow, laborious and requires highly trained staff. Other approaches used in drug discovery are high throughput but have low information content. These include ligand displacement assays, where a labelled ligand bound to the receptor of interest is in competition for binding and can be displaced by an unlabelled test compound. This technique requires previous knowledge of the ligand-binding site and a labelled ligand directed to this site. This method is inefficient at detecting allosteric modulators binding at a weakly coupled site.
Another method involves measurements of radioactive flux using tracers such as 86Rb+
for K+ channels 22Na+ for Na+ channels and 45Caz+ for Ca2+ channels. This technique requires large quantities of radioactive tracer with all the associated problems of exposure to radiations and of disposal of the radioactive waste. TeSample transfer system using this technique are time consuming, requiring long incubation periods with the radioactive tracer. Ion efflux studies can also be carried out using non-radiactive substitute ions, cells are loaded with ions during an incubation period, efflux of ions is detected by atomic adsorbtion spectroscopy.
FLIPR (Fluorometric Imaging Plate Reader) assays use fluorescent ion sensitive or membrane potential sensitive dyes, cells expressing the receptor of interest are placed or cultured in a microtiter plate, voltage sensitive channels are opened by depolarization of the cell with KCl or addition of a channel opener such as veratradine for Na+
channels (see for example [1]). Commonly used dyes are sensitive to calcium requiring that the signal is "converted to a calcium readout" the plate is scanned by a scanning laser and changes in the fluorescent readout indicate whether a test compound is affecting an ion channel of interest. Another technique VIPR (Voltage/lon Probe Reader, Aurora Discovery, San Diego, CA, USA) uses FRET (Fluorescence Resonance Energy Transfer) based membrane potential sensitive dyes to indicate changes in cell membrane potential.
The major drawback of with these two systems is that they require identification and cloning of the gene coding for the ion channel of interest and its expression in a host cell line. They also require a single layer of adherent cells in each well of the microtiter plate.
Moreover, cells are not voltage clamped and measurements are not possible on channels that display strong voltage dependency (rectification) and difficult on fast activating and inactivating channels. These techniques are also unsuitable for ligand-gated channels that strongly desensitize.

Another alternative for automatic measurement is provided for example by the Biacore technology. A main difference, however, is that this technology requires purified proteins that are anchored on a substrate. Detection is done by surface plasmon resonance (see for example [2, 3]). As described in these publications, it should be emphasized, however, that this technology is not applicable to examine living cells.

Recently several partly automated systems have become available to study the effect of test compounds on the electrophysiological properties of cells and/or on specific membrane proteins.
Automated patch clamp (APC), machines, perform patch clamp recordings on single cells immobilised on a planar electrode or inside a glass electrode. These electrodes are disposable and are used only once. Many of these machines are able to record from multiple cells in parallel, giving them a high throughput which makes them highly suited to the application of screening multiple compounds on ion channel function.
These machines also require the cloning off the gene for the ion channel of interest and it's expression in a host cell line. These cells must be in liquid suspension.
OpusXpress (Molecular Devices) is a semi-automated machine capable of maldng simultaneous recordings from eight Xenopus oocytes in parallel, loading of the oocytes is performed by the operator, impalement and voltage clamping is automated. This system (like the APCs) employs the conventional system of applying liquid to an immobilised cell. Application of liquid to the cells is performed by a modified liquid handling robot. A
test compound solution is applied to voltage-clamped cells in a perfusion stream, and is aspirated into the waste at the end of the measurement, to give place to another test compound solution. However, each volume of test solution can only be used once, which seriously limits the number of teSample transfer system in the case of substances that are not available in large amounts.

One way to circumvent the above problems would consist in moving a cell that is maintained under voltage clamp between the different test compound solutions, which are contained in a recepticle. The feasibility of moving Xenopus oocytes between different aqueous solutions has been described by Chang and Weiss [4], but no electrophysiological measurements were attempted using this method and the movement was done manually. Moreover, the method described in the article has a major limitation:
when the cell crosses the liquid-air interface, it experiences important mechanical forces.
Such forces will cause damage to cells that are impaled with intracellular electrodes and the absence of surrounding liquid will prevent maintenance of voltage recording or voltage clamp due to loss of contact with the extracellular ground. In the case of a tissue sample these forces will cause damage and or drying of the sample. Forces will also cause movement of the sample, in relation to the recording electrodes or optical measuring device, disrupting the recording.

Summary of the invention A purpose of the invention is to develop an automated system for electrophysiological measurements on voltage-clamped cells that are exposed to different test solutions, wherein said test solutions can be retrieved after the measurements to be either reused for other measurements or subjected to further analysis.

To this effect the present invention describes a sample transfer system that has the ability to insert or extract sample(s) from a medium, e.g. a liquid, while allowing assaying sample or liquid separately or in combination.

The invention also relates also to a method to transfer a sample from one solution to another while protecting the sample from forces encountered at the air liquid interface.
Moreover, this device can be fully automated and work in an unattended manner.

In one embodiment of the invention a cell is immobilized and electrodes inserted for electrophysiological recordings. The cell can be transferred from one solution to another, while being continuously maintained in voltage clamp and protected from the mechanical stress that occurs when crossing the air-liquid interface. Exposure to mechanical forces at the liquid-air interface during the transfer from one microwell to another is avoided by keeping the cell surrounded at all times with a thin layer of liquid. This is achieved for example by placing an object, such as a spiral, or a ring around the cell, which can also immobilize the cell and can serve as a ground electrode or by moving the cell through a layer of inert hydrophobic liquid.

One advantage of the sample transfer system according to the invention is that it only requires very small amounts of each test compound. If the automated system is used in combination with microtiter plates, the volume of each test compound solution can be as low as 30 microlitres. Standard well plates can be used (to date test have been realized with 96 and 384 microwells). Moreover, after the sample is removed firom the well, the liquid remains in the microwell and can be reused for measurements on another sample.
Contamination between wells can be avoided by rinsing the sample in fresh solution either in a perfusion chamber or in a microwell. Reducing the volume that is maintained around the sample minimizes dilution of the test solution. Measurements in the Xenopus oocyte recording example (presented below) indicate that 20 or more cells can be probed in a single well that contains 45 l of solution before significant dilution of the test solution (> 10%) is observed.

Another advantage of the sample transfer system over other presently existing automated systems is that the liquid can be recovered after the measurement and be reused or analysed, for example for molecules secreted by the sample during the experiment.

An additional benefit is that labelled test compounds can be used without contaminating the whole apparatus. This allows combination of electrophysiological measurements with biochemical experiments at the single cell level.

Another advantage is that several compounds can be tested on the same saxnple, allowing direct comparison of the amplitude of the elicited response. Conversely, the system allows measuring the individual response of several different samples in the same liquid, permitting to easily calculate mean responses.

The sample transfer system according to the invention can be used in a wide range of applications that require transfer of samples (eg. cells, biological or non-biologic material) into different liquids present in small volume and with minimal mechanical forces applied to the tested sample and allowing testing of the sample or liquid using a wide range of techniques. Examples presented below illustrate, in a non-exhaustive way, the adequacy of this method for delicate measurements such as electrophysiological recording while testing cells in different solutions.

For example the sample transfer system according to the invention may be advantageously used in an automated system for electrophysiological fluorometric or biochemical measurements on cells exposed to different solutions Liquid handling has become a field of large development with liquid handlers that have the capacity of pumping or injecting solutions from single or multiple heads. However, despite numerous efforts produced by many companies (i.e.
Gilson, Tecan, Beckman, ...) liquid handling remains difficult and often sheer forces applied to the tested sample or other mechanical problem related to fluid handling can result in undesired side effects.

Difficulties in handling small liquid volumes and managing with care samples of tiny dimensions is well illustrated by a demanding example such as electrophysiological recording of a single cell while changing the bathing solution. The sample transfer system according to the invention is designed to carry out measurement from one or many samples at the same time. The sample transfer system is well suited for low to medium and high throughput analysis. Typically if the measurement requires a minute and assuming continuous work, single head measurement will be limited to about measures/day. Use of multiple head with 12 or 96 sample holders proportionally extends measurements to respectively 17000 and 138000 measures/day.

A list of possible, but not exhaustive, applications includes:
Electrophysiology on oocytes, membrane patches or cells Electrophysiology and biochemistry on tissue slices Electrophysiology and/or biochemistry on organs, parasites or other small living organisms Intracellular injections Biochemical measurements on adherent cells or non adherent cells Ligand fishing, using cells, tissue samples or solid substrates Fluorescence measurements using single or multiple samples Spectrophotometry on liquid samples Testing of different solutions using probes or biosensor Analysis of Tissues Analysis of Membranes Analysis of Biopsies Detection of antibodies RNA or DNA binding Protein blotting Cell culture Differential display analyses of the content of the solution used for incubation Cell fusion Chemical reactions An important feature of the sample transfer system according to the invention is its capacity of challenging multiple samples with a minimum and reusable volume of test solution. Recovery of the test solution is possible at the end of the experiments. This additional feature opens new strategies such as "ligand fishing".

A few examples of applications for which the sample transfer system according to the invention is well suited are described in more details below.

a) Electrophysiology The identification of membrane proteins, including but not limited to voltage-dependent ion channels, ligand-gated ion channels or metabotropic receptors and their role in physiological processes and disease states has underlined the importance of developing new drugs targeted at these proteins. In particular, there is a need for developing new compounds that affect the functional properties of membrane proteins, and for developing methods to test the action of such compounds on their respective targets.

The effect of individual compounds or mixtures of compounds such as natural extracts or chemical libraries on the electrophysiological properties of cells and/or on specific membrane proteins can be measured in Xenopus oocytes or other cells. Cultured or freshly dissociated cells expressing the channels of interest are manually voltage clamped and the cell is then exposed to a test solution applied in a perfusion stream that is then aspirated to the waste. However, such manual methods are tedious and time-consuming, and do not allow for rapid screening of large numbers of test compounds. The development of APCs has partially automated this process, however, each volume of test solution can be used only once and cannot be conserved for further testing or use.
The sample transfer system allows to continuously record the electrophysiological activity of a cell while transferring it between different test solutions.

b) Photometry The effects of different solutions on cells loaded with ratiometric or non-ratiometric ion or voltage sensitive dyes can be measured using the sample transfer system.
Cells loaded with dye are contained in the sample holder and transferred between different wells containing different test compounds. A light-conducting device, such as an optical fiber is held on the sample holder close to the sample and the changes in emitted light in the different solutions are measured by suitable measuring apparatus connected to the other end of the optical fiber.

c) Studies of efJ'lux or influx wlth labeled probe The sample transfer system enables samples loaded with radiolabeled or non radiolabeled substances to be transferred between different microwells filled with desired solutions.
Measurement of the substance efflux into the solution can be determined by subsequent analysis of the microwell liquid. Alternatively, samples can be transferred into liquid containing labeled substances, and influx into the sample can be measured by analysis of the solution or the sample content.
Fluorescent probes or any adequate reporter molecule can be equally used instead of the radiolabeled substance.

d) Studies of efflux or influx of untabeled substances Moving the sample of interest offers multiple advantages in biological applications.
Given the small volume size of the incubation chambers (within tens of microliters) it is possible to examine substances released by a sample. For example, brain slices, are known to release neurotransmitters in the extracellular space upon electrical or chemical stimulation. Detection of the neurotransmitters released in the microwells can be done using appropriate detection methods such as voltametry, high pressure liquid chromatography (HPLC), mass spectrometry (MS) spectrophotrometry etc.
Although release measurements systems are available, they are often cumbersome and difficultly amenable to automation, especially when in the experimental paradigm requires electrical stimulation or culture conditions.
The sample transfer system opens new possibility of challenging the effects of drugs or any chemical on the substance released in response to a given stimulus.
Measurements can be done either in singular or multiple mode depending upon the design of the sampler holder.
Similarly, the sample transfer system can be used to detect influx of substances into the sample. Monitoring of the microwell solution content or determination of the substance in the specimen can be used to quantify the substance influx.

e) Ligand fishing The concept of "ligand fishing" consiSample transfer system in placing a sample (or multiple samples) that contains a target (binding protein, ion channel, receptor or equivalent) into a small volume of liquid that contains a mixture of potential ligands.
Specific binding of any of these molecules to the sample will cause i) a reduction of the amount of that molecule in the liquid and ii) binding of the molecule on the sample.
Detection of the ligands that have been depleted from the original solution or alternatively that are bound to the sample will allow to screen large compound libraries such as those encountered in venom fractionation or testing of chemical libraries . Such detection can for example be performed by differential display analyses using mass spectrometry and proteomic techniques, or any other detection system including but not limited to immunoassays, fluoresence, radioactivity, spectrophotometry, etc.
The sample can also be non-living material such as proteins, receptor fragments or antibodies anchored or attached to a solid substrate.

P Binding of labelled compound A technique analogous to ligand fishing can be designed for labelled compounds. In this application the ligand of interest is tagged, for instance with a fluorescent reporter, binding of a molecule to the ligand of interest can, for instance, be monitored by fluorescence measurement. Monitoring can be done in a continuous or discontinuous way depending upon the type of ineasurements.

, fl Light measurement detection of ligand binding The use of FRET is an alternative and elegant technique for the quantification of molecular interaction. A fluorescent donor emits light that is absorbed by an acceptor when the distance between the two molecules is appropriate. Measurements of the amount of transmitted, emitted, or absorbed light can be used to determine distance changes in a given molecule (typically a protein).
Insertion of light guides in the recording head can allow FRET measurements to be performed either in discrete or continuous way during the sample transfer system operation. Alterna.tively, given its mechanical stability the sample can be monitored using a more conventional optical set of lenses.
Typical example of application could be found in measurements of protein activation by a tagged ligand or if the protein contains both the donor and acceptor of fluorescence such as that found in "chameleon proteins". Binding of the ligand causes a structural change in the protein, which changes the distance between the donor and acceptor groups.

Sample Holder The sample transfer system allows to efficiently transfer the sample (or multiple samples) in different test solutions with precision and automatic handling. Such application requires a specific sample holder that has low adsorption coefficients for the ligand of interest and is non-toxic for the sample.
The sample-holder serves four purposes: i) mechanical holding of the sample, ie.
immobilisation. ii) maintenance of a liquid layer around the sample, iii) protection from the mechanical forces of passing through the liquid-air interface. iv) grounding of the electrical circuit in the case of electrical measurement from the sample.

Depending upon the applica.tion, single or multiple sample-holders can be used on the sample transfer system. Multiple sample-holders can be used to increase the measurement throughput by enabling measurements to be carried out in parallel.

In the case of biological samples the sample holder can also supply oxygenation to the tissue. Negative pressure can also be applied through the sample holder to maintain strong contact between the sample and the sample holder, eg. In the case of extra.cellular electrophysiological recording from tissue slices and samples.

For the Xenopus oocyte electrophysiology recording example the sample-holder serves three purposes: i) mechanical holding of the cell. ii) maintenance of a liquid layer around the oocyte, and protection from the mechanical forces of passing through the liquid-air interface iii) grounding of the electrical circuit.
Specific sample holders can be designed for cell culture of adherent or non-adherent cells, tissue slices, brain slices that can be stimulated during or recorded during solution exposure etc.

Recording of cell activity often requires maintaining the cells in a physiological environment that mimics oxygenation, temperature and other biological parameters found in the body. This is typically achieved in tissue culture conditions with cells maintained in appropriately sized incubators.

The small footprint of the sample transfer system is well suited for "in incubator"
recordings and can therefore be used in long-term studies that will otherwise not be possible. Moreover, it could be used in sterile environment and thereby open multiple possibilities of long-term recordings of biological activities.

Detailed description of the invention The invention will be described in a more detailed manner below together with the following figures :

Figure 1 shows a picture and a corresponding drawing of a sample holder and microelectrodes for electrophysiological recording.
Figure 2 shows a picture and a corresponding drawing of a sample holder containing a Xenopus oocyte with microelectrodes for electrophysiological recording.
Figure 3 shows a picture and a corresponding drawing of a Xenopus oocyte in a sample holder in the recording configuration, with glass microelectrodes inserted in a perfusion stream.
Figure 4 represents a sequence of events involved in an automated oocyte loading.
Figure 5 shows an example of a concentration response curve for inhibition of human alpha 7 nicotinic acetylcholine receptors by methyllycaconitine (MLA).

The sample transfer system is preferably based on a three X-Y-Z movement that is controlled by a computer. This positional system displaces the microwells in relation to the sample or vice versa to incubate the sample in a given liquid. The sample transfer system is controlled by a proprietary software.

In one embodiment (see figures 1 to 3) a Xenopus oocyte 2 is placed in a small basket or appropriately shaped support 1. In the basked shaped version, the support is made from a spiral of coated metal wire, however this can be any shape and can also be uncoated. The coating minimises the interaction of the metal with the sample 2. This support also forms the ground electrode for the recording circuit through the open extremity of the wire. This support immobilizes the oocyte 2 and allows penetration by electrodes 3,4. To ensure reliable positioning of the oocyte 2 the spiral has a conical shape with the larger opening at the top and a 5 degree angle. The spiral support also protects the oocyte 2 from the mechanical forces of passing through the liquid-air interface and the support allows liquid exchange around the cell.

In another embodiment, cells are aspirated using a standard 10 l pipette tip with microelectrodes positioned on either side. A syringe pump under computer control aspirates the cell and holds it firmly at the pipette tip. In one embodiment, the ground electrode is shaped into a spiral or a ring with a diameter slightly larger than that of the cell, and is positioned around the cell, causing the formation, by capillary forces, of a thin layer of aqueous solution around the oocytes.

Penetration of the cell from above by microelectrodes, allows electrophysiological measurements to be made using standard configurations (current clamp or voltage clamp). Voltage clamping is performed by an amplifier also under computer control (i.e.
Geneclamp 500 from Axon instnunent). Ground electrodes are placed in the vicinity of the cell. Test solutions are deposited in microwells and the cell, which is firmly held by the pipette tip, is moved between wells using an X-Y-Z positioner controlled by a computer. In another embodiment the microplate is moved relative to the sample holder in the X, Y and Z directions. The saxnple holder remains stationary, thus minimising possible vibration of the sample holder.
In another embodiment, a layer of an inert, hydrophobic biocompatible fluid, for example mineral oil, is disposed on top of the test compound solution baths and the cell is moved through this fluid from one bath to another without passing the liquid/air interface.

The transfer of the oocyte from a microplate with conical wells into the sample holder is automated and controlled by the software. Loading of the oocyte can be started by the user or can be triggered by pre-determined criteria in the experimental protocol. The sequence of events for oocyte loading is shown in fig. 4. Unloading is also automated and is the reverse of the loading procedure.
The sequence illustrated in figure 4 comprises the following steps :

1.) Microelectrodes are positioned above the empty sample holder.
2.) XYZ table, positions oocyte contained in a conical well below the oocyte loading tube. Sample holder moves horizontally to allow oocyte loading tube to descend.
3.) Loading tube descends close to oocyte.
4.) Oocyte is aspirated into loading tube and loading tube moves up.

5.) Sample holder moves horizontally into position below loading tube.

6.) Loading tube descends above sample holder and the oocyte is expelled from loading tube into the sample holder by gentle pressure.

7.) Loading tube moves up and sample holder moves horizontally into position below electrodes.

8.) Electrodes descend to impale the oocyte.

9.) The impaled oocyte and the sample holder are illustrated iin the configuration for electrophysiological recording. The assembly can now be moved through the liquid air interface without damage or movement of the sample or disturbance of the recording configuration.

Demonstration of feasibility Examples A Xenopus oocyte expressing human 0 nicotinic acetylcholine receptors was placed in the sample holder and impaled with two electrode to establish voltage clamp.
The cell was then briefly challenged with an acetylcholine test pulse in a perfusion chamber and its response recorded. The test pulse and recording lasted 10 s and the cell was then automatically transferred into a microwell from a 96 microtiter plate containing the competitive inhibitor methyllycaconitine. The cell was incubated for 10 s in a given concentration of methyllycaconitine and its response to acetylcholine was tested upon return into the perfusion chamber. As expected a progressive reduction of the amplitude of the response was observed as the cell was placed in increasing concentrations of methyllycaconitine. The entire procedure was fully automated and upon completion of a cycle of exposure to progressive concentrations of inhibitor, the program restarted its recording sequence. Continuous recordings were performed for more than 10 sequences each of which included 10 transfers of the sample. Total recording time lasted roughly two hours. This example illustrates that cells can be transferred more than a 100 times without detectable damage to the cell, modification of the holding current or the cell conditions. In addition this example illustrates that dilution caused by the liquid layer surrounding the cell is negligible.
An example of concentration response curve for inhibition of human alpha 7 nicotinic acetylcholine receptors by methyllycaconitine (MLA) is shown on figure 5.
Oocytes were transferred between wells containing different concentrations of MLA, oocytes were incubated in wells for 10 seconds before currents were elicited by acetylcholine applied in a perfusion stream. Inset shows recorded currents.

References 1. Sullivan E, Tucker EM and Dale IL, Measurement of [Ca2+] using the Fluorometric Imaging Plate Reader (FLIPR). Methods Mol Biol 114: 125-33, 1999.

2. Schmidt EK, Liebermann T, Kreiter M, Jonczyk A, Naumann R, Offenhausser A, Neumann E, Kukol A, Maelicke A and Knoll W, Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.
Biosens Bioelectron 13(6): 585-91, 1998.

3. Yu YY, Van Wie BJ, Koch AR, Moffett DF and Davis WC, Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance. Anal Biochem 263(2): 158-68, 1998.

4. Chang Y and Weiss DS, Channel opening locks agonist onto the GABAC
receptor. Nat Neurosci 2(3): 219-25, 1999.

Claims (11)

1. Sample transfer system comprising a sample holding means (1) having at least a fixing zone adapted to fix a sample (2), characterized by the fact that it furthermore comprises a sample protecting means designed in such a way that a fixed sample (2) would not be in direct contact with the environment in which the sample (2) is located during its transfer.

2. Sample transfer system according to claim 1 which is adapted for moving a sample (2) from one liquid solution to another wherein said sample protecting means comprises a layer of inert hydrophobic biocompatible liquid, said liquid being chosen so as the tension at the liquid-sample interface is weaker than the tension at the air-sample interface.

3. Sample transfer system according to claim 2 wherein said liquid is mineral oil.

4. Sample transfer system according to claim 1 wherein the sample-protecting means consists of an appropriately-shaped object that is designed in such a way that, when placed around or close to a sample (2), it would allows the formation of a layer of aqueous solution around the sample by capillary forces, thus reducing the forces on the sample (2) when crossing the liquid-air interface.

5. Sample transfer system according to claim 4 wherein said sample-holding means (1) comprises a supporting basket or cylinder adapted to be placed around a sample (2).

6. Sample transfer system according to claim 4 wherein said sample-holding means (1) is a spiral adapted to be placed around a sample (2).

7. Sample transfer system according to claim 4 wherein said sample-holding means (1) is a ring or a series of rings or appropriately shaped device adapted to be placed around a sample (2).

8. Sample transfer system according to anyone of the previous claims wherein said sample holding means (1) also comprises other functional means such as measuring means.

9. Automated system for carrying out physiological measurements, comprising measuring electrode(s) (3,4) , a ground electrode and a stage with a microwell plate, all being computer operated, said automated system being characterized by the fact that it comprises a sample-transfer system as defined in anyone of the previous claims.

10. Automated system according to claim 9 furthermore comprising a needle adapted for carrying out micro-injection in a sample (2).

11. Method of use of a system according to anyone of claims 1 to 8 characterized by the following steps :
- extracting a sample (2)from a first medium and protecting said sample from the environment surrounding said first medium, - transferring the sample (2) from the first medium to another location.