Patent Grant
7169609
1. Field of the Invention
The present invention relates generally to instrumentation and methods for manipulating and studying electrical properties of epithelial cells, intact biological membranes, and tissues.
2. Description of the Related Art
The Ussing chamber is named after Hans H. Ussing, who pioneered the concept of measuring ion flux across epithelial tissues via electrical measurements in the 1950s. See Ussing, H. H. & Zerahn, K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Scand. 23: 110–127, hereby expressly incorporated by reference in its entirety.
Ussing's original studies used intact frog skin, but over the years, the Ussing chamber has become a preferred tool to study transport across a variety of epithelial cells, intact biological membranes, and tissues. More recently, progress has been made in the ability to grow primary epithelial cells or immortalized cell lines on a porous supporting membrane. Under appropriate culture conditions, these cells grow to confluence, establish polarity, and can form tight junctions between cells, creating a high resistance monolayer (ca.≧0.3 kohm/cm2) suitable for transepithelial measurements. The ability to use primary cells or engineered cell lines allows the biophysical and pharmacological study of epithelial function, including effects on ion channels or transporters. Despite its utility and diverse applications, the experiments remain laborious and time-consuming. This limits the utility of this technique for modern research methods, including screening of molecules or proteins for effects on ion transport.
A typical Ussing chamber is shown in
As described above, the typical Ussing chamber experiment is a time-consuming, cumbersome, and labor-intensive process which includes (1) zeroing the electrodes to compensate for the solution resistance, (2) mounting one Snapwell™ on the insert, (3) installing the insert into the chamber, (4) inserting the electrodes, (5) adding solutions and reagents, (6) manipulating the electronics manual interface, and (7) collecting the data. Silver/silver chloride electrodes also wear out, and rebuilding these compound electrodes usually involves a cumbersome process of handling melted agar. A typical Ussing experiment takes several hours, yet provides only one data set, as only one Snapwell™ can be tested at a time. In the context of drug screening, where it is often desirable to screen hundreds or thousands of compounds, such throughput is unacceptably low. Even in the scenario of a secondary screen, or the profiling of medicinal chemistry compounds, this throughput of one data point in several hours is still too low to satisfy the need to test a number of compounds at various concentrations in order to calculate an effective concentration, for example, when obtaining a dose response profile. What is needed in the art is a Ussing chamber apparatus and method for its use that allows greater throughput.
One aspect of the invention is a multiwell plate assembly containing: a first tray containing an array of sample wells, wherein each sample well contains an electrode having an electrical connection that passes through an opening in a wall of the sample well; a second tray containing a plurality of cell layers such that the second tray can be coupled to the first tray to form a plurality of assay chambers such that each assay chamber contains: a first compartment; a second compartment; and at least one intact or permeabilized cell layer separating the first compartment from the second compartment.
Another aspect of the invention is a method of forming a multiwell plate assembly including: providing a first tray containing a plurality of sample wells, each sample well of the plurality of sample wells containing one or more electrodes; and substantially simultaneously placing a plurality of cell layers into the plurality of sample wells.
Another aspect of the invention is a method of characterizing the biological activity of a candidate compound including: placing a first tray of a plurality of wells having cell layers affixed to the wells into a second tray of a plurality of wells with electrodes mounted therein such that the trays form respective pairs of compartments separated by the cell layers; placing electrodes in the plurality of wells of the first tray; exposing one or more cells of the layer of cells to the candidate compound; monitoring an electrical property with the electrodes wherein the property is indicative of a biological activity of the compound.
Another aspect of the invention is an assay apparatus containing a multiwell plate having a plurality of wells, each well having a top opening and a bottom panel, wherein at least some of the wells have one or more other openings in the bottom panel.
Another aspect of the invention is an assay apparatus containing: a first multiwell plate having a plurality of wells, each well having a top opening and a bottom panel; a second multiwell plate having a plurality of wells that are aligned with the plurality of wells of the first multiwell plate and are dimensioned such that the plurality of wells on the second multiwell plate fit into the top openings of the plurality of wells of the first multiwell plate to create dual-compartment wells; a first set of electrodes extending into the plurality of wells of the first multiwell plate; and a second set of electrodes extending into the plurality of wells of the second multiwell plate.
Another aspect of the invention is a multiwell assay apparatus containing: a pair of adjacent multiwell plates positioned relative to each other to form a plurality of dual-compartment wells; a pair of printed circuit boards sandwiching the pair of adjacent multiwell plates; and electrodes extending from each of the printed circuit boards and into at least some of the dual-compartment wells.
Another aspect of the invention is a multi-channel voltage clamp for a plurality of dual-compartment assays, the multi-channel voltage clamp containing: a plurality of voltage sensors coupled to corresponding ones of the plurality of dual-compartment assays, each voltage sensor having an output dependent on a voltage difference between the different compartments of the dual-compartment assays to which each voltage sensor is coupled; a digitally programmable controller receiving as inputs a plurality of signals, each of the signals dependent on a corresponding voltage sensor, the programmable controller also providing a plurality of outputs; a plurality of servo amplifiers, each servo amplifier receiving a first signal dependent on the output of a corresponding voltage sensor and a second signal dependent on one of the programmable controller outputs; wherein each servo amplifier is configured to produce an output dependent on changes in the voltage difference between the different compartments of a corresponding dual-compartment assays.
Another aspect of the invention is an assay apparatus containing: a regular array of dual-compartment assays; a corresponding regular array of electrodes extending into both compartments of the dual-compartment assays; multi-channel digitally programmable electronic control and sensing circuitry configured to substantially simultaneously apply signals to at least some of the electrodes and sense signals from at least some of the electrodes.
Multi-well plates (or trays) are widely used in experiments in which it is desirable to perform numerous assays in parallel.
Some embodiments of the present invention include an array of Ussing chambers. Some embodiments feature a first multiwell plate having a plurality of wells and a second multiwell plate having a plurality of wells wherein the plates are dimensioned so that the wells of the second plate can be aligned and placed into the wells of the first plate so as to create dual-compartment wells;
In some advantageous embodiments, an array of Ussing chambers is designed using commercially available multi-well plates that have been modified in certain ways described more fully below. Various improvements to electronics and electrode design are also included in some embodiments of the present invention. By conducting experiments in parallel and reducing the number of individual chambers that need to be handled, some embodiments of the invention can increase the throughput and simplify the execution of transepithelial measurements from cell cultures. In some embodiments, a Ussing chamber array is interfaced with liquid handling hardware, electronic controls, and/or software to allow experimental manipulation and/or data analysis.
When the upper tray 50 and lower tray 52 are brought together, a plurality of Ussing chambers is formed. As in a standard Ussing chamber, each assay chamber contains a first compartment 10 and a second compartment 12 which are separated by a cell layer 18. In this example, the upper well is the second compartment 12 and the lower well (minus the volume displaced by the upper well) is the first compartment 10. In some embodiments, the assembly process can be performed so that each Ussing chamber of the array is formed at substantially the same time as all the others. This can be achieved by the substantially simultaneous placement of the all the cell layer membranes 18 (which reside on the upper tray 50) into the wells of the lower tray 52.
Each compartment can be filled with a fluid that contains ions that will serve as a medium for ion flux across the cell layer membrane 18. The fluid, and any other desired reagents, can be added either before or after the trays are brought together to form the plurality of chambers. Adding reagents to the lower wells after the trays are brought together is easier if pre-formed holes are included in the upper tray.
Ions which are particularly useful for Ussing chamber work include sodium, potassium, calcium, bicarbonate, phosphate, and chloride. The ion concentration of the first compartment may be different than that of the second compartment. In such case, the ion gradient can thus induce an ion flux across the cell layer membrane. In some embodiments, multiple gradients can be created using more than one species of ion. An ion concentration gradient may change over time, either because ions in one compartment have moved to the other compartment, or because of chemical or biological processes occurring in a compartment that consume or generate ions. Ion concentration may also be altered by the addition of one or more reagents to a compartment. The concentrations of different species of ions can vary independently of one another. At any given time, the concentrations of a particular species of ion in the first and second compartments may be different, or may be substantially equal, depending on the requirements of the assay being performed.
As shown in
It will be appreciated that the wells of the tray, once the electrodes are inserted, should be water-tight. A fluid leak from an assay compartment can compromise the assay, require additional clean-up, and possibly damage equipment. A water-tight seal can be created by making the electrodes the exact same size as the openings to form a tight press fit, or by using a sealing agent (such as an adhesive polymer) to fill any gaps between the electrodes and the sides of the openings. A gasket or other device for creating a water-tight seal can also be used and has been found advantageous in some embodiments.
As shown in
It will generally be advantageous if each Ussing chamber in the array is wired separately to these modules using its own channel or group of channels so that each Ussing chamber can be controlled, and its own output monitored, independently of the other Ussing chambers in the array. One useful design that has been discovered is to use a first printed circuit board (PCB) adjacent to the upper tray and a second PCB adjacent to the lower tray. In such a design, the PCBs can be constructed so that they contain an array of electrodes which match up spatially with the array of wells on the trays. The PCB that matches the upper tray can be placed on top of the upper tray so that electrodes extend down into the wells of the upper tray. With regard to the lower tray, it is particularly advantageous to combine the lower tray with a PCB and to use electrodes that extend from the PCB, up through the bottom of the lower tray, and then into the wells of the lower tray. As above, it is advantageous to construct the lower tray electrodes assembly in a manner such that the wells of the lower tray do not leak. Accordingly, it has been found to be advantageous to fasten the lower tray and the lower tray PCB together with a gasket between them to prevent leakage.
Some embodiments of the present invention employ multi-well plates which are commercially available from companies such as Corning, Becton-Dickinson, and Millipore. Some of these plates are designed for measuring compound permeability in Caco2 assay systems. See Corning Costar Transwell Permeable Support Selection and Use Guide, Web document rev. 7/02, hereby expressly incorporated by reference in its entirety.
Some embodiments of the present invention use Transwell™ plates from Corning, the typical specifications of these particular plates are as follows. The plates have 24 wells arranged in a rectangular array of the same footprint as a standard microtiter plate. Each plate consists of three parts: i) a bottom part with 24 cylindrical wells; ii) a middle part consisting of 24 Transwells™, each of which is a cup whose bottom is a microporous membrane support on which epithelial cells can grow; and iii) a lid.
Some embodiments of the present invention involve modifying the bottom part and middle part of a Transwell™ plate assembly so that when they are brought together, an array of Ussing chambers is formed.
The electrodes shown in
An alternative fabrication process can be used for making electrode assemblies in KCl/agar. For example, the electrodes can be built inside a structure into which a mixture of KCl and melted agar is poured. However, the simplicity of fabrication and regeneration of bare AgCl electrodes makes their use generally preferred to that of KCl/agar.
Nevertheless, compound electrodes with KCl/agar are sometimes advantageous when the biological applications call for changes in chloride concentrations during the experiment. In such a case, a design as shown in
Regenerating these compound electrodes will typically require more work than regenerating their agar-less counterparts. Typically, regeneration will involve either de-soldering the electrodes from the printed-circuit board, or re-melting the KCl/agar and pouring it out of the terminal blocks. The re-melting can be achieved by dipping the electrode assemblies into hot water.
The use of a miniaturized arrangement can lead to several substantial advantages in terms of throughput, compound usage, and utility. For example, cells can be cultured simultaneously in 24 Transwells™ and 24 Ussing experiments can be run at the same time. As the area of the Transwell™ membrane support is only ⅓ that of a Snapwell™ support, fewer cells would be needed per data point; this is particularly advantageous if primary culture cells of human origin are used. Moreover, since the volumes of the bottom well and the Transwell's cup are only 1.2 and 0.25 mL, respectively, consumption of reagents and especially test compounds can be significantly reduced when compared to the 3-mL volumes of the traditional Ussing compartments. In terms of utility, the smaller surface area of the monolayer can allow more rapid voltage clamping and increased sensitivity. Standard Ussing chambers are often hampered by the poor resolution of the voltage-step activation of ion channel activity due to the large, slow capacitive current transient associated with the voltage-step command. This can be especially problematic when studying fast-activating or fast-inactivating ion channels. Miniaturizing the Ussing chamber-recording set-up can reduce this capacitive current and increase the utility of the Ussing chamber. In addition, an alternating headstage that switches between a high resistance (50 Gohm) and low resistance (50 Mohm) feedback resistor can be used to first rapidly charge the membrane followed by the high resolution recording of current flux. A capacitive-headstage amplifier can also be used, as it can rapidly charge the monolayer capacitance. Finally, circuits capable of compensating for the capacitance can be added to reduce the duration in which the output of the amplifier is saturated, during which the monolayer cannot be adequately voltage-clamped. Finally, the reduced size of the monolayer can help to reduce the background current noise, which in turn can allow for better resolution of small conductance ion channels or low channel expression levels.
For each channel, an electronic circuit as shown schematically in
It is one important aspect of this part of the system that the circuit is configured to be entirely under computer control. In contrast, control of commercially-available Ussing voltage clamps is entirely manual. User inputs are entered via front panel dials, switches, and knobs. Typically, each channel requires a 7×22 cm panel; the front panel of a state-of-the-art 8-channel voltage clamp is 60×22 cm. To set up all eight channels requires extensive of manual work; a 24-channel version would be prohibitively unwieldy.
Referring now to
In one embodiment, to set the system up, the controller 72 begins by activating the relay (via the digital output in
Manual Ussing voltage clamps can also produce periodic voltage pulses to test the cell layer's electrical resistance; these voltage pulses can be biphasic. This can be achieved by adding a pulse generator whose output is added to the clamp voltage. This generally adds complexity to the circuitry and requires additional manual knobs and dials on the front panel that the user has to manipulate. In some embodiments of the present invention, however, these periodic test pulses are produced by the same digital-to-analog circuitry that the computer uses to set the clamp voltage.
In some embodiments, the controller 72 is provided with a display and user input devices such as a keyboard and mouse to control the sensing and driving circuits as shown in
It is possible to use manual pipettes to add and remove fluids from a 24-Transwell™ plate. However, there are at least two advantages of an automated pipetter that are worth considering. First, a typical plastic disposable pipette tip is quite large when compared to the size of a well when using 24-well Transwell™ plates since the electrodes will take up some room. To avoid disturbing the cell layer, it is generally advantageous to pipette against the side of the well, and not directly onto the layer. Such a procedure is very difficult using disposable pipette tips because of mechanical clearance problems. Second, even though most Ussing work produces slow signals on the order of tens of minutes, it is still best to synchronize all 24 channels so that well-to-well comparison is not undermined by issues such as differential aging of cell samples. Manual pipetting does not allow synchronous addition of reagents to all 24 wells.
Accordingly, some embodiments of the present invention utilize an automated pipetter.
The miniaturization strategy outlined here can be extended to higher densities. Transwell™-type plates also exist in 96-well format. Since the Ag/AgCl electrodes can be very thin metallic wires, they can be made small enough to fit into the wells of a 96-well plate. An automated liquid-handling device would also be advantageous at this density since manual pipetting can be a major source of human error. One main advantage of a 96-well Ussing chamber is higher throughput. In addition to that, however, higher density also leads to a further reduction in cell and reagent consumption. There would also be a significant reduction in the capacitance of the cell layer, which could allow for faster electrical kinetics.
It is also possible to use one pair of electrodes for both voltage measurement and current injection. In this scenario, the electronics circuit quickly switches the electrodes from the voltage sensor to the current source and back. With an analog switch, this can be done quickly enough to maintain a frequency response of 5–10 KHz. An advantage of this is that instead of four electrodes, only two will be needed, which considerably reduces the required mechanical clearance. This would open up the possibility of using 384-well or even higher density plates to perform Ussing experiments. Reducing the size of the monolayers by miniaturization can also help to reduce the capacitance of the cell layer allowing for faster signals to be detected. Further, instead of clamping the voltage across the cell layer, it is also possible to clamp the current. For example, as the current that flows across the cell layer changes because the layer's resistance changes, a current of the appropriate size and polarity can be injected to restore the total current to its initial value. The injected current reflects the resistance change undergone by the cell layer. Again, such a circuit can be computer-controlled.
Some embodiments of the present invention have broad utility for functional analysis of ion transport proteins in both basic research and pharmaceutical drug discovery using a variety of cell types. Basic research applications can include elucidation of biological mechanisms underlying normal function and disease states. Pharmaceutical applications can include screening of test compounds for both effects on specific transport proteins or general epithelial cell function. Functional analysis can be performed on cellular transport proteins, including ligand-gated channels (such as P2X, NMDA, GluR, and Ach), second-messenger operated channels (such as CFTR), voltage-gated channels and electrogenic transporters and pumps. For ligand-gated channels, the automated pipetter can be used to quickly and simultaneously add ligands to all 24 (or more) chambers to control the channels. Voltage-gated channels can be opened by rapidly changing the clamping voltage so as to cause channel opening and current flow. For some types of work, a 1-KHz frequency response of the circuit may not be sufficient to detect certain types of fast current changes. In such cases, however, the electronic design can be optimized to obtain a 10-fold improvement to permit such detection. In some embodiments, the same instrument can be used for both of these modes of action. Some embodiments of the present invention can also be used to study the response of epithelial cell cultures to other signaling molecules such as peptides and proteins acting through receptors or signaling pathways. For example, epithelia are known to regulate ion transport in response to various stimuli including inflammatory mediators. See Danahay, H et al., Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells. Am. J. Physiol (Lung) 282:L226–L236, 2002, which is hereby expressly incorporated by reference in its entirety. Some embodiments of the present invention can be used to study the response of the epithelial monolayer. For example, agents known to damage or stress cells would be expected to cause a loss of integrity of the monolayer, which would be detected as a decrease in resistance. See Duff, T et al., Transepithelial resistance and inulin permeability as endpoints for in vitro nephrotoxicity testing. Altern Lab Anim. 30 Suppl 2:53–9 (2002), which is hereby expressly incorporated by reference in its entirety.
Utility of the Ussing array was demonstrated using a Fischer Rat Thyroid (FRT) epithelial cell line expressing a mutant form of the CFTR (Cystic Fibrosis Transmembrane Regulator) gene. CFTR encodes a protein kinase A-regulated chloride channel called CFTR (cystic fibrosis transmembrane regulator). Mutations in CFTR result in defective expression and/or function of the CFTR protein and result in cystic fibrosis. A high-throughput assay for CFTR function in epithelial cells is of interest for testing compounds that could improve the expression and/or function of CFTR. FRT cells engineered to carry the mutant ΔF508-CFTR in their membranes were grown on the microporous supports of 24-Transwell™ plates.
This experimental setup using a 24-Transwell™ plate, bare Ag/AgCl electrodes, and computer-controlled voltage clamp produces experimental results that are identical in most aspects to those obtained with a traditional Ussing chamber driven by a manual voltage clamp. One noteworthy difference, however, is the amplitude of the current increase. The increase is only one third of that obtained from a traditional Ussing chamber. This is expected, however, since the cell layer area used with the Transwell™ (0.3 cm2) is about one third of the cell layer area in a traditional Ussing chamber (1.1 cm2). Taken together, the two experiments shown here demonstrate that this novel high-throughput Ussing technology will be useful for both screening the activities of compounds, and ranking their potencies.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4055799 | Coster et al. | Oct 1977 | A |
| 5643742 | Malin et al. | Jul 1997 | A |
| 5738826 | Lloyd | Apr 1998 | A |
| 5972694 | Mathus | Oct 1999 | A |
| 6488829 | Schroeder et al. | Dec 2002 | B1 |
| 6936462 | Owen et al. | Aug 2005 | B1 |
| 20020195337 | Osipchuk et al. | Dec 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| 101 17 723 | Oct 2002 | DE |
| 2 844 052 | Mar 2004 | FR |
| WO 9428111 | Dec 1994 | WO |
| WO 9966329 | Dec 1999 | WO |
| WO 03085484 | Oct 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20050221274 A1 | Oct 2005 | US |
