Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2000-06-05
2003-04-01
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S029000, C435S297200, C435S287100, C435S288400, C435S288700, C436S063000
Reexamination Certificate
active
06541243
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related in general to holding chambers for carrying out identification of targets in the drug discovery process and, in particular, to a perfusion chamber for measuring electrophysiological responses from frog oocytes.
2. Description of the Related Art
The modern process of drug discovery involves a number of distinct steps. Various possible sources for drugs are first investigated to generate libraries of compounds to test for potential activity. Such compounds are preferably produced using combinatorial chemistry, and are typically synthesized from theoretical considerations or on the basis of templates derived from natural products harvested from around the world and previously tested for activity. These libraries of compounds are then screened to assess their effects on particular targets of interest. As used in the art, “targets” are the entities acted upon by drugs, normally proteins and receptors that transmit information within and between cells and tissues. Targets are first identified by locating them within any biochemical pathway that is relevant to a disease, and they are then validated by showing that their modification produces an effect on the disease.
These libraries of compounds are screened through assays that involve a predetermined protocol for a biochemical reaction to occur that leads to definitive information about the activity of the screened compound on the target. When an active compound is found (normally called a “hit” in the art), second-pass screenings are conducted to test for low toxicity and good chemical and physical properties. The compounds that successfully emerge from these multiple screenings (called “leads”) are then tested on animals to determine their physiological effects and, if warranted, are finally tested on humans through clinical trials.
The ability to develop screening assays rapidly and to screen compound libraries at high throughput is becoming increasingly critical in the discovery of new drugs, which is now a large-scale industrial activity. The explosion of data made available from the Human Genome Project coupled with advances in chemical synthesis has produced a great demand for ever-higher screening rates to test potentially therapeutic compounds. Therefore, there is a growing need for integrated laboratory systems that assess large numbers of compounds quickly.
Of particular relevance to the present invention are assays conducted on Xenopus frog oocytes, which are uniquely suitable for screening of ion channels linked to a variety of diseases. Using conventional voltage clamping across the membrane of the oocyte, the voltage dependence of ion channel activity in the oocyte cell is assessed by measuring current changes produced in response to exposure to multiple test solutions. Testing of an oocyte cell under voltage-clamped conditions, a well known technique in the art, is carried out in a batch operation in a chamber designed to support an individual oocyte being perfused with a test solution. The cell membrane is pierced with two microelectrodes of a voltage-clamp amplifier capable of recording current variations in response to voltage step changes or to the application of compounds under constant-voltage conditions. A conventional two-electrode voltage-clamp system
10
is illustrated schematically in
FIG. 1
, where numerals
12
and
14
refer to a voltage-recording microelectrode and a current-passing microelectrode, respectively, inserted through the membrane
16
of an oocyte cell C. The membrane potential V
m
is recorded by a unit-gain buffer amplifier
18
connected to the microelectrode
12
. The membrane potential V
m
is compared to a control potential V
c
in a high-gain differential amplifier
20
(with gain &mgr;) producing a voltage output V
&egr;
proportional to the difference &egr; between V
m
and V
c
. The voltage V
&egr;
at the output of the differential amplifier
20
forces current to flow through the current-passing microelectrode
14
into the oocyte cell C, such as to drive the error &egr; to zero and maintain the membrane voltage clamped at V
c
.
The circuit is completed through a ground
22
across the cell membrane, which in the schematic is modeled by impedance and capacitance values R
m
and C
m
, respectively.
The primary concerns of designs of perfusion chambers for oocytes are the isolation of the cell in a stationary condition and the ability to expose it to the test solution of interest. Some chamber designs have involved trapping and flooding the oocyte in a retaining structure, such as in the mesh of a web material suspended above the bottom of the chamber. Once so restrained, the oocyte is connected to the voltage-clamp microelectrodes and perfused with test solution in a batch operation.
Most prior-art perfusion chambers are directed to particular tissues and cell layers, rather than individual cell material. See, for example, U.S. Pat. Nos. 4,762,794, 5,043,260, and 5,565,353.
In addition, prior-art perfusion chambers are designed for carrying out tests using individual workstations, performing one experiment at a time. The configuration of the chamber often impedes direct access to the oocyte, thereby complicating automatic insertion of the electrodes. The structure supporting the oocyte does not always permit exposure of its entire membrane to the test solution, which is an important test factor for pharmacological studies of voltage-gated channels, for instance).
Moreover, considerable skill and manipulation is required to place the oocyte in the appropriate position within the chamber, which is prohibitive for automated, rapid-throughput, parallel-testing applications. Finally, the geometries of the structures used to support the oocytes in prior-art chambers have not been optimized to reduce damage to the cells; and the delivery of perfusion solutions has not been engineered for multiple, sequential testing in a continuous operation. Accordingly, the prior-art chambers are not well suited for the high-throughput, electronically manipulated, automated-system needs of today's pharmaceutical industry. This invention provides a simple solution to address these needs.
BRIEF SUMMARY OF THE INVENTION
The primary objective of this invention is a perfusion chamber suitable for sequential testing of an animal cell through successive exposures to multiple perfusion solutions in an automated, continuous system.
Another objective is a chamber that permits the continuous perfusion of the cell in an environment wherein its entire membrane is exposed to the test solution.
Another goal of the invention is a design particularly suitable for the testing of oocytes, especially Xenopus oocytes.
Still another objective is a perfusion chamber design that can be adapted for parallel testing of multiple oocytes in a high-throughput testing system.
Another goal is a perfusion chamber that is suitable for implementation within an automated voltage-clamp and solution-delivery system.
Yet another object is a system that can be implemented using conventional voltage-clamp hardware and software, modified only to the extent necessary to meet the design parameters of the chamber of the invention.
Still another goal is a method of perfusion that facilitates the rapid, sequential testing of an oocyte with multiple test solutions on a continuous basis.
A final objective is a system that can be implemented economically according to the above stated criteria.
Therefore, according to these and other objectives, one aspect of the present invention lies in a perfusion chamber that includes a porous oocyte support structure. A continuously sloped top surface and a receiving well in the support structure produce the automatic entrapment of the underside of the oocyte, thereby localizing the cell in a predetermined fixed position within the reach of dedicated voltage-clamp microelectrodes. The test solution is delivered continuously at the top of the chamber, above the oocyte, and withdrawn from the bottom of the chamber, below the oo
Harris Eric W.
Kildal Maurice A.
Lanthorn Thomas H.
Axon Instruments, Inc.
Beisner William H.
Durando Antonio R.
Durando Birdwell & Janke, PLLC
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