Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-03-06
2001-07-31
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S284100, C435S286500, C435S283100, C435S287100
Reexamination Certificate
active
06268168
ABSTRACT:
MICROFICHE APPENDIX
One microfiche appendix is filed with this application. Microfiche appendix A contains a total of 1 microfiche and 18 frames.
BACKGROUND
1. Field of the Invention
The present invention relates to apparatuses for data acquisition and perfusion control in the analysis of cellular physiology and electrophysiology and to methods for automated perfusion and membrane voltage and current measurement for physiological and electrophysiological analysis.
2. Description of the Background
Cell membranes communicate information from the extracellular environment by means of receptor and channel proteins located within the cell membrane. Receptor proteins are gated by molecules which can bind to the receptor and signal that a binding event has taken place, often by triggering the opening of ion channels through which ions such as sodium and chloride ions can flow. Ionic flux across a cell membrane generates electrical current that can be measured with appropriate recording equipment. Electrophysiological analysis is widely used today to study the pharmacology and biophysics of membrane proteins.
An expression system utilizing unfertilized eggs, or oocytes, taken from the South African clawed frog,
Xenopus laevis
, is a preferred material for electrophysiological studies of receptor and ion channel function. Xenopus oocytes have the ability to synthesize functional proteins when microinjected with exogenous mRNA or cDNA constructs.
In electrophysiological analysis, an oocyte is electrically connected to intracellular voltage and current measuring and clamping devices. Detection of an electrophysiological response may comprise steps of applying appropriate receptor ligands and adjusting the holding potential manually and measuring any changes in membrane voltage or current.
Recently, electrophysiological analysis of Xenopus oocytes has been actively applied to many fields. In particular, electrophysiological analysis has been used for the study of membrane protein function, such as the function and pharmacology of membrane receptors, voltage-gated ion channels, molecular transporters and ion pumps. Defined combinations of recombinant subunits, chimeric proteins, or mutagenized constructs can be efficiently reconstituted in the oocyte membrane for electrophysiological analysis. For such analysis, the oocyte response may be monitored using intracellular recording, patch clamp and internal perfusion techniques.
It has been difficult to achieve a highly reproducible and reliable assay or to achieve quantitative analysis of electrophysiological response by conventional manual pension and membrane potential measurement techniques. These techniques have many shortcomings because of variabilities due to human errors, operator fatigue and inconsistencies between operators, and less than optimal reproducibility and reliability. Further, the perfusion and detection steps typically require long and complicated manual manipulations which create additional problems. The cultured cell becomes less viable with time and it is difficult to control the temperature and oxygen tension. The limited dexterity of even the most experienced operator limits the number of experiments may be performed on one cell. Reliance on human operators has resulted in reaction times that are considerably longer than theoretically possible.
Conventional systems for analysis of cells have attempted to address some of the problems of automated cell analysis. These systems have suffered generally from inability to individually measure a physiological response of a cell. Examples of systems that do not address individual physiological measurements include Kearney, Engström, Fränzl al. and Capco et al.
Kearney (U.S. Pat. No. 5,424,209), discloses a system for culturing and testing of cells. This culturing and testing system was designed for the culturing and testing of cell populations and not individual cells. Engström (U.S. Pat. No. 5,312,731) discloses a method and apparatus for studying a reaction pattern of a cell or cell aggregate during perfusion with different media. The system is limited to analysis of cell response of a through transmission microscopy. Fränzl et al., (U.S. Pat. No. 5,432,086) discloses an apparatus for the automatic monitoring of microorganism culture. The system is limited to the monitoring of microorganism growth and multiplication by an-impedance measuring process. Capco et aL, (U.S. Pat. No. 4,983,527) discloses a method for detection of tumor promoting compounds. Amphibian oocytes are contacted to a tumor promoting compound and the oocytes are examined visually to detect a change in the size of the light/dark hemisphere of the oocyte. Capco's disclosed method is limited to contacting the oocytes to one solution comprising a candidate tumor promoting compound.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel apparatus and methods for the study of membrane physiology.
One embodiment of the invention is directed to cellular physiology workstations that enable automated execution of experimental protocols for electrophysiological experiments and for the development of more complex protocols based on extended recording sessions. As currently developed for oocyte electrophysiology, the apparatus comprises one or more custom-built recording chambers, a perfusion control system designed for rapid application of about 2 to about 16 or more solutions under automated control, software-based virtual instrumentation developed to automate the execution of experimental protocols, and a data acquisition and control platform which integrates the entire system. The system is fully customizable through a sophisticated object-oriented programming language and can be easily adapted to applications such as patch clamp electrophysiology, calcium imaging studies, confocal microscopy and other applications where perfusion control and data acquisition need to be tightly integrated.
Another embodiment of the invention is directed to apparatus for reproducibly detecting the electrical response of a cell to an agent. The apparatus comprises a plurality of recording chambers. Each chamber is designed to contain one or more cells such as, for example, one or more Xenopus oocytes. Means are provided to perfuse each recording chamber with a plurality of perfusion solutions. Each perfusion solution may contain a different concentration of one or more agent. A plurality of electrodes such as, for example, a voltage measuring electrode, a current injecting electrode or a glass patch electrode, may be connected to each cell to measure the electrical response of the cell to the presence, absence or change in concentration of the agent. The electrical response may also be measured at various holding potentials.
Another embodiment of the invention is directed to automated apparatuses for electrophysiological measurement which comprises injecting means, such as a needle, for delivering an injection solution into the cell. The injection solution may comprise a second agent, a protein, a nucleic acid or a combination thereof. The nucleic acid may be, for example, DNA, RNA or PNA. PNAs, peptide nucleic acids or protein nucleic acids, are synthetic polymers capable of hybridizing in a sequence specific manner with natural nucleic acids.
Another embodiment of the invention is directed to methods for reproducibly detecting a physiological response of a cell to a agent. A cell such as, for example, a Xenopus oocyte, is perfused using an automated perfusion system with a plurality of solutions, which may comprise different concentrations of one or more agents, and the electrophysiological response of the cell measured. The automated perfusion control system may be, for example, a gravity fed flow through perfusion system. The automated perfusion control system may have an optimized lag time of less than about 100 milliseconds and a rise time of less than about 140 milliseconds such as less than about 70 milliseconds.
Another
Farb David H.
Gibbs Terrell T.
Yaghoubi Nader
Farrell Kevin M.
Leary Louise N.
Trustees of Boston University
LandOfFree
Cellular physiology workstations for automated data... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cellular physiology workstations for automated data..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cellular physiology workstations for automated data... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2557144