Chemistry: analytical and immunological testing – Blood gas
Reexamination Certificate
1999-10-14
2002-05-28
Weber, Jon P. (Department: 1641)
Chemistry: analytical and immunological testing
Blood gas
C436S002000
Reexamination Certificate
active
06395555
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and apparatus for determining the effect of a drug on viable animal or plant cells in culture.
BACKGROUND OF THE INVENTION
The in vitro screening of the effects of drugs on human or other live animal cells requires techniques in which large numbers of samples can be measured with minimal consumption of labor and time. Current technology involves growing the cells in culture in multi-well plates that permit the medium to be changed as required and the cells measured using automated plate readers. The evaluation of drug effects generally involves treatment of the cells with a selected drug or combination of drugs as a single administration or over a predetermined time course. This is followed by a determination of the viability of the cells in response to the drug, and an assessment of the efficacy and safety of the drug.
Unfortunately, many of the assessment techniques currently available are subjective determinations, although they are typically based upon objective measurements. For example, cells are typically stained with a selected dye or dyes or by some other staining technique to determine whether the cells are alive or dead following exposure to the test drug. However, in some cases the dyes and staining techniques introduce their own set of variables into the assessment. Although useful, a dye may also introduce certain disadvantages to the evaluation of a drug, e.g., the dye, even if biocompatible, could affect the speed, accuracy, toxicity, and visible coloration of the medium. Regardless of whether the affect is a disadvantage or an enhancement to the effect of the drug being tested, the simple fact that unknown variable could affect the outcome makes the final determination of the drug analysis subjective and of questionable reliability.
Cellular oxygen consumption is a reliable measure of cell physiology. Viable cells require continuous consumption of oxygen in order to survive, metabolize, grow and divide. Under constant temperature and culture conditions, the rate of oxygen consumption for a particular cell type is proportional to the number of cells. Thus, the rate of oxygen consumption provides an accurate measure of the number of live cells. Changes in cell number by either cell death or growth inhibition can be measured. Although others have attempted to measure oxygen concentrations to provide information regarding tissues and other in vivo environments, e.g., Vanderkooi et al,
J. Biol. Chem.,
262 (12):5476-5482 (April 1987); U.S. Pat. Nos. 4,476,870; 4,947,850; 5,186,173; 5,515,864, there has remained a need in the art for methods and apparatus that will more quickly, more accurately, and more economically determine the effect of drugs on a variety of tissues and cell types in a reliable and reproducible manner.
SUMMARY OF THE INVENTION
The present inventors have responded to the need for an improved, reliable and fast way of testing the effect of a drug or drugs on a variety of tissues and cells types by developing methods and apparatus that utilize the rate of respiration of a selected population of cells to provide a measure of the metabolic disturbance (uncoupling, inhibition, stimulation) of the cells in response to exposure to the selected drug or drugs.
In accordance with one aspect of the present invention, there is provided a method for determining the effect of a drug on attached cultures of cells comprising the following steps. A phosphorescent compound is dissolved in a culture medium containing an attached culture of cells, wherein quenching constant and lifetime at zero oxygen of the compound are known or previously determined at a selected, constant temperature. A drug, whose effect on the cells is to be tested or determined, is introduced into the culture medium either before the medium is added to the cells or while the medium is in situ over the cells. Then, the cell culture, comprising the cells, cell culture medium, phosphorescent compound and test drug, is illuminated with a pulsed or modulated excitation light at an intensity and frequency sufficient to cause the phosphorescent compound to emit a measurable phosphorescence. The emitted phosphorescence is then measured; and the phosphorescence lifetime and oxygen concentration gradient in the medium are calculated, thereby permitting a fast and reproducible objective determination of the effect of the drug on the respiration rate of the cells at the constant temperature.
In a preferred embodiment of the invention, the phosphorescent compound is selected or prepared which does not chemically react with the cells or the culture medium, nor does it affect the growth, viability or morbidity of the cells.
In additional embodiments of the invention, one or more of the preceding steps may be repeated as necessary to provide the measurements needed to calculate a phosphorescence distribution profile for the drug being tested. In preferred embodiments, the illumination, measurement and calculation steps are repeated for same cell culture, or multiple matched cell cultures are established to permit comparisons of a variety of drugs, drug combinations, or drug concentrations.
In a preferred embodiment of the invention, the attached cells are animal cells. The cells can be primary or secondary cultures, differentiated or undifferentiated, transformed, transfected, engineered or recombinant cells, or the like, as applicable to the drug or substance being tested. The cells can be attached as confluent monolayers, as actively dividing cells, or at any point in their life cycle. The method is designed to negate the metabolic status of the cells before the drug is added, by the use of mathematical constants based on matched control cell cultures in the calculation of the resulting respiratory rate of the cells in response to the added drug.
In another embodiment of the invention, the method comprises determining (i) the mean partial pressure of oxygen in the culture medium, and (ii) any change in partial pressure, thereby permitting the determination of any change in the respiration rate of the cells in response to the added drug. The oxygen distribution or concentration gradient is calculated for the entire depth of the culture medium, extending from the attached cell layer to the air/medium interface. Determination of the distribution curve of oxygen concentrations throughout the culture medium, permits the determination of respiration rate of the cells. Moreover, the change of mean partial pressure is used to determine morbidity of the cells, growth of the cells, or metabolic alteration of the cells, in response to the drug.
In yet another embodiment of the invention, the excitation light is provided by a light source selected from the group consisting of flash lamp, pulsed light emitting diode, and pulsed laser to illuminate the culture medium at a selected frequency.
In an additional embodiment of the invention, the emitted phosphorescence is measured by a time domain device or by a frequency domain device. In preferred embodiments the emitted phosphorescence is measured by means of a device, such as a photomultiplier, an avalanche photodiode, or a photodiode.
In yet another embodiment of the invention, the measured phosphorescence values are converted into digital values.
In a further embodiment, the rate of oxygen consumption of the cells is calculated by a process which comprises reconstructing the oxygen concentration gradient by deconvoluting the distribution of phosphorescence lifetime data into an underlying distribution of exponentials. Reconstruction of the oxygen concentration gradient permits determination of the rate of oxygen consumption of the cells following exposure to the test drug.
In certain embodiments of the invention, the phosphorescent compound is a porphyrin compound, preferably having the formula:
wherein R
1
is a hydrogen atom or a substituted or unsubstituted aryl; R
2
and R
3
are independently hydrogen or are linked together to form substituted or unsubstituted aryl; and M is a metal. In preferred embodimen
Vinogradov Sergei A.
Wilson David F.
Dilworth Paxson LLP
McConathy Evelyn H.
Patten Patricia
Weber Jon P.
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