Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-12-18
2003-07-29
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007230, C435S040510
Reexamination Certificate
active
06599694
ABSTRACT:
FIELD OF THE INVENTION
The systems and methods described herein provide for image capturing of living, dead, or fixed cells or cell fractions used to identify information about substances used on the cells or information about the cells themselves. Accordingly, the present invention can enable researchers and scientists to identify promising candidates in the search for new and better treatments and medicines, for example, in drug discovery and development. The principles enumerated herein may, with equal facility, be applied to other applications, including but not limited to use in environmental applications such as determining chemical toxicities and other non-pharmaceutical toxicology uses.
BACKGROUND OF THE INVENTION
Purified substances having a desirable combination bio-active properties are rare and often difficult to identify. Recent advances in traditional organic chemistry and the development of rapid combinatorial chemistry techniques have increased the number of compounds that researchers can test for a specific biological activity (e.g., binding to a target). Unfortunately, the vast majority of “hits” generated by such techniques do not possess the right combination of properties to qualify as therapeutic compounds. When these substances are subjected to low throughput cellular and animal tests to establish their therapeutic usefulness, they are typically found to fail in some regard. Unfortunately, such tests are time consuming and costly, thus limiting the number of substances that can be tested. In a like regard, the few hits that do possess the right combination of properties avoid recognition until after the throughput tests are conducted. With better early evaluation techniques, such promising candidates could be identified earlier in the development process and put on a fast track to the marketplace.
There have been some attempts to use image acquisition techniques to screen a large number of substances based upon biological cell information. One such attempt is described in International Application No. WO 98/38490 in the names of Dunlay, et al. Dunlay et al. generally describes a conventional image acquisition system. This conventional system collects and saves cellular images based on certain criteria that are predefined. Unfortunately, this system is has only a limited ability to predict a therapeutic usefulness of particular compounds or other agents.
One difficulty in predicting the clinical effectiveness of any agent is determining what concurrent effects it produces in normal cells, diseased versions of the normal cells, and other related cells. Diseases such as cancer often involve the interaction of various cell types such as cancerous epithelial cells and their stromal cells. During development of a potential new therapeutic, most research at the early stages focuses separately on the diseased cells or normal cells. To the extent that both cell types interact in producing or maintaining a disease state, there is no systematically rigorous technique for evaluating how a potential therapeutic affects their interaction.
What is needed therefore is a technique for quickly and quantitatively evaluating the affect of a potential therapeutic on a combination of various cell types that interact to produce or maintain a biological condition (e.g., cancer).
SUMMARY OF THE INVENTION
This need may be addressed by quantitatively analyzing images of two different cell types that interact in producing and/or maintaining a disease state or other biological condition. The two separate cell types are exposed to an agent or stimulus suspected of influencing the biological condition (e.g., the agent might be a potential therapeutic for treating a cancer). Typically, though not necessarily, the two different cell types are co-cultured or otherwise allowed to interact with one another before and during exposure to the agent. The images of the cells show how the agent separately affects each of the cell types. Specifically, the images show how the phenotype of each type changes (or does not change) upon exposure to the agent. In the context of this invention, the concept of a phenotype encompasses visual indicators showing viability, migration patterns, growth rates, extracellular matrix depositions, etc. The method generates quantitative phenotypes of the cells of cell types by quantitatively analyzing the cell images, usually via an automatic procedure. The quantitative phenotypes typically take the form of a group of scalar or vector descriptors that together provide a “fingerprint.” The descriptors may be size values, positions, morphological values, intensity distributions, etc.
One aspect of the invention provides a method of evaluating the effect of interactions between distinct cell types. The method may be characterized by the following sequence: (a) providing a first cell culture of a first cell type and a second cell culture of a second cell type in a microenvironment; (b) imaging the first and second cell types after exposure to the agent; and (c) quantitatively evaluating one or more images obtained in (b) to identify any effects of the agent. To this end, the method employs quantitative representations of the phenotypes of the cells in the first and second cell cultures. This may show how the effects of the agent are mediated by interactions between the first and second cell cultures. The microenvironment mentioned above is typically a contained environment in which the cells of the first and second cell cultures share a common medium, thereby allowing the first and second cell types to interact in the common medium. In alternative embodiments, the cells of the distinct cell types are separately cultured and imaged. During the process, one of the cell types may be exposed to factors produced the other.
Frequently, a method of this invention includes separate operations of exposing the cells of the microenvironment to the agent and then imaging the first and second cell types. Thereafter, in determining how the agent affects quantitative representations of phenotypes of the cells, the system may be able to predict the effect of the agent in treating the biological condition of interest. The quantitative representation typically includes two or more scalar values or vectors that characterize morphological and/or compositional features of a cell.
The agents or stimuli considered for use with methods of this invention include a wide variety of perturbing influences, and, in some cases where the two cell types interact in particularly interesting ways, may even constitute the absence of a perturbation. Examples of agents contemplated for use with this invention will be discussed below. In many important applications, the agent is a biological material or chemical compound such as a drug candidate.
Many different biological conditions may be analyzed with the methods of this invention. Diseases are an important class of biological conditions. Specific examples of biological conditions that may be analyzed using this invention include cancers, Type I diabetes, Type II diabetes, neurodegenerative diseases, cardiovascular diseases, vascular disease, auto-immune diseases. In certain cases, the biological condition is normal unperturbed functioning of an organ or tissue and the agent causes one or more of the cell types to become abnormal.
The first and second cell types used with this invention are chosen to shed light on a particular biological condition. As mentioned, many of these cell types interact with one another to produce and/or maintain the biological condition. For example, where the biological condition is a cancer, the first cell type may be a cancerous epithelial cell type and the second cell type may be a mesenchymal (stromal) cell type, with both cell types taken from the same tissue or organ.
In one embodiment, the method involves applying the agent to both cancerous epithelial cells and either endothelial or stromal cells from the same tissue or organ. Then both cell types are imaged and the resulting images are evaluated to identify changes in a
Beyer Weaver & Thomas LLP
Brusca John S.
Cytokinetics Inc.
Stevens Lauren L.
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