Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-09-28
2002-05-28
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S030000
Reexamination Certificate
active
06395505
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention relates to methods and apparatus for cell activity assay (CAA) investigation of chemotaxis, migration, invasion, angiogenesis, growth, proliferation, differentiation, or interaction of cells in response to various chemical environments.
2. Background of the Invention
Chemotaxis is the directional movement (migration) of biological cells or organisms in response to concentration gradients of chemicals. Invasion is the movement (migration) of cells into or through a barrier. Tumor invasion is such action initiated by cancer cells into or through biological tissue in vivo, or, into or through extra cellular matrix proteins, e.g., collagen or matrigel, into or through barriers made of other substances, in vitro. Angiogenesis is the migration and formation of capillary blood vessels by endothelial cells. Growth is the increase in the size, form, or complexity of cells. Proliferation is growth of cells by cell division. Differentiation is the process by which cells change from a less specialized to a more specialized state usually associated with different functional roles and the expression of new and different traits. Interaction of cells is the alteration of cell behavior such as movement, invasion, angiogenesis, growth, proliferation, or differentiation in response to the presence and action of nearby cells of the same or different type. These activities and similar activities are referred to herein collectively as “cell activity,” and the apparatus employed to do the assays is referred to herein as “cell activity assay apparatus.”
One kind of single-site conventional cell activity assay apparatus referred to variously in the literature as “chemotaxis chambers,” “Boyden chambers,” “Boyden chemotaxis chambers,” “blind well chambers,” or “microchemotaxis chambers,” comprises two compartments separated by a membrane, with one or both of the compartments open to air. Multi-site apparatus are referred to as “multi-well chemotaxis chambers,” or “multi-well Boyden chambers,” and have the same basic site structure but have multiple sites. (See U.S. Pat. Nos. 5,210,021 and 5,302,515) Assays employing this kind of apparatus pipette cells suspended in media into the upper compartments, and pipette chemotactic factors and controls into the bottom compartments. The chemotactic factors can be used in various dilutions to get a dose-response curve. The controls are generally of three kinds: (a) negative, when the same media that is used to suspend the cells is also used below the membrane, (b) chemokinetic, when a chemotactic factor is placed at the same concentration in the media with the cells and in the well on the opposite side of the membrane, and (c) positive, when a known chemoattractant is placed in the bottom wells. Chemokinetic controls allow the user to distinguish heightened random activity of the cells, due to contact with the chemotactic factor, from directional response in a concentration gradient of that chemotactic factor.
Cell activity assay apparatus can also be used to measure the response of cells of different origins—e.g., immune cells obtained from patients suffering from diseases—to a chemotactic factor of known chemotactic activity. In this case the cells in question are interrogated by both a negative control and a known chemotactic factor to see if the differential response is depressed or normal.
Chemotactic activity is measured by establishing a stable concentration gradient in the cell activity assay apparatus; incubating it for a predetermined time; and then counting the cells that have migrated through the membrane (or into the membrane). A comparison is then made between the activity of the cells in a concentration gradient of the chemotactic factor being tested, and the activity of the cells in the absence of the concentration gradient.
In one type of cell activity assay apparatus and method, the chemotactic response is measured by physically counting the number of cells on the membrane surface closest to the chamber containing the chemical agent. An example of this type of cell activity assay apparatus is described in U.S. Pat. No. 5,210,021 (Goodwin, Jr.), which is hereby incorporated by reference. One prior art method of obtaining quantitative data is to remove the membrane from the cell activity assay apparatus, remove the cells from the membrane surface closest to the chamber containing the original cell suspension, fix and stain the remaining cells, and then observe and count the stained cells under a microscope. Because of the time and expense associated with examining the entire membrane, only representative areas of the membrane are counted, rendering results less accurate than would otherwise be the case if the entire membrane were examined and counted.
Cell activity assays using a disposable ninety-six well microplate format, for example the ChemoTx™ System (available from Neuro Probe, Inc., Gaithersburg, Md.), is amenable to different methods of quantification of results. The manual staining and counting method described above can be used, but is not recommended due to the time involved. A preferred method is to centrifuge the microplate with filter attached, such that, the cells that have migrated through the filter are deposited onto the bottom of the lower wells. The cells are then stained with MTT, MTS (available from Promega, Madison, Wis.), or similar dye, and then read in a standard automated laboratory densitometric reader (sometimes referred to as an Elisa plate reader).
Another method of obtaining quantitative data with this apparatus is to dye the cells with a fluorescent material, e.g., Calcein AM (available from Molecular Probes, Eugene, Oreg.); centrifuge the migrated cells into the microplate; and count cells with an automatic fluorescence plate reader (e.g. Cytofluor available from PE Biosystems, Foster City, Calif., Victor
2
available from EG&G Wallac, Gaithersburg, Md., or fmax available from Molecular Devices, Sunnyvale, Calif.). The automatic plate reader excites the fluorescent dye in the migrated cells with one wavelength of light and reads the light emitted at a second wavelength. Alternatively, the cells that have not migrated are removed from the top of each site, and the plate with framed membrane attached is read in the automatic fluorescent plate reader without spinning the cells into the plate, thereby counting the cells that have fallen off the filter into the lower well as well as those on the bottom of the membrane and in the pores of the membrane.
In another type of chemotaxis apparatus, illustrated by U.S. Pat. No. 5,601,997 (Tchao), the chemotactic response is also measured by labeling the cells with a fluorescent dye, as above. However, in Tchao the membrane is made of film opaque to the excitation and emission wavelengths of the fluorescent dye so that the cells on one side of the membrane can be counted without removing the cells from the opposite side. Tchao's method is an example of a kinetic assay. In such assays, the side of the membrane toward which the cells are migrating is illuminated with the excitation wavelength of the dye, and the cells on that side are periodically counted by measuring the intensity of light emitted in the emission wavelength. This gives the researcher data on the rate at which cells are moving through the membrane. The membrane must be opaque because the researcher cannot remove the cells from the side from which they originated without ending the assay, which makes a kinetic study impossible.
DEFINITIONS & ABBREVIATIONS
Abbreviations
1. “Electromagnetic radiation” is herein abbreviated to “ER.”
2. “Pore diameter” is herein abbreviated to “pd.”
3. “Membrane thickness” is herein abbreviated to “mt.”
4. “Radius of curvature” is herein abbreviated to “rc.”
5. “High throughput screening” is herein abbreviated to “HTS.”
6. “Cell-based high throughput screening” is herein abbreviated to “CBHTS.”
7. “Nanometer” is herein abbreviated to “nm.”
8. “microliters” is herein abbreviated to “&mgr;l.”
9. “micrograms” is herein abbr
Gitomer Ralph
Neuro Probe Incorporated
Shaw Pittman LLP
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