Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-01-11
2003-08-05
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S304200, C422S105000
Reexamination Certificate
active
06602701
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to devices and methods for analyzing cellular growth in vitro.
BACKGROUND OF THE INVENTION
Tumors grow through two primary processes: proliferation and invasion. Proliferative growth represents the increase in size of the central tumor mass through the division of cells. Invasive growth occurs in tissues in the regions adjacent to and around the central tumor mass. In the invasion process, individual tumor cells detach from the central tumor mass and begin to actively move through the surrounding, non-tumorous tissue, either by compression or enzymatic degradation. The presumably highly branched cells formed by these invading cells represent a dynamically evolving network pattern. The chains formed in the invasive network can be as thin as a single cell in width. Further, the invading cells are significantly elongated along the direction in which they are traveling.
Malignant tumors such as highly malignant brain tumors (e.g., gliomas and glioblastoma multiform) have several features such as proliferation, invasion, central necrosis/apoptosis and neo-vascularization. Tumors outside the central nervous system show extensive metastasis by way of the blood circulatory and lymphatic systems.
Several in vitro assays have been described that are designed to measure either cell proliferation, migration, or invasion. For example, a cell colony/spheroid-agarose assay uses cell suspensions within, or multicellular tumor spheroids placed on top of agarose (in a cell culture dish), which is then covered with cell culture medium. Carlsson J.,
Int. J. Cancer,
20:129-136, 1977. The assay is designed to study the growth dynamics of the cell colonies or spheroid. The medium superlayer distributes growth factors and growth limiting factors produced by the tumor, without preserving their important regional concentration differences (i.e., higher concentration around the tumor). Also, the superlayer has to be changed routinely, altering the environmental setting.
A 2D migration assay can be used to describe the movement of a cell population from a central area in an expanding circle. Giese A.,
Neurosurgery,
37:294-302, 1995. The cells are placed in a cell culture dish and covered with medium. Aside from the edge of the dish, there is no mechanical confinement and no chemo-gradients can be established due to “equalizing” in the medium superlayer.
Invasiveness assays (e.g., commercially available through Costar® as the 24-well Transwell™ System) use a medium-supplemented, two-chamber system, which is designed to detect cell migration between the two chambers. Repesh L. A.,
Invasion Metastasis,
9:192-208, 1989. The insert (top chamber) has a polycarbonate membrane on the bottom, which has a number of pores (8 &mgr;m size). After a certain number of cells are placed onto this membrane, they start to move through the pores and drop into the lower chamber where they either start anchorage-independent growth or eventually attach. After an observation period the cell number in both chambers is counted (Coulter Counter System®) and a ratio indicates the specific invasive potential of the cell line used. A variation of the assay uses a Matrigel® layer on top of the polycarbonate membrane to investigate the enzymatic activity of the cells to digest their way towards the pores.
A spheroid-fetal rat brain aggregate assay uses rat brain aggregates co-cultured on a medium/agar-layer and covered with a cell-culture medium that is changed routinely. Khoshyomn S.,
J. Neuro
-
Oncology,
38:1-10, 1998. The migration capacity of the tumor cells is determined by the destruction of the rat brain aggregate, not by the direct measurement of cell branches.
SUMMARY OF THE INVENTION
The invention is based on the discovery that cells can be grown in vitro in an enclosed device that allows for a three-dimensional measurement of both their proliferative and invasive properties. By growing the cells in an enclosed matrix that resembles the environment the cells confront in vivo, the cells can divide, invade, and form branched networks as they are thought to do in living tissue, e.g., in an individual. Propagating the cells in vitro in this manner allows for the imaging and temporal-spatial analysis of cells and cellular behavior that cannot be easily achieved when the cells are grown inside an organism. The methods and devices of the invention are particularly useful for studying the growth of tumor cells in vitro. The assay devices of the invention can thus be used as model systems to study cancer biology and to evaluate the efficacy of anti-cancer therapeutics.
In general, the invention features an assay device for measuring the proliferation and/or invasion of cells, e.g., tumor cells. The device includes a test chamber and a first delivery chamber arranged to contact the test chamber. The device can also include a control chamber, e.g., arranged to contact the first delivery chamber or a second delivery chamber. The first delivery chamber includes a wall with an opening to enable fluid communication between the first delivery chamber and the test chamber. The device also includes a hollow cylinder enclosing a lumen and arranged within the first delivery chamber, the cylinder including a wall with a hole that can be aligned with the opening in the first delivery chamber wall to enable fluid communication between the cylinder lumen and the test chamber.
The assay device can further include a cover that sealingly contacts the delivery chamber, the test chamber, and the control chamber, if present. The assay device can also include a moveable interior wall that is arranged within the test chamber to be moved laterally within the test chamber, e.g., by turning screws located in holes in an outer wall of the test chamber. The assay device optionally includes a second moveable interior wall that is arranged within the control chamber to be moved laterally within the control chamber, e.g., by turning screws located in holes in an outer wall of the control chamber.
The invention also features an assay device that includes a plurality of cylinders, each having a hole that can be aligned with the opening in the delivery chamber wall to enable fluid communication between the cylinder and the test chamber, wherein the cylinders are interchangeable and each has a hole of a different size. In addition, the assay device can also include a second delivery chamber arranged to contact the control chamber, e.g., in the same manner that the first delivery chamber is arranged to contact the test chamber. This would allow the control chamber to be exposed to a control fluid, as compared to a test fluid in the test chamber. The control chamber can include a moveable wall that is arranged to move within the control chamber.
The test chamber of an assay device can include an outer wall with an opening to enable fluid communication between the test chamber and the exterior of the assay device. An assay device can also include a hollow insert constructed to fit within the test chamber. The hollow insert can contain a moveable wall that is arranged to move within the insert.
In another embodiment, the invention features an assay system for measuring the proliferation and/or invasion of cells. The assay system includes an assay device of the invention, a pump having an input and an output, a first conduit that connects one end of the cylinder to the pump input, and a second conduit that connects a second end of the cylinder to the pump output to permit flow of fluid, e.g., a liquid or gas, from the pump, through the cylinder in the delivery chamber of the device, and back to the pump.
The assay system can also include an injection port connected to a conduit that permits the introduction of substances into the system, e.g., by microinjection. Additionally, the assay system can include a device of the invention that includes a first moveable interior wall that is arranged within the test chamber to be moved laterally within the test chamber, e.g., by turning screws located in holes in an outer wall of
Chiocca E. Antonio
Deisboeck Thomas S.
Wright John D.
Beisner William H.
The General Hospital Corporation
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