Point source diffusion cell activity assay

#1. Field of the Invention

#The present invention relates to methods and apparatus for high content screening (HCS) and high throughput screening (HTS). In particular, the present invention relates to cell activity assays (CAA) involving chemotaxis, migration, angiogenesis, growth, proliferation, and other cell activity based on, for example, morphology, shape, and movement of cells. The present invention also relates to cell activity assays involving changes internal to cells such as differentiation, alteration of metabolic rate, and movement of molecules within a cell initiated by activation of receptors in the cell membrane. The present invention also relates to cell activity assays involving the interaction of cells in response to various chemical environments, and the interaction of different cell types with one another. The present invention also relates to assays involving the penetration of cell layers by chemical compounds or other entities, and to assays for ascertaining the diffusion rate of members of a compound library through various confluent cell layers, e.g., endothelial or epithelial cell layers.

#2. Background of the Invention

#FIG. 1 is a schematic diagram of a cross-section of one site of a multi-site assay apparatus having a single film, according to an embodiment of the present invention.

#FIG. 2 is a schematic diagram of a top view of the assay apparatus of FIG. 1, showing a concentration gradient, according to an embodiment of the present invention.

#FIG. 3 is a schematic diagram of a cross-section of one site of a multi-site assay apparatus having two films, according to an embodiment of the present invention.

#FIG. 1 illustrates one site of a multi-site assay apparatus 100 according to an embodiment of the present invention. As shown, assay apparatus 100 includes a film 102, an opening 110 through film 102, cell suspension media 104 disposed on a first side of film 102 over opening 110, cells 108 of a cell suspension settled on the first side of film 102, and test compound solution 106 disposed on a second side of film 102 over opening 110. The test compound solution 106 initially contacts cell suspension media 104 at the meniscus 114 of cell suspension media 104 formed over opening 110 on the second side of film 102. Optionally, as shown in FIG. 1, film 102 could have hydrophobic coatings 116 to help keep cell suspension media 104 and test compound solution 106 in place.

#After the initial contact between cell suspension media 104 and test compound solution 106, the test compound solution 106 diffuses into cell suspension media 104 through opening 110, and to the first side of film 102. The test compound solution 106 diffuses into cell suspension 104 from opening 110, thereby creating a concentration gradient emanating concentrically from opening 110. As this diffusion occurs, the test compound solution 106 reaches the cells 108 nearest opening 110 first and then eventually, over time, reaches the cells 108 farther away from opening 110. During this diffusion, apparatus 100 is periodically read to determine whether and when the cells 108 are responsive to the test compound solution 106. If the cells 108 are responsive, the effects are perceptible in stages, as the test compound solution 106 radially diffuses from opening 110. In this manner, the progressive response by cells 108 can yield both quantitative and kinetic data.

#An embodiment of the present invention provides a method for performing a cell activity assay using the exemplary apparatus 100 shown in FIG. 1. According to this method, a cell suspension (containing cells suspended in a cell suspension media) is deposited onto the first side of film 102 over opening 110. The apparatus is then incubated, which allows the cells 108 to settle and adhere to the first side of film 102. The sites can then be read and recorded to provide a baseline reading of cells 108, or this step can be done immediately after the test solution is introduced.