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Method for using division arrested cells in screening assays

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Method for using division arrested cells in screening assays

Division arrested cells are used in screening assays to determine the effect of a substance of interest on the cells. The division arrested cells can be used in drug screening assays, signal transduction assays, and are especially useful in large scale, high throughput assays.
Related Terms: Drug Screening

Browse recent Life Technologies Corporation patents - Carlsbad, CA, US
Inventors: Thomas LIVELLI, Zhong Zhong, Mark Federici, Mei Cong
USPTO Applicaton #: #20120276545 - Class: 435 613 (USPTO) - 11/01/12 - Class 435 

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The Patent Description & Claims data below is from USPTO Patent Application 20120276545, Method for using division arrested cells in screening assays.

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This application is a continuation of U.S. patent application Ser. No. 13/102,917, filed May 6, 2011, which is a continuation of U.S. patent application Ser. No. 12/685,223, filed Jan. 11, 2010, now U.S. Pat. No. 7,960,101, which is a continuation of U.S. patent application Ser. No. 12/138,218, filed Jun. 12, 2008, now abandoned, which is a continuation of U.S. patent application Ser. No. 11/363,983, filed Feb. 27, 2006, now abandoned, which is a divisional of U.S. patent application Ser. No. 10/251,467, filed Sep. 20, 2002, now U.S. Pat. No. 7,045,281, which applications are entirely incorporated herein by reference.


This invention relates to methods for screening for substances of interest. More particularly, it relates to screening assays which utilize cells in a division arrested state which, nonetheless, function effectively in assays where dividing cells would normally be used. The advantages of the system will be seen in the disclosure which follows.


Cell based screening assays are tools well known to biologists. In the assays, one investigates compounds of interest to determine, e.g. if the compounds modulate one or more biological processes of interest.

Among the cell based systems which are used are those which measure reporter activity, calcium activity assay, and so forth. These, and all other cell based assays are encompassed by the invention.

One area where cell based screening assays have become widely accepted is the high throughput analysis of materials for use as pharmaceuticals. These assays are useful and desirable because compounds which are identified initially in biochemical assays have been known to fail as drug candidates later in the development process. The reasons for this are many. In some cases, the compound does not permeate the cell readily. In others, target binding capability is not predictive of the target modulating function, a feature that is, ultimately, a requirement of drug functions. Cell based screening assays are useful in that they address a number of problems associated with animal model testing (e.g., high expense, intensive labor, long assay period). High throughput cell based screening assays can be scaled up via technologies such as “FLIPR,” “Leedseeker,” “VIPR,” and fluorescent, high speed cell-imaging.

In addition to assays such as those discussed supra, other commonly used, cell based assays involve enzyme-reporter systems, cell activity assays with a fluorescent or colorimetric readout, and so forth. An example of such an assay is a Ca2+mobilization assay to measure G-protein coupled receptor activity with the dye “Fluo-4.”

In addition to the advantages set forth supra, cell based assays have a distinct advantage in that they permit the user to determine the functional outcome, of the use of compounds. Properly designed assays also permit the artisan to select against the toxic compounds, when screening for active ones.

Carrying out high throughput, cell based assays present unique challenges to users. Unlike pure biochemical reagent like enzymes, proteins, and membrane receptor preparations, cells are live dynamic entities. Preparation and cultivation have to be tied to the actual screening process.

Actively managed cell culture cycles have a recovery phase, when they are split from near confluent cultures, followed by an early log growth phase, then a mid log, and a late log phase, leading to a stationary phase if the culture is allowed to become confluent. Variances of the cellular processes and protein components at these different stages of growth and replication occur constantly during these cell cultures as cells cycle through mitosis. These variances must be expected to affect biological assays, and be a factor in the common phenomenon of variability in high throughput assays. One, but by no means the only example of this, relates to the length of time over which assays are run. There is generally an 8-36 hour period following the seeding of cells during which the assay is run. The cells in the particular culture go through different phases of their culture cycle during this time, and it is not usual for the cells to be at the same point in the cycle at the same time.

Further, the miniaturization of cell based screening assays is progressing, with smaller and smaller numbers of cells being used. As this occurs, sensitivity of the assay to variability increases rapidly and dramatically.

The critical factors of a good cell based screening assay are (i) a well validated target, (ii) a sensitive readout, and (iii) extremely high consistency of the cells that are used. The invention which is set forth in the disclosure which follows addresses this third issue. The consistent performance of the cells in an assay can be greatly affected by changes in the level of target expression as a result of increasing cell passage number. In addition, a well characterized population of cells can be division arrested, cryopreserved as a cell bank, and plated for an assay without any additional passaging of the cells. In fact, division arrested cells may be plated and used over a period of up to five (5) days without any further handling of the cells and without a significant change in cell based responses.

Division arrested cells have been used in the art. Exemplary of this are Cho, et al., Biochem. Mol. Biol. Int. 42:949 56 (1997); Yao, et al., Mol. Pharmacol 57:422 30 (1997); Fueger, et al., J. Nucl. Med. 42:1856 62 (2001); Muller, et al., J. Exp. Med. 188: 2033 45 (1998), and Kharbanda, et al., Nature 376 (6543):785-8 (1995). All of these references are incorporated by reference. Review of these, as well as other references will show that these studies concern characterization of the cells, rather than their use in assays of the type described herein, such as drug discovery, screening assays, and/or signal transduction assays, especially when these are carried out on a large scale, high throughput basis as is required for industrial application.

These, as well as other features of the invention will be elaborated upon in the disclosure which follows.


FIGS. 1A-1C set forth FACS scans depicting secrotonin induction of Ca++ mobilization on division arrested cells, which are set forth in example 1.

FIG. 2 shows the result of experiments, described in detail in example 2. It shows carbachol induced, Ca2+ mobilization, observed via changes in fluorescent emission ratios of Fura2 loaded into cells that were induced.

FIG. 3 summarizes the result of experiments set forth in example 3, involving cells where division was arrested by irradiation, and which were treated with isoproterenol and reporter gene changes were measured.

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Application #
US 20120276545 A1
Publish Date
Document #
File Date
Other USPTO Classes
435 29
International Class

Drug Screening

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