Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-10-26
2004-04-20
Brumback, Brenda (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S287100, C435S288400, C435S288500
Reexamination Certificate
active
06723523
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in general, to novel methods for measuring cell movement and to methods for assessing the impact of a variety of factors on the ability of cells to move. In particular, the present invention relates to systems and methods for measuring cell movement and the effect of various chemical species on that phenomenon by monitoring one or more electrical parameters of sensing electrodes that are sensitive to the interaction of cells with the electrode surface.
BACKGROUND OF THE INVENTION
Cells move from place to place in multicellular organisms for a variety of reasons. For example, cell movement occurs during organogenesis, movement is essential to inflammatory immune responses, and movement of neoplastic cells permits metastasis to secondary sites. This movement can arise from the intrinsic characteristics of the cell, or it can be initiated, enhanced or otherwise affected by the presence of external chemical stimuli. Stimuli can be divided into two classes: those that stimulate cell movement without a specific directional aspect (chemokinesis), and those stimuli that enhance directional cell movement according to the location of external cues (chemotaxis).
Cell movement is a critical component of both normal immune function and the dysfunctional immune responses associated with certain disease conditions such as asthma, chronic inflammation and autoimmune disease. Often this movement is characterized as chemotactic, and is initiated in response to the presence of one or more of a set of chemoattractants. These chemoattractant species (e.g., chemokines and activated components of the complement cascade) can be produced by the body in response to a variety of stimuli. Additionally, cells of the immune system are capable of rapid and vigorous responses to stimuli provided by infectious microorganisms (e.g., f-met-leu-phe and other formulated peptides that are products of bacterial protein degradation). In the context of an infection or other inflammation, these signals cause the influx of cells (notably macrophages and neutrophils) at the site of inflammation. The common mechanism of action for these signals is to engage specific receptors on the surface of the cell. Following ligand/receptor engagement, one or more signal transduction cascades are initiated, and the cell responds by specific activation of genes and the movement of the cell along the gradient. Still unknown is the means by which these cells sense the gradient, and the actual mechanisms by which they move through the cellular environment to arrive at the source of the chemoattractant.
From a practical standpoint, studies that identify new chemokines and other attractants, that characterize the signal transduction cascade and the differentiation of the responding cell, as well as studies that characterize the environment in which movement occurs, will each provide potential avenues of therapeutic manipulation. Assays that measure cellular movement in response to a chemotactic gradient offer the ability to assess individual elements along the length of the path from initiation of the response to the cellular accomplishment of the movement.
Quantitative and qualitative measurement of cell movement can be important to the characterization of biological responses, such as those mentioned above, as well as to many others. The rational design of therapeutic strategies for clinical intervention in these systems can theoretically depend upon manipulation of cellular motility: increasing it when a more robust response is desired, and diminishing the influx of cells to reduce their contributions to the response. For example, pharmacological manipulations of cell accumulation in the airways has been found to be an effective treatment for some forms of asthma, and interference with cellular movement through the vascular epithelia can diminish some the inflammatory damage associated with ischemia/reperfusion injury. Vigorous research efforts are currently underway in many biotechnology and pharmaceutical laboratories to discover novel therapeutics with the capacity to affect cell movement. For example, it is understood in the art that a potential therapeutic approach is to use inhibitors of signal transduction to manipulate chemotactic responsiveness, and many investigations are currently under way to assess the viability of such an approach in the treatment of a number of disease conditions. Essential to these investigations is the capacity to make qualitative and quantitative observations of cell movement in response to chemotactic stimuli, as well as mediation of that response by inhibitors or enhancers of chemotactic response.
In the prior art, measurement of cell movement directed by chemotactic agents has been accomplished in several ways. A “small-population” assay can optically measure the movement of cells in an initial localized deposit of these cells in a chemotactic gradient that exists in proximity to the cells. Variations of the Boyden chamber assay (Boyden, S.,
Journal of Experimental Medicine
115: 453 (1953)) are currently the most commonly used. In these assays, the cells are placed on a microporous membrane over a source of chemotactic agent. As the cells detect the higher concentrations of chemotactic agent that diffuse from the source, they migrate through the membrane to its underside. Migrating cells are usually statically detected by manual and optically aided methods on the reverse side of the membrane after staining. The quantity of responding cells is usually determined as an endpoint assay at a predetermined time-point. Thus, assays of this sort are usually capable of a semi-quantitative measure of the number of cells from an initial cell population that travel across a membrane in response to a perceived gradient of a chemoattractive agent. An advantage of this type of technique is the ability to perform many simultaneous assays, as multi-well plates in a two-dimensional array may be effectively utilized. However, a major limitation of a Boyden-type assay is that the chemical gradient sensed by the cells is very steep and dissipates rapidly. Essentially, there is a high concentration of chemoattractant on one side of the separating membrane and none on the other. In addition, it is also difficult to visualize the movement of cells through the membrane in this chemotactic environment. Finally, quantitation of the number of cells to move in response to the chemotactic stimulus is limited by traditional cell-counting methodologies and other errors inherent in the system such as the loss of migrated cells from the underside of the membrane where counting occurs.
Another technique used to measure chemotaxis is to track cell movement by video microscopy in a Zigmond or Dunn chamber. In these assays, the movement of cells is recorded as they respond to an aqueous gradient of chemoattractant formed between two closely spaced glass surfaces. This assay suffers from serous drawbacks in that it is more difficult to set up, only a small number of cells can be analyzed at one time, and the assay cannot be easily multiplexed.
The under-agarose chemotactic assay (Nelson, R. D., et al.
Journal of Immunology
115: 1650-1656 (1975)) provides a different approach from that offered in other, Boyden-type assays. In the under-agarose assay, a planar layer of agarose gel is cast in a cell culture dish. Multiple wells are cut in the agarose layer with a device such as a stainless steel punch. In a typical assay, multiple sets of three wells are punched in a linear array. In the middle well of the three-well set, a portion of cell suspension is added to the well. In one of the adjoining wells, a solution of a chemotactic agent is added. In the third well, a suitable control solution is added. The assembly is allowed to incubate at an appropriate temperature for a pre-determined period of time after which the cells are fixed with the agarose layer in place by the addition of suitable fixing agents such as absolute methanol. After fixation, the gel layer is removed and the p
Knecht David A.
Lynes Michael A.
Brumback Brenda
McCarter & English LLP
University of Connecticut
Winston Randall
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