Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-07-12
2004-11-23
Tate, Christopher R. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S030000
Reexamination Certificate
active
06821747
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to the suppression of non-biological motion of a cell. More specifically, the present invention is related to the suppression of non-biological motion of a cell having a viscosity enhancement medium, such as methyl cellulose.
BACKGROUND OF THE INVENTION
Cell motility plays an important role in numerous cellular biological processes, for example immune response and modulation, stem cell engraftment in bone marrow transplantation, wound healing, biomaterials compatibility, tissue engineering, tumor metastasis, myocardial angiogenesis and tumor anti-angiogenesis, to name some areas of commercial interest with relevance for improving human health. In all of these cases, the measurement of cell motility in vitro provides a basis for better understanding the biology of the process and for testing the effects of pharmaceuticals or other therapeutic approaches with potential for assisting or inhibiting the process of cell motility.
Time-lapse imaging provides the most direct and informative method for analysis of motility in vitro, particularly for adherent cell types. Velocity and changes in velocity over time, direction of motion, persistence (tendency for motion in one direction), frequency of directional changes, frequency of stopping and starting, time spent in motion and at rest, total distance traveled—these are some of the parameters accessible to the automated time-lapse method of analysis that are not accessible by other means. The capability for dissecting out such features of motion is important for determining mechanisms of interaction of potentially therapeutic compounds, because different aspects of motion can be affected, depending upon the molecular pathway(s) involved (Ware, Wells, and Lauffenburger, 1998).
With time-lapse video, short-lived effects or transient effects of added compounds on motility can be readily quantified through comparison to baseline values up to the moment of compound addition. In cases of chemotactic behavior, the response may arise through signaling of “differential” receptors, i.e. receptors that transmit intracellular signals only when ligand concentration changes (Dunn, 1990). In such cases, the response of the individual cell may depend upon the recent prehistory of the cell; time-lapse analysis reveals such behavior patterns.
Of particular interest to us is the possibility of screening for very short-lived secreted products on the basis of changes in migration patterns or morphology or phenotypic marker expression of cells in the immediate vicinity of transfected or otherwise engineered “secretor cells.” Such short-lived products may exist and have important roles in physiological processes, but being short-lived, they would not be readily detectible due to their instability under normal circumstances. Genes for such products could be transfected into “secretor cells” that would express and secrete these products continuously in culture. Changes in motility or other phenotypic indicators of nearby cells would reveal the activity of such compounds. Examples of such compounds might include chemotactic agents, i.e. compounds that induce directed migration in cells. Such compounds guide cells to sites of relevant physiological interactions, for example in coordinating interaction of T cells, dendritic cells, and B cells in peripheral lymphoid tissues for immune response and in guiding neuronal cell synaptic connections during development of the nervous system.
In addition to the analysis of chemotactic responses to short-lived products, as described above, where in situ secretion from living cells would be necessary, we are also interested in analysis of chemotactic responses to more stable compounds, such as chemokines. Such compounds would be released in the vicinity of responding cells by non-biological methods, for example by impregnating gelatin beads or small microvessels or by application of chemotactic compounds to the culture surface.
In some cases, the migratory response to extracellular signaling molecules is linked to changes in cell adhesion molecules and in cell surface markers (phenotype). Moreover, it would be desirable to determine whether specific subpopulations of cells of similar phenotype show similar specific responses in motile behavior toward various stimuli. Linking surface marker phenotype analysis with motile behavior can feasibly be accomplished in parallel with imaging and intelligent image analysis. These goals hold tremendous potential as tools for investigative biology as well as screening of potentially therapeutic compounds.
The present invention addresses the development of capabilities for automated video time-lapse analysis of biological motility of adherent or non-adherent cells of all types. T lymphocytes from peripheral blood are used as a model system here. The category “non-adherent” pertains broadly to cells of the hematopoietic system, including both lymphoid and myeloid lineages. Non-adherent cells can also exist in non-hematopoietic systems, such as freshly isolated myoblasts and certain cell lines (e.g. adapted HeLa (cervical adenocarcinoma) and PC-3 (prostate adenocarcinoma) cells, Colo 205 (colorectal adenocarcinoma), KNRK (normal kidney), RF-1 (gastric adenocarcinoma), Colo 587 (pancreatic adenocarcinoma), and others). Although some hematopoietic cells, most notably monocyte/macrophages and dendritic cells, adhere to tissue culture plastic, most hematopoietic cell types exhibit weak or transient attachment dependent upon added factors, e.g. phytohemaglutinin (PHA), serum components, fibronectin, or immobilized antibodies such as anti-CD3 for T lymphocytes.
Although the non-adherence of blood cells in vivo is implicit, the non-adherent nature in vitro of many types of hematopoietic cells is not so readily accepted. Some investigators hold that T-cells, for example, develop an adherent phenotype upon in vitro activation (consultant, personal communications). Most theories of cell migration and motility require the involvement of molecular attachment of cell adhesion molecules to the surface, for example through integrin-mediated binding to fibronectin (DiMilla et al., 1993; Lauffenburger, 1996; Maheshwari et al., 1999), and there is as yet no satisfactory theory for how non-adherent cells migrate. Nevertheless, observations over the course of numerous experiments, including round-the-clock imaging of CD34+lin-cord blood cells and their progeny, and experiments with naïve and prestimulated peripheral blood T lymphocytes, indicate that hematopoietic cells are highly animated and highly motile. However, it has also become clear that major components of the migratory “behavior” of these cells are non-biological influences of gravity and micro-turbulence, probably due to thermal convection. Convincing evidence for non-biological motion includes observation of dead (propidium iodide positive) cells moving separately in parallel with live cells. Similarly, the movement of beads and particles, the “flocking” or “herding” of live cells for no apparent cause, and finally “forward and reverse” tilting of the entire microscope by less than 3° leave no question that the biological adherence of these cells is relatively weak in comparison to ambient factors such as gravity and turbulence. Yet, as described below, when these ambient factors are controlled, adherence-independent biological motion is clearly evident, and this motion is sensitive to the influence of relevant biological compounds.
Some examples presented in the literature of time-lapse video analysis of hematopoietic cell migration patterns represent, instead, typical examples of environmentally induced “ambient motility” (Crisa et al., 1996; Francis et al., 1997). The cited patterns are similar to those observed repeatedly in a variety of culture vessels with different types of hematopoietic cells, including, even, dead ones. In one of these reported studies, video time-lapse images were used to support an observed arrest of T-cell migration with anti-VLA4 or anti-VLA5 specific antib
Automated Cell, Inc.
Schwartz Ansel M.
Tate Christopher R.
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