U.S. patent application number 10/971261 was filed with the patent office on 2005-05-19 for suppression of non-biological motion.
Invention is credited to Bahnson, Alfred Blalock.
Application Number | 20050106557 10/971261 |
Document ID | / |
Family ID | 25418648 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106557 |
Kind Code |
A1 |
Bahnson, Alfred Blalock |
May 19, 2005 |
Suppression of non-biological motion
Abstract
A method for analyzing a cell or cells by suppressing
non-biological movement. The method includes the steps of placing
the cell or cells in a solution having a viscosity enhancement
medium. There can be the step of measuring the motility of the
cell, or other desired attributes of the cell or cells.
Inventors: |
Bahnson, Alfred Blalock;
(Pittsburgh, PA) |
Correspondence
Address: |
Ansel M. Schwartz
Suite 304
201 N. Craig Street
Pittsburgh
PA
15213
US
|
Family ID: |
25418648 |
Appl. No.: |
10/971261 |
Filed: |
October 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10971261 |
Oct 23, 2004 |
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09904144 |
Jul 12, 2001 |
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6821747 |
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Current U.S.
Class: |
435/4 ;
514/57 |
Current CPC
Class: |
G01N 15/1463 20130101;
G01N 2015/1497 20130101; C12Q 1/24 20130101; G01N 2015/1075
20130101; G01N 2015/1006 20130101; B01L 9/00 20130101; B01L 7/02
20130101 |
Class at
Publication: |
435/004 ;
514/057 |
International
Class: |
C12Q 001/00; A61K
038/16; A61K 031/716 |
Claims
What is claimed is:
1. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution
having a viscosity enhancement medium; and measuring the motility
of the cell in the solution.
2. A method as described in claim 1 wherein the viscosity
enhancement medium is methyl cellulose.
3. A method as described in claim 1 wherein the viscosity
enhancement medium is hyaluronic acid or chondroitin sulfate or
cellulose ester or poly sacharide.
4. A method as described in claim 1 wherein multiple cells are
measured in parallel.
5. A method as described in claim 1 wherein the placing step
includes the step of placing the cell in the solution of between
0.1% to 0.2% by total volume of methyl cellulose for 2D analysis of
motility.
6. A method as described in claim 2 wherein the placing step
includes the step of placing the methyl cellulose solution having a
concentration of between 0.1% and 1.2% methyl cellulose onto cells
in culture medium to provide a layer of methyl cellulose-containing
medium for 2D analysis of motility.
7. A method as described in claim 6 wherein the placing step
includes the step of placing the cell in the solution having a
viscosity of 100-5000 centipoise.
8. A method as described in claim 1 wherein the placing step
includes the step of placing cells in solution having a
concentration of between 0.3% to 2.5% weight per volume methyl
cellulose for analysis of motility in 3D.
9. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and measuring the motility of the cell in the solution when there
is no attachment of the cell involved.
10. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and identifying and quantifying short lived effects or transient
effects of added moiety on motility of the cell in the
solution.
11. A method for analyzing a cell comprising the steps of: placing
the cell in a solution having a viscosity of about 100-5000
centipose; and performing two-dimensional or three-dimensional
migration analysis on the cell in the solution.
12. A method for analyzing a cell comprising the steps of: placing
the cell in a solution having a viscosity of about 100-5000
centipose; and analyzing migration of the cell in the solution
which occurs without adherence.
13. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and controlling ambient motion of the cell in the solution as a
reproducible method for analysis of motion in a 2D or 3D
environment with non-adherent cells.
14. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and analyzing 3D motion of the cell in the solution in the absence
of a solid matrix upon which the cell can attach.
15. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and suppressing the ambient non-biological motion of the cell in
the solution on a 2D surface when there is no attachment involved
of the cell.
16. A method as described in claim 1 wherein the placing step
includes the step of placing the cell in the solution of between 1%
to 5% by total volume of methyl cellulose and a concentration of
between 0.08% and 0.12% of methyl cellulose.
17. A method for analyzing a cell comprising the steps of: placing
the cell in a solution having a viscosity of about 100-5000
centipose; and measuring motility of the cell in the solution,
where surface attachment by the cell is not utilized.
18. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and forming a thin film in the solution whose viscosity resists
brownian and other non-biological sources of motion but does not
interfere with active cell biological motion.
19. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
adding a protein or other biological or chemical moiety to the
solution; and analyzing the effect of the protein on cell motility,
morphology, phenotype, division rate, cell death, or blebbing or
disease state.
20. A method as described in claim 23 wherein the protein is a
human protein, antibody, growth factor, cytokine, kinase or
protease.
21. A method as described in claim 23 wherein the protein is
transduced or transfected into the cell.
22. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
adding a protein to the solution; and analyzing the protein
function regarding the cell using cell motility as an analytical
marker.
23. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and placing methyl cellulose in the solution to reduce ambient
motion of the cell in the solution and eliminate convective
motion.
24. A method for suppressing non-biological movement of a cell
comprising the steps of: placing the cell in a solution; and
forming a layer of methyl cellulose 34 to 137 Um thick in the
solution.
25. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and using methyl cellulose in the solution for stopping the effects
of gravity on the cell in the solution.
26. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and using methyl cellulose in the solution for reducing or
eliminating the effects of micro-turbulances due to thermal
convection in the solution.
27. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and introducing methyl cellulose in the
solution for stopping motion of the cells due to mechanical
movement of a plate on which the cells are disposed.
28. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
or reducing the effects of gravity on the cell.
29. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for reducing
the effects of micro-turbulences due to thermal convection.
30. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
motion of the cells due to mechanical movement of the plate.
31. A method for analyzing a cell by suppressing non-biological
movement comprising the steps of: placing the cell in a solution;
and using methyl cellulose or any viscous fluid to separate
biological motility from ambient motility.
32. A method for analyzing cells comprising the steps of: placing
the cells in a solution; and measuring biological cell motility for
adherent or nonadherent cells in the solution.
33. A method for analyzing cells comprising the steps of: placing
the cells in a solution; and measuring biological motility of both
adherent and nonadherent cells using visible and fluorescent
images.
34. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and measuring swimming vs moving of cells
in the solution in a 2D plane, as cells move up into a viscous
layer of the solution.
35. A method for analyzing a cell comprising the steps of: placing
the cell in a solution; and measuring the effect tilt has on cell
motion, by changing the angle a plate is tilted on which the cell
is disposed and looking for changes in motion or cell attachment of
the cell.
36. A method for analyzing a cell comprising the steps of: placing
the cell in a solution having methyl cellulose; removing the methyl
cellulose from the solution; treating the cell with a desired
material; and reintroducing the methyl cellulose into the
solution.
37. A method for analyzing cells comprising the steps of: placing
the cells in a solution; and identifying specific subpopulations of
cells of similar phenotype which show similar specific responses in
motile behavior toward various stimuli.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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.
[0003] 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).
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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 nave 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.degree. 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.
[0010] 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 antibodies (Crisa et
al., 1996). However, the "arrest" of migration observed after
antibody addition was timed in each case with the cessation of an
initial wave of unidirectional motion lasting for 2.5 hours. In
other words, the "arrest" may have occurred without added antibody
due to transient and variable ambient motion. In numerous
experiments, such ambient motion has been observed as cells
initially settle downhill into lower areas of the well. Motion
stops when the majority of cells have passed beyond the viewfield.
Given such problems, and despite the appeal of video time-lapse
imaging for gathering otherwise unobtainable information relating
to detailed characteristics of cell migration, there are as yet no
validated methods described in the literature for 2 dimensional
migration analysis of non-adherent cells.
[0011] A method for video time-lapse three-dimensional (3D)
analysis of T cell migration has been reported (Friedl, Noble, and
Zanker, 1995). This method is based upon the use of 3D collagen
gels and does not allow for analysis of motion that is achieved
apart from surface adhesion. These authors distinguish 3D from 2D
analysis and state, "Onto two-dimensional surfaces coated with ECM
components, nonactivated peripheral T cells do barely adhere and
are therefore incapable of migration. However, the incorporation of
these cells into a 3D collagen environment leads to the onset of
spontaneous migration; this results in the rapid and persistent
tyrosine phosphorylation of FAK, implicating FAK in T cell
migration." (Entschladen et al., 1997). This quote confirms the
generally held assumption that without adherence, there is no
migration. No explanation is offered as to why T cells do not
adhere to ECM (extracellular matrix) components coated onto a 2D
surface, i.e. tissue culture plastic, and yet T cells do apparently
adhere when incorporated into a 3D collagen matrix.
[0012] It is suggested that the failure to adequately control
ambient motion is the reason why a validated, reproducible method
has not yet been put forth for analysis of motion in a 2D
environment with non-adherent cells. In the presence of a very
slight tilt (less that 3.degree.), motion is observed to trend
downward, and if slight convection is present, live cells are
observed to follow the direction of flow of particles and dead
cells. This ambient motion is superimposed upon their active
motility. Perhaps upon observing this, other researchers recognize
first that there is no strong adherence, and then it may be assumed
that all "residual" motion is thermal or biologically irrelevant.
However, as described more fully below, clearly relevant biological
motion is seen using methyl cellulose in 2D cultures.
[0013] Likewise, no method has been presented for analysis of 3D
motion in the absence of a solid matrix (e.g. collagen) upon which
cells can attach. However, 3D motion among T cells in the absence
of a solid support has been observed using methyl cellulose at a
concentration of 1.2% (see below) . Also, 3D motion in video
time-lapse images of myeloid cell subpopulations in typical CFU-GM
cultures has been observed using methyl cellulose at a
concentration of approximately 0.9%.
[0014] Methyl cellulose has been widely applied for the purpose of
growing "colonies" of cells. Colonies are small clumps or groups of
cells; they are presumed to arise from a single cell, and are used
as a measure of "colony forming units" (CFU). The ability of a cell
to form a colony is equated with its being a "progenitor" cell, and
so CFU type assays are also known as progenitor cell assays. The
methyl cellulose allows the formation of a colony to proceed over a
one to two-week period in culture without mixing or disruption of
the cell positions.
[0015] In summary, there are apparently no validated methods in the
literature for analysis of migration of T cells or other
non-adherent cells on a 2D surface or for analysis of migration in
3 dimensions when there is no solid matrix on which the cells can
attach. When methyl cellulose is used, this dissolved compound is
not considered to provide attachment surfaces for the cells to
adhere to. There may be molecular attachment involved, but there is
no apparent requirement for it, because the cells move in medium
alone without methyl cellulose where, in the absence of strong
ambient motion, they can be observed to "swim" just the same as in
methyl cellulose-containing solution. In medium alone, frequently
it is difficult to distinguish biological from thermal and other
types of ambient motion, and in many cases the ambient motion is
not of uniform direction across the viewfield, nor is it constant
over time. Therefore, methods to mathematically "correct" for
ambient motion will have noise (uncertainty in precision)
associated with them, and in many cases this noise will be greater
than the magnitude of biological motion. With methyl cellulose, a
physical method for suppression of ambient non-biological motion on
a 2D surface has been developed when there is no attachment
involved.
[0016] Interestingly, as reviewed by Wilkinson (Wilkinson, 1990),
prior to development of the filter assay (see below), "many
beautiful studies" of leukocyte motion were made using video
photography and these studies "laid the foundations on which
contemporary studies are based." But due to the degree of technical
difficulty, these visual methods were abandoned when the Boyden
filter assay, now commonly known as the "Boyden chamber" (Boyden,
1962; Falk, Goodwin, and Leonard, 1980) or "Transwell.TM. migration
assay" (Bleul et al., 1996), was introduced. The vast majority of
current motility and chemotaxis investigation is conducted using
this method, whereby cells are added to a chamber separated by a
membrane from a second chamber containing medium with test
compounds. The cells migrate through small well-defined pores into
the lower chamber, and after a specified interval, they are counted
and compared with background numbers of cells migrating into
chambers without added compounds. While an abundance of valuable
data has been obtained using the Boyden chamber, this method "also
had the drawback that it was now possible to spend a research
career studying leukocyte chemotaxis without ever looking at a
moving cell or, indeed, knowing its front from its back 1/4",
according to Wilkinson (Wilkinson, 1990). Also with introduction of
the filter method, many of the clear distinctions regarding
chemotaxis, chemokinesis, contact guidance, direction reactions,
and other forms of locomoter reactions became blurred.
Nevertheless, the filter assay is seeing tremendous application in
the discovery of a large family of chemotactic compounds known as
"chemokines" (Allavena et al., 1999; Hedrick and Zlotnik, 1999),
which hold interest for therapeutic application both in terms of
the ligands themselves and in terms of their receptors as targets
for small drug molecules. The answers to questions about the exact
role of each of these chemokines in the host immune response will
be better answered with analytical approaches such as described by
the present invention.
[0017] In the 1970's, the "under-agarose assay" was introduced. An
agarose layer was poured over a glass slide to form a gel, then
holes were carefully bored and the agarose plugs were removed down
to the surface of the glass slide (Nelson, Quie, and Simmons,
1975). Cells were introduced into one hole and chemotactic
compounds or control substances could be introduced into the other
holes. Over time, the cells were seen to migrate between the
agarose and the glass toward a chemotactic compound at a faster
rate than toward a neutral control substance. Whereas the Boyden
filter assay yields only relative cell numbers, corresponding to
relative chemotactic strength, the "under-agarose assay" yielded a
distance traveled over time for the cell migration front. This
distance was originally compared to the distance traveled by the
control front on the other side of the well toward the neutral
compound to derive an index of migration. This simplistic approach
to migration analysis stimulated valuable mathematical treatment of
the problem (Farrell, Daniele, and Lauffenburger, 1990;
Lauffenburger, Rothman, and Zigmond, 1983; Nagahata et al., 1991;
Rothman and Lauffenburger, 1983; Rupnick et al., 1988; Stickle,
Lauffenburger, and Zigmond, 1984), which has provided the
mathematical framework for much current thinking in this area.
However, the method is subject to variability depending upon how
the holes are bored, perhaps due to lifting of the agarose from the
glass surface allowing cells to migrate along with channeling fluid
rather than through biological motility.
[0018] Another method in current use is measurement of the
infiltration of lymphocytes into a 3 dimensional collagen gel
(Friedl, Noble, and Zanker, 1995; Nikolai et al., 1998; Nikolai G,
1995). Cells are cultured in contact with the gel surface, and
after an elapsed time period, cells migrating to a certain depth
are counted. This number correlates to cytokinetic activity.
[0019] Although all of these methods provide a quantitative measure
of migratory activity, their shortcomings leave many aspects of the
migratory behavior hidden from the investigator.
SUMMARY OF THE INVENTION
[0020] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution having a viscosity
enhancement medium. There is the step of measuring the motility of
the cell. Multiple cells can be measured in parallel.
[0021] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
measuring the motility of the cell in the solution when there is no
attachment of the cell involved.
[0022] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
identifying and quantifying short lived effects or transient
effects of added moiety on motility of the cell in the
solution.
[0023] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution having a viscosity of
about 100-5000 centipose. There is the step of screening for
short-lived secreted products from the cell as a function of
changes in migration patterns or morphology or phenotypic marker
expression of the cell adjacent to transected or otherwise
engineered secretor cells or the cells themselves.
[0024] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of linking surface marker phenotype analysis of the cell
in the solution with motile behavior of the cell in the
solution.
[0025] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution having a viscosity of about 100-5000 centipose. There is
the step of linking surface marker phenotype analysis of adherent
and non-adherent cells in the solution with motile behavior of the
adherent and non-adherent cells in the solution.
[0026] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of performing two-dimensional or three-dimensional
migration analysis on the cell in the solution.
[0027] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of analyzing migration of the cell in the solution which
occurs without adherence.
[0028] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
controlling ambient motion of the cell in the solution as a
reproducible method for analysis of motion in a 2D or 3D
environment with non-adherent cells.
[0029] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
analyzing 3D motion of the cell in the solution in the absence of a
solid matrix upon which the cell can attach.
[0030] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
suppressing the ambient non-biological motion of the cell in the
solution on a 2D surface when there is no attachment involved of
the cell.
[0031] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000. There is the step of
measuring motility of the cell in the solution, where surface
attachment by the cell is not utilized.
[0032] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
forming a thin film in the solution whose viscosity resists
Brownian and other non-biological sources of motion but does not
interfere with active cell biological motion.
[0033] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
adding a protein or other biological or chemical moiety to the
solution. There is the step of analyzing the effect of the protein
on cell motility, morphology, phenotype, division rate, cell death,
or blebbing or disease state.
[0034] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
adding a protein to the solution. There is the step of analyzing
the protein function regarding the cell using cell motility as an
analytical marker.
[0035] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
placing methyl cellulose in the solution to reduce ambient motion
of the cell in the solution and eliminate convective motion.
[0036] The present invention pertains to a method for suppressing
non-biological movement of a cell. The method comprises the steps
of placing the cell in a solution. There is the step of forming a
layer of methyl cellulose 34 to 137 Um thick in the solution.
[0037] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose in the solution for stopping the effects of
gravity on the cell in the solution.
[0038] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose in the solution for reducing or eliminating
the effects of micro-turbulances due to thermal convection in the
solution.
[0039] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing methyl cellulose in the
solution for stopping motion of the cells due to mechanical
movement of a plate on which the cells are disposed.
[0040] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
or reducing the effects of gravity on the cell.
[0041] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for reducing
the effects of micro-turbulences due to thermal convection.
[0042] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
motion of the cells due to mechanical movement of the plate.
[0043] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose or any viscous fluid to separate biological
motility from ambient motility.
[0044] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
measuring biological cell motility with the cell in the
solution.
[0045] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of measuring biological cell motility
for adherent or nonadherent cells in the solution.
[0046] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of measuring biological motility of
both adherent and nonadherent cells using visible and fluorescent
images.
[0047] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of linking a surface marker of the cell
in the solution by phenotype analysis with motile behavior. The
linking step an include the step of linking a surface marker of the
cell in the solution by phenotype with motile behavior.
[0048] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring swimming vs moving of
cells in the solution in a 2D plane, as cells move up into a
viscous layer of the solution.
[0049] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring attachment of the cell to
a surface in the solution, by dispensing fluid into the solution
and looking for a location where the cell detaches from the
surface.
[0050] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring the effect tilt has on
cell motion, by changing the angle a plate is tilted on which the
cell is disposed and looking for changes in motion or cell
attachment of the cell.
[0051] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of removing methyl cellulose effects in
the solution by mixing the solution and diluting the methyl
cellulose.
[0052] The present invention pertains to a method for analyzing a
cell. The method comprises the steps placing the cell in a solution
having methyl cellulose. There is the step of removing the methyl
cellulose from the solution. There is the step of treating the cell
with a desired material. There is the step of reintroducing the
methyl cellulose into the solution.
[0053] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of between 100-5000 centipose. There is
the step of measuring cell division, morphology, cell phenotype,
disease state of the cell, or cell death. There can also be the
step of measuring the motility of the cell.
[0054] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of holding a cell intact for
suppressing motility for division detection of the cell in the
solution.
[0055] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of analyzing migratory response of the
cell to extracellular signaling molecules linked to changes in cell
adhesion molecules and in cell surface markers (phenotype).
[0056] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of identifying specific subpopulations
of cells of similar phenotype which show similar specific responses
in motile behavior toward various stimuli.
[0057] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of identifying and separating specific
subpopulations of the cells based on cell phenotype, morphology,
motility, cell proliferation, cell death, or disease state.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] In the accompanying drawings, the preferred embodiment of
the invention and preferred methods of practicing the invention are
illustrated in which:
[0059] FIG. 1 is plots of average curvilinear velocity for all cell
tracks in each viewfield over time demonstrate IL-2 effect on T
cell motility.
[0060] FIGS. 2a and 2b are a comparison of average curvilinear
velocity (Vcl), a scalar quantity, with straight line velocity
(Vsl), a vector, for cells in wells with and without methyl
cellulose.
[0061] FIGS. 3a-3d show concentration of methyl cellulose for
effective suppression of ambient motion.
[0062] FIGS. 4a and 4b show volumes of 1 ul to 4 ul of full
strength methyl cellulose stock are equally effective for
suppression of ambient motion.
[0063] FIGS. 5a and 5b show T cell tracks in wells containing
methyl cellulose and FIGS. 5c and 5d show T cell tracks without
methyl cellulose indicating the path traveled by T cells over the
course of analysis; ambient motion vs. random biological
motion.
[0064] FIGS. 6a and 6b show the effect of large volume of methyl
cellulose upon T cell motility characteristics.
[0065] FIGS. 7a and 7b show cells swept from a view field in some
wells, and not others, respectively, without methyl cellulose
present from the period over which the data were analyzed in FIG.
6.
[0066] FIGS. 7c and 7d show view fields where cells remained in
view with methyl cellulose present from the period over which the
data were analyzed in FIG. 6.
[0067] FIGS. 8a and 8b show a fluorescent image and a corresponding
view in visible light, respectively.
[0068] FIGS. 8c and 8d show a fluorescent image and a corresponding
view in visible light, respectively.
DETAILED DESCRIPTION
[0069] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution having a viscosity
enhancement medium. There is the step of measuring the motility of
the cell. Multiple cells can be measured in parallel.
[0070] The viscosity enhancement medium can be methyl cellulose.
The viscosity enhancement medium can be hyaluronic acid or
chondroitin sulfate or cellulose ester or poly sacharide.
[0071] The placing step can include the step of placing the cell in
the solution of between 0.1% to 0.2% by total volume of methyl
cellulose for 2D analysis of motility. The methyl cellulose
solution can have a concentration of between 0.1% and 1.2% methyl
cellulose onto cells in culture medium to provide a layer of methyl
cellulose-containing medium for 2D analysis of motility. The
placing step can include the step of placing the cell in the
solution having a viscosity of 100-5000 centipoise. The placing
step can include the step of placing cells in solution having a
concentration of between 0.3% to 2.5% weight per volume methyl
cellulose for analysis of motility in 3D.
[0072] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
measuring the motility of the cell in the solution when there is no
attachment of the cell involved.
[0073] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
identifying and quantifying short lived effects or transient
effects of added moiety on motility of the cell in the
solution.
[0074] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution having a viscosity of
about 100-5000 centipose. There is the step of screening for
short-lived secreted products from the cell as a function of
changes in migration patterns or morphology or phenotypic marker
expression of the cell adjacent to transfected or otherwise
engineered secretor cells or the cells themselves.
[0075] The screening step can include the step of screening for
short-lived secreted products from the cell as a function of
changes in migration patterns or morphology or phenotypic marker
expression of the cell adjacent to transfected or otherwise
engineered secretor cells.
[0076] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of linking surface marker phenotype analysis of the cell
in the solution with motile behavior of the cell in the
solution.
[0077] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution having a viscosity of about 100-5000 centipose. There is
the step of linking surface marker phenotype analysis of adherent
and non-adherent cells in the solution with motile behavior of the
adherent and non-adherent cells in the solution.
[0078] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of performing two-dimensional or three-dimensional
migration analysis on the cell in the solution.
[0079] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000 centipose. There is
the step of analyzing migration of the cell in the solution which
occurs without adherence.
[0080] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
controlling ambient motion of the cell in the solution as a
reproducible method for analysis of motion in a 2D or 3D
environment with non-adherent cells.
[0081] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
analyzing 3D motion of the cell in the solution in the absence of a
solid matrix upon which the cell can attach.
[0082] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
suppressing the ambient non-biological motion of the cell in the
solution on a 2D surface when there is no attachment involved of
the cell. The placing step can include the step of placing the cell
in the solution of between 1% to 5% by total volume of methyl
cellulose and a concentration of between 0.08% and 0.12% of methyl
cellulose.
[0083] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of about 100-5000. There is the step of
measuring motility of the cell in the solution, where surface
attachment by the cell is not utilized.
[0084] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
forming a thin film in the solution whose viscosity resists
Brownian and other non-biological sources of motion but does not
interfere with active cell biological motion.
[0085] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
adding a protein or other biological or chemical moiety to the
solution. There is the step of analyzing the effect of the protein
on cell motility, morphology, phenotype, division rate, cell death,
or blebbing or disease state.
[0086] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
adding a protein to the solution. The protein can be a human
protein, antibody, growth factor, cytokine, kinase or protease. The
protein can be added to the well or transduced or transfected into
the cell using known adenovirus or viral methods. There is the step
of analyzing the protein function regarding the cell using cell
motility as an analytical marker.
[0087] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
placing methyl cellulose in the solution to reduce ambient motion
of the cell in the solution and eliminate convective motion.
[0088] The present invention pertains to a method for suppressing
non-biological movement of a cell. The method comprises the steps
of placing the cell in a solution. There is the step of forming a
layer of methyl cellulose 34 to 137 Um thick in the solution.
[0089] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose in the solution for stopping the effects of
gravity on the cell in the solution.
[0090] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose in the solution for reducing or eliminating
the effects of micro-turbulances due to thermal convection in the
solution.
[0091] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing methyl cellulose in the
solution for stopping motion of the cells due to mechanical
movement of a plate on which the cells are disposed.
[0092] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
or reducing the effects of gravity on the cell.
[0093] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for reducing
the effects of micro-turbulences- due to thermal convection.
[0094] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of introducing a viscous fluid having a
viscosity of about 100-5000 centipose in the solution for stopping
motion of the cells due to mechanical movement of the plate.
[0095] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps of placing the cell in a solution. There is the step of
using methyl cellulose or any viscous fluid to separate biological
motility from ambient motility.
[0096] The present invention pertains to a method for analyzing a
cell by suppressing non-biological movement. The method comprises
the steps placing the cell in a solution. There is the step of
measuring biological cell motility with the cell in the
solution.
[0097] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of measuring biological cell motility
for adherent or nonadherent cells in the solution.
[0098] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of measuring biological motility of
both adherent and nonadherent cells using visible and fluorescent
images.
[0099] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of linking a surface marker of the cell
in the solution by phenotype analysis with motile behavior. The
linking step an include the step of linking a surface marker of the
cell in the solution by phenotype with motile behavior.
[0100] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring swimming vs moving of
cells in the solution in a 2D plane, as cells move up into a
viscous layer of the solution.
[0101] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring attachment of the cell to
a surface in the solution, by dispensing fluid into the solution
and looking for a location where the cell detaches from the
surface.
[0102] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of measuring the effect tilt has on
cell motion, by changing the angle a plate is tilted on which the
cell is disposed and looking for changes in motion or cell
attachment of the cell.
[0103] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of removing methyl cellulose effects in
the solution by mixing the solution and diluting the methyl
cellulose.
[0104] The present invention pertains to a method for analyzing a
cell. The method comprises the steps placing the cell in a solution
having methyl cellulose. There is the step of removing the methyl
cellulose from the solution. There is the step of treating the cell
with a desired material. There is the step of reintroducing the
methyl cellulose into the solution.
[0105] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution having a viscosity of between 100-5000 centipose. There is
the step of measuring cell division, morphology, cell phenotype,
disease state of the cell, or cell death. There can also be the
step of measuring the motility of the cell.
[0106] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of holding a cell intact for
suppressing motility for division detection of the cell in the
solution.
[0107] The present invention pertains to a method for analyzing a
cell. The method comprises the steps of placing the cell in a
solution. There is the step of analyzing migratory response of the
cell to extracellular signaling molecules linked to changes in cell
adhesion molecules and in cell surface markers (phenotype).
[0108] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of identifying specific subpopulations
of cells of similar phenotype which show similar specific responses
in motile behavior toward various stimuli.
[0109] The present invention pertains to a method for analyzing
cells. The method comprises the steps of placing the cells in a
solution. There is the step of identifying and separating specific
subpopulations of the cells based on cell phenotype, morphology,
motility, cell proliferation, cell death, or disease state.
[0110] In the operation of the invention, methyl cellulose is a
common name for solutions of methyl cellulose in cell culture
medium. Methyl cellulose added to medium increases the viscosity so
that convection and mixing are greatly suppressed at the level of
the cells.
[0111] Stem Cell Technologies in Vancouver has a line of products
that incorporate methyl cellulose for the purpose of colony assays.
These products are generally known under the trade name MethoCult.
The line includes the "base" to which other components may be
added. This base consists of a sterile 2.6% solution of methyl
cellulose in Iscove's Modified Dulbecco's Medium (IMDM). The
specification of the methyl cellulose used is as follows: a 2%
aqueous solution at 20.degree. C. has a viscosity of 4,000
centipoises. IMDM is one of several varieties of nutrient solution
used to grow cells.
[0112] The MethoCult "base" has been utilized for studies with T
cell migration analysis. USP Grade has the same viscosity
specification, as for example catalog # M0512 from Sigma-Aldrich in
St. Louis, Mo. The Sigma catalog lists 5 different grades of
viscosity of 2% solutions at 20.degree. C.: from 15 centipoises
through 25, 400, 1,500 to 4,000 centipoises. The methyl cellulose
should have a viscosity of 4,000 centipoises in aqueous solutions
at 20.degree. C.
[0113] Practical ranges of concentration that work for the purpose
of migration analysis of nonadherent cells are shown to lie within
approximate concentrations of between 0.1% and 1.2% when the methyl
cellulose solution is layered onto cells at the bottom of the well.
For adherent cell motility, methyl cellulose is not necessary, but
it would be necessary for chemotaxis determination. It is possible
that formulation of higher methyl cellulose concentrations up to
approximately 2.5% would become advantageous for certain layering
purposes. For example, when exchanging the medium above a thin
methyl cellulose solution layer, the resistance to dilution might
be better with higher methyl cellulose concentrations, i.e. it
could be more durable than lower concentrations.
[0114] When using a layer of methyl cellulose solution added to
cells, the volume of methyl cellulose solutions needed to prevent
ambient migration of non-adherent cells depends upon the size of
the containers. For 96 well plates, where typically 100 ul volumes
of medium are used, the volume of methyl cellulose required can be
as low as 1 ul. For general purposes, 4 to 5 ul have been used
routinely in 96 well plates. It can be generalized that between 1
to 5% of the total volume is effective for the methyl cellulose
solution. This can be applied to any size of sample wells from 6
well plates to 1536 well plates, and plates of even higher density
(9600+) well plates. In some circumstances, much larger volumes
might be advantageous, as when investigating the 3 dimensional
motility of non-adherent cells, or perhaps when using large vessels
where convection and mechanical mixing would disrupt the thin
layer. Adherent cells are cells that strongly attach to a surface
such as the well bottom. These cells are not significantly effected
by methocel or the influence of particles in a well. Cells that are
considered nonadherent, such as stem cells, have minimal attachment
or cyclical attachment to a surface. This minimal attachment is
easily overcome by other forces such as gravity or particle
movement in the fluid environment. If the viscosity of the fluid
environment is slightly increased, the cells move freely on the
bottom of the well, with no effect from motion of particle or dead
cells in the same environment. The motion of nonadherent cells can
be due to minimal attachment and reattachment of the cell to the
surface or, under certain conditions, the cells will swim. That is,
the cells will move without any attachment to the surface. This can
be accomplished if the viscosity of the medium is high enough. In
that case, the nonadherent cells will actually swim up, off the
surface of the well in the more viscous fluid.
[0115] The rationale for using small volumes of methyl cellulose
was based upon a number of expectations. First was the expectation
that the higher specific gravity of concentrated solution in
comparison to medium would result in rapid settling of the added
material to the bottom of the well and that the high viscosity of
the solution would help to maintain the concentration as it settled
through the medium. Next was the expectation that upon reaching the
bottom, the material would spread, still undiluted, into a thin
film whose viscosity would resist Brownian and other non-biological
sources of motion such as the gravitational downhill trend of cells
on tilted surfaces and thermal convection. Furthermore, this
spreading was observed not to significantly push or sweep cells
along in front of its spread. Finally, the viscosity was not
expected to interfere with active cell-biological motion. The
latter expectation was based upon previous observations of CFU-GM
colonies. (The CFU-GM colony assay is prepared in a homogeneous
solution of methyl cellulose (approximately 0.9%)). In these CFU-GM
time-lapse images, alongside colonies of non-motile cells, were
other cells migrating without apparent interference through the
methyl cellulose in 3 dimensions. Some cell phenotypes are seen to
move extremely rapidly. These observations dispel a presumed
requirement for surface attachment in the mechanism of motility
used by these hematopoietic cells.
[0116] It is more convenient to mix the methyl cellulose into a
homogeneous solution with the cells and culture medium prior to or
during the cell plating step. In this case, the final concentration
range is restricted to between approximately 0.1% to 0.2% final
methyl cellulose concentration. At lower concentrations, ambient
motion is not suppressed, and at higher concentrations, actively
motile cells can lift off from the surface and migrate in 3
dimensions out of focus into the overlying medium.
[0117] The motion of a cell off of the bottom of a well is observed
as a cell that moves out of focus in that plane of focus in a well.
For 3 dimensional analysis, a focal stack at a given position is
preferred. The stage moves to a position at a specified time and
takes a number of images at different focal planes, usually 5 to 10
images. The images are processed and cells that are in focus are
identified, by characteristics such as cell area. Cells that are
out of focus will appear larger than cells in focus. The motion in
3D is accomplished by tracking cells that are in focus through the
focal planes vs time.
[0118] Methyl cellulose and other viscosity enhancing compounds in
solution provide an environment for cell culture that is
fundamentally different from the environment produced by gels, such
as the agarose gel used in the "under-agarose assay" (Nelson, Quie,
and Simmins 1975) or collagen gels used in the 3D collagen gel
assay (Friedl, Noble, and Zanker 1995). Gels are "easier to
recognize than to define" (Jordon Lloyd, D. Colloid Chemistry;
Alexander, J., Ed.; The Chemical Catalog Co.: New York, 1926; Vol.
1, p767), but may generally be recognized by having 1) "a
continuous structure with macroscopic dimensions that is permanent
on the time scale of an analytical experiment and (2) is solid-like
in its rheological behavior" (Pierre Terech and Richard G. Weiss,
Low Molecular Mass Gelators of Organic Liquids and the Properties
of Their Gels. Chem. Rev., 97 (8), 3133-3160, 1997.) The molecules
in a gel are generally cross-linked in a complex, three-dimensional
network that immobilizes the liquid component, so that even though
the liquid component, e.g. water, may account for over 99% of the
weight of the gel, the "solidlike" behavior conveys a property such
that no discernable flow or change is observable over long periods
of time. Common gels used in biological studies are gelatin, a
protein, and agarose, a type of polysaccharide. These gels are
formed by dissolving the solid gelator molecules in warm water;
upon cooling, inter-molecular cross-linking occurs and a solidlike
state is attained at temperatures below the "gelation" temperature.
In this case, the process is reversible and such gels may be
redissolved by heating to the "melting" temperature. Gels may also
be formed by chemical polymerization of smaller subunits under
suitable conditions, such as the forming of polyacrylamide gels for
protein or DNA analysis or the forming of collagen gels upon pH
adjustment of a collagen solution. The polymerization process may
or may not be reversible. As in the case of hard-boiled eggs, a
third process for forming a gel is the irreversible denaturation of
soluble protein.
[0119] Clarification of the distinction between a gel and a viscous
solution is necessary for two reasons. First, because the present
invention does not rely upon the "solidlike" property of a gel to
control ambient motion. Instead, the control of viscosity is
distinguished by maintaining a liquid state in which cells are free
to move in any direction at any time without restriction by, or
dependence upon, solidlike linkages or networks. Therefore the
present invention reveals migration without requirements for
attachment or support and without the interference of a matrix that
could confine movement to channels or other small interruptions or
openings within a complex network. For this reason, the present
invention provides a highly suitable environment for observing and
analyzing directional characteristics of cell migration and the
effects of compounds upon such directional behavior. Secondly, the
distinction is important because gels may be used to confine
compounds for the purpose of analysis of the effects of such
compounds on cells in the nearby vicinity. For example, a compound
may be impregnated within a gel that has been cooled or polymerized
into the solidlike state. A small volume of such material can be
introduced into the cell culture for the purpose of analysis of the
effects of the impregnating compound on nearby cells. In the
present invention, the analysis of these cells may involve control
of ambient motion in the vicinity of the gel in order to prevent
disruption of a concentration gradient that becomes established as
the compound diffuses out from the impregnated gel into the
surrounding medium. Control of micro-convection using for example
methyl cellulose, permits the establishment of a stable gradient
while permitting free motion of the cells upon which the gradient
is producing stimulatory or inhibitory effects.
[0120] One test of methyl cellulose included a major goal for
development of an automated assay for motility: the assay must be
sensitive to changes in motility brought about by cytokines that
are known to stimulate T cells.
[0121] Lymphocytes were isolated from freshly drawn venous blood
containing acid citrate dextrose anticoagulant by density gradient
centrifugation over Lymphoprep (Gibco/BRL). Cultures were depleted
of adherent monocytes by incubation (37.degree. C., 5% CO2,
humidified) in tissue culture flasks in complete medium consisting
of 10% fetal bovine serum (FBS, Gibco/BRL), in RPMI containing 200
mM 1-glutamine, penicillin and streptomycin. The non-adherent cells
from monocyte-depleted cultures were stimulated for three days in
complete medium containing 5 ug/ml phytohemagluttinin (PHA, Sigma).
PHA stimulated cells were then washed and resuspended in complete
medium containing 5 ng/ml interleukin-2 (IL-2, R&D Systems).
After at least 2 days of pre-stimulation in IL-2, cells were washed
three times, resuspended in complete medium, and "rested" by
overnight incubation prior to analysis of response to cytokine
mixtures.
[0122] For plating into 384 well plates, rested cells were
suspended in mixtures of fresh medium containing cytokines at
indicated concentration(s) with cells at a density (4 to
5.times.10{circumflex over ( )}4/ml) such that approximately 1200
to 1500 cells were seeded in 30 ul into each well of the 384-well
plate. Preliminary experiments were performed using 100 ul of cell
suspension at similar cell densities in 96 well plates as described
in the text. For these experiments, methylcellulose (2.6% stock in
IMDM, catalog #4100, Stem Cell Technologies, Vancouver, BC) was
diluted in complete medium (1/2, unless otherwise indicated) and
was added to the well as a small volume (1-4 ul) after cells had
settled to the bottom surface. For routine assay, methyl cellulose
was mixed homogeneously with the cell suspension at a final
concentration of 0.2% ({fraction (1/12)} dilution of stock).
Propidium iodide (2 ug/ml) was added for identification of dead
cells. Uniform settling of cells was assisted by brief
centrifugation (2 min@500 g) of the plate in a micro-titer plate
carrier.
[0123] Visible and fluorescent time-lapse images were acquired at
1.5 minute intervals using a Nikon TE-300 epi-fluorescence inverted
microscope with an automated stage that returns precisely to a
pre-selected viewfield centrally located within each well. The
digital camera is a cooled CCD SenSys (Photometrics). Magnification
was through a 10.times. objective with an 0.6.times. high
resolution reduction lens (Diagnostic Instruments). The
z-coordinate settings (focus) were determined at the outset of each
experiment and automatically reset for each well position
throughout the experiment. Fluorescence was obtained using a
mercury or xenon source lamp with a 555 nm single band exciter
filter (Chroma #86555, Chroma Technology Corp.), a Sedat Quad
multiband beamsplitter (Chroma #86100) and a 4 color emission
filter (Chroma #84101). Fluorescence exposures were typically 250
msec. The system employs an customer designed incubated chamber
that seals on top of the movable heated stage and maintains
temperature (37.degree. C.), humidity (>95%) and CO.sub.2 (5%)
throughout the experimental period. Generally, experiments were
performed in triplicate groups using up to 22 wells within a group
and with each group being imaged continuously for one hour or more
before switching to the next group in a cycling manner over periods
of up to three days. Custom software allows the operator to step
through selection of imaging sites, to assign them to groups, and
to select variables for exposure settings, binning, gain, scan
intervals, and so forth. Images are stored as 8 bit with JPEG
compression and 2.times.2 binning.
[0124] A variety of mathematical algorithms are used to detect live
objects and suppress background artifacts within each gray scale
image through a process of image enhancement, topological analysis
and object contour extraction. Segment shapes which satisfy
morphological and topological criteria are used to form final
binary object images for both the visible and fluorescent image
sets. Fluorescent objects, representing dead cells, are subtracted
from the visible objects where the two coincide and the resulting
binary image set is used to develop the motion dynamics of live
objects. A track represents the path of migration of an object;
multiple tracks are built automatically based upon probability
fields for assigning links between objects from one image to the
next. Statistical parameters are developed from these links that
summarize the motion analyzed in terms of velocity, direction of
travel, tendency toward a straight line, frequency and magnitude of
changes in direction along a smoothed curve, and percentage of
objects moving within the entire view-field or region of interest.
These statistics may be further averaged over time and between
imaging sites to assay and compare the effects of added compounds
on migratory rates. Frequently presented parameters include
"curvilinear velocity" calculated as the average over twenty scans
of the distance per scan traveled by the tracked cell divided by
the time between scans. For purposes of detecting ambient motion,
"straight line velocity" is used; this represents the straight line
distance from the center of a tracked object between one image and
another image twenty scans later divided by the elapsed time over
twenty scans. Since this parameter is a vector, its average among
the many cells in each view-field tends toward zero (0) when the
directions are random because vectors in opposite directions cancel
each other. However, when ambient motion is present, the additive
effect of many cells moving in a similar direction is readily
apparent as an increase in this parameter.
[0125] The more important mathematical parameters calculated each
cell include:
[0126] Vinst.sub.k(i)--instantaneous speed of a track at a specific
scan number
[0127] Vavg_inst.sub.k(i)--instantaneous speed of a smoothed track
at a specific scan number
[0128] Vcl--curvilinear velocity, characteristics of the whole
track
[0129] Vsl--straightline velocity, characteristics of the whole
track
[0130] Vavg--curvilinear velocity of smoothed track,
characteristics of the whole track
[0131] Linearity--the measure of how straight cell is moving,
characteristics of the whole track.
[0132] Straightness--the same meaning as Linearity but the smoothed
track is used instead of real. Allows to exclude the fast direction
fluctuations from measurements
[0133] ALHmean--the mean beating amplitude, the measure of
oscillating component of cell movement. Characteristics of the
whole track.
[0134] ALHmax--the maximum beating amplitude, the measure of
oscillating component of cell movement. Characteristics of the
whole track
[0135] BCF--beat cross frequency, the measure of oscillating
component of cell movement. Characteristics of the whole track
[0136] Circular Radius--measure of circular component of the cell
motion If he track passes some criteria, it is approximated by the
circle (using least square fit) and the radius of this circle
becomes the Circ. Radius. Characteristics of the whole track
[0137] Referring now to the drawings wherein like reference
numerals refer to similar or identical parts throughout the several
views, and more specifically to FIG. 1 thereof, there is shown the
results of analysis of T cell motility in response to stimulation
by the cytokine, interleukin-2 (IL-2). A cytokine is a type of
protein.
[0138] For this experiment, prestimulated cells (3 days PHA, 2 days
IL-2; see above) were washed and resuspended in complete medium and
were plated into a 96-well plate using approximately 5,000 cells
per well. After the cells had settled to the surface, 4 ul of
methyl cellulose solution (Stem Cell Technologies #H4230) was added
and the plate was installed on the automated stage of the inverted
microscope and imaging was begun (time=0). Imaging continued during
the initial 15 hour nonstimulatory "rest" period in medium without
cytokine, at which time IL-2 was added (final concentration 100
ng/ml) and imaging was resumed. Curvilinear velocity (Vcl) is shown
for duplicate wells to which IL-2 was added (upper, diamonds and
squares) in comparison to a control well to which no IL-2 was added
(lower, triangles). The added IL-2 induced an immediate increase in
T cell velocity that was sustained throughout the subsequent 24
hour period shown here.
[0139] In previous experiments, motility response was often
obscured by large unpredictable fluctuations. Abbreviated analysis
of control wells without methyl cellulose from this experiment
indicated that such fluctuation was occurring in this experiment
also (FIG. 2). FIGS. 2A and 2B compare average curvilinear velocity
(Vcl) with straight line velocity (Vsl) for cells in wells with and
without methyl cellulose. Vcl is a scalar quantity representing the
average magnitude of velocities of all cells in the view, whereas
Vsl averages both the magnitude and the direction of cells moving
in the view. Vsl is a useful indicator of ambient motion because it
becomes large when cells move in a similar direction and small when
cells move randomly. Ambient motion is characterized by
fluctuations over time of cells moving in a similar direction
resulting in variable large Vsl values as shown here for samples
without methyl cellulose (FIG. 2b, top four curves). On the other
hand, samples with methyl cellulose exhibit random motion as
characterized by consistently low Vsl values throughout the
analysis period. Only 200 scans are shown (.sup..about.5 hours) and
only 1/4 of the original 10.times. image was analyzed in order to
reduce processing time.
[0140] Ambient motion is observed if no methylcel is present.
Particles and dead cells move together as though they were motile.
A live cell in a well will also move along with the particles. This
motion, ambient motion, is caused by fluid motion in the well. The
fluid motion is produced by changes in temperature setting up
convection, or micro turbulences that sweep smaller particles
around, pulling the cells along. If the plate is moved,
mechanically, the particles, since they are not the same density as
the fluid will move at different rates than the fluid and the
cells. This motion will also be seen, it's ambient motion. If the
plate is tilted, the more dense particles will move down hill,
again causing the cells to move along with the particles. With the
correct concentration of methocel present, the turbulence is
minimized, and motion due to mechanical motion of the well/cell
does not cause particles to move around. Since it is possible to
observe wells with and without methocel in the same experiment, the
effect that ambient motility has on cell `biological` motility can
also be detected. If the plate is tilted, the particles and dead
cells will stay put, giving the user a method to measure the effect
that gravity has on the true cell motion, not influenced by ambient
motion. The amount of tilt is also a measure of attachment of the
cell in the environment. Other changes in the environment, such as
a surface treatment in the well or an addition of a protein in the
fluid can be studied to see the effect of cell attachment in a
tilted well, independent of the effects of ambient motility.
[0141] These data demonstrate that methyl cellulose provides the
desired effects of reducing ambient motion without preventing
biological motion, including responsiveness to IL-2.
[0142] Having demonstrated the potential usefulness of methyl
cellulose, the effective concentration range was examined. In order
to determine the minimum methyl cellulose concentration at which
ambient motion suppression is effective, a dilution series of
methyl cellulose in medium was prepared and added to a series of
wells in a 96 well plate. As in the first experiment, a volume of 4
ul of methyl cellulose was used. Larger volumes were also tested in
order to provide assurance that dilution of smaller volumes was not
resulting in inaccuracy in the assumed concentration at the surface
level and to compare the durability of thinner versus thicker
methyl cellulose layers. Dilution is expected due to mixing at the
time of layering upon the bottom and due to diffusion over the
course of the experiment. The results for this experiment are shown
in FIG. 3. In each case, Vsl is plotted over the course of the 15
hour experiment (x axis expressed in scans, where each scan is
approximately 3 minutes). Methyl cellulose dilutions of {fraction
(1/128)} (0.01% methyl cellulose) and {fraction (1/64)} (0.02%
methyl cellulose) were not effective, but some suppression of
ambient motion is observed at {fraction (1/32)} dilution (0.04%
methyl cellulose) for all volumes (FIG. 3A-D). At {fraction (1/16)}
dilution (0.08% methyl cellulose), a 4 ul volume is somewhat
effective for about 8 hours; a 4 ul volume at 1/8 dilution (0.15%
methyl cellulose) is effective for at least 15 hours (FIG. 3A).
Presumably the increased ambient motion at {fraction (1/16)}
dilution after 8 hours results from dilution by diffusion and from
convective "currents" that may be set up due to heating
differentials within a well; these same currents at the culture
surface are likely to cause ambient motion of cells, but methyl
cellulose prevents this.
[0143] With a larger volume of 10 ul of methyl cellulose, the
{fraction (1/16)} dilution level (0.08% methyl cellulose) is seen
to be effective in preventing ambient motion throughout the 15 hour
experimental time span, and a {fraction (1/32)} dilution (0.04%
methyl cellulose) appears to show brief inhibition (FIG. 3B). The
effectiveness of the {fraction (1/16)} dilution is further
supported using 80 ul of the methyl cellulose dilution series added
to T cells in 20 ul of complete medium. In this case, even
homogenization with the overlying medium would not significantly
alter the concentrations (FIG. 3C).
[0144] A separate experiment was designed to determine the minimum
volume of methyl cellulose needed to suppress ambient motion.
Volumes of 1, 2, 3, and 4 ul of stock solution (1.2% methyl
cellulose) were added to wells containing 100 ul of complete medium
in a 96 well plate. (For 384-well, 1536-well, and other plate
sizes, corresponding volumes consist of approximately 1 to 4% of
the total volumes of medium.) As in other experiments, suspended T
cells were allowed to settle prior to methyl cellulose
addition.
[0145] Results in terms of Vcl and Vsl (FIGS. 4A and 4B,
respectively) show that volumes of 1 ul to 4 ul of ethyl cellulose
stock (1.2% methyl cellulose) are equally effective for suppression
of ambient motion. Methyl cellulose-containing samples at 1 ul, 2
ul, 3 ul, and 4 ul (Duplicates B and C05, 06, 07 and 08,
respectively) were compared with duplicate samples without methyl
cellulose (B04 and C04). Extreme ambient motion differs widely
between duplicate samples without methyl cellulose, while all
volumes of methyl cellulose are effective at allowing random motion
(Vcl .sup..about.2 um/min, A-Top Panel) and suppressing ambient
motion (Vsl.sup..about.0, B-Bottom Panel). As expected, duplicate
wells without methyl cellulose show drastically elevated values for
Vcl and Vsl in comparison to samples with methyl cellulose.
Examination of Vsl indicates that a major portion of this velocity
of tracked cells in this case is unidirectional. That is, since
values for Vcl and Vsl are similar, most of the observed Vcl must
be unidirectional for the samples without methyl cellulose.
[0146] On the other hand, for all 4 volumes of methyl cellulose in
this experiment, comparison of plots for Vcl with those for Vsl
indicates that most of the motion is random directional because Vsl
values remain near zero throughout the period of analysis.
Therefore for Vcl, average levels of 2 to 3 um/min represent
biological motility. This conclusion is clearly evident in FIG. 5,
where green lines indicate the path of tracked cells over a
representative period of time during this experiment comparing
cells in a typical well without methyl cellulose (left panel) with
a well containing cells with a 1 ul volume of added methyl
cellulose (right panel). Without methyl cellulose, cells are moving
uniformly down to the right, whereas with 1 ul of methyl cellulose,
cells are moving in random paths characteristic of biological
motion. Here the tracks have been superimposed upon one viewfield
image. Sequences of images of T cells were acquired every 2 minutes
and were analyzed automatically using custom software. On the
average throughout the period analyzed, there were about 170 cells
per field, 40% of which were motile.
[0147] For one of the non-methyl cellulose-containing wells, the
Vsl approaches zero halfway through the analysis period (FIG. 4B).
For this sample, the average Vcl is practically identical in
comparison to all of the methyl cellulose-containing wells (FIG.
4A), and thus in this example, T cell motility in complete medium
is no different than T cell motility in the presence of full
strength methyl cellulose stock solution (1.2% methyl cellulose) at
volumes up to 4 ul. This example demonstrates no significant effect
of methyl cellulose on T cell motility in comparison to motion in
medium alone when ambient motion is not present.
[0148] The calculated height of the methyl cellulose layer using
volumes from 1 to 4 ul in a 96 well plate is shown in Table 1.
1 TABLE 1 Volume of methyl cellulose (ul) Height of methyl added to
100 ul cellulose medium (um) 0 -- 1 34 2 69 3 103 4 137
[0149] These data indicate that as little as 1 ul of full strength
methyl cellulose is effective for suppression of ambient motion in
96 well plates and that greater precision may be expected for
measurement of motility parameters due to reduced ambient motion
using methyl cellulose.
[0150] In an experiment designed to test for possible effects of
methyl cellulose on T cell motility, 4 ul and 40 ul volumes of full
strength (1.2%) methyl cellulose and 40 ul volumes of 1/3 diluted
(0.4%) methyl cellulose were added to T cells in 96 well plates.
Plots of curvilinear velocity (Vcl) are shown in FIG. 6. Although
the 4 ul volume layer of full strength (1.2%) methyl cellulose
shows similar T cell curvilinear velocity to the plastic without
methyl cellulose (uppermost curves), 40 ul volumes of full strength
(1.2% methyl cellulose) and diluted (1/3; 0.4% methyl cellulose)
methyl cellulose show an apparent reduction in velocity in
comparison to the 4 ul volume layer and plastic without methyl
cellulose. Possible interpretations for this effect include the
differing composition of methyl cellulose mixtures in comparison to
complete medium, and the possibility that with larger volumes, T
cells are deprived of factors and/or nutrients that are necessary
for "normal" behavior. Also, when watching time-lapse sequences of
images with 40 ul of full strength methyl cellulose, it is apparent
that when cells move, they are capable of moving upwards
vertically. They then go out of focus and out of view. Thus the
more rapidly moving cells are being selected against in terms of
cells that remain in the viewfield.
[0151] FIG. 7 shows examples from this experiment of view fields
analyzed, with the cell tracks represented by green lines. FIGS. 7
shows tracks of cells from the period over which the data were
analyzed in FIG. 6. When methyl cellulose was not present (top two
panels), cells were readily swept from the viewfield at different
times; it was then possible for selected cells to replace them
yielding fewer cells of higher motility than in viewfields with
methyl cellulose (lower panels) where cells remained in view from
the beginning and greater numbers of non-motile cells remained. In
the case of 40 ul of non-diluted methyl cellulose (lower right
panel), cells were observed to move in 3 dimensions, and so moving
cells frequently left the viewfield as they went out-of-focus.
These and other biases make it difficult to determine the extent to
which methyl cellulose might affect T cell motility. Two wells
without methyl cellulose (FIGS. 7A & B--plastic) present the
extent of variation observed without methyl cellulose, whereas with
methyl cellulose (FIGS. 7C & D), similar numbers of cells are
distributed across the viewfield and these cells have remained from
initial images. Tracks show similar lengths in FIG. 7C (40 ul
diluted methyl cellulose) in comparison to FIG. 7B (plastic),
however there are more stationary cells remaining in FIG. 7C; such
cells having been swept out of the viewfield due to ambient motion
in FIG. 7B. In FIG. 7D, many of the motile cells have escaped into
the vertical direction and are therefore out-of-focus and not
tracked. Further experiments with a dilution series of methyl
cellulose may bring answers to the question of effects of methyl
cellulose on T cell motility. In any case, it is evident that cell
motion is not adversely affected by smaller volumes of methyl
cellulose and that suppression of the otherwise dominant effects of
ambient motion is required for measuring motility of non adherent
cells.
[0152] The method and procedure of adding a small volume of methyl
cellulose as a layer on top of settled cells leaves a relatively
wide margin of approximately 0.1% to 1.2% methyl cellulose
concentrations for effective inhibition of ambient motion.
Alternatively, it is convenient and satisfactory to mix methyl
cellulose homogeneously in solution with the cell culture medium in
which the cells are suspended and allowed to settle. In this case
the effective concentration range is restricted to approximately
0.1% to 0.2% for 2D analysis of motility. At concentrations lower
than 0.1%, ambient motion is not sufficiently suppressed, and at
concentrations higher than 0.2%, cells begin to migrate upwards
from the surface into 3 dimensions. Analysis of motility in 3
dimensions is more complex than analysis in 2 dimensions. In 2
dimensions cells are imaged within a single plane of focus, whereas
in 3 dimensions multiple focal planes must be imaged. Therefore 3D
analysis must integrate the tracking of motion of objects between
adjacent focal planes. Methyl cellulose provides considerable
advantage in enabling 2D analysis of non-attachment mediated
motility.
[0153] The addition of methyl cellulose to the culture medium is
also an effective method for enabling the analysis of chemotaxis,
or directional migration of cells toward an attractive compound.
Chemcotaxis analysis is demonstrated by the establishment of
chemical gradients as shown in FIGS. 8a-8d using fluorescent dye
labeled dextran as markers. The left hand panels show fluorescent
images and the right hand panels show the corresponding view in
visible light. The dye markers, of 70 kilo-Dalton (kD) molecular
weight (FIG. 8A) and 10 kD molecular weight (FIG. 8B), span a
molecular weight range similar to that of bioactive chemotactic
compounds, and provide a visual reference for gauging the slope and
strength of the gradient of both the dyes and experimental
nonvisible chemotactic compounds. In FIG. 8A, the dye may be seen
to diffuse from within the impregnated semi-solid agarose gel held
in place within a small cylinder of Teflon tubing (PTFE
0.047"OD.times.0.015"ID). The agarose (Agarose GenAR, low gel
temperature, cat# 7720, Mallinckrodt) contained within the tubing
is approximately 1% concentration and the dyes consist of Oregon
Green 488 conjugated dextran (70 kd cat# D-7173; 10 kd cat# D-7171,
Molecular Probles) at approximately 1 mg/ml concentration in the
gel dissolved in Dulbecco's phosphate buffered saline (DPBS cat#
14190-144, GibcoBRL).
[0154] In FIG. 8A it is evident that the agarose "plug" within the
hollow cylinder of tubing is confined to the left-hand side of the
lumen and that a small air bubble effectively blocks the right side
(compare fluorescent image (8A--left) with visible image
(8A--right)). Based on this observation, the tubing in FIG. 8B was
created so that the agarose plug was positioned at the far right
end (out of view) and was intentionally "capped" on this side with
a small bubble of air (out of view). The left-hand side of the tube
was filled with culture medium containing methyl cellulose similar
in strength (approximately 0.8% methyl cellulose) to that in which
the cells were suspended within the well. This configuration
provides a method to slow the rate of diffusion from the tube
opening in comparison to cutting the tube flush with the gel.
Moreover, since the cells are capable of migrating in 3 dimensions
in 0.8% methyl cellulose, the open section of tubing filled with
methyl cellulose-containing culture medium supports migrating cells
and creates a trap for chemotactic cells migrating upwards into the
chemotactic gradient using 3D motility analysis methods.
[0155] Analysis for both attractive and repulsive interactions on
cells from chemotactic gradients would be performed using either 2D
or 3D motility analysis by comparison of the magnitude and
direction of the cell migration vector field with that of the
chemical gradient.
[0156] U.S. Pat. No. 6,008,010, incorporated by reference herein,
describes a system that can also be used to perform the embodiments
described herein.
[0157] Although the invention has been described in detail in the
foregoing embodiments for the purpose of illustration, it is to be
understood that such detail is solely for that purpose and that
variations can be made therein by those skilled in the art without
departing from the spirit and scope of the invention except as it
may be described by the following claims.
* * * * *