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Patent 2503186 Summary

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(12) Patent Application: (11) CA 2503186
(54) English Title: BIOLOGICAL ASSAYS USING GRADIENTS FORMED IN MICROFLUIDIC SYSTEMS
(54) French Title: TESTS BIOLOGIQUES UTILISANT DES GRADIENTS FORMES DANS DES SYSTEMES MICROFLUIDIQUES
Status: Deemed Abandoned and Beyond the Period of Reinstatement - Pending Response to Notice of Disregarded Communication
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12M 1/00 (2006.01)
  • C12M 1/34 (2006.01)
  • C12Q 1/02 (2006.01)
(72) Inventors :
  • KIRK, GREGORY L. (United States of America)
  • KIM, ENOCH (United States of America)
  • OSTUNI, EMANUELE (United States of America)
  • SCHUELLER, OLIVIER (United States of America)
  • SWEETNAM, PAUL (United States of America)
(73) Owners :
  • SURFACE LOGIX, INC.
(71) Applicants :
  • SURFACE LOGIX, INC. (United States of America)
(74) Agent: BORDEN LADNER GERVAIS LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2003-10-21
(87) Open to Public Inspection: 2004-05-06
Examination requested: 2008-10-14
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2003/033146
(87) International Publication Number: WO 2004038367
(85) National Entry: 2005-04-20

(30) Application Priority Data:
Application No. Country/Territory Date
60/419,976 (United States of America) 2002-10-22
60/419,980 (United States of America) 2002-10-22

Abstracts

English Abstract


The present invention discloses a device for monitoring chemotaxis or
chemoinvasion. The present invention further provides a flexible assay system
and numerous assays that can be used to test biological interactions and
systems. Laminar flow gradients are employed that mimic gradient situations
present in vivo.


French Abstract

L'invention concerne un dispositif de suivi de chimiotaxie ou de chimio-invasion. Elle concerne aussi un système de test flexible et de nombreux tests pouvant être utilisés afin de tester des systèmes et des interactions biologiques. On utilise à cette fin des gradients d'écoulement laminaire qui miment les situations de gradient présentes in vivo.

Claims

Note: Claims are shown in the official language in which they were submitted.


We claim:
A method of monitoring chemotaxis or chemoinvasion comprising:
providing a device including a housing defining a plurality of chambers
therein,
each of the plurality of chambers including:
a first well region including at least one first well;
a second well region including at least one second well; and
a channel region including at least one channel connecting the first well
region and
the second well region with one another;
introducing at least one soluble test substance in the at least one first well
or the at
least one channel;
forming a dynamic solution concentration gradient along a longitudinal axis of
the
chamber; and
introducing cells in the at least one second well or the at least one channel;
and
monitoring chemotaxis or chemoinvasion of the cells.
2. The method of claim 1, wherein the at least one channel contains a gel
matrix.
3. The method of claim 1, wherein forming a dynamic solution concentration
gradient
comprises:
introducing a first fluid stream having a first concentration of a first
substance in at
least one of the plurality of chambers; and
introducing a second fluid stream having a second concentration of a second
substance in at least one of the plurality of chambers, wherein the first and
second
concentrations are different from one another.
4. The method of claim 3, wherein the first and second substances are the
same.
5. The method of claim 3, wherein the first and second substances are
different.
6. The method of claim 3, wherein the first fluid stream and the second fluid
stream
converge into a single third fluid stream that is in fluid communication with
at least one of
the plurality of chambers, wherein the third fluid stream comprises a
concentration
46

gradient of the first and second substances, the concentration gradient being
substantially
perpendicular to the direction of flow of the third fluid stream.
7. The method of claim 3, wherein the first and second streams converge into a
single
third stream, the single third stream than diverges into separate fourth,
fifth, and sixth
streams, and the fourth, fifth, and sixth streams then re-converge into a
single seventh
stream, the single seventh stream in fluid communication with at least one of
the plurality
of chambers under conditions of substantially laminar flow.
8. The method of claim 1, wherein introducing cells in the at least one second
well or
the at least one channel comprises patterning cells on the at least one
channel along the
longitudinal axis of the chamber, and introducing a soluble test substance in
the at least
one first well or the at least one channel comprises introducing a soluble
test substance in
the at least one channel by laminar flow.
9. The method of claim 1, wherein introducing cells in the at least one second
well or
the at least one channel comprises placing a single cell type in the at least
one channel.
10. The method of claim 1, wherein introducing cells in the at least one
second well or
the at least one channel comprises placing a mixture of cell types in the at
least one
channel.
11. The method of claim 1, wherein the at least one channel is a plurality of
channels
and introducing cells in the at least one second well or the at least one
channel comprises
introducing a different cell type in each of the plurality of channels.
12. The method of claim 11, wherein introducing a different cell type in each
of the
plurality of channels comprises introducing the different cells type at
different
concentrations in each of the plurality of channels.
13. The method of claim 11, wherein introducing a different cell type in each
of the
plurality of channels comprises introducing the different cells type in the
same
concentrations in each of the plurality of channels.
47

14. The method of claim 1, wherein the at least one channel is a plurality of
channels
and introducing cells in the at least one second well or the at least one
channel comprises
introducing a single cell type in each of the plurality of channels.
15. The method of claim 14, wherein introducing a single cell type in each of
the
plurality of channels comprises introducing the single cell type at different
concentrations
in each of the plurality of channels.
16. The method of claim 14, wherein introducing a single cell type in each of
the
plurality of channels comprises introducing the single cell type at the same
concentrations
in each of the plurality of channels.
17. The method of claim 1, wherein the at least one channel is a plurality of
channels
and introducing cells in the at least one second well or the at least one
channel comprises
introducing a mixture of cells in each of the plurality of channels.
18. The method of claim 17, wherein introducing the mixture of cells in each
of the
plurality of channels comprises introducing the same mixture or different
mixture of cells
in each of the plurality of channels.
19. The method of claim 1, wherein the at least one channel is a plurality of
channels
and the at least one soluble test substance is a plurality of test substances
and introducing
at least one soluble test substance in the at least one first well or the at
least one channel
comprises introducing a different one of the plurality of test substances in
each of the
plurality of channels.
20. The method of claim 19, wherein introducing a different one of the
plurality of test
substances in each of the plurality of channels comprises introducing a
different one of the
plurality of test substances in each of the plurality of channels at different
concentrations.
48

21. The method of claim 19, wherein introducing a different one of the
plurality of test
substances in each of the plurality of channels comprises introducing a
different one of the
plurality of test substances in each of the plurality of channels at the same
concentrations.
22. The method of claim 1, wherein the at least one channel is a plurality of
channels
and the at least one soluble test substance is a plurality of the same soluble
test substances
and introducing at least one soluble test substance in the at least one first
well or the at
least one channel comprises introducing one of the plurality of the same test
substances in
each of the plurality of channels.
23. The method of claim 22, wherein introducing one of the plurality of the
same
soluble test substances in each of the plurality of channels comprises
introducing one of
the plurality of the same soluble test substances in each of the plurality of
channels at
different concentrations.
24. The method of claim 22, wherein introducing one of the plurality of the
same
soluble test substances in each of the plurality of channels comprises
introducing one of
the plurality of the same soluble test substances in each of the plurality of
channels at the
same concentrations.
25. The method of claim 1, wherein the housing comprises a support member and
a
top member mounted to the support member by being placed in substantially
fluid-tight,
conformal contact with the support member.
49

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
BIOLOGICAL ASSAYS USING GRADIENTS FORMED IN MICROFLUIDIC
SYSTEMS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Applications
No.
60/419,980 and 60/419,976 filed on October 22, 2002, the contents of which are
incorporated by reference herein.
FIELD OF THE INVENTION
to The present invention relates generally to device for monitoring chemotaxis
and/or
chemoinvasion. The present invention also relates generally to biological
assays
performed in gradients formed in microfluidic systems.
BACKGROUND
15 Test devices, such as those used in cell migration are well known. Such
devices
are disclosed for example in U.S. Patent Numbers 6,329,164, 6,238,874, and
5,302,515.
Three processes involved in cell migration are chemotaxis, haptotaxis, and
chemoinvasion. Chemotaxis is defined as the movement of cells induced by a
concentration gradient of a soluble chemotactic stimulus. Haptotaxis is
defined as the
2o movement of cells in response to a concentration gradient of a substrate-
bound stimulus.
Chemoinvasion is defined as the movement of cells into and/or through a
barrier or gel
matrix. The study of cell migration and the effects of external stimuli on
such behavior
are prevalent throughout contemporary biological research. Generally, this
research
involves exposing a cell to external stimuli and studying the cell's reaction.
By placing a
25 living cell into various envirornnents and exposing it to different
external stimuli, both the
internal workings of the cell and the effects of the external stimuli on the
cell can be
measured, recorded, and better understood.
A cell's migration in response to a chemical stimulus is a particularly
important
consideration for understanding various disease processes and accordingly
developing and
30 evaluating therapeutic candidates for these diseases. By documenting the
cell migration of
a cell or a group of cells in response to a chemical stimulus, such as a
therapeutic agent,
the effectiveness of the chemical stimulus can be better understood.
Typically, studies of
disease processes in various medical fields, such as oncology, immunology,
angiogenesis,

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-wound healing, and neurobiology involve analyzing the chemotactic and
invasive
properties of living cells. For example, in the field of oncology, cell
migration is an
important consideration in understanding the process of metastasis. During
metastasis,
cancer cells of a typical solid tumor must loosen their adhesion to
neighboring cells,
escape from the tissue of origin, invade other tissues by degrading the
tissues'
extracellular matrix until reaching a blood or lymphatic vessel, cross the
basal lamina and
endothelial lining of the vessel to enter circulation, exit from circulation
elsewhere in the
body, and survive and proliferate in the new environment in which they
ultimately reside.
Therefore, studying the cancer cells' migration may aid in understanding the
process of
metastasis and developing therapeutic agents that potentially inhibit this
process. In the
inflammatory disease field, cell migration is also an important consideration
in
understanding the inflammatory response. During the inflammation response,
leukocytes
migrate to the damaged tissue area and assist in fighting the infection or
healing the
wound. The leukocytes migrate through the capillary adhering to the
endothelial cells
lining the capillary. The leukocytes then squeeze between the endothelial
cells and use
digestive enzymes to crawl across the basal lamina. Therefore, studying the
leukocytes
migrating across the endothelial cells and invading the basal lamina may aid
in
understanding the inflammation process and developing therapeutic agents that
inhibit this
process in inflammatory diseases such as adult respiratory distress sydrome
CARDS),
2o rheumotoid arthritis, and inflammatory skin diseases. Cell migration is
also an important
consideration in the field of angiogenesis. When a capillary sprouts from an
existing
small vessel, an endothelial cell initially extends from the wall of the
existing small vessel
generating a new capillary branch and pseudopodia guide the growth of the
capillary
sprout into the surrounding connective tissue. New growth of these capillaries
enables
cancerous growths to enlarge and spread and contributes, for example, to the
blindness
that can accompany diabetes. Conversely, lack of capillary production can
contribute to
tissue death in cardiac muscle after, for example, a heart attack. Therefore
studying the
migration of endothelial cells as new capillaries form from existing
capillaries may aid in
understanding angiogenesis and optimizing drugs that block vessel growth or
improve
vessel function. In addition, studying cell migration can also provide insight
into the
processes of tissue regeneration, organ transplantation, autoimmune diseases,
and many
other degenerative diseases and conditions.
2

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Cell migration assays are often used in conducting these types of research.
Commercially available devices for creating such assays are sometimes based on
or employ a transwell system (a vessel partitioned by a thin porous membrane
to form an
upper compartment and a lower compartment). To study cell chemotaxis, cells
are placed
in the upper compartment and a migratory stimulus is placed in the lower
compartment.
After a sufficient incubation period, the cells are fixed, stained, and
counted to study the
effects of the stimulus on cell chemotaxis across the membrane.
To study chemoinvasion, a uniform layer of a MATRIGELTM matrix is placed over
the membrane to occlude the pores of the membrane. Cells are seeded onto the
to MATRIGELTM matrix in the upper compartment and a chemoattractant is placed
in the
lower compartment. Invasive cells attach to and invade the matrix passing
through the
porous membrane. Non-invasive cells do not migrate through the occluded pores.
After a
sufficient incubation period, the cells may be fixed, stained, and counted to
study the
effects of the stimulus on cell invasion across the membrane.
The use of transwells has several shortcomings. Assays employing transwells
require a labor-intensive protocol that is not readily adaptable to high-
throughput
screening and processing. Because of the configuration of a transwell system,
it is
difficult to integrate with existing robotic liquid handling systems and
automatic image
acquisition systems. Therefore, much of the screening and processing, such as
counting
2o cells, is done manually which is a time-consuming, tedious, and expensive
process. Cell
counting is also subjective and often involves statistical approximations.
Specifically, due
to the time and expense associated with examining an entire filter, only
randomly selected
representative areas may be counted. Moreover, even when these areas are
counted, a
technician must exercise his or her judgment when accounting for a cell that
has only
partially migrated through the filter.
Transwell-based assays have intrinsic limitations imposed by the thin
membranes
utilized in transwell systems. The membrane is only 50-30 microns (~.m) thick,
and a
chemical concentration gradient that forms across the membrane is transient
and lasts for a
short period. If a cell chemotaxis assay requires the chemotactic gradient to
be generated
over a long distance (>100-200~,m) and to be stable over at least two hours,
currently
available transwell assays cannot be satisfactorily performed.
Notwithstanding the above, perhaps the most significant disadvantage of
transwells is the lack of real-time observation of chemotaxis and
chemoinvasion. In

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particular, the changes in cell morphology during chemotaxis cannot be
observed in real-
time with the use of transwells. In transwells, when the cells are fixed to a
slide, as
required for observation, they are killed. Consequently, once a cell is
observed it can no
longer be reintroduced into the assay or studied at subsequent periods of
exposure to a test
agent. Therefore, in order to study the progress of a cell and the changes in
a cell's
morphology in response to a test agent, it is necessary to run concurrent
samples that may
be slated for observation at various time periods before and after the
introduction of the
test agent. In light of the multiple samples required for each test, in
addition to the
positive and negative controls required to obtain reliable data, a single
chemotaxis assay
to can require dozens of filters, each of which needs to be individually
examined and
counted-an onerous and time-consuming task.
More recently, devices for measuring chemotaxis and chemoinvasion have become
available which employ a configuration in which two wells are horizontally
offset with
respect to one another. This configuration of a device was introduced by Sally
Zigmond
in 1977 and, hereafter referred to as the "Zigmond device," consists of a 25
millimeters
(mm) x 75mm glass slide with two grooves 4 mm wide and lmm deep, separated by
a
lmm bridge. One of the grooves is filled with an attractant and the other
groove is filled
with a control solution, thus forming a concentration gradient across the
bridge. Cells are
then added to the other groove. Two holes are provided at each end of the
slide to accept
2o pin clamps. The clamps hold a cover glass in place during incubation and
observation of
the cells. Because of the size and configuration of the Zigmond chamber, it
does not allow
integration with existing robotic liquid handling systems and automatic image
acquisition
systems. Further, as with transwell-based systems, the changes in cell
morphology during
chemotaxis cannot be observed in real-time with the use of the Zigmond chamber
as the
cells are fixed to a slide for observation. In addition, the pin clamps must
be assembled
with an allen wrench and thus the device requires extra handling, positioning,
and
alignment before performing the assay. Such handling and positioning of the
cover glass
on the glass slide, as well as the rigidity of the cover glass, can
potentially damage or
interfere any surface treatment on the bridge.
3o A chemotaxis device attempting to solve the problem of lack of real-time
observation is the "Dunn chamber." The Dunn chamber consists of a specially
constructed
microslide with a central circular sink and a concentric annular moat. In an
assay using a
Dunn chamber, cells migrate on a coverslip, which is placed inverted on the
Dunn
4

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chamber, towards a chemotactic stimulus. The cells are monitored over-night
using a
phase-contrast microscope fitted with a video camera connected to a computer
with an
image-grabber board.
In addition to the problems of rigidity of the coverslip and the lack of
integration
into existing robotic liquid handling systems, a major problem with the Dunn
chamber
assay is that only a very small number of cells are monitored (typically ten).
The average
behavior of this very small sample may not be typical of the population as a
whole. A
second major problem is that replication is very restricted. Each control
chamber and each
treatment chamber must be viewed in separate microscopes, each one similarly
equipped
to with camera and computer.
Another chemotaxis device known in the art is disclosed in United States
Patent
Number 6,238,874 to Jarnigan et. al. (the '874 patent). The '874 patent
discloses various
embodiments of test devices that may be used to monitor chemotaxis. However,
disadvantageously, the devices in Jarnagin et al. can not be easily sealed or
assembled or
15 peeled and disassembled. Thus, it is difficult to maintain surfaces that
are prepared
chemically or biologically during assembly. The test devices of the '874
patent are
therefore more suited for one-time use. Also, disassembly and collection of
cells is
difficult to do without damage to the cells or without disturbing the cell
positions.
The prior art has failed to provide a test device, such as a device for
monitoring
2o chemotaxis and/or chemoinvasion, which device is easily assembled and
dissembled. In
addition, the prior art has failed to provide a test device for monitoring
cell migration,
which is not limited to measuring the effects of chemoattractants,
chemorepellants and
chemostimulants on chemotaxis/chemoinvasion.
25 SUMMARY OF THE INVENTION
The present invention provides a method of monitoring chemotaxis or
chemoinvasion comprising providing a device including a housing defining a
plurality of
chambers therein, each of the plurality of chambers including a first well
region including
at least one first well, a second well region including at least one second
well, and a
3o channel region including at least one channel connecting the first well
region and the
second well region with one another. The method further includes introducing
at least one
soluble test substance in the at least one first well or the at least one
channel and forming a
dynamic solution concentration gradient along a longitudinal axis of the
chamber. The

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method includes introducing a first sample comprising cells in the at least
one second well
or the at least one channel and then
monitoring chemotaxis or chemoinvasion of the cells.
The present invention provides for the optional inclusion of a gel matrix in
the
chamlel(s) of the above-mentioned embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
The various features of the invention will best be appreciated by simultaneous
reference to the description that follows and the accompanying drawings, in
which:
io Figure lA is a top, perspective view, in partial cross section, of a
portion of an
embodiment of test device according to the present invention;
Figure 1B is a top, perspective view of an embodiment of a test device of the
present invention;
Figure 1C is a side-elevational view of a longitudinal cross section of one of
the
15 chambers of the test device of Figure 1B;
Figure 2A is a schematic outline depicting a top plan view of an alternative
embodiment of a chamber defined in a test device of the present invention,
where the
channel region defines a single channel;
Figure 2B is a schematic outline depicting a top plan view of the embodiment
of
2o the chambers defined in the embodiment of the test device according to
Figure 1B, where
the channel region defines a single channel;
Figure 2C is a figure similar to Figure 2A, showing an alternative embodiment
of a
chamber defined in a test device of the present invention, where the channel
region defines
a single channel;
25 Figure 3A is a figure similar to Figures 2A, showing an alternative
embodiment of
a chamber defined in a test device of the present invention, where the channel
region
defines a plurality of channels having identical lengths;
Figure 3B is a figure similar to Figure 3A, showing a channel region defining
a
plurality of channels having lengths that increase from one side of the
chamber to another
30 side of the chamber;
Figure 3C is a figure similar to Figure 3A, showing a channel region defining
a
plurality of channels having widths that increase from one side of the chamber
to another
side of the chamber;

CA 02503186 2005-04-20
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Figure 4A is a figure similar to Figure 1B showing an alternative embodiment
of a
test device according to the present invention;
Figure 4B is an enlarged, schematic, top plan view of a channel of Figure 4A
showing cells on the sides of the channel;
Figures 5 and 6 are views similar to Figure 2A, showing an alternative
embodiment of a chamber defined in a test device of the present invention,
where the
wells are trapezoidal in a top plan view thereof;
Figure 7 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where the chamber
is in the form
of a figure 8 in a top plan view thereof;
Figure 8 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where one well is
rectangular
and the other well circular in a top plan view of the device;
Figure 9 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where the first
well region and
the second well region each define a plurality of wells, and where the chamzel
region
defines a plurality of channels joining respective wells of each well region;
Figure 10 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where the channel
region defines
a plurality of channels joining respective wells of each well region;
Figure 11 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where the first
well region has a
plurality of wells and a respective capillary for each well, the channel
region has a single
channel, and the second well region has a single well;
Figure 12 is a side, cross-sectional view of an embodiment of a portion of the
support member according to the present invention, the portion of the support
member
being shown along a longitudinal axis of a chamber according to the present
invention;
Figure 13 is an isometric view of a collective system according to one
embodiment
of the present invention;
Figure 14 is a view similar to Figure 2A, showing an alternative embodiment of
a
chamber defined in a test device of the present invention, where the first
well region
includes a plurality of wells interconnected by a networlc of capillaries,
where the channel
region includes a single channel, and where the second well includes a single
well;

CA 02503186 2005-04-20
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Figure 15 is a block diagram of an automated analysis system according to an
embodiment of the present invention;
Figure 16 is a flow diagram of a method according to an embodiment of the
present invention;
Figure 17 illustrates exemplary image data on which the method of Figure 16
may
operate ;
Figure 18 illustrates a histogr am that may be obtained from the image data of
Figure 17;
Figure 19 illustrates exemplary image data;
to Figure 20 is a histogram that may be obtained from exemplary image data of
Figure 19;
Figure 21 depicts the results of an experiment involving the creation of a
concentration gradient of TNF-a via laminar flow. The TNF-a was delivered to a
confluent "lawn" of endothelial cells. The endothelial cells that were
contacted by the
15 TNF-a were activated and thus are able to bind the leukocytes. Leukocytes
were then
delivered to the endothelial cells. As is demonstrated in the figure, the
leukocytes bound
to the area of the endothelial cells that received high concentrations of TNF-
a whereas
those areas not exposed to TNF-a or exposed to very little TNF-a did not bind
leukocytes;
and
2o Figure 22 depicts an exemplary microfluidic device for creating a laminar
flow
gradient.
DETAILED DESCRIPTION OF THE EMBODIMENTS
As shown in Figure lA, according to one embodiment of the present invention, a
25 device 10 to moW for chemotaxis/chemoinvasion includes a housing l0a
comprising a
support member 16 and a top member 11 mounted to the support member 16 by
being
placed in substantially fluid-tight, conformal contact with the support member
16. In the
context of the present invention, "conformal contact" means substantially form-
fitting,
substantially fluid-tight contact. The support member 16 and the top member 11
are
3o configured such that they together define a discrete chamber 12 as shown.
Preferably
chemotaxis/chemoinvasion device 10 comprises a plurality of discrete chambers,
as shown
by way of example in the embodiment of Figure 1B. The discrete chamber 12
includes a
first well region 13a including at least one first well 13 and second well
region 14a

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including at least one second well 14, the second well region further being
horizontally
offset with respect to the first well region in a test orientation of the
device. The "test
orientation" of the device is meant to refer to a spatial orientation of the
device during
testing. As shown in Figure 1C, device 10 further includes a channel region
15a including
at least one channel 15 connecting the first well region 13a and the second
well region 14
a with one another. W the embodiments of Figures lA-2C, each well region
includes a
single well, and the channel region includes a single channel. As seen in
Figure 1C, each
well is defined by a through-hole in top member 1 l, corresponding to well 13
and well 14
respectively, and by an upper surface U of support member 16. In particular,
the sides of
each well 13 and 14 are defined by the walls of the through holes in the top
member 11,
and the bottoms of well 13 and 14 are defined by the upper surface U of
support member
16. It is noted that in the context of the present invention, "top," "bottom,"
"upper" and
"side" are defined relative to the test orientation of the device. As seen
collectively in
Figures lA and 1C, a length L of channel region 15a is defined in a direction
of the
longitudinal axis of channel region 15a; a depth D of channel region 15a is
defined in a
direction normal to upper surface U of support member 16; a width W is defined
in a
direction normal to the length L and depth D of channel region 15a. According
to one
embodiment of the present invention, the chamber's first well 13 is adapted to
receive a
test agent that is a soluble test substance and/or immobilized test
biomolecules, which
2o potentially affects chemotaxis/chemoinvasion. Biomolecules include, but not
limited to,
DNA, RNA, proteins, peptides, carbohydrates, cells, chemicals, biochemicals,
and small
molecules. The chamber's second well 14 is adapted to receive a biological
sample of
cells. Immobilized biomolecules are biomolecules that are attracted to support
member 16
with an attractive force stronger than the attractive forces that are present
in the
environment surrounding the support member, such as solvating and turbulent
forces
present in a fluid medium. Non-limiting examples of the test agent include
chemorepellants, chemotactic inhibitors, and chemoattractants, such as growth
factors,
cytokines, chemokines, nutrients, small molecules, and peptides.
Alternatively, the
chamber's first well 13 is adapted to receive a biological sample of cells and
the
3o chamber's second well 14 is adapted to receive a test agent.
In one embodiment of the present invention, when a soluble test substance is
used
as the test agent, channel region 15a preferably contains a gel matrix. The
gel matrix
allows the formation of a solution concentration gradient from first well
region 13a

CA 02503186 2005-04-20
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towards second well region 14a as the solute diffuses from an area of higher
concentration
(well region 13a) through a semi-permeable matrix (the gel matrix) to an area
of lower
concentration (well region 14a). If the soluble test substance comprises a
chemoattractant,
in order for the cells to migrate through the matrix in the direction of the
solution
concentration gradient towards well region 13a, the cells must degrade this
matrix by
releasing enzymes such as matrix metalloproteases. This cell chemotaxis and
invasion
may be subsequently observed, measured, and recorded.
In one embodiment of the present invention, utilizing immobilized biomolecules
as
the test agent, the biomolecules are preferably immobilized or bound on the
portion of
l0 support member 16 underlying channel region 15a and underlying through hole
for well
region 13a. The concentration of biomolecules decreases along the longitudinal
axis of
the device from well region 13a towards well region 14a forming a surface
concentration
gradient of immobilized biomolecules and the biological sample of cells
potentially
responds to tlus surface gradient. This cell haptotaxis may be subsequently
observed,
15 measured, and recorded.
With respect to particular specifications of device 10, top member 11 is made
of a
material that is adapted to effect conformal contact, preferably reversible
conformal
contact, with support member 16. According to embodiments of the present
invention, the
flexibility of such a material, among other things, allows the top member to
form-fittingly
20 adhere to the upper surface U of support member 16 in such a way as to form
a
substantially fluid-tight seal therewith. The conformal contact should
preferably be strong
enough to prevent slippage of the top member on the support member surface.
Where the
confonnal contact is reversible, the top member may be made of a material
having the
structural integrity to allow the top member to be removed by a simple peeling
process.
25 This would allow top member 11 to be removed and cells at certain positions
collected.
Preferably, the peeling process does not disturb any surface treatment or cell
positions of
support member 16. Physical striations, pockets, SAMs, gels, peptides,
antibodies, or
carbohydrates can be placed on support member 16 and the top member 11
subsequently
can be placed over support member 16 without any damage to these structures.
3o Additionally, the substantially fluid-tight seal effected between top
member 11 and
support member 16 by virtue of the conformal contact of top member 11 with
support
member 16 prevents fluid from leaking from one chamber to an adjacent chamber,
and
also prevents contaminants from entering the wells. The seal preferably occurs
essentially

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
instantaneously without the necessity to maintain external pressure. The
conformal
contact obviates the need to use a sealing agent to seal top member 11 to
support member
16. Although embodiments of the present invention encompass use of a sealing
agent, the
fact that such a use is obviated according to a preferred embodiment provides
a cost-
saving, time-saving alternative, and further eliminates a risk of
contamination of each
chamber 12 by a sealing agent. Preferably, the top member 11 is made of a
material that
does not degrade and is not easily damaged by virtue of being used in multiple
tests, and
that affords considerable variability in the top member's configuration during
manufacture
of the same. More preferably, the material may be selected for allowing the
top member
to to be made using photolithography. In a preferred embodiment, the material
comprises an
elastomer such as silicone, natural or synthetic rubber, or polyurethane. In a
more
preferred embodiment, the material is polydimethylsiloxane ("PDMS")
In another embodiment of the present invention, device 10 includes a housing
defining a chamber, the chamber including a first well region including at
least one first
15 well; a second well region including at least one second well; and a
channel region
including a plurality of channels comzecting the first well region and the
second well
region with one another. The second well region is preferably horizontally
offset with
respect to the first well region is a test orientation of the device.
According to a preferred embodiment of a method of the present invention,
2o standard photolithographic procedures can be used to produce a silicon
master that is the
negative image of any desired configuration of top member 11. For example, the
dimensions of chambers 12, such as the size of well regions 13a and 14a, or
the length of
channel region 15a, can be altered to fit any advantageous specification. Once
a suitable
design for the master is chosen and the master is fabricated according to such
a design, the
25 material is either spin cast, inj ected, or poured over the master and
cured. Once the mold
is created, this process may be repeated as often as necessary. This process
not only
provides great flexibility in the top member's design, it also allows the top
members to be
massively replicated. The present invention also contemplates different
methods of
fabricating device 10 including, for example, e-beam lithography, laser-
assisted etching,
3o and transfer printing.
Device 10 preferably fits in the footprint of an industry standard microtiter
plate.
As such, device 10 preferably has the same outer dimensions and overall size
of an
industry standard microtiter plate. Additionally, when chamber 12 comprises a
plurality
11

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
of chambers, either the chambers 12 themselves, or the wells of each chamber
12, may
have the same pitch of an industry standard microtiter plate. The term "pitch"
used herein
refers to the distance between respective vertical centerlines between
adjacent chambers or
adjacent wells in the test orientation of the device. The embodiment of device
10, shown
in Figure 1B, comprises 48 chambers designed in the format of a standard 96-
well plate,
with each well fitting in the space of each macrowell of the plate. The size
and number of
the plurality of chambers 12 can correspond to the footprint of standard 24-,
96-, 384-,
768- and 1536-well microtiter plates. For example, for a 96 well microtiter
plate, device
may comprise 48 chambers 12 and therefore 48 experiments can be conducted, and
for
l0 a 384 well microtiter plate, the device may comprise 192 chambers 12, and
therefore 192
experiments can be conducted. The present invention also contemplates any
other number
of chambers and/or wells disposed in any suitable configuration. For example,
if pitch or
footprint standards change or new applications demand new dimensions, then
device 10
may easily be changed to meet these new dimensions. By conforming to the exact
dimension and specification of standard microtiter plates, embodiments of
device 10
would advantageously fit into existing infrastructure of fluid handling,
storage,
registration, and detection. Embodiments of device 10, therefore, may be
conducive to
high throughput screening as they may allow robotic fluid handling and
automated
detection and data analysis. Top member 11 may additionally take on several
different
variations and embodiments. Depending on the test parameters, such as, for
example,
where chemotaxis or chemoinvasion are to be monitored, the cell type, cell
number, or
distance over which chemotaxis or chemoinvasion is required, chamber 12 of top
member
11 may have various embodiments of which a few exemplary embodiments are
discussed
herein. With respect to a discrete chamber 12, the shape, dimensions,
location, surface
treatment, and numbers of channels in channel region 15a and the shape and
number of
wells 13 and 14 may vary.
Regarding the shape of channel region 15a, each channel 15 in the channel
region
15a is not limited to a particular cross-sectional shape, as taken in a plane
perpendicular to
its longitudinal axis. For example, the cross section of any given channel 15
can be
3o hexagonal, circular, semicircular, ellipsoidal, rectangular, square, or any
other polygonal
or curved shape.
Regarding the dimensions of a channel 15, the length L of a given channel 15
can
vary based on various test parameters. For instance, the length L of a given
channel 15
12

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may vary in relation to the distance over which chemotaxis is required to
occur. For
example, the length L of a given channel 15 can range from about 3 ~.m to
about 1 g mm in
order to allow cells sufficient distance to travel and therefore sufficient
opportunity to
observe cell chemotaxis and chemoinvasion. The width W and depth D of a given
channel
15 may also vary as a function of various test parameters. For examples, the
width W and
depth D of a given channel 15 may vary, in a chemotaxis/chemoinvasion device,
depending on the size of the cell being studied and whether a gel matrix is
added to the
given channel 15. Generally, a given channel 15's width W and depth D may be
approximately in the range of the diameter of the cell being assayed. To
discount random
l0 cellular movement, at least one of the depth D or width W of a given
channel 15 should
preferably be smaller than the diameter of the cell when no gel matrix is
placed in the
given channel 15 so that when the cells are 'activated, they can "squeeze"
themselves
through the given channel toward the test agent. If a given channel 15
contains a gel
matrix, then, the depth D and width W of the given channel 15 may be greater
than the
diameter of the cell being assayed. Refernng by way of example to the
embodiments of
Figures lA-2C, if suspension cells such as leukocytes, which are about 3-12 ~m
in
diameter, are in well 14 and channel 15 contains no gel, then the width W of
channel 15
should range from about 3 microns to about 20 ~,m, and the depth D of channel
15 should
range from about 3 microns to about 20 ~,m but at least either the depth D or
width W of
2o channel 15 should be smaller than the diameter of the cell. If leukocytes
are in well 14
and channel 15 contains a gel matrix, then the width W of charnel 15 should
range from
about 20 to about 100 ~,m and the depth D should range from about 20 ~.m to
about 100
~,m, and both the width W and depth D of channel 15 can be greater than the
diameter of
the cell assayed. Similarly, if adherent cells, such as endothelial cells
which are 3-10
microns in diameter before adherence, are in well 14 and channel 15 contains
no gel, then
the width W and depth D of channel 15 can range from about 3 to about 20 ~.m,
but at
least either the width W or depth D of channel 15 should be smaller than the
diameter of
the cell assayed. If adherent cells are in well 14 and channel 15 contains a
gel matrix then
the width W and depth D of channel 15 should range from about 20 ~m to about
200 ~.m
3o and both the width W and depth D of channel 15 can be greater than the
diameter of the
cell assayed.
13

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WO 2004/038367 PCT/US2003/033146
As seen in Figures 2A-2C, channel 15 may connect the first well 13 to the
second
well 14 at respective sides of the wells, as shown in Figures. 2A and 2C or at
a central
region of the wells, as shown in Figure 2B.
The number of channels in channel region 15a between well regions 13a and 14a
can also vary. Channel region 15a may include a plurality of channels, as
shown by way
of example in Figures 3A-3C. As seen in Figure 3A, in a preferred
configuration, the
length L of each channel 15i-n between well 13 and well 14 is identical. In
another
embodiment as seen in Figure 3B, the length L of each channel 15i-15n of
chasmel region
15a increases in the direction of well 14, starting from channel 15i in the
side portion 12a
of chamber 12 to chaimel 15n in the side portion 12b of chamber 12. In one
embodiment,
as seen in Fig. 3B, the length L of each successive channel in the plurality
of channels 15
of chamber 12 increases in a direction of a width W of the channels with
respect to a
preceding one of the plurality of channels such that respective channel inlets
at one of the
first well region and the second well region, such as well region 13a as
shown, are aligned
along the direction of the width W of the channels. Although, in this
configuration, the
cells traveling in any particular channel will exit the channels and enter
well 14 at points
longitudinally offset with respect to one another, the section of channel
region 15a closest
to well region 13a is positioned so that cells ultimately entering the
different channels will
be aligned in a direction of the width W of the channels so that there is no
longitudinal
2o offset between them. Therefore, in comparing two adjacent channels, a first
group of cells
entering channel 15i has an entry position that is not longitudinally offset
with respect to a
second different group of cells entering channel 15j, but the first group of
cells exiting
channel 15i has an exit point longitudinally offset from the second group of
cells exiting
channel 15j. In a different embodiment of the present invention illustrated in
Figure 3C,
the width W of each channel 15i-15n increases starting from channel 15i in the
side
portion 12a of chamber 12 to channel 15n in the side portion 12b of chamber
12.
Preferably, the width W or depth D of each successive channel of the plurality
of channels
increases in a direction of a width W of the channels with respect to a
preceding one of the
plurality of channels. Alternatively, a depth D of each successive channel
could increase
3o (not shown) along a direction of the width W of the channels. It is
understood to those
skilled in the art, that various embodiments altering the dimensions of the
channels in the
channel region 15a are within the scope of the present invention. For example,
the length
of the channels 15i-15n need not increase in a continuous manner from channel
15i to 15n
14

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
as illustrated in Figure 3B. Instead, channel 15i-15n may have varying lengths
following
no particular order or pattern.
With respect to surface treatment of a given channel 15, to simulate ira vivo
conditions where cells are surrounded by other cells, the lateral walls of a
given channel
15 may be coated with cells, such as endothelial cells 40 as seen in Figure
4B. Non-
limiting examples of endothelial cells include human umbilical vein
endothelial cells or
high endothelial venule cells. hi another embodiment, a given channel 15 is
filled with a
gel matrix such as gelatin, agarose, collagen, fibrin, any natural or
synthetic extracellular
proteinous matrix or basal membrane mimic including, but not limited to
MATRIGELTM
l0 (Becton Dickenson Labware), or ECM GEL, (Sigma, St. Louis, Mo.), or other
hydrogels
containing certain factors such as cytokines, growth factors, antibodies,
ligands for cell
surface receptors, or chemokines. Preferably, the gel has a substantially high
water
content and is porous. enough to allow cell chemotaxis and invasion. As
mentioned above,
when the test agent comprising a soluble test substance is placed in well 13,
the gel
facilitates formation of a solution concentration gradient along the
longitudinal axis of
chamber 12. Additionally, adding a gel matrix to a given channel 15 simulates
the natural
environment in the body, as enzyme degradation through extracellular matrix is
a crucial
step in the invasive process.
According to the present invention, the individual wells of each well region
13a
orl4a may have any shape and size. For example, the top plan contour of a
given well
may be circular, as shown in Figures lA-ZC, or trapezoidal as shown in Figures
5 and 6.
Alternatively, the top plan contour of a given chamber may be generally in the
shape of a
"figure ~" as shown in Figure 7. Preferably when using a soluble test
substance as the test
agent, the shape of well 13 is such that soluble test substance is readily
able to access the
channel 15 and thereby form the necessary solution concentration gradient
along the
length L of channel 15. Preferably, the shape of well 14 is such that cells
are not deferred,
detained, or hindered from migrating out of the first well 14, for example, by
accumulating in a corner, side or other dead space of well 14. Although
possible
accumulation of cells in a dead space of well 14 is not restricted to any
particular cell
3o number, there exists a greater likelihood of cells accumulating in a corner
of well 14 if a
large number of cells are used. Therefore to maximize accessibility to the
concentration
gradient and to minimize the "wasting" of cells when a large cell sample is
utilized, it is
important that the shape of well 14 be such that a sufficiently high
percentage of cells,

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
particularly the cells in the area of well 14 furthest from channel 15, are
capable of
migrating out of well 14. In a different embodiment that also addresses the
problem of the
wasting of cells, well 14 may be shaped such that all cells have a higher
probability of
accessing the concentration gradient. For example as seen in Figure 8, the
length LW of
well 14 in a top plan view thereof is minimized to decrease the surface area
of the well. As
such, the cells are closer to the concentration gradient formed in channel 15.
In a preferred
embodiment, the LW of well 14 in a top plan view thereof is about lmm to about
2mm.
In addition to variations of components of a discrete chamber 12, the present
invention also contemplates variations in the overall chamber 12 as well as
variations from
to chamber to chamber. With respect to the overall chamber 12, in one
embodiment, the
chambers 12 are sized so that a complete chamber 12 fits into the area
normally required
for a single well of a 96-well plate. In this configuration, 96 different
assays could be
performed in a 96-well plate. In another embodiment, the 1:1 ratio of a first
well to
second well, as present in the aforementioned embodiments, is altered by
modifying
15 chamber 12. For example as seen in Figure 9, device 10 includes a chamber
12 having a
first well region 13a having a plurality of first wells 102, 103, 104 and 105
connected to
one another, a second well region 14a having a plurality of wells 106, 107,
108 and 109,
and a channel region 15 a having a plurality of channels 15 connecting
respective ones of
the first wells to respective ones of the second wells. Each well of the first
well region
20 13a may receive the same test agent, and each well of the second well
region 14a may
receive a different cell type. Alternatively, each well of the first well
region 13a may
receive a different test agent, and each well of the second well region 14a
may receive the
same cell type. This configuration allows several different cell types or
different test
agents to be tested simultaneously. In an alternative embodiment as seen in
Figure 10,
25 each channel 15 of chamlel region 15a comprises subchannels as shown. This
arrangement not only allows several different cell types or test agents to be
tested
simultaneously but also generates several tests of each test agent or cell
type.
Figure 11 illustrates an alternative chamber configuration of a
chemotaxis/chemoinvasion device according to an alternative embodiment of the
present
3o invention. In this embodiment, chamber 12 comprises a first well region 13a
connected by
a channel region 15a including a single channel 15 to a second well region 14a
including a
single well 14. The first well region contains a plurality of first wells,
17a, 18a, and 19a
and a plurality of capillaries, a first perimeter capillary 17, a center
capillary 18, and a
16

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
second perimeter capillary 19 connected to respective ones of the plurality of
first wells.
All three of the capillaries converge at a junction into channel 15, which is
connected with
the second well region 14a. Well region 13a is not limited to containing only
three
capillaries and can contain any number of additional capillaries (not shown).
First wells
17a-19a may, for example, be adapted to receive solutions of test
biomolecules, which are
allowed to flow into channel 15 and adsorb nonspecifically to the regions of
the surface
over which the solution containing the test biomolecules flows. First wells
17a-19a are
also adapted to subsequently receive cells.
With respect to variations from chamber to chamber, in one embodiment, the
to length L of each channel 15 increases along one or more dimensions of top
member 11
from one chamber to the adjacent chamber. In an alternative embodiment, all
chambers
12 have channel 15 of the same length L. The width W of each channel 15 can
also vary
and can increase along one or more dimensions of top member 11 from one
chamber to
the adjacent chamber. In an alternative embodiment, all chambers 12 have
channel 15 of
15 the same width W. Figure 4A is a top plan view of an embodiment of the
present
invention where, within top member 11, different chambers have various channel
sizes
and shapes, such sizes and shapes being in no particular order, pattern, or
arrangement. By
employing this varied configuration, the best channel region design for a
given test may be
obtained. In other words, where the optimal channel region design is
determined, a new
2o assay plate configured solely to those specifications may be employed.
Support member 16 of device 10 provides a support upon which top member 11
rests and can be made of any material suitable for this function. Suitable
materials are
known in the art such as glass, polystyrene, polycarbonate, PMMA,
polyacrylates, and
other plastics. Where device 10 is a chemotaxis, haptotaxis and/or
chemoinvasion device,
25 it is preferable that support member 16 comprise a material that is
compatible with cells
that may be placed on the surface of support member 16. Suitable materials may
include
standard materials used in cell biology, such as glass, ceramics, metals,
polystyrene,
polycarbonate, polypropylene, as well as other plastics including polymeric
thin films. A
preferred material is glass with a thickness of about 0.1 to about 2 mm, as
this may allow
3o the viewing of the cells with optical microscopy techniques.
Similar to top member 11, support member 16 can have several different
embodiments. In particular, the configuration and surface treatment of support
member 16
may vary.
17

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As seen in a side view of support member 16 in Figure 12, the upper surface U
of
support member 16, which underlies top member 11, may be sloped at
predetermined
regions thereof with respect to a horizontal plane at less than a 90°
angle. In the shown
embodiment, the predetermined regions correspond to bottom surfaces of
respective wells,
surface 16a corresponding to a bottom surface of a well 13, and surface 16 b
corresponding to a bottom surface of well 14. Surface 16c, in turn,
corresponds to a
bottom surface of channel 15. In this embodiment, the given configuration
facilitates
suspended cells flowing in the direction of the downward slope of top surface
16b of
support member 16 to become more readily exposed to the concentration
gradient. If a
to soluble test substance is used as the test agent in well 13 of device 10,
then top surface 16a
of support member 16 may also be downwardly sloped with respect to a
horizontal plane
at less than a 90° angle to facilitate exposure of the test substance
to channel 15 in order to
facilitate formation of the solution concentration gradient.
Support member 16 may also have a treatment on or embedded into its surface.
15 This treatment may serve numerous functions, including, for example,
facilitating the
placement, adhesion or movement of cells being studied, and simulating i~z
vivo
conditions. Numerous surface configurations and chemicals may be used alone or
in
conjunction for this treatment.
For example, in one embodiment support member 16 includes a patterned self
20 assembled monolayer (SAM) on a gold surface or other suitable material.
SAMs are
monolayers typically formed of molecules each having a functional group that
selectively
attaches to a particular surface, the remainder of each molecule interacting
with
neighboring molecules in the monolayer to form a relatively ordered array. By
using
SAMs, various controls of biological interactions may be employed. For
example, SAMs
25 may be arrayed or modified with vaxious "head groups" to produce "islands"
of
biospecific surfaces surrounded by areas of bio-inert head groups. Further,
SAMs may be
modified to have "switchable surfaces" that may be designed to capture a cell
and then be
subsequently modified to release the captured cell. Moreover, it may also be
desirable to
utilize a bioinert support member material to resist non-specific adsorption
of cells,
3o proteins, or any other biological material. Consequently, the use of SAMs
on support
member 16 may be advantageous.
The present invention also contemplates, as seen in Figure 13, the use of any
system known in the art to detect and analyze cell chemotaxis and
chemoinvasion. In
18

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WO 2004/038367 PCT/US2003/033146
particular, the present invention contemplates the use of any system known in
the art to
visualize changes in cell morphology as cells move across channel 15, to
measure the
distance cells travel in channel 15, and to quantify the number of cells that
travel to
particular points in charmel 15. As such the present invention contemplates
both "real-
time" and "end-point" analysis of chemotaxis and chemoinvasion. In one
embodiment,
the device 122 includes an observation system 120 and a controller 121. The
controller
121 is in communication with the observation system 120 via line 122. The
controller 121
and observation system 120 may be positioned and programmed to observe,
record, and
analyze chemotaxis and chemoinvasion of the cells in the device. The
observation system
l0 120 may be any of numerous systems, including a microscope, a high-speed
video camera,
and an array of individual sensors. Nonlimiting examples of microscopes
include phase-
contrast, fluorescence, luminescence, differential-interference-contrast, dark
field,
confocal laser-scanning, digital deconvolution, and video microscopes. Each of
these
embodiments may view or sense the movement and behavior of the cells before,
during,
15 and after the test agent is introduced. At the same time, the observation
system 120 may
generate signals for the controller 121 to interpret and analyze. This
analysis can include
determining the physical movement of the cells over time as well as their
change in shape,
activity level or any other observable characteristic. In each instance, the
conduct of the
cells being studied may be observed in real time, at a later time, or both.
The observation
2o system 120 and controller 121 may provide for real-time observation via a
monitor. They
may also provide for subsequent playback via a recording system of some kind
either
integrated with these components or coupled to them. For example, in one
embodiment,
cell behavior during the desired period of observation is recorded on VHS
format
videotape through a standard video camera positioned in the vertical ocular
tubes of a
25 triocular compound microscope or in the body of an inverted microscope and
attached to a
high quality video recorder. The video recorder is then played into a
digitization means,
e.g., PCI frame grabber, for the conversion of analog data to digital form.
The electronic
readable (digitized) data is then accessed and processed by an appropriate
dynamic image
analysis system, such as that disclosed in U.S. Patent No. 5,655,028 expressly
3o incorporated in its entirety herein by reference. Such a system is
commercially available
under the trademark DIAS~ from Solltech Inc. (Oakland, Iowa). Software capable
of
assisting in discriminating cells from debris and other detection artifacts
that might be
present in the sample should be particularly advantageous. In either case,
these
19

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WO 2004/038367 PCT/US2003/033146
components may also analyze the cells as they progress through their reaction
to the test
agent.
11z one embodiment, the present invention contemplates the use of an automated
analysis system, as illustrated in Figure 15, to analyze data measuring the
distance cells
travel in channel 15, and to quantify the number of cells that travel to
particular points in
channel 15. Figure 15 is a block diagram of an automated analysis system 100
including,
for example, an image preprocessing stage 110, an obj ect identification stage
120 and a
migration analysis stage 130. The image preprocessing stage 110 may receive
digital
image data of chamber 12 from a digital camera or other imaging apparatus as
described
to above. The data typically includes a plurality of image samples at various
spatial
locations (called, "pixels" for short) and may be provided as color or
grayscale data. The
image preprocessing stage 110 may alter the captured image data to permit
algorithms of
the other stages to operate on it. The object identification stage 120 may
identify objects
from within the image data. Various objects may be identified based on the
test to be
15 performed. For example, the object identifier may identify channels 15,
cells or cell
groups from within the image data. The migration analysis stage 130 may
perform the
migration analysis designated for testing.
Figure 15 illustrates a number of blocks that may be included within the image
preprocessing stage 110. Essentially, the image preprocessing stage 110
counteracts
20 image artifacts that may be present in the captured image data as a result
of imperfections
in the imager or the device. In one embodiment, the image preprocessing stage
110 may
include an image equalization block 140. The equalization 140 may find
application in
embodiments where sample values of captured image data do not occupy the full
quantization range available for the data. For example, an 8-bit grayscale
system permits
25 256 different quantization levels for input data (0-255). Due to
imperfections in the
imaging process, it is possible that pixel values may be limited to a narrow
range, say the
first 20 quantization levels (0-20). The equalization 140 may re-scale sample
values to
ensure that they occupy the full range available in the 8-bit system.
In another embodiment, the equalization block 140 may re-scale sample values
3o based on a color or wavelength. Conventional cellular analysis techniques
often cause
cells to appear in predetermined colors or with predetermined wavelengths,
which permits
them to be distinguished from other materials captured by the imager. For
example, in
fluorescent applications, cells emit light at predetermined wavelengths. In
nuclear

CA 02503186 2005-04-20
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staining applications, cell nuclei are dyed with a material that causes them
to appear in the
image data with predetermined colors. The equalization block 140 may re-scale
sample
values having components that coincide with these expected colors or
wavelengths. In so
doing, the equalization block 140 effectively filters out other colors or
wavelengths, a
consequence that may be advantageous in later image processing.
Image rotation is another image artifact that may occur from imperfect imaging
apparatus. Although the channels 15 are likely to be generally aligned with
columns and
rows of pixels in the image data, further analysis may be facilitated if the
aligmnent is
improved. Accordingly, in an embodiment, the image preprocessing stage 110 may
to include an image alignment block 150 that rotates the captured image data
to counteract
this artifact. Once the rotation artifact has been removed from the captured
image data,
then image from individual chamlels 15 are likely to coincide with a regular
row or
column array of pixel data.
Figure 16 illustrates a method of operation for the image alignment block 150
15 according to an embodiment of the present invention and described in
connection with
exemplary image data illustrated in Figure 17. In the example of Figure 17,
chamlels 15
are aligned generally with rows of image data but for the rotation artifact.
To counteract
the rotation artifact, the image preprocessor may identify a band of image
data coinciding
with a boundary between second well 14 and the channels 15 themselves (block
1010). In
2o the case of Figure 17, the band may constitute column 310. Generally, the
area of second
well 14 will be bright relative to the area of channels 15 due the greater
number of cells
present therein. Thus, a histogram of image data values along a presumed
direction of the
channels 15 may appear as shown in Figure 18. The band 310 may be identified
from an
abrupt change in image data values along this direction.
25 Having identified a column of image data to be considered, the column 310
may be
split into two boundary boxes 320, 330 (block 1020). By summing the intensity
of the
image data in each of the two boundary boxes a~ld comparing summed values to
each
other, an orientation of the rotation artifact may be determined (blocks 1030,
1040). In the
example of Figure 17, the rotation artifact causes more of second well 14 to
fall within the
3o area of boundary box 320 than of boundary box 330 (a clockwise artifact).
The image
data may be rotated counterclockwise until the summed values of each boundary
box 320,
330 become balanced.
21

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Thus, if the image intensity of the first bounding box is greater than that of
the
second bounding box 330, the image data may be rotated in a first direction
(block 1050).
If the image intensity of the second bounding box 330 is greater than that if
the first
bounding box 320, the image data may be rotated in a second direction (block
1060). And
when the image intensities are balanced, the method 1000 may conclude; the
rotation
artifact has been corrected.
Returning to Figure 15, the image preprocessing stage 110 also may process the
captured image data by cropping the image to the area occupied by chamlels 15
themselves (block 160). As described, each test bed may include a pair of
wells
to interconnected by a plurality of channels. For much of the migration
analysis, it is
sufficient to measure cellular movement or activity within channels 15 only.
Activity in
second well 14 or the first well 13 need not be considered. In such an
embodiment, the
image preprocessing stage 110 may crop the image data to remove pixels that
lie outside
channels 15.
15 The image preprocessing stage 110 also may include a thresholding block
170,
performing threshold detection upon the image data. The thresholding block 170
may
truncate to zero any sample having a re-scaled value that fails to exceed a
predetermined
threshold. Such thresholding is useful to remove noise from the captured image
data. In
an embodiment, the thresholding block 170 may be integrated with the
equalization block
20 140 discussed above. It need not be present as a separate element. In some
embodiments,
particularly those where the equalization block 140 scales pixel values
according to
wavelength components, the thresholding block 170 may be omitted altogether.
An
output of the image preprocessing stage 110 may be input to the obj ect
identification stage
120.
25 The object identification stage 120 identifies objects from within the
image data,
including the channels themselves and, optionally, individual cells. According
to an
embodiment, in a fluorescent system, channels 15 may be identified by
developing a
histogram of the fluorescent light along a major axis in the system (block
180). Figure 19
illustrates image data that may have been determined from the example of
Figure 17. The
3o major axis may coincide with the boundary between the well adapted to
receive cells and
the channel region. Light intensity from within channel region 15a area may be
summed
along this axis, yielding a data set represented in Figure 19. In a second
stage, the data set
is "dilated" (block 190). Dilation may be achieved by applying a high pass
filter to the
22

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
data set or any other analogous technique. Figure 20 illustrates the data set
of Figure 19
having been subject to dilation.
From the data set of Figure 20, the channels may be identified. Candidate
channel
15 positions may be identified to coincide with relative maximums of the data
set.
Alternatively, candidate positions of boundaries between channels 15 may be
determined
from relative minimums from within the data set of Figure 20. A final set of
channel 15
positions may be determined from a set of parameters known about channel
region 15a
itself. For example, if channels 15 are known to have been provided with a
regular
spacing among channels 15, any candidate channel 15 position that would
violate the
l0 spacing can be eliminated from consideration.
Returning to Figure 15, in addition to identifying chamlels 15, individual
cells may
be identified within the image data (block 200). In an application where cells
are marked
with nuclear staining, identification of individual cells merely requires an
image processor
to identify and count the number of marked nuclei. The nuclei appear is a
number of dots
15 of a predetermined color. hi an application using fluorescing cells,
identification of
individual cells becomes more complicated. Individual cells can be identified
relatively
easily; they appear as objects of relatively uniform area in the image data.
Identifying a
number of cells clustered together becomes more difficult. hl this case, the
number of
cells may be determined from the area or radius of the cluster in the image
data. The
20 cluster is likely to appear in the image having some area or cluster
radius. By comparing
the cluster's area or radius to the area or radius of an individual cell, the
number of cells
may be interpolated. Of course, identification of individual cells may be
omitted
depending upon the requirements of the migration analysis.
The final stage in the image processing system is the migration analysis 130
itself.
25 In one embodiment, coordinate data of each cell in the channels 15 may be
gathered and
recorded. However, some testing need not be so complicated. In a first
embodiment, it
may be sufficient merely to identify the number of cells present in channel
15. In this
case, identification of individual cells may be avoided by merely summing
quantities of
fluorescent light detected in each channel 15. From this measurement, the
number of cells
3o may be derived without investing the processing expense of identifying
individual cells.
The foregoing description presents image analysis that is relevant to a single
channel 15 to
be tested. Of course, depending upon the requirements of the migration
analysis 130, it
may be desired to generate image samples of a number of different channels 15.
Further,
23

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it may be desirable to generate image samples of a single channel 15 at
different times.
The image processing described above may be repeated for different channels 15
and
different times to accommodate for such test scenarios.
According to an embodiment, the image processing may account for
manufacturing defects of individual channels 15. During image processing,
manufacturing defects may prevent cell migrations into a channel 15. In an
embodiment,
when the system 100 counts a number of cells in the channel 15 (or derives the
number
from identified cell locations), it may compare the number to an expectation
threshold. If
the number is below the expectation threshold, the system 100 may exclude the
channel 15
to from migration analysis. In practice, this expectation threshold may be
established as a
minimum number of cells that are likely to enter a properly configured cell
given the test
conditions being analyzed under the migration analysis. If the actual number
of cells falls
below this threshold, it may lead to a conclusion that channel 15 blocking
conditions may
be present.
The foregoing operations and processes of the analysis system 100 may be
performed by general purpose processing apparatus, such as computers,
workstations or
servers, executing software. Alternatively, some of the operations or
processes may be
provided in a digital signal processor or application specific integrated
circuit
(colloquially, an "ASIC"). Additionally, these operations and processes,
particularly those
associated with image preprocessing, may be distributed in processors of a
digital
microscope system. Such variations are fully within the scope of the present
invention.
The present invention also contemplates the use of the aforementioned
embodiments of device 10 to assay various elements of
chemotaxis/chemoinvasion. In
general, the present invention provides for a first assay comprising high
throughput
screening of test agents to determine whether they influence
chemotaxis/chemoinvasion.
Test agents generally comprise either soluble test substances or immobilized
test
biomolecules and are generally placed in first well region 13a of chamber 12
of device 10.
After determining which test agents influence chemotaxis/chemoinvasion, by
acting as
chemoattractants and promoting or initiating chemotaxis/chemoinvasion, by
acting as
chemorepellants and repelling chemotaxis/chemoinvasion or by acting as
inhibitors and
halting or inhibiting chemotaxis/chemoinvasion, then a second assay can be
performed
screening test compounds. The test compounds generally comprise therapeutics
or
chemotaxis/chemoinvasion inhibitors and are generally introduced in second
well region
24

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WO 2004/038367 PCT/US2003/033146
14a, which contains a biological sample of cells. The test compounds are
screened to
determine if and how they influence the cells' migration in response to the
test agents.
In particular, a chemotaxis/chemoinvasion assay according to an embodiment of
the present invention involves a device 10 including a housing comprising a
top member
11 mounted to a support member 16. The top member and the support member are
configured such that they together define a discrete assay chamber 12. The
discrete assay
chamber 12 includes a first well region 13a connected by a channel 15 to a
second well
region 14a. The first well region 13a includes at least one first well 13,
each of the at least
one first well 13 being adapted to receive a test agent therein. The second
well region 14a
to includes at least one second well 14 horizontally offset with respect to
the first well region
13a in a test orientation of the device, each of the at least one second well
14 being
adapted to receive a cell sample therein. Channel 15 includes at least one
channel
connecting the first well region 13a and the second well region 14a to one
another. The
test agent received in first well 13 is a soluble test substance and/or
immobilized test
15 biomolecules. When the test agent comprises immobilized test biomolecules,
the
biomolecules are immobilized on an upper surface U of support member 16
constituting
the bottom surface of well region 13a as well as on upper surface U of support
member 16
constituting the bottom surface of channel region 15a.
Nonlimiting examples of biological samples of cells include lymphocytes,
2o monocytes, leukocytes, macrophages, mast cells, T-cells, B-cells,
neutrophils, basophils,
eosinophils, fibroblasts, endothelial cells, epithelial cells, neurons, tumor
cells, motile
gametes, motile forms of bacteria, and fungi, cells involved in metastasis,
and any other
types of cells involved in response to inflammation, injury, or infection.
Well region 14a
may receive only one cell type or any combination of the above-referenced
exemplary cell
25 types. For example, as described above, it is often desirable to provide a
mixed cell
population to more effectively create an environment similar to ifz vioo
conditions. Well
region 14a may also receive cells at a particular cell cycle phase. For
example, well
region 14a may receive lymphocytes in Gl phase or Go phase.
Nonlimiting examples of soluble test substances include chemoattractants,
3o chemorepellants, or chemotactic inhibitors. As explained above,
chemoattractants are
chemotactic substances that attract cells and once placed in well region 14a,
cause cells to
migrate towards well region 14a. Chemorepellents are chemotactic substances
that repel
cells and once placed in well region 14a, cause cells to migrate away from
well region

CA 02503186 2005-04-20
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14a. Chemotactic inhibitors are chemotactic substances that inhibit or stop
chemotaxis
and once placed in well region 14a, cause cells to have inhibited migration or
no migration
from well region 14a. Non-limiting examples of chemoattractants include
hormones such
as T3 and T4, epinephrine and vasopressin; immunological agents such as
interleukein-2,
epidermal growth factor and monoclonal antibodies; growth factors; peptides;
small
molecules; and cells. Cells may act as chemoattractants by releasing
chemotactic factors.
For example, in one embodiment, a sample including cancer cells may be added
to well
13. A sample including a different cell type may be added to well 14. As the
cancer cells
grow they may release factors that act as chemoattractants attracting the
cells in well 14 to
to migrate towards well 13. In another embodiment, endothelial cells are added
to well 13
and activated by adding a chemoattractant such as TNF-a or IL-1 to well 13.
Leukocytes
are added to well 14 and may be attracted to the endothelial cells in well 14.
Non-limiting examples of chemorepellants include irritants such as
benzalkonium
chloride, propylene glycol, methanol, acetone, sodium dodecyl sulfate,
hydrogen peroxide,
15 1-butanol, ethanol, and dimethylsulfoxide; and toxins such as cyanide,
carbonylcyanide
chlorophenylhydrazone, endotoxins and bacterial lipopolysaccharides; viruses;
pathogens;
and pyrogens.
Nonlimiting examples of immobilized biomolecules include chemoattractants,
chemorepellants, and chemotactic inhibitors as described above. Further non-
limiting
20 examples of immobilized chemoattactants include chemokines, cytokines, and
small
molecules. Further non-limiting examples of chemoattractants include IL-8, GCP-
2, GRO-
a, GRO-(3, MGSA-[3, MGSA-y, PF4, ENA-78, GCP-2, NAP-2, IL-8, IP10, I-309, I-
TAC,
SDF-1, BLC, BRAK, bolekine, ELC, LKTN-1, SCM-1[3, MIG, MCAF, LD7a, eotaxin, ,
IP-110, HCC-1, HCC-2, Lkn-1, HCC-4, LARC, LEC, DC-CKI, PARC, AMAC-l, MIP-
25 2(3, ELC, exodus-3, ARC, exodus-1, 6Ckine, exodus 2, STCP-1, MPIF-l, MPIF-
2,
Eotaxin-2, TECK, Eotaxin-3, ILC, ITAC, BCA-1, MIP-la, MIP-1(3, MIP-3a, MIP-
3(3,
MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, RANTES, eotaxin-l, eotaxin-2, TARC, MDC,
TECK, CTACK, SLC, lymphotactin, and fractalkine; and other cells. Further non-
limiting examples of chemorepellants include receptor agonists and other
cells.
3o In order to perform a test, such as a chemotaxis and/or chemoinvasion assay
utilizing a soluble test substance, the test device 10 is first fabricated. A
preferred
embodiment of the method of making the device according to the present
invention will
now be described. A master that is the negative of top-plate 11 is fabricated
by standard
26

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
photolithographic procedures. A predetermined material is spin coated or
injection
molded onto the master. The predetermined material is then cured, peeled off
the master to
comprise top member 11 and placed onto support member 16.
A rigid frame with the standard microtiter footprint is preferably placed
around the
outer perimeter of top member 11. In one embodiment, a gel matrix is poured
into well
region 13a and allowed to flow into channel region 15a. After the gel matrix
sets, excess
gel is removed from well regions 13a and 14a. In another embodiment, no gel
matrix is
added to channel region 15a. Subsequently, a biological sample of cells is
placed in well
region 14a and a test substance is placed in well region 13a. In one
embodiment, a low
to concentration of a test substance is placed in well region 14a in order to
activate the cells
and expedite the beginning of the assay. Alternatively, depending on the cells
being
studied and the soluble test substance being used, the soluble test substance
may be
introduced during or after the cells have been placed in well region 14a. Once
the soluble
test substance has been introduced, by the process of diffusion, a solution
concentration
15 gradient of the test substance forms along the longitudinal axis of channel
region 15a from
well region 13a containing the test agent towards well region 14a containing
the biological
sample of cells. A secondary effect of this solution gradient is the formation
of a
physisorbed (immobilized) gradient. When this solution gradient is
established, some
fraction of the solute of the test substance may adsorb onto support member
16. This
2o adsorbed layer of test solute may also contribute to chemotaxis and
chemoinvasion. The
biological sample of cells may respond to this concentration gradient and
migrate towards
the higher concentration of the test substance, migrate away from the higher
concentration
of the test substance, or exhibit inhibited movement in response to the higher
concentration of the test substance. It is through this chemotaxis in response
to the
25 gradient, that the chemotactic influence of the chemotactic substance can
be measured.
Chemotaxis is assayed by measuring the distance the cells travel and the
amount of time
the cells take to reach a predetermined point in the channel region 15a or the
distance the
cells travel and the amount of time the cells take to reach a certain point in
well region 14a
(in the case of a chemorepellant that causes cells to move away from the
chemotactic
30 substance).
Utilizing an alternative embodiment of device 10 containing an alternative
design
of chamber 12, a solution concentration gradient is formed using a network of
microfluidic channel regions. In this embodiment as seen in Figure 14, first
well region
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WO 2004/038367 PCT/US2003/033146
well region 13a of chamber 12 has first wells, 20, 21, and 22, connected by a
network of
microfluidic capillaries 23 to channel 15. In particular, first well region
13a includes a
plurality of first wells connected by a plurality of capillaries 24 connected
to respective
ones of the plurality of first wells and a plurality of subcapillaries 25
branched off such
that each of the plurality of subcapillaries is connected to each of the
plurality of
capillaries at one end thereof and to channel 15 at another end thereof. Each
first well, 20,
21, and 22 receives a different concentration of soluble test substance. After
the three first
wells, 20, 21, and 22 are simultaneously infused with the three different
concentrations of
soluble test substance, the solution streams travel down the network of
channel regions,
to continuously splitting, mixing and recombining. After several generations
of branched
subcapillaries, each subcapillary containing different proportions of soluble
test substances
are merged into a single channel 15, forming a concentration gradient across
channel 15,
perpendicular to the flow direction.
According to another embodiment of the present invention to monitor
haptotaxis,
biomolecules are immobilized onto support member 16, preferably on the portion
of upper
surface U constituting the bottom surface of channel 15 and of well region 13a
in any one
of the embodiments of the test device of the present invention, such as the
embodiments
shown in Figures lA-14. The concentration of biomolecules increases or
decreases along
the longitudinal axis of the device from the upper surface of support member
16
constituting the bottom surface of well region 13a towards the upper surface U
of support
member 16 constituting the bottom surface of well region 14a thus forming a
surface
gradient. After the test biomolecules are immobilized on support member 16,
the top
member is placed onto support member 16 and a rigid frame with the standard
microtiter
footprint is placed around the outer perimeter of top member 11 and cells are
added to
well region 14a. In an alternative embodiment, after the test biomolecules are
irmnobilized on support member 16 and the top member is placed over support
member
16, a gel matrix is added to channel region 15a. Cells are subsequently added
to well
region 14a. The biological sample of cells potentially respond to the
concentration
gradient of immobilized biomolecules and migrates towards the higher
concentrations of
the test biomolecules, away from the higher concentrations of the test
biomolecules, or
exhibit inhibited migration in response to the higher concentrations of the
test
biomolecules. The surface gradient can increase linearly or as a squared,
cubed, or
28

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
logarithmic ftinction or in any surface profile that can be approximated in
steps up or
down.
The test biomolecules can be attached to and form surface gradients on the
upper
surface U of support member 16 by vaa.-ious specific or non-specific
approaches known in
the art as described in K. Efimenlco and J. Genzer, "How to Prepare Tunable
Planar
Molecular Chemical Gradient," 13 Applied Materials, 2001, No. 20, October 16;
U.S
Patent No. 5,514, incorporated herein by reference. For example, microcontact
printing
techniques, or any other method known in the art, can be used to immobilize on
upper
surface U of support member 16 a layer of SAMs presenting hexadecallethiol.
Support
to member 16 is then exposed to high energy light through a photolithographic
mask of the
desired gradient micropattern or a grayscale mask with continuous gradations
from white
to black. When the mask is removed, a surface gradient of SAMs presenting
hexandecanethiol remains. Support member 16 is then immersed in a solution of
ethylene
glycol terminated alkanethiol. The regions of support member 16 with SAMs
presenting
hexadecanethiol will rapidly adsorb biomolecules and the regions of the
support member
with SAMs presenting oligomers of the ethylene glycol group will resist
adsorption of
protein. Support member 16 is then immersed in a solution of the desired test
biomolecules and the biomolecules rapidly adsorb only to the regions of
support member
16 containing SAMs presenting hexadecanethiol creating a surface gradient of
2o immobilized biomolecules.
In another embodiment, the test biomolecules are immobilized on the support
member 16 and a surface concentration gradient forms after the top member 11
has been
placed over support member 16 in any one of the embodiments of the test device
of the
present invention, such as the embodiments shown in Figures lA-14. In this
embodiment,
discrete concentrations of solution containing test biomolecules are
consecutively placed
in well region 14a and allowed to adsorb non-specifically to support member
16. For
example, first, a 1 milligram/milliliter (mg/ml) of solution can first be
placed in well
region 14a; second, a 1 microgram/milliliter (~,g/ml) solution can be placed
in well region
14a; last, a lnanogram/milliliter (ng/ml) solution of test biomolecules can be
placed in
3o well region 14a. The differing concentrations of test biomolecules in
solution result in
differing amounts of adsorption on support member 16.
Utilizing an alternative embodiment of device 10 containing an alternative
design
of chamber 12 as seen in Figure 11, an immobilized biomolecular surface
gradient is
29

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WO 2004/038367 PCT/US2003/033146
formed based on the concept of laminar flow of multiple parallel liquid
streams, a method
known in the art. Based on this concept, when two or more streams with low
Reynolds
numbers are joined into a single stream, also with a low Reynolds number, the
combined
streams flow parallel to each other without turbulent mixing. According to one
embodiment, a solution of chemotactic biomolecules is placed in 17a and 19a
and a
protein solution is placed in 18a. The solutions are allowed to flow into
channel region
15a under the influence of gentle aspiration at well region 14a. Biomolecules
adsorb
nonspecifically to the regions of the surface over which the solution
containing the
biomolecules flows forming a surface gradient. The wells are then filled with
a
1o suspension of cells and potential haptotaxis of the cells towards the
increasing
concentration gradient of biomolecules is observed and monitored. See
~enerally, S.
Takayama et al., "Patterning Cells and their Environment Using Multiple
Laminar Fluid
Flows in Capillary Networks" Pro. Natl. Acad. Sci. USA, Vol. 96, pp. 5545-
5548, May
1999.
The present invention also contemplates an assay using both a soluble and
surface
gradient to determine whether the soluble test substance or the immobilized
test
biomolecules more heavily influence cell migration. In this embodiment, an
assay is
performed by forming a surface gradient as described above, an assay is
performed by
forming a solution gradient as described above, an assay is performed by
forming both
2o types of gradients and the results of all three assays are compared. With
respect to the
combined gradient assay, test biomolecules are immobilized on the upper
surface U of
support member 16 constituting the bottom surface of well region 13a and on
the upper
surface of support member 16 underlying channel region 15a and the
concentration of
biomolecules decreases along the longitudinal axis of chamber 12 from well
region 13a to
well region 14a, in any one of the embodiments of the test device of the
present invention,
such as the embodiments shown in Figures lA-14. Additionally, a soluble test
substance
is added to well region 13a. Such an embodiment creates surface and soluble
chemotactic
concentration gradients that decrease in the same direction. If the combined
concentration
gradients have a synergistic effect on cell migration, then both gradients
should be used in
screening both the cell receptor binding the chemotactic ligands of the
soluble
chemotactic substance and the cell receptor binding the immobilized
biomolecules. Both
types of receptors are identified as important and therapeutic agents that
target both these
receptors or a combination of therapeutic agents, one targeting one receptor
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CA 02503186 2005-04-20
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targeting the other receptor can be screened. If the combined concentration
gradients do
not have a synergistic effect, then the individual gradient that more strongly
promotes cell
migration can be identified and the cell receptor that binds to the
chemotactic ligands of
the test agent forming the gradient can be targeted.
Identifying optimal chemotactic ligand and receptor pairs is important in
understanding the biological pathways implicated in cell migration and
developing
therapeutic agents that target these pathways. Accordingly, the present
invention
generally provides using chemotactic test agents to determine which
chemotactic receptors
expressed on a cell's surface most heavily influence chemotaxis and/or
chemoinvasion. In
to one embodiment, the present invention provides for lugh throughput
screening of a class
of chemoattractants known to attract a particular cell type having a receptor
on the cell's
surface for each chemoattractant within this class in order to identify which
receptor is
more strongly implicated in the chemotaxis and/or chemoinvasion process. After
identifying this receptor, the present invention contemplates high-throughput
screening of
15 therapeutic agents that potentially block this receptor or bind to this
receptor, depending
on whether chemotaxis and/or chemoinvasion is desired to be promoted or
prevented. In
another embodiment, the present invention provides for high throughput
screening of
different chemoattractants known to bind to the same receptor on a particular
cell type's
surface, in order to determine which chemoattractant ligand/receptor pair more
heavily
2o influences chemotaxis and/or chemoinvasion. After identifying this
ligand/receptor pair,
the present invention contemplates high throughput screening of therapeutic
agents that
target this receptor and either block or activate this receptor depending one
whether
chemotaxis and/or chemoinvasion is desired to be promoted or prevented.
The present invention also contemplates high-throughput screening of a class
of
25 chemotactic inhibitors known to inhibit chemotaxis of a particular cell
type having various
chemotactic receptors on the cell's surface in order identify which receptor
is more
strongly implicated in the chemotaxis and chemoinvasion process. After
identifying this
receptor, the present invention provides for high throughput screening of
therapeutic
agents that potentially block this receptor as well (if such action is
desired).
3o In one embodiment of the present invention, an assay is performed to
determine
whether a test compound inhibits cancer cell invasion. In this embodiment,
untreated
cancer cells are placed in well region 14a and a test agent is placed in well
region 13a of
chamber 12 in any one of the embodiments of the test device of the present
invention,
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such as the embodiments shown in Figures lA-14. Cell chemotaxis and invasion
is
measured and recorded. After a suitable test agent is identified (one that
chemically
attracts the cancer cells) another assay is run in chamber 12. In this
subsequent assay,
cancer cells are placed in well region 14a and a test compound, for example, a
therapeutic,
is also placed in well region 14a. In another embodiment, the test compound is
also
placed in channel region 15a. If a gel matrix is to be added to channel region
15a, the test
compound can be mixed with the gel matrix before the gel is contacted with
channel
region 15a during fabrication of device 10. A subsequent sample of the test
agent
identified in the first assay is placed in well region 13a and the chemotaxis
and invasion of
l0 the cells treated with the test compound is compared to the chemotaxis and
invasion of the
cells not treated with the test compound. The test compound's anti-cancer
potential is
measured by whether the treated cancer cells have a slower chemotaxis and
invasion rate
than the untreated cancer cells. .
With respect to another exemplary use of the chemotaxis and chemoinvasion
device of the present invention, the device can be used to assay cells'
response to the
inflammatory response. A local infection or injury in any tissue of the body
attracts
leukocytes into the damaged tissue as part of the inflammatory response. The
inflammatory response is mediated by a variety of signaling molecules produced
within
the damaged tissue site by mast cells, platelets, nerve endings and
leukocytes. Some of
2o these mediators act on capillary endothelial cells, causing them to loosen
their attachments
to their neighboring endothelial cells so that the capillary becomes more
permeable. The
endothelial cells are also stimulated to express cell-surface molecules that
recognize
specific carbohydrates that are present on the surface of leukocytes in the
blood and cause
these leukocytes to adhere to the endothelial cells. Other mediators released
from the
damaged tissue act as chemoattractants, causing the bound leukocytes to
migrate between
the capillary endothelial cells into the damaged tissue. To study leukocyte
chemotaxis, in
one embodiment, channel region 15a is treated to simulate conditions in a
human blood
capillary during the inflammatory response. For example, the side walls of
channel region
15a are coated with endothelial cells expressing cell surface molecules such
as selectins,
for example as shov~m in Fig. 4B. Leukocytes are then added to well region 14a
and a
known chemoattractant is added to well region 13a in any one of the
embodiments of the
test device of the present invention, such as the embodiments shown in Figures
lA-14.
Other suitable cell types that can be added to well region 14a are
neutrophils, monocytes,
32

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
T and B lymphocytes, macrophages or other cell types involved in response to
injury or
inflammation. The leukocytes' chemotaxis across channel region 15a towards
well region
13a is observed. Depending on the type of infection to be studied, different
categories of
leukocytes can be used. For example, in one embodiment studying cell
chemotaxis in
response to a bacterial infection, well region 14a receives neutrophils. In
another
embodiment studying cell chemotaxis in response to a viral infection, well
region 14a
receives T-cells.
In another embodiment simulating the process of angiogenesis, it is known in
the
art that growth factors applied to the cornea induce the growth of new blood
vessels from
l0 the rim of highly vascularized tissue surrounding the cornea towards the
sparsely
vascularized center of the cornea. Therefore in another exemplary assay
utilizing the
chemotaxis and chemoinvasion device, cells from corneal tissue are placed in
well region
13a and endothelial cells are placed in well region 14a in any one of the
embodiments of
the test device of the present invention, such as the embodiments shown in
Figures lA-14.
15 A growth factor is added to well region 13a and chemotaxis of the
endothelial cells is
observed, measured and recorded. Alternatively, since angiogenesis is also
important in
tumor growth (in order to supply oxygen and nutrients to the tumor mass),
instead of
adding growth factor to well region 13a, cancer cells from corneal tissue that
produce
angiogenic factors such as vascular endothelial growth factor (VEGF) could be
added to
2o well region 13a and normal endothelial cells added to well region 14a. In a
different
embodiment also related to the study of angiogenesis, mast cells, macrophages,
and fat
cells that release fibroblast growth factor during tissue repair,
inflammation, and tissue
growth are placed in well region 13a and endothelial cells are placed in well
region 14a.
Since during angiogenesis, a capillary sprout grows into surrounding
connective tissue, to
25 further simulate conditions ifZ viv~, channel region 15a can be filled with
a gel matrix.
There are several variations and embodiments of the aforementioned assays. One
embodiment involves the number of channels connecting well region 13a and well
region
14a of chamber 12 of device 10. In one embodiment, such as the ones shown in
Figures
3A-3C, there are multiple channels connecting well region 13a to well region
14a. By
3o using multiple channels, multiple assays can be performed simultaneously
using one
biological sample of cells. In such an embodiment, all assays are performed
under
uniform and consistent conditions and therefore provide statistically more
accurate results.
For example, each assay begins with exactly the same number of potentially
migratory
33

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
cells and exactly the same concentration of test agent. Once a concentration
gradient
forms, each assay is exposed to the gradient for the same period of time.
These multiple
channels also provide redundancy in case of failure in the assay.
Another embodiment of the cell invasion and chemotaxis assay of the present
invention involves the placement of cells in well region 14a of chamber 12 in
any one of
the embodiments of the test device of the present invention, such as the
embodiments
shown in Figures 1A-14. The cells may be patterned in a specific array on the
upper
surface U of support member 16 constituting the bottom surface of well region
14a or may
simply be deposited in no specific pattern or arrangement in well region 14a.
If the cells
to are patterned in a specific array on the upper surface of support member 16
constituting
the bottom surface of well region 14a, then preferably, during the fabrication
of device 10,
the upper surface of support member 16 constituting the bottom surface of well
region 14a
is first patterned with cells and then top member 11 is placed over support
member 16. It
is desirable to monitor cellular movement from a predetermined "starting"
position to
accurately measure the distance and time periods the cells travel. As such, in
one
embodiment, the cells are immobilized or patterned upon the support member
underlying
the first well in such a manner that the cells' viability is maintained and
their position is
definable so that chemotaxis and invasion may be observed. There are several
teclnuques
known in the art to immobilize and pattern the cells into discreet arrays onto
the support
2o member. A preferred technique is described in copending application no.
60/330,456. In
one embodiment, a cell position patterning member is used to pattern the cells
into
definable areas onto the upper surface U of support member 16 constituting the
bottom
surface of well region 14a of top member 11.
If, for example, top member 11 is fabricated in the footprint of a standard 96-
well
microtiter plate such that wells 13 and 14 correspond to the size and shape of
the
macrowells of the microtiter plate (not shown), then the cell position pattern
member has
outlined areas which correspond to the size and shape of wells 13 and 14 and
therefore
correspond to the size and shape of the macrowells of the microtiter plate.
Each outlined
area has micro through holes through which the cells will be patterned. In
order to pattern
3o the cells, the cell position patterning member iscontacted with support
member 16 and the
outlined areas of the cell position patterning member are aligned with portion
of upper
surface U of support member 16 that constitutes the bottom surface of well
region 14a,
and will ultimately correspond to well region 14a once top member 11 is
contacted with
34

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
support member 16. Cells are then deposited over the cell position patterning
member and
filter through the micro through holes of the patterning member onto the
support member
underlying the areas corresponding to through-holes corresponding to second
well regions
14a of chambers 12. Top member 11 is then placed over support member 16 such
that
through-holes 14a are placed over the area of support member 16 in which the
cells are
patterned. These patterning steps result in discrete arrays of cells in well
region 14a.
Preferably, the cell position patterning member comprises an elastomeric
material
such as PDMS. Using PDMS for the patterning member provides a substantially
fluid-
tight seal between the patterning member and the support member. This
substantially
to fluid-tight seal is preferable between these two components because cells
placed in the
wells are less likely to infiltrate adjoining wells if such a seal exists
between the patterning
member and the support member. The arrangement of the micro through holes of
the
patterning member may be rectangular, hexagonal, or another array resulting in
the cells
being patterned in these respective shapes. The width of each micro-through
hole may be
15 varied according to cell types and desired number of cells to be patterned.
For example, if
the width of both cell and micro through hole is 10 microns, only one cell
will deposit
through each micro through hole. Thus, in this example, if the width of micro
through hole
is 100 microns up to approximately 100 cells may be deposited.
The present invention also contemplates the patterning of more than one cell
type
20 on the upper surface of support member 16 constituting the bottom surface
of well region
14a in any one of the embodiments of the test device of the present invention,
such as the
embodiments shown in Figures lA-14. Since cells of one type in vivo rarely
exist in
isolation and are instead in contact and communication with other cell types,
it is desirable
to have a system in which cells can be assayed in an environment more like
that of the
25 body. For example, since cancer cells are never found in isolation, but
rather surrounded
by normal cells, an assay designed to test the effect of a drug on cancer
cells would be
more accurate if the cancer cells in the assay were surrounded by normal
cells. In testing
an anti-cancer drug, cancer cells may be patterned on the upper surface of
support member
16 constituting the bottom surface of well region 14a in any given one of the
embodiments
30 of the test device of the present invention, such as the embodiments of
Figs. lA-14, and
then through a separate patterning procedure, the cancer cells may be
surrounded by
stromal cells. To pattern two different cell types on the upper surface of
support member
16 constituting the bottom surface of well region 14a, a micro cell position
patterning

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
member, as described above, is contacted with support member 16 and the
outlined areas
of the cell position patterning member are aligned with the portion of upper
surface U of
support member 16 that constitutes the bottom surface of well region 14a, and
will
ultimately correspond to well region 14a once top member 11 is contacted with
support
member 16. Cells of a first type may then be deposited over the cell position
patterning
member and filter through the micro through holes of the patterning member
onto the
portion of the upper surface U of support member 16 constituting the bottom
surface of
well region 14a. The micro cell position patterning member may then be removed
from
support member 16. A macro cell position patterning member with outlined areas
that
correspond to the size and shape of wells 13 and 14 and may therefore
correspond to the
size and shape of the macrowells of a 96 well microtiter plate. The macro cell
position
patterning member has macro through holes. A macro through hole of the macro
cell
position patterning member encompasses an area larger than the surface area
defined by a
micro through hole of the micro cell position patterning member, but smaller
than the
surface area defined by well region 14a of chamber 12. The macro cell position
patterning
member may then be contacted with support member 16. Cells of a second type
may then
be deposited over the macro cell position patterning member and filter through
the macro
through holes of the macro cell position patterning member onto the portion of
upper
surface U of support member 16 constituting the bottom surface of well regions
14a once
2o top member 11 is contacted with support member 16. Such patterning
arrangement may
result in cells of a second type surrounding and "stacking" cells of a first
type. If it is
desired to only have the cells of the second type stack the cells of the first
type, then the
same micro cell position patterning member used to deposit the first cell type
or a different
micro cell position patterning member having the exact same configuration as
the
patterning member used to deposit cells of a first type, may be used to
deposit cells of a
second type. After the cells are patterned on support member 16, top member 11
may be
contacted with support member 16 such that through holes in top member 11
corresponding to the well region 14a encompass the areas patterned with cells.
This
essentially results in cells being immobilized in a specific array within well
region 14a.
3o Notwithstanding how many different cell types are patterned on the upper
surface
of support member 16 constituting the bottom surface of well region 14a, the
cells may be
patterned on the support member through several methods known in the art. For
example,
the cells may be patterned on support member 16 through the use of SAMS. There
are
36

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
several techniques known in the art to pattern cells through the use of SAMs
of which a
few exemplary techniques disclosed in U.S. Patent No. 5,512,131 to Kumar et
al., U.S.
Patent No. 5,620,850 to Bambad et al., U.S. Patent No. 5,721,131 to Rudolph et
al., U.S.
Patent Nos. 5,776,748 and 5,976,826 to Singhvi et al. are incorporated by
reference herein.
Several methods are known in the art to tag the cells in order to observe and
measure the aforementioned parameters. In one embodiment, axl unpurified
sample
containing a cell type of interest is incubated with a staining agent that is
differentially
absorbed by the various cell types. The cells are then placed in well region
14a of
chamber 12 in any given one of the embodiments of the test device of the
present
l0 invention, such as the embodiments of Figs. lA-14. Individual, stained
cells are then
detected based upon color or intensity contrast, using any suitable microscopy
technique(s), and such cells are assigned positional coordinates. In another
embodiment,
an unpurified cell sample is incubated with one or more detectable reporters,
each reporter
capable of selectively binding to a specific cell type of interest and
imparting a
15 characteristic fluorescence to all labeled cells. The sample is then placed
in well region
14a of chamber 12 in any given one of the embodiments of the test device of
the present
invention, such as the embodiments of Figs. lA-14. The sample is then
irradiated with the
appropriate wavelength light and fluorescing cells are detected and assigned
positional
coordinates. One skilled in the art will recognize that a variety of methods
for
2o discriminating selected cells from other components in an unpurified sample
are available.
For example, these methods can include dyes, radioisotopes, fluorescers,
chemiluminescers, beads, enzymes, and antibodies. Specific labeling of cell
types can be
accomplished, for example, utilizing fluorescently-labeled antibodies. The
process of
labeling cells is well known in the art as is the variety of fluorescent dyes
that may be used
25 for labeling particular cell types.
Cells of a chosen type may be also differentiated in a mixed-cell population,
for
example, using a detectable reporter or a selected combination of detectable
reporters that
selectively and/or preferentially bind to such cells. Labeling may be
accomplished, for
example, using monoclonal antibodies that bind selectively to expressed CDs,
antigens,
30 receptors, and the like. Examples of tumor cell antigens include CD13 and
CD33 present
on myeloid cells; CD10 and CD19 present on B-cells; and CD2, CDS, and CD7
present on
T-cells. One of skill in the art will recognize that numerous maxkers are
available that
identify various known cell markers. Moreover, additional markers are
continually being
37

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
discovered. Any such markers, whether known now or discovered in the future,
that are
useful in labeling cells may be exploited in practicing the invention.
Since few, if any markers are absolutely specific to only a single type of
cell, it
may be desirable to label at least two markers, each with a different label,
for each chosen
cell type. Detection of multiple labels for each chosen cell type should help
to ensure that
the chemotaxis and chemoinvasion analysis is limited only to the cells of
interest.
The present invention further provides a test device comprising: support
means;
means mounted to the support means for defining a discrete chamber with the
support
means by being placed in fluid-tight, confonnal contact with the support
means. The
l0 discrete chamber includes a first well region including at least one first
well; a second well
region including at least one second well, the second well region further
being horizontally
offset with respect to the first well region in a test orientation of the
device; and a channel
region including at least one channel coimecting the first well region and the
second well
region with one another. An example of the support means comprises the support
member
15 16 shown in Figures lA, 1B, 12 and 13, while an example of the means
mounted to the
support means comprises the top member 11 shown in Figures lA-11, 13 and 14.
Other
such means would be well known by persons skilled in the art.
In another embodiment, the present invention provides methods of assaying and
studying biological phenomenon that either depend on or react to gradient
formation
20 and/or flow conditions. Such biological phenomenon include many of the
processes in the
body such as cell-surface interactions such as that occurring during leukocyte
adhesion
and rolling. In addition, studies involving chemotaxis, haptotaxis and cell
migration will
be better served with assays that are able to study such cell movement in the
presence of
gradients andlor flow conditions.
25 Various types of gradients are useful in the study of biological systems.
Such
useful gradients include static gradients, which have concentrations that are
fixed, or set or
substantially fixed or set. One example of a static gradient is a gradients of
immobilized
molecules on a surface. Non-limiting examples of static gradients include the
use of
differing concentrations of immobilized biomolecules (proteins, antibodies,
nucleic acids,
30 and the like) or immobilized chemical moieties (drugs and small molecules).
Other useful
gradients include dynamic gradients, which have concentrations that may be
varied. One
example of a dynamic gradient is a gradient of fluid streams having molecules
in varying
concentrations. Non-limiting examples of fluid gradients include the use of
fluid streams
38

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
containing biomolecules such as growth factors, toxins, enzymes, proteins,
antibodies,
carbohydrates, drugs or other chemical and small molecules in varying
concentrations.
In one embodiment of the present invention, a dynamic/solution based gradient
is
created by laminar flow technology. Laminar flow technology typically involves
two or
more fluid streams from two or more different sources. These fluid streams are
brought
together into a single stream and are made to flow parallel to each other
without turbulent
mixing. Fluids with different characteristics such as varying low Reynolds
numbers will
flow side by side and will not mix in the absence of turbulence. Since the
fluids do not
mix, they create pseudo-channels (pseudo by the fact that there is no physical
separation
to between the fluids). The generation of solution and surface gradients is
discussed in U.S.
patent application 2002/0113095 and an article, Jeon, Noo Li, et al.,
Langmuir, 16, 8311-
8316 (2000). Both of these references are herein incorporated by reference in
their
entirety.
In these references a PDMS microfluidic device was used to generate a gradient
through a microfluidic network of capillaries. Solutions containing different
chemicals
were introduced into three separate inlets and allowed to flow through the
network of
capillaries. The fluid streams were repeatedly combined, mixed, and split to
yield distinct
mixtures with distinct compositions in each of the branching channels. When
all of the
branches were recombined, a concentration gradient was established across the
outlet
2o channel, perpendicular to the flow direction. See Figure 22.
By combining the devices of the present invention with the formation of a
dynamic
gradient, a vast number of assay parameters can be generated by altering any
portion of
the device. For example, by combining the device as disclosed herein with cell
patterning
techniques, along with the introduction of a dynamic gradient, various
conditions can be
created to test numerous biological interactions. Further, the device and
assays may be
useful in drug discovery and drug testing as many cells and biological
materials behave
differently ex vivo when not exposed to gradients than compared to when the
cells or
biological materials are present ira vivo and thus exposed to gradients and
flow conditions.
Accordingly, in one embodiment of the present invention, cells can be
patterned
3o across the channel. Cell patterning can be achieved by methods known in the
art, as well
as disclosed in the present invention (such as, but not limited to,
microcontact printing or
by the use of elastomeric stencils). A solution containing any desired
biomolecule or
chemical/drug can then be flowed across the patterned cells. Additionally, the
cells could
39

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
be first treated by a biomolecule such as an activator to more closely
recreate a biological
system, and then be subsequently exposed to a chemical or drug. By creating a
gradient,
such as by laminar flow, different amounts of biomolecules or chemicals/drugs
can be
delivered to the patterned cells and thus the effect of concentration of each
biomolecule or
chemical/drug be tested simultaneously against each other. This side by side,
same time
comparison thus reduces the variability of assay to assay conditions.
Creating dynamic gradients with laminar flow in combination with the devices
of
the present invention provides numerous assay configurations. For example, by
varying
the combinations of the cells on the surface, the biomolecule in the channels
and the
to compounds in the channel, one can create a vast multitude of assays.
With respect to immobilized cells or other immobilized biomolecules such as
proteins, antibodies, nucleic acids, etc. different assay configurations are
possible. In one
embodiment, a single cell type is immobilized throughout the entire channel
region. In
another embodiment, a mixture of cell types are immobilized, one cell type per
region. In
another embodiment, a mixture of cell types is immobilized throughout the
entire channel
region. This may be advantageous in monitoring cell-cell interactions. In yet
another
embodiment, different cell types are immobilized in each different region.
W addition to the various immobilization schemes, further assay design
flexibility
centers around the biomolecules present in the channels. For example, in one
embodiment, one type of biomolecule is present in each channel at the same
concentration. In another embodiment, one type of biomolecule is present in
each channel
at differing concentrations. In another embodiment, different biomolecules are
present in
each channel. hl another embodiment, there is a mixture of biomolecules in
each channel.
Each channel may have the same mixture or a different mixture. When the
mixture is the
same, the ratios or concentrations of the different biomolecules may be
different in each
chamlel.
Likewise with respect to compounds, such as drugs or test substances, the
present
invention provides flexibility in assay design. For example, in one embodiment
a single
compound is present in all the channels at the same concentration throughout.
In another
3o embodiment, the same compound is present in all the channels but each
channel has a
different concentration of that compound. In another embodiment, each channel
has a
different compound. In another embodiment, there is more than one compound.
When
there is more than one compound, each channel may have the same mixture of
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CA 02503186 2005-04-20
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or may have a different mixture of compounds. Further, when the mixtures of
the
compounds are the same, each channel may receive a different concentration of
that
mixture. Yet, even further, each channel may receive the mixture of the
compounds, with
each channel having a different ratio of compounds to each other.
Such assay systems can be used to test among many numerous biological
interactions, the effects of chemical or drugs on cells or other biomolecules.
For example,
one may use the device and the assays of the present invention to measure the
IC50 of a
compound by using a laminar flow gradient of a compound present from a low
concentration to a high concentration flowed across immobilized biomolecules.
to From the foregoing, it will be observed that numerous modifications and
variations
can be effected without departing from the true spirit and scope of the novel
concept of the
present invention. For example, different embodiments of a device of the
present invention
may be combined. Embodiments of the present invention further contemplate
different
types of assays, for example, an assay wherein the test agent comprises a
buffer solution
15 instead of a chemotactic agent. In such an assay, cell migration through
channel region
15a in observed in the absence of a chemotactic gradient.
It will be appreciated that the present disclosure is intended to set forth
the
exemplifications of the invention, and the exemplifications set forth are not
intended to
limit the invention to the specific embodiments illustrated. The disclosure is
intended to
2o cover by the appended claims all such modifications as fall within the
spirit and scope of
the claims.
EXAMPLES
25 EXAMPLE 1: PROCEDURE FOR FABRICATION OF CHEMO1NVASION DEVICE
A silicon wafer (6 inches) is spin coated with photoresist (SU8-50) at 2000rpm
for
45 seconds. After baking the wafer on a hot plate at 115°C for 10
minutes, the wafer is
allowed to cool to room temperature. A mask aligner (EVG620) is used to expose
the
photoresist film through a photomask. Exposure of 45 seconds is followed by
another hard
3o bake at 115° C for 10 minutes. The silicon wafer is allowed to cool
to room temperature
for over 30 minutes. The uncrosslinked photoresist is removed using propylene
glycol
methyl ether acetate (PGMEA). The wafer is dried under a stream of nitrogen,
and the
patterned photoresist is ready for subsequent processing.
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In one embodiment, the patterned photoresist is spin-coated with another layer
of
SU8-100 at 1500 rpm for 45 seconds. A mask aligner is used to selectively
expose
macrofeatures (i.e. wells) of the top member but not expose channel regions
connecting
the wells and other areas of the top member. After post exposure processing
and
photoresist removal, the master contains multiple layered features. This step
may be
repeated to introduce macro-features on the master, which have the height of
approximately 3mm.
When a PDMS prepolylner is cast against the master, it faithfully replicates
the
features in the master. When casting, PDMS is added in an amount slightly
lower than the
to height of the macrofeatures. After curing the PDMS for four hours at 65
degrees C, the
PDMS is peeled off the silicon master and thoroughly cleaned with soap and
water and
rinsed with 100% ethanol. A glass support member is also cleaned and rinsed
with
ethanol. The PDMS membrane and glass support member axe plasma oxidized for 1
minute with the sides that would be bonded together facing upward. The PDMS
15 membrane is then placed onto the glass support member and pressure is
applied to remove
any air bubbles that may have formed between the PDMS membrane and the glass
support
member. The assembled device is then cooled to 4°C. Within 15 minutes
of the plasma
oxidation of the PDMS membrane and the glass support member, 20 microliters
(p.l) of
Matrigel (any other hydrogel may be used) is poured into the first well and
allowed to
2o flow into the capillaries. The device is placed at room temperature for 15
minutes to set
the Matrigel. Excess gel is then removed from the wells of the top member
using a
vacuum and a Pasteur pipette.
EXAMPLE 2: CELL CHEMOINVASION ASSAY
25 Placement of Cells and Test agent in Chamber
The first and second wells of a chamber of a top member are filled with
phosphate
buffered saline solution, PBS. The bottom of the second well may be treated
with
fibronectin (lmg/ml) or other extracellular matrix protein for 30 minutes,
followed by
washing twice with PBS. After aspirating PBS, astrocytoma cells (LT87-MG) are
plated in
3o SOp,I of freshly warmed medium in the second well (25,000 cells per well of
a 24-well
plate, in volume of SOuI of solution per well). The cells deposit through the
second well of
the chamber, and attach to the bottom of the second well.
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CA 02503186 2005-04-20
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Cells are left to attach and spread in the second well overnight in a
37°C incubator.
At the start of the experiment, the cell medium is exchanged for fresh serum-
free medium.
10~,g of bFGF (basic fibroblast growth factor) per ml of medium is added to
the first well
of each chamber.
Image Acquisition and Data Analysis
Digital Images are taken on a Zeiss inverted microscope using AXIOCAMTM.
Data was analyzed on AXIOVISIONTM software. Time-lapsed images are taken every
day
at the same time for four days.
1o EXAMPLE 3: CELL CHEMOINVASION INHIBITION ASSAY USING SOLUTION
GRADIENT
Placement of Cells and Test agent in Chambers
With respect to three chambers, the wells of each chamber of a top member are
filled with PBS. The bottom of the second wells may be treated with
fibronectin (1mg/ml)
15 or other extracellular matrix protein for 30 minutes, followed by washing
twice with PBS.
After aspirating PBS, U87-MG cells are plated in 50,1 of freshly warmed medium
in the
second wells (10,000 cells per well of a 24-well plate, in volume of 50,1 of
medium per
well). The cells deposit through the second wells of each chamber, and adhere
to the
bottom of the second wells.
2o Cells are left to attach and spread in the second wells overnight in a
37°C
incubator. At the start of the experiment, the cell medium is exchanged for
fresh serum-
free medium or 1 % serum. 1 ~,g of bFGF (basic fibroblast growth factor) per
ml of medium
is added to the first wells of the chamber. A solution gradient is allowed to
form for one
hour.
25 With respect to the three different chambers, 100 ~,M of LY294002 are
placed in
the second well of chamber #1, 10~.M LY294002of are placed in the second well
of
chamber #2, and 1.O~,M of LY294002 are placed in the second well of chamber
#3.
Image Acquisition and Data Analysis
Digital Images are taken on a Zeiss inverted microscope using AXIOCAMTM
30 Data was analyzed on AXIOVISIONTM software. Time-lapsed images are taken
every day
at the same time for four days.
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WO 2004/038367 PCT/US2003/033146
EXAMPLE 4: IMMOBILIZATION OF BIOMOLECULES ON SUPPORT MEMBER
After assembling the device as described above, the channel regions are filled
with
ethanolic solution containing (CH3CH20)3Si (CHz)3NHz. After 20 minutes at room
temperature, the channel regions are washed off using ethanol. The device is
incubated at
105°C for one hour to crosslink the siloxane monolayer formed on the
support member.
The device is washed with ethanol to remove residues. The channel regions are
filled with
a solution of diisocyanate, either hexamethylene diisocyanate or tolyl
diisocyanate (1 % in
acetonitrile or N-methyl pyrrolidinone). The diisocyanate is allowed to react
for two hours
with the terminal amino groups of the siloxane monolayer formed on the support
member.
to The diisocyanate is washed off. The channel regions are filled with lmg/ml
solution of
heparan sulfate or other sulfated carbohydrates (for example, di-acetylated
form of
heparin, heparin fragments, lectins containing sulfated sugars, etc.) The
heparan sulfate is
allowed to react with the support member to form immobilized species. The
heparan
sulfate solution and other reagents are washed off. A chemokine solution (any
chemokine
is from CC, CXC, CX3C, or XC families may be used) is introduced into the
channel region.
By electrostatic interaction, chemokines that have higher pI (~9-10) adsorb
onto the
negatively charged sulfated support member.
EXAMPLE 5: CHEMOTAXIS INHIBITION ASSAY USING SURFACE GRADIENT
2o Two wells are filled with SOp,I of PBS, and hydrostatic pressure is allowed
to
equalize. 5~,1 of anti-hisx6 antibody are added to the first well and 5~.1 of
buffer are added
to the second well to equalize hydrostatic pressure. By diffusion, the
antibody
concentration forms a gradient from the first well to the second well. After 2
hours at
room temperature, the two wells are washed off by adding SOp,I of buffer to
the second
25 well and removing 50,1 from the first well. By physisorption, the solution
gradient is
transferred onto a surface thereby forming a surface gradient. A solution of
IL-8
(recombinant human IL-8 with a HISx6 fusion tag, R+D systems, catalog No. 968-
IL) at
concentration of 25~.g/ml is added to the channel regions. The solution is
allowed to
incubate for 30 minutes at room temperature. Excess IL-8 chemokine is washed
off and
3o the surface is decorated with bound IL-8. Neutrophils(freshly isolated from
a healthy
donor) are added to the second well. Typically 20,000-100,000 cells are added
in volume
ranging from 10-550,1. Neutrophils are allowed to adhere to the support member
and
44

CA 02503186 2005-04-20
WO 2004/038367 PCT/US2003/033146
allowed to migrate towards the higher concentration of IL-8. Inhibition of
migration is
achieved by adding polyclonal antibody against IL-8.
EXAMPLE 6: SELECTIVE ACTIVATION OF ENDOTHELIAL CELLS BY
DELIVERY OF TNF-a IN A GRADIENT CREATED BY LAMINAR FLOW
The surface of a device of the present invention was coated with endothelial
cells
and allowed to grow to confluence (to create a "lawn" of cells). TNF-a was
delivered to
the lawn of endothelial cells via laminar flow to "activate" the endothelial
cells. Each
stream of solutions containing TNF-a were at different concentrations, thus
creating a
to gradient perpendicular to the channel. This gradient effectively delivered
TNF-a to the
lawn of endothelial cells at different concentrations at different positions
on the lawn of
cells. Leukocytes were then flowed over the lawn of activated endothelial
cells. Only
those endothelial cells that were activated by TNF-a provide suitable
"attachment" sites
for the leukocytes. The leukocytes did not attach equally to the entire lawn,
but attached
to the areas of the endothelial cell lawn that had been exposed to high
concentrations of
TNF-a and did not attach to those areas of the lawn that had been exposed to
low
concentrations of TNF-a, or those areas not exposed to TNF-a at all. These
results
indicate that there was indeed a creation of a concentration gradient of TNF-a
by the
laminar flow. See figure 21.

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

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Event History

Description Date
Inactive: IPC deactivated 2012-01-07
Inactive: IPC removed 2011-12-08
Inactive: IPC assigned 2011-12-08
Inactive: First IPC assigned 2011-12-08
Inactive: IPC removed 2011-12-08
Inactive: IPC removed 2011-12-08
Inactive: IPC removed 2011-12-08
Inactive: IPC removed 2011-12-08
Time Limit for Reversal Expired 2011-10-21
Application Not Reinstated by Deadline 2011-10-21
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2010-10-21
Letter Sent 2010-04-29
Reinstatement Requirements Deemed Compliant for All Abandonment Reasons 2010-04-13
Inactive: IPC expired 2010-01-01
Deemed Abandoned - Failure to Respond to Maintenance Fee Notice 2009-10-21
Letter Sent 2008-11-12
Request for Examination Requirements Determined Compliant 2008-10-14
All Requirements for Examination Determined Compliant 2008-10-14
Request for Examination Received 2008-10-14
Inactive: IPRP received 2006-04-27
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: IPC from MCD 2006-03-12
Inactive: Cover page published 2005-07-19
Inactive: First IPC assigned 2005-07-17
Inactive: Notice - National entry - No RFE 2005-07-15
Letter Sent 2005-07-15
Application Received - PCT 2005-05-09
National Entry Requirements Determined Compliant 2005-04-20
National Entry Requirements Determined Compliant 2005-04-20
Application Published (Open to Public Inspection) 2004-05-06

Abandonment History

Abandonment Date Reason Reinstatement Date
2010-10-21
2009-10-21

Maintenance Fee

The last payment was received on 2010-04-13

Note : If the full payment has not been received on or before the date indicated, a further fee may be required which may be one of the following

  • the reinstatement fee;
  • the late payment fee; or
  • additional fee to reverse deemed expiry.

Please refer to the CIPO Patent Fees web page to see all current fee amounts.

Fee History

Fee Type Anniversary Year Due Date Paid Date
Registration of a document 2005-04-20
Basic national fee - standard 2005-04-20
MF (application, 2nd anniv.) - standard 02 2005-10-21 2005-10-12
MF (application, 3rd anniv.) - standard 03 2006-10-23 2006-10-23
MF (application, 4th anniv.) - standard 04 2007-10-22 2007-10-22
Request for examination - standard 2008-10-14
MF (application, 5th anniv.) - standard 05 2008-10-21 2008-10-21
Reinstatement 2010-04-13
MF (application, 6th anniv.) - standard 06 2009-10-21 2010-04-13
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
SURFACE LOGIX, INC.
Past Owners on Record
EMANUELE OSTUNI
ENOCH KIM
GREGORY L. KIRK
OLIVIER SCHUELLER
PAUL SWEETNAM
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2005-04-20 45 2,973
Drawings 2005-04-20 19 391
Claims 2005-04-20 4 175
Abstract 2005-04-20 1 54
Cover Page 2005-07-19 1 30
Reminder of maintenance fee due 2005-07-18 1 109
Notice of National Entry 2005-07-15 1 191
Courtesy - Certificate of registration (related document(s)) 2005-07-15 1 114
Reminder - Request for Examination 2008-06-25 1 119
Acknowledgement of Request for Examination 2008-11-12 1 190
Courtesy - Abandonment Letter (Maintenance Fee) 2009-12-16 1 172
Notice of Reinstatement 2010-04-29 1 163
Courtesy - Abandonment Letter (Maintenance Fee) 2010-12-16 1 173
PCT 2005-04-20 3 101
PCT 2003-10-21 1 41
PCT 2003-10-21 1 40
PCT 2005-04-21 3 167