U.S. patent application number 13/651522 was filed with the patent office on 2013-03-21 for method for studying floating, living cells.
This patent application is currently assigned to Seng Enterprises Ltd.. The applicant listed for this patent is Seng Enterprises Ltd.. Invention is credited to Mordechai DEUTSCH.
Application Number | 20130071914 13/651522 |
Document ID | / |
Family ID | 36602667 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130071914 |
Kind Code |
A1 |
DEUTSCH; Mordechai |
March 21, 2013 |
METHOD FOR STUDYING FLOATING, LIVING CELLS
Abstract
Disclosed is a method of studying floating living cell by
confining the cells floating in an aqueous medium in picowells
located on a bottom surface of a top wall of a chamber. Disclosed
is also a picowell-bearing device for the study of cells comprising
a chamber having at least one picowell on a bottom surface of the
chamber. Also disclosed is a picowell-bearing device for the study
of cells comprising a chamber having at least one picowell on an
internal surface and at least one moveable wall, a force applicator
configured to removeably apply a substantially uniformly
distributed force around the periphery of the moveable wall in a
direction to retain the moveable wall secured to the chamber.
Inventors: |
DEUTSCH; Mordechai; (Moshav
Olesh, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seng Enterprises Ltd.; |
Larnaca |
|
CY |
|
|
Assignee: |
Seng Enterprises Ltd.
Larnaca
CY
|
Family ID: |
36602667 |
Appl. No.: |
13/651522 |
Filed: |
October 15, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12084462 |
May 1, 2008 |
8288120 |
|
|
PCT/IL2006/000483 |
Apr 20, 2006 |
|
|
|
13651522 |
|
|
|
|
60734287 |
Nov 8, 2005 |
|
|
|
60734286 |
Nov 8, 2005 |
|
|
|
60732827 |
Nov 3, 2005 |
|
|
|
Current U.S.
Class: |
435/286.5 ;
435/283.1 |
Current CPC
Class: |
B01J 2219/00317
20130101; B01J 2219/00286 20130101; B01J 2219/00743 20130101; B01L
2200/0668 20130101; C12M 23/12 20130101; B01L 3/502761 20130101;
B01J 19/0046 20130101; B01L 2200/0689 20130101; B01L 2400/0406
20130101; B01J 2219/00585 20130101; B01L 2300/0822 20130101 |
Class at
Publication: |
435/286.5 ;
435/283.1 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/36 20060101 C12M001/36 |
Claims
1. A picowell-bearing device for the study of living cells,
comprising: a) a chamber having a first wall and a second wall,
respectively defining top and bottom walls for the chamber, wherein
each wall has a first surface inside the chamber and a second
surface outside the chamber; and b) a first picowell inside the
chamber, a bottom of which is on the first surface of the top wall,
wherein the device is configured such that the picowell is oriented
with an open end of the facing downward when a living cell therein
is being studied.
2. The device of claim 1, wherein the first picowell is comprised
in a first picowell array, all the picowells of which have their
open ends facing downward when living cells therein are being
studied.
3. The device of claim 1, further comprising a second picowell
inside the chamber, a bottom of which is on the top surface of the
bottom wall of the chamber, and which has an open end facing upward
when a living cell therein is being studied.
4. The device of claim 3, wherein the second picowell is comprised
in a second picowell array, all the picowells of which have their
open ends facing upward when living cells therein are being
studied.
5. The device of claim 1, further comprising at least one fluid
inlet path located below the open end of the first picowell in
fluid communication with the chamber when a living cell in the
first picowell is being studied.
6. The device of claim 5, wherein the fluid inlet path is a
capillary channel located below the open end of the first
picowell.
7. The device of claim 1, further comprising at least one fluid
outlet path in fluid communication with the chamber when a living
cell in the first picowell is being studied configured for removal
of liquid from the chamber.
8. The device of claim 7, wherein the fluid outlet path is a
capillary channel located below the open end of the first
picowell.
9. The device of claim 8, including a further fluid outlet path
configured for removal of gas from the chamber.
10. The device of claim 1, wherein the top wall of the chamber is
defined, at least in part, by a component that carries the first
picowell.
11. The device of claim 2, wherein the top wall of the chamber is
defined, at least in part, by a component that carries the first
picowell array.
12. The device of claim 1, further comprising a base that defines,
at least in part, the bottom wall of the chamber.
13. The device of claim 12, further including a body positioned
above the base, to which the first picowell array is attached.
14. The device of claim 13, wherein the base is moveable relative
to the body to provide access to the interior of the chamber.
15. The device of claim 13, wherein the body includes transversely
spaced longitudinally extending portions that define a sliding path
for movement of the base.
16. The device of claim 13, further including a base holder that
cooperates with the body to retain the base in contact with a
bottom surface of the body, while permitting sliding motion of the
base.
17. The device of claim 16, wherein the contact between the base
and the body is such as to substantially prevent leakage of fluid
from the chamber.
18. The device of claim 13, wherein a bottom surface of the body
includes a hollow in which the first picowell array is located.
19. The device of claim 13, wherein the interior of the chamber is
in communication with a fluid inlet path and a fluid outlet path
formed by opposite ends of a capillary channel defined by a portion
of the body and a portion of the base.
20. The device of claim 18, further including a downwardly
extending portion of the body on which the hollow is located, and
which cooperates with the base to form at least part of the
capillary channel.
21. The device of claim 20, wherein the portion of the capillary
channel downstream of the first picowell array provides a waste
removal path.
22. The device of claim 21, further including a waste reservoir at
the downstream end of the waste removal path portion of the
capillary channel.
23. The device of claim 16, wherein the base holder is configured
to resiliently engage the device body to retain the base in place
vertically between device body and base holder.
24. The device of claim 18, further including a liquid inlet
opening, wherein the base and the liquid inlet opening cooperate to
form a liquid reservoir in fluid communication with the capillary
channel.
25. The device of claim 24, wherein the liquid inlet opening is
formed by a hole through the device body.
26. The device of claim 2, wherein the picowells of the first
picowell array are porous.
27. The device of claim 26, further comprising a second chamber
located above the picowell-containing chamber when the device is
oriented for study of cells in the picowell array, wherein the
second chamber is, in fluid communication with the
picowell-containing chamber through the pores of the picowells
comprised in the first picowell array.
28. The device of claim 27, further including an air vent in the
second chamber, wherein the upper chamber is configured to allow
escape through the air vent of excess gas accumulated in the upper
chamber after introduction of the medium and living cells into the
device.
29. The device of claim 1, wherein the chamber includes at least
one element that is moveable to provide access to the interior of
the chamber; and wherein the device further includes a force
applicator operable to apply a force to the moveable element to
retain the moveable wall secured to the chamber.
30. The device of claim 29, wherein the force is a magnetic
force.
31. The device of claim 29, wherein the force applicator includes a
magnet located at least in proximity to the periphery of the
moveable wall.
32. The device of claim 29, wherein the force applicator includes
threads on the moveable wall.
33. The device of claim 29, wherein the force applicator includes a
rigid force distributor contacting the periphery of the moveable
wall.
34. The device of claim 29, wherein the force is applied
substantially uniformly around the periphery of the moveable
wall.
35. The device of claim 21, further including a body of absorbent
material in fluid communication with the waste removal path.
36. The device of claim 1, further including a medium loading
opening in fluid communication with the chamber, and a removable
component to substantially seal the medium loading opening
subsequent to introducing a fluid medium and at least one living
cell into the chamber for study.
37. The device of claim 2, wherein the first picowell array is an
integral part of the bottom surface of the top wall of the
chamber.
38. The device of claim 3, wherein the bottom wall of the chamber
includes a hollow on the surface thereof inside the chamber that
contains the second picowell or the second picowell array.
39. A picowell-bearing device for the study of living cells that
have a density lower than that of a medium in which they are
contained, comprising: a) a chamber having a top and a bottom wall,
each wall having a first surface inside the chamber, and a second
surface outside the chamber; b) a first picowell array on the first
surface of the top wall, wherein the picowells comprised in the
picowell array are open at the ends that face the bottom wall of
the chamber when cells in the picowells are being studied, whereby
cells introduced into the chamber that have a density lower than
that of a medium in which they are contained float upward and are
trapped in the picowells during study; c) a fluid reservoir coupled
to the chamber by an inlet flow path; d) an outlet flow path from
the chamber; e) a first flow control element that controls the rate
of liquid flow from the fluid reservoir to the chamber; f) a gas
reservoir coupled to the inlet flow path; and g) a second flow
control element that controls the rate of gas flow from the gas
reservoir to the inlet flow path.
40. The device of claim 39, further including a controller that
controls the first and second flow control elements.
41. The device of claim 40, wherein the controller includes a
microprocessor.
42. The device of claim 40, further including sensors connected to
the controller for the temperature of the medium in the chamber,
and for the flow rate and gas content of the liquid in the inlet
flow path.
43. The device of claim 40, further including a heater that
controls the temperature of a medium in the chamber; and a
temperature sensor connected to the controller that determines the
temperature of the medium in the chamber, wherein the controller is
operable to control the heater to maintain a desired temperature of
the medium.
44. The device of claim 39, wherein fluid exiting the chamber
through the outlet flow path is fed back to reenter the chamber
through the inlet flow path.
45. The device of claim 39, further including a second picowell
array on the first surface of the bottom wall of the chamber,
wherein the picowells comprised in the second picowell array are
open at the ends that face the top wall of the chamber when cells
in the second picowell array are being studied, whereby cells
introduced into the chamber that have a density higher than that of
a medium in which they are contained sink downward and are trapped
in the picowells of the second picowell array during study.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/084,462 filed on May 1, 2008, which is a U.S. National
Phase of PCT Patent Application No. PCT/IL2006/000483 filed on Apr.
20, 2006, which claims the benefit of priority under 35 USC
.sctn.119(e) of U.S. Provisional Patent Application Nos. 60/734,287
and 60/734,286, both filed on Nov. 8, 2005, and 60/732,827 filed on
Nov. 3, 2005. The contents of the above applications are all
incorporated by reference as if fully set forth herein in their
entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to the field of cellular
biology and more particularly, to a device and method for the study
of cells. Specifically, the present invention is of a device
including a picowell array for the study of cells as well as of a
device configured for the study of cells that float in aqueous
media.
[0003] Combinatorial methods in chemistry, cellular biology and
biochemistry are essential for the near simultaneous preparation of
multitudes of active entities such as molecules. Once such a
multitude of molecules is prepared, it is necessary to study the
effect of each one of the active entities on a living organism.
[0004] The study of the effects of stimuli such as exposure to
active entities on living organisms is preferably initially
performed on living cells. Since cell-functions include many
interrelated pathways, cycles and chemical reactions, the study of
an aggregate of cells, whether a homogenous or a heterogeneous
aggregate, does not provide sufficiently detailed or interpretable
results: rather a comprehensive study of the biological activity of
an active entity may be advantageously performed by examining the
effect of the active entity on a single isolated living cell. Thus,
the use of single-cell assays is one of the most important tools
for understanding biological systems and the influence thereupon of
various stimuli such as exposure to active entities.
[0005] The combinatorial preparation of a multitudes of active
entities coupled with the necessity of studying the effect of each
one of the active entities on living organisms using single-cell
assays, requires the development of high-throughput single live
cell assays. There is a need for the study of real-time responses
to stimuli in large and heterogeneous cell populations at an
individual cell level. In such studies it is essential to have the
ability to define multiple characteristics of each individual cell,
as well as the individual cell response to the experimental
stimulus of interest.
[0006] In the art, various methods and devices for studying living
cells are known.
[0007] One method of studying cells involves placing cells on a
bottom surface of a vessel and observing the behavior of the cell
in response to stimuli. Typically used vessels include slides with
recesses and Petri dishes. To allow for simultaneous study of
distinct groups of cells exposed to similar or different stimuli,
multiwell plates are most commonly used. A multiwell plates is
substantially a group of standard sized individual vessels
physically associated in a standard way allowing for simplified
simultaneous or sequential studies of separated groups of cells.
Multiwell plates having 6, 12, 24, 48, 96, 384 or even 1536 wells
on a standard ca. 8.5 cm by ca. 12.5 cm footprint are well known in
the art. Such multiwell plates are provided with an 2 n by 3 n
array of rectangular packed wells. The diameter of the wells of a
plate depends on the number of wells and is generally greater than
about 250 microns (for a 1536 well plate). The volume of the wells
depends on the number of wells and the depth thereof but generally
is greater than 5.times.10.sup.-6 liter (for a 1536 well plate).
The standardization of the multiwell plate format is a great
advantage for researchers, allowing the use of standardized
products including robotic handling devices, automated sample
handlers, sample dispensers, plate readers, observation components,
plate washers, software and such accessories as multifilters.
[0008] When a vessel having a planar bottom surface is used to
study cells, the cells are most often studied as a group having an
aggregate of properties of the individual cells. Since the cells
are studied as a group, in such studies the identity of individual
cells is not important. Such studies are of limited utility due to
the fact that naturally occuring cell populations are rarely
homogenous and often it is the heterogenity and the differences of
behavior of cells that is of interest.
[0009] Efforts have been made to use vessels having a planar bottom
surface to study cells as individuals but such efforts are plagued
with many difficulties. A first difficulty is that cells have a
tendency to clump together in variably sized groups at random
locations, and often stack one on top of the other. The clumping
and stacking of cells together makes it virtually impossible to
delineate the borders of one cell from another, see discussion in
unpublished PCT Patent Application No. IL2005/000719 of the
inventor, rendering it virtually impossible to identify which cell
has a given behavior. Further, the fact that cells are randomly
distributed over a featureless surface coupled to the fact that
cells often move due to currents makes it virtually impossible to
definitely identify a specific cell without continuous observation
of the cell. These factors further render high-throughput imaging
(for example for morphological studies) challenging as acquiring an
individual cell and focusing thereon is extremely difficult. Such
variability in location also makes high-throughput signal
processing (for example, detection of light emitted by a single
cell through fluorescent processes) challenging as light must be
gathered from the entire area of the well, decreasing the signal to
noise ratio. Further, a cell held in a well of a multiwell plate
well can be physically or chemically manipulated (for example,
isolation or movement of a single selected cell or single type of
cell, changing media or introducing active entities) only with
difficulty. Further, addition of a reagent to a well or even the
slightest incidental jostling of the vessel causes cells held
therein to move randomly leading to the loss of identity of
individual cells and rendering experiments difficult to perform,
limited in scope and slow. Further, cell behavior is influenced by
contact with other cells. If, to avoid the above difficulties, a
multiwell plate holds only one cell per well, loading of the plate
is very low (about 1536 cells in 65 cm.sup.2, or 24 cells
cm.sup.-2). Thus, flat-bottomed vessels are in general only
suitable for the study of homogenous or heterogenous aggregates of
cells as a group.
[0010] Flat-bottomed vessels are also unsuitable for the study of
cells undergoing apoptosis. It is known to study biological
processes by exposing a monolayer of cells adhering to the bottom
of a flat-bottomed vessel to a stimulus that initiates apoptosis.
Once a cell begins apoptosis, the adhesion of the cell to the
bottom of the vessel is no longer sufficient: the cell detaches
from the bottom and is carried away by incidental fluid currents in
the vessel. The cell is no longer observable and its identity
lost.
[0011] Flat-bottomed vessels are also unsuitable for the study of
non-adhering cells. Just as cells undergoing apoptosis, in
flat-bottomed vessels non-adhering cells can be studied as
individuals only with difficulty. This is a major disadvantage as
non-adhering cells are crucial for research in drug discovery, stem
cell therapy, cancer and immunological diseases detection,
diagnosis and therapy. For example, blood contains seven
heterogeneous types of non-adherent cells, all which perform
essential functions, from carrying oxygen to providing immunity
against disease.
[0012] It is known to study cells in a vessel having a planar
bottom surface provided with an array of cell-localizing features.
In such vessels, each cell is held in a specific location that is
individually addressable allowing the identity of a given cell to
be retained even without continuous observation, see for example
Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 1996,
25, 55-78; Craighead et al., J. Vac. Sci. Technol. 1982, 20, 316;
Singhvi et al., Science 1994, 264, 696-698; Aplin and Hughes,
Analyt. Biochem. 1981, 113, 144-148, U.S. Pat. No. 4,729,949, U.S.
Pat. No. 5,324,591, U.S. Pat. No. 6,103,479 and PCT Patent
Application No. US99/04473 published as WO 99/45357. Many such
devices deform the shape of the cells or require cell binding or
adhesion to a vessel surface, adversely effecting the results of
performed studies
[0013] In PCT patent applications IL2001/00992 published as
WO2003/035824 ("Interactive transparent individual cells biochip
processor"), IL2004/000571 published as WO2004/113492 ("Improved
materials for constructing cell-chips, cell-chip covers, cell-chip
coats, processed cell-chips and uses thereof"), IL2004/000194
published as WO2004/077009 ("A method and device for manipulating
individual small objects"), IL2004/000661 published as
WO2005/007796 ("Improved multiwell plate"), and unpublished
IL2005/000801 ("Device for the study of cells") all of the inventor
or applicant, are provided picowell-bearing devices. A
picowell-bearing device is a device for the study of cells that has
at least one picowell-bearing component. A picowell-bearing
component is a component having at least one, but generally a
plurality of picowells, each picowell configured to hold at least
one cell. In such devices, individual cells are generally held in a
substantially natural state, preferably unadhered, in individual
adressable picowells, preferably each cell alone in an individual
picowell. In the art, a picowell-bearing component of a
picowell-bearing device is often a chip, a plate or other
substantially planar component. Such a component is termed a
"carrier".
[0014] A picowell, as the name suggests, is small well-shaped
feature (including cavities, dimples, depressions, tubes and
enclosures) configured to localize cells in well-defined locations
on the bottom surface of a vessel. Since cells range in size from
about 1 microns to about 100 (or even more) microns diameter there
is no single picowell size that is appropriate for holding a single
cell of any type. That said, the dimensions of the typical
individual picowell in the picowell-bearing components known in the
art have dimensions of between about 1 microns up to about 200
microns or even up to about 250 microns, depending on the exact
implementation. For example, a device designed for the study of
single isolated 20 micron cells typically has picowells of
dimensions of about 20 microns. In other cases, larger picowells
are used to study the interactions of a few cells held together in
one picowell. For example, a 200 or even 250 micron picowell is
recognized as being useful for the study of the interactions of two
or three cells, see PCT Patent Application No. IL01/00992 published
as WO 03/035824.
[0015] A feature that increases the utility of picowell-bearing
devices is that each individual picowell is individually
addressable. By individual addressability is meant that each
picowell is identifiable from amongst the other picowells of a
picowell array allowing each picowell to be registered, found,
observed or studied as desired. For example, while cells are held
in picowells of a picowell-bearing component, each cell is
characterized and the respective picowell wherein that cell is held
is noted. When desired, the observation component of the
picowell-bearing device is directed to observe the picowell where a
specific cell is held. One method of implementing individual
addressability is by the use of fiducial points or other features
(such as signs or labels), generally on the picowell-bearing
component. Another method of implementing individual addressability
is by arranging the picowells in a picowell-array and finding a
specific picowell by counting. Another method of implementing
individual addressability is by providing a dedicated observation
component for each picowell. Another method is by observing a whole
picowell array substantially in its entirety.
[0016] An additional feature that increases the utility of
picowell-bearing devices is that the picowells of the picowell
array are close together, preferably being juxtaposed, that is, the
area occupied by a picowell-array is substantially entirely made up
of picowells with little or no inter picowell area, see FIG. 1, a
reproduction of a photograph of a picowell-array from above, such
as described in PCT Patent Application No. IL01/00992. In FIG. 1 is
seen a part of a hexagonally packed picowell array 10 including a
plurality of hexagonal picowells 12, some holding living cells 14.
It is seen that the inter picowell areas 16 make up only a minor
percentage of the total area of picowell-array 10. Preferably the
wells are knife-edged, that is the interwell surfaces are
substantially non-existent. Such a feature allows simple loading of
cells by sedimentation, allows very high-loading (expressed in
units of cells per unit area), avoids stacking of cells, and
clearly separates and prevents physical contact between cells held
in adjacent picowells 12.
[0017] An additional feature that increases the utility of
embodiments of picowell-bearing devices is the use of optical
properties of the picowell array such as of the picowell bottom
surface or picowell walls for automatic delineation of picowell
borders in an image of the picowell array, allowing high throughput
image acquisition, image processing and experimental analysis as
described in PCT patent application IL2005/000719 published as
WO2006/003664 of the inventor.
[0018] The use of the picowell-bearing devices described above
allows large number of cells to be studied as individuals. In
embodiments of picowell-bearing devices such as described in PCT
patent applications IL2001/00992 published as WO2003/035824,
complex experiments involving serial addition of reagents and the
like are performed with dedicated microfluidics systems including
flow generators such as pumps or syringes. In embodiments described
therein, a picowell array is located in a substantially sealed
chamber, the chamber in fluid communication with at least one fluid
inlet and at least one fluid outlet. In embodiments, cells are
loaded onto the picowell array by sedimentation: a moveable wall of
the chamber in which the picowell array is located is moved, a
solution of cells is introduced through the consequent opening, the
cells allowed to settle in the picowells, and the moveable wall
replaced so as to reseal the chamber.
[0019] Despite the unparalleled utility of such picowell-bearing
devices, such devices have a number of limitations. A first
limitation is that the devices are generally difficult to use by an
unskilled person and are not completely suitable for integration
with a robotics system for automatised use. A second limitation is
that the devices are relatively complex to assemble. A third
limitation is that the flow rate of liquids through picowell-array
holding chamber chamber is relatively limited. If a flow rate is
too high, pressure in the chamber increases to the point where the
lid is lifted upwards, allowing leakage of liquid from under the
lid, or even detached. In embodiments this limitation is overcome
by permanently securing all chamber walls, for example by using an
adhesive so that no wall is moveable. In such embodiments, cells
are loaded onto the picowell array through a fluid inlet, a time
consuming step that is less efficient than loading of the picowell
array by cell sedimentation.
[0020] Many of the above limitations are overcome by the teachings
of unpublished PCT patent application IL2005/000801 of the
inventor. In embodiments of the device taught therein, a picowell
array is located inside a depression on a plateau, the plateau
provided with a moveable wall that is substantially a lid, the
space between the lid and the plateau defining a capillary channel.
For use, cells are loaded by sedimentation when the lid is open and
the lid subsequently shut. Liquid added at one end of the lid is
transported past the picowell array through the capillary channel
defined between the lid and the plateau. Embodiments of the device
are remarkably simple to manufacture and simple for use, even by
unskilled practitioners. A disadvantage of embodiments of such
devices is that the flow rate and total volume of liquid that can
be added is limited.
[0021] An additional disadvantage of the devices known in the art
including the discussed above is that the devices are not
exceptionally suited for the studies of cells, such as adipocytes,
that have a density lower than the aqueous medium in which the
cells are found and therefore float.
[0022] It would be highly advantageous to have a device for the
study of cells not having at least some of the disadvantages of the
prior art.
SUMMARY OF THE INVENTION
[0023] Embodiments of the present invention successfully addresses
at least some of the shortcomings of the prior art by providing a
picowell-bearing device for the study of cells.
[0024] Aspects of the present invention provide a picowell-bearing
device for the study of floating cells and methods of using the
device, including downwardly facing picowells inside a chamber
(generally on the bottom surface of a top wall of the chamber e.g.,
the ceiling of the chamber). Cells that are less dense than a
medium found in the chamber float upwards to contact the bottom
surface and to enter the picowells, in a manner analogous to
non-floating cells that settle into picowells disposed on a top
surface of a bottom wall constituting a floor of a chamber of a
picowell-bearing device as known in the art.
[0025] Aspects of the present invention provide device for the
study of cells including at least one picowell in a chamber, the
chamber having at least one moveable wall and a force applicator
configured to removeably apply a substantially uniformly
distributed force around the periphery of the moveable wall in a
direction to retain the moveable wall secured to the chamber. In
embodiments, such a device allows introduction of a continuous flow
of liquids and active entities such as nutrients and reagents at
relatively high pressures with a reduced chance of leakage.
Embodiments of a device implementing this aspect of the present
invention include microincubators where a plurality of living cells
are held in individual picowells.
[0026] Thus, according to the teachings of the present invention
there is provided a method of studying floating living cells,
comprising: a) confining living cells floating in an aqueous medium
to picowells located on a bottom surface of a top wall of a
chamber; and b) observing the living cells. In embodiments of the
present invention, the picowells are arrayed in a picowell array.
In embodiments of the present invention, the top wall is fashioned
from a substantially transparent material in the vicinity of the
picowells and the observing is through the top wall. In embodiments
of the present invention, during the observing, an active entity is
introduced into the chamber.
[0027] According to the teachings of the present invention there is
also provided a picowell-bearing device for the study of living
cells, comprising: a) a chamber; b) a picowell-bearing component
constituting a top wall of the chamber; and c) at least one
picowell (preferably a plurality of picowells, preferably or a
picowell array) inside the chamber on a bottom surface of the top
wall. In embodiments of the present invention the bottom surface is
substantially planar. In embodiments of the present invention, the
at least one picowell is located in a hollow on the bottom surface
of the top wall. In embodiments of the present invention, the at
least one picowell is a feature on the bottom surface of the top
wall.
[0028] In embodiments, a device of the present invention comprises
a second picowell-bearing component constituting a bottom wall of
the chamber having at least one picowell (preferably a plurality of
picowells, preferably or a picowell array) inside the chamber on
the top surface of the bottom wall. In embodiments of the present
invention the top surface is substantially planar. In embodiments
of the present invention, the at least one picowell is located in a
hollow on the top surface of the bottom wall. In embodiments of the
present invention, the at least one picowell is a feature on the
top surface of the bottom wall.
[0029] In embodiments of the present invention, the device further
comprises at least one fluid inlet in fluid communication with the
chamber, preferably configured for introduction of a liquid into
the chamber. In embodiments of the present invention, a fluid inlet
is a capillary channel.
[0030] In embodiments of the present invention, the device further
comprises at least one fluid outlet in fluid communication with the
chamber. In embodiments of the present invention, a fluid outlet is
configured for removal of a liquid from the chamber. In embodiments
of the present invention, a fluid outlet is a capillary channel. In
embodiments of the present invention, a fluid outlet is configured
for removal of a gas from the chamber, for example, air trapped in
the chamber, especially in the at least one picowell, when liquid
is introduced into the chamber.
[0031] In embodiments, the chamber of a device of the present
invention is defined, in part, by a device body. In embodiments,
the device body is a picowell-bearing component. In embodiments,
the picowell-bearing component and the device body are distinct
components.
[0032] In embodiments of the present invention, the device further
comprises a base constituting a bottom wall of the chamber. In
embodiments of the present invention, the base is configured to
contact a bottom surface of the device body substantially in
parallel to the bottom surface of the device body and wherein the
at least one picowell is located in a hollow on the bottom surface
of the device body. In embodiments of the present invention, a
contact region between the bottom surface of the device body and
the base constitutes at least one capillary channel in fluid
communication with the chamber. In embodiments of the present
invention, a contact region between the bottom surface of the
device body and the base constitutes at least two capillary
channels in fluid communication with the chamber. In embodiments of
the present invention, the bottom surface of the device body
comprises a plateau in which the hollow is located, wherein at
least part of the plateau constitutes at least part of the
capillary channel.
[0033] In embodiments, a device of the present invention includes a
hole through the bottom surface of the device body in fluid
communication with the base. In embodiments of the present
invention, the hole constitutes a liquid reservoir in fluid
communication with the capillary channel. In embodiments of the
present invention, in at least one position of the bottom surface,
the base protrudes relative to the device body constituting a
liquid reservoir in capillary communication with a capillary
channel.
[0034] In embodiments of the present invention, the base is
removable, that is, is separable from the device body. In
embodiments of the present invention, the base is moveable relative
to the device body. In embodiments of the present invention, the
base is moveable substantially in parallel to the bottom surface of
the device body.
[0035] In embodiments of the present invention, the base is
slidingly associated with the device body. In embodiments of the
present invention, the device body comprises two rails, the at
least one picowell is located between the two rails and the base is
slidingly associated with the two rails. In embodiments of the
present invention, the at least one picowell is located in a hollow
between the two rails. In embodiments of the present invention, the
base and an area of the device body between the two rails
substantially define a capillary channel. In embodiments of the
present invention, the rails are substantially grooves in walls
protruding from the bottom surface.
[0036] In embodiments of the present invention, at least one
picowell is porous so as to allow fluid flow therethrough. In
embodiments of the present invention, the device comprises an upper
chamber in fluid communication with the chamber through the pores
of the picowells.
[0037] In embodiments of the present invention, the device body is
fashioned from a substantially transparent material in the vicinity
of the at least one picowell allowing observation of cells held in
the picowell therethrough.
[0038] According to the teachings of the present invention there is
also provided a method of studying floating living cells,
comprising: a) providing a chamber including at least one opening,
a top wall with a bottom surface and at least one picowell
(preferably a plurality of picowells, preferably or a picowell
array) on the bottom surface; b) introducing an medium into the
chamber through an opening; c) introducing a living cell
(preferably a plurality of living cells) of a first type into the
chamber through the opening; and d) orienting the chamber so that
the bottom surface faces substantially downwards, whereby the at
least one living cell floats upwards in the medium to be held in a
picowell.
[0039] Generally, the living cell or cells of the first type are
introduced into the chamber together with the medium. In
embodiments of the present invention, the cells and the medium are
introduced separately.
[0040] In embodiments of the present invention, the medium is
introduced when the chamber is already oriented so that the bottom
surface faces substantially downwards.
[0041] In embodiments of the present invention, the medium is first
introduced and subsequently the chamber oriented so that the bottom
surface faces substantially downwards. In embodiments of the
present invention, subsequent to introduction of the medium into
the chamber, the opening through which the medium is introduced is
substantially closed with a closing component. In embodiments, the
closing component constitutes a part of a fluid inlet in fluid
communication with the chamber.
[0042] In embodiments of the present invention, the chamber
includes a bottom wall with a top surface and at least one picowell
(preferably a plurality of picowells, preferably or a picowell
array) on the top surface and the method further comprises: e)
introducing a living cell of a second type into the chamber through
a the opening, the living cell of the second type being denser than
the medium whereby the at least one living cell of the second type
settles into a the picowell on the top surface for study.
[0043] Generally, the living cell or cells of the second type are
introduced into the chamber together with the medium. In
embodiments of the present invention, the cells of the second type
and the medium are introduced separately.
[0044] In embodiments of the present invention, the living cells
that are held in the picowells are observed, for example, to study
cell development or reaction to stimuli such as contact with an
active entity. To this end, in embodiments of the present
invention, the top wall is fashioned from a substantially
transparent material in the vicinity of the at least one picowell
and the observing is through the top wall, through the bottom wall,
or through both the top and bottom wall.
[0045] According to the teachings of the present invention there is
also provided a picowell-bearing device for the study of living
cells, comprising: a) a chamber having at least one picowell
(preferably a plurality of picowells, preferably or a picowell
array) on an internal surface and at least one moveable wall; b) a
liquid inlet into the chamber; c) a liquid outlet from the chamber;
d) a force applicator configured to removeably apply a
substantially uniformly distributed force around the periphery of
the moveable wall in a direction to retain the moveable wall
secured to the chamber.
[0046] In embodiments of the present invention, the at least one
picowell is located in a hollow on the internal surface. In
embodiments of the present invention, the at least one picowell is
a feature on the internal surface.
[0047] In embodiments of the present invention, the internal
surface is an upper surface of a wall (e.g., the bottom wall) of
the chamber. In embodiments of the present invention, the internal
surface is a bottom surface of a wall (e.g., the top wall) of the
chamber, substantially as described hereinabove.
[0048] In embodiments of the present invention, the moveable wall
includes the at least one picowell. In embodiments of the present
invention, the at least one picowell is disposed on a non-moveable,
wall.
[0049] In embodiments the force applied is a magnetic force. In
such embodiments, the force applicator generally includes a magnet,
preferably a magnet at least part of which is located in proximity
of the periphery of the moveable wall.
[0050] In embodiments of the present invention, the force
applicator includes threads on the moveable wall. For example, in
embodiments threads are located on the edge of a disk-shaped
moveable wall and matching threads provided on the sides of an
opening into the chamber so that the moveable wall can be screwed
in and out of the opening.
[0051] In embodiments of the present invention, the force
applicator includes a rigid force distributor contacting the
periphery of the moveable wall.
[0052] In embodiments of the present invention, the device further
comprises a flow generator, e.g., a syringe, a pump, a peristaltic
pump) functionally associated with the liquid inlet. Preferably,
such a flow generator is a variable flow generator configured to
vary the rate of flow of liquids through the liquid inlet.
[0053] In embodiments of the present invention, the device further
comprises a liquid temperature controller, to cool, to heat or
both, medium in the chamber. In embodiments of the present
invention, such a liquid temperature controller is functionally
associated with the flow generator and/or the chamber and/or the
liquid inlet and/or the liquid outlet.
[0054] In embodiments of the present invention, a device is
functionally associated with a pH-determining component for
determining the pH of liquid entering the device through a liquid
inlet, in the chamber or exiting the device from the liquid outlet.
In embodiments of the present invention, a device is functionally
associated with a gas content determining component for determining
the content of a gas, such as carbon dioxide or oxygen, in a liquid
entering the device through a liquid inlet, in the chamber or
exiting the device from the liquid inlet.
[0055] In embodiments of the present invention, a device is
functionally associated with a component for varying the content of
a gas, such as carbon dioxide or oxygen, in a liquid entering the
device through a liquid inlet.
[0056] In embodiments of the present invention, the device body is
fashioned from a substantially transparent material in the vicinity
of the at least one picowell allowing observation of cells held in
the picowell therethrough.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0058] In the drawings:
[0059] FIG. 1 (prior art) is a reproduction of a photograph of a
cell-populated well-array of a device body of a picowell-bearing
device of PCT Patent Application No. IL01/00992;
[0060] FIGS. 2A-2E depict an embodiment of a device of the present
invention including a picowell array on a bottom surface of an
upper wall of a chamber, and capillary channels and a liquid inlet
hole in fluid communication with the picowell array;
[0061] FIG. 2F depicts an embodiment of a device of the present
invention including a picowell array on a bottom surface of an
upper wall of a chamber and capillary channels in fluid
communication with the picowell array;
[0062] FIG. 2G depicts an embodiment of a device of the present
invention including a picowell array on a bottom surface of an
upper wall of a chamber and capillary channels in fluid
communication with the picowell array;
[0063] FIGS. 3A-3H depict the use of the device of FIG. 2A-2E;
[0064] FIGS. 4A-4D depict an embodiment of a device of the present
invention including a picowell array on a bottom surface of a fixed
top wall of a chamber;
[0065] FIGS. 5A and 5B depict embodiments of devices of the present
invention including a porous picowell array on a bottom surface of
a top wall of a first chamber and a second chamber above the first
chamber;
[0066] FIG. 6 depicts an embodiment of a device of the present
invention including a picowell array on a bottom surface of a
removeable top wall of a chamber;
[0067] FIGS. 7A-7D depict an embodiment of a device of the present
invention including a chamber with a removeable top wall and a
picowell array on the top surface of the bottom wall of the
chamber;
[0068] FIG. 7E depicts an embodiment of a device of the present
invention having two picowell arrays in a chamber, one on the top
surface of a bottom wall and one on the bottom surface of a top
wall of a chamber;
[0069] FIG. 8 depicts an embodiment of a device of the present
invention including a chamber with a removeable top wall and a
picowell array on the top surface of the bottom wall of the
chamber;
[0070] FIG. 9 depicts an embodiment of a device of the present
invention including a chamber with a removeable top wall and a
picowell array on the top surface of the bottom wall of the
chamber;
[0071] FIGS. 10A-10C depict an embodiment of a device of the
present invention including a chamber with a removeable top wall
and a picowell array on the top surface of the bottom wall of the
chamber; and
[0072] FIG. 10D depicts an embodiment of a device of the present
invention integrated with other components to constitute a
microincubator.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0073] The present invention is of a method of studying floating
living cells by confining the cells in picowells. The present
invention is also of picowell-bearing devices for the study of
cells that are less dense than the medium in which the cells are
found, generally including at least one downward facing picowell on
a bottom surface of a top wall of a chamber of the picowell-bearing
device.
[0074] The long duration (days) study of cells using
picowell-bearing devices is often desired. Such long duration study
requires replenishment, preferably continuous replenishment, of
medium to avoid that the changes in the medium (e.g., gas,
nutrient, waste matter content) effect the experiment. Prior art
devices provided with capillary channels for inlet and outlet of
liquids have the disadvantages of limited total liquid that can be
added and limited flow rates making performance of long duration
studies difficult. Prior art devices having a picowell-containing
chamber with a moveable wall allow introduction of cells directly
into the chamber enabling the use of extremely small quantities of
cells and uniform distribution by sedimentation. However, in such
prior art devices, liquid introduced into the chamber often leaks
through the moveable wall/chamber joints. In such prior art
devices, if the liquid pressure applied by a flow generator (such
as a pump) is too high or if capillary forces are sufficient, the
moveable wall may disengage from the chamber. As both joint leakage
and moveable wall disengagement can occur at any time during an
experiment, the maximal duration of an experiment is limited.
[0075] On the other hand, prior art devices having
picowell-containing chambers with permanently secured walls do not
allow introduction of cells directly into the chamber, a fact that
makes experiments more complex and difficult to perform.
[0076] Thus, an aspect of the present invention is also of
picowell-bearing devices with at least one moveable wall and a
force applicator configured to apply a substantially uniformly
distributed force around the periphery of the moveable wall to
retain the moveable wall secured to and sealing the chamber the
chamber. Embodiments of such devices of the present invention allow
introduction of a continuous flow of liquids into and out of the
chamber without leaks at the joints of the moveable wall
simplifying the performance of long duration studies.
[0077] The principles and uses of the teachings of the present
invention may be better understood with reference to the
accompanying description, figures and examples. In the figures,
like reference numerals refer to like parts throughout.
[0078] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth herein. The invention
can be implemented with other embodiments and can be practiced or
carried out in various ways. It is also understood that the
phraseology and terminology employed herein is for descriptive
purpose and should not be regarded as limiting.
[0079] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include techniques
from the fields of biology, chemistry, engineering, material
sciences and physics. Such techniques are thoroughly explained in
the literature.
[0080] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. In
addition, the descriptions, materials, methods, and examples are
illustrative only and not intended to be limiting. Methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention.
[0081] As used herein, the terms "comprising" and "including" or
grammatical variants thereof are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof. This term encompasses the terms
"consisting of" and "consisting essentially of".
[0082] The phrase "consisting essentially of" or grammatical
variants thereof when used herein are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof but only if the additional features,
integers, steps, components or groups thereof do not materially
alter the basic and novel characteristics of the claimed
composition, device or method.
[0083] As used herein, "a" or "an" mean "at least one" or "one or
more". The use of the phrase "one or more" herein does not alter
this intended meaning of "a" or "an".
[0084] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
Implementation of the methods of the present invention involves
performing or completing selected tasks or steps manually,
automatically, or a combination thereof.
[0085] Herein, the term "active entity" is understood to include
chemical, biological or pharmaceutical entities including any
natural or synthetic chemical or biological substance that
influences a cell with which the entity is in contact. Typical
active entities include but are not limited to active
pharmaceutical ingredients, antibodies, antigens, biological
materials, chemical materials, chromatogenic compounds, drugs,
enzymes, fluorescent probes, immunogenes, indicators, ligands,
nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
[0086] Herein, by "indicator" is meant any active entity that upon
interaction with some stimulus produces an observable effect. In
the context of the present invention, by stimulus is meant, for
example, a specific compound, (such as a molecule) released by a
cell and by observable effect is meant, for example, a visible
effect or a signal, for example a change in color or emission of
light.
[0087] Herein, by "picowell array" is meant a group of two or more
picowells, preferably a plurality of picowells, preferably a
plurality of picowells arranged in an orderly fashion. Typical
arrangements include hexagonal arrays (picowells arrayed in
staggered rows so that each picowell has six equidistant
neighboring picowells such as depicted in FIG. 1) or rectangular
arrays (picowells arrayed in rows so that each picowell has four
equidistant neighboring picowells in a cross shape).
[0088] Some embodiments of the present invention include components
that are transparent or are made of a transparent material. By
"transparent" is meant that the component or material is
substantially transparent to at least one wavelength of light
(preferably a range of wavelengths) in at least part of the visible
light spectrum, the ultraviolet light spectrum and/or of infrared
radiation, preferably the visible light spectrum.
[0089] It is important to note that some aspects of embodiments of
the present invention are related to embodiments of picowell
bearing devices known in the art, especially picowell-bearing
devices described by the inventor in PCT patent applications
IL2001/00992 published as WO2003/035824 ("Interactive transparent
individual cells biochip processor"), IL2004/000571 published as
WO2004/113492 ("Improved materials for constructing cell-chips,
cell-chip covers, cell-chip coats, processed cell-chips and uses
thereof"), IL2004/000194 published as WO2004/077009 ("A method and
device for manipulating individual small objects"), IL2004/000661
published as WO2005/007796 ("Improved multiwell plate"), and
unpublished IL2005/000801 ("Device for the study of cells") which
are all included by reference as if fully set forth herein.
[0090] According to a method of the present invention, living cells
floating in an aqueous medium are confined in picowells, preferably
in a picowell array, located on a bottom surface of a top wall of a
chamber. Once confined, the living cells are observed. Preferably,
the top wall of the chamber is fashioned from a substantially
transparent material in the vicinity of the picowells allowing
observation of the cells through the top wall. Generally, the
observed cells are subjected to some stimulus, for example,
introduction of an active entity into the chamber, allowing the
study of the response of the cells to the active entity or
stimulus. As the cells are confined in picowells, it is possible to
study the behavior of each individual cell in response to the
stimuli.
[0091] One device useful in implementing the method is a
picowell-bearing device of the present invention.
[0092] An embodiment of a picowell-bearing device of the present
invention is picowell-bearing device 18 depicted in FIGS. 2A-2E.
Device 18 consists essentially of three physically separate
components: a device body 20 (including an attached
picowell-bearing component, carrier 44), a sliding base 22 and a
base holder 24, depicted, inter alia, in FIG. 2A (from below
without sliding base 22 and base holder 24), 2B (from below,
assembled), 2C (in cross section across A-A), 2D (in cross section
across B-B) and 2E (from above, assembled). Suitable materials from
which to fashion the components of device 18 are known in the art,
are preferably transparent, and include for example, polycarbonate
or glass. A picowell-array 10 is found in a hollow 26 found on
plateau 28 on bottom surface 30 of device body 20, see FIG. 2A.
Flanking plateau 26 are gaps 32. At each of the two sides of device
body 20 are rails 34, each rail including a rail ledge 36 and a
rail wall 38. Passing through plateau 26 from bottom surface 30 to
upper surface 40 of device body 20 and upstream of picowell array
10 is a liquid inlet hole 42. Picowell array 10 is a feature on a
picowell bearing component, carrier 44, see FIG. 2C. Carrier 44 is
attached to device body 20 (for example by welding or adhesive) to
the rim of hollow 26 so that picowell array 10 faces downwards.
Carrier 44 is preferably made of substantially transparent
material, e.g., polycarbonate or glass, allowing observation of
cells therethrough.
[0093] Sliding base 22 is configured to fit device body 20
substantially parallel to bottom surface 30. Base holder 24 is
configured to fit up against sliding base 22 and to engage device
body 20 (e.g., by "snapping into place") so as to press sliding
base 22 up against plateau 28 and rail ledges 36 of device body 20,
see FIG. 2B. When held in place by base holder 24, sliding base 22
is laterally confined by rail walls 38, vertically confined between
device body 20 and base holder 24 but is axially slidable relative
to device body 20 in parallel to bottom surface 30. The interface
between the bottom surface of plateau 28 and the top surface of
sliding base 22 defines a space of capillary thickness flanked by
gaps 32, see FIG. 2D. The space of capillary thickness between
liquid inlet hole 42 and hollow 26 constitutes a capillary channel
46 and the space of capillary thickness between downstream of
hollow 26 constitutes a waste capillary channel 48 and a waste
reservoir 50. In device 18, the border between waste capillary
channel 48 and waste reservoir 50 is not clearly delineated: waste
capillary channel 48 is the downstream region of plateau 28
proximate to picowell array 10 while waste reservoir 50 is
substantially a broader and longer section of plateau 28 downstream
from waste capillary channel 48.
[0094] When sliding base 22 is held in place substantially in
parallel with upper surface 28 and covering the opening of hollow
26 (FIG. 2C), a chamber is defined in which carrier 44 is
substantially a top wall, sliding base 22 is a bottom wall and the
wall of hollow 26 is a side wall.
[0095] When sliding base 22 is held in place substantially in
parallel with upper surface 28 and covering the bottom opening of
liquid inlet hole 42 (see FIG. 2B), liquid inlet hole 42 together
with sliding base 22 constitutes a liquid reservoir in fluid
communication with capillary channel 46. A drop of liquid placed in
liquid inlet hole 42 from upper surface 40 (see FIG. 2E) contacts
the top surface of sliding base 22 and is drawn by capillary action
through capillary channel 46, into hollow 26, under and past
picowell-array 10, out through waste capillary channel 48 and out
to waste reservoir 50. Thus, in device 18, picowell-array 10 is in
fluid communication with liquid inlet hole 42, that constitutes a
liquid reservoir, through capillary channel 46. Picowell array 10
is in fluid communication with waste reservoir 50 through waste
capillary channel 48.
[0096] A device such as device 18 is useful in the study of cells
that are less dense than the medium in which the cells are found
and therefore float, in accordance with the teachings of the
present invention, as depicted in FIG. 3 and discussed
hereinbelow.
[0097] For use, sliding base 22 of picowell-bearing device 18 is
positioned at a location wherein a chamber 54 defined by carrier
44, the walls of hollow 26 and sliding base 22 is accessible, that
is the bottom side of chamber 54 is uncovered and constitutes an
opening into chamber 54, see FIG. 3A.
[0098] In this state, device 18 is held so that upper surface 40 of
device body 20 faces substantially downwards and lower surface 30
of device body 20 faces substantially upwards. A medium and cells
56 that are less dense than the medium (e.g., adipocytes) are
loaded into chamber 54 for example using a pipette 58, see FIGS. 3A
and 3B. Subsequently sliding base 22 is slid along rails 34 so as
to cover both hollow 26 and liquid inlet hole 42, substantially
closing the opening into chamber 54, see FIG. 3C. Cells 56 float
upwards in chamber 54, see FIG. 3D.
[0099] Device 18 is turned over and oriented so that upper surface
40 of device body 20 faces substantially upwards and lower surface
30 of device body 20 faces substantially downwards, as depicted in
FIG. 3E. Cells 56 float upwards to enter picowells 12 of picowell
array 10 made up of a hexagonal array of tightly packed knife-edged
picowells 12. Each cell 56 enters an individual hexagonal picowell
36, as depicted in FIG. 3F.
[0100] Subsequently, as depicted in FIG. 3E, a pipette 58 may be
used to add a cell washing solution 60 (e.g., PBS, DMEM or RPMI) to
liquid inlet hole 42. From liquid inlet hole 42, cell washing
solution 60 is transported by capillary action through capillary
channel 46, into chamber 54 and out through waste capillary channel
48 to waste reservoir 50, thus washing living cells 56 held in
picowells 12.
[0101] Subsequently, a pipette 58 is used to add a cell staining
solution (e.g., fluorescein diacetate (1 micromolar), acridine
orange (5 micromolar), rhodamine 123 (1 micromolar), FDA, PI,
Annexin V, anti-GlycophorinA antibody labeled with allophycocanin
for the study of GFP expression) to liquid inlet hole 42. Cell
staining solution is transported from liquid inlet hole 42, into
chamber 54 and out through waste capillary channel 48 to waste
reservoir 50, thus washing living cells 56 held in picowells 12,
thereby staining living cells 56 held in picowells 12. During
transport of the cell staining solution, cell-staining solution 62
displaces cell-washing solution 60, as depicted in FIG. 3G.
[0102] When sufficient time has passed for cell staining, living
cells 56 held in picowells 12 of picowell array 10 of
picowell-bearing device 18 are examined using an observation device
64, in FIG. 3H an Olympus BX61 motorized research microscope
(Olympus America Inc., Melville, N.Y., USA). As carrier 44 is
substantially transparent, observation is performed from above,
through carrier 44 which constitutes the top wall of chamber
54.
[0103] In embodiments, picowell-bearing device 18 of the present
invention is transferred to an incubator for a period of time,
before, during or after living cells 56 are examined using
observation device 64.
[0104] As described above, medium and living cells 56 are
introduced together into chamber 54. In embodiments of the present
invention, medium and cells are not introduced together, for
example living cells 56 are introduced first and subsequently
medium, or first medium is introduced and subsequently living cells
56.
[0105] As described above and depicted in FIG. 3H, in embodiments
of the present invention living cells 56 held in picowells 12 of
picowell array 10 are observed from above. In embodiments of the
present invention (not depicted), cells 56 are observed from below
by an observation device positioned below picowell-bearing device
18. In such embodiments, sliding base 22 is made of substantially
transparent material, e.g., polycarbonate or glass.
[0106] Described hereinabove is an experiment where cells 56 held
in a picowell array 10 of a device 18 of the present invention
undergo only two treatment steps: a first washing step with cell
washing solution 60 and a second staining step with cell staining
solution 62. Using a device of the present invention such as 18, a
greater number of steps are performed if desired. In the embodiment
of the present invention described hereinabove, the volume of waste
reservoir 50 (and, if present, the volume of absorbent element 52
functionally-associated therewith) ultimately determines the
maximal number of such steps that can be performed.
[0107] Described hereinabove is an experiment where cells 56 held
in a picowell array 10 of a device 18 of the present invention
undergo only two types of treatment: washing and staining. In
embodiments of the present invention, cells held in picowells of a
picowell array of a device of the present invention undergo other
types of treatments including washing steps and/or staining steps
and/or exposure to solutions containing active entities such as
drug candidates, selective toxins, active pharmaceutical
ingredients and the like.
[0108] In a picowell-bearing device 18 including a device body 20,
the area of plateau 28 that constitutes a part of waste reservoir
50 and waste capillary channel 48 is relatively large compared to
the area of plateau 28 that constitutes a part of and capillary
channel 54. Thus when sliding base 22 is in place, the volume of
waste reservoir 50 and waste capillary channel 48 is significantly
larger than the volume of capillary channel 46. Liquid placed in
liquid inlet hole 42 is therefore efficiently drawn through
capillary channel 46 past hollow 26 and picowell array 10 to waste
reservoir 50. When waste reservoir 50 is filled to capacity,
capillary flow past picowell array 10 effectively ceases. Thus, in
FIGS. 2A and 2B, device 18 is depicted having an absorbent element
52 (such as a piece of absorbent filter paper) in fluid
communication with waste reservoir 50. Fluid that passes to waste
capillary channel 48 is drawn into absorbent element 52.
[0109] In picowell-bearing device 10, waste capillary channel 48 is
a single capillary channel that is substantially defined by the
interface of sliding base 22 and plateau 28: sliding base 22 is a
component of waste capillary channel 48. In embodiments of the
present invention, there is more than one waste capillary channel
48. In embodiments of the present invention (not depicted), one or
more waste capillary channels 48 are channels inside device body 20
and are not defined in part by a base 22.
[0110] In picowell-bearing device 18, capillary channel 46 is a
single capillary channel through which a liquid reservoir (liquid
inlet hole 42) and picowell array 10 are in fluid communication
that is substantially defined by the interface of sliding base 22
and plateau 28: sliding base 22 is a component of capillary channel
46. In embodiments of the present invention, a device comprises
more than one capillary channel 46. In embodiments of the present
invention (not depicted), one or more capillary channels 46 are
channels inside a device body 20 and are not defined in part by a
base 22.
[0111] In picowell-bearing device 18, sliding base 22 covers the
bottom of liquid inlet hole 42 and also forms capillary channel 46
that provides fluid communication between liquid inlet hole 42 and
picowell array 10 contained within chamber 54. Liquid placed inside
liquid inlet hole 42 is drawn by capillary action through capillary
channel 46 into chamber 54 and out through waste capillary channel
48. In embodiments of the present invention, such as device 66
depicted in FIG. 2F, sliding base 22 is configured to slide
outwards so as to protrude relative to the end of device body 20.
The space between upper surface 68 of sliding base 22 and bottom
surface 30 of plateau 28 of device body 20 defines, as described
above for device 18, a capillary channel 46 providing capillary
communication between upper surface 68 of protruding end 70 of
sliding base 22 (constituting a liquid reservoir) and chamber 54
including picowell array 10.
[0112] In picowell-bearing device 18, sliding base 22 is configured
to rest against and substantially in parallel with bottom surface
30 of device body 20, substantially contacting plateau 28 and rail
ledges 36. When in place, sliding base 22 is laterally confined by
rail walls 38, vertically confined by base holder 24 but is axially
slidable in parallel to bottom surface 30. In embodiments of the
present invention, see FIG. 2G, a sliding base 22 is configured to
slidingly engage grooves 69 in rails so that when in place, sliding
base 22 is laterally and vertically confined by the grooves but is
axially slidable in parallel to bottom surface 30. Such an
embodiment is analogous to a picowell-bearing device provided with
a sliding lid fitting in grooves taught in unpublished PCT patent
application IL2005/000801 of the inventor.
[0113] An additional embodiment of a device of the present
invention, 72, is depicted in FIGS. 4A-4D. Device 72 comprises a
device body 20 including a chamber 54 provided with a medium
loading opening 74, a liquid inlet 76 and a liquid outlet 78. On
bottom surface 80 of top wall 82 of chamber 54 is a picowell array
10. Device 72 further comprises a plug 84, configured to sealingly
engage medium loading opening 74. Preferably, all walls of device
72 are substantially entirely of a transparent material.
[0114] Device 18 of the present invention described above is
provided with sliding base 22 as a moveable wall configured to
substantially define and seal chamber 54. In contrast, device 72 is
provided with a removable component, plug 84, to substantially seal
medium loading opening 74 subsequent to introduction of medium into
chamber 54.
[0115] Device 18 of the present invention described above is
provided with a picowell-array bearing carrier 44 that is fixedly
attached to device body 20. In contrast picowell array 10 of device
72 is an integral feature of bottom surface 80 of top wall 82 of
chamber 54.
[0116] For use, a medium and living cells 56 that are less dense
than the medium are introduced (together or separately) into
chamber 54 through medium loading opening 74, FIG. 4A. Opening 74
is sealed using plug 84, FIG. 4B. Generally, a certain amount of
air remains trapped between the inner surface of plug 84 and the
surface of medium added. Device 72 is oriented by tilting at an
angle so that picowell array 10 slopes upwards from medium loading
opening 74 towards an excess gas storage section 86 of chamber 54,
FIG. 4C. Trapped air rolls upwards past picowell array 10 to be
trapped in excess gas storage section 86. Cells 56 float slowly
upwards along picowell array 10. When a cell 56 encounters an empty
picowell, the cell enters the picowell and is held therein. When a
cell 56 encounters an occupied picowell, the cell floats upwards
further. In such away, dense packing of picowell array 10 with
cells 56 is achieved. When sufficient cells 56 introduced into
device 72 are trapped in picowell array 10, device 72 is oriented
so that picowell array 10 is substantially facing downwards, FIG.
4D. Cells 56 held in picowell array 10 are then observed as
described above. Generally, for the performance of experiments and
the study of cells, a flow generator, such as a pump or syringe, is
attached to liquid inlet 76.
[0117] Two additional embodiments of a device of the present
invention, 88 and 90, are depicted in cross-section in FIGS. 5A and
5B, respectively. Amongst other features, devices 88 and 90 are
provided with a porous picowell array 10 configured to allow the
escape of trapped gases. Devices 88 and 90 comprise a chamber 54
provided with a liquid inlet 76 and a liquid outlet 78. On bottom
surface 80 of top wall 82 of chamber 54 is a picowell array 10. In
both devices 88 and 90, top wall 82 separates chamber 54 from upper
chamber 92. Top wall 82 and consequently picowell array 10 are
porous, allowing the flow of fluids (both gases and liquids)
between chamber 54 and upper chamber 92 therethrough. Typically the
pores through top wall 82 are relatively large, e.g., up to about
15 microns as floating cells such as adipocytes are generally of
between 25 to 75 microns in diameter.
[0118] In both device 88 and device 90, the picowells of picowell
array 10 are porous so as to allow fluid flow therethrough, so that
there is fluid communication between chamber 54 and upper chamber
92, allowing air caught in picowells 12 to pass into upper chamber
92. In device 88 both liquid inlet 76 and liquid outlet 78 are in
direct fluid communication with chamber 54. In device 88, upper
chamber 92 has an upwardly sloping top wall that terminates in
excess gas storage section 86 provided with a vent 94 as a fluid
outlet for trapped gases. In device 90, liquid inlet 76 is in
direct fluid communication with chamber 54 but liquid outlet 78 is
in fluid communication with chamber 54 through top wall 82 and
upper chamber 92.
[0119] For the performance of experiments and the study of cells, a
flow generator such as a pump or syringe is attached to liquid
inlet 76 of a device 88 or 90 as taught in embodiments of PCT
patent application IL2001/00992 published as WO2003/035824 of the
inventor. The device is oriented so that bottom surface 80 of top
wall 82 of chamber 54 faces downwards. Medium and living cells 56
that are less dense than the medium are introduced (together or
separately) into chamber 54 through liquid inlet 76. Cells 56
entering chamber 54 through liquid inlet 76 float upwards in the
medium to be held in picowells of picowell array 10. Air in chamber
54 may be evacuated in any of a number of ways including being
forced out through liquid outlet 78 and being passed through pores
in top wall 82 into upper chamber 92.
[0120] In device 88, air in upper chamber 92 follows the slope of
the upper wall of upper chamber 92 to excess gas storage section 86
and out through vent 94. As a result there are substantially no air
bubbles trapped in picowells 12 that may potentially interfere with
observation of cells 56 held therein. Cells 56 held in picowell
array 10 may then observed and/or manipulated as described
above.
[0121] In device 90, air and medium are forced into upper chamber
92 through pores of top wall 82, follow the slope of the upper wall
of upper chamber 92 and may be removed from upper chamber 92
through liquid outlet 78. As a result there are no air bubbles
trapped in picowells that potentially interfere with observation of
cells 56 held therein. Cells 56 held in picowell array 10 are then
observed as described above.
[0122] In a preferred embodiment, device 88 and device 90 both
include at least three components. A first component is
substantially top wall 82 which constitutes a carrier, preferably
of transparent material such as polycarbonate or glass, having
picowell array 10 as a feature molded on bottom surface 80. An
exceptionally suitable material from which to fashion porous top
wall 82 is porous polytetrafluoroethylene available from Porex
Corporation (Fairburn, Ga., USA) especially a
polytetrafluoroethylene having an index of refraction close to that
of water. A second component is a device body 20, preferably of
transparent material such as polycarbonate or glass, including a
base 96, an inlet 76, side walls (not depicted), in the case of
device 88 outlet 78, and having an opening with a rim configured to
receive porous top wall 82. Preferably, top wall 82 and device body
20 are attached with the use of adhesive (e.g., light-cured
adhesive) or welding. A third component, preferably of a
transparent material such as glass or polycarbonate is
substantially the walls that define upper chamber 92, and in the
case of device 90 also outlet 78, that is configured to attach, for
example by "snap-fit", to the first and second components.
[0123] An additional embodiment of a device of the present
invention, 96, is depicted in an exploded view of a side
cross-section in FIG. 6. In FIG. 6 are depicted two individual
components of device 96, a carrier 44 and a device body 20.
[0124] Carrier 44 is made of transparent material, is substantially
planar and has a bottom surface 80. To bottom surface 80 is
attached (for example using adhesive) a ring-shaped upper retaining
magnet 98 around the periphery of carrier 20 surrounding a picowell
array 10 which is a feature on bottom surface 80 of carrier 20.
[0125] Device body 20, preferably of transparent material, is seen
in cross section in FIG. 6, substantially includes a base 96 and
side walls (not depicted) that define a chamber 54 with a top
opening. In fluid communication with chamber 54 are an inlet 76 and
an outlet 78. Around the periphery of rim 100 of the top opening of
chamber 54 is attached (for example using adhesive) a ring-shaped
lower retaining magnet 102 having similar dimensions but a polarity
opposite that of upper retaining magnet 98.
[0126] Similarly to devices 18, 66, 72, 88 and 90, device 96 is
provided with a picowell array 10 on a bottom surface 80 of a
carrier 44 and bottom surface 80 constitutes an upper wall of
chamber 54. Similarly to devices 72, 88 and 90, device 96 is
provided with non-capillary liquid inlet 76 and liquid outlet 78 in
fluid communication with chamber 54 and for use generally requires
attachment of a flow generator to liquid inlet 76. Similarly to
devices 18 and 66, chamber 54 of device 96 includes a moveable
wall.
[0127] However, unlike devices 18 and 66 where the moveable wall is
a sliding base component, in device 96 the moveable wall is
removable carrier 44 that constitutes the upper wall of chamber
54.
[0128] Magnets 98 and 102 are configured to secure carrier 44 to
device body 20 so as to substantially seal chamber 54. Magnets 98
and 102 together constitute a force applicator that, when
contacted, together apply a substantially uniformly distributed
force around the periphery of carrier 44 in a perpendicular
direction so as to retain carrier 44 secured to device body 20 and
thus to chamber 54.
[0129] For the performance of experiments and the study of cells
using a device 96, a flow generator such as a pump or syringe is
attached to liquid inlet 76 of a device 96 as taught in embodiments
of PCT patent application IL2001/00992 published as WO2003/035824
of the inventor. Carrier 44 is removed, allowing access to chamber
54 through the top opening. Medium and living cells 56 that are
less dense than the medium are introduced (together or separately)
into chamber 54 through the top opening. Carrier 44 is placed in
proximity of the top opening so that magnets 98 and 102 are
mutually attracted, holding carrier 44 firmly in place. Device 96
is oriented so that bottom surface 80 of carrier 44 faces
downwards. Cells 56 in chamber 54 floating in the medium in chamber
54 enter picowells of picowell array 10 and are held therein.
[0130] Cells 56 held in picowell array 10 are then observed as
described above and in the art, including during and subsequent to
introduction of active entities through inlet 76. As noted above,
device 96 is configured to allow simple loading of cells 56 into
picowells and to allow performance of long-duration experiments
free of leaks even with relatively high pressure and continuous
inflow of liquids through inlet 76 due to the use of a force
applicator that applies a substantially uniformly distributed force
around the periphery of carrier 44 to retain carrier 44 secured to
device body 20 and sealing chamber 54.
[0131] An additional embodiment of a device of the present
invention, 104, is depicted in an exploded view in perspective in
FIG. 7A (to scale), an exploded view of a side cross-section in
FIG. 7B (not to scale), a close-up showing details of device body
20 from the top in FIG. 7C (to scale) and an assembled view in
perspective in FIG. 7D (to scale). Unlike previously discussed
devices 18, 66, 72, 88, 90 and 96, device 104 may not be
exclusively configured for the study of floating cells in
accordance with the teachings of the present invention.
[0132] Device 104 essentially consists of eight components: a
carrier 44 as a picowell bearing component, a device body 20, a
liquid inlet 76, a liquid outlet 78, a lower retaining magnet 102,
a gasket 106, a chamber cover 108 and an upper retaining magnet
98.
[0133] Carrier 44, is a substantially planar component including a
picowell array 10 as a feature on an upper surface, similar to
carrier 44 of device 18 depicted in FIG. 2C. Carrier 44 is
preferably made of substantially transparent material, e.g.,
polycarbonate or glass, allowing observation of cells
therethrough.
[0134] Carrier 44 is attached to the periphery of the bottom rim of
a hollow 26 (for example by welding or adhesive) to device body 20
so that picowell array 10 faces upwards defining a chamber 54 with
an opening 120 at the top, the walls of hollow 26 as sides and
carrier 44 as a bottom surface. Preferably, device body 20 is
fashioned from a transparent material.
[0135] Liquid inlet 76 and liquid outlet 78 are essentially tubes
that may be fashioned from for example of PEEK polymer attached,
for example using adhesive, inside grooves 112 located on the top
surface of device body 20. A first end of inlet 76 and outlet 78
emerges into chamber 54 while a second end is provided with
connector 110 allowing simple, quick and leak-free attachment to a
liquid flow generator.
[0136] Lower retaining magnet 102 is substantially a flat thin ring
attached, for example using adhesive, to device body 20 in a broad
circular groove 114 located on the top surface of device body 20
around the periphery of hollow 26.
[0137] Gasket 106 is configured to rest in a circular groove 116
located on the top surface of device body 20 surrounding hollow 26
and surrounded by broad circular groove 114.
[0138] Chamber cover 108 is substantially planar disk made of
substantially transparent material. Chamber cover 108 constitutes a
removable top wall of chamber 54.
[0139] Upper retaining magnet 98 is substantially a flat thin ring
of substantially the same size and shape of lower retaining magnet
102.
[0140] Device 104 is substantially similar to device 96 depicted in
FIG. 6. For example, both device 96 and device 104 are provided
with a carrier 44 as a picowell-bearing component. However, whereas
in device 96 carrier 44 constitutes a moveable top wall of a
chamber 54 for the study of floating cells, in device 104 carrier
44 constitutes a fixed bottom wall of a chamber 54 for the study of
cells that settle in a medium.
[0141] Further, in both device 96 and device 104, magnets 98 and
102 are configured to secure a respective removable top wall of
chamber 54 (carrier 20 in device 96 and chamber cover 108 in device
104). In both devices magnets 98 and 102 together constitute a
force applicator that, when contacted, together apply a
substantially uniformly distributed force around the periphery of
the respective moveable wall in a direction to retain the moveable
wall secured to chamber 54. However, whereas in device 96 upper
retaining magnet 98 is attached to the bottom surface of carrier 44
(which constitutes a removable top wall of chamber 54) and makes
direct contact with lower retaining magnet 102, in device 104
chamber cover 108 (which constitutes a removable top wall of
chamber 54) is sandwiched between upper retaining magnet 98 and
lower retaining magnet 102.
[0142] Performance of experiments using device 104 is substantially
analogous to performance of experiments using device 96. A flow
generator such as a pump or syringe is attached to connector 110 of
liquid inlet 76 of device 104 as taught in embodiments of PCT
patent application IL2001/00992 published as WO2003/035824 of the
inventor. Upper retaining magnet 98 and chamber cover 108 are
removed, allowing access to chamber 54 through top opening 120.
Medium and living cells 56 that are denser than the medium are
introduced (together or separately) into chamber 54 through top
opening 120. Cells 56 settle by sedimentation into picowells of
picowell array 10. Gasket 106 is placed in groove 116. Chamber
cover 108 is placed on top of gasket 106 and lower retaining magnet
102. Upper retaining magnet 98 is placed on top of chamber cover
108 so that magnet 98 and 102 are mutually attracted, holding
chamber cover 108 firmly in place. The force applied on chamber
cover 108 by magnets 98 and 102 compresses gasket 106 ensuring a
tight seal.
[0143] Cells 56 held in picowell array 10 may then be observed
and/or manipulated as described above and in the art, including
during and subsequent to introduction of active entities through
inlet 76. As discussed above, device 104 is configured to allow
simple loading of cells 56 into picowells of picowell array 10 and
to allow performance of long-duration experiments free of leaks
even with relatively high pressure and continuous inflow of liquids
through inlet 76 due to the use of a force applicator that applies
a substantially uniformly distributed force around the periphery of
chamber cover 108 to retain chamber cover 108 secured to chamber
54.
[0144] Both device 96 and device 104 use two mutually attracting
magnets as components of a force applicator to retain a chamber 54
substantially sealed. In embodiments of devices of the present
invention that resemble devices 96 and 104, one of the two magnets
is replaced with an appropriately shaped component of ferromagnetic
material.
[0145] An additional embodiment of a device of the present
invention, 113, is depicted in side cross-section in FIG. 7E. In
FIG. 7E are depicted six individual components of device 113: a
carrier 44, a device body 20, a gasket 106, a lower retaining
magnet 102, a picowell-bearing chamber cover 115 and an upper
retaining magnet 98.
[0146] Carrier 44, is a substantially planar component including a
first picowell array 10a as a feature on an upper surface, similar
to carrier 44 of device 18 depicted in FIG. 2C. Carrier 44 is
preferably made of substantially transparent material, e.g.,
polycarbonate or glass, allowing observation of cells
therethrough.
[0147] Carrier 44 is attached to the periphery of the bottom rim of
a hollow 26 (for example by welding or adhesive) to device body 20
so that first picowell array 10a faces upwards defining a chamber
54 with an opening 120 at the top, the walls of hollow 26 as sides
and carrier 44 as a bottom surface. Preferably, device body 20 is
fashioned from a transparent material. Passing through device body
20 and emerging out into chamber 54 are a liquid inlet and a liquid
outlet such as depicted for device 104 in FIGS. 7A, 7B, 7C and 7D.
Lower retaining magnet 102, gasket 106 and upper retaining magnet
98 are substantially identical to the equivalent components of
device 104 in FIGS. 7A, 7B, 7C and 7D.
[0148] Picowell-bearing chamber cover 115 is substantially planar
disk made of transparent material and like chamber cover 108 of
device 104 constitutes a removable top wall of chamber 54. However,
as a feature integrally formed to the bottom surface of
picowell-bearing chamber cover 115 is a second picowell array
10b.
[0149] The fact that device 113 is provided with two picowell
arrays 15a and 15b allows device 113 to be used for the
simultaneous study of floating cells and cells that settle in a
medium, as well as the interaction therebetween.
[0150] Performance of experiments using device 115 is substantially
analogous to performance of experiments using device 96 and device
104. A flow generator such as a pump or syringe is attached to a
liquid inlet of device 115 as taught in embodiments of PCT patent
application IL2001/00992 published as WO2003/035824 of the
inventor. Upper retaining magnet 98 and picowell-bearing chamber
cover 115 are removed, allowing access to chamber 54 through top
opening 120. Medium and living cells 56 are introduced into chamber
54 (together or separately) through top opening 120. Gasket 106 is
placed in a groove 116. Picowell-bearing chamber cover 115 is
placed on top of gasket 106 and lower retaining magnet 102 so that
guide lips 117 contact gasket 106, thus centering picowell-bearing
chamber cover 115 in chamber 54. Cells of a first type that float
in the medium float upwards to be held in picowells of second
picowell array 10b. Cells denser than the medium settle by
sedimentation into picowells of first picowell array 10a. Upper
retaining magnet 98 is placed on top of picowell-bearing chamber
cover 115 so that magnets 98 and 102 are mutually attracted,
holding picowell-bearing chamber cover 115 firmly in place. The
force applied on picowell-bearing chamber cover 115 by magnets 98
and 102 compresses gasket 106 ensuring a tight seal.
[0151] Cells held in picowell arrays 10a and 10b are then observed
as described above and in the art, including during and subsequent
to introduction of active entities through the liquid inlet. As
discussed above, device 104 is configured to allow simple loading
of cells into picowells of picowell arrays 10a and 10b and to allow
performance of long-duration experiments free of leaks even with
relatively high pressure and continuous inflow of liquids through a
liquid inlet due to the use of a force applicator that applies a
substantially uniformly distributed force around the periphery of
picowell-bearing chamber cover 115 to retain picowell-bearing
chamber cover 115 secured to chamber 54.
[0152] An additional embodiment of a device of the present
invention, 118, is depicted in side cross-section in FIG. 8. In
FIG. 8 are depicted four individual components of device 118: a
carrier 44, a device body 20, a gasket 106 and a chamber cover
108.
[0153] Carrier 44 is a substantially planar component including a
picowell array 10 as a feature on an upper surface, similar to
carrier 44 of device 18 depicted in FIG. 2C. Carrier 44 is
preferably made of substantially transparent material, e.g.,
polycarbonate or glass, allowing observation of cells
therethrough.
[0154] Carrier 44 is attached to the periphery of the bottom rim of
a hollow 26 (for example by welding or adhesive) to device body 20
so that picowell array 10 faces upwards defining a chamber 54 with
a circular opening 120 at the top, the walls of hollow 26 as sides
and carrier 44 as a bottom surface. Preferably, device body 20 is
fashioned from a transparent material. Passing through device body
20 and emerging out into chamber 54 are liquid inlet 76 and liquid
outlet 78. The walls of opening 120 are provided with threads
122.
[0155] Gasket 106 is configured to rest inside hollow 26 on a ledge
located at the bottom of opening 120.
[0156] Chamber cover 108 is a substantially planar disk made of
transparent material. Chamber cover 108 constitutes a removable top
wall of chamber 54. On the sides of chamber cover 108 are threads
124 configured to mate with threads 122 of the walls of opening
120.
[0157] Device 118 is substantially similar to device 104 depicted
in FIGS. 7A-7D. A significant difference is in the nature of the
respective force applicators.
[0158] In device 104 magnets 98 and 102 together constitute a force
applicator that, when contacted together apply a substantially
uniformly distributed force around the periphery of chamber cover
108, the removable top wall of chamber 54, in a direction to retain
chamber cover 108 secured to chamber 54. In contrast, in device 118
threads 122 and 124 together constitute a force applicator
configured to apply a substantially uniformly distributed force
around the periphery of chamber cover 108, the removable wall of
chamber 54 in a direction to retain chamber cover 108 secured to
chamber 54.
[0159] Performance of experiments using device 118 is substantially
analogous to performance of experiments using device 104. A flow
generator such as a pump or syringe is attached to liquid inlet 76
of device 118. Chamber cover 108 is removed, allowing access to
chamber 54 through top opening 120. Medium and living cells 56 that
are denser than the medium are introduced (together or separately)
into chamber 54 through top opening 120. Cells 56 settle by
sedimentation into picowells of picowell array 10. Gasket 106 is
placed on the ledge inside hollow 26. Chamber cover 108 is placed
over opening 120 and rotated so that threads 122 and threads 124
mate. Chamber cover 108 is screwed into opening 120 until gasket
106 is sufficiently compressed to ensure a substantially leak proof
seal.
[0160] Cells 56 held in picowell array 10 may be then observed
and/or manipulated as described above and in the art, including
during and subsequent to introduction of active entities through
inlet 76. As discussed above, device 118 is configured to allow
simple loading of cells 56 into picowells of picowell array 10 and
to allow performance of long-duration experiments free of leaks
even with relatively high pressure and continuous inflow of liquids
through inlet 76 due to the use of a force applicator that applies
a substantially uniformly distributed force around the periphery of
chamber cover 108 to retain chamber cover 108 secured to chamber
54.
[0161] An additional embodiment of a device of the present
invention, 126, is depicted in side cross-section in FIG. 9. In
FIG. 9 are depicted five individual components of device 126: a
carrier 44, a device body 20, a gasket 106, a chamber cover 108 and
a rigid force distributor 128.
[0162] Carrier 44, is a substantially planar component including a
picowell array 10 as a feature on an upper surface, similar to
carrier 44 of device 18 depicted in FIG. 2C. Carrier 44 is
preferably made of substantially transparent material, e.g.,
polycarbonate or glass, allowing observation of cells
therethrough.
[0163] Carrier 44 is attached to the periphery of the bottom rim of
a hollow 26 (for example by welding or adhesive) to device body 20
so that picowell array 10 faces upwards defining a chamber 54 with
an opening 120 at the top, the walls of hollow 26 as sides and
carrier 44 as a bottom surface. Preferably, device body 20 is
fashioned from a transparent material. Passing through device body
20 and emerging out into chamber 54 are liquid inlet 76 and liquid
outlet 78.
[0164] Gasket 106 is placed on a ledge located near the rim of
opening 120 so that the top edge of gasket 106 is somewhat higher
than the top surface of device body 20.
[0165] Chamber cover 108 is substantially planar component made of
transparent material. Chamber cover 108 constitutes a removable top
wall of chamber 54.
[0166] Rigid force distributor 128 is a substantially planar rigid
component with a gap 130 through the middle, e.g., a
polytetrafluoroethylene-coated steel washer. Gap 130 through rigid
force distributor 128 is of a size and shape to allow an unimpeded
view therethrough of cells held in picowell array 10 when rigid
force distributor 128 is placed over chamber cover 108, as
described below.
[0167] Device 126 is substantially similar to device 104 depicted
in FIGS. 7A-7D. A significant difference is in the nature of the
respective force applicators. In device 104, magnets 98 and 102
together constitute a force applicator configured to secure a
respective removable top wall of chamber 54 (chamber cover 108), by
applying a substantially uniformly distributed force around the
periphery of the respective moveable wall in a direction to retain
the moveable wall secured to chamber 54. In contrast, in device 126
rigid force distributor 128, together with a force applying
component such as a clamp, tack, press or clip (not depicted),
constitute components of a force applicator configured to apply a
substantially uniformly distributed force around the periphery of
the respective moveable wall (chamber cover 108) in a direction to
retain the moveable wall secured to chamber 54.
[0168] Performance of experiments using device 126 is substantially
analogous to performance of experiments using device 104 depicted
in FIGS. 7A-7D. A flow generator such as a pump or syringe is
attached to liquid inlet 76 of device 126. Chamber cover 108 is
removed, allowing access to chamber 54 through top opening 120.
Medium and living cells 56 that are denser than the medium are
introduced (together or separately) into chamber 54 through top
opening 120. Cells 56 settle by sedimentation into picowells of
picowell array 10. Gasket 106 is placed on the ledge inside hollow
26. Chamber cover 108 is placed over opening 120 and rigid force
distributor 128 is placed over chamber cover 108 so that gap 130
allows an unimpeded view of picowell array 10 through chamber cover
108. A force-applying component such as a clamp, tack, press or
clip (not depicted) is used to apply a force at at least two points
around the perimeter of rigid force distributor 128. The magnitude
of the force applied is sufficient to compress gasket 106 enough to
ensure a substantially leak proof seal. Since rigid force
distributor 128 is rigid, the force applied by force-applying
component is substantially uniformly distributed around the
periphery of chamber cover 108 to retain chamber cover 108 secured
to chamber 54.
[0169] Cells 56 held in picowell array 10 may then be observed
and/or manipulated as described above and in the art, including
during and subsequent to introduction of active entities through
inlet 76. As discussed above, device 126 is configured to allow
simple loading of cells 56 into picowells 12 and to allow
performance of long-duration experiments free of leaks even with
relatively high pressure and continuous inflow of liquids through
inlet 76 due to the use of a force applicator that applies a
substantially uniformly distributed force around the periphery of
chamber cover 108 to retain chamber cover 108 secured to chamber
54.
[0170] An additional embodiment of a device of the present
invention, 132, is depicted in an exploded view in perspective in
FIG. 10A (to scale), a close-up showing details of device body 20
from the top in FIG. 10B (to scale), and a close-up showing details
of device body 20 from the bottom in FIG. 10C. A fully assembled
device 132 integrated in a system 134 is depicted in FIG. 10D.
[0171] Device 134 essentially consists of six components: a carrier
44, a device body 20, a lower retaining magnet 102, a gasket 106,
an upper retaining magnet 98 and a chamber cover 108.
[0172] Carrier 44, is a substantially planar component including a
picowell array 10 as a feature on an upper surface, similar to
carrier 44 of device 18 depicted in FIG. 2C. Carrier 44 is
preferably made of substantially transparent material, e.g.,
polycarbonate or glass, allowing observation of cells
therethrough.
[0173] Carrier 44 is attached to the periphery of the bottom rim of
a hollow 26 (for example by welding or adhesive) to device body 20
so that picowell array 10 faces upwards defining a chamber 54 with
a circular opening 120 at the top, the walls of hollow 26 as sides
and carrier 44 as a bottom surface. Preferably, device body 20 is
fashioned from a transparent material.
[0174] Passing through device body 20 and emerging out into chamber
54 are liquid inlet 76 and liquid outlet 78, see especially FIG.
10C. Embedded inside device body 20 is heating wire 136 and
electrical contacts 138 (see especially FIG. 10A), where heating
wire 136 passes in the proximity of chamber 54. Heating wire 136
acts as a temperature controller, so when electrical current is
passed through heating wire 136, heating wire 136 generates heat,
raising the temperature of medium held in chamber 54.
[0175] Similarly to gasket 106 of device 104 depicted in FIGS.
7A-7D, gasket 106 of device 134 is configured to fit in a circular
groove 116 located on the top surface of device body 20 surrounding
opening 120.
[0176] Similarly to lower retaining magnet 102 of device 96
depicted in FIG. 6, lower retaining magnet 102 of device 134 is
planar and is attached (for example using adhesive) to the upper
surface of device body 20. Lower retaining magnet 102 is provided
with an opening that fits about gasket 106.
[0177] Chamber cover 108 is a substantially planar component made
of a transparent material having substantially the same outer
dimensions of device body 20.
[0178] Similarly to upper retaining magnet 98 of device 96 depicted
in FIG. 6, upper retaining magnet 98 of device 134 is planar and is
attached (for example using adhesive) to the lower surface of
chamber cover 108. Upper retaining magnet 98 is provided with an
opening that fits about gasket 106.
[0179] Similarly to devices 104, 118 and 126, device 134 is
provided with a picowell array 10 on a top surface of a carrier 44
and constitutes a bottom wall of chamber 54. Similarly to devices
96, 104, 118 and 126 is provided with a non-capillary liquid inlet
76 and liquid outlet 78 in fluid communication with chamber 54 and
for use generally requires attachment of a flow generator to liquid
inlet 76. Analogously to device 104 depicted in FIGS. 7A-7D,
chamber cover 108 of device 134 constitutes a removable upper wall
of chamber 54. Analogously to device 96 depicted in FIG. 6, in
device 134 magnets 98 and 102 are configured to secure chamber
cover 108 to device body 20 so as to substantially seal chamber 54.
Magnets 98 and 102 together constitute a force applicator that,
when contacted, together apply a substantially uniformly
distributed force around the periphery of opening 120 of chamber 54
in a perpendicular direction so as to retain chamber cover 108
secured to device body 20 and thus to chamber 54.
[0180] In embodiments, a device 134 is used substantially similarly
as a device 104 described above.
[0181] As discussed above, device 96 (depicted in FIG. 6), device
104 (depicted in FIGS. 7A-7D), device 118 (depicted in FIG. 8),
device 126 (depicted in FIG. 9) and device 132 (depicted in FIGS.
10A-10C) include at least one picowell inside a chamber with at
least one moveable wall that allows performance of long-duration
experiments substantially free of leaks around the respective
moveable wall/chamber interface due to the use of a force
applicator configured to removeably apply a substantially uniformly
distributed force around the periphery of the moveable wall in a
direction to retain the moveable wall secured to the chamber. In
embodiments of the present invention, such devices are integrated
with other components that allow performance of long duration
experiments with living cells and can be considered as miniature
incubators, or microincubators. One such embodiment, depicted in
FIG. 10D, is system 134 including a picowell-bearing device 132 of
the present invention.
[0182] System 134 includes, in addition to picowell-bearing device
132, a flow-generator 140, a liquid reservoir 142, a gas reservoir
144, a gas inlet valve 146, an electrical power supply 148, a
detector array 150 and a controller 152.
[0183] Flow-generator 140 (e.g., a pump such as a peristaltic pump)
is configured to generate a flow of liquids from liquid reservoir
142 into inlet 76 of picowell-bearing device 132. Flow generator
140 is a variable flow generator and can be set to provide varying
flow rates of liquids into inlet 76.
[0184] Liquid reservoir 142 is a vessel configured to hold liquids,
such as medium.
[0185] Gas reservoir 144 is in communication with liquid reservoir
142 through gas inlet valve 146. When gas inlet valve 146 is open,
gas from gas reservoir 144 bubbles through liquid held in liquid
reservoir 142, increasing the content of the gas in the liquid.
Typical gases include oxygen and carbon dioxide.
[0186] Electrical power supply 148 is functionally associated with
heating wire 136 through electrical contacts 138.
[0187] Detector array 150 includes sensors for measuring various
properties of a liquid passing through tube 154 such as oxygen and
carbon dioxide content (e.g., by infrared absorption), temperature,
pH, osomolarity and/or conductivity.
[0188] Controller 152, a computer, is functionally associated with
flow generator 140, gas-inlet valve 146, electrical power supply
148, detector array 150 and user interface 156. Based on stored
data, instructions received through user interface 156 and
measurements received from detector array 150, controller 152
controls the rate of liquid flow from fluid reservoir 142 through
chamber 54 of device 132 by controlling flow generator 140, the gas
content of such a liquid by controlling gas-inlet valve 146 and the
temperature of liquid in chamber 54 by controlling electrical power
supply 148.
[0189] For example, a system such as 134 is assembled. Chamber
cover 108 and associated upper retaining magnet 98 are separated
from device body 20, allowing access to chamber 54 through top
opening 120. Medium and living cells 56 are introduced (together or
separately) into chamber 54 through top opening 120. Cells 56
settle by sedimentation into picowells 12 of picowell array 10.
Gasket 106 is placed in groove 116. Chamber cover 108 and
associated upper retaining magnet 98 are placed in proximity of
device body 20 so that magnets 98 and 102 are mutually attracted,
holding chamber cover 108 firmly in place in accordance with the
present invention. Medium is placed in liquid reservoir 142. A
desired gas, typically carbon dioxide to encourage cell
development, is placed in gas reservoir 144.
[0190] Controller 152 is activated to perform a desired experiment.
Flow generator 140 is activated, producing a flow of medium from
liquid reservoir 142 through chamber 54 that supplies nutrients and
removes waste products from the vicinity of cells 56 held in
picowells of picowell array 10. Controller 152 receives a reading
of the temperature of medium flowing through tube 154 and adjusts
power to heating wire 126 supplied by electrical power supply 148
to ensure that medium in chamber 54 is at a desired temperature.
Controller 152 receives a reading of the carbon dioxide content of
medium flowing through tube 154 and if necessary activates gas
inlet valve 146 to increase the carbon dioxide content of medium in
liquid reservoir 142.
[0191] Cells 56 held in picowell array 10 are then observed as
described above and in the art, including during and subsequent to
introduction of active entities through inlet 76.
[0192] In system 134, medium held in chamber 56 is heated using
heating wires 136 embedded in device body 20. In embodiments,
heating is performed by an external heating element, for example
using a Peltier heating device as a liquid temperature controller
on which a picowell-bearing device rests. In embodiments liquid is
heated, for example using a liquid temperature controller that
heats the liquid in a liquid reservoir 142, in a flow-generator
140, or in tubing 154 connected to a liquid inlet 76 or liquid
outlet 78.
[0193] System 134 is a closed system, that is, liquid exiting
chamber 54 of picowell-bearing device 132 reenters liquid reservoir
142, processed (e.g., by the addition of gas from gas reservoir
144) and then pumped back into chamber 54. Such a configuration
allows for the construction of a compact system. In embodiments of
the present invention, a system is an open system, that is, liquid
exiting a chamber 54 of a picowell-bearing device is not recycled
back into chamber 54, similarly to systems taught in embodiments of
PCT patent applications IL2001/00992 published as WO2003/035824 of
the inventor.
[0194] The picowell-bearing devices of the present invention
described above, 18, 66, 72, 88, 90, 96, 104, 126 and 132 include
one picowell array 10. In embodiments, a device of the present
invention, such as device 118, includes more than one picowell
array. For use, cells are loaded in each one of the more than one
picowell arrays, preferably a different type in each one of
picowell arrays, allowing performance of experiments on more than
one type of cell using one device body.
[0195] The devices of the present invention are picowell-bearing
devices, that is, the devices include at least one picowell,
preferably an array of picowells. Preferably, individual picowells
of a picowell array are individually addressable. For ease of
optical study and observation, it is preferred that the bottoms of
all picowells be substantially coplanar: coplanarity allows for
optical observation of many cells (whether by scanning or
simultaneously using a wide-angle observation component) without
the need for time consuming and technically difficult to implement
refocusing.
[0196] As noted above, components of a device of the present
invention are preferably fashioned of transparent material,
especially in the vicinity of the picowells allowing illumination
and/or observation of cells held in the picowells. In embodiments
of the present invention, the transparent material has an index of
refraction similar to that of water, for example an index of
refraction less than about 1.4, less than about 1.38, less than
about 1.36, less than about 1.35 and even less than about 1.34.
When such a material is used, the material is substantially
invisible.
[0197] Preferably, picowells of a picowell array are juxtaposed. By
juxtaposed is meant that in an area where picowells are found, the
picowells are nearby and side by side so that most of the area is
picowell area and little of the area is inter picowell area.
According to a feature of the present invention, by juxtaposed is
meant that the inter picowell area between two picowells is less
than or equal to 0.35, 0.25, 0.15, 0.10 or even 0.06 of the sum of
the areas of the two picowells. In embodiments of the present
invention it is preferred that the inter picowell area be
substantially zero, that is that the rims of picowells are
substantially knife-edged.
[0198] As the teachings of the present invention are directed to
cellular biology, it is generally preferred that the picowells be
small so as to avoid having a large number of cells held in any one
picowell. Thus, generally, the dimensions of the picowells are
generally up to about 250, 200, 150, 100, 50, 25, 20, 15 or even 10
microns. By dimensions is meant the usual meaning of the word and
is dependent on the shape of the picowell. For example, for
hexagonal or circular picowells, the term dimension refers to
diameter. For square or triangular picowells is meant the longest
dimension of the square or triangle, respectively. The exact
dimensions of individual picowells depends on the type (and
consequently size) of cells to be studied and the types of
experiments and studies that are to be performed. Since different
types of cells have different sizes, generally a picowell array of
the present invention has picowells of a size to accommodate one or
more cells of the type to be studied. In embodiments it is
preferred that an individual picowell be of a size so as to hold no
more than one living cell of a certain size. In embodiments it is
preferred that the picowell be of a size so as to held no more than
a predetermined number of cells of a certain size (e.g., two or
three cells simultaneously).
[0199] In some embodiments of the present invention, picowells are
dimples or depressions on the bottom surface of the picowell array.
In other embodiments the picowells are substantially enclosures of
dimensions so that at least one cell of a certain size is
containable, preferably but not necessarily substantially entirely,
within the enclosure, each enclosure having an opening at the
surface, the opening defined by a first cross section of a size
allowing passage of cell of the certain size The exact dimensions
of the individual enclosures depends on the type (and consequently
size) of cells to be studied and the types of experiments and
studies that are to be performed. The volume of such enclosure
picowells is typically less than 1.56.times.10.sup.-11 liter
(corresponding to the volume of a 250 micron cube), less than
1.times.10.sup.-11 liter (corresponding to the volume of a 200
micron cube), less than 1.times.10.sup.-12 liter (corresponding to
the volume of a 100 micron cube), less than 1.times.10.sup.-13
liter (corresponding to the volume of a 50 micron cube), less than
1.times.10.sup.-14 liter (corresponding to the volume of a 25
micron cube) and even less than 1.times.10.sup.-15 liter
(corresponding to the volume of a 10 micron cube). The area of the
first cross section, corresponding to the size of the opening of a
respective enclosure is typically less than about 62500
micron.sup.2 (corresponding to the area of a 250 micron square),
about 40000 micron.sup.2 (corresponding to the area of a 200 micron
square), 10000 micron.sup.2 (corresponding to the area of a 100
micron square), 2500 micron.sup.2 (corresponding to the area of a
50 micron square), 625 micron.sup.2 (corresponding to the area of a
25 micron square) or even less than about 100 micron.sup.2
(corresponding to the area of a 10 micron square).
[0200] Embodiments of a device of the present invention have
dimensions near those of a standard microscope slide, i.e., 28.4 mm
wide by 76.2 mm long, e.g., devices 18, 66, 104 and 132. For
example, in an embodiment of device 18 the center of picowell array
10 is 18.1 mm from the edge of device body 20. The width between
the two rail walls 38 is 24.5 mm, between the distal edges of gaps
32 19.5 mm, of plateau 28 in the vicinity of capillary channel 46
and of plateau 28 at the end of the waste reservoir region 50 15.2
mm. The thickness of device body 20 is 2.3 mm. The radius of hollow
26 is 1 mm and has a volume of 5.times.10.sup.-6 liter. When
sliding base 22 is in place, the volume between sliding base 22 and
plateau 28 upstream of hollow 26 is about 10 microliter while the
volume between sliding base 22 and plateau 28 downstream of hollow
26 is about 150 microliter.
Methods of Manufacture of a Device of the Present Invention
[0201] In general, manufacture and assembly of a device of the
present invention is well within the ability of one skilled in the
art upon perusal of the description and figures herein using any
suitable method with which one skilled in the art is well
acquainted. Many such methods are described in relation to the
manufacture of picowell-bearing devices in PCT patent applications
IL2001/00992 published as WO2003/035824, IL2004/000571 published as
WO2004/113492, IL2004/000194 published as WO2004/077009,
IL2004/000661 published as WO2005/007796, and unpublished
IL2005/000801. Suitable methods include methods that employ one or
more techniques including but not limited to casting, embossing,
etching, free-form manufacture, injection-molding, microetching,
micromachining, microplating, molding, spin coating, lithography or
photo-lithography.
[0202] In embodiments of the present invention, an entire device of
the present invention and all components thereof are made of one
material. In other embodiments, a device of the present invention
is made up of a number of different materials. In embodiments of
the present invention, individual components, for example a device
body, are made of a number of different materials as a plurality of
layers or as a coated structure.
[0203] In an embodiment of the present invention, the walls of
picowells are integrally formed with a surface of a device body
(e.g., as in device 72). In embodiments including a separate
picowell-bearing component (e.g., a carrier 44) the picowell
bearing component is made of any suitable material, the same or
different than the material from which the respective device body
is made. Suitable materials from which to make components of a
device of the present invention such as a carrier, a device body, a
sliding base and the like include but are not limited to ceramics,
elastomers, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate
glycol, polymers, polyurethane, polymethyl methacrylate,
polystyrene, polytetrafluoroethylene, polyvinyl chloride, rubber,
silicon, silicon oxide, silicon rubber and sol-gels.
[0204] Manufacture of a device body 20 or carrier 44 with
integrally formed picowells that are a feature of the component, of
a device body without integrally formed picowells or another
picowell-bearing component is clear to one skilled in the art upon
perusal of the description herein. A preferred method is analogous
to the methods disclosed by the inventor in PCT Patent Application
No. IL01/00992, in PCT Patent Application No. IL04/00571 and in PCT
Patent Application No. IL04/00661. Such a method includes a)
contacting a precursor material with a template including a
negative of features of the device body or picowell-bearing
component so as to create the desired features in the precursor
material, the features including the picowell array; b) fixing the
features in the precursor material so as to fashion an incipient
device body or picowell-bearing component; and c) processing the
incipient device body or picowell-bearing component so as to
fashion the device body or picowell-bearing component of the
present invention.
[0205] In an embodiment of the present invention, a
picowell-bearing component is fashioned inside a device body by
contacting an appropriate template with a moldable precursor
material when the precursor material is inside a depression. Such
an embodiment is discussed in detail hereinbelow.
[0206] Components of a device of the present invention such as a
device body with integrally formed picowells, a device body without
integrally formed picowells or a picowell-bearing component of the
present invention are preferably made according to the methods
discussed hereinbelow.
[0207] A first method of the present invention for making a
component of the present invention is substantially by contacting a
precursor material with a template, the template having a negative
of some of the features of the component (especially the picowells)
thus creating the features in the precursor material. The features
are subsequently fixed in the precursor material making an
incipient component. After any further required processing of the
incipient component (which may be limited to simply separating the
template from the incipient component), the device is assembled
from the individual components.
[0208] Depending on the precursor material, fixing includes, but is
not limited to, methods such as heating the precursor material,
cooling the precursor material, curing the precursor material,
polymerizing the precursor material, cross-linking the precursor
material, irradiating the precursor material, illuminating the
precursor material, gelling the precursor material, exposing the
precursor material to a fixative and waiting a period of time. By
fixative is meant an agent that causes the precursor material to
change to the fixed state and is used herein as a general term for
such materials as fixatives, hardeners,
polymerization/crosslinking/curing initiators, catalysts and
agents. It is important to note that in some cases a precursor
material is produced by mixing two or more components which
thereafter change to a fixed state, for example, by simply waiting
a period of time.
[0209] In one preferred embodiment of the present invention, the
precursor material is a irreversibly deformable precursor material.
Herein by irreversibly deformable precursor material is meant a
material that does not recover a shape after deformation and so
there is usually no need for a separate action to fix the features
in the precursor material beyond separating the produced component
from the template. In such cases, the precursor material does not
substantially chemically change subsequent to contact with the
template. Examples of suitable irreversibly deformable precursor
materials include waxes, paraffins, plastics, polymers and the
like. In such an embodiment, a preferred template is a stamp, and
the contacting of the template with the precursor material is
substantially stamping the features of the component into the
precursor material, preferably under controlled thermal
conditions.
[0210] In another preferred embodiment of the present invention,
the precursor material is a reversibly deformable precursor
material. Herein by reversibly deformable precursor material is
meant a material that is capable of recovering shape after
deformation and includes gellable fluids, polymerizable materials,
powders, fluids and thermoplastic materials.
[0211] In a preferred embodiment, the reversibly deformable
precursor material is a thermoplastic material at a pliable
temperature. Subsequent to the contacting of the template but
before the contact is finished, the thermoplastic material is
cooled, thus fixing the desired features in the incipient
component.
[0212] In another preferred embodiment, the reversibly deformable
precursor material is a polymerizable material (e.g., a monomer
solution, a crosslinkable polymer, a vulcanizable polymers, a
polymerizable fluids or a thermosetting resin). Subsequent to the
contacting of the template but before the contact is finished, the
polymerizable material is polymerized, thus fixing the desired
features in the incipient component. In such cases, the precursor
material and the material from which the component is made are
chemically dissimilar (for example, have the relationship of
monomer to polymer).
[0213] One preferred polymerizable precursor material is a
non-cured polydimethylsiloxane precursor mixture. A mixture of two
polydimethylsiloxane components (the prepolymer and curing agent)
are mixed together in the desired ratio (preferably about 10:1, but
ratios between about 5:1 and about 20:1 are generally suitable) to
give a polydimethylsiloxane precursor mixture, the mixture degassed
and contacted with the template. The features are fixed by the
curing of the mixture. Curing of polydimethylsiloxane precursor
generally takes place at room temperature for about 24 hours and,
when desired, is accelerated by heating. For example it has been
found that components of the device of the present invention made
of polydimethylsiloxane are ready for further processing within 2
hours when cured at 60.degree. C. or within 15 minutes when cured
at 150.degree. C. A detailed review of methods for the production
of micronic features such as picowells from polydimethylsiloxane
suitable for implementing the teachings of the present invention
are known in the art and discussed, for example, in Ng et al.,
Electrophoresis 2002, 23, 3461-3473 and Duffy et al., Anal. Chem.
1998, 70, 4974-4984.
[0214] Another preferred polymerizable precursor material is
urethane that is polymerized to yield polyurethane.
[0215] Another preferred reversibly deformable precursor material
is a gellable fluid. After the gellable fluid is brought in contact
with the template, the features are fixed by gelling the gellable
fluid to yield a gel. Most preferred are gellable fluids that
produce a hydrogel.
[0216] Gellable fluids known in the art include fluids that gel
upon heating, fluids that gel upon cooling, fluids that gel upon
irradiation or illumination, fluids that gel as a result of contact
with a gelling reagent and fluids that gel after a period of time.
Preferred gellable fluids for implementing the teachings of the
present invention include solutions of agars, agaroses, gelatins,
low melting temperature agaroses, alginates, proteins, protein
polysaccharides, Ca.sup.2+-inducable alginates (especially those
that gel at room temperature) and polysaccharides.
[0217] One preferred gellable fluid is a low-melting temperature
agarose solution. Such a solution is fluid at temperatures that do
not harm living cells (e.g., 20.degree. C.) and gel at low
temperatures that do not harm living cells (e.g., 4.degree. C.). An
exceptionally suitable agarose for implementing the teachings of
the present invention that may be purchased, for example, from
Cambrex Bio Science Rockland Inc. (Rockland, Me., USA) is HGS-LMP
Agarose 0.5% in PBS (Catalogue Nr. 50221).
[0218] Another preferred gellable fluid is an alginate solution
which gels upon contact with a gelling reagent, the preferred
gelling reagent being a solution having a Ca.sup.2+ ion
concentration of greater than about 1.times.10.sup.-6 M. An
exceptionally useful gelling agent is a 20.times.10.sup.-3 M
calcium gluconate solution. Suitable alginate solutions can be
purchased from Pronova Biopolymers (Drammen, Norway) and include,
for example, Protanal LF120 1% in water and Protanal LF200 1% in
water.
[0219] The template having a negative of the features is, for
example, a stamp or a mold, and is generally made of any suitable
material that is more rigid than a respective precursor material.
Suitable materials include but are not limited to reversibly
deformable materials, irreversibly deformable materials, ceramics,
epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, paraffins, polytetrafluoroethylene,
polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon
oxide, silicon rubbers, sol-gels and wax.
[0220] The template is made, for example, using methods with which
one skilled in the art is acquainted such as casting, embossing,
etching, free-form manufacture, injection-molding, microetching,
micromachining, microplating, molding, lithography or
photo-lithography.
[0221] In an embodiment, a device of the present invention is made
by making picowells as described above inside a hollow (such as 26)
of a device body (such as 20). Suitable device bodys include but
are not limited to device bodys made of reversibly deformable
materials, irreversibly deformable materials, ceramics, epoxies,
glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, polystyrene, polytetrafluoroethylene,
polyurethanes, polyvinyl chloride, silicon, silicon oxide, silicon
rubbers and sol-gels. In such a case, a precursor material for
making picowells is placed into the depression of the device body.
A template is then placed inside the depression so as to make
contact with the precursor material and the precursor material is
fixed as described above. Such an embodiment has the advantage that
a device body devoid of picowells of virtually any material is mass
produced. Subsequently, a template is made and used for fixing
picowells of virtually any desired size, number and arrangement in
the device body.
[0222] In another embodiment noted above, a template including the
negative of the desired features excepting picowells is contacted
with the precursor material so as to form a device body with a
depression devoid of picowells. Subsequently, a grid-like
component, constituting substantially the walls of the picowells of
the picowell array, is attached using an appropriate method, for
example, adhesives (for example, light curable adhesives, such as
light curing adhesive 3051 or 3341 manufactured by Henkel Loctite
Deutschland GmbH, Munchen, Germany) or surface treatments such as
anodic bonding, fusion bonding or plasma treatment such as plasma
discharge (exceptionally suitable for attaching
polydimethylsiloxane, see Duffy et al., Anal. Chem. 1998, 70,
4974-4984).
[0223] Another method of making a picowells in a device body
includes photolithography of a photoresist material placed on a
substrate, a commercially available process (for example, from
Micro Resist Technology GmbH, Berlin, Germany) with which one
skilled in the art is well-acquainted. In brief, a template
including the negative of the desired features excepting picowells
is contacted with the precursor material so as to form a device
body with a depression devoid of picowells. Subsequently, a high
aspect ratio photoresist material (e.g., SU-8 thick photoresist
fluid, MicroChem Corporation, Newton Mass., USA) is placed in the
depression as a uniformly thick film. A preferred method of
achieving a uniformly thin film of a photoresist fluid is by spin
coating, that is, the photoresist fluid is placed in the depression
of the device body and the device body rotated about a
perpendicular axis. As a result of the rotation the photoresist
fluid forms a uniformly thick film, typically between about 5
microns and about 20 microns thick. Once a film of uniform
thickness of photoresist material is achieved, the photoresist
material is illuminated through a mask, the mask being
substantially a template or master of the desired picowells.
Developing of the precursor with the selectively fixed film removes
the non-fixed areas of the film. In such a way the picowell array
is made up of a fixed photoresist layer resting on a device body
where the picowells are carved into the photoresist layer and the
bottom of the picowells is the surface of the precursor plate.
Using a photolithography method, picowells having a flat-bottom
surface are easily produced.
[0224] The components of a device of the present invention are made
of any suitable material. Suitable materials include but are not
limited to ceramics, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane, polymers,
polyethylenterephtalate glycol, polymethyl methacrylate,
polystyrene, polytetrafluoroethylene, polyurethanes, polyvinyl
chloride, silicon, silicon oxide and sol-gels.
[0225] Another method of making a device of the present invention
comprises attaching one or more picowell-bearing components to a
precursor plate using an appropriate method, for example, using an
adhesive or a surface treatment such as a plasma treatment, for
example as described above. A preferred picowell-bearing component
is a device body comprising a plurality of picowells disposed on a
surface. Preferred device bodies include those described in PCT
Patent Application No. IL01/00992 or PCT Patent Application No.
IL04/00571.
[0226] Some embodiments of the device of the present invention
comprise picowells where the inside surface of the picowells (with
which held cells potentially make physical contact) is coated with
a layer of some desired coating material, for example a coating
material that influences the proliferation of living cells as
described in PCT Patent Application No. IL04/00571 .
[0227] One skilled in the art is acquainted with many ways and many
coating materials with which to coat an inside surfaces of
picowells of a device of the present invention.
[0228] One preferred method of coating inside surfaces of picowells
of a device of the present invention, applicable to virtually any
device produced by virtually any method, comprises contacting a
precursor fluid with the inside surface of the picowells and
subsequently solidifying the precursor fluid, forming the layer of
the coating material. Depending on the nature of the precursor
fluid, solidifying is performed by any number of methods including
but not limited to heating the precursor fluid, cooling the
precursor fluid, polymerizing the precursor fluid, cross-linking
the precursor fluid, curing the precursor fluid, irradiating the
precursor fluid, illuminating the precursor fluid, gelling the
precursor fluid, exposing the precursor fluid to a fixative or
waiting a period of time.
[0229] One preferred method of coating the inside surfaces of
picowells of a device of the present invention, applicable to
virtually any device produced by virtually any method, is by vapor
deposition. Vapor deposition involves the deposition of materials
such as molecules or atoms onto a surface at low pressures and is
characterized by the production of evenly thin coatings on a
surface, such as the inner surface of a picowell of a device of the
present invention.
[0230] In one embodiment of vapor deposition to the inside surfaces
of picowells of a device of the present invention, the atoms or
molecules that make up the coating material are deposited. In
another embodiment of vapor deposition, the atoms or molecules that
comprise a precursor of the coating material are deposited on the
inside surfaces of the picowells, followed by solidifying the
coating precursor material thereby forming the layer of coating
material. Solidifying of the coating precursor material to form the
layer of coating material is performed by any number of methods
including but not limited to heating the coating precursor
material, cooling the coating precursor material, polymerizing the
coating precursor material, cross-linking the coating precursor
material, curing the coating precursor material, irradiating the
coating precursor material, illuminating the coating precursor
material, gelling the coating precursor material, exposing the
coating precursor material to a fixative and waiting a period of
time.
[0231] A preferred coating material for coating the inside surfaces
of picowells of a device of the present invention is made of
polymerized para-xylylene molecules (or derivatives thereof,
specifically where one or more hydrogens, especially aromatic
hydrogens of either or both aromatic rings are substituted)
deposited by vapor deposition, a coating commercially known as
Parylene.RTM. (available for example from V & P Scientific,
Inc., San Diego, Calif., USA). Parylene.RTM. is preferred not only
for cell proliferation influencing properties but also for the fact
that Parylene.RTM. coatings are bacteria resistant, fungus
resistant, transparent, have a low permeability, acid and base
resistant, uniform, thin (typically 0.1-1 micron) and without voids
even when a coated surface includes configurations with sharp
edges, points, flat surfaces, crevices or exposed internal
surfaces.
[0232] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in
the art. Accordingly, the present invention is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0233] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In case of conflict, the specification herein,
including definitions, prevails. Citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *