U.S. patent application number 13/116577 was filed with the patent office on 2011-12-15 for microfluidic device.
Invention is credited to Marcel Reichen, Nicolas Szita.
Application Number | 20110306081 13/116577 |
Document ID | / |
Family ID | 40230882 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306081 |
Kind Code |
A1 |
Szita; Nicolas ; et
al. |
December 15, 2011 |
Microfluidic Device
Abstract
The invention relates to a microfluidic device including a
chamber having a fluid inlet, a fluid outlet and a sealable port.
In some embodiments, the fluid inlet and the fluid outlet may be
positioned to direct fluid flowing from the fluid inlet to the
fluid outlet through the chamber. Various embodiments may include a
sealable port which may be aligned with the chamber to allow
material to be placed directly into, or removed from, the chamber
from the exterior of the device when the sealable port is open, and
to inhibit and/or prevent fluid escaping through the sealable port
when the port is sealed.
Inventors: |
Szita; Nicolas; (London,
GB) ; Reichen; Marcel; (London, GB) |
Family ID: |
40230882 |
Appl. No.: |
13/116577 |
Filed: |
May 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/GB2009/002778 |
Nov 26, 2009 |
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13116577 |
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Current U.S.
Class: |
435/29 ; 156/230;
285/382; 409/131; 435/289.1; 435/303.1; 435/395 |
Current CPC
Class: |
Y10T 409/303752
20150115; B01L 2300/0874 20130101; B01L 2300/12 20130101; B01L
2200/027 20130101; B01L 2300/0627 20130101; C12M 23/16 20130101;
B01L 2300/10 20130101; B01L 2200/025 20130101; C12M 23/26 20130101;
C12M 29/10 20130101; B01L 3/502715 20130101; B01L 2400/086
20130101; B01L 2200/0689 20130101; B01L 2300/0816 20130101 |
Class at
Publication: |
435/29 ;
435/289.1; 409/131; 435/303.1; 435/395; 285/382; 156/230 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; B32B 37/16 20060101 B32B037/16; C12N 5/02 20060101
C12N005/02; F16L 13/14 20060101 F16L013/14; C12M 1/00 20060101
C12M001/00; B23C 3/00 20060101 B23C003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 26, 2008 |
GB |
0821636.8 |
Claims
1. A microfluidic device comprising a chamber having a fluid inlet,
a fluid outlet and a sealable port, wherein the fluid inlet and the
fluid outlet are positioned to direct fluid flowing from the fluid
inlet to the fluid outlet through the chamber, and wherein the
sealable port is aligned with the chamber to allow material to be
placed directly into, or removed from, the chamber from the
exterior of the device when the sealable port is open, and to
prevent fluid escaping through the sealable port when the port is
sealed.
2. The device of claim 1, further comprising an interconnect system
which comprises: a first component having a conduit to carry fluid
to the fluid inlet or away from the fluid outlet, wherein the first
component is formed of a deformable material, and a second
component having a projecting portion, wherein a conduit passes
through the projecting portion and the second component; wherein
the conduit of the first component is aligned with the conduit of
the second component, wherein the projecting portion of the second
component deforms an area of the first component surrounding the
conduit therein so as to create a seal around the contiguous
conduits of the first and second components, thus preventing any
fluid from escaping as it flows from one conduit to the other
conduit, and wherein the second component is for connecting the
conduit therein to an external fluid source or sink.
3. The device of claim 2, comprising an interconnect system for
each of the fluid inlet and fluid outlet.
4. The device of claim 2, wherein the interconnect system or
systems each further comprises a guide positioned on the first
component around the conduit therein and which mates with the
projecting portion of the second component to align the conduit of
the first component with the conduit of the second component.
5. The device of claim 1, wherein the base of the chamber is formed
from a substrate for supporting biological material.
6. The device of claim 5, wherein the substrate is a standard glass
or polystyrene microscopy slide or culture plate and the chamber is
formed on at least a portion of the substrate.
7. The device of claim 5, wherein the substrate is detachable from
the device.
8. The device of claim 1 wherein the device is used for culturing
cells.
9. The device of claim 1, further comprising a housing.
10. The device of claim 1, wherein the fluid inlet and the fluid
outlet are positioned on opposite sides of the chamber.
11. The device of claim 1, wherein the fluid inlet and the fluid
outlet are positioned so that a material containment portion of the
chamber is substantially unaffected by the flow of fluid through
the chamber.
12. The device of claim 1, wherein the fluid inlet and/or the fluid
outlet each form at least about 20% of the area of one side of the
chamber.
13. The device of claim 1, wherein the fluid inlet and the fluid
outlet are aligned with the top of the chamber.
14. The device of claim 1, wherein the fluid inlet and fluid outlet
comprise one or more flow restrictors.
15. The device of claim 1, further comprising a conduit to carry
fluid to the fluid inlet and a conduit to carry fluid away from the
fluid outlet, wherein each conduit contains one or more flow
dividers.
16. The device of claim 1, wherein the sealable port forms a lid of
the chamber.
17. The device of claim 1, wherein the fluid is a liquid and the
sealable port comprises a gas permeable membrane to allow gas such
as oxygen to pass into the chamber.
18. The device of claim 1, wherein the device further comprises a
heater.
19. The device of claim 1, wherein the device further comprises a
sensor.
20. The use of the device of claim 1 for culturing cells or
performing cell-based assays.
21. An interconnect system for sealably connecting two fluid
carrying conduits, the system comprising: a first component having
a conduit and being formed of a deformable material; and a second
component having a projecting portion, wherein a conduit passes
through the projecting portion and the first component; wherein, in
use, the conduit of the first component is aligned with the conduit
of the second component and a force is applied to the second
component so that the projecting portion deforms an area of the
first component surrounding the conduit therein so as to create a
seal around the contiguous conduits of the first and second
components, thus preventing any fluid from escaping as it flows
from one conduit to the other conduit.
22. The interconnect system of claim 21, for connecting a conduit
in a microfluidic device to an external fluid carrying conduit.
23. The interconnect system of claim 21 wherein the interconnect
system further comprises a guide positioned on the first component
around the conduit therein and which mates with the projecting
portion of the second component to align the conduit of the first
component with the conduit of the second component.
24. A microfluidic device comprising a chamber having a fluid
inlet, a fluid outlet and a substrate for supporting biological
material, the fluid inlet and the fluid outlet being positioned to
direct fluid flowing from the fluid inlet to the fluid outlet
through the chamber, and wherein the substrate forms the base of
the chamber.
25. The device of claim 24, wherein the substrate is a standard
glass or polystyrene microscopy slide or culture plate and the
chamber is formed on at least a portion of the substrate.
26. The device of claim 24 wherein the substrate is detachable from
the device.
27. The device of claim 24, further comprising an interconnect
system which comprises: a first component having a conduit
therethrough to carry fluid to the fluid inlet or away from the
fluid outlet, wherein the first component is formed of a deformable
material, and a second component having a projecting portion,
wherein a conduit passes through the projecting portion and the
second component; wherein the conduit of the first component is
aligned with the conduit of the second component, wherein the
projecting portion of the second component deforms an area of the
first component surrounding the conduit therein so as to create a
seal around the contiguous conduits of the first and second
components, thus preventing any fluid from escaping as it flows
from one conduit to the other conduit, and wherein the second
component is for connecting the conduit therein to an external
fluid source or sink.
28. The device of claim 27, comprising an interconnect system for
each of the fluid inlet and fluid outlet.
29. The device of claim 27 wherein the interconnect system or
systems each further comprises a guide positioned on the first
component around the conduit therein and which mates with the
projecting portion of the second component to align the conduit of
the first component with the conduit of the second component.
30. A method of fabricating a microfluidic chip, the method
comprising the steps of:-- a) forming a mould defining features of
the microfluidic chip; b) pouring a curable polymer into the mould;
c) curing the polymer to form a cured polymer sheet; d) releasing
the cured polymer sheet from the mould; e) forming a membrane
having a base layer and a overlying cured polymer layer; f) bonding
the cured polymer sheet to the membrane; and g) removing the base
layer of the membrane to release the microfluidic chip.
31. The method of claim 30, wherein the same curable polymer is
used in steps b) and e).
32. The method of claim 30 wherein the polymer is
polydimethylsiloxane (PDMS).
33. The method of claim 30, wherein step a) is carried out by a
milling process.
34. The method of claim 30 wherein the PDMS in step b) is a 10:1
base to curing agent mixture.
35. The method of claim 30 wherein a covering sheet is clamped on
top of the mould prior to the curing process.
36. The method of claim 30 wherein the base layer of the membrane
is a silanised silicon wafer and the overlying curable polymer
layer is a PDMS layer.
37. The method of claim 36, wherein the PDMS layer is spin coated
on the silanised wafer at 500 rpm for 50 seconds to obtain a
thickness of substantially 120 micrometres.
38. The method of claim 30 wherein the cured polymer is bonded to
the membrane by plasma bonding.
39. The method of claim 30 wherein a microfluidic chamber is formed
in the microfluidic chip following step g).
40. The method of claim 30 wherein the PDMS is cured in an oven at
80.degree. C. for one hour.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part application of
international patent application Ser. No. PCT/GB2009/002778 filed
Nov. 26, 2009, which published as PCT Publication No.
WO/2010/061201 on Jun. 3, 2010, which claims benefit of GB patent
application Serial No. 0821636.8 filed Nov. 26, 2008.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appin cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to analytical devices and, in
particular, relates to microfluidic devices such as bioreactors
used for cell culture.
BACKGROUND OF THE INVENTION
[0004] Miniaturised total analysis systems (".mu.TAS") were
proposed as a novel concept for chemical sensing in 1990 [1],
creating the field of microfluidics and leading to the vision of
lab-on-a-chip. .mu.TAS integrates all steps required in chemical
analysis--sampling, pre-processing, and measurement, etc.--into a
single device via miniaturisation, resulting in improved
selectivity and detection limit compared to conventional sensors
[1]. A significant amount of research has been devoted to the
development of microfluidics technology and applications of .mu.TAS
devices over the past decade [2-5]. Common analytical assays,
including polymerase chain reaction (PCR) [6-9], DNA analyses and
sequencing [10-13], protein separations [14-18], immunoassay
[19-24], and intra- and inter-cellular analysis [25-29] have been
reduced in size and fabricated in a centimetre-scale chip. The
reduction in the size of the analytical processes has many
advantages including rapid analysis, less sample amount, and
smaller size [1-5]. The flushing of cells can also potentially lead
to unwanted dissociation of cell colonies.
[0005] Although there have been many successes, an important hurdle
that still needs to be cleared is the connection between the
micro-components of a device and the macro-environment of the
world. This part of the device is often referred to as the
macro-to-micro interface [30], interconnect [31-34], or
world-to-chip interface [35-39]. The difficulty results from the
fact that samples and reagents are typically transferred in
quantities of microlitres (.mu.L) to millilitres (or even litres)
whereas microfluidic devices consume only nanolitres (nL) or
picolitres of samples/reagents due to the size of reaction chambers
and channels, which typically have dimensions on the order of
microns. This problem must be overcome for microfluidic devices to
be successful, especially for high-throughput applications where
manual manipulation is not economical and the macro-to-micro
interface must be developed.
[0006] Microfluidic devices have also been developed for use in a
broad range of cell biology applications [40]. Generally, in these
devices a constant perfusion system is used to provide the cells
with an adequate supply of medium in order to provide the required
nutrient requirements and oxygen supply to keep the cells healthy
[41]. However, the problem with using a constant perfusion or flow
of medium across the cells is that the cells can be exposed to high
shear stress which can be detrimental to the normal functioning of
the cells. This is especially the case for highly sensitive cells
such as human embryonic stem cells (hESC). Further, if high flow
rates are used for the perfusion, cells may be washed out of the
microfluidic device by the medium.
[0007] Another problem associated with existing microfluidic
devices used in cell culture is that it is often difficult to
accurately and carefully introduce cells into the cell culture
chamber of the microfluidic device. For example, some devices flush
the cells into the microfluidic chamber from upstream inlets. This
leads to an undefined number of cells in the chamber. The flushing
of cells can also potentially affect the phenotype of the cells as
a result of exposure to high shear stress.
[0008] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0009] The present invention provides a microfluidic device which
may include a chamber having a fluid inlet, a fluid outlet and a
sealable port. In some embodiments, the fluid inlet and the fluid
outlet may be positioned to direct fluid flowing from the fluid
inlet to the fluid outlet through the chamber. The sealable port
may be aligned with the chamber to allow insertion of material into
the chamber or extraction of material from the chamber when the
sealable port is open, and to inhibit and/or prevent fluid from
escaping through the sealable port when the port is sealed.
[0010] In some embodiments, a microfluidic device may include an
interconnect system having a first component which includes a
conduit to carry fluid to the fluid inlet or away from the fluid
outlet. Some embodiments may include a first component formed of a
deformable material. Some embodiments of a microfluidic device may
include a second component having a projecting portion. In some
embodiments, a conduit passes through the projecting portion and
the second component. The conduits of the first and second
components may be aligned. wherein the projecting portion of the
second component deforms an area of the first component surrounding
the conduit therein so as to create a seal around the contiguous
conduits of the first and second components, thus preventing any
fluid from escaping as it flows from one conduit to the other
conduit, and wherein the second component is for connecting the
conduit therein to an external fluid source or sink.
[0011] Some embodiments may include an interconnect system for each
of the fluid inlet and fluid outlet. In various embodiments, the
interconnect system or systems may include a guide positioned
around the conduit on the first component and may mate with the
projecting portion of the second component to align the conduits of
the first and second components.
[0012] In some embodiments, the base of the chamber of a
microfluidic device is formed from a substrate for supporting
biological material. Some embodiments of substrates may include,
but are not limited to glass (e.g., glass slide), plastic, (e.g.,
polystyrene microscopy slide), culture plate and/or any material
known in the art. The chamber may be formed on at least a portion
of the substrate. In some embodiments, the substrate may be
detachable from the device.
[0013] Embodiments of the microfluidic device may be used for
culturing cells. 20. In various embodiments, the device may be used
for culturing cells and/or performing cell-based assays. Some
embodiments may include a housing.
[0014] In some embodiments, the fluid inlet and the fluid outlet
may be positioned on opposite sides of the chamber. Various
embodiments include a fluid inlet and a fluid outlet which are
positioned so that a material containment portion of the chamber is
substantially unaffected by the flow of fluid through the chamber.
In some embodiments, the fluid inlet and/or the fluid outlet each
form at least about 20% of the area of one side of the chamber.
Embodiments may include a fluid inlet and/or fluid outlet aligned
with the top of the chamber. In some embodiments, wherein the fluid
inlet and fluid outlet may include one or more flow
restrictors.
[0015] Embodiments may include a conduit to carry fluid to the
fluid inlet and a conduit to carry fluid away from the fluid
outlet. Conduits may include one or more flow dividers.
[0016] In some embodiments, the sealable port may form a lid of the
chamber. Some embodiments may include a liquid as the fluid. In
various embodiments, the sealable port may include a gas permeable
membrane to allow gas such as oxygen to pass into the chamber.
[0017] In some embodiments, the microfluidic device may include a
heater and/or sensor.
[0018] In some embodiments an interconnect system for sealably
connecting two fluid carrying conduits may include a first
component having a conduit formed of a deformable material; and a
second component having a projecting portion having a conduit which
passes through the projecting portion and the first component. In
various embodiments, during use, the conduit of the first component
is aligned with the conduit of the second component. In some
embodiments, when force is applied to the second component the
projecting portion deforms an area of the first component
surrounding the conduit to create a seal around the contiguous
conduits of the first and second components, thus inhibiting and/or
preventing any fluid from escaping as it flows from one conduit to
the other conduit.
[0019] Some embodiments may include an interconnect system
configurable to connect and/or connecting a conduit in a
microfluidic device to an external fluid carrying conduit.
[0020] In some embodiments, the interconnect system may include a
guide positioned on the first component around the conduit therein
and which mates with the projecting portion of the second component
to align the conduit of the first component with the conduit of the
second component.
[0021] Some embodiments of a microfluidic device may include a
chamber having a fluid inlet, a fluid outlet and a substrate for
supporting biological material, the fluid inlet and the fluid
outlet being positioned to direct fluid flowing from the fluid
inlet to the fluid outlet through the chamber. In some embodiments,
the substrate forms the base of the chamber of the microfluidic
device. Some embodiments of substrates may include, but are not
limited to glass (e.g., glass slide), plastic, (e.g., polystyrene
microscopy slide), culture plate and/or any material known in the
art. The chamber may be formed on at least a portion of the
substrate. In some embodiments, the substrate may be detachable
from the device.
[0022] In some embodiments, a microfluidic device may include an
interconnect system having a first component with a conduit to
carry fluid to the fluid inlet or away from the fluid outlet. Some
embodiments may include a first component formed of a deformable
material. Various embodiments may include a second component having
a projecting portion and a conduit passing through the projecting
portion and the second component. In some embodiments, the conduit
of the first component may be aligned with the conduit of the
second component, and the projecting portion of the second
component may deform an area of the first component surrounding the
conduit to create a seal around the contiguous conduits of the
first and second components, thus inhibiting and/or preventing any
fluid from escaping as it flows from one conduit to the other
conduit. In some embodiments, the second component may be used to
connect the conduit therein to an external fluid source or sink.
The microfluidic device may include an interconnect system for each
of the fluid inlet and fluid outlet. Some embodiments may include
one or more interconnect systems having a guide positioned on the
first component around the conduit which mates with the projecting
portion of the second component to align the conduits of the first
and components.
[0023] In some embodiments, a method of fabricating a microfluidic
chip may include forming a mould defining features of the
microfluidic chip; pouring a curable polymer into the mould; curing
the polymer to form a cured polymer sheet; releasing the cured
polymer sheet from the mould; forming a membrane having a base
layer and a overlying cured polymer layer; bonding the cured
polymer sheet to the membrane; and removing the base layer of the
membrane to release the microfluidic chip. Some embodiments may
utilize the same curable polymer for pouring the curable polymer
into the mould and overlying a cured polymer layer. In various
embodiments, the polymer is polydimethylsiloxane (PDMS). Some
embodiments may include forming a mould defining features of the
microfluidic chip using a milling process. In some embodiments, the
PDMS used in pouring polymer into the mould is used in a 10:1 base
to curing agent mixture. Some embodiments include clamping a
covering sheet on top of the mould prior to the curing process.
[0024] In some embodiments, the base layer of the membrane is a
silanised silicon wafer and the overlying curable polymer layer is
a PDMS layer. Various embodiments may include spin coating the PDMS
layer on the silanised wafer at 500 rpm for 50 seconds. In some
embodiments, the spin coated PDMS layer may obtain a thickness of
substantially 120 micrometres.
[0025] In some embodiments, the cured polymer is bonded to the
membrane by plasma bonding. In various embodiment, after removing
the base layer of the membrane to release the microfluidic chip, a
microfluidic chamber is formed in the microfluidic chip. In some
embodiments, the PDMS is cured in an oven at about 80.degree. C.
for about one hour.
[0026] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0027] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0028] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings.
[0030] FIG. 1A depicts an exploded view of a microfluidic perfusion
bioreactor (MPB).
[0031] FIG. 1B is a schematic of an embodiment of a microfluidic
chip depicting the channel arrangement.
[0032] FIG. 1C is a cross-sectional perspective view along line "C"
of FIG. 1B depicting a section of a microfluidic chip.
[0033] FIG. 2 is an exploded view of an embodiment of a
microfluidic perfusion bioreactor (MPB) in a similar manner to FIG.
1A.
[0034] FIG. 3 shows a cross-sectional view of a microfluidic
perfusion bioreactor taken along a plane in the interconnect
region.
[0035] FIG. 4 depicts a cross-sectional view of an alternative
embodiment of a microfluidic perfusion bioreactor taken along a
plane in the interconnect region.
[0036] FIG. 5 depicts an embodiment of a fabrication process of a
mould and a microfluidic chip created in the mould.
[0037] FIG. 6 is a schematic of the procedure for the mircrofusion
perfusion bioreactor during seeding and perfusion.
[0038] FIGS. 7A-C depict three cross sectional views of a
microfluidic device and, in particular, the sealable port. FIG. 7A
depicts a microfluidic device with the sealable port 51 open. FIG.
7B depicts a microfluidic device with the sealable port 51 closed.
Figure C depicts a microfluidic device with the sealable port 51
closed in which the cross section is taken perpendicular to that in
FIGS. 7A and 7B.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined in the
appended claims.
[0040] In some embodiments, a microfluidic device may include a
microfluidic perfusion bioreactor and/or a microfluidic chip.
[0041] The present invention will now be described by way of
example only with reference to the figures.
[0042] FIG. 1A shows solid models of a microfluidic perfusion
bioreactor ("MPB"). FIGS. 1B and 1C depict a microfluidic chip 5.
FIG. 1A shows an exploded view of the MPB with its components such
as a lid 1, interconnects 2 for tubing, a top plate 3, a gasket 4,
a microfluidic chip 5, a cell culture slide 6 and a bottom frame 7.
The lid, the top plate and the bottom frame were fabricated in
polycarbonate. In some embodiments, any material known in the art
may be used to fabricate the lid, top plate and/or the bottom
frame. The gasket and the microfluidic chip were made of PDMS. The
interconnects were made of aluminium. The cell culture slide was
tissue culture polystyrene. FIG. 1B shows a schematic of the
channel arrangement of the microfluidic chip with an inlet/outlet
port 8, flow dividers 9, flow restrictors 10 and a cell culture
chamber body 11 allowing access to the cell culture slide for cell
seeding, which has an area of 4 mm.times.13 mm. The dashed circle
around the inlet and outlet port depicts the sealing area 8' of the
cylinder of the interconnects. FIG. 1C shows a section in the
middle of the microfluidic chip taken through the chamber. The
height between the lower solid line and the dashed line depicts the
raised inlet channels, whereas the upper solid line approximately
depicts the culture chamber height, when the MPB was in perfusion
configuration. Some embodiments may include MPBs similar in scale
to the devices depicted in FIGS. 1A-1C. In various embodiments, a
MPB may vary in size from the scale shown in FIGS. 1A-1C.
[0043] FIG. 2 shows an embodiment of solid models of a microfluidic
perfusion bioreactor (MPB) similar manner to the MPB depicted in
FIG. 1. FIG. 2 shows an exploded view of the MPB with its
components such as a lid 12, interconnects for tubing (shown as 2
in FIG. 1A), a top plate 13, a gasket 14, a microfluidic chip 15
which is formed from two parts: a manifold layer 18; and a membrane
19, a cell culture slide 16 and a bottom frame 17.
[0044] FIG. 3 shows a cross section taken through a MPB in the
interconnect region. This figure shows projecting portion 20 on the
interconnect 21 pressing into and deforming the microfluidic chip
22 in order to create a seal with microfluidic chip inhibiting
and/or preventing leakage. In some embodiments, projecting portion
20 may be a cylinder, nipple and/or any other suitable structure
known in the art.
[0045] FIG. 4 shows a cross section of an alternative embodiment of
a MPB in the interconnect region. In this embodiment, the
projection 23 is on the top plate 24 of the MPB rather than on the
interconnect itself The interconnect 26 is mounted on the top plate
and a seal is formed between the top plate and interconnect using a
rubberised O-ring 25.
[0046] FIG. 5 shows a fabrication process of a mould and a
microfluidic chip created in the mould. As shown in step (1) of
FIG. 5, sheet 27 may be provided. Sheet 27 may include but is not
limited to metals, alloys, such as Dural, and/or any material known
in the art. As shown in FIG. 5, in step (2) sheet 27 was machined
with a micromilling machine 28 to form a mould 32. In step (3) PDMS
was poured 29 into the mould and then degassed. The PDMS was
allowed to cure 30. As shown in step (4), a polycarbonate sheet 31
was placed on top of the mould 32 and clamped together.
Concurrently, a silanised silicon wafer 36 was spin coated with
PDMS 35 to form a membrane 34. The PDMS-coated wafer 34 and the
clamped mould were then cured for 1 hour at 80.degree. C. in an
oven. Step (5) depicts the microfluidic manifold layer 33 released
from the mould and the culture chamber body was cut out. In step
(6), the microfluidic manifold layer 33 and the PDMS membrane 34
were exposed to an air plasma and immediately brought into contact
for bonding to form structure 37. As shown in step (7). the
membrane at the bottom of the culture chamber body was cut out and
the microfluidic chip 38 was cut to shape and released from the
wafer. In some embodiments, the representations depicted in FIG. 5
are not to scale.
[0047] FIG. 6 shows a schematic of the procedure for the MPB in
seeding and perfusion configuration. On day 0, MPB 40 and a one
well dish 41 were coated with 0.1% gelatine prior to seeding of the
feeder layer. As shown in step (1) 20,000 inactivated murine
embryonic fibroblasts (MEFs) 39 were seeded with a pipette directly
into the cell culture chamber of the MPB and left over night to
attach to the surface in an incubator. Step 2 of FIG. 6 depicts
Petri dish 43 fitted with spacers 42 for gas exchange used to
accommodate the MPB . The Petri dish with the MPB was kept in an
incubator at 37.degree. C. and 5% CO2 (b). On day 1, MEF medium was
removed and hESC medium added 30 minutes before hESC colonies 45
were seeded. As shown in step (3) hESC colonies 45 were added into
the culture area with a pipette (not shown), the Petri dish
subsequently closed and the MPB placed back into an incubator 44.
On day 2, shown in step (5) medium was aspirated and the culture
chamber closed with a lid 47. Tubing 46, 48 was connected to the
MPB and perfusion started using a syringe pump for two days in an
incubator. On day 4 as shown in step (6), medium was aspirated,
cells fixed and stained with pluripotency markers 49 to assess the
effect of seeding and perfusion of hESC in a MPB. In some
embodiment, dimensions of the various parts shown in FIG. 6 may
vary.
[0048] FIGS. 7A-C show three cross sectional views of a
microfluidic device and, in particular, the sealable port 51. FIG.
7A depicts a microfluidic device 22 with the sealable port 51 open.
FIG. 7B shows a microfluidic device 22 with the sealable port 51
closed. FIG. 7C depicts a microfluidic device 22 with the sealable
port 51 closed in which the cross section is taken perpendicular to
that of FIGS. 7A and 7B.
[0049] In some embodiments, a microfluidic device may include a
chamber having a fluid inlet, a fluid outlet and a sealable port.
The fluid inlet and the fluid outlet may be positioned to direct
fluid flowing from the fluid inlet to the fluid outlet through the
chamber. The sealable port may be aligned with the chamber to allow
insertion of material into the chamber or extraction of material
from the chamber when the sealable port is open, and to inhibit
and/or prevent fluid from escaping through the sealable port when
the port is sealed.
[0050] The sealable port allows material to be placed directly into
or removed from the chamber of the device. For example, the
sealable port permits easy and gentle seeding of cells and
extra-cellular matrix (ECM) compounds into the chamber, the
perfusion of the cells and, subsequently, their easy and gentle
uptake from the chamber. Alternatively, the port allows the loading
and removal of beads or polymer monoliths, for example, for
enzymatic assays. The placement of material directly into the
chamber and the removal of material from the chamber of the device
is done from the exterior of the device, for example, manually
using a pipette or other suitable device. This means that the
material can be placed directly into the chamber in the precise
position that is required for carrying out a particular function.
An advantage of the sealable port is that it allows a
pre-determined and/or an exact quantity of material to be placed in
the chamber which may be crucial for accurately performing assays
or tests. Further, this avoids the problems associated with
flushing the material into the chamber from an upstream inlet. For
example, the problem of high undefined shear stress on the cells as
they are flushed into the chamber is avoided. Further, the sealable
port may improve the macro-to-micro interface as the port can be
opened and closed repeatedly so that different materials can be
inserted into and removed from the chamber at will. This provides
the device with greater flexibility which enhances the achievable
degree of complexity and thus the degree of functionality.
[0051] In the context of this invention, the term "microfluidic
device" means a miniaturised device through which fluids flow in a
controlled manner and in which fluids are geometrically constrained
to a small, typically sub-millimeter, scale. Generally, channels in
a microfluidic device have dimensions in the order of tens to
hundreds of microns and it is through these channels that fluids,
normally liquids, flow. A person skilled in the art would
appreciate what is meant by this term.
[0052] The chamber can be any suitable size and shape so that it
can carry out its particular function. For example, in one
embodiment, the device is used for culturing cells. Therefore, the
chamber is sized so that it can contain a number of cells whilst
still allowing fluid to flow through the chamber from the fluid
inlet to the fluid outlet in a laminar flow fashion. In such an
embodiment, the volume of the chamber may be between about 1
mm.sup.3 and about 150 mm.sup.3. More preferably, the volume of the
chamber will be between about 1 mm.sup.3 and about 50 mm.sup.3,
more preferably still, between about 10 mm.sup.3 and about 40
mm.sup.3, and most preferably, between about 20 mm.sup.3 and about
30 mm.sup.3. A suitable chamber might have a length of between
about 1 mm and 8 mm, a width of between about 5 mm and about 18 mm
and a height of between about 0 2 mm and about 1 mm. A preferred
chamber has a length of about 4 mm, a width of about 13 mm and a
height of about 0.5 mm. A chamber of this size is particular useful
for culturing hESC on a bed of feeder cells. The chamber may be any
suitable shape. For example, it may be cuboidal or disc shaped.
Preferably, the chamber is cuboidal. Preferably, the chamber has a
cross sectional area perpendicular to the flow of fluid of at least
about 4 mm.sup.2, more preferably, at least about 5 mm.sup.2 and,
most preferably, at least about 6 mm.sup.2. As discussed below,
this helps to reduce the flow velocity and shear stress in the
chamber.
[0053] In some embodiments, the size of the chamber can have an
important effect on the conditions inside the chamber. As fluid
flows from the fluid inlet to the fluid outlet, the material in the
chamber will experience a shear stress as a result of the flow
velocity of the fluid around and over the material. Given a
constant flowrate, increasing a channel's height and width
decreases shear stress as the flow velocity is decreased due to the
greater cross sectional area of the channel. Therefore, for the
microfluidic device, the larger the chamber dimensions
perpendicular to the flow of the fluid, the lower the shear stress
that the material in the chamber experiences due to a decreased
flow velocity. For cell cultures, the flowrate can be important as
it must be high enough to ensure that the cells obtain enough
nutrients, such as oxygen, from the medium in order to keep them
healthy. By having a chamber with a large area perpendicular to the
flow of fluid, it is possible to have a relatively high flowrate
but a relatively low flow velocity and, therefore, shear stress.
Therefore, the size of the chamber is an important
consideration.
[0054] The above description relates to the microfluidic device
having the sealable port in a sealed position. In one embodiment,
the volume defined by the chamber may be greater when the sealable
port is not in the sealed position, i.e. when the sealable port is
open. This can be achieved by the sealable port having a protrusion
which fills part of the volume of the chamber. The advantage of
this is that, in certain circumstances, the sealable port can be
left in an open position, so that the chamber has a larger volume.
For example, when the microfluidic device is used for cell culture,
cells can be seeded into the chamber. Having the port open allows
more medium to be contained in the chamber, thereby ensuring that
the cells are kept in a viable condition. The sealable port can be
sealed at a later stage.
[0055] In some embodiments, the fluid inlet and the fluid outlet
are positioned in such a way so that fluid flowing from the fluid
inlet to the fluid outlet may be directed through the chamber. As a
result of the fluid flowing through the chamber, material placed in
the chamber will come into contact with the fluid as the fluid is
passing through the chamber. This ensures that the material is
exposed to any substance (e.g. chemicals, reagents, nutrients,
enzymes, antibodies, etc.) contained in the fluid. Preferably, the
fluid inlet and the fluid outlet are positioned on opposite sides
of the chamber. In some embodiments, the fluid inlet and outlet are
positioned on the largest face or surface of the chamber. This may
ensure that any change in the fluid composition entering the
chamber, for example, the introduction of a chemical, is quickly
dispersed to the whole chamber and also to any material contained
therein. Further, having the inlet and outlet positioned on
opposite sides of the chamber may improve or maintain the
homogeneity of the flow.
[0056] In a preferred embodiment, the fluid inlet and the fluid
outlet are positioned so that a material containment portion of the
chamber is substantially unaffected by the flow of fluid through
the chamber. The material containment portion of the chamber is
simply a portion of the chamber which is for containing material
which is placed in the chamber. For example, this may simply be the
bottom of the chamber. Material placed in the containment portion
of the chamber is substantially unaffected by the flow of the fluid
in that it is not subjected to a significant shear stress as a
result of the flow of the fluid. The flow of fluid is not directed
through this containment portion. This is important for cell
culture and, in particular, for sensitive cell types like hESC
(i.e., human embryonic stem cells). For example, the fluid inlet
and the fluid outlet may both be positioned in a top portion of the
chamber. In some embodiments, the fluid inlet and fluid outlet are
positioned in the top three quarters of the chamber. In this way,
material placed in a bottom portion, for example, the bottom
quarter of the chamber, is not substantially affected by the flow
of fluid as the majority of the flow passes over the top of the
material, thus reducing the shear stress that the material
experiences. In some embodiments, the fluid inlet and fluid outlet
are positioned opposite each other on the side walls of the
chamber. Preferably, they are positioned in the top half of the
chamber. In various embodiments, the fluid inlet and outlet may be
positioned about 120 .mu.m above the base of the chamber.
Preferably, the fluid inlet and the fluid outlet are aligned with
the top of the chamber. When the material containment portion is at
the base of the chamber, the fluid inlet and outlet may be
positioned in range from between about 10 .mu.m to about 1 mm above
the base of the chamber and, preferably, in a range between about
50 .mu.m to about 300 .mu.m above the base of the chamber.
Effectively, this gives a material containment portion having a
depth in a range from about 10 .mu.m to about 1 mm and, preferably,
having a depth in a range from about 50 .mu.m to about 300
.mu.m.
[0057] The reason behind this is that in microfluidic systems fluid
flows in a substantially laminar manner. Therefore, material not
directly in the path of the flow experiences a much reduced flow
velocity and so a much reduced shear stress. Preferably, material
placed in the containment portion of the chamber which is
substantially unaffected by the flow of fluid experiences a shear
stress of less than about 0.001 dyne/cm2 and, more preferably, less
than about 0.0001 dyne/cm2.
[0058] The fluid inlet and fluid outlet can be any suitable conduit
or opening to allow fluid to enter and exit the chamber. A person
skilled in the art would be fully aware of standard fluid inlets
and fluid outlets used in microfluidics which could be used in the
present invention. Preferably, a liquid such as culture medium pass
through the chamber from the fluid inlet to the fluid outlet.
[0059] The fluid inlet and fluid outlet can be any suitable size or
shape. In one embodiment, the fluid inlet, fluid outlet or both are
relatively wide or large compared to the chamber. For example, the
fluid inlet and/or fluid outlet may have a width which is the same
as the width of the chamber. The advantage of having a relatively
large fluid inlet and/or outlet is that, for a given flowrate, the
flow velocity of the fluid entering the chamber will be relatively
low so that the contents of the chamber experience a relatively low
shear stress. Preferably, when the chamber is cuboidal, the fluid
inlet forms at least about 10% of the area of one side of the
chamber. More preferably, the fluid inlet forms at least about 15%
of the area of one side of the chamber, more preferably still, at
least about 20% and, even more preferably, at least about 30%.
Alternatively, the fluid inlet may form between about 10% and about
70% of the area of one side of the chamber, more preferably,
between about 15% and about 60% and, even more preferably, between
about 20% and about 50%. The fluid outlet and the fluid inlet may
be the same size and shape or may be different. The above values
and ranges for the size of the fluid inlet are equally applicable
to the size of the fluid outlet.
[0060] Where the chamber has a curved outer wall, for example where
it is disc shaped, the fluid inlet may be positioned anywhere on
the curved wall. Where the chamber is cuboidal, that is its outer
wall has a number of flat faces joined at the edges of the cuboid,
the fluid inlet may be positioned on one of the faces, or over an
edge joining two faces. Preferably, it is positioned on one of the
faces. Preferably, it is positioned on the widest face. The outlet
may be similarly positioned.
[0061] The device may have a plurality of fluid inlets and/or fluid
outlets. These may be the same size or different sizes. They may
carry the same fluid or they may carry fluids with different
compositions. If there is a plurality of fluid inlets and/or
outlets the above paragraph relating to the area of the chamber
side that is formed by the fluid inlet/outlet, relates to the
plurality of inlets/outlets, i.e. the fluid inlets preferably form
at least about 30% of the area of one side of the chamber, etc.
[0062] In one embodiment, a conduit carries fluid to the fluid
inlet. Preferably, the conduit increases in cross sectional area as
it approaches the fluid inlet. For example, both the height and
width of the conduit may increase to form a cone shape. Preferably,
the conduit only increases in width as it approaches the fluid
inlet. This increase in cross sectional area has the effect of
decreasing the fluid velocity in the conduit so that when the fluid
enters the chamber through the inlet, the material in the chamber
is not subjected to a high shear stress. This is especially the
case in microfluidics where the conduit may have dimensions of tens
or hundreds of microns. For example, the conduit may increase in
width from about 200 .mu.m to about 13 mm where it enters the
chamber. Where the conduit increases in width or size, the fluid
inlet is preferably positioned on the widest face of the chamber.
The fluid outlet can also have a similar feature so that a conduit
decreases in cross sectional area as it becomes more distant from
the fluid outlet.
[0063] In a preferred embodiment, the fluid inlet, the fluid outlet
or both comprise one or more flow restrictors. These are thin
members which partially obstruct the fluid inlet/outlet so that a
plurality of channels are formed in the fluid inlet/outlet and
which have the effect of at least partially homogenising the flow
velocity of the fluid across the entire width or area of the fluid
inlet/outlet. This has the effect of at least partially
homogenising the shear stress profile across the chamber. This is
especially important where the fluid inlet, fluid outlet or both
are relatively wide or large compared to the chamber. Preferably,
there is a plurality of flow restrictors. The larger the number of
flow restrictors, the more homogenous the flow velocity, and
therefore the shear stress profile, will be. Preferably, the flow
restrictors are equally spaced in the fluid inlet/outlet so that
the channels formed thereby are of equal size. This helps to ensure
that the flow velocity is as uniform as possible.
[0064] Generally, in mircofluidic devices, fluid is carried to a
fluid inlet and away from a fluid outlet in channels or conduits
which are of several microns to hundreds of microns in size. In
some embodiments, the device may include a conduit to carry fluid
to the fluid inlet and a conduit to carry fluid away from the fluid
outlet, one or both of the conduits may contain one or more flow
dividers. In some embodiments, one or both of the conduits contain
a plurality of flow dividers. Flow dividers work in a similar
manner to flow restrictors and result in the fluid having a more
uniform flow velocity when it reaches the fluid inlet, thus
resulting in the fluid having a more uniform flow velocity as it
enters the chamber. Preferably, the flow dividers are positioned in
the portion of the conduit which increases in size as it approaches
the fluid inlet. Similarly, they can be positioned in the
decreasing conduits leaving the fluid outlet.
[0065] The sealable port is aligned with the chamber to allow
insertion of material into the chamber or extraction of material
from the chamber when the sealable port is open, and to inhibit
and/or prevent fluid escaping through the sealable port when the
port is sealed. Preferably, the fluid is liquid. The sealable port
can be any suitable size or shape as long as it allows easy
insertion and extraction of material into and out of the chamber.
The size of the port will depend, in part, on the size of the
chamber. The port can be positioned at any suitable point in the
chamber. Preferably, the port is in an uppermost portion of the
chamber. In one embodiment, the sealable port forms a lid of the
chamber so that the uppermost portion of the chamber is formed by
the port. The port may be sealed in any suitable way. For example,
the port may be sealed using a gasket formed from a deformable
material such as rubber or silicone.
[0066] In one embodiment in which liquid passes through the
chamber, the sealable port comprises a gas permeable membrane to
allow gas such as oxygen to pass into the chamber. In this
embodiment, when liquid such as culture medium is not flowing
through the chamber, oxygen can pass into the chamber so that any
cells have the required level of oxygen to keep them healthy. In
such an embodiment, the sealable port stops any liquid escaping
from (or entering) the chamber but allows gas to pass into the
chamber.
[0067] In some embodiments of the invention, the chamber, fluid
inlet and outlet, and any conduits connected to the fluid inlet and
outlet may be encapsulated. For example, they may be encapsulated
in a frame or housing. However, in one embodiment, a conduit which
connects to the fluid inlet may be made of a gas permeable
material, such as PDMS, and at least partially exposed to allow gas
to enter the conduit and any liquid contained therein. In such an
embodiment, the conduit is for carrying liquid to the fluid inlet.
This allows gas such as oxygen to enter the liquid being carried in
the conduit so that the gas passes into the chamber with the
liquid.
[0068] The base of the chamber can be formed from a substrate for
supporting biological material. The substrate can be any suitable
tissue culture substrate such as glass or polystyrene. Preferably,
the substrate is a standard substrate and the chamber is formed on
at least a portion of the substrate. For example, suitable standard
substrates are glass or polystyrene microscopy slides or culture
plates. Suitable culture plates may comprise wells or may not.
Preferably, the substrate is detachable from the device. This
allows rapid exchange of the substrate or allows the substrate to
be removed so that any material on the substrate can be analysed
more easily
[0069] The substrate allows biological material to be attached
thereto. For example, cells, antibodies, proteins such as enzymes
and ECM compounds can be attached to the substrate.
[0070] The fact that the substrate is detachable from the device
facilitates the comparison of microfluidic assays with traditional
assays, for example, the comparison of traditional cell culturing
techniques with microfluidic cell culturing using the same
substrate material. Further, the attachment and detachment of the
substrate simplifies pre- and post-processing steps which may have
to be conducted at another location using conventional larger-scale
equipment and which would necessitate the transport of the
substrate to and from this other location. The use of a standard
substrate, such as a glass or polystyrene microscope slide, makes
pre- and post-processing steps much more convenient as the standard
substrate can be used directly with conventional larger-scale
equipment such as a microscope or plate reader. In one embodiment,
the device can be used directly with a microscope so that it is not
necessary to detach the substrate to view the biological material
attached thereto. This could be done by making the device from
transparent material.
[0071] The device may include an interconnect system having a first
component having a conduit therethrough to carry fluid to the fluid
inlet or away from the fluid outlet, wherein the first component is
formed of a deformable material, and a second component having a
projecting portion, wherein a conduit passes through the projecting
portion and the second component. In some embodiments, the conduit
of the first component is aligned with the conduit of the second
component, and the projecting portion of the second component
deforms an area of the first component surrounding the conduit
therein so as to create a seal around the contiguous conduits of
the first and second components, thus inhibiting and/or preventing
any fluid from escaping as it flows from one conduit to the other
conduit, and wherein the second component is for connecting the
conduit therein to an external fluid source or sink.
[0072] Preferably, the device comprises an interconnect system for
each of the fluid inlet and fluid outlet.
[0073] The advantage of such an interconnect system is that it
allows the device to be easily and robustly connected to external
fluid sources in a leak-free manner. This vastly improves the
macro-to-micro interface. The second component can easily be
standardised to allow easy linkage with standard equipment, for
example, `robotised` liquid handling platforms.
[0074] The first component is made of a deformable material. This
can be any suitable deformable material such as rubber or silicone
(e.g. poly(dimethylsiloxane) (PDMS)). The material must be
sufficiently deformable to allow the second component to deform it
and create a seal therewith.
[0075] The second component is for connecting the conduit therein
to an external fluid source or sink. This can be any suitable fluid
source or sink and can be connected in any suitable way. Such
connections are well known to those skilled in the art. For
example, the second component can have a thread on the inside of
the conduit to allow it to be connected to commonly available
tubing connectors such as Upchurch fingertight units. This allows
fluid from an external source to enter the device, pass through the
chamber and exit the device. The second component can be made of
any suitable material. For example, the second component may be
made of aluminium.
[0076] The projecting portion can be any suitable size or shape so
that it can create a seal with the first member to allow fluid to
pass from one conduit into the other conduit in a leak free manner.
Preferably, the projecting portion is cylindrical in shape so that
the conduit passes through the longitudinal axis of the cylinder. A
cylindrical shape creates a better seal and it is easier to
fabricate.
[0077] The conduit in the first and second components can be the
same size or different sizes. The cross sectional area of the
conduits may change along the length of the components. Preferably,
the conduit of the first component has a cross sectional dimension
of about 1 mm to about 2 mm. For example, the conduit may have a
diameter of about 1 mm to about 2 mm and, more preferably, about
1.2 mm to about 1.4 mm.
[0078] Preferably, the interconnect system further comprises a
guide positioned on the first component around the conduit therein
and which mates with the projecting portion of the second component
to align the conduit of the first component with the conduit of the
second component. The advantage of the guide is that the two
components are self aligning which makes it very easy to correctly
connect the two components to align the conduits.
[0079] The guide may be any suitable guide for aligning the
conduits of the two components. For example, the guide may comprise
an opening which is substantially the same size and shape as the
projecting portion of the second component so that the projecting
portion slots into the guide in a similar manner to a plug and
socket.
[0080] The microfluidic device may further comprise a heater, such
as an electroheater like indium tin oxide, to allow the chamber and
its contents to be heated to and maintained at a predetermined
temperature. Therefore, when cells are being cultured in the
chamber, the chamber can be kept at a suitable temperature rather
than being kept in an incubator. Alternatively, the device may
comprise a heater to heat the fluid before it reaches the fluid
inlet so that fluid entering the chamber has been heated to a
predetermined temperature. The heater may be integrated into the
device. For example, the heater may be integrated into the
substrate of the device in embodiments in which a substrate is
present.
[0081] The device may further comprise immobilised optical sensors
or biosensors. Such sensors, in particular, the optical sensors,
could be integrated into the second component of the interconnect
system provided they are made of a transparent thermoplastic
polymers.
[0082] The device may comprise a housing which contains the other
elements or to which the other elements are attached. For example,
in one embodiment, a microfluidic chip defines the chamber, the
fluid inlets and outlets and any conduits connected to the fluid
inlet and/or outlet. The housing contains the microfluidic chip and
has suitable openings to allow access to the chamber for the
sealable port and also to the conduits and/or fluid inlet and
outlet. This housing can be a standard size and can accommodates
the interconnect systems and the substrate. This can give a
standard housing comprising all the necessary elements to provides
the macro-to-micro interface for the microfluidic device.
Customised microfluidic chips can then be placed in the standard
housing according to the particular function of the device allowing
easy connection to the sealable port, interconnect system,
substrate, etc. In this respect, the housing should allow the
device to be assembled and disassembled repeatedly. The housing may
be made of any suitable material. For example, the housing may be
made of aluminium. Alternatively, the housing may be made of a
transparent material.
[0083] The present invention also provides an interconnect system
for sealably connecting two fluid carrying conduits, the system
comprising: [0084] a first component having a conduit therethrough
and being formed of a deformable material; and [0085] a second
component having a projecting portion, wherein a conduit passes
through the projecting portion and the first component; [0086]
wherein, in use, the conduit of the first component is aligned with
the conduit of the second component and a force is applied to the
second component so that the projecting portion deforms an area of
the first component surrounding the conduit therein so as to create
a seal around the contiguous conduits of the first and second
components, thus preventing any fluid from escaping as it flows
from one conduit to the other conduit.
[0087] Preferably, the interconnect system is for connecting a
conduit in a microfluidic device to an external fluid carrying
conduit.
[0088] Preferably, the interconnect system further comprises a
guide positioned on the first component around the conduit therein
and which mates with the projecting portion of the second component
to align the conduit of the first component with the conduit of the
second component.
[0089] Other features of the interconnect system are as described
above.
[0090] The present invention also provides a microfluidic device
comprising a chamber having a fluid inlet, a fluid outlet and a
substrate for supporting biological material, the fluid inlet and
the fluid outlet being positioned to direct fluid flowing from the
fluid inlet to the fluid outlet through the chamber.
[0091] Other features of this device are as described above in
relation to the device comprising the sealable port.
[0092] The present invention also provides a method of fabricating
a microfluidic chip, the method comprising the steps of: a) forming
a mould defining features of the microfluidic chip; b) pouring a
curable polymer into the mould; c) curing the polymer to form a
cured polymer sheet; d) releasing the cured polymer sheet from the
mould; e) forming a membrane having a base layer and a overlying
cured polymer layer; f) bonding the cured polymer sheet to the
membrane; and g) removing the base layer of the membrane to release
the microfluidic chip. The same curable polymer can be used in
steps b) and e), and can be any suitable polymer such as silicone
or polyurethane. Preferably the polymer is polydimethylsiloxane
(PDMS). Preferably step a) is carried out by a milling process.
[0093] Advantageously, the PDMS in step b) is a 10:1 base to curing
agent mixture. In this case, the PDMS is degassed prior to the
pouring step. In a preferred embodiment, a covering sheet is
clamped on top of the mould prior to the curing process.
Preferably, the PDMS is cured in an oven at 80.degree. C. for one
hour.
[0094] Advantageously, the base layer of the membrane is a
silanised silicon wafer and the overlying curable polymer layer is
a PDMS layer. The PDMS layer may be spin coated on the silanised
wafer at 500 rpm for 50 seconds to obtain a thickness of
substantially 120 micrometres. Preferably, the membrane is cured in
an oven at 80.degree. C. for one hour.
[0095] Conveniently, the cured polymer is bonded to the membrane by
plasma bonding.
[0096] In a preferred embodiment, a microfluidic chamber is formed
in the microfluidic chip following step 7 depicted in FIG. 5.
[0097] Where the microfluidic device is used for culturing cells,
for example, human embryonic stem cells (hESC), it can be used to
study various properties of the cells under different medium
perfusion conditions. For example, the impact of oxygen on
expansion and differentiation of hESC can be determined. The use of
the microfluidic device could also be integrated with post-process
cell preparation.
[0098] Microfluidic cell culture systems, such as the present
invention, operate with significantly fewer resources. They can
also be parallelised so that multiple microfluidic devices can be
combined into a single system. Further, the use of the microfluidic
device can be automated. For example, automated pulse-free medium
perfusion of cells can be performed by an automated system for
execution of cell re-feed schedules. Alternatively, constant medium
perfusion can be performed at different flow rates in a plurality
of devices. This is applicable to execute fully-automated
differentiation and expansion studies. Further, multiplexing of
devices can be used for parallelised execution of cell-based
assays.
[0099] The present invention will be further illustrated in the
following Examples which are given for illustration purposes only
and are not intended to limit the invention in any way.
EXAMPLES
Example 1
[0100] Design of a Microfluidic Perfusion Bioreactor
[0101] As shown in FIG. 1A, to integrate a tissue culture
polystyrene slide 6, a standard adherent cell culture material,
into a microfluidic perfusion bioreactor, a clamp designed with
integrated fluidic interconnects 2 was proposed, where a cell
culture slide 6 and a microfluidic chip 5 were disposed between a
bottom frame 7 and a top plate 3.
[0102] The clamp was held together by screws, where the soft
microfluidic chip formed a seal between the culture slide and the
interconnects in the top plate. In some embodiments, any fastening
mechanism known in the art may be used to hold the MPB together. As
shown in FIG. 1A, the top plate 3 included two pockets to hold the
gasket 4 and the microfluidic chip 5 and which allowed alignment of
the microfluidic chip with the inlet and outlet interconnects 2.
Interconnects 2 were mounted on the top plate 3 with screws and a
portion of the interconnects extend through a hole in the top plate
3. A projection 20 (shown in FIG. 3) at the bottom of the
interconnect was pressed against the microfluidic chip and formed a
tight seal 8' (shown as dotted circle in FIG. 1B) around the inlet
and outlet port 8 (shown in FIG. 1B), when the MPB was assembled.
(shown in FIG. 3)
[0103] The projection stood out approximately 80 .mu.m into the
microfluidic chip pocket from the top plate to assure that the
cylinder is pressed reliably against the microfluidic chip, when
clamped.
[0104] The interconnects had on the top side a thread, which
allowed the use of commonly available tubing connectors, (such as
Upchurch fingertight units).
[0105] An alternative embodiment of the interconnect is shown in
FIG. 4. In this embodiment the projection 23 is on the top plate 24
of the MPB rather than on the interconnect itself The interconnect
26 is mounted on the top plate and a seal is formed between the top
plate and interconnect using a rubberised O-ring 25. Alternatively,
the interconnect can be formed integrally with the top plate to
allow fabrication of the top plate and interconnect in one
piece.
[0106] The interconnects can be made of aluminium, thermoplastic
polymer and/or other materials known in the art. In some
embodiments, aluminium is preferred.
[0107] To avoid dissociation of hESC colonies during seeding into
the microfluidic perfusion bioreactor, a sealable lid was designed,
enabling two configurations of the MPB. When the lid is not
mounted, the MPB is in a cell seeding configuration. When the lid
is mounted, the MPB is in a perfusion configuration. This allows
co-culture seeding and perfusion of hESC on a feeder layer and the
use of a pipette for simple and accurate seeding into the MPB
(defined colony numbers, cell density).
[0108] As depicted in FIG. 1A, a gasket 4 made out of PDMS was
incorporated into the design to guarantee a leakage free closing of
the MPB after seeding. When the MPB was in seeding configuration,
the height of the gasket and the microfluidic chip allowed the same
surface area to volume ("SAV") ratio as in a one well dish or a
T-flask.
[0109] After successful seeding and attachment of the cells, the
lid 1 (shown in FIG. 1A) could be screwed onto the top plate 3 to
close the MPB. The lid 1 determines the chamber height in the
culture chamber during perfusion. In one embodiment, this is about
500 .mu.m (shown in FIG. 1C, upper solid line). This leads to a
total volume of about 4 mm.times.13 mm.times.0.5 mm=26
microlitres.
[0110] As shown in FIG. 1A, this device enables the use of
disposable polymeric microfluidic chips 5 which can easily be
redesigned and inserted into the standardised housing.
[0111] As depicted in FIGS. 1B-C, in some embodiments, the
microfluidic chip 5 had nineteen flow restrictors 10 on each side
of the cell culture chamber. In various embodiments, the flow
restrictors may have dimensions of about 200 .mu.m wide and about
1000 .mu.m long. In some embodiments, channels between the
restrictors were about 400 .mu.m wide and about 200 .mu.m high. As
shown in FIG. 1b, the inlet 8 was divided into three channels
acting as a microfluidic manifold 9 to create together with the
flow restrictors 10 an even velocity pattern in the culture area
and therefore an even distribution of shear stress within the
culture chamber (in flow direction, y-direction).
[0112] As shown in FIG. 1C, a layer (shown between lower solid and
dashed line) at the bottom of the microfluidic chip elevated the
height of the flow restrictors to above the cell culture portion of
the chamber (in between the solid line and dashed line at the
bottom of the chamber of FIG. 1C), reducing the flow rate and the
hydrodynamic shear stress on the cell culture. FIG. 2 depicts the
layer as membrane 19.
[0113] As shown in FIG. 1B, to accommodate hESC colonies sized up
to 1 mm in diameter, an appropriate sized cell culture chamber body
11 was incorporated. In one embodiment of an MPB, the cell culture
area had a size of 0.52 cm.sup.2.
[0114] FIG. 1A depicts bottom frame 7 had a recess incorporated to
hold different microscope slide formats. The bottom frame was
reinforced underneath the sealing area to avoid excessive bending
of the bottom frame when clamped together. An opening under the
culture chamber area allowed access for inverted microscopes. The
bottom frame was fitted with threads to screw together with the top
plate.
Example 2
[0115] Fabrication and Assembly of the Microfluidic Bioreactor
[0116] All parts and moulds were designed in a 3D CAD system
(SolidWorks 2007, Dassault Systemes SolidWorks, USA). G-code was
generated with a CAM program (MasterCam X2, CNC Software, USA) to
control the milling process on a micro milling machine (M3400E,
Folken Industries, USA).
[0117] To mill the bottom frame 7 and top plate 3 (shown in FIG.
1A), a 3 mm thick poly(carbonate) (PC) sheet (681-637, RS, UK) was
machined (8,000 rpm, 104 mm min.sup.-1 feedrate) using 2 mm
diameter end mills (2 flute standard length, Kyocera Micro Tools,
USA).
[0118] The lid 1 as depicted in FIG. 1A was machined from a 5 mm
thick PC sheet (681-659, RS, UK) using 2 mm diameter and 1 mm
diameter end mills (2 flute standard length, Kyocera Micro Tools,
USA).
[0119] Instead of using a SU-8 process, which creates a master for
PDMS reproduction, the inventors used a micromilling machine to
fabricate moulds for the microfluidic chip (FIG. 5) and the
gasket.
[0120] As shown in FIG. 5, in steps 1 and 2 the mould 32 for the
microfluidic manifold layer was milled 28 (8,000-16,000 rpm, 104 mm
min.sup.-1) in Dural with 2 mm, 1 mm and 200 .mu.m diameter end
mills (2 flute standard length, Kyocera Micro Tools, USA). 2 mm and
1 mm diameter end mills were used to create microfluidic manifolds
or channels. A 200 .mu.m diameter end mill was used to machine the
flow restrictors.
[0121] Poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning,
USA) was mixed in a ratio of 10:1, base to curing agent, and
degassed for 15 minutes. As shown in step 3 of FIG. 5, the PDMS was
poured into the negative Dural mould and thoroughly degassed again
until no air bubbles were visible. A 3 mm thick PC sheet was then
placed carefully on top of the mould as shown in step 4 of FIG. 5
and clamped between two aluminium plates. The clamped stack was
placed in an oven at 80.degree. C. for 1 hour to cure the PDMS.
[0122] After releasing the mould/PC sheet stack from the clamping
plates, the mould together with the polycarbonate sheet was left to
cool. The microfluidic manifold layer was then freed from the mould
with tweezers as shown in FIG. 5, step 5. The culture chamber body
of the microfluidic manifold layer was cut out with a scalpel under
a microscope.
[0123] As depicted in FIG. 1C, the thickness of a PDMS membrane
defined the height of the flow restrictors above the cell culture
chamber (shown as the height between the solid and the dashed
line).
[0124] First, a 4'' silicon wafer (100, P-type, Prolog Semicor,
Ukraine) was silanised (85041C, Sigma-Aldrich, UK) to prevent
subsequent sticking of the PDMS. 200 .mu.l of the trichlorosilane
was pipetted into a vial and placed with the silicon wafer in a
desiccator for 1 hour.
[0125] 5 ml of degassed PDMS was spun with a spin coater (P6708D,
Speciality Coating Systems, USA) on the silanised wafer at 500 rpm
for 50 seconds to obtain a thickness of approximately 120 .mu.m and
placed in an oven at 80.degree. C. for 1 hour.
[0126] To bond the thin PDMS membrane 34 with the PDMS microfluidic
manifold layer 33, an air plasma was used. Before bonding, the
PDMS-coated wafer and the microfluidic manifold layer were rinsed
with ethanol and subsequently dried. Both PDMS layers were then
exposed to air plasma for 90 seconds at 30 W and 500 mTorr
(PDC-002, Harrick Plasma, USA). As shown in step 6 of FIG. 5, the
microfluidic manifold layer and the membrane on the wafer were then
immediately brought into contact for bonding. To further strengthen
the bond, the microfluidic chip 38 was placed in an oven at
80.degree. C. for at least 2 hours.
[0127] Step 7 of FIG. 5 depicts cutting the culture chamber body.
To provide access to the culture slide, the culture chamber body
had to be cut out of the membrane with a scalpel. The microfluidic
chip was cut out after and gently released from the silicon wafer
with a tweezer.
[0128] The interconnect was made of an aluminium block. A thread
was cut into the top for an Upchurch fingertight unit. The bottom
of the interconnect had a 2.08 mm high cylinder (6 mm in diameter)
to form a seal as previously described.
[0129] The dimensions of the mould and the microfluidic chip were
measured with a stylus profilometer (Dektak 8, Veeco Instruments
Company, USA) and the quality of the mould was inspected with a SEM
(XB1540 "Cross-Beam", Carl Zeiss AG, Germany).
[0130] Prior to assembly, all parts of the MPB were autoclaved,
except the cell culture slide. Assembly of the MPB was carried out
in a sterile hood.
[0131] A sterile tissue culture polystyrene slide (16004, Nunc,
Denmark) was placed in the bottom frame. The gasket was placed into
the top plate first, followed by the microfluidic chip. The top
plate with the microfluidic chip was then carefully placed over the
bottom frame with the culture slide and held in place with gently
tightened screws, sealing the entire device.
Example 3
[0132] Cell Culture Maintenance
[0133] Primary murine embryonic fibroblasts (MEF) were maintained
in Dulbecco's Modified Eagle Medium (DMEM) (41965, Invitrogen, USA)
supplemented with sodium pyruvate (11360, Invitrogen, USA), 10%
(v/v) heat inactivated foetal bovine serum (FBS) (10270,
Invitrogen, USA) and 1% (v/v) Modified Eagle Medium Non-Essential
Amino Acids (MEM NEAA) (11140, Invitrogen, USA) and passaged every
3 days into T75 flasks (159910, Nunc, Denmark) in a humidified
environment at 37.degree. C. with 5% CO2.
[0134] To inactivate MEFs, the T75 flasks were aspirated and
replaced with mitomycin C.
[0135] DMEM (11960, Invitrogen, USA) was supplemented with 10%
(v/v) FBS (10808, Invitrogen, USA), 1% (v/v) MEM NEAA (11140,
Invitrogen, USA) and 8 mg mL-1 mitomycin C (M4287, Sigma-Aldrich,
UK) and filtered. 5 mL of mitomycin C solution was added to a T75
flask and incubated for 2 hours at 37.degree. C. The flask was then
aspirated and washed with Dulbecco's phosphate buffer solution
(DPBS) (D1408, Sigma-Aldrich, UK) three times. Inactivated MEFs
were then trypsinized with trypsin:EDTA (T4049, Sigma-Aldrich, UK)
and incubated for 3 minutes. The suspension was spun down and the
supernatant resuspended. T25 flasks (156367, Nunc, Denmark) were
incubated with a 0.1% (v/v) in DPBS gelatine solution (G1890,
Sigma-Aldrich, UK) for 10 minutes at room temperature. The flasks
were aspirated and filled with 15,000 cells cm-2.
[0136] In experiments, the inventors used the Shef-3 cell line
obtained from the UK Stem Cell Bank. Use of the line was approved
by the UK Steering Committee.
[0137] Human ESC (hESC) (Shef-3) were cultivated on a mitomycin-c
inactivated feeder layer of primary MEFs (MEFs<passage 5) in T25
flasks (156367, Nunc, Denmark) as stock with filtered KnockOut DMEM
(10829, Invitrogen, USA) and KnockOut Serum Replacement (10828,
Invitrogen, USA) and supplemented with MEM NEAA (11140, Invitrogen,
USA), L-Glutamin (21051, Invitrogen, USA), .beta.-mercaptoethanol
(M3148, Sigma-Aldrich, UK) and FGF2 (4114-TC, R&D Systems,
USA).
[0138] hESC were passaged in small clumps every 3 days using
collagenase IV (17104, Invitrogen, USA).
[0139] The flasks were incubated with collagenase for 3-5 minutes,
before hESC colonies were scraped off the flask surface and
replated on a MEF feeder layer.
Example 3
[0140] Seeding and Experimental Procedure
[0141] The lid of a 150 mm diameter glass Petri dish (2175553,
Schott, USA) was fitted with three custom made silicone spacers to
enhance gas exchange in an incubator. These Petri dishes were used
to provide a sterile environment for the MPB in seeding
configuration (FIG. 6).
[0142] Prior to seeding, the Petri dishes, pipette tips and tubing
to be used were autoclaved and dried.
[0143] On day 0, 200 .mu.L of 0.1% (v/v) gelatine in DPBS solution
was added into the cell culture area of the MPB and incubated for
10 minutes at room temperature in a sterile laminar flow hood. The
gelatine was then aspirated and the MPB was left to dry for 30
minutes. 20,000 inactivated MEFs were seeded into the cell culture
area of the MPB (shown in FIG. 6, step 1). The customised lid was
put on the Petri dish, which accomodated the MPB, and placed in an
incubator (FIG. 6, step 2).
[0144] To compare the MPB with traditional static tissue culture
methods, three one well dishes (353652, BD Biosciences, USA) were
incubated with 0.1% (v/v) gelatine in DPBS solution (G1890,
Sigma-Aldrich, UK) for 10 minutes, aspirated and then seeded with
40,000 inactivated MEFs per one well dish.
[0145] Inactivated MEFs were counted with a haemacytometer
(0630030, Marienfeld, Germany).
[0146] Before seeding with hESC colonies on day 1, the one well
dishes and the MPB were aspirated and replaced with new hESC medium
at least 30 minutes before transferring hESC colonies.
[0147] When hESC colonies were routinely passaged in the stock
flask, the colonies in medium were transferred into the one well
dishes. A drop with hESC colonies in a small Petri dish (Nunc,
Denmark) was used to transfer colonies for the MPBs. hESC colonies
in the small Petri dish were caught with a 10 .mu.L pipette and
then transferred gently to the culture area in the MPB (FIG. 6,
step 3) and the Petri dish was closed again (FIG. 6, step 4). One
well dishes and MPB were then incubated overnight at 37.degree. C.
in an incubator to allow attachment of the hESC colonies to the
feeder layer.
[0148] On day 2, the hESC colonies had spread and attached to the
feeder layer. The medium in the control dishes was replaced every
24 hours for the entire time of the experiment. Medium in the MPB
was aspirated, the lid for the MPB put on, tubing for medium and
the waste were connected and continous perfusion was started for 48
hours and was stoped on day 4 of the experiment (FIG. 6, step
5).
[0149] The perfusion system consists of a syringe pump (Model 100,
KD Scientific, USA), silastic tubing (R3607, Tygon, USA) with Luer
adapters (Cole-Palmer, USA), autoclavable tubing (R1230, Upchurch
Scientific, USA) with fittings for the custom interconnectors
(P207, Upchurch Scientific, USA) and fittings (F331, Upchurch
Scientific, USA) for the Luer adapters (P659, Upchurch Scientific,
USA), the MPB and a waste bottle. The silicone tubing is gas
permeable and equilibriates in an incubator the medium with oxygen
while perfusing.
Example 3
[0150] Immunocytochemistry
[0151] The hESC colonies were characterised by indirect
immunochemistry. hESC colonies in control wells and MPB were fixed
with 4% (v/v) paraformaldehyde (PDF) in phosphate buffered saline
(PBS) for 20 minutes and washed three times in PBS supplemented
with 10% (v/v) FBS to block non specific binding (FIG. 6, step
6).
[0152] Primary monoclonal antibodies Oct-4 (SC-5279, Santa Cruz,
USA), Tra-1-81 (MAB4381, Chemicon, UK) and SSEA-3 (MAB4303,
Chemicon, UK) were used at a dilution of 1:200 and incubated with
the cells for one hour at 37.degree. C. The cells were then washed
three times with PBS and incubated with secondary antibodies with
excitation wavelengths of 488 nm (A21212, Invitrogen, USA) and 555
nm (A21426, Invitrogen, USA) for an hour at room temperature.
Finally, the cells were stained with DAPI (D1306, Invitrogen,
Carlsbad, Calif., USA). DAPI at a dilution of 1:200 was incubated
with cells at room temperature for 10 minutes. The MPB and one well
dishes were then washed three times with PBS.
[0153] In addition, double staining using Tra-1-81 and SSEA-3
antibodies on the same colony was performed.
Example 3
[0154] Imaging
[0155] For the perfusion experiments and the control wells, we used
an inverted microscope (Nikon Eclipse TE2000-U, Nikon Corporation,
Japan) with a colour microscope camera (Nikon DS-Fil, Nikon
Corporation, Japan) for daily inspection and endpoint assays.
[0156] To enhance the immunostaining contrast, the inventors used
Photoshop (Photoshop CS3, Adobe Inc., USA).
[0157] Results
[0158] It was found that the hESC in the MPB were healthy and
showed no difference compared to the controls demonstrating that
the MPB does not have any detrimental effect on the hESC in any
way. This was demonstrated by the fact that the pluripotency of the
hESC determined by morphology and immunostaining seemed equal or
better than in the static control dish, i.e. the hESC had retained
their pluripotency.
[0159] Discussion
[0160] The above described chip-to-world device offers a robust
method of linking a microfluidic chip with the "macro-world". The
interface includes a loading port, which can easily and repeatedly
be opened and closed. Open, the port permits direct access to a
microfluidic chamber. Once closed, the port is leak-free and
permits perfusion of said chamber. This sealable port permits easy
and gentle seeding of cells and extra-cellular matrix (ECM)
compounds into the microfluidic chamber, the perfusion of the cells
and, subsequently, their easy and gentle uptake from said chamber.
The port also enables the loading and removal of beads or polymer
monoliths, for example for enzymatic assays.
[0161] Furthermore, the device includes robust and leak-free
interconnects for the introduction and collection of solutes
(media, drug compounds) into and from the microfluidic chip. The
interconnects self-align and seal without the need of O-rings to
polymeric microfluidic chips. The location of the interconnects can
easily be reconfigured. This ease of reconfiguration enables the
microfluidic chip to be specifically designed according to the
particular application requirements and independently of
chip-to-world design limitations, thereby facilitating rapid
prototyping of complete microfluidic devices. Moreover, the device
enables the complete encapsulation of a polymeric microfluidic chip
in a multi-layer fashion. Again, the device can be opened and
closed easily and repeatedly. This multi-layer encapsulation not
only enhances the achievable degree of complexity for the
microfluidic chip itself (and thus its degree of functionality),
but also accepts standard glass microscope slides or polystyrene
plates. The use of standard material then facilitates the
comparison of microfluidic assays with traditional assays (for
example the comparison of traditional cell culturing techniques
with microfluidic cell culturing via using the same substrate
material). The facile opening and closing of the encapsulation
enables the insertion and removal of standard material and thereby
simplifies pre- and post-processing steps, which may have to be
conducted and transported to and from conventional larger-scale
equipment. Finally, the interconnects could potentially be
standardised for easy linkage with `robotised` liquid handling
platforms and all materials can be autoclaved, which further
broadens the applicability of the device. The described device
enables the realisation of microfluidic cell culture systems
suitable for drug discovery and drug toxicity testing with minute
amounts of cells, tightly controllable environmental conditions,
and ease of optical interrogation.
[0162] The invention is further described by the following numbered
paragraphs: [0163] 1. A microfluidic device comprising a chamber
having a fluid inlet, a fluid outlet and a sealable port, wherein
the fluid inlet and the fluid outlet are positioned to direct fluid
flowing from the fluid inlet to the fluid outlet through the
chamber, and wherein the sealable port is aligned with the chamber
to allow material to be placed directly into, or removed from, the
chamber from the exterior of the device when the sealable port is
open, and to prevent fluid escaping through the sealable port when
the port is sealed. [0164] 2. The device of claim 1, further
comprising an interconnect system which comprises: [0165] a first
component having a conduit to carry fluid to the fluid inlet or
away from the fluid outlet, wherein the first component is formed
of a deformable material, and [0166] a second component having a
projecting portion, wherein a conduit passes through the projecting
portion and the second component; [0167] wherein the conduit of the
first component is aligned with the conduit of the second
component, wherein the projecting portion of the second component
deforms an area of the first component surrounding the conduit
therein so as to create a seal around the contiguous conduits of
the first and second components, thus preventing any fluid from
escaping as it flows from one conduit to the other conduit, and
wherein the second component is for connecting the conduit therein
to an external fluid source or sink. [0168] 3. The device of claim
2, comprising an interconnect system for each of the fluid inlet
and fluid outlet. [0169] 4. The device of claim 2 or claim 3,
wherein the interconnect system or systems each further comprises a
guide positioned on the first component around the conduit therein
and which mates with the projecting portion of the second component
to align the conduit of the first component with the conduit of the
second component. [0170] 5. The device of any preceding claim,
wherein the base of the chamber is formed from a substrate for
supporting biological material. [0171] 6. The device of claim 5,
wherein the substrate is a standard glass or polystyrene microscopy
slide or culture plate and the chamber is formed on at least a
portion of the substrate. [0172] 7. The device of claim 5 or claim
6, wherein the substrate is detachable from the device. [0173] 8.
The device of any preceding claim for culturing cells. [0174] 9.
The device of any preceding claim, further comprising a housing.
[0175] 10. The device of any preceding claim, wherein the fluid
inlet and the fluid outlet are positioned on opposite sides of the
chamber. [0176] 11. The device of any preceding claim, wherein the
fluid inlet and the fluid outlet are positioned so that a material
containment portion of the chamber is substantially unaffected by
the flow of fluid through the chamber. [0177] 12. The device of any
preceding claim, wherein the fluid inlet and/or the fluid outlet
each form at least about 20% of the area of one side of the
chamber. [0178] 13. The device of any preceding claim, wherein the
fluid inlet and the fluid outlet are aligned with the top of the
chamber. [0179] 14. The device of any preceding claim, wherein the
fluid inlet and fluid outlet comprise one or more flow restrictors.
[0180] 15. The device of any preceding claim, further comprising a
conduit to carry fluid to the fluid inlet and a conduit to carry
fluid away from the fluid outlet, wherein each conduit contains one
or more flow dividers. [0181] 16. The device of any preceding
claim, wherein the sealable port forms a lid of the chamber. [0182]
17. The device of any preceding claim, wherein the fluid is a
liquid and the sealable port comprises a gas permeable membrane to
allow gas such as oxygen to pass into the chamber. [0183] 18. The
device of any preceding claim, wherein the device further comprises
a heater. [0184] 19. The device of any preceding claim, wherein the
device further comprises a sensor. [0185] 20. The use of the device
of any one of claims 1 to 19 for culturing cells or performing
cell-based assays. [0186] 21. An interconnect system for sealably
connecting two fluid carrying conduits, the system comprising:
[0187] a first component having a conduit and being formed of a
deformable material; and [0188] a second component having a
projecting portion, wherein a conduit passes through the projecting
portion and the first component; [0189] wherein, in use, the
conduit of the first component is aligned with the conduit of the
second component and a force is applied to the second component so
that the projecting portion deforms an area of the first component
surrounding the conduit therein so as to create a seal around the
contiguous conduits of the first and second components, thus
preventing any fluid from escaping as it flows from one conduit to
the other conduit. [0190] 22. The interconnect system of claim 21,
for connecting a conduit in a microfluidic device to an external
fluid carrying conduit. [0191] 23. The interconnect system of claim
21 or claim 22, wherein the interconnect system further comprises a
guide positioned on the first component around the conduit therein
and which mates with the projecting portion of the second component
to align the conduit of the first component with the conduit of the
second component. [0192] 24. A microfluidic device comprising a
chamber having a fluid inlet, a fluid outlet and a substrate for
supporting biological material, the fluid inlet and the fluid
outlet being positioned to direct fluid flowing from the fluid
inlet to the fluid outlet through the chamber, and wherein the
substrate forms the base of the chamber. [0193] 25. The device of
claim 24, wherein the substrate is a standard glass or polystyrene
microscopy slide or culture plate and the chamber is formed on at
least a portion of the substrate. [0194] 26. The device of claim 24
or claim 25, wherein the substrate is detachable from the device.
[0195] 27. The device of any one of claims 24 to 26, further
comprising an interconnect system which comprises: [0196] a first
component having a conduit therethrough to carry fluid to the fluid
inlet or away from the fluid outlet, wherein the first component is
formed of a deformable material, and [0197] a second component
having a projecting portion, wherein a conduit passes through the
projecting portion and the second component; [0198] wherein the
conduit of the first component is aligned with the conduit of the
second component, wherein the projecting portion of the second
component deforms an area of the first component surrounding the
conduit therein so as to create a seal around the contiguous
conduits of the first and second components, thus preventing any
fluid from escaping as it flows from one conduit to the other
conduit, and wherein the second component is for connecting the
conduit therein to an external fluid source or sink. [0199] 28. The
device of claim 27, comprising an interconnect system for each of
the fluid inlet and fluid outlet. [0200] 29. The device of claim 27
or claim 28, wherein the interconnect system or systems each
further comprises a guide positioned on the first component around
the conduit therein and which mates with the projecting portion of
the second component to align the conduit of the first component
with the conduit of the second component. [0201] 30. A method of
fabricating a microfluidic chip, the method comprising the steps
of:-- [0202] a) forming a mould defining features of the
microfluidic chip; [0203] b) pouring a curable polymer into the
mould; [0204] c) curing the polymer to form a cured polymer sheet;
[0205] d) releasing the cured polymer sheet from the mould; [0206]
e) forming a membrane having a base layer and a overlying cured
polymer layer; [0207] f) bonding the cured polymer sheet to the
membrane; and [0208] g) removing the base layer of the membrane to
release the microfluidic chip. [0209] 31. The method of claim 30,
wherein the same curable polymer is used in steps b) and e). [0210]
32. The method of claim 30 or claim 31, wherein the polymer is
polydimethylsiloxane (PDMS). [0211] 33. The method of any one of
claims 30 to 32, wherein step a) is carried out by a milling
process. [0212] 34. The method of any one of claims 30 to 33,
wherein the PDMS in step b) is a 10:1 base to curing agent mixture.
[0213] 35. The method of any one of claims 30 to 34, wherein a
covering sheet is clamped on top of the mould prior to the curing
process. [0214] 36. The method of any one of claims 30 to 35,
wherein the base layer of the membrane is a silanised silicon wafer
and the overlying curable polymer layer is a PDMS layer. [0215] 37.
The method of claim 36, wherein the PDMS layer is spin coated on
the silanised wafer at 500 rpm for 50 seconds to obtain a thickness
of substantially 120 micrometres. [0216] 38. The method of any one
of claims 30 to 37, wherein the cured polymer is bonded to the
membrane by plasma bonding. [0217] 39. The method of any one of
claims 30 to 38, wherein a microfluidic chamber is formed in the
microfluidic chip following step g). [0218] 40. The method of any
one of claims 30 to 39, wherein the PDMS is cured in an oven at
80.degree. C. for one hour.
[0219] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
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