U.S. patent application number 15/175749 was filed with the patent office on 2016-11-17 for microfluidic cell culture systems.
The applicant listed for this patent is EMD Millipore Corporation. Invention is credited to Paul J. Hung, Philip J. Lee.
Application Number | 20160333298 15/175749 |
Document ID | / |
Family ID | 45399997 |
Filed Date | 2016-11-17 |
United States Patent
Application |
20160333298 |
Kind Code |
A1 |
Hung; Paul J. ; et
al. |
November 17, 2016 |
Microfluidic Cell Culture Systems
Abstract
A number of novel improved microfluidic configurations and
systems and methods of manufacture and operation.
Inventors: |
Hung; Paul J.; (Berkeley,
CA) ; Lee; Philip J.; (Alameda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMD Millipore Corporation |
Billerica |
MA |
US |
|
|
Family ID: |
45399997 |
Appl. No.: |
15/175749 |
Filed: |
June 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13011857 |
Jan 21, 2011 |
9388374 |
|
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15175749 |
|
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|
|
61367371 |
Jul 23, 2010 |
|
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61297278 |
Jan 21, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0829 20130101;
B01L 2300/10 20130101; B01L 2300/0809 20130101; B01L 2400/0487
20130101; C12M 21/08 20130101; B01L 3/502761 20130101; B01L
2400/0457 20130101; C12M 23/34 20130101; C12M 41/36 20130101; B01L
3/502746 20130101; C12M 23/24 20130101; C12M 23/16 20130101; C12M
29/10 20130101; B01L 2300/0848 20130101; B01L 3/50273 20130101;
C12M 41/12 20130101; C12M 23/40 20130101; B01L 3/502715 20130101;
B01L 2400/086 20130101; B01L 2400/0406 20130101; B01L 2300/0883
20130101; C12M 23/58 20130101; B01L 2300/069 20130101 |
International
Class: |
C12M 3/06 20060101
C12M003/06; B01L 3/00 20060101 B01L003/00; C12M 1/00 20060101
C12M001/00 |
Claims
1. A microfluidic structure comprising: a culture chamber; at least
one first object flow outlet disposed on a wall of said culture
chamber; at least one second object flow outlet disposed on said
wall; at least one object flow inlet disposed on said wall and
between said at least one first object flow outlet and said at
least one second object flow outlet for introducing culture objects
and object flow media into said culture chamber; a flow-around
channel allowing fluidic media into said culture chamber; said
flow-around channel separated from the culture chamber by a
perfusion barrier, wherein said perfusion barrier defines at least
two walls of said culture chamber, wherein one wall of said at
least two walls defines an opposite wall of said culture
chamber.
2. The microfluidic structure of claim 1 further comprising: at
least one separate air diffusion channel adjacent to the culture
chamber and running along most of one side of the culture
chamber.
3. The microfluidic structure of claim 1 further comprising: a
second object flow inlet adjacent to the at least one object flow
inlet providing a split object loading inlet.
4. The microfluidic structure of claim 1 further wherein the
perfusion barrier is configured to prevent a gel from filling said
flow-around channel.
5. The microfluidic structure of claim 1 wherein the flow-around
channel comprises more than one half of the culture chamber.
6. The microfluidic structure of claim 1 further wherein the shape
of the culture chamber is selected from the group consisting of: a
rectangle; a rectangle with one or more rounded edges; a circle;
and a culture chamber with at least one side that is an elliptical
or circular shape.
7. The microfluidic structure of claim 1 further wherein said at
least one object flow inlet is configured to load and hold objects
embedded in a 3D gel or matrix and said perfusion barrier is
configured to provide fluid flow while preventing flow of clogging
by the 3D gel or matrix.
8. The microfluidic structure of claim 1 wherein the flow of cells
is loaded via capillary force from said at least one object flow
inlet and out from the at least one first object flow outlet and
the at least one second object flow outlet away from the at least
one object flow inlet; and wherein a very small amount of flow
exits the culture chamber from the at least one first object flow
outlet and the at least one second object flow outlet, thereby
tending to distribute cells more evenly in the culture chamber.
9. The microfluidic structure of claim 1 wherein the culture
chamber is between about 0.1 millimeters and about 5 millimeters
high; the flow-around channel is between about 10 microns and 100
microns high, with a cross section of between about 50 square
microns and about 10,000 square microns; and the perfusion barrier
provides a perfusion passage between about 1 microns high and about
10 microns high, with a cross section of between about 1 square
microns and about 100 square microns.
10. The microfluidic structure of claim 1 further wherein: the
culture chamber further comprises a glass floor and is configured
to perform the culture of cells in 2D culture using liquid culture
medium or 3D culture using a gel medium, wherein in 2D culture,
cells adhere to said glass floor due to a low culture flow rate
from the at least one object flow inlet to the at least one first
object flow outlet and the at least one second object flow outlet;
and wherein in 3D culture, cells are embedded in a gel and
dispensed into the culture chamber with the gel localized to the
culture chamber by the perfusion barrier, allowing medium to flow
around the gel/perfusion barrier and diffuse in to feed the
cells.
11. The microfluidic structure of claim 1 further comprising a
serpentine channel disposed between said first reservoir and said
flow-around channel, wherein said serpentine channel has a fluidic
resistance, and said fluidic resistance determines said flow rate
of said fluid in said flow-around channel.
12. The microfluidic structure of claim 1 further comprising a
serpentine channel disposed between said flow-around channel and
said second reservoir, wherein said serpentine channel has a
fluidic resistance, and said fluidic resistance determines said
flow rate of said fluid in said flow-around channel.
13. The microfluidic structure of claim 1, wherein a flow rate of
said fluid through said flow-around channel is determined based on
a difference in fluid level between said first reservoir and said
second reservoir.
14. The microfluidic structure of claim 1, wherein said perfusion
barrier defines three walls of said culture chamber.
15. The microfluidic structure of claim 1, wherein said perfusion
barrier encloses said culture chamber.
16. The microfluidic structure of claim 1, wherein said perfusion
barrier comprises a non-linear structure.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/011,857 filed Jan. 21, 2011, which claims
priority from provisional patent applications:
[0002] 61/367,371 filed Jul. 23, 2010
[0003] 61/297,278 filed Jan. 21, 2010
[0004] This application is related to material discussed in one or
more of the following applications, each of which are incorporated
herein by reference for all purposes: provisional patent
application 61/037,297 filed Mar. 17, 2008, provisional patent
application 61/018,882 filed Jan. 3, 2008, U.S. application Ser.
No. 11/994,997, filed Aug. 11, 2008, which is a National Stage
Entry of PCT/US06/26364, filed Jul. 6, 2006 and which claims
priority from provisional patent application 60/773,467 filed 14
Feb. 2006 and from provisional patent application 60/697,449 filed
7 Jul. 2005, U.S. application Ser. No. 12/019,857, filed Jan. 25,
2008, which claims priority to U.S. Provisional Patent Application
No. 60/900,651 filed on Feb. 8, 2007, U.S. application Ser. No.
11/648207, filed Dec. 29, 2006, which claims priority to U.S.
Provisional Patent Application U.S. provisional patent application
No. 60/756,399 filed on Jan. 4, 2006, U.S. application Ser. No.
12/348907, filed 5 Jan. 2009.
COPYRIGHT NOTICE
[0005] Pursuant to 37 C.F.R. 1.71(e), applicants note that a
portion of this disclosure contains material that is subject to
copyright protection (such as, but not limited to, diagrams, device
photographs, or any other aspects of this submission for which
copyright protection is or may be available in any jurisdiction.).
The copyright owner has no objection to the facsimile reproduction
by anyone of the patent document or patent disclosure, as it
appears in the Patent and Trademark Office patent file or records,
but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0006] The invention in various embodiments relates to handling of
micro-objects, such as cells or micro-fabricated particles such as
beads, using microfluidic systems. Particular embodiments involve
configurations that can be used with various standard automated
handling systems and with cells or other objects embedded in a gel.
Other particular embodiments involve configurations that can be
used with an open cell culture chamber.
BACKGROUND OF THE INVENTION
[0007] The discussion of any work, publications, sales, or activity
anywhere in this submission, including in any documents submitted
with this application, shall not be taken as an admission that any
such work constitutes prior art. The discussion of any activity,
work, or publication herein is not an admission that such activity,
work, or publication existed or was known in any particular
jurisdiction.
[0008] Microfluidic cell culture is an important technology for
applications in drug screening, tissue culturing, toxicity
screening, and biologic research and can provide improved
biological function, higher-quality cell-based data, reduced
reagent consumption, and lower cost. High quality molecular and
cellular sample preparations are important for various clinical,
research, and other applications. In vitro samples that closely
represent their in vivo characteristics can potentially benefit a
wide range of molecular and cellular applications. Handling,
characterization, culturing, and visualization of cells or other
biologically or chemically active materials (such as beads coated
with various biological molecules) has become increasingly valued
in the fields of drug discovery, disease diagnoses and analysis,
and a variety of other therapeutic and experimental work.
[0009] Publications and/or patent documents that discuss various
strategies related to cell culture using microfluidic systems and
related activities include the following U.S. patent applications
and non-patent literature, which, along with all citations therein,
are incorporated herein by reference for all purposes. A listing of
these references here does not indicate the references constitute
prior art.
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microenvironment for cell culturing, cell monitoring and
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array methods and apparatus for cell-based screening."
[0012] Fluidigm, Inc. Published Application 20040229349 (Nov. 18,
2004) "Microfluidic particle-analysis systems."
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[0051] Earlier work and patent applications as cited above,
involving at least one of the present inventors, discuss various
configurations, methods, and systems related to microfluidic cell
culture and that work and those publications are is incorporated
herein by reference.
SUMMARY
[0052] The present invention involves various components, systems,
and methods related to improved microfluidic cell culture devices
and systems. In one aspect, the invention involves novel
microfluidic cell culture devices, systems and methods that have
advantages over previously proposed microfluidic structures. In
another aspect, the invention involves novel structures and methods
for integrating multiple microfluidic cell culture units into
various multi cell culture unit systems, such as to a microtiter
well plate structure, such as a standard well plate formats (e.g.,
a 96-well SBS culture plate, or other plate formats, including
plates having 6, 12, 24, 96, 384 or 1536 sample wells, as well as
open bottom standard well plates, allowing for attachment to
microfluidic structures as described herein.). In a further aspect,
the invention involves novel fabrication methods for creating an
array of microfluidic cell culture units or areas suitable for
integration with a well plate. In another aspect, the invention
involves novel systems, methods, and components for an improved
automated high-throughput cell culture and/or screening system
using microfluidic cell culture devices and systems. In other
aspects, the invention involves novel culture chamber designs and
systems for providing effective culture of cells in various
situations, including cells cultured in a gel 3D matrix. In other
aspects, the invention involves novel cell culture chamber designs
and systems allowing use of open-top cell culture chambers with the
invention providing sufficiently controlled flow of culture media
to prevent the media from flowing out of the open top culture area.
In other aspects, the invention involves use of customized or
partly customized well-plates along with one or more standard or
customized well plate loading or handling systems to provide
culture units that in part interface with standard plate designs
and in part skip cells or combine cells into culture units.
[0053] In particular embodiments and examples, design features
include the elimination of tubing and connectors to the plates
themselves, the ability to maintain long-term continuous perfusion
cell culture using a passive gravity-driven flow, the ability to
perform direct analysis on the outlet wells and/or cellular
observation wells or culture wells of the microfluidic plate, the
ability to effectively handle gel culture media, and the ability to
effectively allow open culture wells in microfluidic systems.
[0054] While many of the examples discussed in detail herein are
designed to be used in conjunction with a standard or custom well
plate, the microfluidic structures and culture units and systems
and methods of various configurations as described herein can also
be deployed independently of any well-plate, such as in various
integrated lab-on-a-chip systems that are not configured to be used
in conjunction with well plates or various other microfluidic
devices or systems.
[0055] For purposes of clarity, this discussion refers to devices,
methods, and concepts in terms of specific examples. However, the
invention and aspects thereof may have applications to a variety of
types of devices and systems. It is therefore intended that the
invention not be limited except as provided in the attached claims
and equivalents.
[0056] Furthermore, it is well known in the art that systems and
methods such as described herein can include a variety of different
components and different functions in a modular fashion. Different
embodiments of the invention can include different mixtures of
elements and functions and may group various functions as parts of
various elements. For purposes of clarity, the invention is
described in terms of systems that include many different
innovative components and innovative combinations of innovative
components and known components. No inference should be taken to
limit the invention to combinations containing all of the
innovative components listed in any illustrative embodiment in this
specification. Unless specifically stated otherwise herein, any
combination of elements described herein should be understood to
include every sub-combination of any subset of those elements and
also any sub-combination of any subset of those elements combined
with any other element described herein as would be understood to a
practitioner of skill in the art.
[0057] In some of the drawings and detailed descriptions below, the
present invention is described in terms of the important
independent embodiments of multi-component devices or systems. This
should not be taken to limit various novel aspects of the
invention, which, using the teachings provided herein, can be
applied to a number of other situations. In some of the drawings
and descriptions below, the present invention is described in terms
of a number of specific example embodiments including specific
parameters related to dimensions of structures, pressures or
volumes of liquids, temperatures, electrical values, durations of
time, and the like. Except where so provided in the attached
claims, these parameters are provided as examples and do not limit
the invention, which encompasses other devices or systems with
different dimensions. For purposes of providing a more illuminating
description, particular known fabrication steps, cell handling
steps, reagents, chemical or mechanical process, and other known
components that may be included to make a system or manufacture a
device according to specific embodiments of the invention are given
as examples. It will be understood to those of skill in the art
that except were specifically noted herein otherwise, various known
substitutions can be made in the processes described herein.
[0058] All references, publications, patents, and patent
applications cited in this submission are hereby incorporated by
reference in their entirety for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 is a top view of an example array of cell culture
units provided on a 96-well standard SBS plate according to
specific embodiments of the invention.
[0060] FIG. 2 is an underside view showing one culture unit
occupying three wells in an example array according to specific
embodiments of the invention.
[0061] FIG. 3 illustrates high aspect ratio channels (also referred
to herein as perfusion passages or perfusion barriers) surrounding
cell culture areas in an example array according to specific
embodiments wherein channels between solid structures are
approximately 4 .mu.m wide and 40 .mu.m tall to prevent cells from
growing out. The channels in this example are separated by
approximately 40 .mu.m solid structures.
[0062] FIG. 4A-B are simplified schematic diagrams illustrating in
three dimensions the components of a multi well (e.g., 3)
microfluidic system including a representation of the well frame
according to specific embodiments of the invention.
[0063] FIG. 5 is a simplified side view showing a structure
according to specific embodiments of the invention illustrating two
wells that are used in cell flow and fluid flow.
[0064] FIG. 6A-C illustrate configuration and operation of an
example roughly rectangular cell culture chamber design according
to specific embodiments of the invention.
[0065] FIG. 7A-E illustrate configuration and operation of a second
example roughly rectangular new cell culture chamber design
according to specific embodiments of the invention.
[0066] FIG. 8A-C illustrate configuration and operation of an
example cell culture chamber design for 3D gel cell culture
according to specific embodiments of the invention.
[0067] FIG. 9 is a schematic diagram showing steps from an empty
culture region to performing a cell assay according to specific
embodiments of the invention.
[0068] FIG. 10 illustrates a layout of another type of cell culture
array designed for general cell culture automation according to
specific embodiments of the invention.
[0069] FIG. 11 A-D illustrate a 24 unit "3D culture" plate on a 96
well plate according to specific embodiments of the invention.
[0070] FIG. 12 is a schematic diagram illustrating fluidic
operation of open top cell culture systems according to specific
embodiments of the invention.
[0071] FIG. 13A-C is an illustration of an example 96 well plate
having 32 perfusion units with a dye in the culture medium for
illustrative purposes according to specific embodiments of the
invention: FIG. 13A shows an entire 96-well plate; FIG. 13B shows a
close up illustration of a 3-well perfusion unit view from the top
(from the opening of the cell to the open top culture chamber),
FIG. 13C shows a close up illustration of a 3-well perfusion unit
view from the bottom to more clearly see the microfluidic
structures as described herein which are stained for easier
viewing.
[0072] FIG. 14 is a top view schematic illustration of an example
design of an open top perfusion chamber according to specific
embodiments of the invention. In this example, a 2 mm hole (white)
is cut into 3 mm microfluidic chamber (orange). A narrow perfusion
barrier (green) surrounds the culture chamber to separate flows
from cells. An outer air channel (blue) oxygenates the medium in
the flow channels (gray).
[0073] FIG. 15 is a top view schematic illustration of an example
perfusion unit according to the invention and a cross section side
view schematic of an example design of an open top perfusion
chamber showing representative layers.
[0074] FIG. 16 is a photomicrograph showing a 2D perfusion culture
of MCF-10A cells after 7 days. The left side shows cells in
relation to the open well. Right shows a magnified view of cells in
the culture chamber, demonstrating a confluent monolayer of cells
on the bottom surface.
[0075] FIG. 17 is a photomicrograph showing a 3D perfusion culture
of MCF-10A cells after 7 days. Cells were embedded in BD Matrigel.
Left shows cells in relation to the open well. Right shows a
magnified view of cells in the culture chamber, demonstrating a
clustered 3D aggregate morphology suspended in gel.
[0076] FIG. 18 is a photograph of an open chamber with cells. (Top)
and a close up of the channel structure, showing the open cell
chamber, perfusion barrier ring, flow channel, and outer air
channel (Bottom) according to specific embodiments of the
invention.
[0077] FIG. 19A-B are photographs illustrating an example of an
active control plate according to specific embodiments.
[0078] FIG. 20A-B are schematics illustrating an example of an
active control plate according to specific embodiments.
[0079] FIG. 21A-B are schematics and a photo illustrating an
example of (A) a cross section of the open chamber showing
materials used for construction. The bottom layer is a solid glass
slide. On top is a layer of molded PDMS containing microfluidic
structures as described herein. Also, as described herein, an
acrylic sheet is used in the molding process and the PDMS remains
attached to it. For the open top cell chamber, in specific
embodiments, this acrylic sheet is laser cut, etched, drilled or
otherwise opened to create the open top culture chamber. In
specific embodiments, a ring of Teflon tape or similar hydrophobic
material is placed around the open chamber inside the well to
increase the surface tension, for example by preventing wetting of
the acrylic. Alternatively, the acrylic may be coated or treated or
fabricated to have a higher hydrophobicity. The open chamber is
laser cut in these three layers (prior to attaching the glass
bottom) and (B) a photograph showing the Teflon ring on the top
surface of the device. Clear liquid is filled in the open chamber,
with microchannels filled with red dye.
[0080] FIG. 22A-C shows a top view, side view, and plan view of a
schematic of an example manifold according to specific embodiments
of the invention. In this example, the eight tubing lines to the
right are for compressed air, and each is configured to provide
pressure to a column of cell inlet wells in a microfluidic array.
The left-most line in the figure is for vacuum and connects to an
outer vacuum ring around the manifold. Each column of wells is
generally connected to a single pressure line with wells above
imaging regions skipped.
[0081] FIG. 23 illustrates an example system and manifold for
operating the microfluidic plates according to specific embodiments
of the invention.
[0082] FIG. 24 illustrates a manifold with additional gas line and
an objective lens according to specific embodiments of the
invention.
[0083] FIG. 25 is a graph illustrating an example of flow rate
difference between a surface tension mechanism and a gravity driven
mechanism according to specific embodiments of the invention.
[0084] FIG. 26 is a graph illustrating an example of the extent to
which gravity perfusion rate is responsive to the liquid level
difference between the two upper reservoir wells according to
specific embodiments of the invention.
[0085] FIG. 27 illustrates a top view schematic of an example cell
culture automation system according to specific embodiments of the
invention.
[0086] FIG. 28 is a photograph of an example automated microfluidic
perfusion array system according to specific embodiments of the
invention.
[0087] FIG. 29 illustrates operation steps of a less automated or
prototype system according to specific embodiments of the
invention.
[0088] FIG. 30 is a block diagram showing a representative example
logic device in which various aspects of the present invention may
be embodied.
[0089] FIG. 31 (Table 1) illustrates an example of diseases,
conditions, or states that can evaluated or for which drugs or
other therapies can be tested according to specific embodiments of
the present invention.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
1. Overview
Definitions
[0090] A "particle" refers to biological cells, such as mammalian
or bacterial cells, viral particles, or liposomal or other
particles that may be subject to assay in accordance with the
invention. Such particles have minimum dimensions between about
50-100 nm, and may be as large as 20 microns or more. When used to
describe a cell assay in accordance with the invention, the terms
"particles" and "cells" may be used interchangeably.
[0091] A "microchannel" or "channel" or "flow channel" generally
refers to a micron-scale channel used for fluidically connecting
various components of systems and devices according to specific
embodiments of the invention. A microchannel typically has a
rectangular, e.g., square, or rounded cross-section, with side and
depth dimensions in a preferred embodiment of between 10 and 500
microns, and 10 and 500 microns, respectively. Fluids flowing in
the microchannels may exhibit microfluidic behavior. When used to
refer to a microchannel within the microwell array device of the
invention, the term "microchannel" and "channel" are used
interchangeably. "Flow channel" generally denotes channels designed
for passage of media, reagents, or other fluids or gels and in some
embodiments cells. "Culture channel" or "cell culture channel"
generally denotes a portion of a cell culture structure that cells
are designed to flow through and also remain during cell culture
(though the cells may be localized into a particular culture area
of the culture channel in some embodiments). "Air channel"
generally denotes a roughly micron-scale channel used for allowing
gases, such as air, oxygen enriched mixtures, etc., to pass in
proximity to flow channels or culture areas. "Perfusion channel" is
sometimes used to indicate a flow channel and any perfusion
passages or structures that allow media to perfuse to the culture
area.
[0092] A "perfusion barrier" refers to a combination of solid
structures and perfusion passages that generally separate a flow
channel from a cell culture area or chamber. The perfusion passages
are generally smaller than the microchannel height and/or width
(for example, on the order of 5-50% or on the order of about 10%)
and are designed to keep cells, other culture items, and in some
embodiments gels, from migrating into the flow channels, while
allowing some fluidic flow that is generally of a much higher
fluidic resistance than the fluid flow in the flow channels. In one
example embodiment, the perfusion barrier has a perfusion passage
that is 4 microns high and that otherwise runs most of the length
of the microchannel In other embodiments, a perfusion barrier has
many perfusion passages that are about as high as the microfluidic
channel, but about 4 microns wide. In some configurations, a
perfusion barrier may also be referred to as an "epithelial
barrier."
[0093] A "microfluidics device" refers to a device having various
station or wells connected by micron-scale microchannels in which
fluids will exhibit microfluidic behavior in their flow through the
channels.
[0094] A "microwell array" refers to an array of two or more
microwells formed on a substrate.
[0095] A "device" is a term widely used in the art and encompasses
a broad range of meaning. For example, at its most basic and least
elaborated level, "device" may signify simply a substrate with
features such as channels, chambers and ports. At increasing levels
of elaboration, the "device" may further comprise a substrate
enclosing said features, or other layers having microfluidic
features that operate in concert or independently. At its most
elaborated level, the "device" may comprise a fully functional
substrate mated with an object that facilitates interaction between
the external world and the microfluidic features of the substrate.
Such an object may variously be termed a holder, enclosure,
housing, or similar term, as discussed below. As used herein, the
term "device" refers to any of these embodiments or levels of
elaboration that the context may indicate.
[0096] Microfluidic systems provide a powerful tool to conduct
biological experiments. Recently, elastomer-based microfluidics has
especially gained popularity because of its optical transparency,
gas permeability and simple fabrication methods. However, the
interface with the end-users requires labor-intensive hole punching
through the elastomer, and additional steps of tubing and syringe
pump connection.
[0097] The present invention involves integrated microfluidics used
for various culture and assay applications. The invention further
involves methods of manufacture of microfluidics and components and
a system for automating cell culture using such plates. Advantages
of specific embodiments include use of a standard microtiter plate
format, tubing free cell culture, and a biomimetic liver
microenvironment.
[0098] A system according to specific embodiments of the invention
(for example, using 96-well standard plates) can be operated using
standard techniques and equipment for handling standard microtiter
plates, as are well known in the art. For example, liquid
dispensing is achieved with standard pipette mechanics, and cell
culture and analysis can be made compatible with existing
incubators and plate readers.
[0099] According to further embodiments of the invention, a novel
cell loading system uses a pneumatic manifold and pneumatic
pressure to place cells in the micro culture area. With the
addition of this cell loading system, microfluidic cell culture and
analysis can be fully automated using other automated equipment
that exists for handling standard titer plates.
[0100] In further embodiments, the gravity driven flow culture
configuration utilizes the medium level difference between the
inlet and outlet well as well as engineering the fluidic
resistances to achieve the desirable flow rate in nL/min regime.
This provides the significant advantage of being able to
"passively" flow culture medium for long periods of time (up to 4
days) without the use of bulky external pumps or tubes.
[0101] In further embodiments, the invention involves a
microfluidic system to allow control of the cell culture
environment for long-term time-lapse microscopy of adherent cells.
As the trend towards "systems biology" continues, it will become
increasingly important to study dynamic behavior in individual live
cells as well as to improve the functionality and economics of high
throughput live cell screening. According to specific embodiments
of the invention, the invention provides a multiplexed microfluidic
flow chamber allowing for time-lapse microscopy experimentation
among other assays. The microfluidic chamber uses an artificial
endothelial barrier to separate cells from flow channels. The
device is formatted to a standard well plate, allowing liquid and
cell samples to be directly pipetted into the appropriate inlet
reservoirs using standard equipment. A custom pneumatic flow
controller is then used to load the cells into the culture regions
as well as to switch between different exposure solutions. A
digital software interface can be used to allow a user to program
specific inputs (pulses, ramps, etc.) over time to expose the cells
to complex functions during time-lapse imaging.
[0102] Dynamic responses in living cells are the foundation for
phenomena such as biological signal processing, gene expression
regulation, differentiation, and cell division. In specific
embodiments, the invention involves a system capable of controlling
the cellular micro-environment in a multiplexed format compatible
with current cell culture methods. Cell response can be quantified
using high magnification fluorescence microscopy to derive kinetic
information with sub-cellular resolution. This capability has broad
applications in cellular systems biology where dynamic single cell
response experiments are not currently practical.
2. Microfluidic Culture System and Array
[0103] The applications referenced above discussed a variety of
different cell culture configurations and fabrication techniques.
Portions of the operation of the cell culture areas and materials
are useful as background to the present discussion. In some
examples therein, one or more micro culture areas are connected to
a medium or reagent channel via a grid of fluidic passages (or
diffusion inlets or conduits), wherein the grid comprises a
plurality of intersecting high fluidic resistance perfusion
passages. In one discussed example, passages in the grid are about
1 to 4 .mu.m in height, 25 to 50 .mu.m in length and 5 to 10 .mu.m
in width, the grid allowing for more even diffusion between medium
or reagent channels and the culture area and allowing for easier
manufacturing and more even diffusion. The earlier application
further discussed that the high fluidic resistance ratio between
the microchamber and the perfusion/diffusion passages or grid
(e.g., ratios in the range of about 10:1, 20:1 to 30:1) offers many
advantages for cell culture such as: (1) size exclusion of cells;
(2) localization of cells inside a microchamber; (3) promoting a
uniform fluidic environment for cell growth; (4) ability to
configure arrays of microchambers or culture areas; (4) ease of
fabrication, and (5) manipulation of reagents without an extensive
valve network. Examples were illustrated wherein a grid-like
perfusion barrier can be much shorter than the culture area or can
be near to or at the same height, according to specific embodiments
of the invention and further wherein various configurations for
culture devices were illustrated. The application also discussed a
CAD drawing of a proposed 96-unit microfluidic bioreactor wherein
each well was an SBS standard size (3.5 mm in diameter) in order to
be compatible with existing robotic liquid handling systems and
plate readers. The application also discussed several different
configurations for an artificial sinusoid using both cut passages
and grids and with a flow-around perfusion design.
[0104] FIG. 1 is a top view of an example array of cell culture
units provided on a 96-well standard SBS plate according to
specific embodiments of the invention. In this example, 32 culture
units are provided on a 96-well plate (such as the Society for
Biomolecular Screening (SBS) standard microfluidic bioreactor array
schematic), with wells arranged in 12 columns (shown vertically and
labeled as is standard in the art, 1-12 from top to bottom) by 8
rows (shown horizontally and labeled as is standard in the art, A-H
from left to right). In this example, each cell culture unit
occupies three wells, one for use as a medium inlet, one for use as
a cell inlet/medium outlet, and one for use for cell imaging (which
appears as a dark rectangle in the wells in the figure) and/or for
providing air passages to a cell culture area. In specific
embodiments, each unit can be used as an independent biomimetic
cell. This example is shown for discussion purposes, and any number
of other configurations are possible including configurations are
described and illustrated in this application or as would be
understood or suggested to one of skill in the art having benefit
of the teachings provided herein. FIG. 2 through FIG. 3 show
further details of structures according to specific embodiments of
the invention. For purposes of clarity, each of these figures can
be understood as further detail of the example configuration
discussed above.
[0105] FIG. 2 is an underside view showing one culture unit
occupying three wells in an example array according to specific
embodiments of the invention. In this example, the cell culture
portion visible in the middle well is divided into four blocks,
with each block having four separated cell culture areas (or
channels) surrounded by medium channels used for medium fluidic
passage. In particular embodiments, these four separated cell
culture areas may be referred to as sinusoids or artificial
sinusoids, regardless of whether the far end of the areas has a
rounded shape. Separation into four blocks facilitates air
diffusion through the material that defines the microfluidic
channels (such as silicone elastomer polydime-thylsiloxane (PDMS))
structure into the culture areas. Six air holes to facilitate air
passage are shown.
[0106] FIG. 3 illustrates high aspect ratio channels (also referred
to herein as perfusion passages or perfusion barriers) surrounding
cell culture areas in an example array according to specific
embodiments wherein channels between solid structures are
approximately 4 .mu.m wide and 40 .mu.m tall to prevent cells from
growing out. The channels in this example are separated by
approximately 40 .mu.m solid structures.
[0107] FIG. 4A-B are simplified schematic diagrams illustrating in
three dimensions the components of a multi well (e.g., 3)
microfluidic system including a representation of the well frame
according to specific embodiments of the invention.
[0108] FIG. 5 is a simplified side view showing a structure
according to specific embodiments of the invention illustrating two
wells that are used in cell flow and fluid flow. Both views show
side views of the device and illustrate glass layer 501,
microfluidics layer 502, well layer 503, lower reservoir 504, and
upper reservoir 505.
[0109] Thus, the present invention according to specific
embodiments, can be used in a variety of cell culture systems,
including novel improved microfluidic systems, methods, designs,
devices, and/or configurations as discussed in above referenced
applications and incorporated herein by reference. In a first
aspect, three wells are used for each otherwise independent cell
culture system. In a second aspect, artificial sinusoids with
artificial epithelial barriers are provided with just one
(optionally shared or multiplexed) fluidic inlet and one
(optionally shared or multiplexed) fluidic output, where the medium
output also functions as a cellular input. In a third aspect,
artificial sinusoids with artificial epithelial barriers with just
one fluidic inlet and one fluidic output are divided into blocks
with air channels provided between blocks. In a fourth aspect, air
holes are provided in the well chamber above the cell culture area
of a microfluidic cellular culture array, where the medium output
also functions as a cellular input. In a fifth aspect, a
multiplexed medium inlet structure and multiplexed cellular input
structure are provided to connect inputs and outputs to blocks of
artificial sinusoids. In a sixth aspect, a multiplexed medium inlet
structure and larger shared cellular input structure are provided
to connect inputs and outputs to blocks of artificial sinusoids. In
a seventh aspect, artificial sinusoids are configured with non-open
portions of an epithelial barrier to better localize cells, and
with perfusions inlets surrounding a cell culture area and
optionally also present near a cell inlet area of the sinusoid. In
an eighth aspect, longer artificial sinusoid chambers are
provided.
[0110] As discussed elsewhere, various modifications may be made to
the cell culture area as described above. Various configurations
are possible for the epithelial barrier (or perfusion barrier),
such as a grid-like passage structure. Other variations will be
suggested to those of skill in the art having the teachings
provided herein.
[0111] The structures disclosed above can also be adapted to
systems using more or fewer wells on a standard microtiter well
plate or a fully customized or partially customized plate, such as
those described in referenced documents and in other examples
herein.
3. Modified Cell Culture Chamber
[0112] Plates and systems as described herein can be used with
other configurations of cell culture areas or cell culture chambers
and micro-fluidic flow structures, including one or more of the
novel designs described below.
[0113] FIG. 6A-C illustrate configuration and operation of an
example roughly rectangular cell culture chamber design according
to specific embodiments of the invention. In this example design,
the cell culture area provided is an essentially rectangular cell
culture chamber. The cell culture chamber has cell inlet and outlet
passages E2 shown at the right, and flow outlets El also shown at
the right. In this example, the cell passages are paired, with the
center pair used for cell flow loading and the pairs on either side
used as a cell flow outlet. Multiple separate flow inlets are shown
on the left, labeled A1, A2, B1, B2, C1, C2 and in this example
design the flow inlets have a grid pattern to prevent blockage by
cells. Air diffusion channels are shown surrounding the chamber.
Outlet El provides an outlet for fluid flow that is partially
isolated from the culture chamber.
[0114] FIG. 6B illustrates cell loading in a culture unit as shown
in FIG. 6A. Cells are loaded via a low resistance fluidic path
(with higher resistance in the flow paths). The cells are prevented
from blocking the flow paths by the resistance ratio (the cells
preferentially flow to the cell outlet instead of the flow
channels). The channels in this particular embodiment are arranged
such that the cell in and cell out channels are on the right side
of the chamber. This results in the unique feature where flow of
cells goes into the chamber, makes a 180 degree turn, and flows
out, as illustrated by the sharply curved streamlines shown in FIG.
6B from the Cell In to Cell Out passages.). Thus, according to
specific embodiments of the invention, cells are loaded (via
capillary force) from the center right channel(s) and out from the
top and bottom right channels. A very small amount of flow is
directed towards the side outlet channels (the longer less curved
streamlines shown in FIG. 6B exiting at the left edge of the
chamber). The side flow is not important for cell loading, but
serves to help distribute cells more evenly in the chamber. Because
of the low velocity of the flow, the cells naturally settle onto
the chamber floor without needing any physical barrier. The cell
outlet paths help make the loading symmetric, as well as to
increase the number of cells loaded into the chamber. This loading
mechanism can be used to load cells, particles, beads, gels, gels
with cells, etc.
[0115] FIG. 6C is a photomicrograph showing cells loaded into a
microfluidic chamber as described above.
[0116] FIG. 7A-E illustrate configuration and operation of a second
example roughly rectangular new cell culture chamber design
according to specific embodiments of the invention. This example
design differs only slightly from that of FIG. 6 and all operation
modes described for one of these two designs herein apply to the
other.
[0117] FIG. 7A illustrates cell loading in a culture unit with an
essentially rectangular cell culture chamber using with cell inlet
and outlet passages shown at the right and flow outlets also shown
at the right. In this example, the cell passages are unpaired.
Three unpaired flow inlets are shown on the left and these also
have a grid pattern to prevent blockage by cells. Air diffusion
channels generally are placed near the chamber, though not shown in
this figure.
[0118] FIG. 7B is a photomicrograph showing three different cell
types loaded at four different concentrations of cells loaded into
a microfluidic chamber as described above.
[0119] FIG. 7C is a photomicrograph showing a close-up view of
mouse embryonic stem cells cultured in the microfluidic device as
described above.
[0120] FIG. 7D is a photomicrograph showing cell growth in the
microfluidic device as described above.
[0121] FIG. 7E illustrates creating a gradient in the culture
chamber by flowing 2 (or more) solutions at once according to
specific embodiments of the invention in the microfluidic device as
described above.
4. 3D Cell/Gel Culture Chamber
[0122] FIG. 8A-C illustrate configuration and operation of an
example cell culture chamber design for 3D gel cell culture
according to specific embodiments of the invention. This example
includes a cell/gel perfusion barrier with a cross-hatch perfusion
passage design. The cross hatch design allows cells in a gel matrix
to be flowed into the chamber and allows for perfusion of media.
While the cross-hatch perfusion barrier is presently preferred in
some designs, culture chambers with different perfusion barriers or
no perfusion barriers are also implemented according to specific
embodiments. A flow around channel for media includes an outlet and
inlet both on the same side of the barrier. FIG. 8A illustrates a
general embodiment where the outlet and inlet openings are shown to
the right. FIG. 8B illustrates an inlet channel to the left and
outlet channel to the right, which configuration is better suited
in some example systems using a well plate as described herein.
This figure also provides detailed example dimensions of a sample
design according to specific embodiments of the invention. Thus, in
a further embodiment, a cell culture chamber is modified to allow
easier culture of cells in 3D gel matrix. In this design, a
perfusion barrier separates the cell culture area and the flow
channel as illustrated. The barrier is designed to retain a 3D gel
in the culture chamber. Coupling the barrier with the 3-channel
cell/gel inlet design described above is an important feature that
provides improved performance. By having separate flow
inlets/outlets on each side of the barrier, it is possible to
localize a fluid gel in the culture chamber, and not have it
obstruct the flow channel
[0123] In these embodiments, the invention creates a 3D gel
environment for biologic cell culture, for example using a
temperature sensitive gel culture matrix, such as Matrigel.TM.,
Geltrex.TM., collagen, etc. An example gel is liquid at 4 C, which,
for example polymerizes at room temperature or 37 C. In one example
method, cells are initially mixed with a cell suspension on ice.
The solution is then pipetted into the cell inlet well, and carried
into the microfluidic chamber via capillary flow. In specific
examples, the plate is kept at room temperature. The flow rate
allows sufficient cell/gel solution to fully fill the culture
chamber prior to polymerization. The barrier prevents any of the
gel solution from leaking into the flow channel As the gel warms
up, it polymerizes into a solid mass, with cells embedded. Flow of
media in the flow channel diffuses into the cell culture chamber
(through the gel) and nourishes the cells for long term culture.
This novel design allows the invention to provide a 3D gel culture
system in a microfluidic device while avoiding the problem of
having gel block the flow channels. FIG. 8C illustrates cell/gel
loading that operates generally as described above. FIG. 8D
illustrates a cell/gel culture with a medium exposed through a
perfusion according to specific embodiments of the invention. FIG.
8E illustrates two micrographs showing a culture chamber with an
air channel, flow channel, barrier, and cell region according to
specific embodiments of the invention. In the right portion, the
same region is shown, with a fluorescently labeled gel. Note the
gel fully occupies the cell culture chamber but does not extend
beyond the barrier.
5. Example Device Operation
[0124] FIG. 9 is a schematic diagram showing steps from an empty
culture region to performing a cell assay according to specific
embodiments of the invention. Various novel aspects according to
specific embodiments of the invention simplify these steps and
allow them to be automated.
Cell Loading
[0125] Cell loading in specific embodiments of the invention can
utilize the rapid surface tension flow between the cell inlet and
the flow inlet. In this method, the cell inlet reservoir (upper and
lower) is aspirated of its priming solution. Then, the flow inlet
upper reservoir is aspirated. An amount (e.g., five microliters) of
cell suspension (e.g., trypsinized HeLa human cancer cell line,
5.times.10.sup.5 cells/ml) is dispensed into the cell inlet lower
reservoir. The flow inlet lower reservoir is aspirated, causing
liquid to flow from cell inlet to flow inlet via surface
tension/capillary force. Cell loading in various configurations can
be completed in approximately 2-5 minutes. The cell loading
reservoir is then washed with medium (e.g., Dulbecco's Modified
Eagle's Medium, DMEM) and filled with e.g., 50-100 microliters of
clean medium. At this state, the plate is was placed in a
controlled culture environment for a period (e.g., 37 C, 5%
CO.sub.2 incubator for 2-4 hours) to allow for cell attachment. As
stated above, all dimensions and values are given for illustrative
purposes.
3D Gel System
[0126] In one example system, referred to at times herein as the
3D:M, multiplexed perfusion imaging of cells can be performed in a
3D gel matrix. An example plate contains 24 independent culture
units that can be loaded with cells/gel as a user chooses. In an
example system, each row of the plate (A-H) contains 3 fully
independent flow units (4 wells each), consisting of a medium inlet
(e.g., cols. 1, 5 , 9), a cell culture/imaging well (e.g., cols. 2,
6, 10), cell/gel inlet (cols. 3, 7, 10), and an outlet (cols 4, 8,
12). Air diffusion channels (blue) provide gas transfer to the
cells. The inlets are designed to allow continuous flow of culture
media to the cells at 40 .mu.l/day via a gravity driven process. In
this example, each chamber is 1.5.times.0.5 mm in size, with a
height of 200 .mu.m. The perfusion barrier (green) ensures uniform
nutrient transfer through the gel matrix and a thin cover glass
bottom (170 .mu.m) allows for optimum image quality.
3D Gel Loading
[0127] Two example operations of 3D microfluidic cell culture
according to specific embodiments of the invention are provided
below. In a cells embedded method (using a medium such as BD
Matrigel), the procedure is as follows: (1) Prepare a cell
suspension of 1-5106 cells/ml, depending on the desired cell number
for culture. Optionally, for improved results, resuspend in culture
medium on ice. (2) Mix cell suspension with Matrigel on ice. A 1:1
ratio is recommended, but various dilutions are suitable depending
on desired gel density. Keep on ice until loaded into the
microfluidic plate. (3) Aspirate the flow inlet, cell/gel inlet,
and flow outlet wells. Generally, it is desired to empty the liquid
from the holes at the bottom of the wells. Over aspiration is
preferable avoided, as this may introduce air bubbles. (4) Pipet
5-10 .mu.l of cell/gel mixture into the cell/gel inlet well,
generally keeping the plate at room temperature. Once the cell/gel
mixture is pipetted into the cell/gel inlet hole, capillary flow
will rapidly transport the liquid into the culture chamber, while
the perfusion barrier prevents the cells/gel from leaking into the
flow channel (5) In an example embodiment, after .about.2 minutes
at room temperature, the cells will stop flowing as the gel begins
to polymerize. Optionally, polymerization may be completed by, for
example, placing in a 37.degree. C. incubator for 15 minutes. (6)
Pipet 300 .mu.l of culture medium to the "Flow Inlet" well. This
will initiate gravity driven perfusion towards the flow outlet at a
rate of about 40 .mu.l/day. The flow channel passes next to the
cell culture chamber, and feeds the cells via diffusion as
described above. The minimum barrier dimension is 2 micron,
allowing soluble factors to freely pass through. Diffusion across
the culture chamber occurs in .about.20 minutes. (7) For long term
culture, refill the flow inlet and empty the flow outlet at an
appropriate interval, e.g., every 3 days.
Gel Overlay Method
[0128] In an alternative method, a cell suspension may be loaded
into the plate without gel and the gel can be overlaid immediately,
after cell adhesion, or following a few days of growth. Overlay gel
is placed following steps 3 and 4 above using a gel solution (with
no cells). The gel will flow over the cells and polymerize in the
chamber.
6. Culture Units in Multi-well Plates
[0129] As discussed elsewhere herein, any of the various novel
microfluidic cell culture chambers and associated microfluidic
structures can, according to specific embodiments of the invention,
be integrated with a well titer plate device as is commonly used in
macro cell culturing assays. A number of specific examples are
provided below, though the invention encompasses other systems for
integrating with the microfluidic devices.
[0130] FIG. 10 illustrates a layout of another type of cell culture
array designed for general cell culture automation according to
specific embodiments of the invention. In this design, each culture
unit consists of 4 well positions. The first well is for perfusion
medium, the second well is for cell inlet, the third well is for
imaging the microfluidic chamber, and the fourth well is the
outlet. A cell barrier/perfusion channel localizes cells to the
cell area and improves nutrient transport during continuous
perfusion culture. The low fluidic resistance of the cell inlet to
outlet path enables cells to be rapidly loaded via gravity or
surface tension methods without an external cell loading mechanism.
The high fluidic resistance of the perfusion inlet flow channels
allows long term continuous perfusion of medium via gravity flow
without any external pump mechanism.
[0131] FIG. 11 A-D illustrate a 24 unit "3D culture" plate on a 96
well plate according to specific embodiments of the invention.
According to specific embodiments of the invention, this
configuration is a designed for high-thru-put production work. The
design allows cells to be cultured in various 3D gel matrix media
with continuous perfusion medium exposure for long term cell assay
and cell imaging experiments. In a specific embodiment, using a
standard 96-well format and passive gravity driven perfusion allows
simple integration with existing laboratory equipment.
[0132] In a specific example, a 96-well plate contains 24
independent 3D culture units with microfluidic channels (which are
stained in the Figure for visibility) A single unit with flow
channels stained is shown in FIG. 11C. In an example operation,
media flows from the inlet well past the cultured cells and
collects in the outlet well. Cells and gel are loaded by the user
into the biomimetic cell culture chamber.
[0133] In an example specific system, the cell chamber is designed
to mimic the interstitial tissue environment, with cells embedded
or overlayed in physiologic extracellular matrix (ECM), and fed via
diffusion from a continuously perfused capillary channel The cell
microenvironment enables long term growth in, e.g., a 200 micron
thick gel layer. Oxygenation channels maintain adequate gas
transport, and the glass coverslide bottom allows high quality cell
imaging. The standard layout allows the advanced microfluidic units
to be operated just like a typical 96-well plate. The gravity
driven perfusion design eliminates the need for pump or tubing
connections, as described above.
[0134] In an example system, an expected number of cells per unit
is about 500 cells. An example perfusion rate is 40 ul/day for a
single unit. The cell chamber volume is 150 nL, and the chamber
dimensions are 1.5.times.0.5.times.0.2 mm. The gas diffusion
membrane is 50 um silicone with a bottom surface #1.5 thickness
coverglass.
7. Open Chamber Microfluidic Perfusion Plate for Cell Culture
[0135] In many microfluidic systems, cells generally must be
introduced to the culture chamber via flow from a cell inlet well.
This can hamper use of such devices for cultures that need to
introduce large cells, cell clusters, or tissue samples that do not
transport well in microchannels. In a typical existing open top
well (e.g. 384 or 1586 well plate), cell seeding is easy, but there
is no way to maintain a continuous flow environment to the
cells.
[0136] An open top microfluidic cell culture chamber for continuous
perfusion according to specific embodiments uses the surface
tension of liquid in the open chamber to counteract flow pressure,
thereby preventing liquid from spilling out of the open chamber and
instead flowing to downstream channels. In this aspect, embodiments
allow the combination of cell introduction into an open well and
integrated microfluidic perfusion control.
[0137] An important aspect of the operation of open top systems
according to specific embodiments of the invention is illustrated
in FIG. 12. Fluid mechanics dictates that liquid will move from
regions of high pressure to lower pressure. Pressure differences
can be caused by gravity (difference in liquid level), applied
pressure (from pumps), or surface tension. According to specific
embodiments of the invention, an open top cell culture chamber is
provided such that the surface tension pressure of the liquid above
the open well is higher than the chamber pressure, creating a
surface tension barrier that prevents liquid from flowing up out of
the open chamber. In the figure, the angle of the fluid surface
above the chamber walls is exaggerated for illustration
purposes.
[0138] In the example shown in the schematic of FIG. 12, P.sub.in,
P.sub.st, P.sub.f, P.sub.out are the pressures at the inlet well
(P.sub.in), above the open chamber (P.sub.st), inside the culture
chamber (P.sub.f), and at the outlet well (P.sub.out). R.sub.in,
R.sub.out are the fluidic resistances between the inlet well and
chamber, and between the chamber and outlet well. When
P.sub.st>P.sub.f; and P.sub.in>P.sub.f>P.sub.out ; then
liquid flows from the inlet well to the outlet well with no flow
escaping the chamber into the middle well. The surface tension
force is P.sub.st=2.gamma./R (Young's equation). In a typical
water/plastic situation for a 2 mm diameter chamber, this is
approximately 140 Pa. Gravity pressure from a 1 cm head (height of
standard 96 well plate) is .about.100 Pa (not enough to overcome
surface tension). By the "Ohm's Law" relation, when
P.sub.in>P.sub.out;
P.sub.f=(P.sub.in-P.sub.out)*(R.sub.out+R.sub.in))+P.sub.out. This
means that if R.sub.in is large in relation to total resistance, a
large amount of pressure can be applied to the inlet well without
creating a large pressure at the chamber. In one example design
according to specific embodiments, resistances are such that when 1
atm is applied to the inlet well, the chamber only experiences
.about.50 Pa pressure, not enough to overcome the surface tension
force.
Passive Array Plate
[0139] One example implementation of embodiments according to the
open top aspect is a cell culture array on a standard 96-well
plate, where each unit consists of 3 well positions: a flow inlet
well, an open top chamber, and a flow outlet well. (It will be
understood that in various embodiments, plates with larger or
smaller numbers of wells or units that have more or less than
3-wells may embody the invention.) FIG. 13A-C is an illustration of
an example 96 well plate having 32 perfusion units with a dye in
the culture medium for illustrative purposes according to specific
embodiments of the invention FIG. 13B shows a top view of a single
perfusion unit in an example system. The left well is the inlet,
the center is the open culture chamber, and the right well is the
outlet. A 2 mm open top 310 of the culture chamber is indicated by
the dashed circle.
[0140] FIG. 13C Illustrates a bottom view of a single example
perfusion unit. The serpentine channels shown at the right control
the gravity perfusion rate to be, in one example, approximately 100
microliters/day.
[0141] FIG. 14 is a top view schematic illustration of an example
design of an open top perfusion chamber according to specific
embodiments of the invention. In this example, a 2 mm hole (white)
is cut into 3 mm microfluidic chamber (orange). A narrow perfusion
barrier (green) surrounds the culture chamber to separate flows
from cells. An outer air channel (blue) oxygenates the medium in
the flow channels (gray). FIG. 15 is a top view schematic
illustration of an example perfusion unit according to the
invention and a cross section side view schematic of an example
design of an open top perfusion chamber showing representative
layers. An example open chamber is about 2 mm in diameter with a 1
mm height. FIG. 15 illustrates glass layer 1501, microfluidics
layer 1502, well layer 1503, cell culture reservoir 1504,
microfluidics channels 1505, Teflon ring 1510, and air channel
1506.
[0142] This chamber design supports culture of cells in 2D systems
using liquid culture medium, as well as 3D cultures as described
herein. In 2D culture, cells adhere to the glass floor after being
dispensed directly into the culture region. Perfusion of medium
passes over the cells for long term growth. In the 3D format, cells
are embedded in a gel (such as BD Matrigel), and dispensed into the
culture well. The gel will be localized to the central chamber by
the perfusion barrier, allowing medium to flow around the gel and
diffuse in to feed the cells. FIG. 16 is a photomicrograph showing
a 2D perfusion culture of MCF-10A cells after 7 days. The left side
shows cells in relation to the open well. Right shows a magnified
view of cells in the culture chamber, demonstrating a confluent
monolayer of cells on the bottom surface. FIG. 17 is a
photomicrograph showing a 3D perfusion culture of MCF-10A cells
after 7 days. Cells were embedded in BD Matrigel. Left shows cells
in relation to the open well. Right shows a magnified view of cells
in the culture chamber, demonstrating a clustered 3D aggregate
morphology suspended in gel.
Controlled Perfusion Plate
[0143] A second implementation of the open top design is in an
active control plate. In this configuration, the open culture
chamber is routed to 6 upstream inlet wells, a gravity perfusion
well, and an outlet well. The plate can be sealed to a pneumatic
manifold, allowing pressure driven control of the 6 inlet
solutions. This allows experiments where solutions are quickly
changed over the cells. Pressure driven flow of up to 10 PSI is
possible due to the large resistance region between the inlet and
culture chamber, leading to a pressure near the chamber less than
1/1000.sup.th the input pressure.
[0144] FIG. 18 is a photograph of an open chamber with cells. (Top)
and a close up of the channel structure, showing the open cell
chamber, perfusion barrier ring, flow channel, and outer air
channel (Bottom) according to specific embodiments of the
invention.
[0145] FIG. 19A-B are photographs illustrating an example of an
active control plate according to specific embodiments. FIG. 19A
shows a plate design with 4 independent flow units (rows of the
plate) with 6 inlet solutions, an open chamber, an outlet, and a
gravity flow channel FIG. 19B shows the four open chambers (green
circles), with inlet streams above and outlet streams below the
open chamber. The design allows flows to pass from the inlet to
outlet channels without overflowing the open chamber. The
availability of multiple liquid or reagent inlets provides systems
that are particularly good for live cell imaging and other
experiments and assays in cell biology. In such a system a research
can study cultures of pancreatic or other organ cells, or cancer
cells, to determine how they respond to different drugs or other
stimulus introduced via the inlets. In specific embodiments of this
design, a gravity well is also provides to facilitate maintaining
(e.g., feeding) the cells before experiments are performed.
[0146] FIG. 20A illustrates the layout of the active control plate
with 4 independent units (rows), 6 upstream inlets (A1-A6, B1-B6,
C1-C6, D1-D6), the open chambers (red circles) in a central imaging
window with four culture chambers, a large outlet well (oval, A7,
B7, C7, D7), and gravity perfusion well (last column, A8, B8, C8,
D8). FIG. 20AB is a schematic of the culture chamber showing the 6
inlet channels, 2 mm diameter open culture chamber (red), outlet
(center right), and gravity feed (top and bottom right).
[0147] FIG. 21A-B are schematics and a photo illustrating an
example of (A) a cross section of the open chamber showing
materials used for construction. The bottom layer is a solid glass
slide. On top is a layer of molded PDMS containing microfluidic
structures as described herein. Also, as described herein, an
acrylic sheet is used in the molding process and the PDMS remains
attached to it. For the open top cell chamber, in specific
embodiments, this acrylic sheet is laser cut, etched, drilled or
otherwise opened to create the open top culture chamber. In
specific embodiments, a ring of Teflon tape or similar hydrophobic
material is placed around the open chamber inside the well to
increase the surface tension, for example by preventing wetting of
the acrylic. Alternatively, the acrylic may be coated or treated or
fabricated to have a higher hydrophobicity. The open chamber is
laser cut in these three layers (prior to attaching the glass
bottom) and (B) a photograph showing the Teflon ring on the top
surface of the device. Clear liquid is filled in the open chamber,
with microchannels filled with red dye. The Teflon ring, for
example, increases the hydrophobicity of the top surface. This
increases the surface tension and prevents liquid from spilling out
of the culture chamber. In an example embodiment, the Teflon is
applied during fabrication of the bottom acrylic portion of the
plate. A strip of Teflon is taped onto the acrylic sheet before
laser cutting. The laser cutter creates the open chamber by cutting
through the PDMS, acrylic, and Teflon at the same time. The laser
cutter also preferably cuts the excess Teflon tape around the
outside of the cell chamber opening to create the circular ring.
The end result is a Teflon ring that is a roughly donut shape with
1 mm width and 200 micron thickness. Cutting off the excess Teflon
or otherwise restricting the hydrophobic treatment of the acrylic
to the area just around the culture chamber can facilitate
subsequent joining of the acrylic bottom to an open bottom well
plate. Since cell culture media is hydrophilic, a surface that is
hydrophilic will prevent the liquid from flowing out of the well.
Telfon is an extremely hydrophobic material with a water contact
angle around 120 degrees. Without the Teflon, there is a
liquid/air/acrylic interface, which is less hydrophobic, and more
likely to overflow during experiment.
[0148] It will be understood that other configurations that allow
for an open top culture area are possible. In one example, the
holes through the acrylic may be drilled so that there is a sunken
edge around the culture chamber hole and that sunken edge can be
treated to be hydrophobic while leaving the surface that attaches
to the well portion untreated.
8. Pneumatic Manifold
[0149] While gravity or passive loading is effective for some
microfluidic cell culture devices, in some embodiments, a
proprietary pneumatic manifold, as described herein, is mated to
the plate and pneumatic pressure is applied to the cell inlet area
for cell loading. For particular cell systems, it has been found
that overall cell culture area design can be made more effective
when it is not necessary to allow for passive cell loading.
[0150] FIG. 22A-C shows a top view, side view, and plan view of a
schematic of an example manifold according to specific embodiments
of the invention. In this example, the eight tubing lines to the
right are for compressed air, and each is configured to provide
pressure to a column of cell inlet wells in a microfluidic array.
The left-most line in the figure is for vacuum and connects to an
outer vacuum ring around the manifold. Each column of wells is
generally connected to a single pressure line with wells above
imaging regions skipped. The manifold is placed on top of a
standard well plate. A rubber gasket lies between the plate and
manifold, with holes matching the manifold (not shown). The vacuum
line creates a vacuum in the cavities between the wells, holding
the plate and manifold together. Pressure is applied to the wells
to drive liquid into the microfluidic channels (not shown). A
typical pressure of 1 psi is used, therefore the vacuum strength is
sufficient to maintain an air-tight seal. In one example there are
9 tubing lines to the pressure controller: 8 lines are for
compressed air and 1 line is for vacuum (leftmost). In specific
example embodiments, each column is connected to a single pressure
line. Columns above the cell imaging regions are skipped.
[0151] Pressurized cell loading in a system according to specific
embodiments of the invention has been found to be particularly
effective in preparing cultures of aggregating cells (e.g., solid
tumor, liver, muscle, etc.). Pressurized cell loading also allows
structures with elongated culture regions to be effectively loaded.
Use of a pressurized manifold for cell loading and passive flow for
perfusion operations allows the invention to utilize a fairly
simple two inlet design, without the need for additional inlet
wells and/or valves as used in other designs.
Modified Manifold
[0152] In a further embodiment, a plate manifold includes an
additional "gas line" that is used to bathe the cells in the
microfluidic device with a specified gas environment (for example,
5% CO.sub.2). Other examples include oxygen and nitrogen control,
but any gaseous mixture can be sent to the cells. The gas flows
through the manifold into the sealed wells above the cell culture
area and holes in the microfluidic device enable the gas to flow
into specified microfluidic air channels, as described above. The
gas permeable device layer (PDMS) allows the gas to diffuse into
the culture medium prior to exposing the cells. By continuously
flowing the gas through the microfluidic plate, a stable gas
environment is maintained.
[0153] This provides an optional means for controlling the gas
environment to placing the microfluidic plate into an incubator. In
this modified manifold, the manifold can be used to create a
"micro-incubator" independent of the ambient air.
[0154] FIG. 23 illustrates an example system and manifold for
operating the microfluidic plates according to specific embodiments
of the invention.
Fluid Flow and Operation: Gravity and Surface Tension flow
[0155] The format of the microfluidic plate design allows two
automation-friendly flow modalities dependent on the extent of
dispensing/aspiration. The first is surface tension mediated flow.
In this case, when the lower reservoir is aspirated in either one
of the wells, the capillary force of the fluid/air interface along
with the wetted surfaces (glass, silicone, acrylic) will rapidly
draw liquid in from the opposing well until the lower reservoir is
filled (or in equilibrium with the opposing lower reservoir). This
effect is useful for microfluidic flows as it is only evident when
the reservoir diameter is small and the flow volumes are small. In
an example array design, the lower reservoir wells are 1-2 mm in
diameter, and with a total flow volume of approximately 3-5
microliters. Since the microfluidic channel volume is only 0.2
microliters, this mechanism is well suited for cell loading and
cell exposures.
[0156] The second mechanism is gravity driven perfusion, which is
well suited for longer term flows, as this is dependent on the
liquid level difference and not the reservoir dimensions. According
to specific embodiments of the invention, this may be accomplished
by adding more liquid into one reservoir (typically filling near
the top of the upper reservoir). The fluidic resistance through the
microfluidic channels will determine how long (e.g., 24 hours) to
reach equilibrium between the wells and thus determine how often
wells should be refilled.
[0157] FIG. 25 shows the flow rate difference between the surface
tension mechanism and the gravity driven mechanism. For the surface
tension flow, in an example, 5 microliters was dispensed into the
lower reservoir followed by aspiration of the opposing lower
reservoir. For the gravity flow, a liquid level difference of 2.5
mm was used, with both wells filled into the upper reservoir
portion.
Changing Gravity Flow Rate via Liquid Level
[0158] The gravity perfusion rate is also responsive to the liquid
level difference between the two upper reservoir wells as
illustrated in FIG. 26. This fact allows an automated
dispenser/aspirator to control and maintain a given perfusion flow
rate over a 10-fold range during culture. Here, different liquid
level differences were produced via dispensing volumes and measured
for volumetric flow rate.
Controlling Gravity Perfusion Rate via Plate Tilt Angle
[0159] According to specific embodiments of the invention, the
liquid height difference between the inlet/outlet wells across the
plate can also be precisely controlled using a mechanical tilting
platform. In this implementation, it is possible to maintain a
constant flow rate over time, as well as back-and-forth flow with
different forward and reverse times (i.e. blood flow).
[0160] In an example system, perfusion cell culture can be
initiated by filling the flow inlet reservoir with 200-300
microliters of fresh medium (e.g., DMEM supplemented with 10% fetal
bovine serum) and aspirating the cell inlet upper reservoir. The
liquid level difference between the flow inlet and cell inlet wells
will then cause a continuous gravity driven flow through the
attached cells. For sustained culture, the flow inlet well is
refilled and the cell inlet well aspirated during a period
depending on fluidic resistance and reservoir volumes (e.g., every
12, 24, 36, 48, 72 hours).
Cell Assay and/or Observation
[0161] Cell assay can be performed directly on the microfluidic
cell culture using standard optically based reagent kits (e.g.
fluorescence, absorbance, luminescence, etc.). For example a cell
viability assay utilizing conversion of a substrate to a
fluorescent molecule by live cells has been demonstrated (CellTiter
Blue reagent by Promega Corporation). The reagent is dispensed into
the flow inlet reservoir and exposed to the cells via gravity
perfusion over a period of time (e.g., 21 hours). For faster
introduction of a reagent or other fluid, the new fluid can be
added to the flow inlet reservoir followed by aspiration of the
cell inlet reservoir.
[0162] Data can be collected directly on the cells/liquid in the
microfluidic plate, such as placing the plate into a standard
fluorescence plate reader (e.g., Biotek Instruments Synergy 2
model). In some reactions, the substrate may diffuse into the
outlet medium, and therefore be easily detected in the cell inlet
reservoir. For cell imaging assays, the plate can be placed on a
scanning microscope or high content system. For example, an
automated Olympus IX71 inverted microscope station can be used to
capture viability of cultured liver cells with a 20.times.
objective lens.
[0163] By repeatedly filling/aspirating the wells, cells can be
maintained for long periods of time with minimal effort (e.g.
compared to standard "bioreactors" which require extensive sterile
preparation of large fluid reservoirs that cannot be easily swapped
out during operation).
9. Automated Systems
[0164] FIG. 27 illustrates a top view schematic of an example cell
culture automation system according to specific embodiments of the
invention. Because the plates are designed to be handled using SBS
compliant instruments, various "off-the-shelf" machines can be used
to create an automated system. This schematic shows an example of
how this is accomplished. A robotic arm (plate handler) moves the
microfluidic plates from station to station. An automated incubator
stores the plates at the proper temperature and gas environment for
long term perfusion via gravity flow. The pipettor dispenses
liquids (media, drugs, assay reagents, etc.) to the inlet wells and
removes liquid from the outlet wells. A plate reader is used for
assay. The cell loader is optionally used to introduce the cells to
the microfluidic arrays at the beginning of the experiment. The
cell loader in particular is generally not "off-the-shelf" and
operates by applying pneumatic pressure to specified wells of the
array plate to induce flow. Standard or custom computer software is
available to integrate operations.
[0165] FIG. 28 is a photograph of an example automated microfluidic
perfusion array system according to specific embodiments of the
invention. The basic process includes: 1) removing the plate from
the incubator, 2) removing liquid from the outlet wells via the
pipettor, 3) moving a media/drug storage plate from the "plate
stacks," 4) transferring liquid from the media/drug plate to the
microfluidic plate via the pipettor, 5) placing the microfluidic
plate into the incubator, 6) repeat for each plate, 7) repeat after
specified time interval (e.g. 24 hours).
[0166] FIG. 29 illustrates operation steps of a less automated or
prototype system according to specific embodiments of the
invention. The 96-well plate standard allows the microfluidic
system to be operated using standard techniques and equipment. For
example, liquid dispensing is achieved with standard pipette
mechanics, and cell culture and analysis is compatible with
existing incubators and plate readers. A custom built cell loading
system can be used to load the cells using air pressure as
described above. The gravity driven flow culture configuration
utilizes the medium level difference between the inlet and outlet
well as well as engineering the fluidic resistances to achieve the
desirable flow rate in nL/min regime. This provides the significant
advantage of being able to "passively" flow culture medium for long
periods of time (for example, up to 4 days) without the use of
bulky external pumps.
Integrated Systems
[0167] Integrated systems for the collection and analysis of
cellular and other data as well as for the compilation, storage and
access of the databases of the invention, typically include a
digital computer with software including an instruction set for
sequence searching and/or analysis, and, optionally, one or more of
high-throughput sample control software, image analysis software,
collected data interpretation software, a robotic control armature
for transferring solutions from a source to a destination (such as
a detection device) operably linked to the digital computer, an
input device (e.g., a computer keyboard) for entering subject data
to the digital computer, or to control analysis operations or high
throughput sample transfer by the robotic control armature.
Optionally, the integrated system further comprises valves,
concentration gradients, fluidic multiplexors and/or other
microfluidic structures for interfacing to a microchamber as
described.
[0168] Readily available computational hardware resources using
standard operating systems can be employed and modified according
to the teachings provided herein, e.g., a PC (Intel x86 or Pentium
chip-compatible DOS.TM., OS2.TM., WINDOWS.TM., WINDOWS NT.TM.,
WINDOWS95.TM., WINDOWS98.TM., LINUX, or even Macintosh, Sun or PCs
will suffice) for use in the integrated systems of the invention.
Current art in software technology is adequate to allow
implementation of the methods taught herein on a computer system.
Thus, in specific embodiments, the present invention can comprise a
set of logic instructions (either software, or hardware encoded
instructions) for performing one or more of the methods as taught
herein. For example, software for providing the data and/or
statistical analysis can be constructed by one of skill using a
standard programming language such as Visual Basic, Fortran, Basic,
Java, or the like. Such software can also be constructed utilizing
a variety of statistical programming languages, toolkits, or
libraries.
[0169] FIG. 30 shows an information appliance (or digital device)
700 that may be understood as a logical apparatus that can read
instructions from media 717 and/or network port 719, which can
optionally be connected to server 720 having fixed media 722.
Apparatus 700 can thereafter use those instructions to direct
server or client logic, as understood in the art, to embody aspects
of the invention. One type of logical apparatus that may embody the
invention is a computer system as illustrated in 700, containing
CPU 707, optional input devices 709 and 711, disk drives 715 and
optional monitor 705. Fixed media 717, or fixed media 722 over port
719, may be used to program such a system and may represent a
disk-type optical or magnetic media, magnetic tape, solid state
dynamic or static memory, etc. In specific embodiments, the
invention may be embodied in whole or in part as software recorded
on this fixed media. Communication port 719 may also be used to
initially receive instructions that are used to program such a
system and may represent any type of communication connection.
[0170] Various programming methods and algorithms, including
genetic algorithms and neural networks, can be used to perform
aspects of the data collection, correlation, and storage functions,
as well as other desirable functions, as described herein. In
addition, digital or analog systems such as digital or analog
computer systems can control a variety of other functions such as
the display and/or control of input and output files. Software for
performing the electrical analysis methods of the invention are
also included in the computer systems of the invention.
Other Embodiments
[0171] Although the present invention has been described in terms
of various specific embodiments, it is not intended that the
invention be limited to these embodiments. Modification within the
spirit of the invention will be apparent to those skilled in the
art.
[0172] It is understood that the examples and embodiments described
herein are for illustrative purposes and that various modifications
or changes in light thereof will be suggested by the teachings
herein to persons skilled in the art and are to be included within
the spirit and purview of this application and scope of the
claims.
[0173] All publications, patents, and patent applications cited
herein or filed with this submission, including any references
filed as part of an Information Disclosure Statement, are
incorporated by reference in their entirety.
* * * * *