Note: Descriptions are shown in the official language in which they were submitted.
CA 02631466 2008-06-02
MICROFLUIDIC CELL CULTURE MEDIA
This application claims the benefit of the following U.S. provisional
applications: 60/741,864 filed December 2, 2005; 60/741,665 filed December 2,
2005.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
This invention was made with Government support under at least one
of: NASA award NNC04AA21A; Army Research Office award DAAD19-03-1-
0168; and, National Science Foundation Award BES0238265 The Government has
certain rights to the invention.
BACKGROUND
1. Field of the Invention
The invention relates to microfluidic cell culture media.
2. Discussion
Microfluidic devices allow a user to work with nano- to microliter
volumes of fluids and are useful for reducing reagent consumption, creating
physiologic cell culture environments that better match the fluid-to-cell-
volume
ratios in vivo, and performing experiments that take advantage of low Reynolds
number phenomenon such as subcellular treatment of cells with multiple laminar
streams. Many microfluidic systems are made of polydimethylsiloxane (PDMS)
because of its favorable mechanical properties, optical transparency, and bio-
compatibility.
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SUMMARY
Embodiments of the invention may take the form of a microfluidic
cell culture media for a cellular mass in a microfluidic cell culture device.
The
media provides the cellular mass a therapeutic environment. The cellular mass
has
an active material. The media includes a fluid having an initial concentration
of the
active material. The active material of the fluid is capable of being used by
the
cellular mass for at least a portion of one cell process. The fluid is capable
of
receiving active material released by the cellular mass. The initial
concentration is
less than a therapeutic window of concentration necessary to provide the
cellular
mass a therapeutic environment.
Embodiments of the invention may take the form of a microfluidic
cell culture media for use, during a cell culture period, with a cellular mass
in a
microfluidic cell culture device. The device is configured to at least one of
absorb
a portion of the media and permit a portion of the media to evaporate. The
media
includes a water-based fluid having an initial concentration of an active
material less
than a therapeutic window of concentration necessary to provide the cellular
mass
a therapeutic environment during the cell culture period. The initial
concentration
has a value such that if a predetermined amount of water from the fluid is at
least
one of absorbed and evaporated during the cell culture period, the
concentration of
the active material adjacent the cellular mass will fall within the
therapeutic window
of concentration.
Embodiments of the invention may take the form of a microfluidic
cell culture media for use, during a cell culture period, with a cellular mass
in a
microfluidic cell culture device. The device is configured to at least one of
absorb
a portion of the media and permit a portion of the media to evaporate. The
media
includes a water-based fluid having an initial osmolality less than a
therapeutic
window of osmolality necessary to provide the cellular mass a therapeutic
environment during the cell culture period. The initial osmolality has a value
such
that if a predetermined amount of water from the fluid is at least one of
absorbed
and evaporated during the cell culture period, the osmolality of the fluid
adjacent the
cellular mass will fall within the therapeutic window of osmolality.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 is an exploded, perspective view of a microfluidic cell
culture device;
FIGURE 2a is a top view of a substrate of the system of Figure 1;
FIGURE 2b is a side view, and in cross-section, of the substrate
taken along section line 2b-2b in Figure 2a;
FIGURE 2c is a bottom view of the substrate of Figure 2a;
FIGURE 3a is a side view, partially broken-away and in cross-
section, of a membrane of the system of Figure 1 with a pair of actuator pins
in
engagement with a lower surface of the membrane;
FIGURE 3b is a side view, partially broken-away and in cross-
section, of an alternative embodiment of a membrane of the system of Figure 1
with
a pair of actuator pins spaced away from the lower surface of the membrane;
FIGURE 4a is a side view, partially broken-away and in cross-
section, of an alternative embodiment of the substrate of Figure 1 and the
membrane
of Figure 3b and illustrating two different heights of a biological fluid in a
pair of
reservoirs formed in the substrate;
FIGURE 4b is an enlarged side view, partially broken-away and in
cross-section, of the substrate and membrane of Figure 4a and illustrating the
relative heights of a cell mass and an end portion of a passageway;
FIGURE 4c is another enlarged side view, partially broken-away and
in cross-section, of the substrate and membrane of Figure 4a and illustrating
the
angle of the surface which defmes a reservoir and the relative widths of a
cell mass
and a lower portion of the reservoir;
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FIGURE 5a is an enlarged side view, partially broken-away and in
cross-section, of an alternative embodiment of a substrate and membrane of
Figure
1 and illustrating a cell mass retained in a lower portion of the reservoir
above the
passageway;
FIGURE 5b is another enlarged side view, partially broken-away and
in cross-section, of an alternative embodiment of a substrate and membrane of
Figure 1 and illustrating a cell mass retained in a lower portion of the
reservoir
below the passageway; and
FIGURE 5c is yet another enlarged side view, partially broken-away
and in cross-section, of an alternative embodiment of a substrate and membrane
of
Figure 1 and illustrating a cell mass with a width snialler than the width of
the lower
portion of the reservoir.
DETAILED DESCRIPTION
Figure 1 is an exploded, perspective view of microfluidic cell culture
system or device 10. Device 10 includes substrate 12 configured to receive a
cellular mass, e.g., an embryo, as explained in detail below, non-rigid
membrane
14, locating block 16, and pin actuating device 18.
Figure 2a is a top view of substrate 12. Substrate 12 includes fimnel
22, reservoir 24, and overlay reservoir 26. Bottom portion 28 of funnel 22 is
in
fluid communication with reservoir 24 via microchannel 30. Microchanne130 has
a volume less than 1 microliter. Reservoir 24 includes reservoir openings 32
which
provide openings to microchannel 30 such that fluids may travel between
funne122
and reservoir 24 as explained in detail below.
Figure 2b is a side view, and in cross-section, of substrate 12 taken
along section line 2b-2b in Figure 2a. A portion of microchannel 30 is formed
in
substrate 12 while another portion of microchannel 30 is formed by membrane 14
as described in detail below. Microchanne130, however, may be completely
formed
in substrate 12 or in any other suitable fashion. Microchannel 30 may have a
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square, circular, bell, or any other suitably shaped cross-section. Substrate
12
further includes hydrophilic surface 34 to promote fluid retention within
overlay
reservoir 26.
Fluid may move between funnel 22 and reservoir 24 via localized
deformation of membrane 14. Fluid may also move between funnel 22 and
reservoir 24 under the influence of gravity as explained in detail below.
Substrate 12 may be optically transparent and made from such
materials as plastic, e.g., PDMS, polymethylmethacrylate, polyurethane, or
glass.
Figure 2c is a bottom view of substrate 12. Substrate 12 includes
female locators 36 which assist in locating substrate 12 relative to membrane
14 as
explained in detail below.
Substrate 12 may comprise a thick, e.g., 8mm, PDMS slab,
fabricated by using soft lithography. The PDMS slab may be prepared by casting
a prepolymer (Sylgard 184, Dow-Corning) at a 1:10 curing agent-to-base ratio
against positive relief features. Relief features may comprise SU-8
(MicroChem,
Newton, MA) and be fabricated on a thin, e.g., 200 m, glass wafer by using
backside diffused-light photolithography. The prepolymer may then cure at 60 C
for 60 minutes, and holes may be punched by a sharpened 14-gauge blunt needle.
Substrate 12 may comprise two layers of cured PDMS at a ratio of
1:10 base to curing agent sealed together irreversibly using plasma oxidation
(SPI
supplies, West Chester, PA). Funnel 22 and reservoir 24 are formed in the top
layer. Microchannel 30 is formed in the bottom layer using soft lithography.
Microchannel 30 faces downward and may be sealed against membrane 14 as
explained in detail below.
Figure 3a is a side view, partially broken-away and in cross-section,
of inembrane 14 and pins 54 of pin actuating device 18 (Figure 1). Membrane 14
includes male locators 38 (Figure 1) configured to be received by female
locators
36 of substrate 12 to locate membrane 14 relative to substrate 12.
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Membrane 14 is optically transparent and includes top layer 40, upper
surface 41, middle layer 42, bottom layer 44, and.bottom surface 45. Top layer
40
and bottom layer 42 comprise PDMS. Middle layer 42 comprises parylene. Top
layer 40 and bottom layer 44, alternatively, may comprise any suitable non-
rigid,
bio-compatible polymer such as a non-rigid plastic, e.g., polyurethane, or a
hyrdrogel, e.g., polyvinylalcohol. Middle layer 42, alternatively, may
comprise any
suitable non-rigid polymer such as polyvinylidene chloride or polyurethane.
Top layer 40 and bottom layer 44 may have a combined thickness of
less than 1mm, e.g., 200 m. Middle layer 42 may range in thickness from
2-20 m, e.g., 2-5 m.
Pins 54 of pin actuating device 18 may selectively extend from the
position shown into membrane 14 to locally deform membrane 14 such that at
least
a portion of top layer 40 extends into microchannel 30 (Figure 2b). The
selective
actuation of pins 54 may move a fluid in microchannel 30 or prevent, or
impede, the
movement of the fluid in microchannel 30 as explained in detail below.
Middle layer 42 minimizes evaporation of a fluid, e.g., a water based
fluid, contained within microchanne130 to prevent, for example, undesirable
shifts
in osmolality of the fluid. Middle layer 42 is also resistant to the flow of
at least
one gas, such as oxygen and carbon dioxide, from microchannel 30 and provides
mechanical durability and stability against cracking caused by the selective
actuation
of pins 54. Fatigue from the actuation of pins 54 does not substantially
increase
middle layer's 42 ability to substantially reduce the rate at which a fluid
from
microch.anne130 moves through membrane 14.
Membrane 14 includes female locators (not shown) wh'ich are used
to locate membrane 14 relative to locating block 16 as explained in detail
below.
Membrane 14 may be prepared by spin-coating PDMS onto a 4"
silanized silicon wafer to a thickness of 100 m, curing this layer at 120 C
for 30
minutes, depositing a 2.5 or 5 m thick parylene layer, plasma oxidizing the
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resulting parylene surface for 90 seconds, spin-coating another 100 m thick
layer
of PDMS, and curing for a total thickness of approximately 200 m.
Figure 3b is a side view, partially broken-away and in cross-section,
of an alternative embodiment of membrane 114 and pins 154 of pin actuating
device
118 (not shown). Membrane 114 includes top layer 140, upper surface 141,
bottom
layer 142, and lower surface 145. Top layer 140 comprises PDMS and bottom
layer
142 comprises polyvinylidene chloride. Top layer 140, alternatively, may
comprise
any suitable non-rigid, bio-compatible polymer such as a non-rigid plastic,
e.g.,
polyurethane, or a hyrdrogel, e.g., polyvinylalcohol, whereas bottom layer 142
may
comprise any suitable non-rigid polymer such as polyurethane.
Top layer 140 and bottom layer 142 may have a combined thickness
of less than 1mm, e.g., 200 m.
Bottom layer 142 minimizes evaporation of a fluid, e.g., a water
based fluid, contained within microchannel 30 to prevent, for example,
undesirable
shifts in osmolality of the fluid. Bottom layer 142 is also resistant to the
flow of at
least one gas, such as oxygen and carbon dioxide, from microchannel 30 and
provides mechanical durability and stability against cracking caused by the
selective
actuation of pins 154. Fatigue from the actuation of pins 154 does not
substantially
increase bottom layer's 142 ability to substantially reduce the rate at which
a fluid
from microchannel 30 moves through membrane 114.
Membrane 114 may be prepared by spin-coating freshly mixed 1:10
PDMS onto silanized glass slides (Corning Glass Works, Corning, NY) to a
uniform
thickness of either approximately 1204m or 400 m, curing overnight at 120 C,
and
then adhering polyvinylidene chloride via conformal contact with the PDMS.
Referring to Figure 1, locating block 16 includes pin holes 48 and
male locators 50. Pin holes 48 are configured to receive pins 54 of pin
actuating
device 18. Male locators 50 are configured to be received by the female
locators of
membrane 14 to locate locating block 16 relative to membrane 14. In
particular, by
locating block 16 relative to membrane 14, pin holes 48 are aligned with
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microchanneI 30. Locating block 16 includes female locators (not shown) which
are
used to locate locating block 16 relative to pin actuating device 18 as
explained in
detail below.
Locating block 16 is rigid and optically transparent and made from
such materials as polystyrene, cyclic olefin copolymer, glass, or metal.
Pin actuating device 18 is a Braille-type actuator as described in detail
below. Pins 54 are actuated with a force of 18g. Pins 54, however, may be
actuated with a force ranging from approximate 3g to 300g. Pins 54 may be
actuated, for example, 10 times per second or once a minute. Pins 54 may be
actuated for a period ranging from minutes to weeks. Any suitable tactile
device,
however, may be used.
Pins 54 of pin actuating device 18, when actuated, extend and press
upon membrane 14, restricting or closing microchannel 30. Pins 54 may be
actuated in any suitable fashion such that a fluid flows between funnel 22 and
reservoir 24 via microchannel 30. Pins 54 may also be actuated such that the
fluid
does not move between funnel 22 and reservoir 24 via microchannel 30.
Pin actuating device 18 includes male locators 56. Male locators 56
are configured to be received by female locators 52 of locating block 16 to
align
locating block 16 relative to pin actuating device 18. By aligning locators
46, 56,
pins 54 are aligned with pin holes 48.
Figure 4a is side view, partially broken-away and in cross-section,
of substrate 112 and membrane 114. Reservoir 124 and funnel 122 are in fluid
communication via microchannel 130. Bio-compatible fluid 158 may be
transported
between reservoir 124 and funnel 122 via localized deformation of membrane 114
by pin actuating device 118. D is the difference in height between bio-
compatible
fluid 158 in reservoir 124 and funnel 122.
Funnel 122 and reservoir 124 are further in fluid communication via
upper channel 126. Microchannel 130 has a resistance to fluid flow greater
than
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upper channel 126. Upper channel 126 is defined by a hydrophobic surface to,
for
example, repel bio-compatible fluid 158.
Immiscible fluid 160, e.g., an oil having a density lower than bio-
compatible fluid 158, may move between funnel 122 and reservoir 124 via
channel
126. Immiscible fluid 160 reduces evaporation of bio-compatible fluid 158 and
reduces the flow of oxygen and carbon dioxide into and out bio-compatible
fluid
158. Gravity will act upon immiscible fluid 160 such that the height of
immiscible
fluid 160 in funnel 122 will equal the height of immiscible fluid 160 in
reservoir 124
thereby maintaining the difference in height, D, of bio=compatible fluid 158.
D' is the desired difference in height between bio-compatible fluid
158 in funnel 122 and bio-compatible fluid 158 in reservoir 124 after pin
actuating
device 118, for example, has been used to move bio-compatible fluid 158 from
reservoir 124 to funnel 122. Such a height may provide a desired amount of
fluid
in funnel 122 conducive to cell culturing. As bio-compatible fluid 158 is
moved
from reservoir 124 to funnel 122, immiscible fluid 160 will flow from funnel
122
to reservoir 124 via channel 126 under the influence of gravity such that in
the
absence of deformation of membrane 114 that would cause, for example, bio-
compatible fluid 158 to further move between funnel 122 and reservoir 124 or
prevent bio-compatible fluid 158 from moving between funnel 122 and reservoir
124, immiscible fluid 160 will substantially maintain the difference in height
D'
under the influence of gravity for a desired period of time, e.g.,
approximately 30
minutes. Microchannel 130 and and channel 126 thus from a continuous fluid
path
between funnel 122 and reservoir 124.
Fluid may move between funnel 122 and reservoir 124 in any number
of ways. For example, a pump may pump immiscible fluid 160 from. one of funnel
122 and reservoir 124 to the other of funnel 122 and reservoir 124 thereby
changing
the height of bio-compatible fluid 158.
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Funnel 122 includes upper portion 164 and lower portion 166.
Surface 168 of funnel 122 tapers inwardly from upper portion 164 to lower
portion
166. Furthermore, upper portion 164 has a width greater than lower portion
166.
The shape of funnel 122 facilities the one-step loading and unloading
of cells into and out of lower portion 166. A pipette holding cells may be
inserted
into funnel 122 at an angle such that a user has a substantially unobstructed
view of
lower portion 166. Likewise, a pipette may be inserted into funnel 122 to
remove
cells from lower portion 166 such that a user has a substantially unobstructed
view
of lower portion 166.
Figure 4b is an enlarged side view, partially broken-away and in
cross-section, of funnel 122 and microchannel 130. Lower portion 166 of funnel
122 is configured to receive cellular mass 170. Cellular mass 170 has a
cellular
height H and microchannel 130 has a channel height h. Cellular mass 170 may
be,
for example, a human zygote, a mammalian zygote, a clump of mammalian cells,
or a single mammalian cell. Microchannel 130 is configured such that cellular
mass
170 will not exit lower portion 166 of fannel 122.
Figure 4c is another enlarged side view, partially broken-away and
in cross-section, of funnel 122 and microchannel 130 looking down the length
of
nzicrochannel 130. Cellular mass 170 has a cellular width W and microchannel
130
has a channel width w. Cellular mass 170 also has a cellular length (not
shown).
Microchannel 130 may be configured such that at least one of the channel
height h
and the channel width w is less than at least one of the cellular height H,
the cellular
width W, and the cellular length L.
Angle A is defined by opposite surfaces 168 of funnel 122. Angle
A may range between 30 and 160 inclusive.
At least one of the channel height h and the channel width w may be
less than 2501im or the width of human hair. In the case where cellular mass
170
is a denuded human zygote, at least one of the channel height h and the
channel
width w may be less than 140 m. In the case where cellular mass 170 is a
denuded
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mammalian zygote, at least one of the channel height h and the channel width w
may
be less than 70 m. In the case where cellular mass 170 is a clump of mammalian
cells, at least one of the channel height h and the channel width w may be
less than
50 m. In the case where cellular mass 170 is a single mammalian cell, at least
one
of the channel height h and the channel width w may be less than 5 m.
Figure 5a is an enlarged side view, partially broken-away and in
cross-section, of fannel 222 and microchannel 230. Lower portion 266 is sized
such
that a portion of cellular mass 270 is confined to lower portion 266.
Lower portion 266 may have a width less than 250 m. In the case
where cellular mass 270 is a denuded human zygote, the width may be less than
140 m. In the case where cellular mass 270 is a denuded mammalian zygote, the
width may be less than 70 m. In the case where cellular mass 270 is a clump of
mammalian cells, the width may be less than 50 m. In the case where cellular
mass
270 is a single mammalian cell, the width may be less than 5 m.
Figure 5b is an enlarged side view, partially broken-away and in
cross-section, of funne1322 and microchanne1330. Lower portion 366 is sized
such
that a portion of cellular mass 370 is confined to lower portion 366.
Additionally,
microchannel 330 is above lower portion 366.
Figure 5c is an enlarged side view, partially broken-away and in
cross-section, of funnel 422 and microchannel 430. Lower portion 466 and
microchannel 430 are sized such that portions of cellular mass 470 may be in
either
of lower portion 466 and the portion of microchannel 430 adjacent lower
portion
466.
Microfluidic devices may include a PDMS slab with bell-shaped
microfluidic channel features, a culture media reservoir, and a funnel shaped
well
for culture. The media reservoir and funnel shaped well are connected with the
microfluidic channels. The fannel shaped well may have an approach angle of
approximately 60' to facilitate the one-step loading and unloading of cells
and an
approximately 500 m diameter tip.
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In funnel type wells, cells do not need to be moved to designated
areas. Instead, cells loaded in the funnel remain stationary. The medium or
chemical composition in the funnel can be gradually changed to mimic
conditions
cells experience in vivo. In addition, the dimensions of the channels
connected to
the funnel can be controlled through soft-lithography processes such that
cells are
confined to the funnel. Cells may then be subjected to diverse flow
conditions.
PDMS slabs may be prepared by casting prepolymer (Sylgard 184,
Dow-Corning) at a 1:10 curing agent-to-base ratio against positive relief
features
approximately 30 m in height and 400 m in width. The relief features may
comprise SU-8 (MicroChem, Newton, MA) and be fabricated on a thin glass wafer,
approximately 200 m thick, using backside diffused-light photolithography.
Microfluidic devices may include a tapered well which at its tip has
an opening which communicates with one or a plurality of microchannels. The
well
and microchannels may be filled with fluid. One or more cells, e.g., embryos,
may
be introduced into the well, for example, by pipet. The cells settle to the
bottom,
but are prevented from exiting the well due to them being larger than the
microchannels.
Fluid may be introduced into the well continuously or
discontinuously. The fluid may contain the necessary growth media for the
cells.
In a well with a single hole at the bottom, for example, fluid may be caused
to rise
in the well from the microchannels, introducing extra nutrients, and then to
fall,
removing fluid which now contains exogenous substances, e.g., waste, via the
microchannels.
Introduction and removal of fluid can be made using. conventional
gravity pumps or constant flow gravity driven pumps. Introduction and removal
of
fluid can also be made by outside supplies, such as pumps, or by on-board or
"semi-
on-board" tactile actuator-based pumping systems.
Wells may have inlets at other locations and or heights rather than at
the bottom, so long as the entrance ways are sized such that cells will not
pass into
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the channels. For example, there may be an opening at the bottom of a well and
an
opening near the middle or top, with fluid being supplied at the bottom and
being
removed closer to the top.
Wells may have a polygonal shape whose walls are inclined, in either
a linear or curved fashion, such that cells added to the well have a tendency
to
gravitate toward the bottom and center of the well.
The material in which a well is formed may be, for example,
thermosetting resin, thermoplastic, metal, glass, or ceramic.
Microfluidic devices may include a top layer containing a well, and
constructed of a relatively rigid material so as to provide support for
elastomeric
layers or layers of lesser strength or modulus below. The top layer may
comprise
a hard transparent material, such as glass or polymethylmethacrylate. The well
may
have a low surface roughness ranging, for example, between 5 m Ra and 0.l m
Ra.
The well may penetrate through the top layer, thus having an open,
wide-mouthed end on one side of the top layer, and on the bottom layer, a
relatively
narrow hole which allows fluid communication with microchannels in the second
layer.
The microchannels may be positioned closely with respect to the
opening in the well to minimize misalignment. For example, misalignment should
not exceed 50 m. The second layer may also constitute the bottom layer,
particularly when the microchannels are substantially on top of the second
layer,
e.g, abutting the bottom surface of the top layer.
Microfluidic devices may include microchannels that are, at least in
part, along the bottom of the second layer. A third, or sealing layer may be
applied
thereto. This sealing layer may be rather thin, such that braille-type tactile
actuators
may act as valves and pumps for the various microchannels. By this means, for
example, fluid can be caused to flow or to be pumped in one or both directions
in
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a given microchannel depending upon the valving, whether the valves are on or
off,
and whether a pump is pumping one way or. the other with respect to the
microchannel.
In use, a well is first filled with fluid, e.g., an embryo culture
medium, and one or more embryos added to the well. An oil overlay, produced by
dropping one or two fine drops of oil onto the liquid surface in the well, is
then
provided.
The oil reduces or prevents evaporation of liquid from the well, thus
stabilizing the osmolality, or concentration, of the ingredients therein. In
the
absence of such oil, the liquid may evaporate. The oil overlay also affects
the flow
of air, including specifically oxygen and CO2 into the fluid, and the release
of these
gases from the fluid. The oil may be any compatible oil, for example, a
silicone oil,
a paraffin oil, or a polyethylene oligomer oil. For the same reason, the
second or
third layers, if present, may include, for example, parylene, or other
materials,
which minimize water loss.
The second and third layers may be made of cast elastomer,
particularly when the embodiments employ tactile actuators. If "off-chip"
fluid
supply or valving is used, however, the use of an elastomer is not necessary,
and
other materials, such as cast epoxy, injection molded thermoplastic, or glass,
can
be used. The surface of these materials should be bio-compatible, and if not,
should
be coated appropriately.
Zygotes may be introduced into a well containing a fluid as is
conventionally employed for embryo culture. The fluid in the well is then
covered
with oil and incubated at a suitable temperature. Fluid is directed into and
out of
the well through microchannels continuously or discontinuously, e.g., a back
and
forth type of fluid supply wherein the fluid level in the well increases and
then
decreases cyclically. The growing embryo may be inspected by conventional
optical
microscopy methods, and when judged grown to the proper stage, the embryo is
removed from the well. If the top of the well is larger then the bottom, one-
step
removal is particularly easy and the risk of damage to the embryo is low.
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Microfluidic devices may contain microchannels whose flow
characteristics are to be actively varied and formed in a compressible or
distortable
elastomeric material such as an organopolysiloxane elastomer. Substrates,
however,
may be constructed of hard, e.g., substantially non-elastic material at
portions where
active control is not desired.
Microfluidic devices may contain at least one active portion which
alters the shape or volume of chambers or passageways ("empty space"). Such
active portions include mixing portions, pumping portions, valving portions,
flow
portions, channel or reservoir selection portions, cell crushing portions, and
unclogging portions. These active portions induce some change in the fluid
flow,
fluid characteristics, channel, or reservoir characteristics by exerting a
pressure on
the relevant portions of the microfluidic device, and thus alter the shape or
volume
of the empty space which constitutes these features. The term "empty space"
refers
to the absence of substrate material. In use, the empty space may be filled
with
fluid.
The active portions may be activatable by pressure to close their
respective channels or to restrict the cross-sectional area of the channels to
accomplish the desired active control. To achieve this purpose, the channels
or
reservoirs may be constructed in such a way that modest pressure from the
exterior
of the microfluidic device causes the channels or reservoirs ("microfluidic
features")
to compress, causing local restriction or total closure of the respective
feature.
Walls surrounding the feature and external surfaces may be
elastomeric such that a minor amount of pressure causes an external surface
and,
optionally, the internal feature walls to distort, either reducing cross-
sectional area
at this point or completely closing the feature.
The pressure required to "activate" the active portion(s) of the device
may be supplied by an external tactile device such as a refreshable Braille
display.
The tactile actuator contacts the active portion of the device, and when
energized,
extends and presses upon the deformable elastomer, restricting or closing the
feature
in the active portion.
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Dimensions of the various flow channels and reservoirs may be
determined by volume and flow rate properties. Channels which are designed for
complete closure may be of a depth such that the elastomeric layer between the
microchannel and the actuator can approach the bottom of the channel.
Manufacturing the substrate of elastomeric material facilitates complete
closure, in
general, as does also a cross-section which is rounded, particularly at the
furthest
corners (further from the actuator). The depth will also depend, for example,
on the
extension possible for the actuator's extendable protrusions, e.g., pins.
Thus,
channel depths may vary, for example, from inm to 500 m.
Microfluidic devices may be prepared through the use of a negative
photoresist, for example, SU-8 50 photoresist (Micro Chem Corp., Newton,
Mass.)
The photoresist may be applied to a glass substrate and exposed from the
uncoated
side through a suitable mask. Since the depth of cure is dependant on factors
such
as length of exposure and intensity of the light source, features ranging from
very
thin up to the depth of the photoresist may be created. The unexposed resist
is
removed, leaving a raised pattern on the glass substrate. The curable
elastomer is
cast onto this master and then removed.
The material properties of SU-8 photoresist and the diffuse light from
an inexpensive light source can be employed to generate microstructures and
channels with cross-sectional profiles that are rounded and smooth at the
edges yet
flat at the top, e.g, bell-shaped. Short exposures tend to produce a radiused
top,
while longer exposures tend to produce a flat top with rounded corners. Longer
exposures also tend to produce wider channels. These profiles are ideal for
use as
compressive, deformation-based valves that require complete collapse of the
channel
structure to stop fluid flow. With such channels, Braille-type actuators
produce full
closure of the microchannels, thus producing a very useful valved
microchannel.
Such shapes also lend themselves to produce uniform flow fields, and have good
optical properties as well.
In a typical procedure, a photoresist layer is exposed from the
backside of the substrate through a mask, for example photoplotted film, by
diffused
light generated with an ultraviolet (UV) transilluminator. Bell-shaped cross-
sections
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CA 02631466 2008-06-02
are generated due to the way in which the spherical wavefront created by
diffused
light penetrates into the negative photoresist. The exposure dose dependent
change
in the SU-8 absorption coefficient limits exposure depth at the edges.
The exact cross-sectional shapes and widths of the fabricated
structures may be determined by a combination of photomask feature size,
exposure
time/intensity, resist thickness, and distance between the photomask and
photoresist.
Although backside exposure makes features which are wider than the size defmed
by the photomask and in some cases smaller in height compared to the thickness
of
the original photoresist coating, the change in dimensions of the transferred
patterns
is readily predicted from mask dimensions and exposure time.
The relationship between the width of the photomask patterns and the
photoresist patterns obtained is essentially linear, e.g., slope of 1, beyond
a certain
photomask aperture size. This linear relationship allows straightforward
compensation of the aperture size on the photomask through simple subtraction
of
a constant value. When exposure time is held constant, there is a threshold
aperture
size below which incomplete exposure will cause the microchannel height to be
lower than the original photoresist thickness. Lower exposure doses will make
channels with smoother and more rounded cross-sectional profiles. Light
exposure
doses that are too slow or photoresist thicknesses that are too large,
however, are
insufficient in penetrating through the photoresist, resulting in cross-
sections that are
thinner than the thickness of the original photoresist.
The suitability of bell-shaped cross-section microchannels of 304m
thickness may be evaluated by exerting an external force onto the channel
using a
piezoelectric vertical actuator of commercially available refreshable Braille
display.
Spaces may be left between the membrane and the wall when the channel
cross-section has discontinuous tangents, such as in rectangular cross-
sections. In
contrast, a channel with a bell-shaped cross-section may be fully closed under
the
same conditions. When a Braille pin is pushed against a bell-shaped or
rectangular-shaped cross-section microchannel through a 2004m PDMS membrane,
the bell-shaped channels may be fully closed while the rectangular channels of
the
same width may have considerable leakage.
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When used as deformation-based microfluidic valves, bell-shaped
microchannels may show self-sealing upon compression compared to conventional
rectangular or semi-circular cross-section channels. By way, of example, a
bell-shaped channel, having a width and height of 30 m, may be completely
closed
by an 18 gf-force squeeze of a Braille pin.
Channels that have the bell-shaped cross-sections with gently sloping
sidewalls may not be fabricated by melting resist technology, one of the most
convenient methods to fabricate photomask-definable rounded patterns, because
the
profile is determined by surface tension.
Bell-shaped channels maximize the cross-sectional area within
microfluidic channels without compromising the ability to completely close
channels
upon deformation. Furthermore, bell-shaped cross-sections provide channels
with
flat ceilings and floors, which is advantageous for reducing aberrations in
optical
microscopy and in obtaining flow fields with a more uniform velocity profile
across
the widths of the channel. These advantages of microchannels with bell-shaped
cross-sectional shapes combined with the convenient, inexpensive, and
commercially
available valve actuation mechanism based on refreshable Braille displays will
be
useful for a wide range of microfluidic applications such as microfluidic cell
culture
and analysis systems, biosensors, and on-chip optical devices such as
microlenses.
The extension outwards of tactile actuators should be sufficient for
their desired purpose. Complete closure of a 40 m deep microchannel, for
example, will generally require a 40 m extension, e.g., pin, or more when a
single
actuator is used, and about 201im or more when dual actuators on opposite
sides of
the channel are used.
For peristaltic pumping, mixing, and flow regulation, lesser
extensions relative to channel height are useful. The areal size of the
tactile
activators may vary appropriately with channel width and function, and may
range
from 40 m to about 2mm. Larger and smaller sizes are possible as well.
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A cellular mass, e.g., cells, may be cultured in microchannels and/or
reservoirs within microfluidic devices. Additionally, cells may release
various
products, or active materials, e.g., immuno-modulators, metabolic substrates,
nutrients, and growth factors, during cell culture. Unlike traditional cell
culture
devices, e.g., petri dishes, active materials released by a cell in a
microfluidic cell
culture device may noticeably affect the concentration of the media adjacent
the cell.
This is due, at least in part, to the relatively small volumes, e.g., 500
nanoliters, of
media associated with microfluidic cell culture devices and the relatively
lower
diffusive transport of these active materials away from the media adjacent the
cell,
e.g., away from media within 450 microns of a surface of the cell. The above
circumstances are also true in the presence of convection if convective
transport of
the active materials away from the fluid adjacent the cell is less than
diffusive
transport of the active materials away from the fluid adjacent the cell.
Cells typically prefer a range of concentrations for the active
materials discussed above. In other words, there is a therapeutic window of
concentration for a given active material and a given cell. Within the window,
cell
processes, e.g., growth, are optimized and the media thus provides the cell a
therapeutic environment. In some circumstances, the therapeutic window may be
a single value. In other circumstances, the therapeutic window may be a range
of
values.
Media prepared for use in non-microfluidic cell culture devices
typically have initial concentrations of the materials discussed above falling
within
the respective material's therapeutic window. For example, if an optimum
concentration of lactate in a media in a vicinity of a human embryo is 21.4
millimolars, a media prepared for use in a non-microfluidic cell culture
device
would have an initial concentration of lactate of at least 21.4 millimolars.
That same
media, however, may yield a sub-optimal concentration of lactate in a
microfluidic
cell culture device because the human embryo's net release of lactate would
affect
the concentration of the lactate in the media adjacent the embryo.
Microfluidic cell culture media for use with a cellular mass in a
microfluidic cell culture device may be prepared taking into account that the
cellular
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mass will affect the concentration of the products discussed above in the
media
adjacent the cellular mass. If an optimum concentration of lactate in a media
in a
vicinity of a human embryo is 21.4 millimolars, a media prepared for use in a
microfluidic cell culture device may have an initial concentration of lactate
less than
21.4 millimolars. This lower concentration may be found experimentally. For
example, several cell culture media for use with a fertilized zygote from
fertilization
until the 8 cell stage may be prepared having initial lactate concentrations
at 10, 15,
20, and 21.4 millimolars. Fertilized zygotes would be cultured at each
concentration in a microfluidic cell culture device for 72 hours. At the
expiration
of the 72 hours, zygote development would be evaluated. If it is found that
the
media with an initial concentration of 20.0, millimolars of lactate was
optimal for
zygote development, a media for use with a microfluidic cell culture device
would
be prepared with an initial concentration of lactate of 20.0 millimolars.
A difference between the initial concentrations of lactate in media for
use in a microfluidic cell culture device compared to a non-microfluidic cell
culture
device may indicate that the concentration of the product in the vicinity of a
cell in
the microfluidic device falls within the therapeutic window, e.g., that a
zygote
releases a net amount of lactate which causes the concentration of the lactate
in the
vicinity of the zygote to raise from an initia120.0 millimolars to an optimum
21.4
millimolars.
Microfluidic cell culture media for use with microfluidic cell culture
devices may be prepared for any cell. There are many ingredients in a media
that
are added to provide therapeutic benefit to cells which are also able to be
provided
by cells themselves. In such cases, media formulations where that ingredient
is
provided, in the base media, at concentrations lower than would be required
for
therapeutic benefits but still enable reaching the therapeutic level in the
=vicinity of
cells can be provided. There are also additives that can be added to promote
this
self-production.
A variety of other cell-released molecules can be the subject of
variations in concentrations, for example, immuno-modulators which may include
soluble histocompatibility complexes, insoluble histocompatibility complexes,
and
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CA 02631466 2008-06-02
interferons. Other examples include metabolic products which may include
carbon
dioxide, pyruvate, and carbohydrates, and nutrients which may include
metabolites.
Additives, e.g., lipids, metabolic substrates, and growth factors, may
be added to microfluidic cell culture media to further promote the endogenous
production of some of the products discussed above by a cell. Examples of
additives
include metabolic substrates, e.g., glucose, pyruvate. Examples of lipid
additives
include Vitamin D3, dexamethasone, and glucocorticoid. Examples of metabolic
substrate additives include pyruvate and carbohydrates.
More detailed examples of some of the metabolic molecules are
described below.
Pyruvate is included as a substrate within the range of 0-5 mM for
various culture media. Pyruvate is also a useful additive to enhance
endogeneous
production of carbon dioxide and its media equivalents.
Glucose is another example of a metabolic substrate. The amount of
glucose in cell culture formulations range from 5.5 mM - 55 mM. Many classical
media are supplemented with 5.5 mM D-glucose (normal blood sugar levels in
vivo). Several important media used as the base media for the design of
proprietary
media used in bio-manufacturing and tissue engineering contain diabetic levels
(10mM or over) of glucose supplementation. These levels of glucose require
special
formulation strategies to protect the cells and cell products from glucose
mediated
oxidative and carbonyl stress.
More detailed examples of some of the lipids are described below.
Linoleic acid is often included in the 200 nM - 300 nM range in
various media. Linoleic acid is a precursor to a number of other fatty acids
(prostaglandins, prostacyclins, thromboxanes, phospho-lipids, glycolipids, and
vitamins). These are important constituents of cell structures such as
membranes,
and long-term energy storage. Lipoic acid is included atlO nM - 1.0 uM in some
basal media and many serum-free media. Lipoic acid is often required for the
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CA 02631466 2008-06-02
metabolism of pyruvate. Lipoic acid has been shown to overcome oxidative
stress-
induced insulin-resistance in vitro. Under conditions of oxidative stress,
lipoic acid
may replace insulin as an agent that supports increased glucose uptake. Lipoic
acid
regenerates endogenous antioxidants, removes transition metals from redox
reactions
by chelation, and reacts non-enzymatically (scavenging) with reactive oxygen
species. Lipoic acid is able to increase glutathione (antioxidant) in cells.
Lipoic
acid helps protect cells from glutamate induced apoptosis by reducing
extracellular
L-cystine to L-cysteine. Lipoic acid can scavenge hydroxyl radicals;
hypochlorous
acid; peroxynitrous acid, and singlet oxygen.
More detailed examples of some of the hormones are described
below.
Hydrocortisone(dexamethasone) is contained at 1-20nM or 140-
500nM to increase plating efficiency, improve clonal growth, and stimulate the
production of fibronectin. Insulin is contained at 0.1-IOng/ml or 1-l0ug/ml in
some
media.
Some microfluidic cell culture devices may absorb a portion of the
media and/or permit a portion of the media to evaporate during a cell culture
period.
This absorption and/or evaporation may cause shifts in the concentrations of
certain
products within the prepared media.
In general, the osmolality of the extracellular environment is normally
- 300 mmol/kg. Tolerance to higher osmolalities is cell type dependent. While
Chinese Hamster Ovary (CHO) cells and a variety of hardy cell lines tolerate
and
proliferate under a wide range of osmolality (300-500 mmol/kg), more sensitive
cells such as mammalian gametes and embryos will undergo a development that is
blocked at osmolalities significantly lower or higher than 265 to 285
(mmol/kg).
If cells are placed in a solution of nonpenerating solutes having an
osmolarity of 300 mmol/kg, they will neither swell nor shrink since the water
concentrations in the intra- and extracellular fluid are the same, and the
solutes
cannot leave or enter. Such solutions are said to be isotonic, defined as
having the
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same concentration of nonpenerating solutes as normal extracellular fluid.
Solutions
containing less than 300 mmol/kg of nonpenetrating solutes (hypotonic
solutions)
cause cells to swell because water diffuses into the cell from its higher
concentration
in the extracellular fluid. Solutions containing greater than 300 mmol/kg of
nonpenetrating solutes (hypertonic solutions) cause cells to shrink as water
diffuses
out of the cell into the fluid with the lower water concentration. Therefore,
it is
important to maintain osmolarity levels during cell culture in vitro.
Microfluidic cell culture media for use with a microfludic cell device
may be prepared taking into account that a certain amount of absorption and/or
evaporation may occur. For example, if a solute is to have a concentration
within
a range to yield a therapeutic environment for the cell, the initial
concentration of
the solute in the prepared media would be lower than the range. The value of
the
concentration would be selected taking into account that a certain amount of
the
media will be absorbed and/or evaporated during the cell culturing event.
Evaporation may be detrimental to cell culture in microfluidic chips
because the slight amount of evaporation from the small liquid volumes present
in
microfluidic systems may result in a significant increase in osmolality.
Furthermore, microfluidic chips are especially prone to evaporation due to
their
fluidic compartments having very high surface areas for water to permeate or
evaporate from relative to their volumes. It is well-documented that elevated
osmolality can affect ion balance, cellular growth rate, metabolism, antibody
production rate, signaling, and gene expression.
The solute may be a charged or neutral solute. Charged solutes may
include at least one of sodium chloride, sodium bicarbonate, potassium
chloride, and
organic buffers. Charged solutes may include glutamine, glycine; and taurine.
Charged solutes may include a protein. Neutral solutes may include
carbohyrdrates
such as glucose, trehalose, and galactose. Neutural solutes may include
macromolecules such as polyethylene glycol, polyvinyl alcohol, dextran,
polyvinylpyrrolidone, and polylysine.
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Nonessential amino acids benefit cells through osmoregulation and
cellular signaling. Most mammalian cells require mechanisms for regulatory
volume increase or decrease in response to hormonal (e.g., insulin and
glucagons)
and other stimuli known to alter cellular volume. Additionally,
preimplantation
mouse embryos appear to develop in a somewhat hypertonic environment in
oviductal fluid. In vitro, glycine, alanine, glutamine, and taurine protect
preimplantation embryos from the otherwise detrimental effects of hypertonic
media. For example, increased external osmolality causes an increase in
intracellular glycine accumulation by early mouse embryos cultured from
zygotes
to two-cell embryos. Glycine functions as an intracellular osmolyte in embryos
and
the increase in total accumulated glycine at higher osmolality largely
reflects
increased intracellular concentration, since there is no significant
difference between
the volumes of two-cell embryos arising from culture in 240 vs. 310 mmol/kg.
Intracellular accumulation of glycine seems to account for the osmoprotective
effect
of adding glycine to culture media.
The following examples describe media for use with microfluidic
devices.
Example 1
In microfluidic channels, due to small dimensions and confined
geometry, secreted molecules from a cell which are necessary for normal
function
will stay in the vicinity of the cell and increase the local concentration of
that
molecule. Because of this local increase in concentration, the concentration
of the
molecule in the original basal media can be lower than would be required in a
macroscopic culture system where cell secreted molecules would rapidly diffuse
away or be carried away by convection. Here, the control volume, or media
adjacent the cells, is a volume within which a majority of the secreted
molecules will
be confined in during a given time period such as 600 seconds. The time period
being defined by characteristics of the fluid refresh rate. Considering a
microchannel with a cross section of 100 m in width x 100 m in height and
assuming one dimensional diffusion along the channel length, the diffusion
length,
L for lactate over the 600 second period is 1100 m and the control volume will
be
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CA 02631466 2008-06-02
1001.tm x 1001im x 2200 m = 3*107" m3 = 2.2*10'$ L. Here, lactate diffusion
coefficient at 37 C is 1.1x10'9 mZ/s and diffusion length, L is defined as
approximating diffusion to be mainly one-dimensional along the length of the
channel. In contrast, in conventional static culture dishes, diffusion is
three-
dimensional with no confinement making the control volume approximately 10
times
larger than in the microchannel described above.
Now_ a single bovine embryo in the blastocyst stage has a lactate
production rate of 38.13 pmol/blasto/hr. Thus, 10 blastocysts will produce,
within
600 seconds, approximately 6.36x10'11 mol of lactate. In conventional GI/G2
media
used for culture of bovine embryos, lactate concentration is 5.87 mmol/L in G2
media (G1 has 10mmo1/L). With the given control volume, the amount of lactate
in conventional G2 media is 5.87x10'3 x 2.2*10$ = 1.29x10-10mo1. Therefore,
there
would be an approximately 50% increase in the average lactate concentration in
the
control volume due to secreted lactate from embryos [(6.36x10'")/( 1.29x10'1)
x100= 49.3 %] if the conventional G2 media were used. In contrast, in culture
dishes with a larger control volume, the local increase in average
concentration
would be only 5 %. To account for this localization of embryo produced lactate
within the particular microfluidic device described here, in the microfluidic
optimized media, a reduced concentration of lactate (e.g. 2.5-3.5 nunol/L) is
contained.
Example 2
This is a basal cell culture medium that enables long-term celI culture
inside a microfluidic environment and outside of a CO2 incubator with long-
term
optical imaging. The medium has the following advantageous characteristics 1)
high
stability to light exposure, 2) limited but sufficient pH stability
patticularly in
microfluidic cell cultures, 3) an amount of NaHCO3 that is below the
conventional
therapeutic window but is sufficient to satisfy immediate cellular needs for
carbonates, and 4) additional additives that enhance endogenous production of
carbon dioxide and its media equivalents.
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More specifically, compared to conventional media, the buffers of
this medium were reduced (10 - 20 mM NaHCO3 and 5 - 15 mM HEPES). To
acconunodate this reduction, fluctuations in pH were suppressed by using high
concentrations of sodium pyruvate (2 - 5 mM) to increase endogenous production
of C02; by addition of free base amino acids (1.5 - 3mM L-arginine; 0.7 -
1.4mM L-histidine; 0.5 - 1mM L-cysteine) which also enhance buffering
capacity;
and by using a combination of glucose (10 - 20 niM) with galactose (25 - 50
mM), rather than galactose alone, to help suppress pH drops due to rapid
glycolysis.
Because of the small-volume microfluidic cell culture, endogenously produced
carbon dioxide and equivalents and other cell-generated products were retained
in
the cell vicinity to maintain concentrations of active products within a
therapeautic
window. In addition, the moderate riboflavin (0.2 - 0.5 :M), and phenol red
(10
- 20 :M) concentrations used in the medium improves stability against light
exposure.
In water, C02 (carbon dioxide) spontaneously interconverts between
C02 (carbon dioxide) and H2C03 (carbonic acid). The relative concentrations of
C02, H2C03, and the deprotonated forms HCO3- (bicarbonate) and C03--
(carbonate) depend on pH.
The carbonate ion is a polyatomic anion with the empirical formula
C03-- and is the conjugate base of bicarbonate, HCO3-1, which is the conjugate
base of H2C03 (carbonic acid).
NaHCO3 (sodium bicarbonate) is a salt consisting of the ions Na+
and the bicarbonate anion, HCO3-. The bicarbonate anion forms some hydroxide,
which results in its solutions being mildly alkaline:
HCO3- -> C02 + OH-
In this example, carbon dioxide and its media equivalents are the
active material. Bicarbonates, carbonic acid, and carbonates are media
equivalents
of carbon dioxide.
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Example 3
In conventional cultures, the local concentration of the active material
decreases because of cellular consumption and because replenishing of the
active
material in the cell vicinity occurs mainly by diffusion only. To accommodate
for
this depletion and slow mass transport, the bulk media concentration of that
material
is set at a high concentration so that the decreased local concentration is
still within
the therapeautic window. In active microfluidic systems where there is
efficient
convective transport, the bulk concentration of the active material can be
lowered
because the material may be constantly refreshed around the cell.
Exarnple 4
In many microfluidic manipulations, just the process of transferring
small volumes of media from a large container to an on chip reservoir causes
shifts
in osmolality. For example, transferring 30 microliters of KSOM media from a
bottle to a small shallow microreservoir over several minutes in a biological
safety
cabinet caused the osmolality to immediately shift upwards 15 mmol/kg. Thus in
microfluidic media, the osmolality is decreased from 265 to 250 mmol/kg.
Example 5
To compensate for evaporation from the culture media, the osmolality
of MCDB 131 microfluidic cell culture medium was adjusted to be lower than
EGM2-MV, a proprietary media from Cambrex for human microvascular
endothelial cells (HMVECs) with an osmolality range of 270-290 mmol/kg.
Osmolality levels of MCDB 131 adjusted to be as low as 200 mmol/kg were
experimentally tested to support proliferation and thus provide HMVECs with a
therapeutic environment. Over a five day period, the rate of HMVEC
proliferation
supported by MCDB 131 with an osmolality of 200 mmol/kg was comparable to
what is supported with EGM2-MV.
Evaporation during culture enables media initially with low
osmolality to slightly increase their osmolality yet remain within a
therapeutic range
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CA 02631466 2008-06-02
to support long-term proliferation. On the other hand, evaporation may result
in the
osmolality of culture media at normal levels to increase and leave its
therapeutic
osmolality range to support proliferation.
Example 6
Elevated osmolality can affect ion balance, cellular growth rate,
metabolism, antibody production rate, signaling, and gene expression. In
general,
the osmolality of the extracellular environment is normally - 300 mmol/kg.
Tolerance to higher osmolalities is cell type dependent. More sensitive cells
such
as mammalian gametes and embryos will undergo a development that is blocked at
osmolalities significantly lower or higher than 265 to 285 (mmol/kg).
Compared to conventional cell cultures performed in Petri dishes with
low cell volume to extracellular fluid volume (CV/EV) ratios, microfluidic
environments with large CV/EV ratios have many advantages in terms of cellular
self-conditioning of their surrounding medium. Systems with large CV/EV
ratios,
however, typically also possess large surface to volume (SAV) ratios which
increases the rate of evaporation and presents a challenge, particularly when
using
microfluidic devices made of water vapor permeable materials such as PDMS.
First, by evaporation occurring during the few minutes of handling
between transfer of droplets from a macroscopic bottle of media to a well or
microfluidic device and covering it with oil, osmolility will shift from 265
to 280
(mmol/kg) though media with 265 mmol/kg osmolality is prepared for culturing.
So these losses of water should be compensated by lowering media osmolality
by15
mmol/kg for microfluidic cell culture.
Next, water permeation into microfluidic device over culture period
will cause additional osmolality shifts in media, which will affect cellular
functions
or viability and necessitate a periodical refreshing time, t.
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Using the simple diffusion equation, Am = D= S= P=(1- ) the
t4=t s Ps dJ[
amount of water loss, Om in media or osmolality shifts over culture period
under
given humidity condition can be calculated.
Considering a PDMS microfluidic device which is composed of a
bottom thin membrane for fluidic actuations and a thick (- 8mm) PDMS slab with
microfluidic channel feature with a cross section of 300 m in width x 30 m in
height and 50,000 m in length forming a closed loop for recirculation, channel
area, A is = 1.5x10-5 m2 and channel volume is 4.5x10-10m3. Here this
microchannel is used for studying the effect of autocrine and paracrine
effects on
cells under fluid perfusion conditions over 24 hours and media need to be
refreshed
every 24 hours for each run. This refreshing time can be varied based on
experiment's purpose.
Assuming that most evaporation over 24hours occurs only through
the thicker top PDMS (8mm thickness) because bottom thin PDMS membrane is
coated with parylene and that culture is conducted under a non-humidified
environment (25 %), the amount of water escaping through the top PDMS over 24
hours is 7.85x10-$kg based on the simple diffusion equation. This amount of
water
loss causes osmolality shift from 265 mmol/kg to 320mmol/kg in given channel
volume, 4.5x10'10m3(or 4.5x10'7 kg in water).
If the culture is conducted under a humidified incubator (85 %), the
amount of water escaping is 1.57x10'akg and the resulted osmolality shift is
from
265 mmol/kg to 275 mmol/kg.
The increased amount by 10 - 55mmol/kg should be compensated
by further lowering osmolality in media. Namely, to compensate total losses of
water including loss during handling, lower osmolality media such as 195 -
240mmollkg, which depends on humidity condition, is prepared for microfluidic
cell
culture.
Here approximate values of variables for the simple diffusion
equation are: diffusion coefficient, D= 3x10'9 mZ/s, water solubility
coefficient in
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PDMS,S = 1.04 [(cm),,,9a/cmHg=(cm3)pDMs], saturated vapor pressure, P$ at 37 C
= 6.33x103, P/PS = 0.25(25 % humidity) or 0.85{85 %humidity), dx = 8000x10-6
(m). Here, the humidity at the media surface is considered to be 100% humidity
at
37'C, and the saturated vapor specific volume (VB) at 37 C is V8Z22.94(m3/kg),
pg=0.0436(kg/m3).
Example 7
Many media include scavengers to scavenge harmful products.
Utilization of a microfluidic system incorporating dynamic flow of media can
function as a scavenger of the waste produced by cells by washing the harmful
products away. Thus the concentration of chemical or material scavenger can be
reduced in a microfluidic media. Examples of harmful products include toxic
metals and radicals. Examples of scavengers include ethylenediaminetetraacetic
acid
(EDTA), oil, thiols and ascorbate.
Example 8
Culture media optimized for traditional cell culture in macroscopic
dishes in flasks when applied to microfluidic cell culture do not account for
the high
surface area-to-volume ratios present in microfluidic systems. The surface
area-to-volume ratio is two to three orders of magnitude larger in
microfluidic
systems compared to macroscopic culture dishes and flasks. As a result, there
is
much more surface present in microfluidic systems compared to macroscopic
culture
flasks and dishes for proteins such as immunoglobulins, growth factors,
cytokines,
and other factors necessary to provide a therapeutic cellular environment to
adsorb
onto the surface and deplete the concentrations present in solution.
Consequently,
the high degree of non-specific adsorption of proteins and other 'factors in
microfluidic cell culture systems may cause culture media that had been
optimized
to provide a therapeutic environment in macroscopic dishes and flasks to no
longer
provide a therapeutic environment when applied to microfluidic cell culture
because
the concentrations of the factors have dropped below a threshold level
necessary to
provide a therapeutic environment.
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Optimization of media for microfluidic cell culture requires the
amounts of factors necessary to remain above.a threshold level to provide a
therapeutic environment to be adjusted relative to the levels present in
traditional
cell culture media. For instance, in macroscopic endothelial cell culture, 50
ng/ml
of vascular endothelial factor (VEGF) in culture media is added to provide the
environment to stimulate endothelial celis to form vascular networks which is
known
to be a threshold response. Thus, in microfluidic culture conditions, the
amount of
VEGF added will need to be adjusted to account for increased protein
adsorption
and to ensure that endothelial cells form vascular networks.
Examples of instances when adsorption-mediated depletion of factors
supporting a therapeutic environment is particularly high are: 1) long lengths
(on the
order of mm or cm) between the fluid inlet and the location of cells because
this
length of channel provides much surface for protein adsorption and 2) slow
pumping
conditions where diffusion effects dominate providing more time for proteins
to
adsorb.
Adsorption occurs more rapidly during initial exposure of device
surfaces to media (starts absorbing in less than 1 seconds and will continue
for up
to hours depending on product adsorbing and concentrations.) For some products
such as proteins, depletion by retention onto channel walls will greatly
reduce after
a monolayer of the product is formed on the walls.
Other products (other than proteins) that can be retained by device
include lipids (steroids and some hormones and vitamins). Examples of proteins
include albumin, globulins, growth factors (specifically VEGF and leukemia
inhibitory factor (LIF) and fibroblast growth factor).
Appendix A discloses a handheld recirculation system and customized
media for microfluidic cell culture. Appendix B discloses a device for embryo
culture and use thereof. Appendix C discloses integrated microfluidic control
employing programable tactile actuators. Appendix D discloses a computerized
control method and system for microfluidics and computer program product for
use
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therein. Embodiments of the invention may be used in the devices described in
Appendices A, B, C, and D.
APPENDIX A
Many modifications will be apparent to those skilled in the art, and
are part of the subject matter disclosed herein. The clamping mechanism, for
example, may be replaced or augmented by other clamping mechanisms, including
simple clamps which are separate from but engageable with the fingerplate, or
which
can span the height of the entire device, including the braille display
module.
In similar manner, while the transparent heating element is described
as being fabricated on a glass slide, it will be appreciated that this glass
slide may
be incorporated into a disposable device, become an integrated part thereof
rather
than a separate device. While less favorable, the heating element may also be
disposed directly on the microfluidics chip. The heating unit may also be
patterned
such that only portions of the glass slide or chip are heated, thus conserving
electrical power as well as avoiding heat in areas where heating is not
desired, for
example in fluid storage areas.
In advanced versions of the present lab-on-chip, it is desirable to have
a battery power supply, either one-time use or rechargeable, on the chip
itself,
together with electrical circuitry for controlled operation of the heater
unit, and of
the tactile actuators also, when this is desired. The ability to divorce the
structure
from corded power supplies allows the module to be easily transported to other
stations for testing, analysis, etc., while preserving the microenvironrnent
within the
module.
The subject invention further pertains to PMDS or other elastomeric
silicone structures which incorporate a film, coating, or membrane over all or
only
a portion of the module structure, which serves as a vapor barrier to minimize
evaporation of liquids contained in the channels, reservoirs, etc., of the
devices.
Suitable vapor barriers are, in general, relatively pore free, hydrophobic
films, e.g.
of parylene. In addition, films which are resistant to the flow of oxygen, of
carbon
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dioxide, or both these gases may also be applied to minimize any influence of
the
ambient atmosphere on the conditions established within the_ device. Such
films are
well known from the field of plastic, particularly polyethylene terephthalate,
drink
containers.
APPENDIX B
It has now been surprisingly discovered that embryos may be grown
with good survival rates in an efficient manner by growth at the bottom of a
well
which is in communication with a microchannel device supplying fluid to the
well
proximate its bottom. The bottom opening is sized so as not to allow the
embryo
to enter the channel.
The invention may be described with relation to the accompanying
drawings, many of which illustrate the volumes or hollows, channels, etc.
within the
microfluidics device rather than the walls of the device themselves. As
illustrated,
the best mode of the device is a generally conical well which at its tip has
an
opening which communicates with one or a plurality of microchannels. The well
is filled with fluid, as are the microchannels, and one or more embryos are
introduced into the well, for example by pipet. The embryos settle to the
bottom,
but are prevented from exiting the well due to them being larger than the
holes in
the well.
Fluid may be introduced into the well continuously or
discontinuously, the fluid preferably containing the necessary growth media
for the
embryo. For example, in a well with a single hole at the bottom, fluid may be
caused to rise in the well from the microchannels, introducing extra
nutrients, and
then to fall, removing fluid which now contains exogenous substances (waste)
via
the microchannels.
Introduction and removal of fluid can be made using conventional
gravity pumps, or constant flow gravity driven pumps. Fluid can also be
supplied
by outside supplies such as pumps, etc., or preferably by on-board or "semi-on
board" tactile actuator-based pumping systems.
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The well can also have inlets at other locations and or heights rather
than exclusively at the bottom, so long as the entrance ways to the channels
are sized
such that the embryos will not pass into the channels. For example, there
might be
an opening at the bottom of the well and an opening near the middle or top,
with
fluid being supplied at the bottom, for example, and being removed closer to
the
top.
The well also need not be entirely conical in shape, but is preferably
shaped such that the walls are inclined, regardless of whether linear or
curved such
that the embryo's will have a natural tendency to gravitate toward the bottom
and
center of the well. The material of the well is not overly critical, and may
be
thermosetting resin or thermoplastic, metal, glass, ceramic, etc. In preferred
constructions, the device is a multilayer device, the top layer containing the
well,
and constructed of relatively rigid material so as to provide support for
elastomeric
layers or layers of lesser strength and/or modulus below.
Thus, it is preferable that the top layer be of hard transparent material
such as glass, polymethylmethacrylate, etc. The conical well should have a low
surface roughness, preferably below 5 m Ra, more preferably less than l m Ra,
and yet more preferably less than 0.1 m Ra.
In preferred devices, the conical well penetrates entirely through the
top layer, thus having an open, wide-mouthed end on one side of the top layer,
and
on the bottom this layer, a relatively narrow hole which allows fluid
conununication
with the microchannels in the second layer. The second layer preferably
directly
abuts the first layer, and has one or a plurality of microchannels which are
in fluid
communication with the conical well. It is relatively important that the
channels be
positioned closely with respect to the opening in the well. For example
misalignment should preferably be maximized at 50 m. The second layer may also
constitute the bottom layer, particularly when the microfluid channels are
substantialIy on top of the second layer, i.e. abutting the bottom surface of
the top
layer. However, in preferred devices, the channels are at least in part along
the
bottom of the second layer and a third, or sealing layer is applied thereto.
This
sealing layer is preferably rather thin, such that braille-type tactile
actuators may act
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as valves and pumps for the various microchannels. By this means, for example,
fluid can be caused to flow or to be pumped in only one direction in a given
microchannel, or can be bidirectional flow, depending upon the valving,
whether the
valves are on or off, and whether a pump is pumping one way or the other with
respect to the channel.
In use, the device is first filled with fluid, for example an embryo
culture medium, and one or more embryos added to the well, An oil overlay,
produced by dropping one or two fine drops of oil onto the liquid surface in
the well
is then provided. The oil prevents evaporation of liquid from the well, thus
changing the osmolality, or concentration, of the ingredients therein. It also
affects
the flow of air, including specifically oxygen and CO2 into the fluid, and the
release
of these gases from the fluid. The oil may be any compatible oil, for example
a
silicone oil, a paraffin oil, a polyethylene oligomer oil, etc. For the same
reason,
portions of the apparatus in the second and/or third layers may be coated, for
example with parylene or other coating which minimizes, particularly, water
loss.
The second and third layers are preferably made of cast elastomer,
particularly when the valving and pumping embodiments employing tactile
actuators
are employed. However, if "off-chip" fluid supply, valving, etc. is used, then
use
of an elastomer is not necessary, and other materials such as cast epoxy,
injection
molded thermoplastic, glass, etc., can be used. It is of course recognized
that the
surface of these materials should be compatible with embryo culture, and if
not,
should be coated appropriately.
The process of the subject invention requires introduction of zygote(s)
into the well which contains fluid, preferably a growth fluid as is
conventionally
employed for embryo culture. The fluid in the conical well is then covered
with oil,
preferably mineral oil, and the device incubated at a suitable temperature.
Fluid is
directed into and out of the well through the microchannels continuously or
discontinuously. For example, a back and forth type of fluid supply wherein
the
fluid level in the well increases and then decreases cyclically has been found
most
advantageous. The growing embryo may be inspected by conventional optical
microscopy methods, and when judged grown to the proper stage, the embryo is
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CA 02631466 2008-06-02
removed from the well. Because the top of the well is larger then the bottom,
removal is particularly easy and the risk of damage is low.
APPENDIX C
The microfluidic devices of the present invention contain
microchannels whose flow characteristics are to be actively varied, formed in
a
compressible or distortable elastomeric material. Thus, it is preferred that
substantially the entire microfluidic device be constructed of a flexible
elastomeric
material such as an organopolysiloxane elastomer ("PDMS"), as described
hereinafter. However, the device substrate may also be constructed of hard,
i.e.,
substantially non-elastic material at portions where active control is not
desired,
although such construction generally involves added construction complexity
and
expense. The generally planar devices preferably contain a rigid support of
glass,
silica, rigid plastic, metal, etc, on one side of the device to provide
adequate
support, although in some devices, actuation from both major surfaces may
require
that these supports be absent, or be positioned remote to the elastomeric
device
itself.
The microfluidic devices of the present invention contain at least one
active portion which alters the shape and/or volume of chambers or passageways
("empty space"), particularly fluid flow capabilities of the device. Such
active
portions include, without limitation, mixing portions, pumping portions,
valving
portions, flow portions, channel or reservoir selection portions, cell
crushing
portions, unclogging portions, etc. These active portions all induce some
change
in the fluid flow, fluid characteristics, channel or reservoir
characteristics, etc. by
exerting a pressure on the relevant portions of the device, and thus altering
the shape
and/or volume of the empty space which constitutes these features. The term
"empty space" refers to the absence of substrate material. In use, the empty
space
is usually filled with fluids, microorganisms, etc.
The active portions of the device are activatable by pressure to close
their respective channels or to restrict the cross-sectional area of the
channels to
accomplish the desired active control. To achieve this purpose, the channels,
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CA 02631466 2008-06-02
reservoirs, etc. are constructed in such a way that modest pressure from the
exterior
of the microfluidic device causes the channels; reservoirs, etc.
("microfluidic
features") to compress, causing local restriction or total closure of the
respective
feature. To accomplish this result, the walls within the plane of the device
surrounding the feature are preferably elastomeric, and the external surfaces
(e.g.,
in a planar device, an outside major surface) are necessarily elastomeric,
such that
a minor amount of pressure causes the external surface and optionally the
internal
feature walls to distort, either reducing cross-sectional area at this point
or
completely closing the feature.
The pressure required to "activate" the active portion(s) of the device
is supplied by an external tactile device such as are used in refreshable
Braille
displays. The tactile actuator contacts the active portion of the device, and
when
energized, extends and presses upon the deformable elastomer, restricting or
closing
the feature in the active portion.
Rather than close or restrict a feature by being energized, the tactile
actuator may be manufactured in an extended position, which retracts upon
energizing, or may be applied to the microfluidics device in an energized
state,
closing or restricting the passage, further opening the passage upon de-
energizing.
The preferred actuators at the present time are programmable Braille
display devices such as those previously commercially available from
Telesensory
as the Navigator" Braille Display with Gateway' software which directly
translates screen text into Braille code. These devices generally consist of a
linear
array of "8-dot" cells, each cell and each cell "dot" of which is individually
programmable. Such devices are used by the visually impaired to convert a row
of
text to Braille symbols, one row at a time, for example to "read" a textual
message,
book, etc. These devices are presently preferred because of their ready
commercial
availability. The microfluidic device active portions are designed such that
they will
be positionable below respective actutable "dots" or protrusions on the
Braille
display. Braille displays are available from Handy Tech, Blazie, and Alva,
among
other suppliers.
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CA 02631466 2008-06-02
However, to increase flexibility, it is possible to provide a regular
rectangular array usable with a plurality of microfluidics devices, for
example
having a 10 x 10, 16 x 16, 20 x 100, 100 x 100, or other array. The more close
the
spacing and the higher the number of programmable extendable protrusions, the
greater is the flexibility in design of microdevices. Production of such
devices
follows the methods of construction known in the art. Addressability also
follows
from customary methods. Non-regular arrays, i.e. in patterns having actuators
only
where desired are also possible.
Suitable Braille display devices suitable for non-integral use are
available from Handy Tech Electronik GmbH, Horb, Germany, as the Graphic
Window Professional.TM. (GWP), having an array of 24 x 16 tactile pins.
Pneumatic displays operated by microvalves have been disclosed by Orbital
Research, Inc. said to reduce the cost of Braille tactile cells from 70 $ U.S.
per cell
to Ca. 5-10 $lcell. Piezoelectric actuators are also usable where a
piezoelectric
element replaces the electrorheological fluid, and electrode positioning is
altered
accordingly.
The microfluidic devices of the present invention have many uses.
In cell growth, the nutrients supplied may need to be varied to simulate
availability
in living systems. By providing several supply channels with active portions
to
close or restrict the various channels, supply of nutrients and other fluids
may be
varied at will. An example is a three dimensional scaffolding system to create
bony
tissue, the scaffolding supplied by various nutrients from reservoirs, coupled
with
peristaltic pumping to simulate natural circulation.
A further application involves cell crushing. Cells may be crushed
by transporting them in channels through active portions and actuating channel
closure to crush the cells flowing through the channels. Cell detection may be
achieved, for example, by flow cytometry techniques using transparent
microfluidic
devices and suitable detectors. Embedding optical fibers at various angles to
the
channel can facilitate detection and activation of the appropriate activators.
Similar
detection techniques, coupled with the use of valves to vary the delivery from
a
channel to respective different collection sites or reservoirs can be used to
sort
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CA 02631466 2008-06-02
embryos and microorganisms, including bacteria, fungi, algae, yeast, viruses,
sperm
cells, etc.
Growth of embryos generally require a channel or growth chamber
which is capable of accommodating the embryo and allowing for its subsequent
growth. Such deep channels cannot effectively be closed, however. A
microfluidics
device capable of embryo growth may be fabricated by multiexposure
photolithography, using two masks. First, a large, somewhat rectangular
(2001im
width x 200 m depth) channel, optionally with a larger 200 m deep by 300 m
length and 300 m width growth chamber at one end is fabricated. Merging with
the 2001im x 200 m channel is a smaller channel with a depth of ca. 30 m,
easily
capable of closure by a Braille pin. Exiting the bulbous growth chamber are
one or
more thin (30 m) channels. In operation, embryo and media are introduced into
the
large channel and travel to the bulbous growth chamber. Because the exit
channels
from the growth chamber are very small, the embryo is trapped in the chamber.
The merging channels and exit channels can be used to supply nutrients, etc.,
in any
manner, i.e. continuous, pulsating, reverse flow, etc. The embryo may be
studied
by spectroscopic and/or microscopic methods, and may be removed by separating
the elastomeric layer covering the PDMS body which houses the various
channels.
Construction of fluidic devices is preferably performed by soft
lithography techniques, as described, for example by D. C. Duffy et al., Rapid
Prototyping of Microfluidic Systems in Poly(dimethylsiloxane), ANALYTICAL
CHEMISTRY 70, 4974-4984 (1998). See also, J. R. Anderson et al.,
ANALYTICAL CHEMISTRY 72, 3158-64 (2000); and M. A. Unger et al.,
SCIENCE 288, 113-16 (2000). Addition-curable RTV-2 silicone elastomers such
as SYLGARD® 184, Dow Coming Co., can be used for this purpose.
The dimensions of the various flow channels, reservoirs, growth
chambers, etc. are easily determined by volume and flow rate properties, etc.
Channels which are designed for complete closure must be of a depth such that
the
elastomeric layer between the microchannel and the actuator can approach the
bottom of the channel. Manufacturing the substrate of elastomeric material
facilitates complete closure, in general, as does also a cross-section which
is
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CA 02631466 2008-06-02
rounded, particularly at the furthest corners (further from the actuator). The
depth
will also depend, for example, on the extension possible for the actuator's
extendable protrusions. Thus, channel depths may vary quite a bit. A depth of
less
than 100gm is preferred, more preferably less than 50 m. Channel depths in the
range of 10 m to 40 m are preferred for the majority of applications, but even
very
low channel depths, i.e. lnm are feasible, and depths of 500 m are possible
with
suitable actuators, particularly if partial closure ("partial valving") is
sufficient.
The substrate may be of one layer or a plurality of layers. The
individual layers may be prepared by numerous techniques, including laser
ablation,
plasma etching, wet chemical methods, injection molding, press molding, etc.
However, as indicated previously, casting from curable silicone is most
preferred,
particularly when optical properties are important. Generation of the negative
mold
can be made by numerous methods, all of which are well known to those skilled
in
the art. The silicone is then poured onto the mold, degassed if necessary, and
allowed to cure. Adherence of multiple layers to each other may be
accomplished
by conventional techniques.
A preferred method of manufacture of some devices employs
preparing a master through use of a negative photoresist. SU-8 50 photoresist
from
Micro Chem Corp., Newton, Mass., is preferred. The photoresist may be applied
to a glass substrate and exposed from the uncoated side through a suitable
mask.
Since the depth of cure is dependant on factors such as length of exposure and
intensity of the light source, features ranging from very thin up to the depth
of the
photoresist may be created. The unexposed resist is removed, leaving a raised
pattern on the glass substrate. The curable elastomer is cast onto this master
and
then removed.
The material properties of SU-8 photoresist and the diffuse light from
an inexpensive light source can be employed to generate microstructures and
channels with cross-sectional profiles that are "rounded and smooth" at the
edges
yet flat at the top (i.e. bell-shaped). Short exposures tend to produce a
radiused top,
while longer exposures tend to produce a flat top with rounded corners. Longer
exposures also tend to produce wider channels. These profiles are ideal for
use as
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CA 02631466 2008-06-02
compressive, deformation-based valves that require complete collapse of the
channel
structure to stop fluid flow, as disclosed by M. A. Unger, et al., SCIENCE
2000,
288, 113. With such channels, Braille-type actuators produced full closure of
the
microchannels, thus producing a very useful valved microchannel. Such shapes
also
lend themselves to produce uniform flow fields, and have good optical
properties as
well.
In a typical procedure, a photoresist layer is exposed from the
backside of the substrate through a mask, for example photoplotted film, by
diffused
light generated with an ultraviolet (UV) transilluminator. Bell-shaped cross-
sections
are generated due to the way in which the spherical wavefront created by
diffused
light penetrates into the negative photoresist. The exposure dose dependent
change
in the SU-8 absorption coefficient (3985 m' unexposed to 9700 m' exposed at
365 nm) limits exposure depth at the edges.
The exact cross-sectional shapes and widths of the fabricated
structures are determined by a combination of photomask feature size, exposure
time/intensity, resist thickness, and distance between the photomask and
photoresist.
Although backside exposure makes features which are wider than the size
defined
by the photomask and in some cases smaller in height compared to the thickness
of
the original photoresist coating, the change in dimensions of the transferred
patterns
is readily predicted from mask dimensions and exposure time. The relationship
between the width of the photomask patterns and the photoresist patterns
obtained
is essentially linear (slope of 1) beyond a certain photomask aperture size.
This
linear relationship allows straightforward compensation of the aperture size
on the
photomask through simple subtraction of a constant value. When exposure time
is
held constant, there is a threshold aperture size below which incomplete
exposure
will cause the microchannel height to be lower than the original photoresist
thickness. Lower exposure doses will make channels with smoother and more
rounded cross-sectional profiles. Light exposure doses that are too slow (or
photoresist thicknesses that are too large), however, are insufficient in
penetrating
through the photoresist, resulting in cross-sections that are thinner than the
thickness
of the original photoresist.
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The suitability of bell-shaped cross-section microchannels of 30 m
thickness to be used as deformation-based valves was evaluated by exerting an
external force onto the channel using a piezoelectric vertical actuator of
commercially available refreshable Braille displays. Spaces may be left
between the
membrane and the wall when the channel cross-section has discontinuous
tangents,
such as in rectangular cross-sections. In contrast, a channel with a bell-
shaped
cross-section is fully closed under the same conditions. When a Braille pin is
pushed against a bell-shaped or rectangular-shaped cross-section microchannel
through a 200 m poly(dimethylsiloxane) (PDMS) membrane, the bell-shaped
channels were fully closed while the rectangular channels of the same width
had
considerable leakage.
The technique described is cost- and time-effective compared to other
photolithographic methods for generating well defmed rounded profiles such as
gray-scale mask lithography, or laser beam polymerization because there is no
need
for special equipment such as lasers, collimated light sources (mask aligner),
or
submicron resolution photomasks; it only requires a transilluminator available
in
many biological labs. In addition, the backside exposure technique can
generate
more profiles compared to other soft lithography-based patterning methods such
as
microfluidic mask lithography and the use of patterned laminar flows of
etchant in
an existing microchannel.
When used as deformation-based microfluidic valves, these
bell-shaped microchannels showed improved self-sealing upon compression
compared to conventional rectangular or semi-circular cross-section channels
as
demonstrated by simulations, and by experiments. A bell-shaped channel (width:
30 m; height 30 m) was completely closed by an 18 gf-force squeeze of a
Braille
pin. It is notable that channels that have the bell-shaped cross-sections with
"gently
sloping" sidewalls cannot be fabricated by melting resist technology, one of
the most
convenient methods to fabricate photomask-definable rounded patterns, because
the
profile is determined by surface tension. The bell-shaped channels maximize
the
cross-sectional area within microfluidic channels without compromising the
ability
to completely close channels upon deformation. For example, the channel
cross-section described here is larger than previously reported, pneumatically
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CA 02631466 2008-06-02
actuated deformation-based valves (100 m in width; 201m in height) and may be
more suitable for mammalian cell culture. = Furthermore, the bell-shaped
cross-sections provide channels with flat ceilings and floors, which is
advantageous
for reducing aberrations in optical microscopy and in obtaining flow fields
with a
more uniform velocity profile across the widths of the channel. These
advantages
of microchannels with bell-shaped cross-sectional shapes combined with the
convenient, inexpensive, and commercially available valve actuation mechanism
based on refreshable Braille displays will be useful for a wide range of
microfluidic
applications such as microfluidic cell culture and analysis systems,
biosensors, and
on-chip optical devices such as microlenses.
The extension outwards of the tactile actuators must be sufficient for
their desired purpose. Complete closure of a 40 m deep microchannel, for
example, will generally require a 404m extension ("protrusion") or more when a
single actuator is used, and about 20 m or more when dual actuators on
opposite
sides of the channel are used. For peristaltic pumping, mixing,. and flow
regulation,
lesser extensions relative to channel height are useful. The areal size of the
tactile
activators may vary appropriately with channel width and function (closure,
flow
regulation, pumping, etc.), and may preferably range from 40 m to about 2 mm,
more preferably 0.5 mm to 1.5 mm. Larger and smaller sizes are possible as
well.
The actuators must generate sufficient force. The force generated by one
Braille-type display pin is approximately 176 mN, and in other displays may be
higher or lower.
By use of the present invention, numerous functions can be
implemented on a single device. Use of multiple reservoirs for supply of
nutrients,
growth factors, etc. is possible. The various reservoirs make possible any
combination of fluid supply, i.e. from a single reservoir at a time; or from
any
combination of reservoirs. This is accomplished by establishing fluid
communication with a reservoir by means of a valved microchannel, as
previously
described. By programming the Braille display or actuator array, each
individual
reservoir may be connected with a growth channel or chamber at will. By also
incorporating a plurality of extendable protrusions along a microchannel
supply,
peristaltic pumping may be performed at a variety of flow rates. Uneven,
pulsed
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CA 02631466 2008-06-02
flow typical of vertebrate circulatory systems can easily be created. Despite
the
flexibility which the inventive system offers, construction is
straightforward. The
simplicity of the microfluidics device per se, coupled with a simple,
programmable
external actuator, enables a cost-effective system to be prepared, where the
microfluidic device is relatively inexpensive and disposable, despite its
technological
capabilities.
Combinatorial, regulated flow with multiple pumps and valves that
offer more flexibility in microfluidic cell studies in a laptop to handheld-
sized
system are created by using a grid of tiny actuators on refreshable Braille
displays.
These displays are typically used by the visually impaired as tactile analogs
to
computer monitors. Displays usually contain 20-80 rows of cells, each holding
8
(4 x2) vertically moving pins (~ 1-1.3 mm). Two pins on the same cell may
typically be 2.45 mm apart center to center and 3.8 mm apart on different
cells.
Each pin may have the potential to protrude 0.7 - 1 mm upward using
piezoelectric
mechanisms, and may hold up to - 15-20 cN. Control of Braille pins actuators
is
accomplished by changing a line of text in a computer program. Unique
combinations of Braille pins will protrude depending on the letters displayed
at a
given time. Braille displays are pre-packaged with software, easy to use, and
readily accessible. They are designed for individual use, and range from
walkman
to laptop sizes while using AC or battery power. By using the moving Braille
pins
against channels in elastomeric, transparent rubber, it is possible to deform
channels
and create in situ pumps and valves.
APPENDIX D
Embodiments of microfluidic devices may be suitable for the culture
of a living organism in a fluid. A microfluidic device may control the flow
and
composition of fluids provided to the living organism. The microfluidic device
may
provide laminar, pseudo-multiple laminar or non-laminar flows. The
microfluidic
device may perform physical operations on the living organism. The
microfluidic
device may be used, for example, for general cell culture including cell
washing and
detaclunent, cell seeding and culture. The microfluidic device may be used as
a
microreactor, a tissue culture device, a cell culture device, a cell sorting
device, a
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cell crushing device, a micro flow cytometer, a motile sperm sorter, a micro
carburetor, a micro spectrophotometer, or a microscale tissue engineering
device.
The microfluidic device may includes sensors to determine states or flow
characteristics of elements of the microfluidic device or the passage of
particles in
a channel. The sensors may be, for example, optical, electrical, or
electromechanical sensors.
In one embodiment, a microfluidic device includes microchannels
having flow characteristics that are actively varied and formed in a
compressible or
distortable elastomeric material. In one embodiment, the entire microfluidic
device
is constructed of a flexible elastomeric material, such as an
organopolysiloxane
elastomer ("PDMS"), as described hereinafter. However, the device substrate
may
also be constructed of hard, e.g., substantially non-elastic material at
portions,
where active control is not desired.
The microfluidic devices may contain at least one active portion that
alters the shape and/or volume of chambers or passageways ("empty space"),
particularly fluid flow capabilities of the device. Such active portions
include,
without limitation, mixing portions, pumping portions, valving portions, flow
portions, channel or reservoir selection portions, cell crushing portions, and
unclogging portions. These active portions all induce some change in the fluid
flow,
fluid characteristics, channel or reservoir characteristics, by exerting a
pressure on
the relevant portions of the device, and thus altering the shape and/or volume
of the
empty space which constitutes these features. The term "empty space" refers to
the
absence of substrate material. In use, the empty space is usually filled with
fluids
or microorganisms.
The active portions of the device are activatable by pressure to close
their respective channels or to restrict the cross-sectional area of the
channels to
accomplish the desired active control. To achieve this purpose, the channels,
reservoirs, or other elements are constructed in such a way that modest
pressure
from the exterior of the microfluidic device causes the channels, reservoirs
or other
elements ("microfluidic features") to compress, causing local restriction or
total
closure of the respective feature. To accomplish this result, the walls within
the
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plane of the device surrounding the feature are preferably elastomeric, and
the
external surfaces (e.g., in a planar device, an outside major surface) are
elastomeric,
such that a minor amount of pressure causes the external surface and
optionally the
internal feature walls to distort, either reducing cross-sectional area at
this point or
completely closing the feature.
The pressure used to "activate" the active portion(s) of the device is
supplied by an external tactile device, such as are used in refreshable
Braille
displays of the actuator system. The tactile actuator contacts the active
portion of
the device, and when energized, extends and presses upon the deformable
elastomer,
restricting or closing the feature in the active portion.
In some embodiments, rather than close or restrict a feature by being
energized, the tactile actuator may be manufactured in an extended position,
which
retracts upon energizing, or may be applied to the microfluidic device in an
energized state, closing or restricting the passage, further opening the
passage upon
de-energizing.
A significant iinprovement in the performance, not only of the subject
invention devices, but of other microfluidic devices which use pressure, e.g.,
pneumatic pressure, to activate device features, may be achieved by molding
the
device to include one or more voids adjacent the channel walls. These voids
allow
for more complete closure or distortion of the respective feature.
In one embodiment, the actuator system is a programmable Braille
display that includes a plurality of moveable pins that each engage a
corresponding
element of the microfluidic device to perform a fluidic operation. The
elements of
the microfluidic device include pumps and valves. The pins may be -arranged in
a
regular geometric array. Such arrangement maybe used with different
configurations of the microfluidic device. In this arrangement, some pins may
not
be used for particular microfluidic devices because no element in the device
corresponds to the pin. Alternatively the pins may be selected to correspond
to
elements of a specific or a group of multifluidic devices. Each pin may be
controlled independently, and individually addressable.
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An example of an actuator system is a Telesensory system such as the
Navigator' Braille Display with GatewayT" software, which directly translates
screen text into Braille code. These devices generally comprise a linear array
of
"8-dot" cells, each cell and each cell "dot" of which is individually
programmable.
Such devices are used by the visually impaired to convert a row of text to
Braille
symbols, one row at a time, for example to "read" a textual message or book.
The
microfluidic device active portions are designed such that they will be
positionable
below respective actuable "dots" or protrusions on the Braille display.
Braille
displays are available from Handy Tech, Blazie, and Alva, among other
suppliers.
As will be described below, the system may use various software programs for
controlling the pins of the actuator system by allowing the user to select
processes
to be performed on the organism, and then executing processes from a library.
However, to increase flexibility, it is possible to provide a regular
rectangular array usable with a plurality of microfluidic devices, for example
having
a 10 x 10, 16 x 16, 20 x 100, 100 x 100, or other size array. The closer the
spacing
and the higher the number of programmable extendable protrusions, the greater
is
the flexibility in design of microdevices. Production of such devices follows
the
methods of construction known in the art. Addressability also follows from
customary methods. Non-regular arrays, e.g., in patterns having actuators only
where desired are also possible.
Devices can also be constructed which integrate the tactile actuators
with the microfluidic device. The actuators are still located external to the
microfluidic device itself, but attached or bonded thereto to form an
integrated
whole. Other types of actuator systems may be used, such as a tactile actuator
device, which employs a buildup of an electrorheological fluid, an
electromechanical
Braille-type device employing shape memory wires for displacement between "on"
and "off" portions, devices employing electrorheologic or magnetorheologic
working fluids or gels, a pneumatically operated Braille device, "voice coil"
type
structures, especially those employing strong permanent magnets, devices
employing
shape memory alloys and intrinsically conducting polymer sheets.
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Suitable Braille display devices suitable for non-integral use are
available from Handy Tech Electronik GmbH, Horb, Germany, as the Graphic
Window Professional' (GWP), having an array of 24 x 16 tactile pins.
Piezoelectric actuators are also usable where a piezoelectric element replaces
the
electrorheological fluid, and electrode positioning is altered accordingly.
The microfluidic device has many uses. The software described
herein automates the operation of these uses. In cell growth, the nutrients
supplied
may be varied to simulate availability in living systems. By providing several
supply channels with active portions to close or restrict the various
channels, supply
of nutrients and other fluids may be varied at will. An example is a three
dimensional scaffolding system to create bony tissue, the scaffolding supplied
by
various nutrients from reservoirs, coupled with peristaltic pumping to
simulate
natural circulation.
Another application involves cell crushing. Cells may be crushed by
transporting them in channels through active portions and actuating channel
closure
to crush the cells flowing through the channels. Cell detection may be
achieved, for
example, by flow cytometry techniques using transparent microfluidic devices
and
suitable detectors. Embedding optical fibers at various angles to the channel
can
facilitate detection and activation of the appropriate activators. Similar
detection
techniques, coupled with the use of valves to vary the delivery from a channel
to
respective different collection sites or reservoirs can be used to sort
embryos and
microorganisms, including bacteria, fungi, algae, yeast, viruses, and sperm
cells.
The software controls the actuator system to control the pressure and
thus the opening and closing of the channel and the timing. Depending on the
processes to be performed, the software may address the actuators individually
or
in groups, and in patterns to provide actions, such as a peristaltic pumping
action
or a mixing action with respect to fluid in the channel. The software may
monitor
the sensors of the microfluidic device to selectively control the channel
flow.
As an illustrative example of peristaltic pump formed by three pins
engaging the microfluidic device, a pattern, such as XXO, OXX, OOX, XOX in
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repetition, where X is a closed position and 0 is an open position, to pump
fluid in
a channel may be used. The resultant fluid flow is pulsatile, with transient
movements in both directions. The net movement can be predicted by its linear
relationship to the pattern change frequency, and flow direction can be
switched by
reversing the pattern of actuation.
By use of the present invention, numerous functions can be
implemented on a single device. Use of multiple reservoirs for supply of
nutrients,
growth factors, and the like is possible. The various reservoirs make possible
any
combination of fluid supply, e.g., from a single reservoir at a time, or from
any
combination of reservoirs. This is accomplished by establishing fluid
communication with a reservoir by means of a valved microchannel, as
previously
described. By programming the actuator system, each individual reservoir may
be
connected with a growth channel or chamber at will. By also incorporating a
plurality of extendable protrusions along a microchannel supply, peristaltic
pumping
may be performed at a variety of flow rates. Uneven, pulsed flow typical of
vertebrate circulatory systems can easily be created. Combinatorial, regulated
flow
with multiple pumps and valves that offer more flexibility in microfluidic
cell
studies are created by using a grid of tiny actuators on refreshable Braille
displays
and executed automatically by software in response to user selections of
processes
to be performed.
While embodiments of the invention have been illustrated and
described, it is not intended that these embodiments illustrate and describe
all
possible forms of the invention. Rather, the words used in the specification
are
words of description rather than limitation, and it is understood that various
changes
may be made without departing from the spirit. and scope of the invention.
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