Pumping means for a microfluidic device
FIELD OF THE INVENTION
The present invention relates to a pumping means for a microfluidic device, as well as to a microfluidic device including such a pumping means. The device is primarily intended for use in cell culture, but may be useful in other applications.
BACKGROUND TO THE INVENTION
Microfluidics systems may be used to drive fluid flow for various purposes; one such application is in microfluidics cell cultures. For example, US Patent 8,318,479 describes a culture plate having a triple layer structure of a diaphragm membrane sandwiched between upper and lower plates. The upper plate includes multiple culture wells each of which includes a bioreactor well and a reservoir well connected by fluidic channels allowing recirculation of culture medium within the culture well. This upper plate may also be termed a fluidic manifold. The lower plate includes multiple control channels or lines and valves aligning with locations on the fluidic manifold which can be driven by a pneumatic pump. This lower plate may be termed a control manifold. The diaphragm membrane seals the two manifolds from one another. Actuation of the pneumatic pump moves air or other gas through the channels of the control manifold, causing the diaphragm membrane to deform within the valves. This deformation causes culture medium in the fluidic manifold to be pumped, thereby achieving culture medium circulation within the connected wells.
The plate is typically driven by a pneumatic supply; this may include a dedicated pneumatic driver manifold which connects to the supply and directs gas along further pneumatic channels formed within the driver manifold which connect with outlets formed in the manifold and arranged to align with ports in the control manifold allowing access to the control channels.
Typically circulation is activated in parallel by common pneumatic control lines; for example, one control line may connect multiple valves. The plate described in US 8,318,479 includes three valves per circuit (that is, per bioreactor / reservoir well pair) which are actuated sequentially to allow pumping along the circuit and improve flow between the bioreactor and reservoir. Corresponding valves in each circuit may then be connected to corresponding common control lines so as to synchronise pumping in each well pair, and simplify control.
The present invention describes improvements in microfluidic pumps incorporating diaphragm valves of this and similar types. The invention also describes the disposition of the fluid channels over multiple planes with interconnecting apertures which offer improvements to the manufacture of the microfluidic pumps and channels compared to the plate described in US 8,318,479.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention, there is provided a microfluidic plate comprising: a pneumatic manifold comprising a plurality of pneumatic channels for receiving a gas under pneumatic pressure; a fluidic manifold comprising a plurality of fluid channels for receiving a fluid; a flexible diaphragm membrane located between the pneumatic and fluidic manifolds, such that the control channels and fluid channels are pneumatically isolated from one another; and at least one fluid pump formed by the pneumatic channels, fluid channels, and diaphragm membrane, wherein the fluid pump comprises: at least first, second, and third sequential valve chambers formed by corresponding portions in a pneumatic channel and fluid channel, each such valve chamber being separated by the diaphragm membrane into a pneumatic portion and a fluid portion, and each such portion including a valve seat formed by a cap separated from the membrane and against which said membrane may be urged by application of vacuum or pneumatic pressure; each pneumatic portion being connected to a separate pneumatic channel permitting application of vacuum or pneumatic pressure to the diaphragm membrane within the chamber; each fluid portion being connected to at least one fluid channel permitting entry or exit of fluid to the chamber; and said first, second, and third sequential valve chambers are fluidically connected in sequence via said fluid channels.
Thus, the present invention provides a microfluidic device having a pump made of three sequential chambers (first, second, and third; but for convenience also referred to herein as chambers A, B, and C). In preferred embodiments each fluid portion is connected to at least a pair of fluid channels, such that fluid may flow one way along each; but in some embodiments a single fluid channel may be used for both forward and reverse flow in connection with the same fluid portion. It will be understood that the following description generally assumes that each chamber is connected to two fluid channels, and at least one pneumatic channel, each located in the appropriate portion of the chamber; although where appropriate the skilled person may envisage a single fluid channel per chamber. Assuming that the pump is intended to move fluid from chamber A to B to C, chamber A will have one fluid channel as a pump inlet (from, for example, a previous microfluidic component), and one fluid channel as an outlet connecting it to chamber B. Chamber B in turn is fluidically connected to chamber A and chamber C, while chamber C is fluidically connected to chamber B and to a pump outlet. In such an embodiment the pump inlet and pump outlet fluid channels will not connect the chambers together (although they may in some embodiments connect to another pump).
Note that the pneumatic manifold and the pneumatic channels may also be referred to herein as a control manifold and control channels, as the actuation of these elements controls the pump.
In preferred embodiments, either or both (and preferably both) of the caps of the pneumatic and fluid portions are generally in the form of a spherical cap (that is, a portion of a sphere cut off by a plane). Minor deviations from a spherical cap form may be present, but in general the intention is that the cap approximates the normal shape that a circular diaphragm membrane would adopt when being placed under pressure.
Preferably the spherical cap has a relatively shallow depth (ie, height of the cap) and a relatively wide radius. A high aspect ratio of the radius to the height of the cap minimises the amount of stretch required of the membrane to achieve a given volume displacement. Preferably both caps of the pneumatic and fluid portions of a given valve chamber are identical (albeit the may be inverted symmetrically about the plane defined by the diaphragm membrane). In preferred embodiments, all caps of a given first, second, and third valve chamber are similar in shape; although as described below in some embodiments the relative size of valve chambers (and hence caps) may vary.
Preferably each cap comprises a central aperture to connect with a pneumatic or fluid channel as appropriate. The central aperture is preferably circular.
In some embodiments, the second valve chamber has a greater volume than each of the first and third chambers. The first and third chambers may be of similar or identical volumes. As described below, this may in some embodiments assist in pumping by reducing extraneous movement of fluid during a pump cycle.
In some embodiments, the fluid channels connecting any or preferably all of the first, second, and third sequential valve chambers are located at least partly in a plane displaced from that of the portion of the fluidic manifold which is adjacent the diaphragm membrane. This is intended in part to aid in pneumatic isolation of the chambers, as weakness in or around the seal with the diaphragm membrane could permit pneumatic leakage between pneumatic and fluid channels if both are entirely along the plane of the membrane. In embodiments, at least a portion of said fluid channels is located in said displaced plane and extends parallel to the plane of the portion of the fluidic manifold which is adjacent the diaphragm membrane. In more preferred embodiments, a further portion of said fluid channels is located in the plane of the portion of the fluidic manifold which is adjacent the diaphragm membrane; that is, a single fluid channel is located in two separated but parallel planes. These separated portions of the fluid channel may be connected by, for example, a further portion extending perpendicularly to said planes -that is, the fluid channel has a stepped configuration, being located primarily in two parallel planes. In more preferred embodiments, the portion of the fluid channel in said displaced plane connects to an aperture in the centre of the valve cap of the fluid portion of the relevant valve. The portion of the fluid channel adjacent the diaphragm membrane may connect to a peripheral aperture in the valve cap of the fluid portion of the relevant valve. Similar arrangements and locations of the connections may be applied to fluid connections extending outside the pump (for example, at the inlet and outlet of the pump).
In preferred embodiments, the microfluidic plate is a cell culture plate. In such embodiments, the fluid plate may include a plurality of culture wells within which culture medium may be circulated by pumping action. The fluid plate may include a plurality of bioreactor and reservoir well pairs connected by a fluid channel; each of these well pairs is otherwise fluidically separate from other pairs, and together forms an individual culture region. Cells may be cultured in the bioreactor well, with the reservoir well including additional culture medium; the pump is located in the fluid channel, so as to pump fluid between the wells. A bioreactor well may further comprise a cell culture insert or scaffold, on which cells may be cultured.
A further aspect of the invention provides a microfluidic system comprising a microfluidic plate as described herein, in combination with a pneumatic supply. The system may further comprise computing means for controlling operation of the pneumatic supply, so as to actuate the valves in a predetermined manner (for example, the timing, order, frequency, and magnitude of valve actuation may each be predetermined).
Embodiments of the invention may further comprise incubation apparatus for maintaining the system under preferred environmental conditions; for example, conditions suitable for cell culture.
A further aspect of the invention provides a fluid pump for use in a microfluidic device, the fluid pump being formed by pneumatic channels, fluid channels, and a diaphragm membrane, wherein the fluid pump comprises: at least first, second, and third sequential valve chambers formed by corresponding portions in a pneumatic channel and fluid channel, each such valve chamber being separated by the diaphragm membrane into a pneumatic portion and a fluid portion, and each such portion including a valve seat formed by a cap separated from the membrane and against which said membrane may be urged by application of vacuum or pneumatic pressure; each pneumatic portion being connected to a separate pneumatic channel permitting application of vacuum or pneumatic pressure to the diaphragm membrane within the chamber; each fluid portion being connected to at least one fluid channel permitting entry or exit of fluid to the chamber; and said first, second, and third sequential valve chambers are fluidically connected in sequence via said fluid channels.
Other features of the first aspect of the invention may also be applied to this aspect.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a top view of a microfluidic cell culture plate.
Figure 2 shows an exploded view of a microfluidic cell culture plate.
Figures 3 and 4 show upper and lower face views of a fluidic plate.
Figure 5 shows a portion of a microfluidic pump from the fluidic plate in more detail.
Figure 6 shows an alternate embodiment of a portion of a microfluidic pump, having a single fluid channel for each chamber.
Figures 7 and 8 show further variations of a portion of a microfluidic pump, in which the interface of the fluid channels with the chamber may be varied.
DETAILED DESCRIPTION OF THE INVENTION
In general terms, the present invention provides a microfluidic plate in which diaphragm pumps driven pneumatically are used to circulate fluid. An embodiment of the invention will be described here with reference to a cell culture device, as illustrated in Figures 1- 5. Variations of the pump are shown in Figure 6-8.
The plate described herein is part of a microfluidic system which may include two separate components: a cell culture plate 10, and a pneumatic drive manifold (not illustrated) which receives the plate 10. The cell culture plate 10 is formed of multiple layers making up a fluidic manifold 12, a control or pneumatic manifold 14, and a diaphragm pump membrane 16. In this example, each manifold 12, 14 itself is made of multiple layers: the fluidic manifold 12 includes a top plate 18, a fluidic membrane 20, and a fluidic plate 22. The control manifold includes a pneumatic plate 24, a pneumatic membrane 26, and a bottom plate 28.
Perhaps seen best in Figure 1, the top plate 18 includes multiple cell culture wells 30, each of which includes a reservoir well 32 and a bioreactor well 34 which are connected by a fluid circulation channel (not seen in Figure 1, but formed below the culture well 30 in the top plate 18 and in combination with the fluidic plate 22). Each culture well 30 is fluidically isolated from the other culture wells, so forming a single system within which fluid may circulate when pumped. The fluidic membrane 20 serves both to bond together the top plate 18 and fluidic plate 22, and to seal the plates together.
The control manifold 14 includes ports in the bottom plate 28 which allow gas to access the control channels in the pneumatic plate 24; in use, this gas may be provided by the pneumatic drive manifold which engages with this plate, and which itself will include pneumatic channels aligned with these ports. As with the top plate 18, the pneumatic membrane 26 serves to bond and seal the bottom plate 28 and the pneumatic plate 24.
Both the fluidic 12 and control 14 manifolds can be fabricated e.g. by micromechanical milling out of polymers such as polysulphone. This can be cost effective in small batch fabrication. In large volume fabrication, mass replication techniques such as injection molding and materials e.g. cyclic olefin copolymer (COC), can be used. The membrane material can be e.g. elastomeric cyclic olefin copolymer (COO). The membranes can be e.g. bonded to the fluidic and control manifold by plasma oxidizing the mating surfaces and immediately pressing the parts together. Alternatively, the components may simply be held together by pressure applied by, eg, clips, screws, and so on.
The cell culture plate 10 has diaphragm valves forming a pump between the control manifold and the fluidic manifold. The valves are created by sandwiching a monolithic elastomer membrane 16 between fluidic 12 and control 14 manifolds. A valve is created where a control channel (in the control manifold) crosses a fluidic channel (in the fluidic manifold). In the present example, an individual pump is formed from three valves.
Figures 3 and 4 show upper and lower faces of the fluidic plate 22 respectively, which in use forms part of the fluidic manifold. Of particular interest here are the pump elements 50 which extend between the upper and lower faces of the plate. The lower face will seat against the pump membrane 16 (as shown in figure 2), while the upper face includes fluidic channels 52 which form a part of the culture well circuits 30, 32, 34 described with reference to Figure 1. Each pump element 50 corresponds to a culture well 30. The pump element 50 is shown in more detail in Figure 5. It will be understood that the pump as a whole will be formed of this element 50 located in the fluidic plate 22, and a corresponding element (not shown) located in the pneumatic plate 24; although the corresponding pneumatic element may include somewhat different connections, in that each pneumatic valve element has a single connection to a pneumatic channel.
The fluid is moved by a pump consisting of three interconnected chambers, formed by the spherical caps 52, 54, 56 of the two pump elements. Each chamber contains an elastic membrane (ie, pump membrane 16) which divides the chamber into a distinct fluid portion and an air portion. The membrane seals against each portion of the chamber preventing the air and fluid from mixing.
The fluid portion of each chamber contains two channels 58, 60 (indicated only for one of the spherical caps 54) to enable the fluid to flow into and out of the fluid portion of the chamber. The air portion of each chamber has a single channel which permits air to flow into and out of the air portion of the chamber. The air channel is connected to a solenoid valve and pneumatic supply external to the microfluidic device which allow the air pressure in each chamber to be independently controlled.
Note that where the fluid channel 58 intersects the spherical cap 56, the change in channel profile may result in a reduced fluid flow cross-section. For this reason, in some embodiments it may be advantageous to modify the shape of the channel 58 at and near the intersection to optimise fluid flow. Two possible solutions are shown in Figures 7 and 8. The inlet channel should be tailored to optimise the flow into and out of the chamber by maintaining a constant cross-section and providing a smoother transition between the channel and spherical cap. This transition should aim to minimise the deviation of form from the spherical cap so as to maintain the symmetry between the two halves of the chamber. In one embodiment (shown in Figure 8) this has been achieved through the use of a kidney-shaped channel following the periphery of the spherical cap. Alternatively (Figure 7), use may be made of a connecting funnel shaped channel of constant cross-section which transitions between the two shapes.
Enlarged views of the intersection are also shown in the Figures.
When the air portion of the chamber is subject to vacuum, the membrane displaces into the air portion of the chamber. This causes fluid to be drawn into the fluid portion of the chamber 54 from the fluid inlet channel 58. When the air portion of the chamber is subject to positive pressure, the membrane displaces the fluid in the fluid portion of the chamber. This causes fluid to be displaced out of the fluid portion of the chamber 54 into the fluid outlet channel 60. The shape and size of the chamber, the material and mechanical properties of the membrane, and the positive and vacuum pressures are selected so as to ensure that the membrane fully contacts the peripheral surfaces of the chamber so as to displace a volume defined by the sweeping movement of the membrane. The motion of the membrane and relative change in size of the fluid portion of the chamber also permits the chamber to prime effectively when fluid is first introduced into the fluid channels.
The preferred shape of each portion of the chamber is a spherical cap 52 of shallow depth and wide footprint diameter. The spherical cap shape is preferred as it closely resembles the free state of the membrane 16 under pressure. The high aspect ratio of the cap 52 minimises the amount of stretch required of the membrane 16 to achieve a given volume displacement. Reducing the stretch minimises the effect of non-elastic behaviour in the membrane 16 (e.g. creep, stress relaxation, compression set) and reduces the amount of pressure required to deform the membrane 16 into the peripheral surface of the chamber (thus maximising the amount of pressure available for displacing the fluid itself or provide a seal between the membrane and the chamber surfaces when the chamber is closed). Similar chamber shapes symmetrical about the membrane plane are preferred as this ensures that the deformation pattern of the membrane is similar when the membrane is deformed into either portion of the chamber thus further reducing the effect of non-elastic behaviour in the membrane (e.g. wrinkles due to non-symmetrical stretching).
For both portions of the chamber the preferred shape and position of one of the channels 60 is a circular hole 62 at the centre of the spherical cap 54. This position is preferred as it has the greatest clearance between the membrane and peripheral surfaces of the chamber during the membrane displacement. This maximises the cross-sectional area available to flow out of the chamber whilst minimising the effective flow path length of the fluid (e.g. lowest fluid resistance). It also minimises the potential occurrence of the membrane sealing over the channel before the membrane is fully displaced.
To allow the chambers (A, B, C) to achieve net fluid movement in the desired direction, the chambers must be actuated in a sequence. The preferred sequence for delivering fluid from the inlet to the outlet of the pump is: Chamber Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7/ Step 1 A Closed Open Open Closed Closed Closed Closed B Closed Closed Open Open Open Closed Closed C Closed Closed Closed Closed Open Open Closed This sequence allows the delivery of a finite volume of fluid from the inlet to the outlet of the pump chambers. Due to the deterministic deflection of the membrane (full contact with the chamber walls in both directions), the volume of chamber B delivered determines the volume of fluid volume displaced from the inlet to the outlet by the pump after one full cycle of the above sequence. The volumes of chamber A and C are drawn from and delivered to the inlet and outlet fluid of the pump respectively, so have no net effect on the volume delivered. Their primary function is to control the flow direction.
Due to the intended behaviour of the chambers described above, it is often desirable to reduce the volumes of chamber A and C relative to the volume of chamber B to reduce the extraneous movement of fluid during a pump cycle.
Furthermore, the above chamber sequence also ensures that at least one chamber is always closed during the sequence. The closure prevents the unintended flow of fluid in the opposite direction when the fluid at the pump outlet has a higher pressure than the inlet of the chambers.
In microfluidic devices where there is significant flow resistance in the fluid path due to small channel sizes or downstream pressure, or where the switching time between the positive pressure and vacuum is appreciable due to the chamber volume, it may be desirable to introduce a delay between each step to allow the membrane time to fully displace. For example, it may be that a small delay, 50 milliseconds or less is preferred, is introduced in between the sequential steps to allow the membrane to displace and fill or empty the chamber of fluid.
To pneumatically isolate each chamber, a continuous seal is required around the pneumatic portion of each chamber. Adjacent fluidic portions will be fluidically connected, as described. In many instances, this fluidic connection may be implemented by utilising a connecting channel at the membrane interface (that is, the portion of the fluidic plate 22 which is in contact with the diaphragm membrane 16). However, the seal profile becomes open at the intersection with the channel resulting in a potential pneumatic leak path between chambers leading to indeterminate membrane actuation. To prevent this, it is preferred that at least a portion of the connecting fluidic channel occurs in a plane different to that of the membrane interface. It is preferred that this plane is parallel to that of the membrane interface (for example, on the other side of the fluidic plate 22 which does not contact the diaphragm membrane 16). This arrangement simplifies the manufacture of these features through methods such as CNC milling and injection moulding. The preference is to minimise the length of connecting track on the membrane interface and connect at least one end of the connecting channel to a hole in the centre of the fluid portion of the chamber so that the area of membrane potentially exposed to the air in the chamber & channel arrangement is minimised. The parallel plane may be sealed through another membrane or alternative methods (e.g. laser welding, solvent bonding, adhesive bonding) with a further (non-membrane) sealing surface. This arrangement is illustrated in Figure 5, which shows the central hole 62 connecting to a perpendicular portion 60 of a channel, in turn leading to a parallel portion 64 which in use will be displaced from the plane of the membrane interface. This in turn connects to a further perpendicular portion 66 which connects to a portion 58 which is parallel to and in the plane of the membrane interface.
In the portion of the pump which is on the air side of the membrane interface, a similar arrangement is present but without the parallel portions 58 and perpendicular portions 66, such that only the perpendicular portions 60 provide connections to the pneumatic channels.
Alternate pumping sequences may of course be used if desired. The precise pumping sequence may be selected depending on the intended use and the preferred characteristics; the pneumatic driver may be connected to a pneumatic supply and a computing device programmed to actuate individual pneumatic channels as desired.
For example, a travelling wave pumping sequence may be used, which simultaneously creates opposed displacement in adjacent chambers so that the fluid volume is transferred between chambers due to favourable pressure gradients. In this manner fluid is drawn wholly from the closing of the adjacent chamber rather than from the inlet or outlet of the pump system. A lack of synchronisation between the adjacent chambers can result in a chamber becoming "locked" at a partial fill/emptying state due to premature closing of the adjacent chamber, so it is important to avoid such events. The sequence is: Chamber Step 1 Step 2 Step 3 Step 4 Step 5/ Step 1 A Closed Open Closed Closed Closed B Closed Closed Open Closed Closed C Closed Closed Closed Open Closed Another example is a combined opening & closing pumping sequence. This relies on simultaneous opening of chambers A & B and/or simultaneous closing of chambers B & C to reduce the number of steps. This has the effect of increasing the magnitude and/or duration of the favourable pressure gradient which can shorten the time required to complete the sequence. However, this requires that the fluid flow restrictions within the pump system be low compared to the inlet/outlet restrictions so that chamber B may fully fill or empty before chambers A or C respectively. In pump systems where this is not the case, chamber B will only achieve a partial level of fill or emptying causing the flow rate to be uncorrelated to the volume of chamber B. The sequence is: Chamber Step 1 Step 2 Step 3 Step 4 Step 5/ Step1 A Closed Open Closed Closed Closed B Closed Open Open Open Closed C Closed Closed Closed Open Closed As the flow direction in this instance is determined by the actuation order of chambers A and C, chamber B may be designed to have only a single inlet/outlet channel for the fluid portion. This is illustrated in Figure 6. Here the second chamber has a single fluid channel acting as both inlet and outlet, and connecting to a common fluid channel which in turn links to the first and third chambers. Those first and third chambers may in embodiments have two fluid channels each, one being an inlet or outlet from the pump as a whole. This embodiment is simpler for the manufacture of the pump system, but results in a reversal of the fluid flow direction in the channel. This reversal may allow air bubbles or solid material in the fluid to become more easily entrained and block the fluid flow in the inletloutlet channel of chamber B, hence it is not preferred.
The inclusion of a pumping means within a microfluidic device enables applications for the device to be used in medical, chemical, and biological assays. Its inclusion within the confines of the device helps to miniaturise the footprint of the device and increase its ease of handling and portability. The use of pneumatic actuation also enables the microfluidic pump to be used for applications where it is desirable to actuate the device in a non-contact means so that a degree of separation may be maintained between the fluid and other system elements, for reasons of sterility or to avoid potential cross-contamination.