Pneumatically dampened microfluidic device

Abstract

A microfluidic system is described herein comprising a microfluidic plate and a pneumatic driver manifold. The microfluidic plate comprises a control manifold (14) with a plurality of control channels for receiving a gas under pneumatic pressure, a fluidic manifold (12) comprising a plurality of fluid channels, a plurality of valve chambers from by either or both of the control and fluid channels and a flexible diaphragm membrane (16). The pneumatic driver manifold is configured to engage with the control manifold of the microfluidic plate and provide gas to the control channels. The driver manifold also comprises a pneumatic reservoir (60a,b,c) upstream of the pneumatic channels and arranged to reduce the rate of change of gas pressure propagating to the control channels. A pneumatic driver manifold is also described.

Description

[0001] Pneumatically Dampened Microfluidic Device

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a microfluidic device including a pneumatic dampening system to reduce pulsatility in pumping. The device is primarily intended for use in cell culture, but may be useful in other applications where reduction in pulsatility is desirable.

[0004] BACKGROUND TO THE INVENTION

[0005] 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.

[0006] 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.

[0007] 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 I 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. This fluid flow could be recirculatory in nature.

[0008] As a consequence of this pumping arrangement, the flow of culture medium is naturally pulsatile to some extent, synchronised with the sequential actuation of the valves. While pulsatile flows can be important in cell culture for various reasons, instability or too much pulsatility may lead to changes in cell behaviour or function. As such, control of the pulsatility in the flow is important. Various mechanisms have been proposed to do this. Pressure pumps alone may not be reliable, as they can be prone to overshooting the desired pressure, sudden pressure changes in the system, or delayed response to feedback mechanisms due to internal pressure regulation by the controller. One of the main mechanisms to control pulsatility is to incorporate dampening mechanisms in the fluidic flow (that is, in the culture medium or culture medium circuit itself). This may be achieved for example by including an additional relatively large reservoir for the fluid; via inertial damping of the fluid flow; or by incorporating longer fluid flow channels to reduce the impact of pulsatility. However, these mechanisms may suffer from various drawbacks, potentially including the need to use larger volumes of culture medium than desirable; the introduction of dead space into the fluid system; and the requirement for more complex designs of microfluidic devices.

[0009] SUMMARY OF THE INVENTION

[0010] According to a first aspect of the present invention, there is provided a microfluidic system comprising: a) a microfluidic plate comprising a control manifold comprising a plurality of control channels for receiving a gas under pneumatic pressure; a fluidic manifold comprising a plurality of fluid channels for receiving a fluid; a plurality of valve chambers formed by either or both of the control and fluid channels; a flexible diaphragm membrane located between the control and fluidic manifolds and contacting the valve chambers, such that the control channels and fluid channels are pneumatically isolated from one another; wherein the diaphragm membrane is arranged to be deflected into or out of the valve chambers by said gas under pneumatic pressure, to thereby pump fluid in the fluid channels; and b) a pneumatic driver manifold configured to engage with the control manifold of the microfluidic plate and provide gas to the control channels, said driver manifold comprising a plurality of pneumatic channels having outlets arranged to align with ports of the control manifold when engaged, and said driver manifold further comprises a pneumatic reservoir upstream of the pneumatic channels and arranged to reduce the rate of change of gas pressure propagating to the control channels.

[0011] This microfluidic system incorporates the pneumatic reservoir in order to act as a dampening mechanism in the pneumatic flow of the device. The reduction in rate of change of gas pressure means that the movement of the diaphragm membrane is slowed (given that the change of pressure from positive to negative takes place more slowly), thereby reducing the pressure of the pump and extending the time required for the pump to fully displace. This has the effect of reducing the peak flow rate of the fluid in the pump stroke, and so reduces pulsatility in the fluid. Any reduction in rate of change of pressure should, of course, be determined with respect to a corresponding system without the pneumatic reservoir. This may be determined experimentally, or may be calculated based on known techniques. It may be helpful to consider a corresponding system without pneumatic reservoir as effectively having a near-instantaneous change from positive to negative pressure within the control channels, and hence also movement of the diaphragm. Reducing the rate of change of pressure will slow down movement of the diaphragm, leading to less abrupt changes of state, resulting in reduced pulsatility. Such a system also sets a theoretical upper limit for any such reduced rate of change - the rate of change should preferably not be reduced beyond the cycling time of the system (ie, the time taken for the valve to complete one full cycle), as otherwise the valve may have insufficient time to completely fill or empty if peak pressure or vacuum is not reached as a result.

[0012] One advantage of implementing this system in a pneumatic reservoir, rather than in the fluid itself, is that this pneumatic dampening introduces less dead volume in the fluid system and leads to more consistent (less pulsatile) shear stresses within the system than corresponding fluidic dampening methods. These features are particularly important when the microfluidic system is intended for use in cell culture, in which shear stresses can lead to uncontrolled changes in cell cultures which may reduce the function or performance of the cells in the culture or potentially damage the cells. In preferred embodiments, the pneumatic driver manifold is a separate device from other elements of the system. In particular, the microfluidic plate may be designed so as to be a consumable element, while the driver manifold may be intended for longer term reuse with multiple consumables. This may be particularly beneficial when the system is intended for use as a cell culture system.

[0013] The preferred form of the pneumatic reservoir may vary depending on specific requirements of the user. In particular, the reservoir may be designed to provide a specific pneumatic resistance and volume to achieve a given reduction in rate of change. The preferred reduction in rate of change may also be in part determined by the desired net flow rate and cycling time of the valves; as noted above, if the rate of change is reduced beyond the cycling time the valves may not fully fill or empty, so fluid flow rate will be reduced. The skilled person will be aware of suitable techniques which may be used to design such a reservoir. For example, the present inventors have used a lumped- element model (similar to the one described in Zbigniew Kaminski (2017) A simplified lumped parameter model for pneumatic tubes, Mathematical and Computer Modelling of Dynamical Systems, 23:5, 523-535, DOI: 10.1080 / 13873954.2017.1280512) to calculate the reduction in rate of change based on an assumed pneumatic resistance and volume based on the design. However, it is conceivable that this could be done through other forms of simulation such as computational fluid dynamics or other forms of numerically solving the Navier-Stokes equations. Lastly, the results can also be determined experimentally by measuring the pressure decay time in a physical system using a suitable pressure transducer.

[0014] In some embodiments, some contribution to the reduction in rate of change of pressure may also be provided by creating “choked flow” through suitable selection of charging / discharging (positive / negative) pressures above a critical pressure ratio defined by the specific heat ratio of the gas. When choked, the charging / discharging flow cannot exceed the choked flow velocity as the gas at the choke becomes sonic in which condition increased pressure gradient across the choke does not result in further increases in flow. The choked flow condition remains until the pressure ratio between the source and pneumatic reservoir does not exceed the critical pressure ratio. To maximise the effect of the choke for the purposes of this invention, it may be desirable to locate the choke point at the inlet of the pneumatic reservoir so as to maximise the volume and thus the amount of gas required to achieve sufficient pressure change to lower the pressure ratio across the choke point to below the critical pressure ratio. The pneumatic reservoir itself does not include any valves or other control elements (which are present in the control and / or fluid channels, while the pneumatic channels can be considered to have control elements in the form of outlets aligning with the control channels). In some embodiments, it may be convenient to consider the pneumatic reservoir as ending immediately adjacent the first outlet formed by the pneumatic channels, since the whole volume prior to the first outlet will assist in the pneumatic damping effect. Alternatively, in some embodiments the system is designed to allow simultaneous actuation of a number of valves by provision of a number of parallel pneumatic channels (and corresponding control channels, fluidic channels) which are provided for from a common source. This is most readily achieved by bifurcation or multifurcation of a number of parallel channels from a single supply; in such embodiments, it may be convenient to consider the pneumatic reservoir as ending immediately prior to such bifurcation or multifurcation. Given that the volume of the pneumatic channels themselves may have some influence on the rate of change of pressure, in some embodiments it may be important to arrange that any bifurcated / multifurcated path is kept a similar length across all branches so as not to create differing flow resistances.

[0015] In preferred embodiments, the pneumatic reservoir may take the form of one or more elongate channels of generally the same or similar cross section as the pneumatic channels. The length of the reservoir channels, however, is preferably significantly longer than the length of the pneumatic channels. For example, the reservoir channel is preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times longer than the pneumatic channels. In some embodiments the reservoir channel may be around 10-100 times longer than the pneumatic channels. Forming the reservoir from channels has the advantage that a long channel can be provided on a pneumatic driver manifold without requiring an increase in manifold size, simply by looping the channel around within a particular volume.

[0016] In embodiments the driver manifold may itself be multilayer. Preferably each such layer may include either or both pneumatic channels and pneumatic reservoir. This construction allows firstly a larger pneumatic reservoir (having longer channels, if present) and multiple pneumatic channels arranged in separate planes, so providing greater flexibility in microfluidic device design and in particular the inclusion of more outlets and / or valves than would otherwise be possible with a single monolithic manifold. In a preferred embodiment, the pneumatic driver manifold has three layers, although more may be possible in some embodiments. A first layer may provide a connection to a pneumatic supply, as well as pneumatic reservoir and pneumatic channels. A second layer may provide additional pneumatic reservoir and pneumatic channels. The third layer may provide outlets for the pneumatic channels. These layers will of course be pneumatically connected where appropriate.

[0017] 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 of the microfluidic pumps and valves. 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; valves of the device are aligned with the well pairs so as to pump fluid around the system. A bioreactor well may further comprise a cell culture insert or scaffold, on which cells may be cultured.

[0018] Embodiments of the invention may include multiple control channels each having a plurality of valves, with each control channel being configured to be capable of being actuated separately. This allows simultaneous actuation of each valve on a given control channel, with sequential actuation of each separate control channel. Such an arrangement is preferred as it permits directional pumping to be achieved by, for example, sequentially actuating valves across the length of a cell culture well. In a preferred embodiment, the device includes three control channels, arranged to provide three valves per culture well or bioreactor I reservoir well pair. Where multiple control channels are provided, so too may multiple separate pneumatic reservoirs; preferably one for each channel. Here a single control channel may be considered to include bifurcating or otherwise branching channels connected to a common source; such branching channels would be able to be actuated as a common unit.

[0019] The system may further comprise 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). Aspects of the invention may further comprise incubation apparatus for maintaining the system under preferred environmental conditions; for example, conditions suitable for cell culture.

[0020] A further aspect of the invention provides a pneumatic driver manifold configured to engage with and provide gas to a microfluidic plate, said driver manifold comprising a plurality of pneumatic channels having outlets arranged to align with ports of a microfluidic plate when engaged, said driver manifold further comprising a pneumatic reservoir upstream of the pneumatic channels and arranged to reduce the rate of change of gas pressure propagating to the microfluidic plate.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 shows a top view of a microfluidic cell culture plate.

[0023] Figure 2 shows an exploded view of a microfluidic cell culture plate.

[0024] Figure 3 shows a plan view of a pneumatic drive manifold.

[0025] Figure 4 shows an exploded view of a pneumatic drive manifold.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] In general terms, the present invention provides a microfluidic system in which pneumatic damping is used to reduce pulsatility arising in fluid circulation from the use of diaphragm pumps driven pneumatically. An embodiment of the invention will be described here with reference to a cell culture device, as illustrated in Figures 1-4.

[0028] The system here includes two separate components: a cell culture plate 10, and a pneumatic drive manifold 50 which receives the plate 10. The cell culture plate 10 is formed of multiple layers making up a fluidic manifold 12, a control 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. The drive manifold 50 also has a multilayer construction, as shown in Figure 4. 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.

[0029] The control manifold 14 includes ports in the bottom plate 28 which allow gas to access the control channels in the pneumatic plate 24. As with the top plate 18, the pneumatic membrane 26 serves to bond and seal the bottom plate 28 and the pneumatic plate 24.

[0030] Both the fluidic 12 and control 14 manifolds can be fabricated e.g. by micromechanical milling out of polymers such as polysulphone (PSU) or polymethylmethacrylate (PMMA). 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), polymethylmethacrylate (PMMA), polystyrene (PS) or polycarbonate (PC) can be used. The membrane material can be e.g. cyclic olefin copolymer (COC) elastomer, polyurethane (Pll) elastomer, polydimethylsiloxane (PDMS), or any other suitable material. 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. In some embodiments the various layers of the plate 10 may be retained together with clips or screws, rather than bonding layers together.

[0031] 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). By applying vacuum to the control channels in the control manifold, the elastomer membrane 16 is deflected down, the valves are opened, and cell culture medium fills the valve above the membrane 16. Applying positive pressure forces the membrane against the valve and the cell culture medium out of the valves. In the present example, three valves are provided for each paired bioreactor / reservoir well within the relevant fluidic channel. The valves of each triplet are operated in a cycle in order to direct fluid flow in a single direction around the fluidic circuit. Further details of the operation and design of cell culture plates of this general type can be found in, for example, US Patent 8,318,479.

[0032] The cell culture plate 10 is intended for use together with a driver manifold 50, which in turn connects to a pneumatic supply via a connector 52. In this example the connector 52 includes three air inlets 52a, b, c, corresponding to the three valves provided for each paired bioreactor / reservoir well, to permit each valve to be operated in series. The driver manifold 50 has a triple layer structure. The top layer 50a includes a number of openings 54 which, in use, align with corresponding ports provided on the lower surface of the control manifold 14 and connect the pneumatic system of the driver manifold 50 with the control channels of the control manifold 14. The middle layer 50b includes further apertures 56 aligned with certain of the openings 54 and which extend through to the bottom layer 50c. These in turn align with pneumatic channels 58a, 58b formed in the bottom layer 50c. Additional pneumatic channels 58c are provided in the middle layer 50b, which align with the remaining openings 54 of the top layer.

[0033] In addition to the pneumatic channels 58a, b, c, the middle and bottom layers 50b, c each include a pneumatic reservoir 60a, b, c in communication with and located upstream of the corresponding pneumatic channels 58a, b, c. Each such reservoir 60 a, b, c comprises a serpentine channel having a significantly greater length than the connected pneumatic channel, and extending from the channel to one of the air inlets 52a, b, c. No apertures or openings are provided in these reservoirs 60a, b, c; and they each provide a single channel up until the point of bifurcation or multifurcation of the pneumatic channels 58a, b, c, after which point apertures 56 or openings 54 allow communication with the control manifold.

[0034] In use, the presence of these pneumatic reservoirs results in a reduced rate of change of pressure reaching the diaphragm pumps of the cell culture plate. The degree of reduction may be determined during the design phase by selecting pneumatic reservoirs having particular pneumatic resistance and volume. This reduction in turn reduces the drive pressure of the fluid in the pump, and extends the time required for the pump to fully displace. This reduces the peak flow rate of the fluid in the pump, thereby reducing pulsatility in the system.

Claims

CLAIMS:

1. A microfluidic system comprising: a) a microfluidic plate comprising a control manifold comprising a plurality of control channels for receiving a gas under pneumatic pressure; a fluidic manifold comprising a plurality of fluid channels for receiving a fluid; a plurality of valve chambers formed by either or both of the control and fluid channels; a flexible diaphragm membrane located between the control and fluidic manifolds and contacting the valve chambers, such that the control channels and fluid channels are pneumatically isolated from one another; wherein the diaphragm membrane is arranged to be deflected into or out of the valve chambers by said gas under pneumatic pressure, to thereby pump fluid in the fluid channels; and b) a pneumatic driver manifold configured to engage with the control manifold of the microfluidic plate and provide gas to the control channels, said driver manifold comprising a plurality of pneumatic channels having outlets arranged to align with ports of the control manifold when engaged, and said driver manifold further comprises a pneumatic reservoir upstream of the pneumatic channels and arranged to reduce the rate of change of gas pressure propagating to the control channels.

2. The microfluidic system according to claim 1 , wherein the pneumatic driver manifold is a separate device from other elements in the system.

3. The microfluidic system according to claim 1 or claim 2, wherein the pneumatic reservoir comprises one or more elongate channels of generally the same or similar cross section as the pneumatic channels.

4. The microfluidic system according claim 3, wherein the reservoir channel is at least 10-100 times longer than the pneumatic channels.

5. The microfluidic system according to any preceding claim wherein the driver manifold is multilayer.

6. The microfluidic system according to claim 5 wherein the pneumatic driver manifold has three layers, wherein a first layer provides a connection to a pneumatic supply, as well as pneumatic reservoir and pneumatic channels; a second layer provides additional pneumatic reservoir and pneumatic channels; and a third layer provides outlets for the pneumatic channels.

7. The microfluidic system according to any preceding claim wherein the microfluidic plate is a cell culture plate.

8. The microfluidic system according to claim 7 wherein the fluid plate comprises a plurality of culture wells within which culture medium may be circulated by pumping action.

9. The microfluidic system according to claim 7 or claim 8 wherein the fluid plate comprises a plurality of bioreactor and reservoir well pairs connected by a fluid channel, in which each of these well pairs is otherwise fluidically separate from other pairs, and together forms an individual culture region.

10. The microfluidic system according to any preceding claim wherein the control manifold comprises multiple control channels each having a plurality of valves, with each control channel being configured to be capable of being actuated separately.

11. The microfluidic system according to claim 10 wherein multiple separate pneumatic reservoirs are provided; preferably one for each channel.

12. The microfluidic system according to any preceding claim further comprising a pneumatic supply.

13. A pneumatic driver manifold configured to engage with and provide gas to a microfluidic plate, said driver manifold comprising a plurality of pneumatic channels having outlets arranged to align with ports of a microfluidic plate when engaged, said driver manifold further comprising a pneumatic reservoir upstream of the pneumatic channels and arranged to reduce the rate of change of gas pressure propagating to the microfluidic plate.

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