Patent Description:
Microfluidic technologies enable users to culture cells in a more physiological three-dimensional (3D) microenvironment, offering the capabilities of high-resolution real-time imaging, multiple communicating cells types and control over flow and gradients. International Patent Application No <CIT> describes Three-Dimensional Microfluidic Platforms and Methods of Use Thereof. The material used to make these prior art devices is polydimethylsiloxane (PDMS), a moldable silicone that is optically clear and gas permeable. PDMS is commonly used for rapid prototyping with soft lithography processes to produce microfluidic devices. However, PDMS is not an ideal material for cell-based applications for reasons outlined in detail by <NPL>. In brief, some of the disadvantages include:.

The present invention was developed with a view to providing a three dimensional microfluidic platform made of plastics material that is less susceptible to the problems of the prior art devices made of PDMS. The microfluidic platform of the present invention may also incorporate a number of other advantageous features that improve its functionality. Document <CIT> discloses a microfluidic platform for investigating cell-based interactions, the platform comprising: a chip base made of a suitable plastics material with the appropriate optical properties, the chip base having a plurality of ports in fluid communication with a microfluidic channel for containing a culture medium in which cells are held. It discloses as well a method of manufacturing a microfluidic platform for investigating cell-based interactions, the method comprising the steps of: moulding a chip base from a suitable plastics material with appropriate optical properties, the chip base having a plurality of ports in fluid communication with a microfluidic channel for containing a culture medium in which cells are held.

References to prior art documents in this specification are provided for illustrative purposes only and are not to be taken as an admission that such prior art is part of the common general knowledge in Singapore or elsewhere.

According to the present invention there is provided a microfluidic platform for investigating cell-based interactions according to claim <NUM>.

Preferably the chip base is made from an engineering plastics material that can be injection molded and is optically clear. Typically the plastics material is selected from the group consisting of: polycarbonates (PC), polystyrenes (PS), polyethylenes (PE), cyclic olefin co-polymers (COC), cyclic olefin polymers (COP). Preferably the platform further comprises a gas permeable laminate bonded to a bottom surface of the chip base.

Preferably the gas permeable laminate is made from a polymer with low bulk density. Typically the polymer of low bulk density is selected from the group consisting of: polymethylpentene (PMP) and poly(<NUM>-trimethylsilyl-<NUM>-propyne) (PTMSP), or from polymethylated polymers such as polymethylated poly(diphenylacetylene), or from polymers that achieve sufficient gas permeability through other means. Typically the laminate is bonded to the chip base by heat lamination, solvent bonding, adhesion (with wet or dry adhesives), or by other means depending on the specific materials used for the chip base and laminate respectively. Preferably the gas permeable laminate is optically clear.

Typically the chip base has a plurality of microfluidic channels of elongate configuration arranged in a linear array, each microfluidic channel being substantially parallel to an adjacent channel. Preferably each microfluidic channel has a pair of ports, one port provided at each end respectively. Preferably the ports all open onto an upper surface of the chip base. Preferably the microfluidic channels are arranged into pairs with a third microfluidic channel provided there between, the third channel being arranged so as to permit controlled fluid communication between the pair of microfluidic channels and the third microfluidic channel. Typically the third microfluidic channel is filled with a hydrogel or other extracellular matrix. Preferably all of the microfluidic channels are formed in a bottom surface of the chip base and the gas permeable laminate bonded to the bottom surface of the chip base encloses the channels.

Advantageously the chip base is formed with a plurality of reservoirs molded into the upper surface thereof which are not in fluid communication with the ports, wherein, in use, each reservoir is adapted to hold sterile water, a hydrogel or other substance to create a humid environment around the device.

Typically the chip base is of generally elongate, rectangular configuration, with the ports arranged along its respective longitudinal edges.

According to the present invention there is provided a method of manufacturing a microfluidic platform for investigating cell-based interactions according to claim <NUM>.

Preferably the step of molding the chip base involves injection molding using an engineering plastics material that is optically clear. Typically the plastics material is selected from the group consisting of: polycarbonates (PC), polystyrenes (PS), polyethylenes (PE), cyclic olefin co-polymers (COC), and cyclic olefin polymers (COP).

Typically the method further comprises the step of bonding a gas permeable laminate to a bottom surface of the chip base.

Preferably the gas permeable laminate is optically clear and is made from a polymer with low bulk density. Typically the polymer of low bulk density is selected from the group consisting of: polymethylpentene (PMP) and poly(<NUM>-trimethylsilyl-<NUM>-propyne) (PTMSP), polymethylated polymers such as polymethylated poly(diphenylacetylene), or polymers that achieve sufficient gas permeability through other means.

Typically the step of bonding the laminate to the chip base involves laminating the laminate to the chip base by heat lamination. Alternatively the step of bonding the laminate to the chip base involves solvent bonding, adhesion bonding (with wet or dry adhesives), or other bonding means depending on the specific materials used for the chip base and laminate respectively.

According to the present invention there is provided a microfluidic platform for investigating cell-based interactions, the platform comprising:
a chip base having a plurality of ports in fluid communication with a microfluidic channel for containing a fluid culture medium in which cells are held, each port having an internal inlet, connecting the port with the microfluidic channel, and a trough for containing a small reservoir of the culture medium adjacent to the inlet wherein, in use, culture medium can be aspirated from the microfluidic channel via the trough rather than directly via the internal inlet.

In one embodiment the inlet is provided centrally of the port and the trough is of annular configuration surrounding the inlet.

Typically the bottom of the trough is of semicircular cross-section.

Preferably the ports of the microfluidic platform are designed as modular attachment interfaces.

Advantageously the ports are adapted to receive a universal modular luer connector for attaching standard luer fittings, such as tubing connectors and syringe pumps to the microfluidic platform.

Advantageously a plurality of the microfluidic chips can be received and held in a single microplate holder. Preferably the holder comprises a plurality of internal reservoirs provided in an upper surface thereof, which are not in fluid communication with the chips, wherein, in use, each reservoir is adapted to hold sterile water, a hydrogel or other substance to create a humid environment around the chips.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Likewise the word "preferably" or variations such as "preferred", will be understood to imply that a stated integer or group of integers is desirable but not essential to the working of the invention.

The nature of the invention will be better understood from the following detailed description of several specific embodiments of the microfluidic platform, given by way of example only, with reference to the accompanying drawings, in which:.

A preferred embodiment of a microfluidic platform <NUM> for investigating cell-based interactions in accordance with the invention, as illustrated in <FIG>, comprises a chip base <NUM> made of a suitable plastics material with the appropriate optical properties. The chip base <NUM> has a plurality of ports <NUM> in fluid communication with a microfluidic channel <NUM> for containing a culture medium <NUM> in which cells are held.

Typically the chip base <NUM> has a plurality of microfluidic channels <NUM> of elongate configuration arranged in a linear array, each microfluidic channel <NUM> being substantially parallel to an adjacent channel, as can be seen most clearly in <FIG>. Each microfluidic channel <NUM> has first and second ports <NUM> provided at each end respectively, as can be seen most clearly in <FIG>. Preferably the ports <NUM> all open onto an upper surface of the chip base <NUM>. Typically the chip base <NUM> is of generally elongate, rectangular configuration, with the ports <NUM> arranged along its respective longitudinal edges. Typical dimensions of the chip base <NUM> are <NUM> in length, <NUM> in width, and <NUM> in depth. The microfluidic channels <NUM> are typically <NUM> microns deep.

Preferably the microfluidic channels <NUM> are arranged into pairs 16a, 16b with a third microfluidic channel 16c provided there between, as illustrated in <FIG>. The third channel 16c is arranged so as to permit controlled fluid communication between the pair of microfluidic channels 16a, 16b and the third microfluidic channel 16c. Typically the third microfluidic channel 16c is filled with a hydrogel <NUM> or other extracellular matrix.

Preferably the chip base <NUM> is made from an engineering plastics material that can be injection molded and is optically clear. Typically the plastics material is selected from the group consisting of (but not limited to): polycarbonates (PC), polystyrenes (PS), polyethylenes (PE), cyclic olefin co-polymers (COC), cyclic olefin polymers (COP).

Plastics such as polycarbonate, polystyrene, etc. have historically been used to make cell culture devices in large scales. Traditional cell culture devices are flasks or wells that have large air head spaces and media volumes, so gas exchange is easily achieved. However, microfluidic devices consist of small volumes in sealed channels, and gas exchange becomes a limiting factor as most plastics are gas impermeable. This limitation may be been overcome by combining the plastic chip base <NUM> with a gas permeable laminate <NUM>.

Preferably the gas permeable laminate <NUM> is optically clear and is made from a polymer with low bulk density. Typically the polymer of low bulk density is selected from the group consisting of: polymethylpentene (PMP) and poly(<NUM>-trimethylsilyl-<NUM>-propyne) (PTMSP), or from polymethylated polymers such as polymethylated poly(diphenylacetylene), or from polymers that achieve sufficient gas permeability through other means. Preferably all of the microfluidic channels <NUM> are formed in a bottom surface of the chip base <NUM> and the gas permeable laminate <NUM> is bonded to the bottom surface of the chip base to enclose the channels <NUM> as shown in <FIG> and <FIG>. Typically the laminate <NUM> is bonded to the chip base <NUM> by heat lamination, solvent bonding, adhesion (with wet or dry adhesives), or by other means depending on the specific materials used for the chip base <NUM> and laminate <NUM> respectively.

It is also possible to make the chip base <NUM> entirely out of the gas permeable polymer. Doing so may have advantages in providing a simpler lamination process, since both the laminate <NUM> and chip base <NUM> would then have identical material properties. However there may not be a significant gain in oxygen availability because the oxygen has to diffuse across a thick chip base (in the order of millimeters) versus a thin laminate (in the order of tens to hundreds of micrometers). The specialised gas permeable plastics may also have material properties that make them unsuitable for injection molding. For these reasons, in the preferred embodiment the chip base <NUM> is fabricated out of standard injection moldable plastics and the device is laminated with a thin gas permeable laminate.

The microfluidic platform or chip <NUM> of the present invention is capable of replicating the in vivo behavior of cells in a culture system. Applications of the microfluidic platform or chip <NUM> may include (but are not limited to):.

Ease of use is a critical differentiator for the academic RandD customer segment. Besides the inconvenience of PDMS chip fabrication, users face other usage difficulties including:.

A number of innovations have been incorporated into the preferred embodiment of the microfluidic platform or chip <NUM> to overcome the usage difficulties outlined above. These additional innovations will now be described in detail.

Prior art microfluidic port designs are cylindrical in shape, leading directly into the channels (see <FIG>). Vacuum suction during the changing of culture medium can lead to cells being sucked out of the channels. An improved port design involves making internal troughs that are deeper than internal inlets (see <FIG>). Each port <NUM> has an internal inlet <NUM>, connecting the port <NUM> with the microfluidic channel <NUM>, and a trough <NUM> for containing a small reservoir of culture medium fluid adjacent to the inlet <NUM> wherein, in use, culture medium can be aspirated from the microfluidic channel <NUM> via the trough <NUM> rather than directly via the internal inlet <NUM>.

In the illustrated embodiment the inlet <NUM> is provided centrally of the port <NUM> and the trough <NUM> is of annular configuration surrounding the inlet in a concentric circle (as shown in cross-section in <FIG>). Alternatively, the trough <NUM> may be of a different configuration yet still placed adjacent to the inlet <NUM>. Typically the bottom of the trough is of semicircular cross-section.

The application of a vacuum through a glass/pipette tip <NUM> placed in the trough <NUM>, as shown in <FIG>, results in the removal of culture medium fluid, stopping when the medium in the trough is completely removed. Due to the higher height of the internal inlet <NUM>, the culture medium and cells in the channel would not be affected by the vacuum aspiration, no matter how long the pipette tip is kept in the trough <NUM>. Fresh medium can then be added to the port on one side of the channel <NUM> (the 'upstream' port), and allowed to flow through the channel, replacing the old medium. As a consequence of surface tension effects in microfluidic systems, a small volume of fresh media may need to be added to the downstream port, so that surface tension at the downstream inlet can be overcome to allow incoming flow.

The chip channel ports <NUM> are preferably designed as modular attachment interfaces. AIM's universal luer lock connector <NUM> (as shown in <FIG>) enables users to attach standard luer fittings (e.g. for attaching tubing connectors and syringe pumps) to the microfluidic chip <NUM>. Future accessories developed by AIM may connect directly to the ports <NUM> or via the universal connectors. <FIG> shows a plurality of the modular connectors <NUM> connected to respective ports <NUM> of the microfluidic platform <NUM>. <FIG> shows both a connector for luer slip and luer lock connections (left), and connectors attached to luer slip and luer lock syringes (right).

Prior art approaches by other manufacturers are based on separate components built directly onto the chip. The connector components protrude out of the chip and are included in the chip by default. This present modular design has two important advantages:.

Advantageously a plurality of the microfluidic chips <NUM> can be received and held in a single microplate holder <NUM>, as shown in <FIG> and <FIG>. The illustrated embodiment of the microplate holder <NUM> comprises a tray <NUM>, having side walls and a substantially planar base, and a plurality of compartments <NUM> provided in connection therewith. Each compartment <NUM> in the tray <NUM> is adapted to receive a microfluidic chip <NUM> therein. In the illustrated embodiment the tray <NUM> is designed to receive up to three of the fluidic chips <NUM> therein. Preferably the holder <NUM> further comprises a cover <NUM> which is received on top of the tray to enclose the microfluidic chips <NUM> therein. Advantageously the cover <NUM> is substantially transparent. Advantageously a plurality of the holders <NUM> is also stackable.

Standard form factors such as microscope slides and microtiter plates are prevalent in the biological and pharmaceutical research industries. Both the chips and holders are designed to comply with these existing standards so that the devices fit into existing workflows. The holders will also fit onto standard microscopy platforms and are stackable to maximise working space in cell culture incubators. The holders are designed to position the chip channel ports <NUM> to comply with SBS/ANSI standards for microtiter plates, so that they will be compatible with automated plate filling/handling systems. Such systems are made to fill wells in microplates and require the fill positions to be accurately located. A further advantage of this design approach is that devices suitable for manual operation in academic laboratories can also be deployed in an industrial, automated setting.

Users of microfluidic systems often have to place their devices in humidity chambers to limit evaporation. Advantageously both the chips <NUM> and holders <NUM> have built-in reservoirs (see <FIG>) that can be filled with sterile water, hydrogels (e.g. agarose, polyacrylamide, etc.) or other substances to create a humid environment around the device (and within the holder). This approach does away with the need to set up a separate humidity chamber. It also facilitates easy handling and retention of humid conditions when transferring the devices onto imaging platforms, since the humidification function is built into the chips and holders themselves.

As can be seen most clearly in <FIG>, the chip base <NUM> is formed with a plurality of reservoirs <NUM> molded into an upper surface thereof. The reservoirs <NUM> are not in fluid communication with the ports <NUM>. In use, each reservoir can be used to hold sterile water, a hydrogel or other substance to create a humid environment around the device.

Likewise the holder <NUM> further comprises a plurality of internal reservoirs <NUM> provided within the tray <NUM>, as can be seen most clearly in <FIG>. The reservoirs <NUM> are separate from and not in fluid communication with the chips <NUM>. Hence, in use, each reservoir <NUM> can be used to hold sterile water, a hydrogel or other substance to create a humid environment around the chips <NUM>.

Now that preferred embodiments of the microfluidic platform have been described in detail, it will be apparent that it provides a number of advantages over the prior art, including the following:.

Claim 1:
A microfluidic platform (<NUM>) for investigating cell-based interactions, the platform comprising:
a chip base (<NUM>) made of a suitable plastics material with the appropriate optical properties, the chip base (<NUM>) having a plurality of ports (<NUM>) in fluid communication with a microfluidic channel (<NUM>) for containing a culture medium in which cells are held;
wherein each port (<NUM>) has an internal inlet (<NUM>), connecting the port (<NUM>) with the microfluidic channel (<NUM>), and a trough (<NUM>) for containing a small reservoir of culture medium fluid adjacent to the inlet (<NUM>), wherein, in use, culture medium can be aspirated from the microfluidic channel via the trough rather than directly via the internal inlet and each port has an upper reservoir above said trough and said internal inlet, wherein the trough (<NUM>) is deeper than the internal inlet (<NUM>).