Abstract
The devices, methods and systems are described for providing controlled amounts of gas, gas pressure and vacuum to microfluidic devices the culturing of cells under flow conditions. The devices, methods, and systems contemplated here may also be used to control the environment surrounding the microfluidic devices; offer user control over experiments comprising microfluidic devices, such as the ability to directly or remotely control experiment conditions; and comprise information aggregation and transmission, such that experimental data may be collected, stored, aggregated and transmitted to users.
Claims
1. A method of delivering a gas mixture to at least one microfluidic device, comprising the steps: a) providing 1) an apparatus comprising i) a gas mixer configured to mix gas from at least two gas sources into a gas mixture, ii) at least one pneumatic pressure generator configured to generate at least one pneumatic pressure and iii) conduits configured to deliver said gas mixture and said at least one pneumatic pressure to 2) a culture module comprising a moveable pressure manifold, and 3) at least one microfluidic device; b) mixing gas from at least two gas sources to generate a gas mixture within said apparatus; c) generating at least one pneumatic pressure within said apparatus; and d) delivering said gas mixture and said at least one pneumatic pressure from said apparatus to said culture module and said at least one microfluidic device, wherein said at least one pneumatic pressure actuates movement of said moveable pressure manifold in said culture module to establish a pneumatic connection with said at least one microfluidic device, and wherein said gas mixture provides culture conditions in said at least one microfluidic device.
2. The method of claim 1, wherein one of said at least two gas sources is ambient air.
3. The method of claim 2, wherein said mixing comprises mixing said ambient air with gas from a second gas source.
4. The method of claim 3, wherein said apparatus further comprises a gas tank adapted as said second gas source.
5. The method of claim 2, wherein said gas mixture comprises a mixture of air and CO2.
6. The method of claim 1, wherein said at least one pneumatic pressure comprises vacuum pressure.
7. The method of claim 1, wherein said gas mixture is delivered to said at least one microfluidic device via said culture module.
8. The method of claim 1, wherein said at least one pneumatic pressure is delivered to said at least one microfluidic device via said culture module.
9. The method of claim 1, wherein said at least one microfluidic device comprises living cells.
10. The method of claim 1, further comprising pressurizing said gas mixture prior to said delivering in step d).
11. The method of claim 1, further comprising e) generating fluid flow within said at least one microfluidic device.
12. A system of delivering a gas mixture to at least one microfluidic device, comprising: 1) an apparatus comprising i) a gas mixer configured to mix gas from at least two gas sources into a gas mixture, ii) at least one pneumatic pressure generator configured to generate at least one pneumatic pressure and iii) conduits configured to deliver said gas mixture and said at least one pneumatic pressure to 2) a culture module comprising a moveable pressure manifold and 3) at least one microfluidic device; wherein said at least one pneumatic pressures is configured to actuate a movement of said moveable pressure manifold in said culture module, and wherein said gas mixture is configured to provide culture conditions in said at least one microfluidic device.
13. The system of claim 12, wherein one of said at least two gas sources is ambient air.
14. The system of claim 13, wherein said gas mixer is configured to mix said ambient air with gas from a second gas source.
15. The system of claim 12, wherein said apparatus further comprises a gas tank adapted as said second gas source.
16. The system of claim 14, wherein said gas mixture comprises a mixture of air and CO2.
17. The system of claim 12, wherein said at least one pneumatic pressure comprises vacuum pressure.
18. The system of claim 12, wherein said at least one microfluidic device comprises living cells.
19. The system of claim 12, wherein said apparatus further comprises a means to pressurize said gas mixture.
20. The system of claim 12, further comprising at least one fluid present within said at least one microfluidic device.
21. The system of claim 20, wherein said at least one pneumatic pressure is adapted to generate flow in said at least one fluid.
22. The system of claim 20, wherein said gas mixture is adapted to generate flow in said at least one fluid.
23. The system of claim 20, further comprising at least one fluid reservoir containing at least a portion of said fluid.
24. The system of claim 23, wherein said at least one pneumatic pressure is adapted to be in communication with said at least one reservoir.
25. The system of claim 23, wherein said gas mixture is adapted to be in communication with said at least one reservoir.
26. A method of delivering a gas mixture to at least one microfluidic device, comprising the steps: a) providing 1) an apparatus comprising i) a gas mixer configured to mix gas from at least two gas sources into a gas mixture, ii) at least one pneumatic pressure generator configured to generate at least one pneumatic pressure and iii) conduits configured to deliver said gas mixture and said at least one pneumatic pressure to 2) a culture module and 3) at least one microfluidic device comprising cells on a membrane; b) mixing gas from at least two gas sources to generate a gas mixture within said apparatus; c) generating at least one pneumatic pressure within said apparatus; and d) delivering said gas mixture and said at least one pneumatic pressure from said apparatus to said culture module and said at least one microfluidic device, wherein said at least one pneumatic pressure actuates a movement of said membrane in said microfluidic device, and wherein said gas mixture provides culture conditions in said at least one microfluidic device.
27. The method of claim 26, wherein said membrane is stretched.
28. The method of claim 26, wherein one of said at least two gas sources is ambient air.
29. The method of claim 28, wherein said mixing comprises mixing said ambient air with gas from a second gas source.
30. The method of claim 26, wherein said at least one pneumatic pressure comprises vacuum pressure.
31. The method of claim 26, wherein said gas mixture is delivered to said at least one microfluidic device via said culture module.
32. The method of claim 26, wherein said at least one pneumatic pressure is delivered to said at least one microfluidic device via said culture module.
33. A system of delivering a gas mixture to at least one microfluidic device, comprising: 1) an apparatus comprising i) a gas mixer configured to mix gas from at least two gas sources into a gas mixture, ii) at least one pneumatic pressure generator configured to generate at least one pneumatic pressure and iii) conduits configured to deliver said gas mixture and said at least one pneumatic pressure to 2) a culture module and 3) at least one microfluidic device comprising cells on a membrane; wherein said at least one pneumatic pressures is configured to actuate a movement of said membrane in said at least one microfluidic device, and wherein said gas mixture is configured to provide culture conditions in said at least one microfluidic device.
34. The system of claim 33, wherein one of said at least two gas sources is ambient air.
35. The system of claim 34, wherein said gas mixer is configured to mix said ambient air with gas from a second gas source.
36. The system of claim 35, wherein said gas mixture comprises a mixture of air and CO2.
37. The system of claim 33, wherein said apparatus further comprises a gas tank adapted as said second gas source.
38. The system of claim 33, wherein said at least one pneumatic pressure comprises vacuum pressure.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIG. 1A is an exploded view of one embodiment of the perfusion manifold assembly (also called the perfusion disposable or “pod”) showing the cover (or cover assembly) off of the reservoirs (the reservoir body can be made of acrylic, for example), the reservoirs positioned above the backplane, the backplane in fluidic communication with the reservoirs, the skirt with a side track for engaging a representative microfluidic device or “chip” (which can be fabricated out of plastic, such as PDMS, for example) having one or more inlet, outlet and (optional) vacuum ports, and one or more microchannels, the chip shown next to (but not in) one embodiment of a chip carrier (which can be fabricated out of a themioplastic polymer, such as acrylonitrile butadiene styrene (ABS), for example), the carrier being configured to support and carrier the chip, e.g. dimensioned so that the chip fits within a cavity. FIG. 1B shows the same embodiment of the perfusion manifold assembly with the cover on and over the reservoirs, and the chip inside the chip carrier fully linked to the skirt of the perfusion manifold assembly, and thereby in fluidic communication with the reservoirs. In one embodiment, each chip has two inputs, two outputs and (optionally) two connections for the vacuum stretch. In one embodiment, putting the chip in fluidic communication connects all six in one action, rather than connecting them one at a time. FIG. 1C is an exploded view of one embodiment of the perfusion manifold assembly (before the components have been assembled) comprising reservoirs positioned over a fluidic backplane (comprising a fluid resistor), that is fluidically sealed with a capping layer and is positioned over a skirt, with each piece dimensioned to fit over the next. In one embodiment, the skirt comprises structure (e.g. made of polymer) that borders or defines two open spaces, one of the spaces configured to receive the carrier with the chip inside. In one embodiment, the skirt has structure that completely surrounds one open space and two “arms” that extend outwardly that define a second open space for receiving the carrier. In one embodiment, the two arms have side tracks for slidably engaging the carrier edges.
(2) FIG. 2A is an exploded view of one embodiment of the cover assembly comprising a pressure cover or pressure lid. In the illustrated embodiment, the pressure lid comprises a plurality of ports (e.g. through-hole ports) associated with filters and corresponding holes in a gasket. The illustrated design of the holes in the gasket is intended to permit the gasket to aid in retaining the illustrated filters in position. In alternative embodiments, gasket openings may employ a shape different from openings in the lid. For example, the gasket can be shaped to follow the contour of one or more reservoirs with which it is intended to form a fluidic or pressure seal. In some embodiments, a plurality of gaskets may be employed. FIG. 2B shows the same embodiment of the cover assembly illustrated in FIG. 2A with the filters and gasket positioned within (and under) the cover.
(3) FIG. 3A shows one embodiment of the microfluidic device or chip, showing two channels, each with an inlet and outlet port, as well as (optional) vacuum ports. FIG. 3B is a topside schematic of an alternative embodiment of the perfusion disposable or “pod” featuring the transparent (or translucent) cover over the reservoirs, with the chip inserted. The chip can be seeded with cells and then placed in a carrier for insertion into the perfusion disposable.
(4) FIG. 4A shows a side view of one embodiment of a chip carrier (with the chip inside) approaching (but not yet engaging) a side track of a skirt of one embodiment of the perfusion manifold assembly, the carrier aligned at an angle matching an angled front end portion of the side track, the carrier comprising a retention mechanism configured as a upwardly protecting clip. Without being bound by theory, a suitably large angle permits chip engagement without smearing or premature engagement of liquid droplets present on the chip and/or the perfusion manifold assembly during the insertion and alignment processes. FIG. 4B shows a side view of one embodiment of a chip carrier (with the chip inside) engaging a side track of a skirt of one embodiment of (but not yet linked to) the perfusion manifold assembly. FIG. 4C shows a side view of one embodiment of a chip carrier (with the chip inside) fully engaging a side track of a skirt of one embodiment of (but not yet linked to) the perfusion manifold assembly (with an arrow showing the necessary direction of movement to get a snap fit whereby the retention mechanism will engage to prevent movement). FIG. 4D shows a side view of one embodiment of a chip carrier (with the chip inside) detachably linked to the perfusion manifold assembly, where the retention mechanism is engaged to prevent movement.
(5) FIG. 5 is a schematic of one embodiment of a work flow (with arrows showing each progressive step), where the chip is linked (e.g. snapped in) to a disposable perfusion manifold assembly (“perfusion disposable”), which in turn is positioned with other assemblies on a culture module, which is placed in an incubator. In one embodiment, this is a process or method, with each link, connection and positioning being steps done in any order or done simultaneously. In one embodiment, the present invention contemplates that the gas mixer and pressurizer is coupled to and in communication with one or more culture modules in the incubator, or one or more components of one or more culture modules in an incubator, e.g. providing pressurized gas to the pressure manifold of one or more culture modules.
(6) FIG. 6 is a schematic of another embodiment showing the tray (or rack) and sub-tray (or nest) for transporting and inserting the perfusion disposables (PDs) into the pressure module, which has a user interface on the outside of the housing.
(7) FIG. 7A is a schematic of the interior of one embodiment of the pressure module (in an open position), showing the positioning of the tray (or rack), sub-tray (or nest), perfusion disposables (PDs) under a pressure manifold (but not engaging it, so the clearance is sufficient to remove them), with the actuation assembly (including the pneumatic cylinder) above. Three microfluidic devices or perfusion disposables are shown to illustrate, although more (e.g. 6, 9 or 12) are typically used at once. FIG. 7B is a schematic of the interior of one embodiment of the pressure module (in a closed position), showing the positioning of the tray (or rack), sub-tray (or nest), perfusion disposables (PDs) under the pressure manifold (and engaging it), with the actuation assembly (including the pneumatic cylinder) above. Again, three microfluidic devices or perfusion disposables are shown to illustrate, although more (e.g. 6, 9 or 12) are typically used at once.
(8) FIG. 8 is a schematic of one embodiment of a connection scheme comprising a tube connecting manifold permitting four culture modules (three are shown) to be connected inside a single incubator using one or more hub modules (the two circles provide magnified views of a first end and second end of the connections).
(9) FIG. 9 is a schematic of another embodiment of a connection scheme wherein one embodiment of a gas mixer and pressurizer apparatus is connected to a plurality of culture modules in an incubator.
(10) FIG. 10 is a schematic of a control panel of one embodiment of a gas mixer and pressurizer apparatus.
(11) FIG. 11A-B shows two sources of CO.sub.2 gas for one embodiment of the gas mixer and pressurizer apparatus. FIG. 11A shows the CO.sub.2 input line from an external CO.sub.2 tank (the tank is not shown). FIG. 11B shows a CO.sub.2 canister attached to the back of the gas mixer and pressurizer apparatus (the canister is shown).
(12) FIG. 12A-C shows various views of one embodiment of a drip tray. FIG. 12A shows the drip tray opened on the gas mixer and pressurizer apparatus. FIG. 12B is an enlarged view of the drip tray in isolation. FIG. 12C shows the drip tray in a closed position such that it is flush with the edge of the gas mixer and pressurizer apparatus.
(13) FIG. 13 is a schematic showing the overall relationships and functions of one embodiment of the gas mixer and pressurizer apparatus when connected to an external CO.sub.2 tank, which provides 100% CO.sub.2 that is mixed with room air and pressurized. The pressurized gas is passed through the incubator wall to a hub and the gas is sent to the control lines of the pressure manifold (in the culture module) for pressurized flow of fluid (e.g. culture fluid) to the individual pods. The pressurized gas is also used, in one embodiment, to control the movement of the actuation assembly (or component thereof, such as the cylinder) in relationship to the pressure manifold (in the culture module). Finally, in one embodiment, the gas mixer and pressurizer apparatus also has a vacuum pump that allows for control of the (optional) stretching of the membrane within the microfluidic device or chip.
(14) FIG. 14 is a piping and instrumentation diagram of one embodiment of a gas mixer and pressurizer apparatus showing the internal conduits, regulators, switches, pumps and vessels in the process flow, along with the external input and output conduits, together with the instrumentation and control devices, where CO.sub.2 from a supply source is mixed with air to achieve a gas mixture, e.g. 5% CO.sub.2 gas. FIG. 14 also shows a vacuum functionality/capability for the gas mixer and pressurizer apparatus by virtue of a vacuum pump.
DESCRIPTION OF THE INVENTION
(15) Devices, methods and systems are contemplated to provide controlled amounts of gas, gas pressure and vacuum to microfluidic devices culturing cells under flow conditions. In one embodiment, a gas mixer and pressurizer apparatus provides a gas mixture, e.g. 5% CO.sub.2, to culture module comprising a plurality of perfusion manifold assemblies or “pods.” In one embodiment, pressurized gas from the gas mixer and pressurizer apparatus is sent to the control lines of the pressure manifold (in the culture module) for pressurized flow of fluid (e.g. culture fluid, blood, serum or other fluid, or combinations of fluids) to the individual pods. The pressurized gas is also used, in one embodiment, to control the movement of the cylinder (52) of the pressure manifold (50) in the culture module (30), as shown in the figures. Finally, in one embodiment, the gas mixer and pressurizer apparatus also has a vacuum pump that allows for control of the (optional) stretching of the membrane within the microfluidic device or chip. In this manner, the gas mixer and pressurizer apparatus (3) provides three functions at one time.
(16) In one embodiment (as shown in FIGS. 1A, 1B and 1C), the perfusion manifold assembly (10) comprises i) a cover or lid (11) configured to serve as to top of ii) one or more fluid reservoirs (12), iii) a capping layer (13) under said fluid reservoir(s), iv) a fluidic backplane (14) under, and in fluidic communication with, said fluid reservoir(s), said fluidic backplane comprising a fluidic resistor, and v) a projecting member or skirt (15) for engaging the microfluidic device (16) or chip which is preferably positioned in a carrier (17), the chip having one or more microchannels (1) and in fluidic communication with one or more ports (2). The assembly can be used with or without the lid or cover. Other embodiments lack a skirt or projecting member. In one embodiment, the carrier (17) has a tab or other gripping platform (18), a retention mechanism such as a clip (19), and a visualization cutout (20) for imaging the chip. The cutout (20) can enable placing a carrier (e.g. a carrier engaged with the perfusion manifold assembly or “pod” or not so engaged) onto a microscope or other inspection device, allowing the chips to be observed without having to remove the chip from the carrier. In one embodiment, the fluidic resistor comprises a series of switchbacks or serpentine fluid channels.
(17) FIG. 3A shows one embodiment of the microfluidic device or chip (16), showing two microchannels (1), each with an inlet and outlet port (2), as well as (optional) vacuum ports. FIG. 3B is a topside schematic of an alternative embodiment of the perfusion disposable or “pod” (10) featuring the transparent (or translucent) cover (11) over the reservoirs, with the chip (16) inserted. The chip (16) can be seeded with cells and then placed in a carrier (17) for insertion into the perfusion disposable (10).
(18) In one embodiment (FIGS. 2A and 2B), the cover or lid comprises ports such as through-hole ports (36) that are engaged by corresponding pressure points on the pressure surface of the culture module. These ports (36), when engaged, transmit applied pressure inward through the cover and through a gasket (37) and apply the pressure to the fluid in the reservoirs (12) of the perfusion manifold assembly (10). Thus, in this embodiment, pressure is applied through the lid (11) and the lid seals against the reservoir(s). For example, when on applies 1 kPa, this nominal pressure results, in one embodiment, in a flow rate of approximately 30-40 uL/hr. Alternatively, these ports (36), when engaged, move inward on the cover so as to contact the gaskets (i.e. the ports act essentially like plungers).
(19) In one embodiment, the cover or lid is made of polycarbonate. In one embodiment, each through-hole port is associated with a filter (38) (e.g. a 0.2 um filter). In one embodiment, the filters are aligned with holes (39) in a gasket (37) positioned underneath the cover.
(20) In one embodiment, the lid includes a port (35) that allows pneumatic (e.g. vacuum) control of (optional) chip stretching to be communicated through the lid (see FIGS. 2A-2B). It is not intended that the lid be limited to communicating only pneumatic pressure; it is contemplated that the lid can communicate additionally fluidic or electrical interfaces.
(21) In one embodiment, the microfluidic device (16) is detachably linked with the perfusion manifold assembly (10) by a clipping mechanism that temporarily “locks” the microfluidic device, including organ-on-chip devices, in place (FIGS. 4A, 4B, 4C and 4D). In one embodiment, the clipping or “snap fitting” involves a projection (19) on the carrier (17) which serves as a retention mechanism when the microfluidic device (16) is positioned. In one embodiment, the clipping mechanism is similar to the interlocking plastic design of a Lego™ chip and comprises a straight-down clip, friction fit, radial-compression fit or combination thereof. However, in another embodiment, the clipping mechanism is triggered only after the microfluidic device, or more preferably, the carrier (17) comprising the microfluidic device (16), engages the perfusion manifold assembly (or cartridge) on a guide rail, side slot, internal or external track (5) or other mechanism that provides a stable glide path for the device as it is conveyed (e.g. by machine or by hand) into position. The guide rail, side slot, internal or external track (5) or other mechanism can be, but need not be, strictly linear and can be positioned in a projecting member or skirt (15) attached to the main body of the perfusion manifold assembly (10). In one embodiment, the beginning portion of the guide rail (5) (or side slot, internal or external track or other mechanism) comprises an angled slide (7) which provides a larger opening for easier initial positioning, followed by a linear or essentially linear portion (8). In one embodiment, the end portion (9) (close to the corresponding ports of the assembly) of an otherwise linear (or essentially linear) guide rail (5) (or side slot, internal track or other mechanism) is angled (or curves) upward so that there is a combination of linear movement (e.g. initially) and upward movement to achieve linking.
(22) Once a microfluidic device (or “chip”) (16) has docked with the perfusion manifold assembly (10), the assembly-chip combination can be placed into an incubator (31) (typically set at a temperature above room temperature, e.g. 37° C.), or more preferably, into a culture module (30) capable of holding a plurality of assembly-chip combinations, the culture module configured to fit on an incubator shelf (see FIG. 5).
(23) FIG. 6 is a schematic of another embodiment of the culture module (30) showing the tray (or rack) (32) and sub-tray (or nest) (47) for transporting and inserting the perfusion disposables (10) into the culture module, which has two openings (48, 49) in the housing to receive the trays, and a user interface (46) to control the process of engaging the perfusion disposables and applying pressure. A typical incubator (not shown) can hold up to six modules (30).
(24) FIG. 7A is a schematic of the interior of one embodiment of the module (i.e. the housing has been removed), showing the pressure manifold (50) in an open position, with the positioning of the tray or rack (32), sub-tray or nest (47), perfusion disposables (10) under the pressure manifold (50) but not engaging it (so the clearance is sufficient to remove them), with the actuation assembly (51) including the pneumatic cylinder (52) above. FIG. 7B is a schematic of the interior of one embodiment of the module (i.e. the housing has been removed), showing the pressure manifold (50) in a closed position, with the positioning of the tray or rack (32), sub-tray or nest (47), perfusion disposables (10) under the pressure manifold (50) and engaging it, with the actuation assembly (51) including the pneumatic cylinder (52) above. The pressure manifold (50) simultaneously engages all of the perfusion disposables (10) while media perfusion is required or needed. Independent control of the flow rate in the top and bottom channels of the chip (16) can be achieved. The pressure manifold (50) can disengage (without complicated fluid disconnects) as desired to allow removal of the trays (32) or nests (47) for imaging or other tasks. In one embodiment, the pressure manifold (50) can simultaneously disengage from a plurality of perfusion manifold assemblies. In one embodiment, the perfusion disposables (10) are not rigidly fixed inside the nests (47), allowing them to locate relative to the pressure manifold (50) as it closes. In a preferred embodiment, integrated alignment features in the pressure manifold (50) provide guidance for each perfusion disposable (10).
(25) FIG. 8 is a schematic of one embodiment of a connection scheme comprising a tube connecting manifold (82) permitting four culture modules (30) (three are shown) to be connected inside a single incubator (31) using one or more hub modules (the two circles provide magnified views of a first end (83) and second end (84) of the connections).
(26) FIG. 9 is a schematic of another embodiment of a connection scheme wherein one embodiment of a gas mixer and pressurizer apparatus (3) is connected to and in communication with a plurality of culture modules (30) in an incubator (31). Going from left to right, FIG. 9 shows a single culture module (30) with the power cables (26) in an enlarged view, followed by a vacuum hub (27A) and a gas hub (27B). In this embodiment, the gas mixer and pressurizer apparatus (3) provides electricity to the culture modules (30) via the power cables (26). There is an enlarged view associated with the vacuum hub (27A) showing the vacuum port (23A) and the associated vacuum (out) connector line (23B) (which includes a filter). There is also an enlarged view associated with the gas hub (27B) showing the gas port (24A) and the associated mixer gas (out) connector line (24B) (which includes a filter). Still in reference to FIG. 9, three culture modules (30) are shown in an incubator (31) which is in communication with the gas mixer and pressurizer apparatus (3), which is associated with two enlarged views, one showing the culture module power cables (26) connected to the back of the gas mixer and pressurizer (3), and one showing panel with the alarm silence button (25), the CO.sub.2 input (22A), the vacuum (out) connector line (23B) and the mixer gas (out) connector line (24B). In one embodiment, the gas mixer and pressurizer apparatus (3) has a Main Status Indicator light (4) that will display one of three states: pulsing blue, green or black (normal), solid red, orange or yellow (the CO.sub.2 canister is low) and pulsing red, orange or yellow with an audible alarm (error). The Alarm Silence Button (25) on the Control Panel can be pushed to turn off the audible alarm.
(27) FIG. 10 is a schematic of a control panel of one embodiment of a gas mixer and pressurizer apparatus (3), showing the CO.sub.2 canister connection (29), the power input (33A) and on/off switch (33B), the power ports (34) to the culture module, the alarm silence button (25), the mixer gas (out) port (24A) along with the associated warning indicator (24C), the vacuum (output) port (23A) along with the associated warning indicator (23C), and the CO.sub.2 input port (22A) along with the associated warning indicator (22C). The various associated warning indicators on the Control Panel provide more detail in the event of an error state. A power cord (not shown) is connected to the power input (33A) and plugged into a wall power outlet (not shown) in order to provide power to the gas mixer and pressurizer apparatus (3), which in turn provides electricity to the culture modules (30).
(28) FIG. 11A-B shows two sources of CO.sub.2 gas for the gas mixer and pressurizer apparatus (3), which is connected to the culture modules (30) in an incubator (31). FIG. 11A shows the CO.sub.2 input line (22B) from an external CO.sub.2 tank (the tank is not shown). FIG. 11B shows a CO.sub.2 canister (28) attached to the back of the gas mixer and pressurizer apparatus (3).
(29) FIG. 12A-C shows various views of one embodiment of the drip tray (40). FIG. 12A shows the drip tray opened on the gas mixer and pressurizer apparatus (3). FIG. 12B is an enlarged view of the drip tray (40) in isolation. FIG. 12C shows the drip tray (40) in a closed position such that it is flush with the edge of the gas mixer and pressurizer apparatus (3).
(30) FIG. 13 is a schematic showing the overall relationships and functions of one embodiment of the gas mixer and pressurizer apparatus (3) when connected to an external CO.sub.2 tank, which provides 100% CO.sub.2 that is mixed with room air and pressurized. The pressurized gas is passed through the incubator wall to a hub and the gas is sent to the control lines of the pressure manifold (inside a culture module) for pressurized flow to the individual pods. In one embodiment, the pressurized gas is also used to control the movement of the cylinder of the pressure manifold. Finally, the gas mixer and pressurizer apparatus (3) also has a vacuum pump that allows for control of the (optional) stretching of the membrane within the microfluidic device or chip.
(31) FIG. 14 is a piping and instrumentation diagram (P&ID) of one embodiment of a gas mixture and pressurizer apparatus (with the additional function of providing a vacuum), showing the internal conduits, regulators (55A-D), switches (56A-C), filters (57A-E), sensors (58A-F), vacuum pump (53), pressure pump (54), buffer tank (59) and vessels in the process flow, along with the external input and output conduits, together with the instrumentation and control devices (60), where CO.sub.2 from a supply source is mixed with air to achieve a gas mixture, e.g. 5% CO.sub.2 gas. In one embodiment, two sources of CO.sub.2 are contemplated, i.e. a CO.sub.2 canister 28 that is attached to the gas mixture and pressurizer apparatus, and a CO.sub.2 supply from an external tank. In one embodiment, a mass flow mixer (61) is used to mix air with the 100% CO.sub.2 to achieve a 5% CO.sub.2 mixture.
(32) In one embodiment, a complex programmable logic device or CPLD (60) is contemplated as a microprocessor for controlling the various pumps, switches, regulators and the like. For example, the CPLD (60) can monitor the various sensors, e.g. a pressure sensor (58D) in order to assess there is sufficient pressure from the pressure pump (54) (see the dashed lines in FIG. 14). This is just one of many examples. By way of another example, the buffer tank (59) (or accumulator tank) allows the pressure pump (54) to be turned off; when the pressure pump (50) is turned off, the switch (56C) if flipped and the mixed gas output comes from the tank (59). Turning the pressure pump (50) off saves the equipment wear and tear damage. The CPLD (60) controls when the pressure pump (50) is running. In this regard, the CPLD (60) is preferably linked to all of the sensors (58A-F). For example, one sensor (58F) may indicate there is not enough output pressure, and the CPLD (60) will respond, e.g. by activating the pressure pump (54). Another sensor (58E) may indicate there is not enough vacuum, and the CPLD (60) will respond, e.g. by activating the vacuum pump (53).
(33) The CPLD (60) controls switch 56A and switch 56B, configuring them appropriately depending on whether the external CO.sub.2 tank or the connected CO.sub.2 canister (28) is providing the 100% CO.sub.2. The gas mixer and pressurizer apparatus can be connected to both CO.sub.2 sources simultaneously. If the pressure from the external CO.sub.2 source drops below 10 psi (as detected by a sensor linked to the CPLD), the apparatus (via the CPLD) will automatically switch to the canister as the CO.sub.2 source.
(34) The gas mixture and pressurizer apparatus, in one embodiment, has one or more system indicator lights controlled by the CPLD (60). In one embodiment, the light (which can be a logo or other design on the surface of the gas mixture and pressurizer) pulses a neutral color (e.g. black, green or blue) during normal operation. In one embodiment, it pulses at a frequency, e.g. pulsing blue at a frequency of every four to ten seconds (which can be adjusted in some embodiments). When there is a problem or error, the light will change to a bright color (e.g. orange, red or yellow). In some embodiment, the bright color will pulse at a rapid frequency (e.g. pulse red at a frequency of approximately every 2 second). In some embodiments, the light will turn red and stay lit until an operator responds. These lighting states are indicative of different error states for the system. One problem that will trigger the change in color is where the CO.sub.2 canister (28) is low. Another problem that will trigger the change in color of the status indicator light is where a pump fails. As noted above, CPLDs are commercially available and programmable.
Example
(35) In one embodiment, the gas mixer and pressurizer apparatus have the following operating technical parameters:
(36) Power Consumption: 105 W
(37) Electrical Power: 100-240 VAC 50-60 Hz
(38) Gas Input Pressure: 10-20 psi from fixed source/3000 psi from 68 gm gas canister
(39) Gas Output Pressure: 40+/−5 psi
(40) Mixed Gas Flow rate: 130 mL/min maximum
(41) Vacuum Output: 73 KPa minimum
(42) Electrical Output: 4 12 VDC, powers up to four culture modules
