MULTIWELL PLATE WITH INTEGRATED STIRRING MECHANISM

Abstract

This invention describes a design for a multiwell plate that contains integrated pumps that are used to stir each well of the plate. The device employs microfluidic logic technology to drive each peristaltic pump. This enables the plates to run autonomously, requiring only a static vacuum supply for power. The devices are entirely constructed out of low-cost polymers, with no electronics, and yet contains simple digital logic circuits to control the pumps. A stack of these plates may be run continuously in a standard cell culture incubator, allowing high-throughput culture of organoids.

Claims

1. An integrated multiwell stirring plate (500) comprising: a. a plate body (501); b. a plurality of wells (502) embedded within the plate body (501); and c. a plurality of pneumatic, peristaltic pumps (510), each pump comprising: i. a pump inlet (504); ii. a pump outlet (506), fluidly connected with one of the wells (502); and iii. a fluid channel (508), fluidly connecting the pump in line between the pump inlet (504) and the pump outlet (506); wherein the pump (510) is configured to pump a fluid through the fluid channel (508) and out of the pump outlet (506) so as to produce a fluid jet (511) into the well (502), and wherein each jet (511) is configured to impart a convective flow (512) of the fluid within the well (502); and d. one or more microfluidic pneumatic control mechanisms (540) configured to control the pumps (510); wherein both the peristaltic pumps (510) and the control mechanisms (540) are embedded and integrated within the plate body (501).

2. The multiwell stirring plate of claim 1, wherein the jet (511) is angled to agitate the fluid in a flow pattern.

3. The multiwell stirring plate of claim 2, wherein the flow pattern is a rotational flow pattern.

4. The multiwell stirring plate of claim 2, wherein the flow pattern is configured for organoid culture.

5. The multiwell stirring plate of claim 1, wherein the pumps (510) are connected with the control mechanisms (540) via pneumatic lines (520).

6. The multiwell stirring plate of claim 1, wherein each pump (510) is configured to be coupled with a pressure source (530) via a single pneumatic connection (525) so as to be powered by a positive or negative pressure.

7. The multiwell stirring plate of claim 6, wherein a speed of the convective flow is directly proportional to strength of the positive or negative pressure.

8. The multiwell stirring plate of claim 1, wherein each well (502) is fluidly connected to multiple pumps (510).

9. The multiwell stirring plate of claim 1, wherein the pump inlet (504) is fluidly connected to the same well (502) as the pump outlet (506), and wherein the pump (510) is configured to recirculate the fluid in a closed loop.

10. The multiwell stirring plate of claim 1, wherein the control mechanism (540) comprises a microfluidic oscillator circuit (542), comprising: a. a plurality of pneumatic channels (544); and b. one or more positive or negative pressure driven pneumatic inverter logic gates (545) connected in a loop by the pneumatic channels (544); wherein each logic gate (545) exhibits a gain.

11. The multiwell stirring plate of claim 10, wherein each pump (510) comprises a plurality of membrane valves (546) in line with the fluid channel (508), each membrane valve (546) comprising: a. a membrane valve control channel (547); b. a membrane valve input channel (548), fluidly connected in line with the fluid channel (508); and c. a membrane valve output channel (549), fluidly connected in line with the fluid channel (508); wherein when positive or negative pressure is applied to the membrane valve control channel (547), the membrane valve (546) opens allowing the fluid to flow from the membrane valve input channel (548) to the membrane valve output channel (549), and wherein when atmospheric pressure is applied to the membrane valve control channel (547), the membrane valve (546) closes.

12. The multiwell stirring plate of claim 10, wherein each of the one or more inverter logic gates (545) further comprises a pull-up resistor channel (560), wherein the pull-up resistor channel (560) comprises a long narrow channel separating the pressure source (530) from the logic gate (545), wherein each pull-up resistor channel (560) has a pull-up resistance that varies as a function of the length of the long narrow channel, and wherein an oscillation frequency of the pressure oscillator circuit (542) varies as a function of the pull-up resistance.

13. An integrated multiwell stirring plate (500) comprising: a. a plate body (501); b. a plurality of wells (502) embedded within the plate body (501); c. a plurality of pneumatic, peristaltic pumps (510), embedded and integrated within the plate body (501), each pump (510) comprising: i. a pump inlet (504); ii. a pump outlet (506), fluidly connected with one of the wells (502); iii. a fluid channel (508), fluidly connecting the pump in line between the pump inlet (504) and the pump outlet (506); and iv. a plurality of fluid valves (546) within the fluid channel (508), the valves (546) configured to move a fluid within the fluid channel (508); wherein the pump (510) is configured to pump the fluid through the fluid channel (508) and out of the pump outlet (506) so as to produce a fluid jet into the well (502), and wherein the jets (511) are configured to impart a convective flow (512) of the fluid within the well (502); and d. one or more microfluidic pneumatic control mechanisms (540), embedded and integrated within the plate body (501), each control mechanism (540) comprising: i. a microfluidic oscillator circuit (542) comprising: 1. an odd number of pneumatic inverter logic gates (545) connected in a closed loop; and 2. a plurality of nodes (550), each node (550) being located between two logic gates (545) in the loop; and ii. a plurality of valve control channels (547), each control channel (547) fluidly connecting one of the nodes (550) with one of the fluid valves (546) such that the positive or negative pressure at the node (550) is configured to operate the valve (546); wherein the control mechanisms (540) are configured to open and close the plurality of fluid valves (546) in a controlled manner so as to cause peristaltic pumping of the fluid within each fluid channel (508).

14. The multiwell stirring plate of claim 15, wherein the entire multiwell stirring plate (500) is configured to be powered and operated by a single pneumatic connection (525) to a positive or negative pressure source (530).

15. The multiwell stirring plate of claim 15, wherein one of the control mechanisms (540) controls multiple pumps (510).

16. A pneumatic peristaltic pump system (600) comprising: a. a microfluidic substrate (601); b. a peristaltic pump (510), embedded and integrated within the substrate (601), the pump (510) comprising: i. a fluid channel (508); ii. a plurality of pump valves (546) within the fluid channel (508), the pump valves (546) configured to move a fluid within the fluid channel (508); c. a microfluidic pneumatic control mechanism (540), embedded and integrated within the substrate (601) and fluidly connected with the pump (510), the control mechanism (540) comprising: i. a microfluidic oscillator circuit (542) comprising: 1. an odd number of pneumatic inverter logic gates (545) connected in a closed loop; and 2. a plurality of nodes (550), each node (550) being located between two logic gates (545) in the loop; ii. a plurality of valve control channels (547), each control channel (547) fluidly connecting one of the nodes (550) with one of the pump valves (546) such that positive or negative pressure at the node (550) is configured to operate the pump valve (546); wherein the control mechanism (540) is configured to open and close the plurality of pump valves (546) in a controlled manner so as to cause peristaltic pumping to move the fluid within the fluid channel (508), and wherein the entire pump system (600) is configured to be powered and operated by a single pneumatic connection (525) to a positive or negative pressure source (530).

17. The pump of claim 16, wherein a rate of the peristaltic pumping is directly proportional to a strength of the pressure source.

18. The pump of claim 16, wherein each logic gate (545) comprises: a. a valve control channel (547), fluidly connected in line with the closed loop of the oscillator circuit (542); b. a valve input channel (548), fluidly connected in line with atmospheric pressure; c. a valve output channel (549), fluidly connected in line with both the pressure source (530) and the closed loop of the oscillator circuit (542); and d. a pull-up resistor channel (560), fluidly connected in line between the pressure source (530) and the rest of the oscillator circuit (542).

19. The pump of claim 16, wherein each pump valve (546) comprises: a. a valve control channel (547); b. a valve input channel (548), fluidly connected in line with the fluid channel (508); and c. a valve output channel (549), fluidly connected in line with the fluid channel (508); wherein when positive or negative pressure is applied to the valve control channel (547), the pump valve (546) opens allowing the fluid to flow from the valve input channel (548) to the valve output channel (549), and wherein when atmospheric pressure is applied to the valve control channel (547), the valve (546) closes.

20. The pump of claim 19, wherein each pneumatic inverter logic gate (545) further comprises a pull-up resistor channel (560) comprising a long narrow channel separating the pressure source (530) from the logic gate (545), wherein the pull-up resistor channel (560) has a pull-up resistance that varies as a function of a length of the long narrow channel, and wherein an oscillation frequency of the ring oscillator circuit (542) varies as a function of the pull-up resistance.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0013] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0014] FIG. 1A shows a schematic drawing of an integrated multiwell stirring plate of the present invention.

[0015] FIG. 1B shows a schematic drawing of a pump system of the present invention.

[0016] FIG. 2A shows a diagram of a pneumatic oscillator circuit with three inverter logic gates.

[0017] FIG. 2B shows a diagram of an oscillator pump, including a three-inverter ring oscillator circuit coupled with three in-line fluid valves for peristaltic pumping of fluids from a fluid inlet through the three fluid valves to a fluid outlet.

[0018] FIG. 3 shows a graphical representation of the output values at nodes 1, 2, and 3 of FIG. 2B and a graphical and diagrammatic representation of the opening and closing of valves A, B, and C of FIG. 2B as a function of time.

[0019] FIG. 4A shows a diagram of a pneumatic membrane valve of the present invention in which the valve is in the closed position, with the membrane in a default position.

[0020] FIG. 4B shows a diagram of a pneumatic membrane valve of the present invention in which the valve is in the opened position, with the membrane in a deformed position.

[0021] FIG. 5 shows an expanded-view diagram of a pneumatic membrane valve of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Following is a list of elements corresponding to a particular element referred to herein:

[0023] 500 Multiwell stirring plate

[0024] 501 Plate body

[0025] 502 Well

[0026] 504 Pump inlet

[0027] 506 Pump outlet

[0028] 508 Fluid channel

[0029] 510 Pump

[0030] 511 Fluid Jet

[0031] 512 Convective flow

[0032] 520 Pneumatic line

[0033] 525 Pneumatic connection

[0034] 530 Pressure source

[0035] 540 Control mechanism

[0036] 542 Oscillator circuit

[0037] 544 Pneumatic channel

[0038] 545 Logic gate

[0039] 546 Pump valve

[0040] 547 Valve control channel

[0041] 548 Valve input channel

[0042] 549 Valve output channel

[0043] 550 Node

[0044] 560 Pull-up resistor channel

[0045] 570 Control valve

[0046] 571 Membrane

[0047] 572 Valve substrate

[0048] 573 Valve seat

[0049] 574 Chamber wall

[0050] 575 Displacement chamber

[0051] 600 Pump system

[0052] 601 Microfluidic substrate

[0053] In one embodiment, the present invention features an integrated multiwell stirring plate (500). As a non-limiting example, the stirring plate (500) may comprise: a plate body (501); a plurality of wells (502); a plurality of pneumatic, peristaltic pumps (510); and one or more microfluidic pneumatic control mechanisms (540) configured to control the pumps (510). In some embodiments, the plurality of wells (502) may be embedded within the plate body (501). In other embodiments, each pump may comprise: a pump inlet (504); a pump outlet (506); and a fluid channel (508). In some embodiments, the pump outlet (506) may be fluidly connected with one of the wells (502). In other embodiments, the fluid channel (508) may fluidly connect the pump in line between the pump inlet (504) and the pump outlet (506). In still other embodiments, the pump (510) may be configured to pump a fluid through the fluid channel (508) and out of the pump outlet (506) so as to produce a fluid jet (511) into the well (502). In yet other embodiments, each jet (511) may be configured to impart a convective flow (512) of the fluid within the well (502). According to one embodiment, both the peristaltic pumps (510) and the control mechanisms (540) may be embedded and integrated within the plate body (501). In another embodiment the peristaltic pumps (510) are embedded and integrated within the plate body (501) and the control mechanisms (540) sit on a separate chip which is attached to the plate body (501).

[0054] In some embodiments, the jet (511) may be angled to agitate the fluid in a flow pattern. As a non-limiting example, the flow pattern may a rotational flow pattern. In other embodiments, the flow pattern may be configured for organoid culture. As a non-limiting example, the flow pattern may have a speed and direction which promotes growth of an organoid culture.

[0055] In an embodiment, the pumps (510) may be connected with the control mechanisms (540) via pneumatic lines (520). In another embodiment, each pump (510) may be configured to be coupled with a pressure source (530) via a single pneumatic connection (525) so as to be powered by a positive or negative pressure. As a non-limiting example, the negative pressure may be a vacuum pressure. In yet another embodiment, a speed of the convective flow may be directly proportional to strength of the positive or negative pressure.

[0056] According to some embodiments, each well (502) may be fluidly connected to multiple pumps (510). In other embodiments, a pump (510) may be connected to multiple wells (520). In some embodiments, a well may be fluidly connected with multiple pump outlets (506). In some other embodiments, the pump inlet (504) may be fluidly connected to the same well (502) as the pump outlet (506), and the pump (510) may be configured to recirculate the fluid in a closed loop. In still other embodiments, the pump (510) may be configured to circulate the fluid from one well (502) or reservoir to another well (502) or reservoir.

[0057] In one embodiment, the control mechanism (540) may comprise a microfluidic oscillator circuit (542). As a non-limiting example, the oscillator circuit may comprise a plurality of pneumatic channels (544); and one or more positive or negative pressure driven pneumatic inverter logic gates (545) connected in a loop by the pneumatic channels (544). In some embodiments, each logic gate (545) may exhibit a gain.

[0058] In some embodiments, each pump (510) may comprise a plurality of membrane valves (546) in line with the fluid channel (508). As a non-limiting example, each membrane valve (546) may comprise: a membrane valve control channel (547); a membrane valve input channel (548); and a membrane valve output channel (549). In one embodiment, the membrane valve input channel (548) may be fluidly connected in line with the fluid channel (508). In another embodiment, the membrane valve output channel (549) may be fluidly connected in line with the fluid channel (508). In yet another embodiment, when positive or negative pressure is applied to the membrane valve control channel (547), the membrane valve (546) may open to allow the fluid to flow from the membrane valve input channel (548) to the membrane valve output channel (549). In still another embodiment, when atmospheric pressure is applied to the membrane valve control channel (547), the membrane valve (546) may close.

[0059] According to one embodiment, each of the one or more inverter logic gates (545) may further comprise a pull-up resistor channel (560). In a further embodiment, the pull-up resistor channel (560) may comprise a long narrow channel separating the pressure source (530) from the logic gate (545). In another further embodiment, each pull-up resistor channel (560) may have a pull-up resistance that varies as a function of the length of the long narrow channel. In still another further embodiment, an oscillation frequency of the pressure oscillator circuit (542) may vary as a function of the pull-up resistance.

[0060] In an embodiment, the present invention may feature an integrated multiwell stirring plate (500). As a non-limiting example, the stirring plate (500) may comprise: a plate body (501); a plurality of wells (502); a plurality of pneumatic, peristaltic pumps (510); and one or more microfluidic pneumatic control mechanisms (540). In one embodiment, the plurality of wells (502) may be embedded within the plate body (501). In another embodiment, the plurality of pneumatic, peristaltic pumps (510) may be embedded and integrated within the plate body (501). As a non-limiting example, each pump (510) may comprise: a pump inlet (504); a pump outlet (506); a fluid channel (508); and a plurality of fluid valves (546) within the fluid channel (508). In some embodiments, the pump outlet (506) may be fluidly connected with one of the wells (502). In other embodiments, the fluid channel (508) may fluidly connect the pump in line between the pump inlet (504) and the pump outlet (506). In still other embodiments, the fluid valves (546) may be configured to move a fluid within the fluid channel (508). In one embodiment, the pump (510) may be configured to pump the fluid through the fluid channel (508) and out of the pump outlet (506) so as to produce a fluid jet into the well (502). In another embodiment, the jets (511) may be configured to impart a convective flow (512) of the fluid within the well (502). In still another embodiment, the control mechanisms (540) may be embedded and integrated within the plate body (501).

[0061] In some embodiments, each control mechanism (540) may comprise a microfluidic oscillator circuit (542) and a plurality of valve control channels (547). As a non-limiting example, the microfluidic oscillator circuit (542) may comprise an odd number of pneumatic inverter logic gates (545) connected in a closed loop; and a plurality of nodes (550), each node (550) being located between two logic gates (545) in the loop. In one embodiment, each control channel (547) may fluidly connect one of the nodes (550) with one of the fluid valves (546) such that the positive or negative pressure at the node (550) is configured to operate the valve (546). In another embodiment, the control mechanisms (540) may be configured to open and close the plurality of fluid valves (546) in a controlled manner so as to cause peristaltic pumping of the fluid within each fluid channel (508).

[0062] In one embodiment, the entire multiwell stirring plate (500) may configured to be powered and operated by a single pneumatic connection (525) to a positive or negative pressure source (530). As a non-limiting example, this configuration may allow the multiwell stirring plate (500) to be stackable. According to another embodiment, one of the control mechanisms (540) may control multiple pumps (510).

[0063] The present invention may feature a pneumatic peristaltic pump system (600). As a non-limiting example, the pump system (600) may comprise: a microfluidic substrate (601); a peristaltic pump (510), embedded and integrated within the substrate (601); and a microfluidic pneumatic control mechanism (540), embedded and integrated within the substrate (601) and fluidly connected with the pump (510). In one embodiment the pump (510) may comprise a fluid channel (508) and a plurality of pump valves (546) within the fluid channel (508). In another embodiment, the pump valves (546) may be configured to move a fluid within the fluid channel (508). In some embodiments, the microfluidic pneumatic control mechanism (540) may comprise: a microfluidic oscillator circuit (542) and a plurality of valve control channels (547). In a further embodiment, the microfluidic oscillator circuit (542) may comprise: an odd number of pneumatic inverter logic gates (545) connected in a closed loop; and a plurality of nodes (550), each node (550) being located between two logic gates (545) in the loop.

[0064] In some embodiments, each control channel (547) may fluidly connect one of the nodes (550) with one of the pump valves (546) such that positive or negative pressure at the node (550) is configured to operate the pump valve (546). In other embodiments, the control mechanism (540) may be configured to open and close the plurality of pump valves (546) in a controlled manner so as to cause peristaltic pumping to move the fluid within the fluid channel (508). In still other embodiments, the entire pump system (600) may be configured to be powered and operated by a single pneumatic connection (525) to a positive or negative pressure source (530). In yet other embodiments, a rate of the peristaltic pumping may be directly proportional to a strength of the pressure source.

[0065] In one embodiment, the pump system (600) is configured to be powered by positive pressure. In another embodiment, the pump system (600) is configured to be powered by negative pressure. To convert the negative pressure powered embodiments into positive pressure embodiments, the vacuum-powered inverter logic gates may be replaced with positive pressure-powered inverter logic gates. One main difference of the two embodiments is that while the vacuum-powered gates are closed at rest, the positive pressure-powered gates are open at rest.

[0066] In one embodiment, each logic gate (545) may comprise: a valve control channel (547); a valve input channel (548); a valve output channel (549); and a pull-up resistor channel (560). In another embodiment, the valve control channel (547) may be fluidly connected in line with the closed loop of the oscillator circuit (542). In still another embodiment, the valve input channel (548) may be fluidly connected in line with atmospheric pressure. In yet another embodiment, the valve output channel (549), may be fluidly connected in line with both the pressure source (530) and the closed loop of the oscillator circuit (542). In some embodiments, the pull-up resistor channel (560) may be fluidly connected in line between the pressure source (530) and the rest of the oscillator circuit (542).

[0067] In some embodiments, each pump valve (546) may comprise: a valve control channel (547); a valve input channel (548), fluidly connected in line with the fluid channel (508); and a valve output channel (549), fluidly connected in line with the fluid channel (508). In other embodiments, when positive or negative pressure is applied to the valve control channel (547), the pump valve (546) may open allowing the fluid to flow from the valve input channel (548) to the valve output channel (549). According to some other embodiments, when atmospheric pressure is applied to the valve control channel (547), the valve (546) may close.

[0068] According to an embodiment, each pneumatic inverter logic gate (545) may further comprise a pull-up resistor channel (560). As a non-limiting example, the pull-up resistor channel (560) may comprise a long narrow channel separating the pressure source (530) from the logic gate (545). In one embodiment, the pull-up resistor channel (560) may have a pull-up resistance that varies as a function of a length of the long narrow channel. In another embodiment, an oscillation frequency of the ring oscillator circuit (542) may vary as a function of the pull-up resistance.

[0069] As used herein, the term about refers to plus or minus 10% of the referenced number.

[0070] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting essentially of or consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting essentially of or consisting of is met.

[0071] The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.