Micro-Incubation Systems for Microfluidic Cell Culture and Methods

• US 20130171679A1
• Filed: 12/03/2012
• Published: 07/04/2013
• Est. Priority Date: 12/03/2011
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Inventors: Lee, Philip J., Gaige, Terry, Ho, Wei Hsuan (Jessie)
• Granted Patent: US 9,206,384 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/146 Employing pressure sensors
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/048 enabling gas exchange, e.g. vents
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/563 Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• CPC Class Codes: B01L 7/00 Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/36 Means for collection or storage of gas; Gas holders
• CPC Class Codes: C12M 23/40 Manifolds; Distribution pieces fluid transfer means B01L3/563
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 29/24 Recirculation of gas
• CPC Class Codes: C12M 41/12 of temperature controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00
• CPC Class Codes: C12M 41/14 : Incubators; Climatic chambers per se B01L1/00
• CPC Class Codes: C12M 41/18 : Heat exchange systems, e.g. heat jackets or outer envelopes
• CPC Class Codes: C12M 41/34 : of gas
• CPC Class Codes: C12M 41/36 : of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/40 : of pressure
• CPC Class Codes: C12M 41/46 : of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12M 41/48 : Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12Q 1/02 : involving viable microorganisms
• CPC Class Codes: G01N 33/5005 : involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/574 : for cancer

Abstract

A micro-incubator manifold for improved microfluidic configurations and systems and methods of manufacture and operation for a manifold and automated microfluidic systems.

Claims

1. A microincubator manifold comprising:

a pneumatic layer carrying one or more pneumatic channels for conveying pressure or gas to a culture plate;

a gasket layer for providing a removable air-tight seal to the culture plate;

a heat exchange module;

wherein said manifold seals to said culture plate thereby enclosing an incubation volume of gas;

the incubation volume of gas circulating between a culture chamber or observation area of the plate and a heat exchange area of the heat exchange module; and

thereby providing a controlled temperature and gas composition above and in communication with the culture chamber or observation area.

2. The manifold of claim 1 further comprising:

one or more pneumatic connections at said pneumatic layer for interfacing with an external pneumatic controller;

one or more electrical connections to said heat exchange module for providing power and/or sensor and/or control connections between said heat exchange module and an outside controller.

3. The manifold of claim 1 and optionally also any combination of claim 2 to the preceding claim further comprising:

a transparent window that sits above said culture area when the manifold is sealed to the plate, thereby containing the incubation volume while allowing for observation of the cell culture chamber.

4. The manifold of claim 1 and optionally also any combination of claim 2 to the preceding claim further comprising:

cells are cultured in one or more microfluidic culture chambers in the plate that are separated from the incubation volume by one or more gas permeable layers.

5. The manifold of claim 1 and optionally also any combination of claim 2 to the preceding claim further comprising:

the heat exchange module having a circulating fan for circulating gases in the incubation volume.

6. The manifold of claim 1 and optionally also any combination of claim 2 to the preceding claim further comprising:

the heat exchange module having a second fan for cooling a portion of a heat exchanger.

9. An apparatus or system according to any combination of any of the above claims further wherein:

the incubation volume is less than about 10 milliliters.

10. An apparatus or system according to any combination of any of the above claims further wherein:

the incubation volume is maintained at about 37 C and about 5% CO₂.

11. An apparatus or system according to any combination of any of the above claims further wherein:

the microfluidic plate has the dimensions and configuration of a standard cell culture plate.

12. An apparatus or system according to any combination of any of the above claims further wherein:

the manifold can be attached or removed thereby the incubation chamber can be created and removed without affecting the culture chamber integrity.

13. An apparatus or system according to any combination of any of the above claims further wherein:

the incubation contents can be controlled and changed independent of microchannel flow.

32. The micro-incubator of claim 1 and optionally also any combination of claims 2 to 6:

further comprising a microfluidic device separable from the manifold, said microfluidic device consisting of;

one or more cell culture regions where cells and media are introduced by flow through one or more microfluidic channels;

one or more inlet wells or paths for introducing cells or media into said culture region;

one or more outlet wells or paths for removing cells or media into said culture region; and

further wherein one or more of the inlet wells or outlet wells are configured to provide gas or pressure or temperature control to the cell culture regions when used with a gas or temperature control device.

33. The system of claim 32 where the microfluidic device is formatted to a standard 96 well plate, with 24 independent units, where each unit occupies 4 well positions designated as flow inlet, cell/gel inlet, invasion chamber, and flow outlet.

34. The system of claim 32 or 33 where flow from the flow inlet to the flow outlet is driven by a gravity flow, e.g., a flow arising from a liquid height difference between the wells.

35. The system of claim 32 through the preceding claim and any combination thereof wherein the chamber materials are optically transparent to allow visualizing cells using light microscopy.

36. The system of claim 32 through the preceding claim and any combination thereof further comprising:

at least one culture unit, the culture unit comprising;

an inlet reservoir with a fluidic connection to the culture medium flow channel inlet;

an outlet reservoir with a fluidic connection to the culture medium flow channel outlet.

37. The system of claim 32 through the preceding claim and any combination thereof further wherein said system is integrated into a standard well plate.

38. The system of claim 32 through the preceding claim and any combination thereof further wherein said system is integrated into a standard well plate with a culture region situated within an open gas area.

39. The system of claim 32 through the preceding claim and any combination thereof further comprising:

a cell culture array on a standard N-well plate, where each culture unit consists of 3 to 16 well positions;

where N equals any standard culture well plate, including 48, 96, 192, 384 well plates.

7. A micro-incubator comprising:

a microfluidic plate comprising a plurality of wells and at least one culture chamber or observation area;

a manifold;

the manifold comprising a heat exchange module;

wherein said manifold seals to said plate thereby enclosing an incubation volume of gas;

the incubation volume of gas circulating between said culture or observation area of said plate and a heat exchange area of the heat exchange module;

thereby providing a controlled temperature and gas composition above and in communication with said culture or observation area.

8. A system for a microincubator comprising:

a manifold for connecting with a microwell culture system, said manifold comprising;

a heating element;

a gas circulation chamber;

wherein upon mating of the heating element and gas circulation chamber provides a controlled temperature and gas composition above and in communication with a cell culture or observation area.

14. A manifold that is attachable to a microfluidic plate comprising:

an onboard circulating feedback heater;

a gas inlet to alter the composition of the circulated air; and

wherein circulating gas is exposed only to cell culture chambers of the microfluidic plate.

15. The manifold of claim 14 further comprising:

at least one vacuum connector allowing vacuum sealing between said manifold and said well plate.

16. The manifold of claim 14 or claim 15 further wherein:

temperature and gas control is independent of flow control.

17. The manifold of claim 14 and optionally also any combination of claim 15 to the preceding claim further wherein:

the manifold interfaces with a plate with the dimensions or outlook of a standard culture plate.

18. The manifold of claim 14 and optionally also any combination of claim 15 to the preceding claim further wherein:

the manifold is optically transparent over the cell culture region allowing microscopy.

19. The manifold of claim 14 and optionally also any combination of claim 15 to the preceding claim further wherein:

the temperature/gas control mechanism has no parts on the bottom side of the microfluidic plate.

20. The manifold of claim 14 and optionally also any combination of claim 15 to the preceding claim further comprising:

a heat exchange module to control temperature and gas environment of the micro-incubation chamber;

an automatic sealing mechanism on one side of the manifold to provide an air-tight seal to a well plate;

one or more connectors to integrate with a flow control mechanism;

wherein the manifold integrates with optical imaging by microscopy or related methods;

wherein the manifold comprises one or more detectors that allow software to perform automated experiments.

21. A microfluidic cell culture system comprising:

at least one culture unit configured on a culture plate comprising;

a microfluidic culture chamber;

a perfusion barrier separating said chamber from one or more medium flow channels;

a plurality of independent inlet channels;

a perfusion well;

an outlet reservoir;

wherein the culture plate can be sealed to a pneumatic manifold, allowing pressure driven control of the independent inlet channels;

so that solutions can be quickly changed over the cells or can be multiplexed or combined in the culture chamber;

wherein the pneumatic manifold further comprises a heat exchange module for providing a circulating heat controlled gas in a contained gas volume over said culture chamber.

22. The system of claim 21 wherein a high fluidic resistance channel or perfusion barrier is used to reduce flow pressure from the flow inlets to the culture chamber.

23. The system of claim 21 or 22 wherein a high fluidic resistance channel or perfusion barrier is used to contain a gel media to the culture chamber.

24. The system of claim 21 and optionally also any combination of claim 22 to the preceding claim wherein a perfusion barrier mostly surrounds the chamber to separate the chamber from the flow channels.

25. The system of claim 21 and optionally also any combination of claim 22 to the preceding claim wherein gravity pressure is used to drive perfusion.

26. A method of culturing cells comprising:

placing cells into a culture chamber, the culture chamber connected to a plurality of microfluidic channels, the culture and channels configured into a culture unit on a standard well plate or a modified well plate;

placing media into different wells of the plate, said well fluidically connected to a plurality of microfluidic perfusion passages, said microfluidic perfusion passages providing perfusion passages through said cell invasion area;

allowing said cells to culture for an appropriate time;

observing and/or assaying cells and or media in said standard well plate by observing invasion of cells into said invasion region.

27. The method of claim 26 further comprising:

interfacing said standard well plate with a pneumatic heating manifold for providing pressure and gas as necessary to drive cells or media into said cell culture areas, said pneumatic manifold also providing a controlled circulating heated gas environment above said culture area.

28. The method of claims 26 and/or 27 further wherein said pneumatic manifold interfaces with said well plate using a vacuum seal.

29. The method of claim 26 and optionally also any combination of claim 27 to the preceding claim further comprising:

using a pipette to introduce and/or remove fluids from said reservoirs, said fluids passively perfusing through said cell culture areas as a result of gravity and/or differential fluid levels; and

or surface tensions.

30. The method of claim 26 and optionally also any combination of claim 27 to the preceding claim further wherein said steps are performed by fully automated robotic equipment.

31. The method of any combination of claims 26 through the above claim further wherein said steps are performed by fully automated robotic equipment and further wherein said combination of pneumatic cell loading and passive perfusion allows simultaneous automatic culture of a large number of plates using one pneumatic manifold and one automated pipettor because plates do not need to be attached to any equipment during perfusion fluid flow.

40. The system of any one or any combination of the above claims further wherein said system is integrated into a standard well plate.

41. The system of any one or any combination of the above claims further wherein said standard well plate is handled by robotic equipment allowing for automated cell culture and analysis, said robotic equipment being in part equipment designed for use with standard well plates.

42. The system of any one or any combination of the above claims further wherein said standard well plate is configured to pneumatically couple with a pneumatic manifold, said manifold providing pneumatic pressure to drive cells into said cell culture areas, said manifold further comprising at least one additional gas line for introducing a desire gas to said culture chambers.

43. The system of any one or any combination of the above claims further wherein:

said cell culture areas are separated into at least three blocks, each block containing at least two cell culture areas;

each block having an air channel adjacent thereto.

44. The system of any one or any combination of the above claims further wherein:

cells are introduced into said cell culture areas using pneumatic pressure, and thereafter, cells are maintained by perfusion of media using a passive gravity driven fluid flow.

49. The device of claim 44 where each unit of the array is fluidically independent.

50. The device of claim 44 wherein the array complies with SBS microtiter plate standards.

51. The device of claim 44 wherein the array contains more than 16 addressable units.

52. The device of claim 44 wherein the array density is more than 2 units per square centimeter.

53. The device of claim 44 wherein the reservoir spacing is less than 5 mm.

54. The device of claim 44 wherein flow is driven by pneumatic pressure.

55. The device of claim 44 wherein flow is driven by a liquid height difference between one or more of the reservoirs.

56. The device of claim 44 wherein flow is driven by surface tension and/or capillary forces.

57. The device of claim 44 wherein the cells are cultured in a 3D matrix.

58. The device of claim 44 wherein the microfluidic passages are made of gas permeable materials.

59. The device of claim 44 wherein at least some portions of the microfluidic passages and the fluidic reservoirs are made of the same material.

45. The system of any one or any combination of the above claims further comprising:

a large fluidic opening directly connected to said cell culture channels to allow for easier cell flow.

46. The system of any one or any combination of the above claims further wherein:

said perfusion passages have a height and/or width smaller than the diameter of cells, wherein said height is between 20-60 microns and said width is between 2-8 microns.

47. The system of any one or any combination of the above claims further wherein:

said perfusion passages have a height and/or width smaller than the diameter of cells, wherein said height is about 40 microns and said width is about 4 microns.

48. The system of any one or any combination of the above claims further comprising:

a plurality of cell culture units;

each of said units comprising at least two fluidic reservoirs; and

a plurality of said units being fluidically addressable in a cell culture array.

60. An automatic handling system for sealing a pneumatic manifold to a culture plate comprising:

a pneumatic linear actuator cylinder (e.g., a piston);

one or more position sensors;

two arms for holding a manifold from the sides, without blocking a view through the top of a manifold;

a plate carriage and plate grippers for holding a culture plate;

said system configured so that movement of the cylinder causes the plate carriage to move horizontally to a position under said manifold;

said system configured so that movement of the cylinder causes the manifold to descend onto the plate.

61. The system of claim 60 further wherein:

the cylinder is attached to the arms so that the cylinder provides both horizontal and vertical motion.

62. The system of any one or combination of claim 60 to the preceding claim further wherein:

positive seal between the manifold and microfluidic plate is accomplished by applying vacuum through the manifold to the interstitial areas of the plate while the necessary initial downward force is applied mechanically.

63. The system of any one or combination of claim 60 to the preceding claim wherein:

the area above the plate remains clear prior to placement of the manifold to allow access by an automated liquid hander.

64. The system of any one or combination of claim 60 to the preceding claim wherein:

the area above the manifold remains clear during operation of the manifold to allow observation of culture areas during operation.

65. The system of any one or combination of claim 60 to the preceding claim further comprising:

one or more vacuum and pressure sensors as well as plate presence and carriage position sensors allow for intelligent software based control and error handling.

66. A automated plate handler generally as illustrated in FIG. 18 and described herein.

67. A automated plate handler comprising:

a receiver for holding a well plate;

a pneumatic manifold with mechanisms for interfacing with at least one well of the well plate and providing one or more of a gas, a positive or negative pressure, temperature control;

an arm for providing an initial downward force pressing the manifold to the well plate;

one or more vacuum mechanisms for completing a seal between the manifold and the plate by applying vacuum to the interstitial areas of the plate between wells;

68. The automated plate handler according to claim 67 further wherein:

the automated plate handler operates as an auto-sealer and plate reader to allow automated handling of the microfluidic plates.

69. The automated plate handler according to claim 67 further wherein:

the area above the manifold is clear to allow access by an automated liquid handler.

70. The automated plate handler according to claim 67 further comprising:

vacuum and pressure sensors as well as plate presence and carriage position sensors.

71. The automated plate handler according to claim 67 further comprising:

a logic controller and tangibly recorder computer instructions to allow for intelligent software based software control and error handling.

72. The automated plate handler according to claim 67 further wherein the plate handler operates as shown in FIG. 18B.

73. The automated plate handler according to claim 67 further wherein a single pneumatic linear actuator (e.g., a piston) provides both horizontal and vertical motion.

74. The automated plate handler according to claim 67 further wherein a single pneumatic linear actuator (e.g., a piston) provides both horizontal and vertical motion and wherein the single operation provides highly precise control of the manifold and plate.

75. The automated plate handler according to claim 67 further wherein the handler holds the plate in place during operation of the pneumatic manifold.