METHOD AND DEVICE FOR ISOLATING CELLS FROM HETEROGENEOUS SOLUTION USING MICROFLUIDIC TRAPPING VORTICES

• US 20130171628A1
• Filed: 09/12/2011
• Published: 07/04/2013
• Est. Priority Date: 09/14/2010
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Di Carlo, Dino, Hur, Soojung C., Mach, Albert J.
• Granted Patent: US 9,133,499 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/082 Active control of flow resistance, e.g. flow controllers
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 29/00 Means for introduction, extraction or recirculation of materials, e.g. pumps pumps per se F04B
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 1/4077 by other techniques involving separation of suspended solids
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

A method of isolating cells includes providing a microfluidic device having at least one microfluidic channel coupled to an inlet and an outlet, the at least one microfluidic channel comprises at least one expansion region disposed along the length thereof. The at least one expansion region is an abrupt increase in a cross-sectional dimension of the at least one microfluidic channel configured to generate a vortex within the at least one expansion region in response to fluid flow. A solution containing a population of cells at least some of which have diameters ≧10 μm flows into the inlet. A portion of cells is trapped within vortex created within the at least one expansion region. The trapped cells may then released from the expansion region.

Claims

1. A method of isolating cells comprising:

providing a microfluidic device having at least one microfluidic channel coupled to an inlet and an outlet, the at least one microfluidic channel comprising at least one expansion region disposed along the length thereof, the at least one expansion region comprising an abrupt increase in a cross-sectional dimension of the at least one microfluidic channel configured to generate a vortex within the at least one expansion region in response to fluid flow;

flowing a solution containing a population of cells into the inlet;

trapping at least some of the cells within the vortex created within the at least one expansion region, the at least some of the cells having diameters ≧

10 μ

m; and

releasing the trapped cells from the plurality expansion regions by reducing the flow rate of solution through the at least one microfluidic channel.

2. The method of claim 1, wherein the solution flowed into the inlet contains a homogenous population of cells.

3. The method of claim 1, wherein the solution flowed into the inlet contains a heterogeneous population of cells.

4. The method of claim 1, wherein the at least one expansion region comprises a leading wall extending at least 45°

with respect to the axis of flow.

5. The method of claim 1, wherein the at least one expansion region has a profile of a rectangle, square, triangle, polygonal, or semi-circle.

6. The method of claim 1, wherein the at least one expansion region comprises an abrupt decrease in the cross-sectional dimension of the at least one microfluidic channel.

7. The method of claim 1, wherein the at least one expansion region comprises a substantially no decrease in the cross-sectional dimension of the at least one microfluidic channel.

8. The method of claim 1, wherein the at least one expansion region comprises a gradual decrease in the cross-sectional dimension of the at least one microfluidic channel.

9. The method of claim 1, wherein the released cells are collected at the outlet.

10. The method of claim 1, wherein cells having a diameter larger than 15 μ

m are trapped within the vortex.

11. The method of claim 1, wherein the trapped cells comprise cancer cells.

12. The method of claim 1, wherein releasing the trapped cells comprises reducing the flow rate of solution through the at least one microfluidic channel to substantially zero and initiating the flow of a flushing solution.

13. The method of claim 3, wherein the heterogeneous population of cells comprises a population containing cancer cells and wherein the cancer cells are trapped within the vortices.

14. The method of claim 4, wherein the solution containing a heterogeneous population of cells comprises one of blood, urine, pleural fluid, and a peritoneal wash.

15. The method of claim 1, wherein the microfluidic device comprises a plurality of microfluidic channels coupled to the inlet and the outlet.

16. The method of claim 1, wherein the plurality of microfluidic channels are arranged in a two-dimensional array.

17. The method of claim 1, wherein the plurality of microfluidic channels are arranged in a three-dimensional array.

18. A method of exchanging solution around isolated cells comprising:

providing a microfluidic device having at least one microfluidic channel coupled to an inlet and an outlet, the at least one microfluidic channel comprising at least one expansion region disposed along the length thereof, the at least one expansion region comprising an abrupt increase in a cross-sectional dimension of the at least one microfluidic channel configured to generate a vortex within the at least one expansion region in response to fluid flow;

flowing a first solution containing a population of cells into the inlet;

trapping at least a portion of the cells within the vortex created within the at least one expansion region; and

flowing one or more solutions different from the first solution into the inlet while continuously maintaining the vortex containing the trapped cells.

19. The method of claim 18, further comprising releasing the trapped cells from the at least one expansion region after exposure to the one or more solutions.

20. The method of claim 19, wherein releasing the trapped cells comprises reducing fluid flow through the microfluidic device.

21. The method of claim 18, further comprising rapidly reducing the flow rate of fluid through the microfluidic device to substantially zero.

22. The method of claim 18, wherein the one or more solutions comprises a solution containing a label selected from the group consisting of a fluorescent label, antibody, nucleic acid dye, and fluorogenic substrate.

23. The method of claim 18, wherein the one or more solutions comprises a fixation agent.

24. The method of claim 18, wherein the one or more solutions comprise a permeabilization agent.

25. The method of claim 19, further comprising flowing a wash solution into the inlet after flowing the one or more solutions and before releasing the trapped cells.

26. The method of claim 18, further comprising collecting the trapped cells.

27. The method of claim 26, wherein the trapped cells have a volumetric concentration that is more than 10 times greater than the volumetric concentration of the cells in the first solution.

28. A method of trapping particles or cells by size comprising:

providing a microfluidic device having at least one microfluidic channel coupled to an inlet and an outlet, the at least one microfluidic channel comprising at least one expansion region disposed along the length thereof, the at least one expansion region comprising an abrupt increase in a cross-sectional dimension of the at least one microfluidic channel configured to generate a vortex within the at least one expansion region in response to fluid flow;

flowing a solution containing a plurality cells or particles into the inlet; and

trapping at least some of the cells or particles within the vortex created within the at least one expansion region, wherein the cells or particles having a size above threshold value are substantially trapped within the vortex and wherein cells or particles having a size below a threshold value substantially pass by the vortex.

29. The method of claim 28, further comprising releasing the trapped cells or particles from the at least one expansion region by reducing the flow rate of solution through the at least one microfluidic channel.

30. The method of claim 28, further comprising rapidly reducing the flow rate of solution through the at least one microfluidic channel to substantially zero.

31. The method of claim 28, wherein the threshold value comprises a diameter of 15 μ

m.

32. The method of claim 28, wherein the at least one expansion region comprises a leading wall extending at least 45°

with respect to the axis of flow.

33. The method of claim 28, wherein the at least one expansion region has a profile of a rectangle, square, triangle, polygonal, or semi-circle.

34. The method of claim 28, wherein the at least one expansion region comprises an abrupt decrease in the cross-sectional dimension of the at least one microfluidic channel.

35. The method of claim 28, wherein the at least one expansion region comprises substantially no decrease in the cross-sectional dimension of the at least one microfluidic channel.

36. The method of claim 28, wherein the at least one expansion region comprises a gradual decrease in the cross-sectional dimension of the at least one microfluidic channel.

37. A microfluidic device comprising:

a substrate containing at least one microfluidic channel coupled to at least one inlet and an outlet, the at least one microfluidic channel comprising at least one expansion region disposed along the length of the at least one microfluidic channel, the at least one expansion region comprising an abrupt increase of at least 80 μ

m in a cross-sectional dimension of the at least one microfluidic channel, the at least one expansion region configured to generate a vortex within the at least one expansion region in response to fluid flow.

38. The microfluidic device of claim 37, wherein the at least one expansion region as a length between 200 μ

m and 2 mm.

39. The microfluidic device of claim 37, further comprising a source of particles configured to flow through the at least one microfluidic channel, the source of particles comprising particles having a diameter ≧

10 μ

m.

40. The microfluidic device of claim 39, wherein the source of particles comprise particles having a diameter ≧

15 μ

m.

41. The microfluidic device of claim 37, further comprising a plurality of expansion regions disposed along the length of the at least one microfluidic channel.

42. The microfluidic device of claim 37, further comprising a plurality of microfluidic channels, wherein each of the plurality of microfluidic channels comprises at least one expansion region.

43. The microfluidic device of claim 40, wherein each of the plurality of microfluidic channels comprises a plurality of expansion regions.

44. The microfluidic device of claim 42, wherein adjacent microfluidic channels are staggered with respect to one another.

45. The microfluidic device of claim 42, wherein the plurality of microfluidic channels are arranged in a two-dimensional array.

46. The microfluidic device of claim 42, wherein the plurality of microfluidic channels are arranged in a three-dimensional array.

47. The microfluidic device of claim 37, wherein the at least one expansion region comprises a leading wall extending at least 45°

with respect to the axis of flow.

48. The microfluidic device of claim 37, wherein the at least one expansion region has a profile of a rectangle, square, triangle, polygonal, or semi-circle.

49. The microfluidic device of claim 37, wherein the at least one expansion region comprises an abrupt decrease in the cross-sectional dimension of the at least one microfluidic channel.

50. The microfluidic device of claim 37, wherein the at least one expansion region comprises substantially no decrease in the cross-sectional dimension of the at least one microfluidic channel.

51. The microfluidic device of claim 37, wherein the at least one expansion region comprises a gradual decrease in the cross-sectional dimension of the at least one microfluidic channel.

52. The microfluidic device of claim 39, wherein the particles comprise cells.

53. A microfluidic system comprising:

a substrate containing at least one microfluidic channel coupled to at least one inlet and an outlet, the at least one microfluidic channel comprising at least one expansion region disposed along the length of the at least one microfluidic channel, the at least one expansion region comprising an abrupt increase in a cross-sectional dimension of the at least one microfluidic channel configured to generate a vortex within the at least one expansion region in response to fluid flow;

at least one pump configured to pump fluid into the at least one inlet containing particles or cells; and

a computer operatively coupled to the at least one pump and configured to adjust the flow rate of fluid passing through the at least one microfluidic channel.

54. The microfluidic system of claim 53, wherein the at least one pump comprises at least one of a syringe pump, pressurized air pump, peristaltic pump, and positive displacement pump.

55. The microfluidic system of claim 53, further comprising a plurality of expansion regions disposed along the length of the at least one microfluidic channel.

56. The microfluidic system of claim 53, further comprising a plurality of microfluidic channels, wherein each of the plurality of microfluidic channels comprises at least one expansion region.

57. The microfluidic system of claim 56, wherein each of the plurality of microfluidic channels comprises a plurality of expansion regions.

58. The microfluidic system of claim 56, wherein adjacent microfluidic channels are staggered with respect to one another.

59. The microfluidic system of claim 56, wherein the plurality of microfluidic channels are arranged in a two-dimensional array.

60. The microfluidic system of claim 56, wherein the plurality of microfluidic channels are arranged in a three-dimensional array.

61. The microfluidic system of claim 53, wherein the at least one expansion region comprises a leading wall extending at least 45°

with respect to the axis of flow.

62. The microfluidic system of claim 53, wherein the at least one expansion region has a profile of a rectangle, square, triangle, polygonal, or semi-circle.

63. The microfluidic system of claim 53, wherein the at least one expansion region comprises an abrupt decrease in the cross-sectional dimension of the at least one microfluidic channel.

64. The microfluidic system of claim 53, wherein the at least one expansion region comprises substantially no decrease in the cross-sectional dimension of the at least one microfluidic channel.

65. The microfluidic system of claim 53, wherein the at least one expansion region comprises a gradual decrease in the cross-sectional dimension of the at least one microfluidic channel.

66. The microfluidic system of claim 53, wherein the width of the at least one microfluidic channel is within the range of 20 μ

m to 200 μ

m.

67. The microfluidic system of claim 53, wherein the at least one expansion region extends away from the at least one microfluidic channel a distance within the range of 80 μ

m to 800 μ

m.

68. The microfluidic system of claim 53, wherein the at least one expansion region extends along the length of the at least one microfluidic channel a distance within the range of 200 μ

m to 2 mm.

69. The microfluidic system of claim 58, wherein adjacent expansion regions are separated by a distance greater than 20 μ

m.

70. The microfluidic system of claim 53, further comprising one or more valves disposed upstream of the at least one inlet, the one or more valves configured to be controlled by the computer.

71. The microfluidic system of claim 53, further comprising one or more valves disposed downstream of the outlet, the one or more valves configured to be controlled by the computer.