Apparatus and method for compound profiling of living cells

• US 6,096,509 A
• Filed: 05/24/1999
• Issued: 08/01/2000
• Est. Priority Date: 08/02/1996
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Axiom Biotechnologies Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Okun, Alex, Okun, Ilya
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/317,793
• Time in Patent Office: 435 Days
• Field of Search: 435/29, 435/4, 435/7.1, 435/283.1, 422/50, 422/68.1, 364/148.05, 364/528.01
• US Class Current: 435/29
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 35/00 Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
• CPC Class Codes: G01N 35/1095 for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00

Abstract

An apparatus and method for real-time measurement of a cellular response of a test compound or series of test compounds (303) on a flowing suspension of cells (349), in which a homogeneous suspension of each member of a series of cell types (349) is combined with a concentration of a test compound (303), directed through a detection zone (355), and a cellular response of the living cells is measured in real time as the cells in the test mixture are flowing through the detection zone (355). The apparatus may be used in automated screening of libraries of compounds, and is capable of real-time variation of concentrations of test and standard compounds and generation of dose/response profiles within a short timespan.

Claims

1. An apparatus for determining the concentration-dependent response of living cells to an agonist, comprising:

a cell suspension input that provides a constant flow of living cells;

an agonist input that provides varying concentrations of an agonist;

a mixing zone, fluidly coupled to said agonist input and said cell suspension input, for mixing said varying concentrations of said agonist with said living cells; and

a detector, fluidly coupled to the mixing zone, for measuring a response of the living cells to the varying concentrations of said agonist as said cells flow past said detector.

2. The apparatus of claim 1, wherein said cell suspension input comprises a means to maintain the cells in suspension.

3. The apparatus of claim 1, wherein said cell suspension input further comprises a flow directing means having an intake port connected to said cell suspension and an output port fluidly connected to said mixing zone.

4. The apparatus of claim 3, wherein said flow directing means is a pump.

5. The apparatus of claim 1, wherein said agonist input comprises at least one reservoir for maintaining an agonist solution.

6. The apparatus of claim 1, wherein said agonist input comprises a multi-well plate for maintaining a plurality of agonist solutions.

7. The apparatus of claim 6, wherein said multi-well plate comprises a plate chosen from 6-well, 12-well, 24-well, 96-well, 33-well and 1536-well plates.

8. The apparatus of claim 1, wherein said agonist input further comprises a proportioning diluting means.

9. The apparatus of claim 8, wherein said proportioning diluting means comprises a first intake port fluidly connected to a reservoir comprising a buffer solution, a second intake port fluidly connected to an agonist reservoir and an output port fluidly connected to said mixing zone for providing a mixture of said buffer solution and said agonist.

10. The apparatus of claim 9, wherein said proportioning diluting means further comprises a 3-way actuating valve that intermittently fluidly couples said first intake port and said second intake port to said output port.

11. The apparatus of claim 10, wherein said actuating valve is controlled by a computer.

12. The apparatus of claim 11, wherein said computer comprises instructions for defining the duty-cycle of said actuating valve.

13. The apparatus of claim 12, wherein said duty-cycle of said actuating valve defines a proportion of said agonist and said buffer solution in said mixture.

14. The apparatus of claim 1, wherein said mixing zone comprises a mixer that mixes said cell suspension with said agonist and said buffer.

15. The apparatus of claim 1, wherein said detector comprises an optical means for measuring changes in the optical properties of said living cells.

16. The apparatus of claim 15, wherein said optical means is a spectrophotometer.

17. The apparatus of claim 15, wherein said optical means is a spectrofluorometer.

18. The apparatus of claim 15, wherein said optical means is a flow cytometer.

19. The apparatus of claim 15, wherein said detector is a microscope.

20. An apparatus for determining the effect of a compound on a population of live cells, comprising:

a cell suspension input that provides a constant flow of living cells;

a first compound input that provides a first compound;

a second compound input that provides varying concentrations of a second compound;

a mixing zone, fluidly coupled to the first compound input, second compound input and cell suspension input, for mixing said varying concentrations of said second compound, said first compound and said living cells; and

a detector, fluidly coupled to the mixing zone, for measuring the response of the living cells to the varying concentrations of said second compound in the presence of said first compound as said cells flow past said detector.

21. The apparatus of claim 20, wherein said first compound is an antagonist of said living cells and said second compound is an agonist of said living cells.

22. The apparatus of claim 20, wherein said first compound is an agonist of said living cells and said second compound is an antagonist of said living cells.

23. The apparatus of claim 20, wherein said cell suspension input comprises a means to maintain the cells in suspension.

24. The apparatus of claim 20, wherein said cell suspension input further comprises a flow directing means having an intake port connected to said cell suspension and an output port fluidly connected to said mixing zone.

25. The apparatus of claim 24, wherein said flow directing means is a pump.

26. The apparatus of claim 20, wherein said first compound input comprises at least one reservoir for maintaining a solution of said first compound.

27. The apparatus of claim 20, wherein said first compound input comprises a multi-well plate for maintaining a plurality of first compound solutions.

28. The apparatus of claim 27, wherein said multi-well plate comprises a plate chosen from 6-well, 12-well, 24-well, 15-well, 33-well and 1536-well plates.

29. The apparatus of claim 20, wherein said second compound input further comprises a proportioning diluting means.

30. The apparatus of claim 29, wherein said proportioning diluting means comprises a first intake port fluidly connected to a reservoir comprising a buffer solution, a second intake port fluidly connected to a second compound reservoir and an output port fluidly connected to said mixing zone for providing a mixture of said buffer solution and said second compound.

31. The apparatus of claim 29, wherein said proportioning diluting means further comprises a 3-way actuating valve that intermittently fluidly couples said first intake port and said second intake port to said output port.

32. The apparatus of claim 31, wherein said actuating valve is controlled by a computer.

33. The apparatus of claim 32, wherein said computer comprises instructions for defining the duty-cycle of said actuating valve.

34. The apparatus of claim 33, wherein said duty-cycle of said actuating valve defines a proportion of said agonist and said buffer solution in said mixture.

35. The apparatus of claim 20, wherein said mixing zone comprises a mixer that mixes said cell suspension with said agonist and said buffer.

36. The apparatus of claim 20, wherein said detector comprises an optical means for measuring changes in the optical properties of said living cells.

37. The apparatus of claim 36, wherein said optical means is selected from the group consisting of:

a spectrophotometer, a spectrofluorometer and a flow cytometer.

38. The apparatus of claim 20, wherein said detector is a microscope.

43. The apparatus of claim 20, wherein said first compound is an agonist of said living cells and said second compound is an antagonist of said living cells.

44. The apparatus of claim 43, wherein said first compound input provides varying concentrations of said agonist.

39. An apparatus for determining the effect of a compound on a population of live cells, comprising:

a cell suspension input that provides a constant flow of living cells;

a first compound input that provides a first compound;

a first mixing zone fluidly coupled to said cell suspension input and said first compound input;

a reaction chamber fluidly connected to said first mixing zone for incubating said living cells with said first compound;

a second mixing zone, fluidly coupled to the reaction chamber and a second compound input; and

a detector, fluidly coupled to said second mixing zone, for measuring the response of the living cells to the presence of said first compound and said second compound as said cells flow past said detector.

40. The apparatus of claim 39, wherein said first compound is an antagonist of said living cells and said second compound is an agonist of said living cells.

41. The apparatus of claim 40, wherein said first compound input provides varying concentrations of said antagonist.

42. The apparatus of claim 40, wherein said second compound input provides varying concentrations of said agonist.

45. The apparatus of claim 40, wherein said second compound input provides varying concentrations of said antagonist.

46. A method for determining a response of living cells to an agonist, comprising:

providing a constant flow of varying concentrations of an agonist;

providing a constant flow of living cells;

combining said flow of varying concentrations of said agonist with said flow of living cells to form a combined flow of said agonist and said living cells;

directing said combined flow of said agonist and said cells to a detector, and measuring a response of the living cells to the varying concentrations of the agonist as said cells flow past said detector.

47. The method of claim 46, wherein providing said flow of varying concentrations of said agonist comprises proportionate diluting of said agonist with a buffer prior to combining said agonist with said cells.

48. The method of claim 47, wherein the proportionate diluting comprises providing a constant volumetric flow rate.

49. The method of claim 47, wherein said proportionate diluting varies with time.

50. The method of claim 46, wherein combining said flow of varying concentrations of said agonist with said flow of living cells comprising introducing said agonist flow and said living cells flow into a mixing zone.

51. The method of claim 46, wherein said combined flow of said agonist and said living cells is directed to said detector through an incubation line.

52. The method of claim 51, wherein said incubation line provides a reaction time of between about 1 second and about 180 seconds between combining said flow in said mixing zone and directing said combined flow passed said detector.

53. The method of claim 46, comprising determining profile parameters of the effect of said varying concentrations of said agonist on said living cells over time.

54. The method of claim 53, comprising determining a concentration-dependent response of said cells to said agonist at said reaction time after combining said agonist with said cells in said mixing zone.

55. The method of claim 54, wherein said response of said cells comprises a change in a spectral characteristic of said cells, induced by said varying concentrations of said agonist.

56. The method of claim 54, comprising determining a dose-response curve of said agonist on said living cells.

57. The method of claim 56, comprising using a computer to determine said profile parameters based on fitting said dose-response curve of said agonist on said living cells, to an equation describing a mechanism whereby said agonist interacts with said cells.

58. A method for determining an effect of a test compound on a population of live cells, comprising:

providing a constant flow of living cells;

providing a constant flow of a constant concentration of a first compound;

providing a constant flow of varying concentrations of a second compound;

combining said constant flow of a constant concentration of said first compound and said constant flow of a varying concentration of said second compound with said constant flow of living cells to form a combined flow;

directing said combined flow to a detector; and

measuring a response over time of the living cells to the varying concentrations of said second compound in the presence of said first compound as said cells flow past said detector.

59. The method of claim 58, wherein said first compound is an antagonist of said living cells and said second compound is an agonist of said living cells.

60. The method of claim 58, wherein said first compound is an agonist of said living cells and said second compound is an antagonist of said living cells.

61. The method of claim 58, wherein providing said flow of varying concentrations of said second compound comprises proportionate diluting of said second compound with a buffer.

62. The method of claim 61, wherein said proportionate diluting comprises providing a constant volumetric flow rate.

63. The method of claim 61, wherein said proportionate diluting varies with time.

64. The method of claim 58, wherein combining said flow of a constant concentration of said first compound with said flow of a varying concentrations of said second compound and with said flow of said living cells comprising introducing said first compound flow, said second compound flow and said living cells flow into a mixing zone.

65. The method of claim 58, wherein said cell response comprises a change in a spectral characteristic of said cells.

66. The method of claim 58, comprising determining profile parameters of the effect of said varying concentrations of said second compound on said living cells over time in the presence of constant concentration of said first compound.

67. The method of claim 66, comprising determining a concentration-dependent response of said cells to said second compound after combining said cells with said first compound and said second compound in said mixing zone.

68. The method of claim 67, wherein said response is between about 1 second and about 180 seconds.

69. The method of claim 66, comprising determining a dose-response curve of said second compound on said living cells in the presence of said constant concentration of said first compound.

70. The method of claim 69, comprising using a computer to determine said profile parameters based on fitting said dose-response curve of said second compound on said living cells in the presence of said constant concentration of said first compound, to an equation describing a mechanism whereby said second compound interacts with said cells in the presence of said first compound.

71. A method for determining an effect of a test compound on a population of live cells, comprising:

providing a constant flow of living cells;

providing a constant flow of an antagonist;

providing a constant flow of an agonist;

combining said flow of said antagonist with said flow of living cells;

combining said combined flow of said antagonist and said cells with an agonist;

directing said combined flow to a detector; and

measuring a response over time of the living cells as said cells flow past said detector.

72. The method of claim 71, comprising providing said flow of said antagonist at varying concentrations, and said flow of said agonist at a constant concentration.

73. The method of claim 71, comprising providing said flow of said antagonist at a constant concentration and said flow of said agonist at varying concentrations.

74. The method of claim 71, wherein combining said flow of said antagonist with said flow of living cells comprising introducing said antagonist flow and said living cells flow into a first mixing zone.

75. The method of claim 74, comprising directing a combined flow of said antagonist and said living cells to a second mixing zone through a first incubation line.

76. The method of claim 75, comprising introducing said flow of said agonist into said second mixing zone.

77. The method of claim 72, wherein measuring the response comprises determining profile parameters of the effect of said varying concentrations of said antagonist on said living cells over time in the presence of constant concentration of said agonist.

78. The method of claim 72, wherein measuring the response comprises determining profile parameters of the effect of said varying concentrations of said agonist on said living cells over time in the presence of a constant concentration of said antagonist.