Apparatus and method for real-time measurement of cellular response

• US 5,804,436 A
• Filed: 08/02/1996
• Issued: 09/08/1998
• 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): Redding, David A.
• Application Number:
  US08/691,356
• Time in Patent Office: 767 Days
• Field of Search: 435/283.1, 435/286.1, 435/286.4, 435/286.5, 435/286.7, 435/287.1, 435/287.3, 435/288.7, 422/81, 422/82.05, 436/63, 436/79, 436/172
• US Class Current: 435/286.1
• 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 on a flowing suspension of cells in which a homogeneous suspension of living cells is combined with a concentration of a test compound, directed through a detection zone, 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. 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 automatically measuring the effect of a plurality of test compounds on living cells, comprising:

a test compound sampler for sequentially and automatically providing samples of multiple test compounds;

a cell suspension source for providing a suspension of living cells;

a mixing chamber, coupled to the test compound sampler and the cell suspension source, for receiving the samples of the test compounds from the test compound sampler, receiving the suspension of living cells from the cell suspension source, and for mixing each test compound with the suspension of living cells so as to provide a respective test solution for each test compound;

a detector, coupled to the mixing chamber, for receiving each respective test solution and for measuring a cellular response of the suspended cells for each said test solution; and

a conduit establishing a flow channel between the mixing chamber and the detector, such that each said test solution flows from the mixing chamber to the detector.

2. The apparatus of claim 1 further comprising a standard compound sampler, coupled to said mixing chamber, for providing a sample of a standard compound having a known effect on said cellular response of said suspended cells, wherein said mixing chamber receives the sample of the standard compound from the standard compound sampler and mixes the standard compound with the suspended cells and said detector measures the cellular response of the suspended cells to the standard compound.

3. The apparatus of claim 2 wherein said mixing chamber simultaneously mixes said test compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

4. The apparatus of claim 2 further comprising:

a first gradient device, coupled to said test compound sampler, for automatically adjusting the concentration level of said test compound transferred to said mixing chamber from said test compound sampler; and

a second gradient device, coupled to said standard compound sampler, for automatically adjusting the concentration level of said standard compound transferred to said mixing chamber from said standard compound sampler.

5. The apparatus of claim 4 further comprising a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to said mixing chamber at an output of the switching valve, for selectively switching the flow of a concentration of said test compound or a concentration of said standard compound or both to the mixing chamber where the test compound and/or the standard compound is then mixed with said suspension of cells.

6. The apparatus of claim 5 further comprising a calibration unit, coupled to said switching valve, wherein the switching valve also selectively switches the flow of a calibration solution provided by the calibration unit into said mixing chamber where the calibration solution is mixed with said suspension of cells.

7. The apparatus of claim 6 wherein said conduit comprises a reaction developing line coupled to the output of said mixing chamber, for receiving a mixture of said cell suspension mixed with either said test compound, said standard compound or said calibration solution, and providing a flow path for the mixture such that there is adequate time for the suspension cells to react with the test compound, the standard compound or the calibration solution, wherein the reaction developing line is further coupled to the input of said detector which receives the mixture from the reaction developing line.

8. The apparatus of claim 7 wherein said detector detects changes in intracellular ion concentration.

9. The apparatus of claim 8 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

10. The apparatus of claim 8 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

11. The apparatus of claim 10 wherein said dye is fluorescent.

12. The apparatus of claim 7 further comprising:

a controller, coupled to said first and second gradient devices, said test compound sampler, said standard compound sampler and said switching valve, for controlling their operation; and

a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to said detector in order to send and receive cell response measurement signals to and from the detector.

13. The apparatus of claim 1 wherein said test compound sampler is an automated robotic sampler capable of selecting a specified test compound from a library of test compounds.

14. The apparatus of claim 13 further comprising:

a controller, coupled to said test compound sampler, for controlling the operation of the test compound sampler; and

a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the test compound sampler from said test compound library.

15. The apparatus of claim 14 further comprising a gradient pump having an input and an output, coupled to said test compound sampler, for adjusting the concentration level of said test compound transferred to said mixing chamber from said test compound sampler, wherein:

said test compound sampler comprises;

a first intake nozzle for receiving said specified test compound;

a second intake nozzle for receiving a buffer solution; and

wherein said gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

16. The apparatus of claim 15 further comprising a standard compound sampler for providing a sample of a standard compound to said mixing chamber.

17. The apparatus of claim 16 wherein said standard compound sampler is an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds.

18. The apparatus of claim 17 further comprising a second gradient pump having an input and an output, coupled to said standard compound sampler, for adjusting the concentration level of said standard compound provided to said mixing chamber from said standard compound sampler, wherein:

said standard compound sampler comprises;

a third intake nozzle for receiving said specified standard compound;

a fourth intake nozzle for receiving a buffer solution; and

wherein said second gradient pump is coupled to the third and fourth intake nozzles and receives specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound.

19. The apparatus of claim 18 further comprising a second mixing chamber coupled to the outputs of the first and second gradient pumps, for receiving and mixing said specified concentrations of said specified test compound and said specified standard compound, such that the output of the second mixing chamber is provided to said first mixing chamber.

20. The apparatus of claim 19 further comprising:

a calibration unit for providing a calibration solution; and

a switching valve, having a first input coupled to said second mixing chamber, a second input coupled to the calibration unit, and an output coupled to said first mixing chamber, for switching between the flow of either a compound mixture from the second mixing chamber or the calibration solution from the calibration unit and then providing the flow to the first mixing chamber where it may be mixed with said cell suspension.

21. The apparatus of claim 20 wherein said calibration unit comprises:

a calibration maximum solution which provides for maximal cell response when mixed with said cell suspension;

a calibration minimum solution which provides for minimal cell response when mixed with said cell suspension;

a diverting valve having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution; and

a pump, coupled to the output of the diverting valve and an input of said switching valve, for pumping either the calibration maximum or calibration minimum solution from the diverting valve into the switching valve.

22. The apparatus of claim 21 further comprising a second pump, coupled to an input of said first mixing chamber, for pumping said suspension of cells from said cell suspension input into the first mixing chamber.

23. The apparatus of claim 22 wherein said conduit comprises a reaction developing line, having an input coupled to an output of said first mixing chamber and a output coupled to an input of said detector, for providing a flow path and a reaction time delay for a mixture received from the first mixing chamber and for providing the mixture to the detector.

24. The apparatus of claim 23 further comprising:

a controller, coupled to said first and second gradient pumps, said test compound sampler, said standard compound sampler and said switching valve, said first and second mixing chambers, said first and second pumps and said diverting valve for controlrmg their operation; and

a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to said detector in order to send and receive cell response measurement signals to and from the detector.

25. The apparatus of claim 24 wherein said detector detects changes in intracellular ion concentration.

26. The apparatus of claim 25 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

27. The apparatus of claim 25 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

28. The apparatus of claim 27 wherein said dye is fluorescent.

29. The apparatus of claim 13 further comprising:

a pump, coupled to the output of said detector, for providing negative pressure to the apparatus;

a proportionating valve, coupled to said test compound sampler, for adjusting the concentration level of said test compound transferred to said mixing chamber from said test compound sampler, wherein said test compound sampler further comprises;

a first intake nozzle for receiving said specified test compound;

a second intake nozzle for receiving a buffer solution; and

the proportionating valve receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

30. The apparatus of claim 29 further comprising:

an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, said standard compound sampler including a third intake nozzle for receiving said specified standard compound and a fourth intake nozzle for receiving a buffer solution; and

a second proportionating valve, coupled to the third and fourth intake nozzles, for receiving specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound.

31. The apparatus of claim 30 further comprising:

a first priming valve, coupled to the output of said first proportionating valve, for receiving said specified concentration of said test compound and providing the test compound to said mixing chamber;

a second priming valve, coupled to the output of said second proportionating valve, for receiving said specified concentration of said standard compound and providing the standard compound to the mixing chamber.

32. The apparatus of claim 31 further comprising:

a calibration unit including a calibration maximum solution which provides for maximal cell response when mixed with said cell suspension and a calibration minimum solution which provides for minimal cell response when mixed with the cell suspension;

a first diverting valve, having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution;

a second diverting valve, having a first input coupled to the output of said mixing chamber and a second input coupled to the output of said first diverting valve, for switching between the flow of either a calibration solution from the first diverting valve or a mixture from the mixing chamber;

a third priming valve, coupled to the output of the second diverting valve, for receiving a mixture from the second diverting valve; and

a second mixing chamber, coupled to the output of the third priming valve, for mixing a mixture provided by the third priming valve with said cell suspension, wherein said cell suspension input and said detector are coupled to the second mixing chamber instead of the first mixing chamber.

33. The apparatus of claim 32 wherein said conduit comprises a reaction developing line, having an input coupled to the output of said second mixing chamber and an output coupled to an input of said detector, for providing a flow path and a reaction time delay for a mixture received from the second mixing chamber before the mixture reaches said detector.

34. The apparatus of claim 33 wherein said cell suspension input comprises:

a cell suspension reservoir;

a buffer reservoir;

a third diverting valve, having a first input coupled to the cell suspension reservoir and a second input coupled to the buffer reservoir, for adjusting the concentration of the cell suspension, wherein the buffer is a diluting agent of the cell suspension; and

a fourth priming valve, coupled to the output of the third diverting valve, for receiving the cell suspension mixture from the third diverting valve and providing this mixture to said second mixing chamber.

35. The apparatus of claim 34 wherein said detector detects changes in intracellular ion concentration.

36. The apparatus of claim 35 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

37. The apparatus of claim 34 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

38. The apparatus of claim 37 wherein said dye is fluorescent.

39. An apparatus for measuring the effect of a test compound on living cells, comprising:

a test compound source;

a cell suspension reservoir providing a suspension of living cells;

a first channel, coupled to the test compound source, through which the test compound can flow;

a second channel, coupled to the cell suspension source, through which the suspension of living cells can flow;

a mixing zone, coupled to the first and second channels, so that the test compound and cell suspension can flow into the mixing zone to provide a desired mixture of the test compound and the cell suspension in said mixing zone;

a fluid flow path through which said mixture can flow from the mixing zone; and

a detector, coupled to the fluid flow path, which measures a cellular response of the suspended cells to the test compound, wherein the desired mixture of the test compound and the cell suspension flows along the fluid flow path from the mixing chamber to the detector.

40. The apparatus of claim 39 further comprising:

a diluent source for providing a diluting agent of the test compound;

a third channel, coupled to the diluent source, through which the diluting agent can flow; and

wherein said mixing zone is coupled to the third channel, to receive the diluting agent so as to provide a desired concentration of said test compound.

41. The apparatus of claim 40 further comprising:

a standard compound source, providing a standard compound having a known effect on said suspension of cells; and

a fourth channel, coupled to the standard compound source, through which the standard compound can flow, wherein said mixing chamber is coupled to the fourth channel, receives the standard compound from the fourth channel, and mixes the standard compound with the suspension of cells; and

wherein said detector measures a cellular response of the suspended cells to the standard compound.

42. The apparatus of claim 41 wherein said mixing chamber simultaneously mixes said test compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

43. The apparatus of claim 42 further comprising a controller, coupled to the first, second, third and fourth channels, which automatically controls a flow of test compound, buffer solution, cell suspension and standard compound through the first, second, third and fourth channels, respectively.

44. The apparatus of claim 39 further comprising a controller, coupled to the first channel, which automatically controls a flow of the test compound through the first channel.

45. An apparatus for measuring the effect of a test compound on living cells, comprising:

a test compound source;

a first gradient device, coupled to the test compound source, for automatically adjusting the concentration level of a test compound transferred to said mixing chamber from said test compound source;

a cell suspension source;

a mixing chamber, coupled to the first gradient device and the cell suspension source, which receives the test compound and a suspension of living cells from the cell suspension source and mixes the test compound with the suspension of living cells; and

a detector, coupled to the mixing chamber, for measuring a cellular response of the suspended cells to the test compound.

46. The apparatus of claim 45 further comprising a standard compound source which provides a standard compound having a known effect on said suspension of cells, wherein said mixing chamber is coupled to the standard compound source, receives the standard compound, and mixes the standard compound with the suspension of cells, and wherein said detector measures a cellular response of the suspended cells to the standard compound.

47. The apparatus of claim 46 wherein said mixing chamber simultaneously mixes said test compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

48. The apparatus of claim 46 further comprising a second gradient device, coupled to said standard compound source, for automatically adjusting the concentration level of said standard compound transferred to said mixing chamber from said suspension of cells.

49. The apparatus of claim 48 further comprising a controller, coupled to the first and second gradient devices, for automatically controlling a flow of the test compound and automatically controlling a flow of the standard compound, into said mixing chamber.

50. The apparatus of claim 45 further comprising a controller, coupled to the first gradient device, for automatically controlling a flow of the test compound into said mixing chamber.