Method and apparatus for discovering, identifying and comparing biological activity mechanisms

• US 20020159625A1
• Filed: 04/02/2002
• Published: 10/31/2002
• Est. Priority Date: 04/02/2001
• Status: Abandoned Application

• Current Assignee: Atto BioScience, Inc. (Becton Dickinson & Co)
• Original Assignee: Cytoprint, Inc. (Becton Dickinson & Co)
• Inventors: Elling, John W.
• Application Number:
  US10/116,640
• Publication Number:
  US 20020159625A1
• Time in Patent Office: Days
• US Class Current: 382/133
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 35/06 Magnetic means C12M35/02 takes precedence
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability

Abstract

Provided herein are methods and devices for the assessment and identification of cellular biological activity mechanisms; the assessment and identification of the changes in cellular biological activity mechanisms caused by cellular perturbations; the assessment and identification of the cellular function, or biological activity mechanisms of genes and gene products and; and the identification of the many genes and their products that collectively act together in a biological mechanism.

Claims

1. A method of identifying the biological mechanisms affected by a selected gene, comprising

a) culturing a first reference cell under reproducible conditions;

b) processing the first reference cell through an assay in the presence of a perturbation;

c) collecting one or more images of the first cell to detect a first cell assay response to the respective perturbation;

d) culturing a second cell under the reproducible conditions of step a), wherein the first reference cell and the second test-cell are the same cell species, and the second test-cell is altered to modify the expression of the protein encoded by the selected gene;

e) processing the second test-cell through the assay of step b) in the presence of the same perturbation;

f) collecting one or more images of the second cell to detect a second test-cell assay response to the respective perturbation;

g) comparing the one or more images obtained of the first reference cell to the one or more images obtained of the second altered test-cell to identify assay response image changes between the first reference cell and the second test-cell, wherein the assay response image changes correspond to the biological mechanisms affected by the selected gene.

2. The method of claim 1, further comprising repeating steps a) through f);

with a multiplicity of perturbations; and

comparing the multiplicity of images obtained of the first reference cell to the multiplicity of images obtained of the second altered test-cell to identify assay response image changes between the first reference cell and the second test-cell, wherein assay response image changes are used to link the biological mechanisms affected by the selected gene with the biological mechanisms affected by the perturbations.

3. The method of claim 1, wherein the perturbation is selected from any one or more of the forces selected from the group consisting of chemical, biological, mechanical, thermal, electromagnetic, gravitational, nuclear, and temporal.

4. The method of claim 3, wherein the perturbation is treatment with a test-compound.

5. The method of claim 4, wherein the test-compound is known to modulate one or more known biological mechanisms.

6. The method of claim 2, wherein the multiplicity of perturbations is treatment of the cells with a multiplicity of test-compounds.

7. The method of claim 6, wherein the multiplicity of test-compounds are each known to modulate one or more known biological mechanisms.

8. The method of claim 1, wherein the first reference cell is labeled with one or more imaging reagents corresponding to the respective assay, and wherein the second test-cell is labeled with the same one or more imaging reagents of step b).

9. The method of claim 1, wherein steps a) through g) are repeated for a multiplicity of different imaging reagents.

10. The method of claim 8, wherein the one or more imaging reagents are selected from any combination of cellular stains and molecular labels.

11. The method of claim 1, wherein the images are digitally converted to features.

12. The method of claim 2, further comprising correlating the assay responses caused by the test-compounds to the biological mechanisms.

13. The method of claim 1, wherein the expression of the protein encoded by the selected gene is suppressed.

14. The method of claim 13, wherein the expression of the protein encoded by the selected gene is suppressed by knocking out the selected gene.

15. The method of claim 1, wherein the expression of the protein encoded by the selected gene is enhanced.

16. The method of claim 1, wherein a series of images are collected over time to assess the temporal behavior of the first and second cells.

17. The method of claim 16, wherein the images are collected after multiple times, during the same assay experiment.

18. The method of claim 16, wherein the cells are fixed prior to collecting the images.

19. The method of claim 16, wherein the images are collected at different times on different assay experiments of the same cell species.

20. The method of claim 1, wherein the images collected are of different assay experiments of same cell type subject to the same perturbation at different quantities.

21. The method of claim 20, wherein the perturbation is a test-compound administered at different concentrations.

22. The method of claim 1, wherein the images are collected from different locations within the first and second cells.

23. The method of claim 1, wherein the images are collected from different locations within the assay container containing the first and second cells.

24. The method of claim 1, wherein the first and second cells are cell lines.

25. The method of claim 1, wherein the assay response image changes are associated with the respective perturbation and stored in a database.

26. The method of claim 1, further comprising repeating steps a) through f);

with a multiplicity of cell types; and

comparing the multiplicity of images obtained of the multiplicity of first reference cells to the multiplicity of images obtained of the multiplicity of second altered test-cells to identify assay response image changes between the multiplicity of first reference cells and the multiplicity of second test-cells, wherein assay response image changes correspond to the biological mechanisms affected by the selected gene in the particular cell type in which a change is detected.

27. The method of claim 2, further comprising repeating steps a) through f);

with a multiplicity of cell types; and

comparing the images obtained of the multiplicity of first reference cell types to the images obtained of the multiplicity of second altered test-cell types to identify assay response image changes that differ between the second test-cell types, wherein assay response image changes correspond to the biological mechanisms affected by the selected gene in the particular cell type.

29. The method of claim 28, further comprising repeating steps a) through g);

with a multiplicity of perturbations.

30. The method of claim 29, further comprising identifying shared patterns of assay response image changes between the multiplicity of perturbations and identifying within the shared patterns, a specific sub-pattern of assay response image changes, wherein the sub-pattern of assay response image changes corresponds to an individual biological mechanism or a subset of all biological mechanisms affected by the subgroup of perturbations.

31. The method of claim 30, wherein the specific sub-pattern of assay response image changes is identified using one or more statistical clustering methods.

32. The method of claim 31, wherein the one or more statistical clustering methods is selected from the group consisting of fuzzy-clustering and multi-domain clustering.

33. The method of claim 28, wherein the perturbation is selected from any one or more of the forces selected from the group consisting of chemical, biological, mechanical, thermal, electromagnetic, gravitational, nuclear, and temporal.

34. The method of claim 33, wherein the perturbation is treatment with a test-compound.

35. The method of claim 34, wherein the test-compound is known to modulate one or more known biological mechanisms.

36. The method of claim 29, wherein the multiplicity of perturbations is treatment of the cells with a multiplicity of test-compounds.

37. The method of claim 36, wherein the multiplicity of test-compounds are each known to modulate one or more known biological mechanisms.

38. The method of claim 28, wherein the first reference cell is labeled with one or more imaging reagents corresponding to the respective assay, and wherein the second test-cell is labeled with the same one or more imaging reagents of step b).

39. The method of claim 28, wherein steps a) through g) are repeated for a multiplicity of different imaging reagents.

40. The method of claim 28, wherein a series of images are collected over time to assess the temporal behavior of the first and second cells.

41. The method of claim 40, wherein the images are collected after multiple times, during the same assay experiment.

42. The method of claim 41, wherein the cells are fixed prior to collecting the images.

43. The method of claim 40, wherein the images are collected at different times on different assay experiments of the same cell species.

44. The method of claim 28, wherein the images collected are of different assay experiments of same cell type subject to the same perturbation at different quantities.

45. The method of claim 44, wherein the perturbation is a test-compound administered at different concentrations.

46. The method of claim 28, wherein the images are collected from different locations within the first and second cells.

47. The method of claim 28, wherein the images are collected from different locations within the first and second cells.

48. The method of claim 28, wherein the images are collected from different locations within the assay container containing the first and second cells.

49. The method of claim 28, wherein the first and second cells are cell lines.

50. The method of claim 28, wherein the assay response image changes are associated with the respective perturbation and stored in a database.

51. An imaging device suitable for conducting the method of claim 1.

28. A method of producing a fingerprint of assay responses caused by a perturbation, comprising

a) culturing a first reference cell under reproducible conditions;

b) processing the first reference cell through a multiplicity of assay experiments in the absence of a perturbation;

c) collecting one or more images of the first reference cell to detect a first cell assay response to the respective assays;

d) culturing a second test-cell under the reproducible conditions of step a), wherein the first reference cell and the second test-cell are the same cell species;

e) processing the second test-cell through the same multiplicity of assay experiments of step b) in the presence of a perturbation;

f) collecting one or more images of the second test-cell to detect a second test-cell assay response to the respective perturbation;

g) comparing the one or more images obtained of the first reference cell to the one or more images obtained of the second test-cell to identify assay response image changes between the first reference cell and the second test-cell, wherein the assay response image changes correspond to a fingerprint of assay responses caused by the perturbation.

52. An imaging device suitable for conducting the method of claim 28.