Methods for high-content drug screening

Patent metadata

Field	Value
Publication number	US-12038430-B2
Application number	US-201917046099-A
Country	US
Kind code	B2
Filing date	Apr 25, 2019
Priority date	Apr 27, 2018
Publication date	Jul 16, 2024
Grant date	Jul 16, 2024

Abstract

Provided herein are methods and systems for screening a candidate agent to determine whether the candidate agent modulates an activity of cultured cells. Compositions for screening a candidate agent are also provided herein.

First claim

What is claimed is:

1. A method of screening a candidate agent to determine whether the candidate agent modulates an activity of cultured cells, the method comprising:
positioning a hydrogel comprising at least one candidate agent in a lumen of a hollow micropillar, wherein the hollow micropillar comprises a first surface having an open end and a second surface having a closed end in contact with a first substrate, wherein the hollow micropillar is orthogonal to the first substrate;
bringing the first surface of the hollow micropillar into communication with a surface of cultured cells on a second substrate to provide an interaction gap between the first surface of the hollow micropillar and the surface of the cultured cells, wherein the first surface of the hollow micropillar is in a spaced apart and in facing relationship with the surface of cultured cells;
adding a solution to the interaction gap such that the at least one candidate agent is released from the hydrogel onto the surface of cultured cells; and
measuring a signal from the cultured cells, wherein the signal indicates whether the at least one candidate agent modulates the activity of the cultured cells.

2. The method of claim 1 , wherein the hydrogel comprises alginate.

3. The method of claim 1 , wherein the at least one candidate agent comprises at least two candidate agents.

4. The method of claim 1 , wherein the at least one candidate agent comprises at least one thousand candidate agents.

5. The method of claim 1 , wherein the positioning comprises filling the lumen of the hollow micropillar such that a surface of the hydrogel in the lumen is coplanar with the first surface of the hollow micropillar.

6. The method of claim 1 , wherein the positioning comprises filling a portion of the lumen of the hollow micropillar.

7. The method of claim 1 , wherein the positioning comprises printing the hydrogel by microinjection in the lumen of the hollow micropillar.

8. The method of claim 1 , wherein the hollow micropillar comprises an inner diameter and an outer diameter.

9. The method of claim 8 , wherein the inner diameter is 50-600 microns.

10. The method of claim 8 , wherein the outer diameter is 60-900 microns.

11. The method of claim 1 , wherein the hollow micropillar comprises a coating layer.

12. The method of claim 11 , wherein the coating layer is hydrophilic.

13. The method of claim 1 , wherein the hollow micropillar has a height of 50-600 microns.

14. The method of claim 1 , wherein the first substrate comprises polydimethylsiloxane.

15. The method of claim 1 , wherein the cultured cells comprise non-epithelial cells.

16. The method of claim 1 , wherein the cultured cells comprise epithelial cells.

17. The method of claim 16 , wherein the cultured cells comprise filter-grown epithelial cells.

18. The method of claim 16 , wherein the epithelial cells express the cystic fibrosis transmembrane conductance regulator chloride (CFTR) channel.

19. The method of claim 1 , wherein the first surface of the hollow micropillar is in a spaced apart and in facing relationship with an apical surface of the cultured cells.

20. The method of claim 1 , wherein the second substrate comprises polydimethylsiloxane.

21. The method of claim 1 , wherein the interaction gap has a width of 10 microns.

22. The method of claim 1 , wherein the interaction gap has a width of 5 microns.

23. The method of claim 1 , wherein the first substrate is held parallel to the second substrate.

24. The method of claim 23 , wherein the first substrate of the hollow micropillar is held parallel to the second substrate by a force.

25. The method of claim 24 , wherein the force is a magnetic force.

26. The method of claim 24 , wherein the force is an electrical force.

27. The method of claim 1 , wherein the first substrate of the hollow micropillar is held parallel to the second substrate by a support device.

28. The method of claim 27 , wherein the support device is a clamp.

29. The method of claim 27 , wherein the support device is a spacer and wherein the spacer is positioned between the first substrate of the hollow micropillar and the second substrate.

30. The method of claim 29 , wherein the spacer has a height greater than the height of the hollow micropillar.

31. The method of claim 29 , wherein the spacer comprises a rectangular shape.

32. The method of claim 1 , wherein the adding comprises adding a buffer solution to the interaction gap.

33. The method of claim 32 , wherein the buffer solution comprises phosphate-buffered saline.

34. The method of claim 32 , wherein the buffer solution comprises chloride.

35. The method of claim 32 , wherein the buffer solution comprises iodide.

36. The method of claim 32 , wherein the buffer solution is manually added.

37. The method of claim 1 , the method further comprising determining an amount of the at least one candidate agent released from the hydrogel onto the surface of cultured cells.

38. The method of claim 37 , wherein the determining comprises performing mass spectrometry.

39. The method of claim 1 , wherein the measuring comprises measuring an optical signal.

40. The method of claim 1 , wherein the measuring comprises measuring a fluorescent signal.

41. The method of claim 1 , wherein the first substrate is in contact with a plurality of hollow micropillars.

42. The method of claim 41 , wherein the plurality of hollow micropillars comprises two or more hollow micropillars.

43. The method of claim 41 , wherein the plurality of hollow micropillars comprises three or more hollow micropillars.

44. The method of claim 41 , wherein the hydrogel comprising the at least one candidate agent is positioned in the lumen of each hollow micropillar of the plurality of hollow micropillars.

45. The method of claim 41 , wherein each hollow micropillar is positioned at a set distance apart such that crosstalk between each hollow micropillar is reduced.

46. The method of claim 45 , wherein crosstalk between each hollow micropillar is less than 2%.

47. The method of claim 45 , wherein the set distance is the same between each hollow micropillar.

48. The method of claim 45 , wherein the set distance is 100-1000 microns.

49. The method of claim 1 , the method further comprising increasing viscosity of the solution added to the interaction gap such that crosstalk is reduced.

50. The method of claim 49 , wherein the increasing viscosity comprises adding methylcellulose to the solution.

Assignees

• Univ California

Inventors

• Verkman Alan S
• Jin Byung-Ju
• Lee Sujin

Classifications

• G01N2800/382

  Cystic fibrosis · CPC title

• B01L2300/0861

  Configuration of multiple channels and/or chambers in a single devices · CPC title

• B01L2300/0819

  Microarrays; Biochips · CPC title

• B01L2200/027

  for microfluidic devices · CPC title

• B01L3/502715

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title