PATHOLOGICALLY RELEVANT TUMOR MICROENVIRONMENTS FOR HIGH-THROUGHPUT DRUG SCREENING

• US 20100056390A1
• Filed: 07/28/2009
• Published: 03/04/2010
• Est. Priority Date: 07/29/2008
• Status: Active Grant

• Current Assignee: Cornell University
• Original Assignee: Cornell University
• Inventors: Fischbach, Claudia
• Granted Patent: US 9,097,702 B2
• Time in Patent Office: Days
• US Class Current: 506/10
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/0036 Nozzles
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00664 Three-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: C40B 60/08 Integrated apparatus specially adapted for both creating and screening libraries
• 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 33/5011 for testing antineoplastic activity

Abstract

A three-dimensional cell culture system is provided comprising one or more cell types of interest incorporated in a natural or synthetic hydrogel. An apparatus for high-throughput drug screening is also provided comprising a plurality of three-dimensional cell culture systems and a dispenser for placing at least one three-dimensional cell culture system into a pre-determined well of a multiwell plate. A method for high-throughput drug screening is also provided that comprises providing a test agent, providing a three-dimensional cell culture system, determining a first cellular response of the cell of interest before culturing in the presence of the agent, culturing the cell of interest in the cell culture system in the presence of the agent, determining a second cellular response of the cell of interest after culturing in the presence of the agent, and comparing the first and second cellular responses.

Claims

1. A three-dimensional cell culture system comprising:

a cell of interest; and

a natural or synthetic hydrogel incorporating the cell of interest.

2. The cell culture system of claim 1 wherein the hydrogel is alginate-based.

3. The cell culture system of claim 1 wherein the incorporating hydrogel forms a roughly spherical bead.

4. The cell culture system of claim 3 wherein the diameter of the bead is 100-3000 μ

m.

5. The cell culture system of claim 1 comprising a cell adhesion peptide modifying the hydrogel.

6. The cell culture system of claim 5 wherein the cell adhesion peptide is RGD, YIGSR, or a derivative or modification thereof.

7. The cell culture system of claim 1 wherein the value for the elastic modulus of the hydrogel is in the range of about E=0.5 kPa to E=100 kPa.

8. The cell culture system of claim 7 wherein the value for the elastic modulus of the hydrogel is about E=0.5-5 kPa, E=5-10 kPa, E=10-20 kPa, E=20-60 kPa, or E=60-100 kPa.

9. The cell culture system of claim 1 wherein the value for the elastic modulus of the hydrogel is controlled by extent of crosslinking of the hydrogel.

10. The cell culture system of claim 1 wherein the hydrogel is crosslinked by chemically mediated crosslinking, ionically mediated crosslinking, thermally mediated crosslinking or photocrosslinking.

11. The cell culture system of claim 1 wherein the cell of interest is selected from the group consisting of a normal cell, benign cell, cancer cell, tumor cell, immortalized cell, genetically engineered cell, stem cell and patient-derived primary cell.

12. The cell culture system of claim 11 wherein the tumor cell or cancer cell is a breast tumor or cancer cell.

13. The cell culture system of claim 1 comprising a second cell of interest of different cell type from the first cell of interest.

14. The cell culture system of claim 13 wherein the second cell of interest is associated with the hydrogel incorporating the cell of interest.

15. An apparatus for high-throughput drug screening comprising:

a plurality of three-dimensional cell culture systems, wherein each cell culture system comprises a cell of interest and a natural or synthetic hydrogel incorporating the cell of interest; and

a first dispenser for placing at least one three-dimensional cell culture system of the plurality into a pre-determined well of a multiwell plate.

16. The apparatus of claim 15 wherein the incorporating hydrogel is alginate-based.

17. The apparatus of claim 15 comprising a movable stage for placing at least one three-dimensional cell culture system into the pre-determined well.

18. The apparatus of claim 17 wherein the movable stage is an X-Y transport stage.

19. The apparatus of claim 15 comprising a second dispenser for placing a test agent into a pre-determined well of a multiwell plate.

20. The apparatus of claim 15 wherein the first dispenser additionally dispenses a test agent into a pre-determined well of a multiwell plate.

21. The apparatus of claim 15 wherein the first dispenser is computer-controlled.

22. The apparatus of claim 19 wherein the second dispenser is computer-controlled.

23. The apparatus of claim 18 wherein the movable stage is computer-controlled.

24. The apparatus of claim 15 wherein the first dispenser is syringe.

25. The apparatus of claim 19 wherein the second dispenser is syringe.

26. A method for screening a test agent comprising:

(a) providing a test agent;

(b) providing a three-dimensional cell culture system comprising a cell of interest and a natural or synthetic hydrogel incorporating the cell of interest;

(c) determining a first cellular response of the cell of interest before culturing in the presence of the agent;

(d) culturing the cell of interest in the cell culture system in the presence of the agent;

(e) determining a second cellular response of the cell of interest after culturing in the presence of the agent; and

(f) comparing the first and second cellular responses.

27. The method of claim 26 wherein a plurality of three-dimensional cell culture systems is provided.

28. The method of claim 27 wherein the cells of interest in the plurality of three-dimensional cell culture systems comprise one or more cell types.

29. The method of claim 26 wherein the agent is a drug, compound, biologically active agent or composition.

30. The method of claim 26 wherein the hydrogel comprises alginate.

31. The method of claim 26 wherein the first or second cellular response is cell growth, proliferation, differentiation or de-differentiation, inhibition of cell growth proliferation, differentiation or de-differentiation, cell death, cell survival, necrosis or inhibition of necrosis, cell-cell interaction, release of a cellular factor, transmitter or agent, release or uptake of an ion, change in cell adhesion, morphology or migration, or a change in an intracellular or extracellular signaling pathway.

32. The method of claim 26 wherein the cell of interest is selected from the group consisting of a normal cell, benign cell, cancer cell, tumor cell, immortalized cell, genetically engineered cell, stem cell and patient-derived primary cell.

33. The method of claim 32 wherein the tumor cell or cancer cell is a breast tumor or cancer cell.

34. The method of claim 26 wherein the cell culture system comprises a second cell of interest of different cell type from the first cell of interest.

35. The method of claim 26 wherein the culturing step (d) comprises dynamically culturing the cell of interest in the cell culture system.

36. The method of claim 26 wherein step (e) comprises the step of releasing the cell of interest from the hydrogel prior to or after determining the second cellular response.

37. The method of claim 36 comprising performing genomic or proteomic analysis on the released cell of interest.

38. A method for testing a response of a cell of interest to a microenvironmental condition of interest comprising:

(a) providing a three-dimensional cell culture system comprising the cell of interest and a natural or synthetic hydrogel incorporating the cell of interest;

(b) determining a first response of the cell of interest before culturing under the microenvironmental condition of interest;

(c) culturing the cell of interest in the cell culture system under the microenvironmental condition of interest;

(d) determining a second response of the cell of interest during, under, or after exposure to, the microenvironmental condition; and

(e) comparing the first response and the second response of the cell of interest.

39. The method of claim 38 wherein the first or second cellular response is cell growth, proliferation, differentiation or de-differentiation, inhibition of cell growth proliferation, differentiation or de-differentiation, cell death, cell survival, necrosis or inhibition of necrosis, cell-cell interaction, release of a cellular factor, transmitter or agent, release or uptake of an ion, change in cell adhesion, morphology or migration, or a change in an intracellular or extracellular signaling pathway.

40. The method of claim 38 wherein the cell of interest is selected from the group consisting of a normal cell, benign cell, cancer cell, tumor cell, immortalized cell, genetically engineered cell, stem cell and patient-derived primary cell.

41. A method for making a three-dimensional cell culture system comprising:

(a) providing a natural or synthetic hydrogel solution;

(b) providing an isolated cell of interest;

(c) suspending the cell in the hydrogel solution; and

(d) incorporating the cell in the hydrogel solution by crosslinking the hydrogel.

42. The method of claim 41 wherein the hydrogel solution is comprises alginate.

43. The method of claim 41 wherein step (d) comprises forming the hydrogel into a roughly spherical bead.

44. The method of claim 43 wherein the diameter of the bead is 100-3000 μ

m.

45. The method of claim 41 wherein the crosslinking of step (d) is selected from the group consisting of chemically mediated crosslinking, ionically mediated crosslinking, thermally mediated crosslinking and photocrosslinking.

46. The method of claim 41 wherein step (d) comprises extruding the suspended cell in the hydrogel solution into a crosslinking solution.

47. The method of claim 46 wherein step (d) comprises the step of extruding the suspension into isotonic CaCl₂.

48. The method of claim 46 wherein the extruding step is performed under coaxial nitrogen flow.

49. The method of claim 41 comprising the step of modifying the hydrogel solution with a cell adhesion peptide prior to step (d).

50. The method of claim 41 wherein the hydrogel solution is 0.5-10% hydrogel.

51. The method of claim 41 wherein the cell density is 0-50×

10⁶ cells/mL hydrogel solution.

52. The method of claim 42 wherein the cell density is 0-50×

10⁶ cells/mL alginate hydrogel solution.

53. The method of claim 41 comprising, after step (d), the step of associating the hydrogel incorporating the cell of interest with a second cell of interest of different cell type.

54. The method of claim 53 comprising pre-coating the hydrogel incorporating the cell of interest with a layer of poly-L-lysine prior to the step of associating the hydrogel with a second cell of interest of different cell type.

55. The method of claim 41 comprising dynamically culturing the incorporated cell of interest.

56. The method of claim 41 wherein the cell of interest is selected from the group consisting of a normal cell, benign cell, cancer cell, tumor cell, immortalized cell, genetically engineered cell, stem cell and patient-derived primary cell.

57. The method of claim 56 wherein the tumor cell or cancer cell is a breast tumor or cancer cell.

58. The method of claim 41 wherein the cell culture system comprises a second cell of interest of different cell type from the first cell of interest.