Pathologically relevant tumor microenvironments for high-throughput drug screening

• US 9,097,702 B2
• Filed: 07/28/2009
• Issued: 08/04/2015
• Est. Priority Date: 07/29/2008
• Status: Active Grant

• Current Assignee: Cornell University
• Original Assignee: Cornell University
• Inventors: Fischbach, Claudia
• Primary Examiner(s): Weber, Jon P
• Assistant Examiner(s): Srivastava, Kailash C
• Application Number:
  US12/510,925
• Publication Number:
  US 20100056390A1
• Time in Patent Office: 2,198 Days
• Field of Search: None
• US Class Current: 1/1
• 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 biomimetic tumor system comprising:

a first cell of interest;

a natural or synthetic hydrogel incorporating the first cell of interest, wherein;

the incorporating hydrogel is crosslinked by chemically mediated crosslinking, ionically mediated crosslinking, thermally mediated crosslinking or photocrosslinking,

wherein the incorporating hydrogel forms a roughly spherical bead,

the value for the elastic modulus of the hydrogel is controlled by the extent of crosslinking of the hydrogel, and

the value for the elastic modulus of the hydrogel is in the range of about E=0.5 kPa to E=100 kPa;

a second cell of interest of different cell type from the first cell of interest, wherein the second cell of interest is dispersed on, attached to, bound to, disposed or coated on the surface of the hydrogel incorporating the first cell of interest.

2. The biomimetic tumor system of claim 1, wherein the hydrogel is alginate-based.

3. The biomimetic tumor system of claim 1, wherein the diameter of the bead is 100-3000 μ

m.

4. The biomimetic tumor system of claim 1, comprising a cell adhesion peptide modifying the hydrogel.

5. The biomimetic tumor system of claim 4, wherein the cell adhesion peptide is RGD, YIGSR [SEQ ID NO:

1], or a derivative or modification thereof.

6. The biomimetic tumor system of claim 1, 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.

7. The biomimetic tumor system of claim 1, wherein the first 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.

8. The biomimetic tumor system of claim 7, wherein the tumor cell or cancer cell is a breast tumor or cancer cell.

9. The biomimetic tumor system of claim 1, wherein the second 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.

10. A method for making a three-dimensional biomimetic tumor system comprising:

(a) providing a natural or synthetic hydrogel solution;

(b) providing an isolated first cell of interest;

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

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

wherein step (d) comprises forming the hydrogel into a roughly spherical bead; and

(e) providing an isolated second cell of interest of a different cell type from the first cell of interest; and

wherein the second cell of interest is dispersed on, attached to, bound to, disposed or coated on the surface of the hydrogel.

11. The method of claim 10 wherein the hydrogel solution is comprises alginate.

12. The method of claim 11 wherein the cell density is 0-50×

106 cells/mL alginate hydrogel solution.

13. The method of claim 10 wherein the diameter of the bead is 100-3000 μ

m.

14. The method of claim 10 wherein the crosslinking of step (d) is selected from the group consisting of chemically mediated crosslinking, ionically mediated crosslinking, thermally mediated crosslinking and photocrosslinking.

15. The method of claim 10 wherein step (d) comprises extruding the suspended cell in the hydrogel solution into a crosslinking solution.

16. The method of claim 15 wherein step (d) comprises the step of extruding the suspension into isotonic CaCl2.

17. The method of claim 15 wherein the extruding step is performed under coaxial nitrogen flow.

18. The method of claim 10 comprising the step of modifying the hydrogel solution with a cell adhesion peptide prior to step (d).

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

20. The method of claim 10 wherein the cell density is 0-50×

106 cells/mL hydrogel solution.

21. The method of claim 10 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.

22. The method of claim 10 comprising dynamically culturing the incorporated cell of interest.

23. The method of claim 10 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.

24. The method of claim 23 wherein the tumor cell or cancer cell is a breast tumor or cancer cell.

25. The method of claim 10 wherein the cell culture system comprises a second cell of interest of different cell type from the first cell of interest.