Microfabricated polymeric vessel mimetics

• US 9,670,447 B2
• Filed: 01/28/2014
• Issued: 06/06/2017
• Est. Priority Date: 01/29/2013
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Das, Chandan, Jaeger, Ashley A., Pursley, Randall H., McQueen, Philip G., Pohida, Thomas J., Gottesman, Michael M., Morgan, Nicole Y.
• Primary Examiner(s): Sweet, Thomas J
• Assistant Examiner(s): Schutte, Wade P
• Application Number:
  US14/166,744
• Publication Number:
  US 20140212967A1
• Time in Patent Office: 1,225 Days
• CPC Class Codes: B29D 99/005 Producing membranes
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

The present disclosure is directed to embodiments of microstructured membranes, methods of fabricating microstructured membranes, bioreactors housing microstructured membranes, and methods of using bioreactors and microstructured membranes. In some embodiments, the present disclosure allows culturing of cellular tissues in an environment which more accurately resembles a native environment. In some more specific embodiments, the present disclosure allows culturing of tumor cells on a membrane having a microfabricated pattern which mimics a native vasculature system.

Claims

1. A microfabricated oxygen permeable membrane comprising a base member having a cell culture support surface with solid oxygen-permeable pillars formed thereon that mimic microvasculature in a three-dimensional cell culture, the pillars having respective top surfaces and side surfaces that are oxygen permeable and through which oxygen can diffuse three-dimensionally between and around the pillars, the pillars having a height of greater than 1 μ

m and less than 1 mm to form blood vessel mimetics that mimic the three-dimensional structure of the microvasculature, the base member and the pillars comprising an oxygen-permeable material, the base member including a bottom surface opposite the cell culture support surface and configured to be exposed to oxygen such that oxygen can diffuse from the bottom surface through the oxygen-permeable material toward the cell culture support surface and three-dimensionally through the pillars toward the top surfaces and side surfaces of the pillars, the base member including a barrier between the pillars that reduces oxygen diffusion through the cell culture surface to preferentially direct oxygen flow diffusing through the membrane into and through the pillars.

2. The membrane of claim 1, wherein the pillars are arranged in a microstructured array.

3. The membrane of claim 2, wherein the pillars are cylindrical and have diameters greater than 10 μ

m and less than 100 μ

m, heights greater than 100 μ

m and less than 500 μ

m, and are spaced apart by 200 μ

m.

4. The membrane of claim 3, wherein the pillars have diameters between 25 μ

m and 100 μ

m, heights between 200 μ

m and 350 μ

m, and a shear modulus sufficient to withstand stresses induced by fabrication and cell culturing processes.

5. The membrane of claim 3, wherein the pillars have heights which are between 2 and 10 times larger than their diameters.

6. The membrane of claim 5, wherein the pillars have heights which are 7.5 times larger than their diameters.

7. The membrane of claim 1 wherein the oxygen-permeable material comprises an organosilicon polymer.

8. The membrane of claim 7 wherein the organosilicon polymer comprises hydrophilicized polydimethylsiloxane (PDMS).

9. The membrane of claim 1 wherein the oxygen-permeable material further comprises:

(a) a hydrogel;

(b) an organosilicon polymer;

(c) a crosslinker and a photoinitiator;

or

(d) some combination of (a), (b), and (c).

10. The membrane of claim 1, further comprising a cell culture bioreactor comprising an input chamber including walls forming a sealed input chamber environment, and an output chamber including walls forming a sealed output chamber environment;

wherein the membrane is supported on a support grid and separates the input chamber from the output chamber, and wherein the cell culture support surface is hydrophilic;

wherein the input chamber is adapted for connection to a source of oxygen;

wherein the output chamber comprises an oxygen sink with an outlet; and

wherein oxygen from the input chamber is capable of preferentially flowing into and through the pillars into the output chamber as the oxygen diffuses through the membrane to oxygenate a cell culture on the cell culture support surface.

11. The membrane of claim 10, further comprising:

a culture substrate on the membrane in the output chamber, the cell culture being in the culture substrate; and

wherein the pillars are covered by the culture substrate such that oxygen can diffuse from the pillars through the cell culture support surface into the culture substrate between and around the pillars.

12. The membrane of claim 11, wherein the cell culture includes isolated human tumor cells.

13. The membrane of claim 12, wherein the tumor cells are ovarian carcinoma cells.

14. The membrane of claim 10, wherein the solid pillars are capable of diffusing oxygen through the solid pillars and between and around the solid pillars in an oxygen gradient that decreases with increasing distance from the solid pillars.

15. The membrane of claim 1, wherein the barrier comprises a substantially oxygen-impermeable material that covers the cell culture support surface but does not cover the pillars to preferentially induce flow of oxygen through the pillars.

16. The membrane of claim 1, wherein the barrier comprises a substantially oxygen-impermeable material having openings aligned with the location of the pillars.

17. The membrane of claim 16, wherein a thickness of the base member is less than or equal to an inter-pillar spacing.

18. The membrane of claim 1, wherein the pillars are solid pillars consisting of a silicone hydrogel.

19. The membrane of claim 1, wherein the pillars are solid pillars consisting of polydimethylsiloxane.

20. The membrane of claim 1, wherein the cell culture support surface is hydrophilic to increase adherence of the cell culture on the top and side surfaces of the pillars.

21. The membrane of claim 1, wherein oxygen can diffuse from central regions of the solid pillars through the side surfaces and the top surfaces of the pillars without reacting with the pillars.

22. The membrane of claim 1, wherein the solid oxygen permeable pillars are hydrophilic hydrogel pillars.

23. The membrane of claim 22, wherein the solid oxygen permeable pillars comprise 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS), methacryloxypropyl terminated polydimethylsiloxane, N,N′

-dimethylacrylamide (DMA), N-vinyl-2-pyrrolidone (NVP), or ethylene glycol dimethacrylate (EGDMA).

24. The membrane of claim 1, further comprising a cell culture bioreactor.

25. A cell culture bioreactor comprising:

the microfabricated oxygen permeable membrane of claim 1 that forms blood vessel mimetics;

an input chamber; and

an output chamber separated from the input chamber by the membrane such that the base surface forms a cell culture surface;

wherein the input chamber is adapted for connection to a source of oxygen;

wherein the output chamber comprises an oxygen sink with an outlet; and

wherein oxygen from the input chamber is capable of flowing preferentially through the pillars into the output chamber to oxygenate the cell culture.

26. The bioreactor of claim 25, further comprising:

a culture substrate on the membrane in the output chamber; and

a cellular material in the culture substrate.

27. The bioreactor of claim 26, wherein the cellular material includes human tumor cells.

28. The bioreactor of claim 25, wherein the microfabricated membrane comprises an organosilicon polymer.

29. A method of culturing cells in the bioreactor of claim 10, comprising:

providing cells to be cultured in a culture substrate on the base surface of the microfabricated oxygen permeable membrane; and

maintaining an oxygen gradient between a second surface of the membrane and a surface of the culture substrate separated from the base surface of the membrane to perfuse oxygen through the pillars into the culture substrate.

30. The method of claim 29, wherein the cellular material comprises tumor cells.

31. The method of claim 29, wherein the microfabricated membrane comprises an organosilicon polymer.

32. A microfabricated oxygen permeable membrane comprising an oxygen-permeable material, the membrane having a hydrophilic cell culture support surface forming an array of solid oxygen permeable pillars that mimic microvasculature in a three-dimensional cell culture, the membrane having a bottom surface opposite the cell culture support surface and configured to be exposed to oxygen such that oxygen can diffuse through the oxygen-permeable material from the bottom surface toward the cell culture support surface, and three-dimensionally through the pillars toward respective top surfaces and side surfaces of the pillars, the pillars having a height of greater than 1 μ

m and less than 1 mm that mimic the three-dimensional structure of the microvasculature.

33. The membrane of claim 32, wherein the solid pillars consist of the oxygen-permeable material.