Ex vivo generated tissue system

Abstract

The present invention relates to methods of generating an ex vivo tissue-like system in a bioreactor system capable of supporting continuous production of, and output of cells and tissues and an ex vivo tissue system made therefrom.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic view of a bioreactor described herein.

FIG. 2 is a depiction of a design for a recirculating flow loop described in the Examples.

FIG. 3 schematically depicts the cross-flow generation of fluid shear in the 3-D bioreactor design

FIGS. 4 A-C depict graphs of cell counts where (A) is sloughed cell harvest output, (B) is Total Sloughed Cell yield in each harvest, and (C) is combined daily sloughed cell harvest.

FIG. 5 shows a cytospin depicting a normal bone marrow morphology obtained from the culture stained with Giemsa orange stain.

FIG. 6A-V depicts various images of scanning electron microscopy (SEM) at day 376, day 268 post re-seeding with FIG. 6A showing a SEM micrograph of a blank matrix.

Claims

1. A method of generating, ex vivo, a matrix of cells that mimics at least one phenotype of a naturally occurring mammalian tissue, the method comprising introducing stem cells, progenitor cells, mature cells or a mixture of these into a bioreactor, the bioreactor comprising:(i) a matrix material configured to receive cells in a first fluid,(ii) a first chamber configured to flow the first fluid including the cells to the matrix material;(iii) a first gas permeable membrane configured to permit a first gas to flow therethrough to the first fluid in the first chamber; and(iv) a gassing chamber operably linked to the first chamber, said gassing chamber providing gas that flows through the first gas permeable membrane;providing a culture media to the bioreactor for a time and under conditions suitable for the cells to arrange on the matrix material of the bioreactor whereby the cells so arranged mimic at least one phenotype of a naturally occurring mammalian tissue.

2. The method according to claim 1, wherein the first chamber of the bioreactor defines a first flow path, and the first flow path extends tangentially to a surface of the matrix material in contact with the first fluid.

3. The method according to claim 2, wherein the bioreactor further comprises: a second chamber configured to flow a second fluid through a second flow path to the matrix material.

4. The method according to claim 3, wherein the matrix material in the bioreactor is disposed between the first and second flow paths.

5. The method according to claim 4, wherein the second flow path in the bioreactor extends tangentially to a surface of the matrix material in contact with the second fluid.

6. The method according to claim 5, wherein the bioreactor further comprises: a second gas permeable membrane configured to permit a second gas to flow therethrough to the second fluid in the second chamber.

7. The method according to claim 1, wherein the bioreactor comprises: a gassing chamber in fluid communication with the first permeable gas member.

8. The method according to claim 1, wherein a support in which the matrix material is disposed in the matrix and the first gas permeable membrane provide a first flow path for the first fluid.

9. The method according to claim 8, wherein the bioreactor further comprises: a gassing chamber in fluid communication with the first permeable gas member, the gassing chamber configured to provide the first gas flowing through the first gas permeable member.

10. The method according to claim 1, wherein the matrix material in the bioreactor allows cells to migrate throughout the material.

11. The method according to claim 1, which further comprises harvesting cells that are released from the matrix material.

12. The method according to claim 11, wherein the cells are harvested from a lower fluid chamber in the bioreactor.

13. The method according to claim 11, which further comprises introducing a media flow through the matrix material of the bioreactor in a manner to cause the release of some of the cells adhered to the matrix material.

14. A method according to claim 11 wherein, the cells harvested are a mixture of different cell types.

15. The method according to claim 1, which further comprises providing a device operably connected to the bioreactor which recirculatates the flow of media through the bioreactor and permits the removal of waste and delivery of fresh media to the bioreactor.

16. The method according to claim 16, wherein the device comprises a reservoir which equilibrates fresh medium with the recycled media prior to being introduced into the bioreactor.

17. The method according to claim 1, wherein the bioreactor comprises one or more ports positioned in a manner to permit collection of cells, media, or both without disturbing the culture.

18. The method according to claim 1, wherein the bioreactor comprises one or more ports positioned in a manner to permit introducing one or more cell culture additives to the bioreactor.

19. The method according to claim 1, wherein cells dissociate from the matrix material and circulate with the media in the bioreactor.

20. The method according to claim 1, wherein the cells are culture for a period of at least one year.

21. A ex vivo generated tissue matrix obtained according to the method of claim

22. A method of providing a therapeutic benefit to a patient, comprising providing the ex vivo generated tissue matrix of claim 21 to the patient to provide a therapeutic benefit thereto.

23. A method of providing a therapeutic benefit to a patient, comprising harvesting the cells cultured according to the method of claim 1 from the bioreactor; andproviding the harvested cultured cells to the patient to provide a therapeutic benefit thereto.

24. A bioreactor, comprising: a matrix material configured to receive cells in a first fluid,a first chamber configured to flow the first fluid including the cells to the matrix material;a gassing chamber operably linked to the first chamber, said gassing chamber providing gas that flows through the first gas permeable membrane; anda first gas permeable membrane configured to permit a first gas to flow therethrough to the first fluid in the first chamber.

25. The bioreactor according to claim 24, wherein the first chamber defines a first flow path, and the first flow path extends tangentially to a surface of the matrix material in contact with the first fluid.

26. The bioreactor according to claim 25, further comprising: a second chamber configured to flow a second fluid through a second flow path to the matrix material.

27. The bioreactor according to claim 26, wherein the matrix material is disposed between the first and second flow paths.

28. The bioreactor according to claim 27, wherein the second flow path extends tangentially to a surface of the matrix material in contact with the second fluid.

29. The bioreactor according to claim 28 further comprising: a second gas permeable membrane configured to permit a second gas to flow therethrough to the second fluid in the second chamber.

30. The bioreactor according to claim 24, further comprising: a gassing chamber in fluid communication with the first permeable gas member.

31. The bioreactor according to claim 24, wherein a support in which the matrix material is disposed and the first gas permeable membrane provide a first flow path for the first fluid.

32. The bioreactor according to claim 31, further comprising: a gassing chamber in fluid communication with the first permeable gas member, the gassing chamber configured to provide the first gas flowing through the first gas permeable member.

33. A method of reproducing cells in the bioreactor according to claim 24, comprising: flowing a first fluid including the cells through a first flow path to a matrix material;immobilizing the cells from the first fluid in the matrix material;delivering a first metabolic gas to the cells in the matrix material through a portion of the first flow path.

34. The method according to claim 33, wherein delivering the first metabolic gas comprises delivering the first metabolic gas to the cells in the matrix material through a first gas permeable membrane that defines a portion of the first flow path.

35. The method according to claim 33, wherein the cells are provided with metabolic gasses and waste removal via diffusion through the gassing membrane.

36. The method according to claim 33, wherein the matrix material allows cells to migrate throughout the material and arrange themselves in a configuration mimicking natural tissue.

37. The method according to claim 33, further comprising harvesting the cells which have released from the scaffold from a lower fluid chamber of the bioreactor.

38. The method according to claim 37, which comprises adjusting the flow of the media flow through the matrix material to release cells from the matrix material.

39. The method according to claim 37, wherein the cells harvested are a mixture of cells of different lineages.

40. The method according to claim 33, which further comprises providing a supporting apparatus operably connected to the bioreactor, which supporting apparatus recirculates media, removes waste, delivers fresh medium or a combination thereof.

41. The method according to claim 33, which further comprises providing a reservoir operably connected to the bioreactor, wherein the reservoir equilibrates media prior to being introduced into the bioreactor.

42. The method according to claim 33, wherein delivering the second metabolic gas comprises delivering the second metabolic gas to the cells in the matrix material through a second gas permeable membrane that defines a portion of the second flow path.

43. The method according to claim 33, further comprising: flowing the first metabolic gas from a chamber through the first gas permeable membrane.