Microencapsulation of Cells in Hydrogels Using Electrostatic Potentials

• US 20080031962A1
• Filed: 10/07/2005
• Published: 02/07/2008
• Est. Priority Date: 10/08/2004
• Status: Active Grant

• Current Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Original Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Inventors: Kinney, Ramsey, Boyan, Barbara, Schwartz, Zvi
• Granted Patent: US 8,202,701 B2
• Time in Patent Office: Days
• US Class Current: 424/490
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0012 Cell encapsulation
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Compositions and methods for producing encapsulated cells having an average diameter of less than about 200 μm are provided. Methods for using the disclosed encapsulated cells are also provided

Claims

1. A method for producing injectable microencapsulated cells comprising:

(a) applying an electrostatic potential to a droplet of cells suspended in a first solution comprising one or more types of monomers, where the electrostatic potential is in an amount sufficient to disrupt the surface tension of the droplet; and

(b) dropping the droplet into a polymerization solution from a distance sufficient to produce a structure encapsulating the cells with an average diameter of less than about 200 μ

m, wherein the polymerization solution comprises a polymerizing agent that promotes the polymerization of the one or more types of monomers.

2. The method of claim 1, wherein the first solution comprises one or more polysaccharides.

3. The method of claim 1, wherein the first solution comprises alginate, agarose, hyaluronic acid, collagen, synthetic monomers, albumin, fibrinogen, fibronectin, vitronectin, laminin, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin, chitosan, heparan, heparan sulfate, or a combination thereof.

4. The method of claim 1, wherein the polymerization agent comprises a divalent cation.

5. The method of claim 1, wherein the polymerization solution comprises at least 20 mM or a polymerizing agent.

6. The method of claim 1, wherein the structure comprises less than about 100 cells.

7. The method of claim 1, wherein the structure comprises between about 60 and about 100 cells.

8. The method of claim 1, wherein the encapsulated cells comprise mammalian cells.

9. The method of claim 8, wherein the mammalian cells comprise differentiated mesenchymal cells, neural cells, endothelial cells, epithelial cells, myoblasts, chondrocytes, myoblasts, osteoblasts, osteoclasts, bone marrow cells, adult stem cells, embryonic stem cells, umbilical cord blood cells, fibroblasts, or a combination thereof.

10. The method of claim 1, wherein the encapsulated cells comprise at least one exogenous nucleic acid.

11. The method of claim 1, wherein the encapsulated cells are viable in culture for about two weeks.

12. The method of claim 1, wherein the number of encapsulated cells does not increase when cultured in vitro or in vivo.

13. The method of claim 1, wherein the structures can withstand shear forces generated during injection of the structures into a host.

14. The method of claim 1, wherein the structure comprises polyamino acids.

15. The method of claim 14, wherein the polyamino acid is selected from the group consisting of poly-L-lysin, poly-L-ornithine, poly-L-arginine, poly-L-asparagine, poly-L-aspartic acid, poly-benzyl-L-aspartate, poly-S-benzyl-L-cysteine, poly-γ

-benzyl-L-glutamate, poly-S-CBZ-L-cysteine, poly-ε

-CBZ-D-lysine, poly-δ

CBZ-DL-ornithine, poly-O-CBZ-L-serine, poly-O-CBZ-D-tyrosine, poly(γ

-ethyl-L-glutamate), poly-D-glutamic acid, polyglycine, poly-γ

-N-hexyl L-glutamate, poly-L-histidine, poly(α

,β

-[N-(2-hydroxyethyl)-DL-aspartamide]), poly-L-hydroxyproline Poly(α

,β

-[N-(3-hydroxypropyl)-DL-aspartamide]), poly-L-isoleucine, poly-L-leucine, poly-D-lysine, poly-L-phenylalanine, poly-L-proline, poly-L-serine, poly-L-threonine, poly-DL-tryptophan, poly-D-tyrosine, or a combination thereof.

45. The method of claim 14, wherein the polyamino acid is selected from the group consisting of poly-L-lysin, poly-L-ornithine, poly-L-arginine, poly-L-asparagine, poly-L-aspartic acid, poly-benzyl-L-aspartate, poly-S-benzyl-L-cysteine, poly-γ

-benzyl-L-glutamate, poly-S-CBZ-L-cysteine, poly-ε

-CBZ-D-lysine, poly-δ

CBZ-DL-ornithine, poly-O-CBZ-L-serine, poly-O-CBZ-D-tyrosine, poly(γ

-ethyl-L-glutamate), poly-D-glutamic acid, polyglycine, poly-γ

-N-hexyl L-glutamate, poly-L-histidine, poly(α

,β

-[N-(2-hydroxyethyl)-DL-aspartamide]), poly-L-hydroxyproline Poly(α

,β

-[N-(3-hydroxypropyl)-DL-aspartamide]), poly-L-isoleucine, poly-L-leucine, poly-D-lysine, poly-L-phenylalanine, poly-L-proline, poly-L-serine, poly-L-threonine, poly-DL-tryptophan, poly-D-tyrosine, or a combination thereof.

52. A cellular array comprising:

(a) a substrate having a plurality of addressable locations; and

(b) a unit of encapsulated cells positioned at each addressable location, wherein each unit of encapsulated cells is produced according to the method of claim 1.

54. A method for treating a host comprising delivering encapsulated cells to host produced according to the method of claim 1.

55. The method of claim 54, wherein the encapsulated cells produce cartilage or cartilage components in the host.

56. The method of claim 54, wherein at least one encapsulated cell expresses a recombinant polypeptide.

57. The method of claim 54, wherein at least one encapsulated cell comprises an exogenous nucleic acid.

58. A method for repairing tissue in a host comprising:

administering encapsulated cells produced by the method of claim 1, wherein the encapsulated cells produce tissue or tissue components in the host.

59. The method of claim 58, wherein the encapsulated cells produce cartilage, smooth muscle, skin, heart tissue, connective tissue, or components thereof.

60. Encapsulated cells produced according to the method of claim 1.

16. A method of positioning a uniform number of mammalian cells at a plurality of locations of a substrate comprising:

(a) applying an electrostatic potential to a succession of droplets of cells suspended in a first solution comprising one or more types of monomers, wherein the electrostatic potential is in an amount sufficient to disrupt the surface tension of each successive droplet;

(b) dropping each droplet into a polymerization solution from a distance sufficient to produce a structure encapsulating a predetermined number of cells, wherein each structure produced comprises the predetermined number of cells plus or minus forty or less cells; and

(c) positioning each structure at an addressable location of the substrate.

17. The method of claim 16, wherein each structure produced comprises the predetermined number of cells plus or minus thirty or less cells.

18. The method of claim 16, wherein each structure produced comprises the predetermined number of cells plus or minus twenty or less cells.

19. The method of claim 16, wherein each structure produced comprises the predetermined number of cells plus or minus ten or less cells.

20. The method of claim 16, wherein each structure produced comprises the predetermined number of cells plus or minus five or less cells.

21. The method of claim 16, wherein the first solution comprises one or more polysaccharides.

22. The method of claim 16, wherein the first solution comprises alginate, agarose, synthetic monomers, or a combination thereof.

23. The method of claim 16, wherein the polymerization solution comprises a polymerizing agent.

24. The method of claim 23, wherein the polymerizing agent comprises a divalent cation.

25. The method of claim 16, wherein the polymerization solution comprises at least 20 mM of a polymerizing agent.

26. The method of claim 16, wherein each structure comprises less than about 100 cells.

27. The method of claim 16, wherein each structure comprises between about 60 and about 100 cells.

28. The method of claim 16, wherein the encapsulated cells comprise mammalian cells.

29. The method of claim 28, wherein the mammalian cells comprise differentiated mesenchymal cells, neural cells, endothelial cells, epithelial cells, myoblasts, chondrocytes, myoblasts, osteoblasts, osteoclasts, bone marrow cells, adult stem cells, embryonic stem cells, umbilical cord blood cells, fibroblasts, or a combination thereof.

30. The method of claim 16, wherein the encapsulated cells comprise at least one exogenous nucleic acid.

31. The method of claim 16, wherein the encapsulated cells are viable in culture for about two weeks.

32. The method of claim 16, wherein the total number of encapsulated cells does not increase when cultured in vitro or in vivo compared to the number of cells originally encapsulated.

33. The method of claim 16, wherein the structure comprises polyamino acids.

34. The method of claim 33, wherein the polyamino acid is selected from the group consisting of poly-L-lysin, poly-L-ornithine, poly-L-arginine, poly-L-asparagine, poly-L-aspartic acid, poly-benzyl-L-aspartate, poly-S-benzyl-L-cysteine, poly-γ

-benzyl-L-glutamate, poly-S-CBZ-L-cysteine, poly-ε

-CBZ-D-lysine, poly-δ

CBZ-DL-ornithine, poly-O-CBZ-L-serine, poly-O-CBZ-D-tyrosine, poly(γ

-ethyl-L-glutamate), poly-D-glutamic acid, polyglycine, poly-γ

-N-hexyl L-glutamate, poly-L-histidine, poly(α

,β

-[N-(2-hydroxyethyl)-DL-aspartamide]), poly-L-hydroxyproline Poly(α

,β

-[N-(3-hydroxypropyl)-DL-aspartamide]), poly-L-isoleucine, poly-L-leucine, poly-D-lysine, poly-L-phenylalanine, poly-L-proline, poly-L-serine, poly-L-threonine, poly-DL-tryptophan, poly-D-tyrosine, or a combination thereof.

53. An array produced by the method of claim 16.

35. A method for selecting a test compound comprising:

(a) contacting encapsulated cells with at least one test compound, where the encapsulated cells are spotted on addressable locations on a substrate and are encapsulated in a structure having a diameter of less than about 200 μ

m; and

(b) selecting the test compound that induces or promotes a change in phenotype of the encapsulated cells contacted with the test compound compared to a control.

36. The method of claim 35, wherein each structure comprise less than about 100 cells.

37. The method of claim 35, wherein the structure comprise between about 60 and about 100 cells.

38. The method of claim 35, wherein the encapsulated cells comprise mammalian cells.

39. The method of claim 35, wherein the mammalian cells comprise differentiated mesenchymal cells, neural cells, endothelial cells, epithelial cells, myoblasts, chondrocytes, myoblasts, osteoblasts, osteoclasts, bone marrow cells, adult stem cells, embryonic stem cells, umbilical cord blood cells, fibroblasts, or a combination thereof.

40. The method of claim 35, wherein the encapsulated cells comprise at least one exogenous nucleic acid.

41. The method of claim 35, wherein the cells are encapsulated in a polysaccharide polymer.

42. The method of claim 41, wherein the polymer comprises alginate, agarose, polyamino acids, or a combination thereof.

43. The method of claim 35, wherein the phenotype change comprises a change in protein synthesis, DNA synthesis, gene expression, color, morphology, size, cell number, cell viability, secretion, or a combination thereof.

44. The method of claim 35, wherein the encapsulated cells comprise polyamino acids.

46. The method of claim 35, wherein the polymerization solution comprises a polymerizing agent.

47. The method of claim 46, wherein the polymerizing agent comprises a divalent cation.

48. The method of claim 47, wherein the divalent cation is calcium.

49. The method of claim 35, wherein the droplet of cells comprises about 60 to about 100 cells suspended in a solution of alginate.

50. The method of claim 35, wherein the polymerization solution comprises at least 20 mM CaCl₂.

51. The method of claim 35, wherein the polymerization solution comprises glucose.

61. A cellular array comprising:

a substrate comprising a plurality of addressable locations;

a unit of encapsulated cells at each addressable location, wherein each unit comprises a predetermined number of cells plus or minus 40 cells or less.

62. A microcapsule comprising a predetermined number of cells and having an average diameter of less than about 200 μ

m.

63. The microcapsule of claim 62, wherein the cells are encapsulated in alginate.

64. The microcapsule of claim 63, further comprising a layer of a polymeric material coating the outer surface of alginate.

65. The microcapsule of claim 64, wherein the outer layer of polymeric material is semi-permeable.

66. The microcapsule of claim 65, wherein the polymeric material comprises polyamino acids.