Microcapsules and composite microreactors for immunoisolation of cells

• US 6,126,936 A
• Filed: 03/10/1995
• Issued: 10/03/2000
• Est. Priority Date: 03/10/1995
• Status: Expired due to Fees

• Current Assignee: Biohybrid Technologies Incorporated
• Original Assignee: Biohybrid Technologies Incorporated
• Inventors: Kuhtreiber, Willem M., Lanza, Robert P., Chick, William L.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US08/402,209
• Time in Patent Office: 2,034 Days
• Field of Search: 435/174, 435/177, 435/178, 435/180, 435/182, 435/240.2, 435/240.23, 435/382, 435/395, 435/397, 424/93.7
• US Class Current: 424/93.7
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0012 Cell encapsulation

Abstract

Microcapsules and composite microreactors are prepared that immunoisolate living cells such as islet cells or genetically engineered cells. A reduced volume microcapsule is formed by coating a gel matrix particle with a polyamino acid of 15,000 daltons or less molecular weight to reduce volume of the particle by at least 30% as compared to volume prior to coating. A composite microreactor includes the microcapsule containing cell embedded in a gel matrix and provides a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from containing the living cell. A double composite microreactor includes an internal particle that includes an internal particle gel matrix containing a living cell and having a coating, a particle that includes the internal particle embedded in a particle gel matrix and a coating, and a gel super matrix in which the particle is embedded. At least one of the coatings is a volume reducing coating of polyamino acid of 15,000 daltons or less molecular weight. The gel matrices may be alginate, the polyamino acid may be polylysine or polyornithine, and at least one of the gel matrices or coatings may be treated by aging for between 2 hours and 14 days. The internal particle, particle and composite microreactor may have diameters respectively between 50 and 700 microns, 400 and 800 microns and 300 and 1500 microns. to be implanted so there is little or no need for immunosuppressant or antifibrotic drugs. A reduced volume microcapsule is formed by coating a gel matrix particle with a polyamino acid of 15,000 daltons or less molecular weight to reduce volume of the particle by at least 30% as compared to volume prior to coating. A composite microreactor is provided by embedding the microcapsule when containing a living cell in a gel matrix to obtain a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from contacting the living cell. A double composite microreactor is formed containing an internal particle including an internal particle gel matrix containing a living cell and having a coating, a particle including the internal particle embedded in a particle gel matrix having a coating, and a gel super matrix in which the particle is embedded. At least one of the coatings is a volume reducing coating of polyamino acid of 15,000 daltons or less molecular weight. The gel matrices may be alginate, the polyamino acid may be polylysine or polyornithine, and at least one of the gel matrices or coatings may be treated by aging for between 2 hours and 14 days. The internal particle, particle and composite microreactor may have diameters respectively between 50 and 700 microns, 400 and 800 microns and 300 and 1500 microns.

Claims

1. A method of making a microcapsule having reduced volume comprising:

(a) forming a component of a microcapsule, wherein said component is a gel particle; and

(b) applying a volume reducing coating of a polyamino acid of 15,000 daltons or less in molecular weight to the component, thereby producing a microcapsule including said component and said coating, wherein said coating reduces the volume of said component by at least 30% relative to the volume of said component prior to coating said component.

2. The method of claim 1, wherein the polyamino acid comprises polylysine.

3. The method of claim 1, wherein the polyamino acid comprises polylysine having a molecular weight of less than 15 kDa.

4. The method of claim 1, wherein said component comprises a living cell encapsulated in a gel matrix which forms said gel particle.

5. The method of claim 4, wherein the living cell is an islet cell.

6. The method of claim 5, wherein said cell is a porcine islet cell.

7. The method of claim 1, wherein the diameter of the microcapsule is less than 4 millimeters.

8. The method of claim 1, wherein the diameter of the gel particle is more than 1 millimeter.

9. The method of claim 1, wherein the diameter of the microcapsule is more than 4 millimeters.

10. The method of claim 1, wherein said living cell is a genetically engineered living cell.

11. The method of claim 1, wherein the polyamino acid comprises polyornithine.

12. A composite microreactor which includes:

(a) an internal particle which includes;

(i) a living cell; and

(ii) an internal particle alginate gel matrix, said living cell being in said matrix;

(b) an internal particle coating on said internal particle, said coating comprising a polyamino acid of 15,000 daltons or less in molecular weight, said coating reducing the volume of said internal particle by greater than 30% relative to the volume of said internal particle prior to coating said internal particle; and

(c) a gel super matrix in which the coated internal particle is embedded;

the composite microreactor providing a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from coming into contact with said living cell.

13. The composite microreactor of claim 12, wherein the polyamino acid comprises polylysine.

14. The composite microreactor of claim 12, wherein the polyamino acid comprises polylysine having a molecular weight of less than 15 kDa.

15. The composite microreactor of claim 12, wherein said cell is an islet cell.

16. The composite microreactor of claim 12, wherein said cell is a porcine islet cell.

17. The composite microreactor of claim 12, wherein the gel super matrix comprises alginate.

18. The composite microreactor of claim 12, wherein the diameter of the internal particle is between 50 and 700 microns and the diameter of the composite microreactor is between 300 and 1500 microns.

19. The composite microreactor of claim 12, wherein the composite microreactor includes between 2 and 100 internal particles.

20. The composite microreactor of claim 12, wherein the internal particle alginate gel matrix is treated by allowing it to age for between 2 hours and 14 days prior to applying the internal particle coating.

21. The composite microreactor of claim 12, wherein the internal particle coating has a lower molecular weight exclusion number than does the gel super matrix.

22. The composite microreactor of claim 12, wherein the diameter of the composite microreactor is less than 4 millimeters.

23. The composite microreactor of claim 12, wherein the diameter of the internal particle is more than 1 millimeter.

24. The composite microreactor of claim 12, wherein the diameter of the composite microreactor is more than 4 millimeters.

25. The composite microreactor of claim 12, wherein said living cell is a genetically engineered living cell.

26. The composite microreactor of claim 12, wherein said polyamino acid comprises polyornithine.

27. The composite microreactor of claim 12, having a single internal particle.

28. A method of implanting a living donor cell into a recipient comprising:

providing the composite microreactor of claim 12, which contains the living donor cell; and

implanting the composite microreactor into said recipient.

29. The method of claim 28, further comprising testing the recipient for antibodies to the living donor cell.

30. The method of claim 28, wherein no adjunctive immunosuppression is administered to the recipient.

31. A double composite microreactor which includes:

(1) an internal particle which includes;

(a) a living cell;

(b) an internal particle alginate gel matrix which contains said living cell; and

(c) an internal particle coating, which is semipermeable, on the internal particle alinate gel matrix;

(2) a particle which includes;

(a) at least one internal particle of (1);

(b) a particle alginate gel matrix in which the internal particle is embedded;

(c) a particle coating on the particle alginate gel matrix; and

(3) a gel super matrix in which the particle of (2) is embedded, wherein at least one of the coating in (1)c and the coating in (2)c is a volume-reducing coating of polyamino acid of 15,000 daltons or less in molecular weight.

32. The double composite microreactor of claim 31, wherein the internal particle coating (1)c is polylysine.

33. The double composite microreactor of claim 31, wherein the internal particle coating (1)c is polylysine having a molecular weight of less than 15 kDa.

34. The double composite microreactor of claim 31, wherein the particle coating (2)c is polylysine.

35. The double composite microreactor of claim 31, wherein the particle coating (2)c is polylysine having a molecular weight of less than 15 kDa.

36. The double composite microreactor of claim 31, wherein the gel super matrix comprises alginate.

37. The double composite microreactor of claim 31, wherein the diameter of the internal particle is between 50 and 700 microns, the diameter of the particle is between 400 and 800 microns, and the diameter of the double composite microreactor is between 300 and 1500 microns.

38. The double composite microreactor of claim 31, wherein said cell is an islet cell.

39. The double composite microreactor of claim 31, wherein said cell is a porcine islet cell.

40. The double composite microreactor of claim 31, wherein the diameter of the double composite microreactor is less than 4 millimeters.

41. The double composite microreactor of claim 31, wherein the diameter of the internal particle is more than 1 millimeter.

42. The double composite microreactor of claim 31, wherein the diameter of the composite microreactor is more than 4 millimeters.

43. The double composite microreactor of claim 31, wherein said living cell is a genetically engineered cell.

44. The double composite microreactor of claim 31, wherein at least one of said internal particle coating and said particle coating comprises polyornithine.

45. The double composite microreactor of claim 31, having a single internal particle and a single particle.

46. The double composite microreactor of claim 31, wherein the volume-reducing coating reduces the volume of at least one of the internal particle alignate gel matrix and the particle alginate gel matrix by 30% or more relative to the volume prior to coating.

47. A double composite microreactor which includes:

(1) an internal particle which includes;

(a) a living cell;

(b) an internal particle gel matrix in which the living cell is embedded;

(c) an internal particle coating enclosing the internal particle gel matrix;

(2) a particle which includes;

(a) at least one internal particle of (1);

(b) a particle gel matrix in which the internal particle is embedded; and

(c) a particle coating enclosing the particle gel matrix; and

(3) a gel super matrix in which the particle of (2) is embedded, wherein at least one of (i) the thickness of the particle gel matrix 2(b) and (ii) the thickness of the gel super matrix is at least 10 microns.

48. The double composite microreactor of claim 47, having a single internal particle and a single particle.

49. A double composite microreactor which includes:

(1) an internal particle which includes;

(a) a living cell;

(b) an internal particle gel matrix containing said living cell; and

(c) an internal particle coating on the internal particle gel matrix;

(2) a particle which includes;

(a) at least one internal particle of (1)

(b) a particle gel matrix in which the internal particle is embedded;

(c) a particle coating on the particle gel matrix; and

(3) a gel super matrix in which the particle of (2) is embedded, wherein at least one of the internal particle gel matrix, the particle gel matrix and the particle has been treated such that no substantial change in shape or volume occurs when combined respectively with the internal particle coating, the particle coating or the gel super matrix.

50. The double composite microreactor of claim 49, wherein said treatment is by aging for between 2 hours and 14 days.

51. The double composite microreactor of claim 49, wherein at least one of said internal particle gel matrix, internal particle tin particle gel matrix, particle coating or gel super matrix has been treated by aging for between 2 hours and 14 days.

52. The double composite microreactor of claim 49, wherein the internal particle coating (1)c is polylysine.

53. The double composite microreactor of claim 49, wherein the internal particle coating (1)c is polylysine having a molecular weight of less than 15 kDa.

54. The double composite microreactor of claim 49, wherein the particle coating (2)c is polylysine.

55. The double composite microreactor of claim 49, wherein the particle coating (2)c is polylysine having a molecular weight of less than 15 kDa.

56. The double composite microreactor of claim 49, wherein said internal particle gel matrix (1)b comprises alginate.

57. The double composite microreactor of claim 49, wherein said particle gel matrix (2)b comprises alginate.

58. The double composite microreactor of claim 49, wherein the living cell is an islet cell.

59. The double composite microreactor of claim 49, wherein the living cell is a porcine islet cell.

60. The double composite microreactor of claim 49, wherein at least one of the internal particle coating and the particle coating comprises a polyamino acid.

61. The double composite microreactor of claim 49, wherein the diameter of the internal particle is between 50 and 700 microns, the diameter of the particle is between 400 and 800 microns, and the diameter of the double composite microreactor is between 300 and 1500 microns.

62. The double composite microreactor of claim 49, wherein the diameter of the double composite microreactor is less than 4 millimeters.

63. The double composite microreactor of claim 49, wherein the diameter of the internal particle is more than 1 millimeter.

64. The double composite microreactor of claim 49, wherein the diameter of the double composite microreactor is more than 4 millimeters.

65. The double composite microreactor of claim 49, wherein said living cell is a genetically engineered living cell.

66. The double composite microreactor of claim 49, wherein at least one of said internal article coating and said particle coating comprises polyornithine.

67. The double composite microreactor of claim 49, having a single internal particle and a single particle.

68. A double composite microreactor which includes:

(1) an internal particle which includes;

(a) a living cell;

(b) an internal particle gel matrix containing said living cell; and

(c) an internal particle coating on the internal particle gel matrix;

(2) a particle which includes;

(a) at least one internal particle of (1)

(b) a particle gel matrix in which the internal particle is embedded;

(c) a particle coating on the particle gel matrix; and

(3) a gel supermatrix in which the particle of (2) is embedded, wherein the double composite microreactor has one or more of the following properties;

the microreactor includes a plurality of internal particles;

the microreactor includes a plurality of particles;

the thickness of the gel supermatrix is at least 20 microns;

or

the thickness of the particle gel matrix is at least 20 microns.

69. The double composite microreactor of claim 68, wherein the internal particle coating is polylysine.

70. The double composite microreactor of claim 68, wherein the internal particle coating is polylysine having a molecular weight of less than 15 kDa.

71. The double composite microreactor of claim 68, wherein the particle coating is polylysine.

72. The double composite microreactor of claim 68, wherein the particle coating is polylysine having a molecular weight of less than 15 kDa.

73. The double composite microreactor of claim 68, wherein said internal particle gel matrix comprises alginate.

74. The double composite microreactor of claim 68, wherein said particle gel matrix comprises alginate.

75. The double composite microreactor of claim 68, wherein the living cell is an islet cell.

76. The double composite microreactor of claim 68, wherein at least one of the internal particle coating and the particle coating comprises a polyamino acid.

77. The double composite microreactor of claim 68, wherein the diameter of the double composite microreactor is less than 4 millimeters.

78. The double composite microreactor of claim 68, wherein the diameter of the internal particle is more than 1 millimeter.

79. The double composite microreactor of claim 68, wherein the diameter of the double composite microreactor is more than 2 millimeters.

80. The double composite microreactor of claim 68, wherein said living cell is a genetically engineered living cell.

81. The double composite microreactor of claim 68, wherein at least one of said internal particle coating and said particle coating comprises polyomithine.

82. The double composite microreactor of claim 68, having a single internal particle and a single particle.