Microcarriers For Stem Cell Culture

• US 20110014693A1
• Filed: 03/17/2009
• Published: 01/20/2011
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Lecina, Marti, Choo, Andre, Oh, Steve, Chen, Allen, Zweigert, Robert, Reuveny, Shaul
• Granted Patent: US 8,716,018 B2
• Time in Patent Office: Days
• US Class Current: 435/363
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0075 using microcarriers
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

We disclose a particle comprising a matrix coated thereon and having a positive charge, the particle being of a size to allow aggregation of primate or human stem cells attached thereto. The particle may comprise a substantially elongate, cylindrical or rod shaped particle having a longest dimension of between 50 μm and 400 μm, such as about 200 μm. It may have a cross sectional dimension of between 20 μm and 30 μm. The particle may comprise a substantially compact or spherical shaped particle having a size of between about 20 μm and about 120 μm, for example about 65 μm. We also disclose a method of propagating primate or human stem cells, the method comprising: providing first and second primate or human stem cells attached to first and second respective particles, allowing the first primate or human stem cell to contact the second primate or human stem cell to form an aggregate of cells and culturing the aggregate to propagate the primate or human stem cells for at least one passage. A method of propagating human embryonic stem cells (hESCs) in long term suspension culture using microcarriers coated in Matrigel or hyaluronic acid is also disclosed. We also disclose a method for differentiating stem cells.

Claims

1. A method of culturing stem cells in suspension culture in vitro, the method comprising:

(i) attaching stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coated in a matrix;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 3 passages,

wherein stem cells in the culture after step (iv) are pluripotent.

2. The method of claim 1 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

3. The method of claim 1 or 2 wherein the stem cells are primate or human.

4. The method of any one of claims 1 to 3 wherein steps (i)-(iii) are repeated through at least 5 passages, or at least 7 passages, or at least 10 passages.

5. The method of any one of claims 1 to 4 wherein the microcarriers are rod-shaped.

6. The method of any one of claims 1 to 5 wherein the matrix comprises an extracellular matrix component.

7. The method of any one of claims 1 to 5 wherein the matrix comprises one or more of Matrigel™

(BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate.

8. The method of any one of claims 1 to 5 wherein the matrix comprises a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

9. The method of any one of claims 1 to 8 wherein the microcarrier comprises or consists of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone.

10. The method of any one of claims 1 to 8 wherein the microcarrier is a macroporous or microporous carboseed microcarrier.

11. The method of any one of claims 1 to 10 wherein the microcarrier is coupled with protamine or polylysine.

12. The method of any one of claims 1 to 11 wherein the microcarrier is positively charged.

13. The method of any one of claims 1 to 12 wherein the microcarrier has a positive surface charge.

14. The method of any one of claims 1 to 13 wherein the microcarrier is hydrophilic.

15. The method of any one of claims 1 to 4 or 6 to 14 wherein the microcarriers have a substantially spherical shape.

16. The method of any one of claims 1 to 15 wherein in step (ii) the stem cells are cultured for a period of time sufficient to expand the number of stem cells in the culture.

17. The method of any one of claims 1 to 16 wherein in each repeat cycle the stem cells of step (i) are obtained from the passaged cells of step (iii) of the preceding repeat cycle.

18. The method of any one of claims 1 to 17 wherein in step (iv), steps (i)-(iii) are repeated through one of:

at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, at least 20 passages, at least 21 passages, at least 22 passages, at least 23 passages, at least 24 passages, at least 25 passages, at least 30 passages, at least 40 passages, at least 50 passages, at least 60 passages, at least 70 passages, at least 80 passages, at least 90 passages, at least 100 passages.

19. The method of any one of claims 1 to 18 wherein for at least 60% of the cycles of steps (i)-(iii) the microcarriers are coated in a matrix.

20. The method of any one of claims 1 to 19 wherein in cycles of steps (i)-(iii) the microcarriers are coated in the same matrix.

21. The method of any one of claims 1 to 20 wherein the matrix is different or absent in first and second consecutive cycles of steps (i)-(iii).

22. The method of any one of claims 1 to 21 wherein after step (iv) at least 60% of the stem cells in the culture are pluripotent.

23. The method of any one of claims 1 to 22 wherein after step (iv) at least 60% of the stem cells in the culture express one, two, three or all of Oct4, SSEA4, TRA-1-60 and Mab84.

24. The method of any one of claims 1 to 23 wherein the method comprises culturing the stem cells in serum free media, or stem cell conditioned media, or feeder cell free conditions.

25. The method of any one of claims 1 to 24 wherein feeder cells are also attached to the microcarriers.

26. The method of any one of claims 1 to 24 wherein the culture further comprises feeder cells attached to microcarriers which are different to the microcarriers to which the stem cells are attached.

27. Pluripotent stem cells obtained by the method of any one of claims 1 to 26.

28. The method of any one of claims 1 to 26 further comprising the step of inducing differentiation of the stem cells obtained after step (iv).

29. The method of claim 28 wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.

30. The method of any one of claims 1 to 26 wherein after step (iv) the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.

31. The method of any one of claims 1 to 26 further comprising the differentiation of pluripotent stem cells, comprising:

(v) attaching pluripotent stem cells obtained after step (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers is coated in a second matrix or is uncoated; and

(vi) culturing the microcarrier-stem cell complexes from (v) in suspension culture under conditions that induce the differentiation of the stem cells.

32. The method of claim 31 wherein the first and second matrix are the same.

33. The method of claim 31 wherein the first and second matrix are different.

34. The method of any one of claims 31 to 33 wherein the first and second microcarriers are the same.

35. The method of any one of claims 31 to 33 wherein the first and second microcarriers are different.

36. The method of any one of claims 31 to 35 wherein the method further comprises:

(vii) attaching differentiated stem cells obtained from step (vi) to a plurality of third microcarriers to form microcarrier-stem cell complexes, wherein the surface of the third microcarriers is coated in a third matrix or is uncoated; and

(viii) culturing the microcarrier-stem cell complexes from (vii) in suspension culture under conditions that induce the further differentiation of the differentiated stem cells.

37. The method of claim 36 wherein the third matrix is different to the first and second matrix.

38. The method of claim 36 wherein the third matrix is the same as one of the first and second matrix.

39. The method of any one of claims 36 to 38 wherein the third microcarriers are different to the first and second microcarriers.

40. The method of any one of claims 36 to 38 wherein the third microcarriers are the same as one of the first and second microcarriers.

41. A differentiated cell obtained by the method of any one of claims 28 to 40.

42. The method of any one of claims 28 to 40 wherein the differentiated cells are cultured to form an embryoid body.

43. An embryoid body obtained by the method of claim 42.

44. A method of culturing stem cells in suspension culture in vitro, the method comprising:

(i) attaching stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coated in Matrigel™

;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 7 passages,

wherein stem cells in the culture after step (iv) are pluripotent, wherein the culture is free of feeder cells, wherein the number of stem cells is expanded between each passage and wherein the stem cells are human or primate embryonic stem cells or human or primate induced pluripotent stem cells.

45. A method of culturing and differentiating stem cells in vitro, the method comprising:

(i) attaching stem cells to a plurality of first microcarriers to form microcarrier-stem cell complexes, wherein the surface of the first microcarriers is coated in a first matrix;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 3 passages,

wherein stem cells in the culture after step (iv) are pluripotent, the method further comprising;

(v) attaching pluripotent stem cells obtained after step (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers is coated in a second matrix or is uncoated; and

(vi) culturing the microcarrier-stem cell complexes from (v) in suspension culture under conditions that induce the differentiation of the stem cells.

46. The method of claim 45 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

47. The method of claim 45 or 46 wherein the stem cells are primate or human.

48. The method of any one of claims 45 to 47 wherein the microcarriers are rod-shaped.

49. The method of any one of claims 45 to 48 wherein the first and second matrix are the same.

50. The method of any one of claims 45 to 48 wherein the first and second matrix are different.

51. The method of any one of claims 45 to 50 wherein the first and second microcarriers are the same.

52. The method of any one of claims 45 to 50 wherein the first and second microcarriers are different.

53. The method of any one of claims 45 to 52 wherein the method further comprises:

(vii) attaching differentiated stem cells obtained from step (vi) to a plurality of third microcarriers to form microcarrier-stem cell complexes, wherein the surface of the third microcarriers is coated in a third matrix or is uncoated; and

(viii) culturing the microcarrier-stem cell complexes from (vii) in suspension culture under conditions that induce the further differentiation of the differentiated stem cells.

54. The method of claim 53 wherein the third matrix is different to the first and second matrix.

55. The method of claim 53 wherein the third matrix is the same as one of the first and second matrix.

56. The method of any one of claims 53 to 55 wherein the third microcarriers are different to the first and second microcarriers.

57. The method of any one of claims 53 to 55 wherein the third microcarriers are the same as one of the first and second microcarriers.

58. A method of differentiating stem cells in vitro, comprising attaching pluripotent stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coated in a matrix or is uncoated, and culturing the microcarrier-stem cell complexes in suspension culture under conditions that induce the differentiation of the stem cells.

59. The method of claim 58 wherein the stem cells are embryonic stem cells, or induced pluripotent stem cells.

60. The method of claim 58 or 59 wherein the stem cells are primate or human.

61. The method of any one of claims 58 to 60 wherein the microcarriers are rod-shaped.

62. The method of any one of claims 58 to 61. wherein the matrix comprises an extracellular matrix component.

63. The method of any one of claims 58 to 61 wherein the matrix comprises one or more of laminin, fibronectin, vitronectin, Matrigel™

(BD Biosciences), hyaluronic acid, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate.

64. The method of any one of claims 58 to 61 wherein the matrix comprises a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

65. The method of any one of claims 58 to 64 wherein the microcarrier comprises or consists of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone.

66. The method of any one of claims 58 to 64 wherein the microcarrier is a macroporous or microporous carboseed microcarrier.

67. The method of any one of claims 58 to 66 wherein the microcarrier is coupled with protamine or polylysine.

68. The method of any one of claims 58 to 67 wherein the microcarrier is positively charged.

69. The method of any one of claims 58 to 68 wherein the microcarrier has a positive surface charge.

70. The method of any one of claims 58 to 69 wherein the microcarrier is hydrophilic.

71. The method of any one of claims 58 to 60 or 62 to 70 wherein the microcarriers have a substantially spherical shape.

72. A method of culturing multipotent stem cells in suspension culture in vitro, the method comprising:

(i) attaching multipotent stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

wherein stem cells in the culture after step (ii) are multipotent.

73. The method of claim 72 wherein in (i) the surface of the microcarriers is coated in a matrix.

74. The method of claim 72 or 73 further comprising the step of inducing differentiation of the stem cells obtained after step (ii).

75. The method of claim 74 wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.

76. The method of any one of claims 72 to 75 wherein after step (ii) the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.

79. Multipotent stem cells obtained by the method of any one of claim 72, 73, 77 or 78.

84. A differentiated cell obtained by the method of any one of claims 74 to 83.

88. The method of any one of claims 72 to 87 wherein the stem cells are adult stem cells, or multipotent stem cells derived from pluripotent stem cells.

89. The method of any one of claims 72 to 88 wherein the microcarriers are rod-shaped.

90. The method of any one of claims 72 to 89 wherein the matrix comprises an extracellular matrix component.

91. The method of any one of claims 72 to 89 wherein the matrix comprises one or more of Matrigel™

(BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate.

92. The method of any one of claims 72 to 89 wherein the matrix comprises a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

93. The method of any one of claims 72 to 92 wherein the microcarrier comprises or consists of one or more of cellulose, dextran, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, silicone.

94. The method of any one of claims 72 to 92 wherein the microcarrier is a macroporous or microporous carboseed microcarrier.

95. The method of any one of claims 72 to 94 wherein the microcarrier is positively charged.

96. The method of any one of claims 72 to 95 wherein the microcarrier has a positive surface charge.

97. The method of any one of claims 72 to 96 wherein in step (ii) the stem cells are cultured for a period of time sufficient to expand the number of stem cells in the culture.

77. A method of culturing multipotent stem cells in suspension culture in vitro, the method comprising:

(i) attaching multipotent stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 2 passages,

wherein stem cells in the culture after step (iv) are multipotent.

78. The method of claim 77 wherein in (i) the surface of the microcarriers is coated in a matrix.

80. The method of claim 77 or 78 further comprising the step of inducing differentiation of the stem cells obtained after step (iv).

81. The method of claim 80 wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.

82. The method of any one of claims 77 to 81 wherein after step (iv) the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.

83. The method of claim 77 further comprising the differentiation of multipotent stem cells, comprising:

(v) attaching multipotent stem cells obtained after step (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers is coated in a second matrix or is uncoated; and

(vi) culturing the microcarrier-stem cell complexes from (v) in suspension culture under conditions that induce the differentiation of the stem cells.

98. The method of any one of claims 77 to 97 wherein in each repeat cycle the stem cells of step (i) are obtained from the passaged cells of step (iii) of the preceding repeat cycle.

99. The method of any one of claims 77 to 98 wherein in step (iv), steps (i)-(iii) are repeated through one of:

at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, at least 20 passages, at least 21 passages, at least 22 passages, at least 23 passages, at least 24 passages, at least 25 passages, at least 30 passages, at least 40 passages, at least 50 passages, at least 60 passages, at least 70 passages, at least 80 passages, at least 90 passages, at least 100 passages.

85. A method of culturing and differentiating multipotent stem cells in vitro, the method comprising:

(i) attaching stem cells to a plurality of first microcarriers to form microcarrier-stem cell complexes;

(ii) culturing the microcarrier-stem cell complexes in suspension culture;

(iii) passaging the cultured cells from (ii); and

(iv) repeating steps (i)-(iii) through at least 2 passages,

wherein stem cells in the culture after step (iv) are multipotent, the method further comprising;

(v) attaching multipotent stem cells obtained after step (iv) to a plurality of second microcarriers to form microcarrier-stem cell complexes, wherein the surface of the second microcarriers is coated in a second matrix or is uncoated; and

(vi) culturing the microcarrier-stem cell complexes from (v) in suspension culture under conditions that induce the differentiation of the stem cells.

86. The method of claim 85 wherein in (i) the surface of the microcarriers is coated in a first matrix.

87. A method of differentiating stem cells in vitro, comprising attaching multipotent stem cells to a plurality of microcarriers to form microcarrier-stem cell complexes, wherein the surface of the microcarriers is coated in a matrix or is uncoated, and culturing the microcarrier-stem cell complexes in suspension culture under conditions that induce the differentiation of the stem cells.

100. Use of a microcarrier coated in a matrix for the propagation and/or differentiation of primate or human stem cells, the microcarrier being chosen from:

DE-52 (Whatman), DE-53 (Whatman), QA-52 (Whatman), TSKgel Tresyl-5Pw (Tosoh) or Toyopearl AF-Tresyl-650 (Tosoh), SM1010 (Blue Membranes) and SH1010 (Blue Membranes).

101. The use of claim 100 wherein the matrix comprises one or more of Matrigel™

(BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate.

102. The use of claim 100 wherein the matrix comprises a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1.

103. A microcarrier suitable for use in the growth and/or differentiation of pluripotent or multipotent cells in in vitro suspension culture, wherein the microcarrier comprises one or more of cellulose, dextran, hydroxylated methacrylate, or collagen, and wherein the microcarrier has an elongate shape and has a longest dimension of less than about 2000 μ

m and a shortest dimension of more than about 10 μ

m, and wherein the surface of the microcarrier is coated in a matrix, and wherein one or a plurality of pluripotent or multipotent cells are attached to the matrix coating.

104. The microcarrier of claim 103 wherein the microcarrier is rod-shaped.

105. The microcarrier of claim 103 or 104 wherein the matrix coating comprises one or more of Matrigel™

(BD Biosciences), hyaluronic acid, laminin, or fibronectin.

106. The microcarrier of any one of claims 103 to 105 wherein the cells are pluripotent cells.

107. The microcarrier of any one of claims 103 to 106 wherein the pluripotent cells are primate or human embryonic stem cells, or induced pluripotent stem cells.

108. The microcarrier of any one of claims 103 to 107 wherein the microcarrier is positively charged.

109. The microcarrier of any one of claims 103 to 108 wherein the microcarrier has a positive surface charge.

110. The microcarrier of any one of claims 103 to 109 having a longest dimension of between 50 μ

m and 400 μ

m.

111. An aggregate comprising two or more microcarriers having pluripotent or multipotent cells attached thereto, each according to any one of claims 103 to 110.

112. Use of a microcarrier according to any one or claims 103 to 110 in the culture of pluripotent or multipotent cells in vitro to generate new cells having pluripotent or multipotent status.

113. Use of a microcarrier according to any one or claims 103 to 110 in the in vitro differentiation of pluripotent or multipotent cells.

114. A method of culturing pluripotent or multipotent cells in vitro to generate new cells having pluripotent or multipotent status, the method comprising culturing a microcarrier according to any one or claims 103 to 110 under conditions suitable for the generation of new cells having pluripotent or multipotent status.

115. A method of differentiating pluripotent or multipotent cells in vitro, the method comprising culturing a microcarrier according to any one or claims 103 to 110 under conditions that induce the differentiation of the pluripotent or multipotent cells.