Method of dynamically culturing embryonic stem cells

• US 20060148078A1
• Filed: 11/30/2003
• Published: 07/06/2006
• Est. Priority Date: 11/29/2002
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Gerecht-Nir, Sharon, Itskovitz-Eldor, Joseph
• Granted Patent: US 7,947,499 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 5/00 Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; plant reproduction by tissue culture techniques A01H4/00
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

he present invention is of a method of dynamically generating human embryoid bodies which can be used for generating lineage specific cells and cell lines. Specifically, the present invention can be used to generate ESC-differentiated cells for cell-replacement therapy.

Claims

1-76. -76. (canceled)

77. A method of dynamically generating embryoid bodies comprising culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in said fluid to thereby obtain dynamically generated embryoid bodies.

79. The method of claim 77, wherein said bioreactor is capable of exerting a microgravitational force selected from the range of 0.05-0.9 G on said cells cultured therein.

80. The method of claim 79, wherein said microgravitational force is 0.1 G.

81. The method of claim 77, wherein said bioreactor is capable of maintaining said embryonic stem cells in a state of free fall.

82. The method of claim 77, wherein said bioreactor is a tubular vessel capable of rotating around a horizontal longitudinal axis.

83. The method of claim 77, wherein said bioreactor is capable of holding a fluid volume selected from the range of 1-500 ml.

84. The method of claim 82, wherein a diameter of said tubular vessel is selected from range of 2.54-15.24 centimeters.

85. The method of claim 84, wherein said culturing of said embryonic stem cells is effected in said tubular vessel under a rotational speed selected from a range of 15-20 rpm.

86. The method of claim 77, wherein said bioreactor enables oxygenation of said fluid via controlled diffusion of dissolved gasses.

87. The method of claim 77, wherein said embryonic stem cells are cultured at an initial concentration of 0.5-0.7×

10⁶ cells per milliliter medium.

88. The method of claim 77, wherein said bioreactor is a slow turning lateral vessel.

89. The method of claim 77, wherein said culturing of said embryonic stem cells is effected in a culture medium selected suitable for embryoid bodies formation.

90. The method of claim 89, wherein said culture medium includes 80% KO-DMEM, 20% serum, 0.5% Penicillin-Streptomycin, 1 mM L-glutamine, 0.1 mM β

-mercaptoethanol and 1% non-essential amino acid stock.

91. The method of claim 77, wherein said culturing is effected over a culturing period of 1-35 days.

92. The method of claim 77, wherein said culturing is effected for 30 days.

78. A method of generating expanded and/or differentiated cells from embryonic stem cells comprising:

(a) culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in said fluid to thereby obtain dynamically generated embryoid bodies;

(b) isolating lineage specific cells from said embryoid bodies, and;

(c) culturing said lineage specific cells under culturing conditions selected suitable for the expansion and/or differentiation of said lineage specific cells to thereby obtain expanded and/or differentiated lineage-specific cells.

93. A cell culture comprising embryoid bodies, wherein at least 90% of said embryoid bodies fall within a diameter size range of 400-800 μ

m.

94. The cell culture of claim 93, wherein at least 85% of said embryoid bodies are devoid of necrotic centers.

95. A method of treating a disorder requiring cell replacement therapy comprising:

(a) culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in said fluid to thereby obtain dynamically generated embryoid bodies;

(b) isolating lineage specific cells from said embryoid bodies;

(c) culturing said lineage specific cells under culturing conditions selected suitable for the expansion and/or differentiation of said lineage specific cells to thereby obtain expanded and/or differentiated lineage-specific cells, and;

(d) administering cells of said expanded and/or differentiated lineage-specific cells to an individual in need thereof thereby treating the disorder requiring cell replacement therapy.

96. The method of claim 95, wherein said bioreactor is capable of exerting a microgravitational force selected from the range of 0.05-0.9 G on said cells cultured therein.

97. The method of claim 96, wherein said microgravitational force is 0.1 G.

98. The method of claim 95, wherein said bioreactor is capable of maintaining said embryonic stem cells in a state of free fall.

99. The method of claim 95, wherein said bioreactor is a tubular vessel having at least one vessel port for transferring materials into and out of said tubular vessel.

100. The method of claim 95, wherein said bioreactor is a slow tuning lateral vessel.

101. The method of claim 95, wherein said culturing of said embryonic stem cells is effected in a culture medium selected suitable for embryoid bodies formation.

102. The method of claim 101, wherein said culture medium includes 80% KO-DMEM, 20% serum, 0.5% Penicillin-Streptomycin, 1 mM L-glutamine, 0.1 mM β

-mercaptoethanol and 1% non-essential amino acid stock.

103. A method of dynamically expanding embryonic stem cells while maintaining the embryonic stem cells in an undifferentiated state, the method comprising culturing embryonic stem cells in a bioreactor selected capable of subjecting cells cultured in a fluid contained therein to randomized gravity vectors without formation of gas bubbles in said fluid to thereby obtain dynamically expanded undifferentiated embryonic stem cell cultures.

104. The method of claim 103, wherein said bioreactor is capable of exerting a microgravitational force on said cells cultured therein.

105. The method of claim 104, wherein said microgravitational force is selected from the range of 0.05-0.9 G.

106. The method of claim 103, wherein said microgravitational force is 0.1 G.

107. The method of claim 103, wherein the embryonic stem cells are attached to a carrier.

108. The method of claim 107, wherein said carrier is coated with a matrix selected capable of supporting a growth of said embryonic stem cells.

109. The method of claim 103, wherein said culturing of said embryonic stem cells is effected in a culture medium selected suitable for expanding embryoid stem cell cultures in the undifferentiated state.