Pluripotent Stem Cell Culture on Micro-Carriers

• US 20150166950A1
• Filed: 02/25/2015
• Published: 06/18/2015
• Est. Priority Date: 11/20/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Nelson, Shelley
• Granted Patent: US 9,969,972 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0075 using microcarriers
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• 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 5/0676 Pancreatic cells

Abstract

The present invention is directed to methods for the growth, expansion and differentiation of pluripotent stem cells on micro-carriers.

Claims

1. A method for the propagation of pluripotent stem cells, comprising the steps of:

a. Attaching a population of pluripotent stem cells to a first volume of micro-carriers,

b. Culturing the pluripotent stem cells on the first volume of micro-carriers,

c. Removing the pluripotent stem cells from the first volume of micro-carriers, and

d. Attaching the population of pluripotent stem cells to a second volume of micro-carriers.

2. The method of claim 1, wherein the steps of culturing, removing and attaching the pluripotent stem cells on micro-carriers are is repeated using subsequent volumes of micro-carriers.

3. The method of claim 1, wherein the first volume of micro-carriers is selected from the group consisting of dextran micro-carriers and polystyrene micro-carriers.

4. The method of claim 1, wherein the second volume of micro-carriers is selected from the group consisting of dextran micro-carriers and polystyrene micro-carriers.

5. The method of claim 1, wherein the pluripotent stem cells are attached to the first volume of micro-carriers in medium containing a Rho kinase inhibitor.

6. The method of claim 1, wherein the pluripotent stem cells are attached to the second volume of micro-carriers in medium containing a Rho kinase inhibitor.

7. The method of claim 1, wherein the pluripotent stem cells are removed from the first volume of micro-carriers by enzymatic treatment.

8. The method of claim 1, wherein the pluripotent stem cells are removed from the second volume of micro-carriers by enzymatic treatment.

9. The method of claim 1, wherein the first volume of micro-carriers is removed from the pluripotent stem cells prior to attaching the cells to the second volume of micro-carriers.

10. A method to differentiate pluripotent stem cells to insulin-expressing cells on micro-carriers comprising the steps of:

a. Attaching the pluripotent stem cells to a volume of micro-carriers,

b. Differentiating the pluripotent stem cells to cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage,

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, and

e. Differentiating the cells expressing markers characteristic of the pancreatic endocrine lineage into insulin-expressing cells.