Methods for culturing human embryonic stem cells

• US 20050158852A1
• Filed: 12/13/2004
• Published: 07/21/2005
• Est. Priority Date: 12/31/2003
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Fan-Chiang, Mei-Hwei, Lin, Chin-Yu, Wang, Mei-Chih, Lin, Hui-Ti
• Granted Patent: US 7,935,527 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• 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

Abstract

The present invention discloses a method for improving growth and survival of single human embryonic stem cells. The method includes the step of obtaining a single undifferentiated HES cell; mixing the single undifferentiated cell with an extracellular matrix (ECM) to encompass the cell; and inoculating the mixture onto feeder cells with a nutrient medium in a growth environment. Therefore the single cells can survive, proliferate and grow in vitro.

Claims

1. A method of culturing single undifferentiated human embryonic stem cells (HES), comprising:

(a) obtaining a single undifferentiated HES cell;

(b) mixing the single cell with an extracellular matrix to form a mixture to encompass the cell; and

(c) inoculating the mixture onto feeder cells with a nutrient medium.

2. The method of claim 1, wherein the human embryonic stem cells are HES-3.

3. The method of claim 1, wherein the extracellular matrix is a human extracellular matrix extracted from human embryonic stem cells.

4. The method of claim 1, wherein the extracellular matrix is Matrigel™

.

5. The method of claim 1, wherein the extracellular matrix is prepared from an isolated matrix component or the combination selected from the group consisting of collagen, elastin, fibronectin, laminin and fibrillin.

6. The method of claim 1, wherein the extracellular matrix is prepared from collagen.

7. The method of claim 1, wherein the feeder cells are selected from the group consisting of primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human fetal muscle cells (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human adult fallopian tube epithelial cells (HAFT) and human marrow stromal cells (HMS).

8. The method of claim 1, wherein the feeder cells are mouse embryonic fibroblasts (MEF), human adult skin cells or human foreskin fibroblasts (HFF).

9. The method of claim 1, which the method of obtaining a single undifferentiated HES cells comprises:

(a) selecting a group of undifferentiated cells from a cell culture ;

and

(b) dissociating the group of undifferentiated cells into single cells

10. The method of claim 9, wherein the dissociation method is enzymatic degradation.

11. The method of claim 10, wherein the enzymatic degradation is using collagenase.

12. The method of claim 1, wherein the method is used for establishing a clonal HES cell line.

13. The method of claim 1, wherein the method is suitable for gene transfection.