Method for producing pluripotent stem cells derived from dental pulp

• US 10,041,039 B2
• Filed: 03/28/2013
• Issued: 08/07/2018
• Est. Priority Date: 03/29/2012
• Status: Active Grant

• Current Assignee: JCR Pharmaceuticals Co., Ltd.
• Original Assignee: JCR Pharmaceuticals Co., Ltd.
• Inventors: Imagawa, Kiwamu
• Primary Examiner(s): Bertoglio, Valarie E
• Application Number:
  US14/388,464
• Publication Number:
  US 20150050731A1
• Time in Patent Office: 1,958 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2535/00 Supports or coatings for cell culture characterised by topography
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0664 Dental pulp stem cells, Dental follicle stem cells

Abstract

Disclosed is a method for producing pluripotent stem cell-enriched human dental pulp-derived cells. The method is characterized in that it includes (a) culturing a dental pulp suspension in a feeder cells-culture vessel containing feeder cells whose proliferative ability is suppressed, on a membrane having micropores that can block passage of the feeder cells and supported in the feeder cells-culture vessel in a manner to prevent the lower side face thereof from contacting with the feeder cells, thereby preventing direct contact with the feeder cells, and, (b) a step for recovering the cells proliferating on the membrane.

Claims

1. A method for production of multipotent stem cell-enriched human dental pulp-derived cells comprising the steps of,

(a) culturing dental pulp-derived cells contained in a dental pulp suspension in a feeder cells-culture vessel containing feeder cells whose proliferative ability is suppressed, on a membrane coated with fibronectin or collagen and having micropores that can block passage of the feeder cells and supported in the feeder cells-culture vessel in a manner to prevent the lower side face thereof from contacting with the feeder cells, thereby preventing direct contact of the dental pulp-derived cells with the feeder cells,

wherein the multipotent stem cells possess the ability to differentiate into chondrocytes and osteoblasts, and are positive for the surface antigen markers CD29, CD44, CD73, CD90, CD105, and CD166, and negative for CD34 and CD45,

wherein the mean diameter of the micropores is 0.2 to 1.2 μ

m, and

wherein the culture is conducted by using Dulbecco'"'"'s modified Eagle medium containing 20% of fetal bovine serum and 3 to 5 mM of L-alanyl-L-glutamine and whose glucose concentration is 5 to 7 mM, and

(b) recovering the cells that have proliferated on the membrane.

2. The method for production according to claim 1 further comprising:

(c) culturing the recovered cells in a feeder cells-culture vessel containing feeder cells whose proliferative ability is suppressed, avoiding direct contact with the feeder cells, and on a membrane having micropores that can block passage of the feeder cells and supported in the feeder cells-culture vessel in order not to contact on the lower side face thereof with the feeder cells, and

(d) recovering the cells that have proliferated on the membrane in (c).

3. The method for production according to claim 1, wherein the membrane is coated with fibronectin.

4. The method for production according to claim 1, wherein the feeder cells are mammalian cells whose proliferative ability is suppressed by mitomycin C.

5. The method for production according to claim 4, wherein the mammalian cells are NIH3T3 cells.

6. The method for production according to claim 1, wherein the culture is conducted by using Dulbecco'"'"'s modified Eagle medium that contains 4 mM L-alanyl-L-glutamine and whose glucose concentration is 5.5 to 5.7 mM.

7. The method for production according to claim 1, further comprising:

adding the recovered cells to a culture vessel at a density of 1000 to 20000 cells/cm²; and

culturing them until 70 to 100% of the bottom face of the culture vessel is occupied by the cells.

8. The method for production according to claim 1, further comprising:

adding the recovered cells to a culture vessel at a density of 5000 to 10000 cells/cm²; and

culturing them until 90 to 100% of the bottom face of the culture vessel is occupied by the cells.

9. The method for production according to claim 1, wherein the mean diameter of the micropores is 0.4 μ

m.