Feeder-free culture method for embryonic stem cells or primate primordial stem cells

• US 7,413,902 B2
• Filed: 12/24/2002
• Issued: 08/19/2008
• Est. Priority Date: 10/23/1997
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Gold, Joseph D., Murai, James T., Chiu, Choy-Pik, West, Michael D., Bodnar, Andrea G., Inokuma, Margaret
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US10/330,873
• Publication Number:
  US 20030175956A1
• Time in Patent Office: 2,065 Days
• Field of Search: 435/366, 435/377
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2510/00 Genetically modified 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

Abstract

Methods and materials for culturing primate-derived primordial stem cells are described. In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from nucleosides and a pyruvate salt.

Claims

1. A method of culturing primate primordial stem (pPS) cells to proliferate in an undifferentiated state, comprising contacting the primate primordial stem cells with an extracellular matrix and a cell culture medium in a growth environment free of feeder cells, wherein the extracellular matrix is a fibroblast matrix, prepared by culturing fibroblasts, lysing the fibroblasts in situ, and then washing what remains after lysis.

2. A method of culturing primate primordial stem (pPS) cells to proliferate in an undifferentiated state, comprising contacting the primate primordial stem cells with an extracellular matrix and a cell culture medium in a growth environment free of feeder cells, wherein the nutrient medium comprises sodium pyruvate and nucleosides, and has a low endotoxin level.

3. A method of culturing primate primordial stem (pPS) cells to proliferate in an undifferentiated state, comprising contacting the primate primordial stem cells with an extracellular matrix and a cell culture medium in a growth environment free of feeder cells, wherein the nutrient medium contains added fibroblast growth factor.

4. A method of culturing primate primordial stem (pPS) cells to proliferate in an undifferentiated state, comprising contacting the primate primordial stem cells with an extracellular matrix and a cell culture medium in a growth environment free of feeder cells, wherein the nutrient medium contains added forskolin.

5. A method of culturing primate primordial stem (pPS) cells to proliferate in an undifferentiated state, comprising contacting the primate primordial stem cells with an extracellular matrix and a cell culture medium in a growth environment free of feeder cells, and further comprising testing the pPS cells for expression of telomerase.