Stem cell culture medium and method of using said medium and the cells

• US 8,940,535 B2
• Filed: 11/04/2004
• Issued: 01/27/2015
• Est. Priority Date: 11/04/2003
• Status: Active Grant

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture, University of Maryland Baltimore County (University of Maryland)
• Inventors: Trisler, G. David, Bever, Christopher T. Jr., Goolsby, James E., Pessac, Bernard M.
• Primary Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US10/982,381
• Publication Number:
  US 20050164380A1
• Time in Patent Office: 3,736 Days
• US Class Current: 435/375
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0662 Stem cells

Abstract

Methods and compositions are provided for isolation of proliferating cells. In particular, the methods enrich stem cells in a mixture of stem cells and non-stem cells, and in some cases the non-stem cells may be differentiated cells. The methods exploit the non-adherent property of stem cells, as opposed to the adherent property of differentiating cells, by serially passaging the suspended cells in liquid media.

Claims

1. A method of isolating CD34+ stem cells from a plurality of bone marrow cells, comprising the steps of:

culturing bone marrow cells in a first liquid cell culture medium that lacks serum, but that comprises interleukin-3, interleukin-6, stem cell factor, and beta-mercaptoethanol;

successively passaging cells in suspension from the first medium to a subsequent liquid cell culture medium that lacks serum, but that comprises interleukin-3, interleukin-6, stem cell factor, and beta-mercaptoethanol, wherein the passaging occurs about once a week or more than once a week and the successive passaging steps occur over the course of three to four weeks; and

isolating the cells in suspension from the final medium.

2. The method of claim 1, wherein the first liquid cell culture medium lacks matrix and feeder cells.

3. The method of claim 1, wherein the subsequent liquid cell culture medium lacks matrix and feeder cells.

4. The method of claim 1, wherein the passaging occurs about once a week.

5. The method of claim 1, wherein the passaging occurs more than once a week.

6. The method of claim 1, wherein the first liquid cell culture medium further comprises Flt-3/Flk-2, Tpo, Shh, Wnt-3a, Kirre, or a mixture thereof.

7. The method of claim 1, wherein the isolated cells express Oct4, Neurofilament H, Sca-1, AA4.1, cKit, CD45, HMBP, Pax-6, HuC/HuD, NeuN, GAD65, CNPase, MOSP, MBP2, galactocerebroside, NG2, or a combination thereof.

8. The method of claim 1, further comprising the step of delivering one or more of the stem cells to an individual.