Methods of expanding human embryonic stem cells

• US 8,592,208 B2
• Filed: 01/07/2013
• Issued: 11/26/2013
• Est. Priority Date: 03/07/2006
• Status: Active Grant

• Current Assignee: Geeta Shroff
• Original Assignee: Geeta Shroff
• Inventors: Shroff, Geeta
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US13/735,281
• Publication Number:
  US 20130122588A1
• Time in Patent Office: 323 Days
• Field of Search: None
• US Class Current: 435/366
• 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 35/48 Reproductive organs
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 27/02 Ophthalmic agents
• 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 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 39/00 : General protective or antinoxious agents
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2501/31 : Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
• CPC Class Codes: C12N 2501/392 : Sexual steroids
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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: G01N 33/5044 : involving specific cell types

Abstract

The present invention relates to a pharmaceutical composition comprising of preparations of human embryonic stem (hES) cells and their derivatives and methods for their transplantation into the human body, wherein transplantation results in the clinical reversal of symptoms, cure, stabilization or arrest of degeneration of a wide variety of presently incurable and terminal medical conditions, diseases and disorders. The invention further relates to novel processes of preparing novel stem cell lines which are free of animal products, feeder cells, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor and conditioned media. This invention further relates to the isolation, culture, maintenance, expansion, differentiation, storage, and preservation of such stem cells.

Claims

1. A method of expanding hES cells free of animal products, feeder cells, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor, and conditioned media, comprising:

(a) introducing hES cells in a cell culture medium consisting of minimal essential medium, a progestin, and a β

-human chorionic gonadotropin (β

hCG) agonist; and

(b) incubating the cells at a temperature of about 34°

C. to about 38°

C. in an environment of about 3.5% to about 6% carbon dioxide for about 12 hours to about 48 hours.

2. The method of claim 1, wherein said cell culture medium is RPMI.

3. The method of claim 1, further comprising:

(c) taking an aliquot of incubated cells of (b) wherein said aliquot contains at least one cell,

(d) resuspending the cells in cell culture medium along with progesterone and β

hCG to the aliquot,

(e) diluting the cells in cell culture medium without progestin and β

hCG agonist, and

(f) incubating the cells at a temperature of about 34°

C. to about 38°

C. in an environment of about 3.5% to about 6% carbon dioxide tor about 12 hours to about 48 hours.

4. The method of claim 1, wherein said incubation is carried out in a water jacketed cell culture incubator.

5. The method of claim 1, wherein said cells in culture medium are incubated in a biocompatible container under substantially anaerobic conditions.

6. The method of claim 5, wherein said cells do not differentiate on proliferation.

7. The method of claim 1, further comprising testing the expanded cells for any contamination.

8. The method of claim 1, wherein said culture process is carried out in biocompatible containers.

9. The method of claim 1, wherein the ratio of the cells to the cell medium is about 1:

3.5 to about 1;

35.

10. The method of claim 1, wherein said incubation is carried out in a substantially anaerobic environment.

11. The method of claim 1, wherein said incubation is carried out in a biocompatible container where the volume is almost completely occupied by the medium and the container is kept in a vertical position.