In vitro techniques for use with stem cells

• US 8,685,724 B2
• Filed: 06/01/2005
• Issued: 04/01/2014
• Est. Priority Date: 06/01/2004
• Status: Active Grant

• Current Assignee: Kwalata Trading Ltd.
• Original Assignee: Kwalata Trading Ltd.
• Inventors: Fulga, Valentin, Porat, Yael, Belkin, Danny, Shimoni-Zalk, Daphna, Porozov, Svetlana
• Primary Examiner(s): Shen, Bin
• Application Number:
  US11/628,488
• Publication Number:
  US 20080220466A1
• Time in Patent Office: 3,226 Days
• Field of Search: 435/325, 435/372
• US Class Current: 435/325
• CPC Class Codes: A61P 13/12 of the kidneys
• 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 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 41/00 Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

A method is provided for use with extracted blood, including (a) applying blood to a first gradient suitable for selecting first-pass cells having a density less than 1.077 g/ml; (b) applying the first-pass cells to a second gradient suitable for selecting second-pass cells having a density between 1.055 and 1.074 g/ml; (c) increasing the number of cells having a density between 1.055 and 1.074 g/ml, by culturing the second-pass cells for a period lasting between 3 and 30 days; and (d) identifying endothelial progenitor cells in the cultured cells. Other embodiments are also described.

Claims

1. A method for use with extracted blood, comprising:

applying blood to a first gradient suitable for selecting first-pass white blood cells having a density less than 1.077 g/ml;

applying the entire first-pass white blood cell fraction to a second gradient suitable for selecting second-pass cells having a density between 1.055 and 1.074 g/ml;

increasing the number of cells having a density between 1.055 and 1.074 g/ml, by culturing the second-pass cells for a period lasting between 1 and 30 days; and

identifying endothelial progenitor cells in the cultured cells.

2. The method according to claim 1, wherein culturing the second-pass cells comprises culturing the second-pass cells for a period lasting between 3 and 30 days.

3. The method according to claim 2, wherein applying the blood to the first gradient comprises applying the blood to a solution including a copolymer of sucrose and epichlorohydrin.

4. The method according to claim 2, wherein applying the first-pass cells to the second gradient comprises applying the first-pass cells to an aqueous solution of iodixanol.

5. The method according to claim 2, wherein applying the first-pass cells to the second gradient comprises applying the first-pass cells to a continuous density solution including polyvinylpyrrolidone-coated silica colloids.

6. The method according to claim 2 wherein increasing the number of cells comprises culturing the cells for a period lasting between 4 and 8 days.

7. The method according to claim 2, wherein culturing the second-pass cells comprises culturing the second-pass cells in a culture medium comprising one or more agents selected from the group consisting of:

autologous serum, VEGF, b-FGF, IGF and heparin.

8. The method according to claim 2, further comprising, subsequently to the selecting of the first-pass cells, dividing the entire first-pass white blood cell fraction into respective first and second portions thereof, and wherein:

applying the first pass-cells comprises applying the first portion of the first-pass white blood cells to the second gradient,

the method further comprises mixing the second portion of the first-pass cells with the second-pass cells having the density of between 1.055 and 1.074 g/ml, and

increasing the number of cells comprises increasing the number of cells following the mixing.

9. The method according to claim 8, wherein dividing the first-pass cells comprises setting the first portion to be larger than the second portion.

10. The method according to claim 8, wherein dividing the first-pass cells comprises setting the first portion to be smaller than the second portion.