In Vitro Techniques For Use With Stem Cells

• US 20080220466A1
• Filed: 06/01/2005
• Published: 09/11/2008
• Est. Priority Date: 06/01/2004
• Status: Active Grant

• Current Assignee: Kwalata Trading Ltd.
• Original Assignee: Kwalata Trading Ltd.
• Inventors: Porat, Yael, Fulga, Valentin, Porozov, Svetlana, Belkin, Danny, Shimoni-Zalk, Daphna
• Granted Patent: US 8,685,724 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• 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 cells having a density less than 1.077 g/ml;

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;

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

identifying endothelial progenitor cells in the cultured cells.

2. The method according to claim 1, wherein applying the blood to the first gradient comprises applying the blood to a solution including a copolymer of sucrose and epichlorohydrin.

3. The method according to claim 1, wherein applying the first-pass cells to the second gradient comprises applying the first-pass cells to an aqueous solution of iodixanol.

4. The method according to claim 1, wherein applying the first-pass cells to the second gradient comprises applying the first-pass cells to a gradual density solution including polyvinlypyrrolidone-coated silica colloids.

5. The method according to claim 1, wherein applying the blood cells to the first gradient comprises applying the blood cells to a Ficoll-like gradient.

7. The method according to claim 1, wherein applying the first-pass cells to the second gradient comprises applying the first-pass cells to a Percoll-like gradient.

6. (canceled)

8-86. -86. (canceled)

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

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

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;

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 progenitor cells in the cultured cells.

88-92. -92. (canceled)

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

applying blood to a gradient to select cells having a first desired density range;

increasing the number of cells having a second desired density ranges by culturing the selected cells for a period lasting between 3 and 30 days; and

identifying progenitor cells in the cultured cells.

94. The method according to claim 93, wherein the progenitor cells include endothelial progenitor cells (EPCs), and wherein identifying the progenitor cells comprises identifying the EPCs.

95. The method according to claim 93, wherein applying the blood to the gradient comprises applying blood to a gradient suitable for selecting cells having a density less than 1.077 g/ml.

96. The method according to claim 93, wherein applying the blood to the gradient comprises applying blood to a gradient suitable for selecting cells having a density between 1.055 and 1.074 g/ml.

97. The method according to claim 93, wherein increasing the number of cells comprises culturing the cells for a period lasting between 4 and 8 days.

98. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to a solution including a copolymer of sucrose and epichlorohydrin.

99. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to a gradual density solution including polyvinylpyrrolidone-coated silica colloids.

100. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to an aqueous solution of iodixanol.

101. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to a Ficoll-like gradient.

102. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to an OptiPrep-like gradient.

103. The method according to claim 93, wherein applying the blood to the gradient comprises applying the blood to a Percoll-like gradient.

104-176. -176. (canceled)

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

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

dividing the first-pass cells into respective first and second portions thereof;

applying the first portion of 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;

mixing the second portion of the first-pass cells with the 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 3 and 30 days; and

identifying progenitor cells in the cultured cells.

178. The method according to claim 177, wherein the progenitor cells include endothelial progenitor cells (EPCs), and wherein identifying the progenitor cells comprises identifying the EPCs.

179. The method according to claim 177, wherein dividing the first-pass cells comprises setting the first portion to be larger than the second portion.

180. The method according to claim 177, wherein dividing the first-pass cells comprises setting the first portion to be smaller than the second portion.