Closed system separation of adherent bone marrow stem cells for regenerative medicine applications

• US 9,169,464 B2
• Filed: 10/15/2010
• Issued: 10/27/2015
• Est. Priority Date: 10/16/2009
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: Rutgers University
• Inventors: Sabaawy, Hatem
• Primary Examiner(s): Lankford, Blaine
• Assistant Examiner(s): BERKE-SCHLESSEL, DAVID W
• Application Number:
  US13/502,174
• Publication Number:
  US 20130101561A1
• Time in Patent Office: 1,838 Days
• Field of Search: None
• US Class Current: 1/1
• 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: C12M 23/14 Bags
• CPC Class Codes: C12M 25/06 Plates; Walls; Drawers; Multilayer plates
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0669 Bone marrow stromal cells; Whole bone marrow isolated stem cells from bone marrow C12N5/0647, C12N5/0663
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

A method for isolating and processing bone marrow derived stem cells, including the steps of: (a) collecting a biological sample containing adherent bone marrow stem cells in a receptacle with interior walls coated with a cell-adherent substrate; (b) incubating the bone marrow cells on the adherent substrate so that a layer of adherent bone marrow stem cells adheres to the substrate; (c) washing any non-adherent cells from the substrate; and (d) collecting the bone marrow stem cell layer. Isolation kits and use of bone marrow cells harvested for cell therapies are also described.

Claims

1. A stem cell isolation kit comprising:

a) a first bag containing a plasma blood expander;

b) a second bag containing an anticoagulant and a growth medium, said second bag comprising an injection port for introduction of a biological sample comprising red blood cells and stem cells;

c) a third bag for collection of sedimented red blood cells from the second bag;

d) a fourth bag comprising an interior surface coated with a cell-adherent surface layer for collection and isolation of adherent stem cells;

e) a fifth bag containing a preservative for storage of adherent stem cells;

wherein said first and second bags are interconnected with each other through sterile tubing for transferring the contents of the first bag to the second bag;

the second and third bags are interconnected with each other through sterile tubing for sedimenting red blood cells from the second bag to the third bag;

the second and fourth bags are interconnected with each other through sterile tubing for transferring of stem cells from the second bag to the fourth bag; and

wherein the fourth and fifth bags are interconnected with each other through sterile tubing for the transfer of adherent stem cells from the fourth to the fifth bag.

2. The kit of claim 1, wherein said growth medium is selected from the group consisting of X-vivo, RPMI-1640, DMEM, and α

MEM, or a combination thereof.

3. The kit of claim 2, wherein said growth is RPMI-1640 or X-vivo medium.

4. The kit of claim 1, further comprising a container of cell detachment solution connected to said fourth bag through sterile tubing.

5. The kit of claim 1, further comprising aspirating means for collecting bone marrow aspirates.

6. The kit of claim 1, wherein said interior surface of said fourth bag is corona discharge treated before being coated with said cell-adherent surface layer.

7. The kit of claim 1, wherein said cell-adherent surface layer comprises a cell-adherent biopolymer, polypeptide, protein or polysaccharide.

8. The kit of claim 7, wherein said cell-adherent surface layer comprises a coating of one or more basement membrane proteins.

9. The kit of claim 8, wherein said basement membrane proteins are selected from the group consisting of fibronectin, collagen, laminin, keratin, fibrin and fibrinogen.

10. The kit of claim 7, wherein said cell-adherent surface layer comprises a coating of one or more polysaccharides selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate and agarose.

11. The kit of claim 7, wherein said cell-adherent surface layer comprises poly-L-lysine.

12. The kit of claim 1, wherein said plasma blood expander in said first bag is hydroxyethyl starch (HES) and said anticoagulant in said second bag is selected from the group consisting of ACD, CPD, CPDA, heparin and sodium citrate.

13. The kit of claim 12, wherein said first bag is graded to allow fixed ratio mixing of hydroxyethyl starch (HES) to an anti-coagulated bone marrow or biological fluid sample in said second bag to reach a final HES concentration of 1.2%.