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

• US 9,770,470 B2
• Filed: 10/08/2015
• Issued: 09/26/2017
• Est. Priority Date: 10/16/2009
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: Rutgers University
• Inventors: Sabaawy, Hatem
• Primary Examiner(s): BERKE-SCHLESSEL, DAVID W
• Application Number:
  US14/878,278
• Publication Number:
  US 20160022739A1
• Time in Patent Office: 719 Days
• Field of Search: None
• 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 method for isolating and processing bone marrow derived stem cells comprising the steps of:

(a) collecting a biological sample comprising adherent bone marrow stem cells;

(b) transferring the biological sample to a first closed receptacle containing an anticoagulant;

(c) adding to the first closed receptacle an amount of plasma blood volume expander effective to separate red blood cells from the biological sample;

(d) transferring the biological sample from the first closed receptacle to a second closed receptacle through interconnected sterile tubing, the second closed receptacle having an interior surface coated with a cell-adherent surface-layer and containing a growth medium;

(e) incubating the adherent bone marrow stem cells in said second closed receptacle for in between about two hours and about five days, wherein the adherent bone marrow stem cells adhere to the cell-adherent surface-layer, forming an adherent bone marrow stem cell layer;

(f) washing the adherent bone marrow stem cell layer, wherein any non-adherent bone marrow stem cells are removed from the adherent bone marrow stem cell layer; and

(g) collecting said adherent bone marrow stem cell layer to create collected bone marrow stem cells.

2. The method of claim 1, wherein said biological sample further comprises umbilical cord blood or bone marrow aspirates.

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

4. The method of claim 3, wherein the interior surface of said second closed receptacle is corona discharge treated prior to coating with said cell-adherent surface layer.

5. The method of claim 3, wherein said cell-adherent surface layer comprises a coating of at least one basement membrane protein.

6. The method of claim 5, wherein said basement membrane protein is selected from the group consisting of fibronectin, collagen, laminin, keratin, fibrin, and fibrinogen.

7. The method of claim 3, wherein said cell-adherent surface layer comprises gelatin.

8. The method of claim 3, wherein said cell-adherent surface layer comprises at least one polysaccharide selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate, and agarose.

9. The method of claim 3, wherein said cell-adherent surface layer comprises poly-L-lysine or poly-D-lysine.

10. The method of claim 1, wherein step (f) further comprises replacing said growth medium at least once.

11. The method of claim 1, wherein step (g) comprises incubating said adherent bone marrow stem cell layer with a cell detachment solution.

12. The method of claim 1, further comprising the step of (h-1) of suspending said collected bone marrow stem cells in a pharmaceutically acceptable saline solution, wherein step (h-1) is performed after step (g).

13. The method of claim 1, further comprising the step of (h-2) of checking the collected bone marrow stem cells for at least of a cell phenotype, viability, and sterility, wherein step (h-2) is performed after step (g).

14. The method of claim 1, wherein the plasma blood volume expander is hydroxyethyl starch (HES) or gelatin polysuccinate.