Mixed cell populations for tissue repair and separation technique for cell processing

• US 8,394,631 B2
• Filed: 12/07/2010
• Issued: 03/12/2013
• Est. Priority Date: 11/03/2006
• Status: Active Grant

• Current Assignee: Vericel Corporation
• Original Assignee: Aastrom Biosciences, Inc.
• Inventors: Hampson, Brian, Goltry, Kristin, Smith, Douglas M., Rowley, Jonathan A., Venturi, Naia
• Primary Examiner(s): Ford, Allison
• Application Number:
  US12/962,378
• Publication Number:
  US 20110124105A1
• Time in Patent Office: 826 Days
• Field of Search: None
• US Class Current: 435/378
• 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: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 39/461 characterised by the cell type used
• CPC Class Codes: A61K 39/464 characterised by the antigen targeted or presented
• 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 13/12 of the kidneys
• 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 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 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• 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 9/04 : 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 2500/30 : Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/32 : Amino acids
• CPC Class Codes: C12N 2500/84 : from mammals
• CPC Class Codes: C12N 2509/00 : Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0645 : Macrophages, e.g. Kuepfer cells in the liver; Monocytes
• 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

Abstract

The present invention provides a fluid exchange cell culture technique and tissue repair cells (TRCs) made by these methods, as well as methods using these cells. The method includes a new wash step which increases the tissue repair properties of the TRCs of the invention. This wash step allows for the production of TRC populations with greater tissue repair and anti-inflammatory capabilities. Embodiments of the present invention include a post-culture process for cultured cells that preferably includes the steps of: a wash process for removing unwanted residual culture components, a volume reduction process, and a harvesting process to remove cultured cells. Preferably, all these steps are performed within a aseptically closed cell culture chamber by implementing a separation method that minimizes mechanical disruption of the cells and is simple to automate. The harvested cells may then be concentrated to a final volume for the intended use. In such embodiments, the final composition is a substantially purified and concentrated cell mixture suspended in a physiologic solution suitable for immediate use in humans without further washing, volume reduction, or processing. Embodiments are also applicable to harvesting (and/or washing) particles within a liquid or solution within a chamber.

Claims

1. A method for processing cells in a biochamber, the method comprising:

a) culturing said cells with culture media for a predetermined time period;

b) displacing the culture media from the biochamber into a waste container by introducing a biocompatible first rinse solution;

c) replacing the first rinse solution with a cell harvest solution comprising an enzyme that allows for the detachment of cells from each other and/or from the biochamber surface;

d) incubating the contents of the biochamber for a predetermined period of time to allow the detachment of cells adhered to each other and/or the biochamber surface, wherein said cells remain settled on the surface;

e) displacing the cell harvest solution into a waste container by introducing a second rinse solution into the biochamber, wherein said cells remain settled on the surface; and

f) agitating the biochamber to bring settled cells into suspension,

wherein said method produces a mixed cell population that is at least 80% viable and at least 5% of the cells express CD90.

2. The method according to claim 1, wherein the second rinse solution comprises a solution capable of being injectable into a human.

3. The method for processing cultured cells according to claim 1, further comprising:

g) draining the solution with suspended cells into a cell collection container.

4. The method for processing cultured cells according to claim 3, further comprising, subsequent to g) draining the solution with suspended cells into a cell collection container:

(h) adding an additional amount of the second rinse solution to the biochamber, and

(i) agitating the biochamber to bring residual settled cells into suspension.

5. The method for processing cultured cells according to claim 4, further comprising:

j) draining the solution with suspended cells into a cell collection container.

6. The method for processing cultured cells according to claim 1, further comprising:

displacing a portion of the second rinse solution with a gas to obtain a predetermined reduced liquid volume in the biochamber.

7. The method according to claim 6 wherein the flow rate for the introduction of solutions and/or gases added to the biochamber is between about 0.03 to about 1.0 volume exchanges/min.

8. The method according to claim 6, wherein the flow rate for the introduction of solutions and/or gases added to the biochamber is between about 0.50 to about 0.75 volume exchanges/min.

9. The method according to claim 1, wherein liquids/solutions or gases are introduced into the biochamber according to a radial plug flow.

10. The method according to claim 1, wherein said cells are derived from mononuclear cells.

11. The method according to claim 10, wherein said mononuclear cells are bone marrow, peripheral blood, umbilical cord blood or fetal liver.