Method for fabricating cell-containing implants

• US 6,886,568 B2
• Filed: 04/04/2001
• Issued: 05/03/2005
• Est. Priority Date: 04/08/1998
• Status: Expired due to Fees

• Current Assignee: Chondros, Inc., Johns Hopkins University
• Original Assignee: Chrondros Incorporated, Johns Hopkins University
• Inventors: Frondoza, Carmelita G., Hungerford, David S., Shikani, Alan H., Fink, David J.
• Primary Examiner(s): McDermott, Corrine
• Assistant Examiner(s): BARRETT, THOMAS C
• Application Number:
  US09/825,632
• Publication Number:
  US 20010014475A1
• Time in Patent Office: 1,490 Days
• Field of Search: 128/898, 424/422, 623/13.17, 623/13.18, 623/23.72
• US Class Current: 128/898
• CPC Class Codes: A61F 2/08 Muscles; Tendons; Ligaments sutures A61B17/04
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 47/36 Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61K 9/5036 Polysaccharides, e.g. gums, alginate; Cyclodextrin
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: C08B 15/005 Crosslinking of cellulose derivatives
• CPC Class Codes: C08B 33/00 Preparation of derivatives of amylose
• CPC Class Codes: C08B 37/00 Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof cellulose D21; microbiological processes C12P
• CPC Class Codes: C08B 37/0021 Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• CPC Class Codes: C08B 37/006 Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• CPC Class Codes: C08H 1/00 Macromolecular products derived from proteins food proteins A23; glue, gelatine C09H
• CPC Class Codes: C08J 2305/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• CPC Class Codes: C08J 3/12 Powdering or granulating
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 2533/70 : Polysaccharides
• CPC Class Codes: C12N 2533/72 : Chitin, chitosan
• CPC Class Codes: C12N 5/0075 : using microcarriers
• CPC Class Codes: C12N 5/0654 : Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/0655 : Chondrocytes; Cartilage

Abstract

Disclosed is a method of growing cells on biodegradable microcarrier particles and more specifically growing chondrocytes for an extended period of time until they aggregate. These aggregated cells can be injected directly or shaped for implantation into the body. In another embodiment of this invention, the cell microcarrier aggregates are grown in a mold that is shaped to conform to the geometry of the desired body part to be replaced. An apparatus for shaping the aggregated cells is disclosed. The aggregated cells can be supplied in a kit.

Claims

1. A method of preparing and then implanting cells comprising the steps of culturing said cells to grow and proliferate on microcarrier particles in a stirred bioreactor under normal gravity conditions for an extended period of time to secrete an extracellular matrix thereby producing a cell-microcarrier particle aggregate and then implanting said cell-microcarrier particle aggregate into a patient to replace and repair damaged or diseased tissue.

2. The method of claim 1 wherein the cells secrete extracellular matrix to create a tissue-like implant.

3. The method of claim 1 wherein the cell-microcarrier aggregates are suspended in a medium suitable for injection for use in repairing or reconstructing missing or diseased tissues, comprising the steps of, (1) isolating the cells to be implanted from donor tissue;

(2) seeding the cells onto a resorbable microcarrier;

(3) culturing the cells on microcarriers to achieve an expansion in the number of cells and the secretion of extracellular matrix;

(4) transferring cell-containing microcarriers to a medium suitable for injection; and

implanting the cell-microcarrier aggregates by injection.

4. The method of claim 3 wherein the cell-microcarrier aggregates are implanted into a cavity or an anatomic site requiring repair or replacement of missing or diseased tissue.

5. The method of claim 3 wherein the cells are chondrocytes.

6. The method of claim 3 wherein the cells are extracellular matrix producing cells selected from chondrocytes;

osteoblasts;

fibroblasts derived from skin, tendon, ligament, meniscus, disk or any other connective tissue;

stem cells derived from skin, tendon, ligament, meniscus, disk, or any other connective tissue;

stem cells derived from bone marrow stroma, muscle, skin or other stem cell-containing tissue;

embryonic stem cells;

or combinations of these cells that may be seeded onto the microcarrier.

7. The method of claim 3 wherein the microcarrier is a biodegradable material.

8. The method of claim 3 wherein the microcarrier is selected from inorganic materials selected from the group of calcium phosphates, calcium carbonates, calcium sulfates or combinations of these materials;

organic materials selected from the group of biopolymers, collagen, gelatin, hyaluronic acid, chemically derived modifications of hyaluronic acid, chitin, chitosan, chitosan derivatives, fibrin, dextran, agarose, or calcium alginate, or synthetic polymeric materials, polylactic acid, polyglycolic acid or copolymers or combinations of the two, polyurethanes, polycarbonates, polycaprolactones, hydrogels such as poly acrylates, poly vinyl alcohols, poly ethylene glycols, or poly ethylene imines;

or particles of tissues such as bone or demineralized bone, cartilage, tendon, ligament, fascia, intestinal mucosa or other connective tissues, or chemically modified derivatives of these materials.

9. The method of claim 3 wherein the fluid medium suitable for injection is selected from isotonic saline for injection, phosphate buffered saline, or Hank'"'"'s balanced salt solution.

10. The method claim 3 wherein the fluid medium suitable for injection contains a material capable of polymerizing or gelling after implantation.

11. The method of claim 6 wherein the material is selected from fibrin glues, collagen, combinations of fibrin/collagen, transglutaminase-catalyzed binding systems, hyaluronic acid, calcium alginate gels, chitosan derivatives capable of gelling at body temperature, hydrogels such as polyacrylates, polyvinyl alcohols, polyethylene glycols, or polyethylene imines, or similar materials with suitable gelling compositions.

12. The method of claim 10 where in the in situ gelling of these materials is initiated by thermal, enzymatic or chemical catalysts, pH or ionic strength changes or photo-initiation procedures.

13. The method of claim 1 wherein the cell-microcarrier aggregates are further cultured inside a mold which has been shaped to configure the geometry of the area of the body receiving the cells for implantation.

14. The method of claim 13 wherein the mold is shaped to produce a form that can be or modified to a desired shape at the time of implantation.

15. The method of claim 13 wherein the cell-microcarrier aggregates are consolidated into an implantable structure for repair or replacement of missing or diseased tissue.

16. The method of claim 13 wherein the cell-microcarrier aggregates are implanted into a cavity or an anatomic site requiring repair or replacement of missing or diseased tissue.

17. The method of claim 1 wherein the cells are chondrocytes.

18. The method of claim 1 wherein the cells are extracellular matrix producing cells selected from chondrocytes;

osteoblasts;

fibroblasts derived from skin, tendon, ligament, meniscus, disk or any other connective tissue;

stem cells derived from skin, tendon, ligament, meniscus, disk or any other connective tissue;

stem cells derived from bone marrow stroma, muscle, skin or other stem cell-containing tissue;

embryonic stem cells;

or combinations of these cells that may be seeded onto the microcarrier.

19. The method of claim 1 wherein the microcarrier is a biodegradable material.

20. The method of claim 1 wherein the microcarrier is selected from inorganic materials such as calcium phosphates, calcium carbonates, calcium sulfates or combinations of these materials;

organic materials including biopolymers selected from the group consisting of collagen, gelatin, hyaluronic acid or chemically derived modifications of hyaluronic acid, chitin, chitosan, chitosan derivatives, fibrin, dextran, agarose, calcium alginate, synthetic polymeric materials selected from the group consisting of polylactic acid, polyglycolic acid or copolymers or combinations of the two, polyurethanes, polycarbonates, polycaprolactones, polyanhydrides, hydrogels selected from the group consisting of polyacrylates, polyvinyl alcohols, polyethylene glycols, polyethylene imines, or particles of tissue such as bone or demineralized bone, cartilage, tendon, ligament, fascia, intestinal mucosa or other connective tissues, or chemically modified derivatives of these materials.

21. The method of claim 1 wherein the cell-microcarrier aggregate is prepared by culturing said cells to grow and proliferate on microcarrier particles for an extended period of time of from about seven to sixty days thereby producing a cell-microcarrier particle aggregate useful for implantation.

22. The method of claim 21 wherein the cells are chondrocytes.

23. The method of claim 21 wherein the cells are selected from chondrocytes;

osteoblasts;

fibroblasts derived from skin, tendon, ligament, meniscus, disk or any other connective tissue;

stem cells derived from bone marrow stroma, muscle, skin or other stem cell-containing tissue;

embryonic stem cells;

or combinations of these cells that may be seeded onto the microcarrier.

24. The method of claim 1 wherein the growth and proliferation is over an extended period of time of about five weeks.

25. The method of claim 1 wherein the growth and proliferation is over an extended period of time of about 28 days.

26. The method of claim 1 wherein the growth and proliferation is over an extended period of time of about 30 days.

27. The method of claim 1 wherein the growth and proliferation is over an extended period of time of about 14-28 days.

28. The method of claim 1 wherein the growth and proliferation is over an extended period of time of about 60 days.

29. A method of preparing and then implanting chondrocytes comprising the steps of culturing said chondrocytes on microcarriers in an implant assembly unit for a period of time under normal gravity conditions by which said chondrocytes secrete and extracellular matrix and aggregate and thus can be shaped or cut to conform to the body area which will receive said chondrocytes and then implanting said chondrocytes in a patient to replace and repair damaged or diseased tissue.

30. The method of claim 29 wherein culturing is carried out in an implant assembly unit.

31. A method of claim 29 wherein the cells are chondrocytes.

32. The method of claim 29 wherein the cells are selected from chondrocytes;

osteoblasts;

fibroblasts derived from skin, tendon, ligament, meniscus, disk or any other connective tissue;

stem cells derived from bone marrow stroma, muscle, skin or other stem cell-containing tissue;

embryonic stem cells;

or combinations of these cells that may be seeded onto the microcarrier.

33. The method of claim 31 wherein the culturing of said cells takes place over one to five weeks.

34. The method of claim 31 wherein the cells are seeded onto the microcarrier at a density of 1 to 4×

10³ cells/cm².