Method for preparation of implantable constructs

• US 8,906,686 B2
• Filed: 04/28/2006
• Issued: 12/09/2014
• Est. Priority Date: 03/22/2002
• Status: Expired due to Fees

• Current Assignee: Histogenics Corporation (Ocugen, Inc.), Takagi Industrial Company Limited
• Original Assignee: Histogenics Corporation (Ocugen, Inc.)
• Inventors: Mizuno, Shuichi, Kusanagi, Akihiko, Tarrant, Laurence J. B., Tokuno, Toshimasa, Smith, Robert Lane
• Primary Examiner(s): Weber, Jon P
• Assistant Examiner(s): Constantine, Charles Zoltan
• Application Number:
  US11/413,419
• Publication Number:
  US 20060216822A1
• Time in Patent Office: 3,147 Days
• Field of Search: None
• US Class Current: 435/395
• CPC Class Codes: A61F 2/28 Bones joints A61F2/30
• CPC Class Codes: A61F 2310/00365 Proteins; Polypeptides; Degradation products thereof
• 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 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• CPC Class Codes: A61L 27/3843 Connective tissue
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61L 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: C12N 2521/00 Culture process characterised by the use of hydrostatic pressure, flow or shear forces
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Neo-cartilage constructs suitable for implantation into a joint cartilage lesion in situ and a method for repair and restoration of function of injured, traumatized, aged or diseased cartilage. The construct comprises at least chondrocytes incorporated into a support matrix processed according to the algorithm comprising variable hydrostatic or atmospheric pressure or non-pressure conditions, variable rate of perfusion, variable medium composition, variable temperature, variable cell density and variable time to which the chondrocytes are subjected.

Claims

1. A method for preparation of an implantable construct for implantation into a cartilage lesion, said method comprising steps:

providing a support matrix comprising a plurality of pores;

suspending chondrocytes in a collagen solution;

seeding said suspended chondrocytes into the pores of said support matrix, wherein said suspended chondrocytes are seeded at a density of about 3 to about 60 million cells/mL of the collagen solution; and

culturing said chondrocytes seeded in the support matrix under conditions sufficient to activate and promote proliferation of the chondrocytes within said support matrix, wherein said conditions comprise;

applying a cyclic hydrostatic pressure from about 0.5 MPa to about 3 MPa above atmospheric pressure, at a frequency about 0.1 Hz to about 2.0 Hz, for about 6 days to about 14 days followed with a resting period at atmospheric pressure for about 7 to about 28 days; and

perfusing said seeded support matrix with a culture medium at a rate of perfusion from about 5 μ

L/min to about 50 μ

L/min.

2. The method of claim 1 wherein said perfusing is further performed under a reduced oxygen concentration from about 1% to about 5%.

3. The method of claim 2 wherein said perfusing is further performed under a carbon dioxide concentration of about 5%.

4. The method of claim 3 wherein said support matrix is prepared from a material selected from the group consisting of Type I collagen, Type II collagen, Type IV collagen, collagen containing proteoglycan, collagen containing glycosaminoglycan, collagen containing glycoprotein, gelatin, agarose, hyaluronin, elastin, fibrin, polymeric fiber made of a polylactic acid, polymeric fiber made of a polyglycolic acid, polycaprolactone, polyamino acid, thermoreversible gelation hydrogel (TRGH), and a combination thereof.

5. The method of claim 4 wherein said material is Type I collagen or Type II collagen.

6. The method of claim 5 wherein said Type I collagen, or Type II collagen is lyophilized.

7. The method of claim 2 wherein the oxygen concentration is about 2%.

8. The method of claim 7 wherein the plurality of pores have a pore size from about 100 to about 300 μ

m.

9. The method of claim 1 wherein said chondrocytes are seeded into said support matrix at a density of about 12 to about 15 million cells/mL of the collagen solution.

10. The method of claim 1 wherein the cyclic hydrostatic pressure is applied at about 3 MPa above atmospheric pressure.

11. The method of claim 1 wherein said cyclic hydrostatic pressure is applied for a period of about 6 days followed by a 12 day resting period.

12. The method of claim 1 wherein the rate of perfusion is about 5 μ

L/min.

13. The method of claim 12 wherein a cell density is about 3 millions cells/mL of collagen solution.