Cell-culture and polymer constructs

• US 20020123142A1
• Filed: 01/03/2002
• Published: 09/05/2002
• Est. Priority Date: 04/08/1998
• Status: Abandoned Application

• Current Assignee: Abraham J. Domb, Afshin Sohrabi, Alan H. Shikani, Carmelita G. Frondoza, David S. Hungerford
• Original Assignee: Abraham J. Domb, Afshin Sohrabi, Alan H. Shikani, Carmelita G. Frondoza, David S. Hungerford
• Inventors: Sohrabi, Afshin, Domb, Abraham J., Frondoza, Carmelita G., Hungerford, David S., Shikani, Alan H.
• Application Number:
  US10/039,718
• Publication Number:
  US 20020123142A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61F 2/08 Muscles; Tendons; Ligaments sutures A61B17/04
• CPC Class Codes: A61F 2/30756 Cartilage endoprostheses A61F2/3603 takes precedence
• 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 9/1652 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 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• 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: 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

Cells grown on a microcarrier are separated from the microcarrier by enzymatically digesting the microcarrier. More specifically, chondrocytes may be grown on dextran microcarrier beadlets and then the beadlets digested using dextranase to separate the chondrocytes from the carrier. Cells can also be grown on chitosan microcarriers to be used for implantation. In addition, cells can be grown on polysaccharide polymers to be used as implant devices. Various polymers serve as scaffolds for cells to be used for implantation. The polymers can be used for cell culture as well as for preparing scaffolds useful for tissue replacement such as cartilage tissue.

Claims

1. In a method of cell culture wherein cells are cultured on microcarrier beadlets the improvement comprising growing the cells on dextran microcarrier beadlets and thereafter separating the cells from the microcarrier beadlets by digestion with dextranase.

2. The method of claim 1 wherein the cells are a member of the group consisting of chondrocytes, oesteocytes, oesteoblasts, monocytes, chondrogenic cells, pluripotential cells, mucosal cells and fibroblasts.

3. The method of claim 2 wherein the cells are chondrocytes or chondrogenic cells.

4. The method of claim 3 wherein after separating the chondrocytes or chondrogenic cells from the microcarrier beadlets the cells are plated on a scaffold or template for growth thereon.

5. The method of claim 4 wherein after the cells are plated on a scaffold or template for growth thereon, the scaffold or template is surgically implanted into the cartilage tissue.

6. The method of claim I wherein after separating the cells from the microcarrier beadlets the cells are injected into a patient in need of said cells.

7. A crosslinked polymer prepared by crosslinking a polysaccharide with a polyamine said polymer to be used for cell culture and preparing scaffolds or templates for tissue replacement.

8. The crosslinked polymer of claim 7 wherein the polysaccharide is selected from the group consisting of arabinogalactan and oxidized arabinogalactan;

and the crosslinking agent is glutamic acid and lysine, albumin, gelatin or lysine.

9. The crosslinked polymer of claim 7 wherein the polysaccharide is selected from the group consisting of dextran, cellulose, arabinogalactan, pollutant and amylose.

10. In a method of cell culture wherein cells are cultured on microcarrier beadlets the improvement comprising growing, the cells on a polyamine cross-linked polysaccharide polymer of claim 7 and thereafter separating the cells from the microcarrier beadlets by digestion with an enzyme compatible with the polysaccharide.

11. The method of claim 10 wherein the cells are a member of the group consisting of chondrocytes, chondrogenic cells, oesteoblasts, monocytes, pluripotential cells, mucosal cells and fibroblasts.

12. The method of claim 11 wherein the cells are chondrocytes or chondrogenic cells.

13. The method of claim 12 wherein after separating the chondrocytes or chondrogenic cells from the microcarrier beadlets the cells are plated on a scaffold or template for growth thereon.

14. The method of claim 13 wherein after the cells are plated on the scaffold or template for growth thereon, the scaffold or template is surgically implanted into the cartilage tissue.

16. The beadlet for cell culture or the scaffold used for cartilage repair of claim 15 wherein the polysaccharide used to prepare the crosslinked polysaccharide derivative is a member selected from the group consisting of dextran, arabingogalactan, pollutant. cellulose and amylose;

with the polysaccharide derivative being crosslinked by a polyamine compound selected from the group consisting of lysine, ethylenediamine, alkylenediamine, phenylenediarnine, xylenediamine, polyethylenimine, gelatin, albumin, fibrinogen and

18. The beadlet for cell culture, or the scaffold used for cartilage repair of claim 17 wherein the polysaccharide used to prepare the crosslinked polysaccharide derivative is a member selected from the group consisting of dextran, arabinogalactan, pollulan, cellulose and amylose;

with the polysaccharide derivative being crosslinked by a polyamine compound selected from the group consisting of lysine, ethylenediamie, alkylenediamine, phenylenediamine, xylenediamine, polyethylenimine, gelatin, albumin, fibrinogen and

19. The method for the surgical repair of cartilage wherein the crosslinked polysaccharide derivative of claim 15 in the form of a scaffold containing chondrocytes thereon is surgically implanted into the cartilage tissue of a patient.

20. The method for the surgical repair of cartilage wherein the crosslinked polysaccharide derivative of claim 17 in the form of a scaffold containing chondrocytes thereon is surgically implanted into the cartilage tissue of a patient.

15. A beadlet for cell culture cell culture or a scaffold used for cartilage repair comprising a crosslinked polysaccharide derivative of the formula

17. A beadlet for cell culture or a scaffold used for cartilage repair comprising a crosslinked polysaccharide derivative of the formula

21. A scaffold or template for cartilage repair comprising a chitosan scaffold or template with chondrocytes incorporated thereon to produce a scaffold or template for cartilage repair having viable chondrocytes.

22. A method for the surgical repair of cartilage tissue wherein the chitosan scaffold or template with chondrocytes of claim 21 is surgically incorporated into the cartilage tissue of a patient.

24. In a surgical procedure for surgically implanting chondrocytes into cartilage tissue wherein the scaffold or template having plated and growing thereon the chondroctyes of claim 23 is surgically implanted into the cartilage for cartilage repair.

25. In the surgical procedure of claim 24 wherein the scaffold or template having plated and growing thereon chondroctyes is surgically implanted into cartilage of the knee.

26. In the surgical procedure of claim 24 wherein the scaffold or template having plated and growing thereon chondrocytes is surgically implanted into cartilage of t he head or neck area.

27. In the surgical procedure of claim 24 wherein the scaffold or template having plated and growing thereon chondrocytes is surgically implanted into the knee of a horse.

30. The combination of claim 29 wherein the chitosan is in the form of a scaffold to be used in transplantation surgery.

23. A scaffold or template for cartilage repair comprising a member of the group consisting of chitosan and a crosslinked polysaccharide prepared by crosslinking said polysaccharide with a polyamine, said member being shaped in the form of a scaffold or template and having plated and growing thereon chondrocytes.

28. In a surgical method for surgically implanting chondrocytes into cartilage tissue comprising the steps of:

1) culturing chondrocytes on dextran microcarrier beadlets and thereafter separating the chondrocytes form the microcarrier beadlets by digestion with dextranase,

2) plating and growing said separated chondrocytes on a scaffold or template manufactured from a crosslinked polysaccharide prepared by crosslinking said polysaccharide with a polyamine or alternatively plating and growing said separated chondrocytes on a scaffold or template comprising chitosan and then

3) surgically implanting into cartilage tissue said scaffold or template having chondrocytes plated and grown thereon, to thereby repair cartilage tissue in a patient requiring cartilage repair.

29. In combination chitosan and a member of the group consisting of osteoblasts and chondrocytes wherein the chitosan serves as a substrate for the growth of said osteoblasts or chondrocytes.

31. A method of culturing and obtaining increased levels of collagen type II chondrocytes comprising culturing the chondrocytes at a low oxygen tension.

32. The method of claim 31 wherein the oxygen tension is about 5%.

33. In a surgical procedure wherein chondrocytes are taken from a patient rapidly multiplied and transplanted into said patient comprising the steps of:

1) obtaining cartilage tissue from a patient,

2) treating the cartilage to obtain chondrocytes therefrom,

3) culturing the chondrocytes to obtain an adequate sample of chondrocytes,

4) plating and growing the chondrocytes onto a chitosan scaffold, and

5) surgically implanting the chondrocytes bearing chitosan scaffold into said patient, wherein the entire surgical procedure is carried out expeditiously with good quality chondrocytes being returned to the patient and the scaffold being reabsorbed leaving in place viable chondrocytes.

34. A method for preparing a scaffold for tissue repair comprising culturing chondrocytes onto a chitosan scaffold wherein the culturing takes place in the presence of integrins.

35. The method of preparing a scaffold for tissue repair comprising culturing chondrocytes onto a chitosan scaffold wherein the culturing takes place at a low oxygen tension under controlled conditions in an antomated spin culture.