Encapsulation of cells in biologic compatible scaffolds by coacervation of charged polymers

• US 20060019362A1
• Filed: 07/26/2004
• Published: 01/26/2006
• Est. Priority Date: 07/26/2004
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Yu, Hanry, Toh, Yi Chin
• Granted Patent: US 7,704,714 B2
• Time in Patent Office: Days
• US Class Current: 435/182
• CPC Class Codes: A61L 27/165 Rubbers
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: A61L 27/44 having a macromolecular matrix
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: A61L 27/58 Materials at least partially resorbable by the body
• CPC Class Codes: C08L 33/12 Homopolymers or copolymers of methyl methacrylate
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

This invention relates to a method for the encapsulation of cells in biologic compatible three dimensional scaffolds and the use of such cells encapsulated in a scaffold. The cells are embedded in a charged polymer that is complex coacervating with an oppositely charged polymer within biologic compatible scaffolds. The polymer complex embedding the cells is forming an ultra thin membrane on the surface of the three dimensional scaffold.

Claims

1. A method for encapsulating at least one cell species in a biologic compatible scaffold comprising:

(a) providing a first substrate coated with a first charged polymer;

(b) placing a scaffold comprising at least one cell species embedded in a second charged polymer having an electrical charge opposite to that onto the first charged polymer on the first substrate;

(c) placing a second substrate coated with a third charged polymer to form a sandwich structure with the first substrate and the scaffold arranged there in between, wherein the first polymer and the third polymer are of the same electrical charge.

2. The method of claim 1, wherein an attachment-dependent cell species is used as the at least one cell species.

3. The method of claim 1, wherein an attachment-independent cell species is used as the at least one cell species.

4. The method of claim 1, wherein charged polymers are used which form polymer complexes being permeable to substances necessary to sustain the normal metabolic functions of the at least one cell species and to products released by the at least one cell species.

5. The method of claim 4, wherein the first charged polymer and the third charged polymer used are identical.

6. The method of claim 5, wherein the second charged polymer is selected from the group consisting of chitosan, polyanionic alginate, negatively or positively charged collagen, carboxymethylcellulose (CMC) and polyanionic alginate.

7. The method of claim 6, wherein the first charged polymer is selected from the group consisting of terpolymer, Ca²⁺, chondroitin sulfate A-chitosan and polycationic poly(L-lysine).

8. The method of claim 6, wherein the positively charged collagen is used as second charged polymer.

9. The method of claim 8, wherein negatively charged terpolymer comprising at least one of acrylic acid and methacrylic acid and at least one of hydroxyethyl methacrylate and hydroxylpropyl methacrylate is used as first charged polymer.

10. The method of claim 9, wherein a terpolymer consisting of 25% 2-hydroxyethyl methacrylate, 25% methacrylic acid and 50% methyl methacrylate (HEMA-MAA-MMA) is used as first charged polymer.

11. The method of claim 9, wherein the degree of cross linking of the polymer complex formed by the reaction of the positively charged collagen with said terpolymer is controlled by varying the reaction time (contact time) between said polymers.

12. The method of claim 1, wherein the degree of cross linking of the polymer complex can be altered by varying the charge density of the polymers.

13. The method of claim 12, wherein a slightly methylated polymer and/or a highly methylated polymer is used, wherein the slightly methylated polymer has a charge density of between 0.9 and 1.7 CE and the highly methylated polymer has a charge density of between 1.7 and 2.5 CE.

14. The method of claim 1, wherein the second substrate coated with the third charged polymer is contacted with the first substrate coated with the first charged polymer and the scaffold arranged there in between for about 30 seconds to about 1 hour.

15. The method of claim 1, wherein the first substrate comprises a material selecting from the group consisting of glass, plastic, silicon, a metal and a metal oxide.

16. The method of claim 2, wherein the at least one cell species is an attachment-dependent cell selected from the group consisting of bone marrow stroma cells, calvarial osteoblasts, Langerhans cells, hepatocytes, chondrocytes, cardiarc myocytes, sinusoidal endothelial cells, dermal fibroblasts, kerotinocytes and oligodendrocytes.

17. The method of claim 3, wherein the at least one cell species is an attachment-independent cell selected from the group of consisting of hematopoetic stem cells, T-lymphocytes, macrophages and neutrophils.

18. A cell species encapsulated in a scaffold, wherein the encapsulation is carried out according to the method of claim 1.

19. The cell species according to claim 18, wherein the pore density of the scaffold varies between 20% and 95%.

20. The cell species according to claim 18, wherein the scaffold comprises a material selected from the group consisting of agarose, polycaprolactone, niobium coated carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, hydroxyapatite-tricalcium phosphate, fibrin, alginate, poly-glycolic acid, carbon nano fibres, polytetrafluoroethylene, polylactic acid and mixtures thereof.

21. The cell species according to claim 18, wherein the scaffold has a regular shape.

22. The cell species according to claim 21, wherein the scaffold has a rectangular shape.

23. The cell species according to claim 22, wherein the largest dimension of the rectangular scaffold is between about 1 mm to about 5 cm.

24. The cell species according to claim 21, wherein the scaffold has the shape of a sphere.

25. The cell species according to claim 24, wherein the spherical scaffold has a diameter of between 1 mm to 5 cm.

26. A scaffold comprising a cell species encapsulated therein, wherein said scaffold is obtained by a method according to claim 1.

27. The use of a scaffold according to claim 26 for a member selected from the group consisting of expansion of autologous cells as in vitro culture system for attachment-dependent and attachment-independent cells.

28. The use of a scaffold according to claim 27, wherein the autologous cells are selected from the group consisting of bone marrow mesenchymal cells, dermal fibroblasts, osteoblasts, chondrocytes and hepatocytes.