Method of forming polysaccharide sponges for cell culture and transplantation

• US 6,334,968 B1
• Filed: 10/13/1999
• Issued: 01/01/2002
• Est. Priority Date: 05/22/1996
• Status: Expired due to Term

• Current Assignee: Ben Gurion University of The Negev
• Original Assignee: Ben Gurion University of The Negev
• Inventors: Cohen, Smadar, Glicklis, Rachel, Shapiro, Lilia
• Primary Examiner(s): Kuhns, Allan R.
• Application Number:
  US09/416,911
• Time in Patent Office: 811 Days
• Field of Search: 264/28, 264/41, 264/344
• US Class Current: 264/28
• CPC Class Codes: A61F 2/105 Skin implants, e.g. artificial skin
• CPC Class Codes: A61F 2210/0004 bioabsorbable
• CPC Class Codes: A61F 2250/0067 Means for introducing or releasing pharmaceutical products into the body
• 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: A61K 9/122 Foams; Dry foams edible foams A61K9/0056
• CPC Class Codes: A61L 27/20 Polysaccharides
• 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 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin
• CPC Class Codes: C08J 2305/02 Dextran; Derivatives thereof
• CPC Class Codes: C08J 9/26 by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
• CPC Class Codes: C08L 5/02 Dextran; Derivatives thereof

Abstract

A polysaccharide sponge characterized by having: (i) an average pore size in the range between about 10 μm to about 300 μm; (ii) an average distance between the pores being the wall thickness of the pores in the range between about 5 μm to about 270 μm; and (iii) an E-modulus of elasticity being a measure of the rigidity of the sponge in the range of about 50 kPa to about 500 kPa.

Claims

1. A process for producing a polysaccharide sponge characterized by having:

(i) an average pore size in the range between about 10 μ

m to about 300 μ

m;

(ii) an average distance between the pores being the wall thickness of the pores in the range between about 5 μ

m to about 270 μ

m; and

(iii) an E-modului of elasticity being a measure of the rigidity of the sponge in the range of about 50 kPa to about 500 kPa, the process comprising;

(a) providing a polysaccharide solution containing about 1% to about 3% w/v polysaccharide in water;

(b) optionally diluting said polysaccharide solution with additional water to obtain a final solution having about 0.5% to about 2% w/v polysaccharide, and subjecting said solution of (a) to gelation, to obtain a polysaccharide gel;

(c) freezing the gel of (b); and

(d) drying the frozen gel of (c) to obtain a polysaccharide sponge.

2. A process according to claim 1, further comprising the addition of a cross-linker to said polysaccharide solution of (a) during the step of gelation (b), said cross-linker being added in an amount to provide a concentration of cross-linker in the final solution being subjected to gelation of between about 0.1% to about 0.3% w/v.

3. A process according to claim 1, wherein said polysaccharide solution of (a) is prepared by dissolving the polysaccharide in powdered form in double distilled water in amounts to yield a concentration between about 1% to about 3% w/v polysaccharide in said solution, said polysaccharide solution being mixed in a homogenizer at about 25000 RPM for about 30 minutes at room temperature.

4. A process according to claim 1, wherein the gelation step (b) is by intensive stirring of the polysaccharide solution in a homogenizer at about 31800 RPM for about 3 minutes, and wherein when a cross-linker is added to the solution, said cross-linker is added very slowly during said intensive stirring of the polysaccharide solution.

5. A process according to claim 1, wherein said polysaccharide comprises an alginate derived from brown sea algae selected from the group consisting of alginate Protanal™

LF 120 (LF

     120) derived from Laminaria hyperhorea, alginate Protanal™

LF 20/60 (LF 20/60) derived from Laminaria hyperborea, alginate MVG™

(MVG) derived from Laminaria hyperborea, alginate Pronatal™

HF 120 (HF

     120) derived from Laminaria hyperborea, alginate Pronatal™

SF 120 (SF

     120) derived from Laminaria hyperborea, alginate Pronatal™

SF 120 RB (SF 120 RB) derived from Laminaria hyperborea, alginate Pronatal™

LF 200 RB (LF 200 RB) derived from Laminaria hyperborea, alginate Manugel™

DMB (DMB) derived from Laminaria hyperhorea, Keltone™

HVCR (HVCR) derived from Macrocystis pyrifera, and Keltone™

LV (LV) derived from Macrocystis pyrifera.

6. A process according to claim 5, wherein in said process, the final solutions subjected to gelation in step (b) are selected from the group consisting of:

(i) LF 120 alginate 1% w/v without cross-linker;

(ii) LF 120 alginate 1% w/v and Ca-Gl 0.1% w/v;

(iii) LF 120 alginate 1% w/v and Ca-Gl 0.2% w/v;

(iv) LF 120 alginate 1% w/v and SrCl₂ 0.15% w/v;

(v) LF 120 alginate 1% w/v and CaCl₂ 0.1% w/v;

(vi) LF 120 alginate 0.5% w/v and Ca-Gl 0.2% w/v;

(vii) LF 20/60 alginate 1% w/v and Ca-Gl 0.2% w/v;

(viii) HVCR alginate 0.5% w/v and Ca-Gl 0.2% w/v; and

(ix) HVCR alginate 1% w/v and Ca-Gl 0.2% w/v.

7. A process according to claim 1 wherein said freezing step (c) is by rapid freezing in a liquid nitrogen bath at about −

80°

C. for about 15 minutes.

8. A process according to claim 1 wherein said freezing step (c) is by slow freezing in a freezer at about −

18°

C. for about 8-24 hours.

9. A process according to claim 1 wherein said drying step (d) is by lyophilization under conditions of about 0.007 mmHg pressure and at about −

60°

C.

10. A process according to claim 4 wherein the final polysaccharide solution with or without cross-linker is poured into a vessel of desired shape before commencement of the intensive stirring of the gelation step (b), said vessel having a shape that is desired for the shape of the polysaccharide sponge.