CA2256327A1 - Polysaccharide sponges for cell culture and transplantation - Google Patents

Polysaccharide sponges for cell culture and transplantation

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Publication number
CA2256327A1
CA2256327A1 CA002256327A CA2256327A CA2256327A1 CA 2256327 A1 CA2256327 A1 CA 2256327A1 CA 002256327 A CA002256327 A CA 002256327A CA 2256327 A CA2256327 A CA 2256327A CA 2256327 A1 CA2256327 A1 CA 2256327A1
Authority
CA
Canada
Prior art keywords
alginate
polysaccharide
sponge
cells
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002256327A
Other languages
French (fr)
Other versions
CA2256327C (en
Inventor
Lilia Shapiro
Rachel Glicklis
Smadar Cohen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ben Gurion University of the Negev Research and Development Authority Ltd
Original Assignee
Ben-Gurion University Of The Negev
Lilia Shapiro
Rachel Glicklis
Smadar Cohen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ben-Gurion University Of The Negev, Lilia Shapiro, Rachel Glicklis, Smadar Cohen filed Critical Ben-Gurion University Of The Negev
Publication of CA2256327A1 publication Critical patent/CA2256327A1/en
Application granted granted Critical
Publication of CA2256327C publication Critical patent/CA2256327C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A61K9/122—Foams; Dry foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2210/00—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2210/0004—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof bioabsorbable
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2250/00—Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2250/0058—Additional features; Implant or prostheses properties not otherwise provided for
    • A61F2250/0067—Means for introducing or releasing pharmaceutical products into the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/02—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 (40)

1. 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.
2. A polysaccharide sponge according to claim 1, wherein said sponge comprises a polysaccharide selected from the group comprising the polyanionic polysaccharides: alginates, gellan, gellan gum, xanthan chitosan, agar, carrageenan and the polycationic polysaccharide: chitosan.
3. A polysaccharide sponge according to claim 1 or claim 2, wherein said sponge comprises an alginate selected from the group of alginates characterised by having: (i) a mannuronic acid (M) residue content in the range of between about 25% and about 65% of total residues; (ii) a guluronic acid (G) residue content in the range of between about 35% and about 75% of total residues; (iii) a M/G ratio of about 1/3 and about 1.86/1;
and (iv) a viscosity of the final alginate solution having 1% w/v alginate, from which the sponge is obtained in the range between about 50 cP to about 800cP.
4. A polysaccharide sponge according to claim 3, wherein said sponge comprises an alginate derived from brown sea algae selected from the group consisting of alginate Protanal TM LF 120 (LF 120) derived from Laminaria hyperborea, alginate Protanal TM LF 20/60 (LF 20/60) derived from Laminaria hyperborea, alginate MVGTM (MVG) derived from Laminaria hyperborea, alginate Pronatal TM HF 120 (HF 120) derived from Laminaria hyperborea, alginate Pronatal TM SF 120 (SF 120) derived from Laminaria hyperborea, alginate Pronatal TM SF 120 RB (SF 120 RB) derived from Laminaria hyperborea, alginate Pronatal TM LF 200 RB (LF
200 RB) delived from Laminaria hyperborea, alginate Manugel TM DMR
(DMB) derived from Laminaria hyperborea, Keltone TM HVCR (HVCR) derived from Macrocystis pyrifera, and Keltone TM LV (LV) derived from MacrocYstis pyrifera.
5. A polysaccharide sponge according to claim 4, wherein said sponge comprises an alginate selected from the group consisting of said LF 120, LF 20/60 and HVCR.
6. A polysaccharide sponge according to any one of claims 3 to 5, wherein said alginate is used in the form of a sodium alginate solution having a concentration of alginate between about 1% to about 3% w/v to provide an alginate concentration between about 0.1% to about 2% w/v in the final solution from which the sponge is obtained.
7. A polysaccharide sponge according to any one of claims 1 to 5, wherein said sponge further comprises a cross-linking agent selected from the group consisting of the salts of calcium, copper, aluminum, magnesium, strontium, barium, tin, zinc, chromium, organic cations, poly(amino acids), poly(ethyleneimine), poly(vinylamine), poly(allylamine), and polysaccharides.
8. A polysaccharide sponge according to claim 7, wherein said sponge further comprises a cross-linking agent selected from the group consisting of calcium chloride (CaCl2), strontium chloride (SrCl2) and calcium gluconate (Ca-Gl).
9. A polysaccharide sponge according to claim 7 or claim 8, wherein said cross-linker is used in the form of a cross-linker solution having a concentration of cross-linker sufficient to provide a cross-linker concentration between about 0.1% to about 0.3% w/v in the final solution from which the sponge is obtained.
10. A polysaccharide sponge according to any one of claims 1 to 9, wherein said sponge is prepared from a polysaccharide solution with or without the addition of a cross-linker.
11. A polysaccharide sponge according to claim 10, wherein said sponge is an alginate sponge prepared from an alginate solution with or without the addition of a cross-linker and wherein said final alginate solution with or without cross-linker from which said sponge is obtained is selected from the group of final solutions, having concentrations of alginate or alginate and cross-linker, 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 0.16% 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.
12. A polysaccharide sponge according to claim 11, wherein said sponge is obtained from a final solution of LF 120 alginate 1% w/v and Ca-Gl cross-linker 0.2% w/v.
13. A polysaccharide sponge according to claim 11, wherein said sponge is obtained from a final solution of HVCR alginate 1% w/v and Ca-Gl cross-linker 0.2% w/v.
14. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for growing mammalian cells in vitro.
15. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for implantation into a patient to replace or repair tissue that has been removed or damaged, wherein said implanted sponge is a substrate, matrix or scaffold for surrounding tissue to invade it, proliferate thereon and replace the damaged or removed tissue, or wherein said implant is an initial substrate for vascularization by the surrounding host tissue and the vascularized implant then serves as a substrate to receive injected cells of choice from the host, or grown in vitro, said injected cells being capable of rapid acclimitization and proliferation on the vascularized sponge to rapidly replace the damaged or removed tissue.
16. A polysaccharide sponge according to any one of claims 1 to 13 for use as an implanted support for therapeutic drug delivery into a desired tissue, said drug delivery being by way of the action of genetically engineered cells or natural cells carried by said sponge and expressing said therapeutic drugs, said cells expressing said drug or expressing regulatory proteins to direct the production of the drug endogenously in said tissue.
17. A polysaccharide sponge according to claim 16, wherein said therapeutic drug expressed by said cells carried in said sponge is a therapeutic protein, wherein said cells express said protein or express regulatory proteins to direct the production of said protein endogenously in the tissue into which said sponge is implanted.
18. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for in vitro culturing of plant cells and algae.
19. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for the delivery to a tissue or organ of genetically engineered viral vectors, non-viral vectors, polymeric microspheres and liposomes all encoding or containing a therapeutic agent for said tissue or organ.
20. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for in vitro fertilization of mammalian oocytes.
21. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for storage of fertilized mammalian oocytes or other mammalian cells cultured in vitro.
22. A polysaccharide sponge according to any one of claims 1 to 13 for use as a matrix, substrate or scaffold for the transplantation of cells grown on or within said sponge in vitro into a tissue of a patient in need of said cells as a result of tissue damage, removal, or dysfunction.
23. A process for producing a polysaccharide sponge according to any one of claims 1-22, comprising:
(a) providing a polysaccharide solution containing about 1% to about 3% w/v polysaccharide in water;
(b) diluting said polysaccharide solution with additional water when desired 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.
24. A process according to claim 23, 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.
25. A process according to claim 23 or 24, 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.
26. A process according to any one of claims 23-25, 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.
27. A process according to any one of claims 23 to 26, wherein said polysaccharide is an alginate of claim 4.
28. A process according to claim 27, 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 0.6% 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.
29. A process according to any one of claims 23-28 wherein said freezing step (c) is by rapid freezing in a liquid nitrogen bath at about -80°C for about 15 minutes.
30. A process according to any one of claims 23-28 wherein said freezing step (c) is by slow freezing in a freezer at about -18°C for about 8-24 hours.
31. A process according to any one of claims 23-30 wherein said drying step (d) is by lyophilization under conditions of about 0.007 mmHg pressure and at about -60°C.
32. A process according to any one of claims 23-31 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.
33. Use of a polysaccharide sponge according to any one of claims 1-13 as a matrix, substrate or scaffold for the in vitro growth of mammalian cells, plant cells, algae, or for the in vitro fertilization of mammalian oocytes.
34. Use of a polysaccharide sponge according to claim 33 for the in vitro growth of fibroblast cells.
35. Use of a polysaccharide sponge according to claim 33 for the in vitro growth of hepatocytes.
36. Use of a polysaccharide sponge according to any one of claims 1 to 13 as a matrix, substrate or scaffold for implantation into a patient according to any one of claims 15-17.
37. Use of a polysaccharide sponge according to any one of claims 1 to 13 as a matrix, substrate or scaffold for the transplantation of cells grown on said sponge in vitro into a tissue of a patient in need of said cells as a result of tissue damage, removal, or dysfunction.
38. Artificial skin comprising a polysaccharide sponge according to any one of claims 1 to 13 and dermal fibroblast cells grown on said sponge in vitro to the stage wherein said cells are in an active proliferating stage suitable for transplantation to a patient in need of said artificial skin.
39. An artificial organ equivalent comprising a polysaccharide sponge according to any one of claims 1 to 13 and representative cells of said organ, said cells having been grown on said sponge in vitro to the stage wherein said cells are fully active and equivalent to the active cells of said organ, said artificial organ being suitable for transplantation or implantation into a patient in need thereof following organ damage.
removal or dysfunction.
40. An artificial organ equivalent according to claim 39 being an artificial liver equivalent, wherein said cells grown on said sponge are hepatocytes at a stage in which said hepatocytes are active and function in an equivalent manner to hepatocytes in vivo and are suitable for transplantation or implantation into a patient suffering from liver dysfunction, damage or at least partial removal.
CA2256327A 1996-05-22 1997-05-21 Polysaccharide sponges for cell culture and transplantation Expired - Lifetime CA2256327C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IL11837696A IL118376A0 (en) 1996-05-22 1996-05-22 Polysaccharide sponges for cell culture and transplantation
IL118376 1996-05-22
PCT/IL1997/000161 WO1997044070A1 (en) 1996-05-22 1997-05-21 Polysaccharide sponges for cell culture and transplantation

Publications (2)

Publication Number Publication Date
CA2256327A1 true CA2256327A1 (en) 1997-11-27
CA2256327C CA2256327C (en) 2012-07-24

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Family Applications (1)

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CA2256327A Expired - Lifetime CA2256327C (en) 1996-05-22 1997-05-21 Polysaccharide sponges for cell culture and transplantation

Country Status (12)

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US (3) US6425918B1 (en)
EP (1) EP0901384B1 (en)
JP (1) JP2000512666A (en)
AT (1) ATE249251T1 (en)
AU (1) AU714647B2 (en)
CA (1) CA2256327C (en)
DE (1) DE69724780T2 (en)
DK (1) DK0901384T3 (en)
ES (1) ES2206707T3 (en)
IL (1) IL118376A0 (en)
PT (1) PT901384E (en)
WO (1) WO1997044070A1 (en)

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