CA2373302A1 - Conditioned cell culture medium compositions and methods of use - Google Patents
Conditioned cell culture medium compositions and methods of use Download PDFInfo
- Publication number
- CA2373302A1 CA2373302A1 CA002373302A CA2373302A CA2373302A1 CA 2373302 A1 CA2373302 A1 CA 2373302A1 CA 002373302 A CA002373302 A CA 002373302A CA 2373302 A CA2373302 A CA 2373302A CA 2373302 A1 CA2373302 A1 CA 2373302A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- medium
- conditioned
- stromal
- tissue
- 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
Links
- 238000000034 method Methods 0.000 title claims abstract 45
- 230000001143 conditioned effect Effects 0.000 title claims abstract 16
- 239000006143 cell culture medium Substances 0.000 title claims abstract 11
- 239000000203 mixture Substances 0.000 title claims abstract 11
- 210000004027 cell Anatomy 0.000 claims abstract 32
- 239000002609 medium Substances 0.000 claims abstract 27
- 239000003636 conditioned culture medium Substances 0.000 claims abstract 14
- 210000002536 stromal cell Anatomy 0.000 claims abstract 14
- 210000004738 parenchymal cell Anatomy 0.000 claims abstract 10
- 210000003527 eukaryotic cell Anatomy 0.000 claims abstract 8
- 239000007788 liquid Substances 0.000 claims abstract 5
- 239000003937 drug carrier Substances 0.000 claims abstract 4
- 210000001671 embryonic stem cell Anatomy 0.000 claims abstract 4
- 210000004185 liver Anatomy 0.000 claims abstract 4
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract 4
- 239000000843 powder Substances 0.000 claims abstract 4
- 239000007787 solid Substances 0.000 claims abstract 4
- 230000035876 healing Effects 0.000 claims abstract 3
- 210000001178 neural stem cell Anatomy 0.000 claims abstract 3
- 239000002674 ointment Substances 0.000 claims abstract 3
- 210000000130 stem cell Anatomy 0.000 claims abstract 3
- 239000012141 concentrate Substances 0.000 claims abstract 2
- 239000003894 surgical glue Substances 0.000 claims abstract 2
- 230000000699 topical effect Effects 0.000 claims abstract 2
- 210000001519 tissue Anatomy 0.000 claims 17
- 239000008194 pharmaceutical composition Substances 0.000 claims 13
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- 108090000623 proteins and genes Proteins 0.000 claims 8
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- -1 lyophilized Substances 0.000 claims 4
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- 108010035532 Collagen Proteins 0.000 claims 3
- 102000008186 Collagen Human genes 0.000 claims 3
- 230000003110 anti-inflammatory effect Effects 0.000 claims 3
- 210000001612 chondrocyte Anatomy 0.000 claims 3
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- 239000002537 cosmetic Substances 0.000 claims 3
- 210000002889 endothelial cell Anatomy 0.000 claims 3
- 210000002950 fibroblast Anatomy 0.000 claims 3
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- 230000002440 hepatic effect Effects 0.000 claims 3
- 210000003630 histaminocyte Anatomy 0.000 claims 3
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- 210000002540 macrophage Anatomy 0.000 claims 3
- 239000000463 material Substances 0.000 claims 3
- 210000001616 monocyte Anatomy 0.000 claims 3
- 230000001537 neural effect Effects 0.000 claims 3
- 210000003668 pericyte Anatomy 0.000 claims 3
- 210000004180 plasmocyte Anatomy 0.000 claims 3
- 230000002792 vascular Effects 0.000 claims 3
- 102000015081 Blood Coagulation Factors Human genes 0.000 claims 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 claims 2
- 102000004127 Cytokines Human genes 0.000 claims 2
- 108090000695 Cytokines Proteins 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 claims 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims 2
- 238000001042 affinity chromatography Methods 0.000 claims 2
- 230000001772 anti-angiogenic effect Effects 0.000 claims 2
- 239000003114 blood coagulation factor Substances 0.000 claims 2
- 239000002775 capsule Substances 0.000 claims 2
- 239000013522 chelant Substances 0.000 claims 2
- 238000004587 chromatography analysis Methods 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 2
- 230000002939 deleterious effect Effects 0.000 claims 2
- 238000005227 gel permeation chromatography Methods 0.000 claims 2
- 239000003102 growth factor Substances 0.000 claims 2
- 230000003779 hair growth Effects 0.000 claims 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims 2
- 238000004191 hydrophobic interaction chromatography Methods 0.000 claims 2
- 238000000338 in vitro Methods 0.000 claims 2
- 230000003834 intracellular effect Effects 0.000 claims 2
- 238000005342 ion exchange Methods 0.000 claims 2
- 239000002184 metal Substances 0.000 claims 2
- 230000003647 oxidation Effects 0.000 claims 2
- 238000007254 oxidation reaction Methods 0.000 claims 2
- 230000001376 precipitating effect Effects 0.000 claims 2
- 102000004169 proteins and genes Human genes 0.000 claims 2
- 238000000746 purification Methods 0.000 claims 2
- 230000001105 regulatory effect Effects 0.000 claims 2
- 150000003839 salts Chemical class 0.000 claims 2
- 238000000926 separation method Methods 0.000 claims 2
- 230000000638 stimulation Effects 0.000 claims 2
- 239000000126 substance Substances 0.000 claims 2
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- 241000124008 Mammalia Species 0.000 claims 1
- 108010050808 Procollagen Proteins 0.000 claims 1
- 230000002378 acidificating effect Effects 0.000 claims 1
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- 102000036639 antigens Human genes 0.000 claims 1
- 108091007433 antigens Proteins 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 210000004556 brain Anatomy 0.000 claims 1
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- 235000015872 dietary supplement Nutrition 0.000 claims 1
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- 210000000936 intestine Anatomy 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000004400 mucous membrane Anatomy 0.000 claims 1
- 230000007935 neutral effect Effects 0.000 claims 1
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- 210000000496 pancreas Anatomy 0.000 claims 1
- 230000008929 regeneration Effects 0.000 claims 1
- 238000011069 regeneration method Methods 0.000 claims 1
- 231100000241 scar Toxicity 0.000 claims 1
- 210000000278 spinal cord Anatomy 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 239000003981 vehicle Substances 0.000 abstract 1
Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/36—Materials or treatment for tissue regeneration for embolization or occlusion, e.g. vaso-occlusive compositions or devices
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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Abstract
Novel products comprising conditioned cell culture medium compositions and methods of use are described. The conditioned cell medium compositions of the invention may be comprised of any known defined or undefined medium and may be conditioned using any eukaryotic cell type. The medium may be conditioned by stromal cells, parenchymal cells, mesenchymal stem cells, liver reserve cells, neural stem cells, pancreatic stem cells and/or embryonic stem cells. Additionally, the cells may be genetically modified. A three-dimensional tissue construct is preferred. Once the cell medium of the invention is conditioned, it may be used in any state. Physical embodiments of the conditioned medium include, but are not limited to, liquid or solid, frozen, lyophilized or dried into a powder. Additionally, the medium is formulated with a pharmaceutically acceptable carrier as a vehicle for internal administration, applied directly to a food item or product, formulated with a salve or ointment for topical applications, or, for example, made into or added to surgical glue to accelerate healing of sutures following invasive procedures. Also, the medium may be further processed to concentrate or reduce one or more factors or components contained within the medium.
Claims (60)
1. A phamaceutical composition comprising conditioned cell culture medium, said medium comprising medium which has previously supported the growth of eukaryotic cells cultured in three-dimensions, and a pharmaceutical carrier.
2. The pharmaceutical composition of claim 1 wherein the conditioned cell culture medium is in the form of a liquid, solid, lyophilized, powder, gel or film.
3. The pharmaceutical composition of claim 1 wherein the composition comprises the reduction or enrichment of one or more extracellular products derived from the conditioned media by protein separation techniques including immunoaffinity chromatography, gel chromatography, ion exchange, metal chelate affinity chromatography, HPLC purification and hydrophobic interaction chromatography.
4. The pharmaceutical composition of claim 3 wherein the extracellular product(s) is an extracellular matrix protein.
5. The pharmaceutical composition of claim 3 wherein the extracellular product(s) is a growth factor, anti-inflammatory mediator, enzyme, cytokine, hormone, clotting factor, regulatory factor, angiogenic factor, or anti-angiogenic factor.
6. The pharmaceutical composition of claim 1 wherein the cells cultured in three-dimensions comprise stromal cells and form a three-dimensional stromal tissue.
7. The three-dimensional stromal tissue of claim 6 comprising stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure.
8. The pharmaceutical composition of claim 7 wherein the framework structure comprises a mesh, sponge or polymerized hydrogel.
9. The pharmaceutical composition of claim 8 wherein the stromal cells comprise mesenchymal stem cells, liver reserve cells, neural stem cells, pancreatic stem cells, fibroblast-like cells, fibroblast cells, chondro-progenitor cells, chondrocytes, embryonic stem cells, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells or any combination thereof.
10. The pharmaceutical composition of claim 9 wherein the stromal cells are human.
11. The pharmaceutical composition of claim 9 or 10 wherein the stromal cells comprise genetically engineered cells.
12. The pharmaceutical composition of claim 6 wherein parenchymal cells are cultured on the three-dimensional stromal tissue to form a tissue-specific three-dimensional tissue construct.
13. The pharmaceutical composition of claim 12 wherein the parenchymal cells comprise skin, hepatic, kidney, neuronal, pancreatic, intestinal, genitourinary, vascular, spleen, bone, bone marrow, or mucosal cells.
14. The pharmaceutical composition of claim 13 wherein the parenchymal cells comprise genetically engineered cells.
15. A food substance with enhanced nutritional content comprising conditioned cell culture medium, said medium comprising medium which has previously supported the growth of eukaryotic cells together with a separate food item for consumption by a mammal.
16. The food substance of claim 15 wherein the separate food item is suitable for human consumption.
17. A nutritional supplement comprising conditioned cell culture medium, said medium comprising medium which has previously supported the growth of eukaryotic cells, together with a carrier suitable for human consumption and where said carrier is in the form of a liquid, tablet or capsule.
18. A method of making a pharmaceutical composition comprising:
(a) culturing eukaroytic cells in three-dimensions in a cell culture medium sufficient to meet the nutritional needs required to grow the cells in vitro;
(b) culturing the cells in vitro until the cell culture medium contains a desired level of extracellular products so that a conditioned medium is formed;
(c) removing the conditioned medium from the cells used to condition the medium; and (d) combining the conditioned medium with a pharmaceutical carrier.
(a) culturing eukaroytic cells in three-dimensions in a cell culture medium sufficient to meet the nutritional needs required to grow the cells in vitro;
(b) culturing the cells in vitro until the cell culture medium contains a desired level of extracellular products so that a conditioned medium is formed;
(c) removing the conditioned medium from the cells used to condition the medium; and (d) combining the conditioned medium with a pharmaceutical carrier.
19. The method of claim 18 wherein the conditioned medium is recovered from a continuous flow system.
20. The method of claim 18 or 19 wherein the conditioned medium is cultured in an aseptic environment.
21. The method claim 18 wherein the conditioned cell medium is processed into the state of a liquid, solid, lyophilized, powder, gel or film.
22. The method of claim 18 wherein the conditioned medium is further processed to concentrate or reduce one or more products contained in the medium.
23. The method of claim 22 wherein the composition comprises the reduction or enrichment of one or more extracellular products derived from the conditioned media by protein separation techniques including immunoaffinity chromatography, gel chromatography, ion exchange, metal chelate affinity chromatography, HPLC
purification and hydrophobic interaction chromatography.
purification and hydrophobic interaction chromatography.
24. The method of claim 22 wherein the extracellular product(s) is an extracellular matrix protein.
25. The method of claim 22 wherein the extracellular product(s) is a growth factor, anti-inflammatory mediator, enzyme, cytokine, hormone, antigen, antibody, clotting factor, regulatory factor, angiogenic factor, or anti-angiogenic factor.
26. The method of claim 18 wherein the cells cultured in three-dimensions comprise stromal cells and form a three-dimensional stromal tissue.
27. The method of claim 26 comprising stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure.
28. The method of claim 27 wherein the three-dimensional framework comprises a mesh, sponge or a polymerized hydrogel.
29. The method of claim 26 or 27 wherein the stromal cells comprise mesenchymal stem cells, liver reserve cells, neural stem cells, pancreatic stem cells, fibroblast-like cells, fibroblast cells. chondro-progenitor cells, chondrocytes, embryonic stem cells, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells or any combination thereof.
30. The method of claim 26 wherein the stromal cells comprise genetically engineered cells.
31. The method of claim 30 wherein the genetically engineered cells are transfected with an exogenous gene under the control of an expression element so that the exogenous gene product is expressed and secreted into the conditioned medium.
32. The method of claim 26 wherein parenchymal cells are cultured on the three-dimensional stromal tissue to form a tissue-specific three-dimensional tissue culture.
33. The method of claim 32 wherein the parenchymal cells are skin, hepatic, kidney, neuronal, pancreatic, intestinal, genitourinary, kidney, spleen, bone, bone marrow, or mucosal cells.
34. The method of claim 33 wherein the parenchymal cells comprise genetically engineered cells.
35. The method of claim 34 wherein the genetically engineered cells are transfected with an exogenous gene under the control of an expression element so that the exogenous gene product is expressed and secreted into the conditioned medium.
36. A method for improved wound or burn healing comprising administering to a patient in need of wound or burn healing treatment a composition comprising cell culture medium, said medium comprising medium which has previously supported the growth of eukaryotic cells cultured in three-dimensions, together with a separate therapeutic component so that the patient exhibits a reduction in the amount of traumatized tissue or scar tissue and further exhibits improved regeneration of healthy tissue at the wound site.
37. The method of claim 36 wherein the wound is a vascular wound.
38. The method of claim 36 wherein the wound is to the brain or spinal cord.
39. The method of claim 36 wherein the wound is to the skin, liver, kidney, pancreas, intestines, spleen, genitourinary tract, bone, bone marrow or mucosal tissue.
40. The method of claim 36 wherein the component is a bandage.
41. The method of claim 36 wherein the component is an ointment or cream and the composition is applied topically.
42. The method of claim 36 wherein component is a surgical glue or wound filler.
43. The method of claim 36 wherein the component is a suture or implant and is coated with the conditioned medium prior to use.
44. The method of claim 36 wherein the component is a pharmaceutical carrier so that the conditioned medium is in the form of an injectable, tablet or capsule.
45. The method of claim 36 wherein the composition further comprises an antibiotic, anti-inflammatory, antiviral, antifungal, hormone, antitumor, analgesic, anesthetic, or any combination thereof.
46. The method of claim 36 wherein the cells cultured in three-dimensions contain stromal cells and form a three-dimensional stromal tissue.
47. The method of claim 46 wherein the three-dimensional stromal tissue comprises stromal cells attached to and substantially enveloping framework composed of a biocompatible, non-living material formed into a three-dimensional structure.
48. The method of claim 47 wherein the framework structure comprises a mesh, sponge or hydrogel.
49. The method of claim 46, wherein the stromal cells are mesenchymal stem cells, fibroblast-like cells, fibroblast cells, chondro-progenitor cells, chondrocytes, embryonic stem cells, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells or any combination thereof.
50. The method of claim 49 wherein the stromal cells are genetically engineered cells.
51. The method of claim 46 wherein parenchymal cells are cultured on the three-dimensional stromal tissue to form a tissue-specific three-dimensional tissue construct.
52. The method of claim 51 wherein the parenchymal cells comprise skin, hepatic, kidney, neuronal, pancreatic, intestinal, genitourinary, vascular, spleen, bone, bone marrow or mucosal cells.
53. The method of claim 52 wherein the parenchymal cells are genetically engineered cells.
54. The method of claim 53 wherein the genetically engineered cells are transfected with an exogenous gene under the control of an expression element so that the exogenous gene product is expressed and secreted into the conditioned medium.
55. A method for the correction of a cosmetic defect comprising administering to a person a composition comprising conditioned cell culture medium, said medium comprising medium which has previously supported the growth of eukaryotic cells cultured in three-dimensions, together with a component useful for cosmetic purposes, so that the person exhibits a cosmetic improvement.
56. A method of inhibiting or reversing the deleterious effects to cells in a person, said effects induced by intracellular oxidation, comprising administering to a person in need of such treatment an application of conditioned cell medium, said medium comprising medium which has previously supported the growth of eukaryotic cells cultured in three-dimensions, so that intracellular oxidation is reduced.
57. The method of claim 56 wherein the deleterious effect is the appearance of aging skin.
58. A method for the stimulation of hair growth comprising topically administering to a person a composition comprising conditioned cell culture medium, said medium comprising medium which has previously the growth of eukaryotic cells cultured in three dimensions, together with a topical carrier, so that the person exhibits an improved stimulation of hair growth.
59. The method of claims 36, 55, 56 and 57 wherein the conditioned cell medium is processed into the form of a liquid, solid, lyophilized, powder, gel or film.
60. A method for isolating collagen, comprising:
(a) precipitating procollagen from medium conditioned by a three-dimensional culture at high salt conditions under neutral pH
conditions;
(b) removing the propeptides under acidic conditions so that the triple helix of the collagen remains intact; and (c) precipitating the collagen at high salt concentrations.
(a) precipitating procollagen from medium conditioned by a three-dimensional culture at high salt conditions under neutral pH
conditions;
(b) removing the propeptides under acidic conditions so that the triple helix of the collagen remains intact; and (c) precipitating the collagen at high salt concentrations.
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PCT/US2000/013016 WO2000069449A2 (en) | 1999-05-14 | 2000-05-12 | Conditioned cell culture medium compositions and methods of use |
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