Conditioned cell culture medium compositions and methods of use

• US 20070077232A1
• Filed: 10/03/2006
• Published: 04/05/2007
• Est. Priority Date: 05/14/1999
• Status: Active Grant

• Current Assignee: Allergan, Inc. (Canada) (Abbvie Incorporated)
• Original Assignee: Skinmedica Incorporated (Abbvie Incorporated)
• Inventors: Ratcliffe, Anthony, Kern, Andreas, Zeltinger, Joan, Horwitz, David, Landeen, Lee, Naughton, Gail, Mansbridge, Jonathan, Pinney, R., Applegate, Mark
• Granted Patent: US 8,361,485 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A23L 33/10 using additives addition of substantially indigestible substances A23L33/21
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 2800/86 Products or compounds obtained by genetic engineering
• CPC Class Codes: A61K 2800/91 Injection
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 35/35 Fat tissue; Adipocytes; Stromal cells; Connective tissues adipose-derived stem cells A61K35/28; collagen A61K38/39
• CPC Class Codes: A61K 35/36 Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells islets of Langerhans A61K35/39
• CPC Class Codes: A61K 35/50 Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 8/02 characterised by special physical form
• CPC Class Codes: A61K 8/0208 Tissues; Wipes; Patches
• CPC Class Codes: A61K 8/64 Proteins; Peptides; Derivatives or degradation products thereof
• CPC Class Codes: A61K 8/65 Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
• CPC Class Codes: A61K 8/985 Skin or skin outgrowth, e.g. hair, nails
• CPC Class Codes: A61K 9/0014 Skin, i.e. galenical aspects of topical compositions non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00
• CPC Class Codes: A61L 2300/414 Growth factors
• CPC Class Codes: A61L 2430/36 : for embolization or occlusion, e.g. vaso-occlusive compositions or devices artificial blood vessels A61L27/507
• CPC Class Codes: A61L 31/044 : Collagen
• CPC Class Codes: A61L 31/047 : Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
• CPC Class Codes: A61L 31/16 : Biologically active materials, e.g. therapeutic substances A61L31/047 takes precedence
• CPC Class Codes: A61P 1/00 : Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 : for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 : for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 : for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 13/12 : of the kidneys
• CPC Class Codes: A61P 15/00 : Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 : Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 : for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/14 : for baldness or alopecia
• CPC Class Codes: A61P 17/16 : Emollients or protectives, e.g. against radiation
• CPC Class Codes: A61P 17/18 : Antioxidants, e.g. antiradicals preparations for protection against sunlight A61Q17/00
• CPC Class Codes: A61P 19/00 : Drugs for skeletal disorders
• CPC Class Codes: A61P 19/08 : for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/00 : Drugs for disorders of the nervous system
• CPC Class Codes: A61P 39/06 : Free radical scavengers or antioxidants
• CPC Class Codes: A61P 41/00 : Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61Q 19/00 : Preparations for care of the skin
• CPC Class Codes: A61Q 19/08 : Anti-ageing preparations
• CPC Class Codes: A61Q 7/00 : Preparations for affecting hair growth
• CPC Class Codes: C12N 2502/1323 : Adult fibroblasts
• CPC Class Codes: C12N 5/0603 : Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 : Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0625 : Epidermal cells, skin cells; Cells of the oral mucosa
• CPC Class Codes: C12N 5/0629 : Keratinocytes; Whole skin
• CPC Class Codes: C12N 5/0656 : Adult fibroblasts
• CPC Class Codes: C12N 5/069 : Vascular Endothelial cells
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00

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

1. A pharmaceutical 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.

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.

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.

56-57. -57. (canceled)

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. (canceled)

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.