METHODS FOR EMBRYONIC STEM CELL CULTURE

• US 20080159994A1
• Filed: 07/30/2007
• Published: 07/03/2008
• Est. Priority Date: 01/28/2005
• Status: Abandoned Application

• Current Assignee: Imperial College Innovations Ltd. (Imperial College London), NovaThera Ltd. (Medcell Bioscience Limited)
• Original Assignee: Imperial College Innovations Ltd. (Imperial College London), NovaThera Ltd. (Medcell Bioscience Limited)
• Inventors: Mantalaris, Sakis, Randle, Wesley
• Application Number:
  US11/830,378
• Publication Number:
  US 20080159994A1
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 27/16 Otologicals
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C12N 2500/42 Organic phosphate, e.g. beta glycerophosphate
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0012 Cell encapsulation
• 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 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth

Abstract

The invention relates to a method of cell culture comprising providing a pluripotent ES cell encapsulated within a support matrix to form a support matrix structure, maintaining the encapsulated cell in 3-D culture in maintenance medium, and optionally differentiating the encapsulated cell in 3-D culture in differentiation medium. The invention further relates to screening methods incorporating the use of encapsulated cells.

Claims

1. A method of cell culture comprising:

(a) providing a human embryonic stem (ES) cell encapsulated within a support matrix to form a support matrix structure, and,

(b) maintaining culture by maintaining the encapsulated cell in 3-D culture in maintenance medium.

2. The method according to claim 1 wherein maintenance culture is carried out in the absence of feeder cells and in the absence of feeder cell conditioned medium.

3. The method according to claim 1 further comprising differentiating the encapsulated cell in 3-D culture in differentiation medium in conditions suitable for cell differentiation.

4. The method according to claim 3 wherein the maintaining and differentiating stages are performed in the same vessel.

5. The method according to claim 3 wherein a stimulus for differentiation is provided.

6. The method according to claim 5, wherein the stimulus for differentiation comprises a stimulus for embryoid body formation.

7. The method according to claim 6, wherein the stimulus for embryoid body formation is removal of, or reduced, exposure to a substance that suppresses differentiation;

and/or, addition of, or increased, exposure to a substance that promotes embryoid body formation.

8. The method according to claim 3, wherein a stimulus for differentiation to a ectodermal, endodermal or mesodermal lineage is provided.

9. The method according to claim 3, wherein a stimulus for differentiation to a mesodermal skeletal lineage is provided.

10. The method according to claim 3, wherein a stimulus for osteogenic or chondrogenic differentiation is provided.

17. The method according to claim 1 wherein a single cell is are provided encapsulated within each support matrix structure.

19. The method according to claim 1 wherein the support matrix structure is in the form of a bead.

34. The method according to claim 1 wherein the support matrix comprises a hydrogel.

35. The method according to claim 1 wherein the support matrix comprises alginate.

36. The method according to claim 34, wherein the support matrix further comprises one or more material selected from the group comprising gelatin, laminin, Bioglass™

, hydroxyapatite, extracellular matrix, an extracellular matrix protein, a growth factor;

an extract from another cell culture, an extract from an osteoblastic culture.

37. The method according to claim 35, wherein the support matrix further comprises one or more material selected from the group comprising gelatin, laminin, Bioglass™

, hydroxyapatite, extracellular matrix, an extracellular matrix protein, a growth factor;

an extract from another cell culture, an extract from an osteoblastic culture.

38. The method according to claim 36 wherein the support matrix further comprises gelatin.

39. The method according to claim 37 wherein the support matrix further comprises gelatin.

40. The method according to claim 1 further comprising freezing the encapsulated cells.

41. The method according to claim 1 further comprising liberation of a cell or cells from the support matrix.

42. A cell or cells obtained by the method according to claim 41.

43. An encapsulated cell or cells obtainable or obtained by the method of claim 1.

45. An encapsulated human ES cell or cells obtained by the method of claim 1.

46. An encapsulated multipotent cell or cells obtained by the method of claim 3.

48. An encapsulated osteogenic, chondrogenic or cardiomyogenic cell or cells obtainable or obtained by the method of claim 3.

50. An encapsulated terminally differentiated cell or cells obtainable or obtained by the method of claim 3.

52. A method of treatment of a subject comprising administration of the cells according to claim 42, to a subject.

53. A method of treatment of a subject for a disease or condition requiring bone reconstruction comprising administration of the encapsulated osteogenic cell or cells according to claim 48 to a subject.

54. A method of treatment of a subject for a disease or condition selected from osteoporosis, bone breaks, bone fractures, bone cancer, osteocarcinoma, osteogenesis imperfecta, Paget'"'"'s disease, fibrous dysplasia, bone disorders associated with hearing loss, hypophosphatasia, myeloma bone disease, osteopetrosis, over-use injury to bone, sports injury to bone, and periodontal (gum) disease, wherein the method comprises administration of the encapsulated osteogenic cell or cells according to claim 48 to a subject

55. A method of treatment of a subject for a disease or condition selected from arthritis, a cartilage disease or disorder, cartilage repair, and rheumatoid and osteoarthritis, wherein the method comprises administration of the encapsulated chondrogenic cell or cells according to claim 48 to a subject

56. A method of reconstructive surgery selected from therapeutic or cosmetic surgery comprising administration of the encapsulated cell or cells according to claim 42 to a subject.

57. A method of reconstructive surgery selected from therapeutic or cosmetic surgery comprising administration of the encapsulated osteogenic cell or cells or the encapsulated chondrogenic cell or cells according claim 48 to a subject.

58. A composition comprising the encapsulated cell or cells according to claim 43 and a pharmaceutically acceptable carrier or diluent.

59. The composition according to claim 58, further comprising a pharmaceutically acceptable carrier.

60. A bone or cartilage tissue derived from the encapsulated cell or cells according to claim 43.

61. A cell scaffold having seeded on or impregnated therein encapsulated cells according to claim 43.

62. A. pre-filled administration device, such as a syringe, containing the composition according to claim 60.

63. A. pre-filled administration device, such as a syringe, containing the composition according to claim 61.

11. A method of cell culture comprising:

(a) providing a single ES cell or a plurality of ES cells encapsulated within a support matrix to form a support matrix structure,

(b) maintaining culture by maintaining the encapsulated cell(s) in 3-D culture in maintenance medium, in conditions suitable for ES cell maintenance,

(c) performing osteogenic differentiation by differentiating the encapsulated cells in 3-D culture in differentiation medium, in conditions suitable for osteogenic differentiation.

12. The method according to claim 11 wherein osteogenic differentiation of the encapsulated cells comprises:

(i) incubating the encapsulated ES cells in 3-D culture in differentiation medium and providing a stimulus for embryoid body formation, then,

(ii) incubating the encapsulated cells generated in (i) in differentiation medium and providing a stimulus for osteogenic differentiation.

13. The method according to claim 11 wherein osteogenic differentiation of the encapsulated cells comprises:

(i) incubating the encapsulated ES cells in 3-D culture in differentiation medium, then,

(ii) incubating the encapsulated cells generated in (i) in differentiation medium and providing a stimulus for osteogenic differentiation.

14. The method according to claim 11 wherein osteogenic differentiation of the encapsulated cells comprises:

incubating the encapsulated ES cells in differentiation medium and providing a stimulus for osteogenic differentiation.

15. The method according to claim 11 wherein the ES cells are of human, non-human primate, equine, canine, bovine, porcine, caprice, ovine, piscine, rodent, murine, or avian origin.

16. The method according to claim 11 wherein the plurality of cells is provided encapsulated within each support matrix structure.

18. The method according to claim 11 wherein in step (a) a plurality of support matrix structures are provided.

44. An encapsulated cell or cells obtainable or obtained by the method of claim 11

47. An encapsulated multipotent cell or cells obtained by the method of claim 11.

49. An encapsulated osteogenic, chondrogenic or cardiomyogenic cell or cells obtainable or obtained by the method of claim 11.

51. An encapsulated terminally differentiated cell or cells obtainable or obtained by the method of claim 11.

20. A method of identifying a cell environment factor that is capable of modulating cell maintenance and/or cell differentiation, wherein the method comprises

(a) providing a human embryonic stem (ES) cell encapsulated within a support matrix to form a support matrix structure,

(b) maintaining culture by maintaining the encapsulated cell in 3-D culture in maintenance medium.

(c) incubating the encapsulated cell in the presence of the cell environment factor, and

(d) assessing the effect of the cell environment factor on cell maintenance and/or differentiation.

21. The method according to claim 20, wherein the cell environment factor is a test compound.

22. The method according to claim 20, wherein the cell environment factor is a test stimulus.

23. The method according to claim 20, wherein the cell environment factor is test medium and/or test conditions.

24. The method according to claim 23, wherein the cell is provided with a test stimulus and the effect of test stimulus on maintenance and/or differentiation of the cell is assessed.

25. The method according to claim 20, wherein in step (a) a plurality of cells is encapsulated within each support matrix structure.

26. The method according to claim 20, wherein in step (a) a single cell is encapsulated within each support matrix structure.

27. The method according to claim 20, wherein encapsulated cells are provided in an array of culture vessels.

28. The method according to claim 27, wherein the array of culture vessels is a multi well or multi tube array.

29. The method according to claim 27, wherein in step (a), a plurality of encapsulated cells is provided in each culture vessel.

30. The method according to claim 27, wherein in step (a), a plurality of support matrix structures are provided in each culture vessel.

31. The method according to claim 27, wherein in step (a), a single encapsulated cell is present in each culture vessel.

32. The method according to claim 20, wherein the support matrix structure is in the form of a bead.

33. The method according to claim 20, wherein the effect on cell maintenance and/or differentiation is assessed by one or more method selected from the group consisting of microscopic examination, detection of a stage-specific antigen or antigens and detection of gene expression.