Organotypic intestinal culture and methods of use thereof

• US 20040175367A1
• Filed: 01/29/2004
• Published: 09/09/2004
• Est. Priority Date: 08/23/2001
• Status: Active Grant

• Current Assignee: The Wistar Institute of Anatomy & Biology (University of Pennsylvania)
• Original Assignee: The Wistar Institute of Anatomy & Biology (University of Pennsylvania)
• Inventors: Herlyn, Meenhard, Kalabis, Jiri
• Granted Patent: US 7,217,570 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/38 Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/365 Endothelin
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/1347 Smooth muscle cells
• CPC Class Codes: C12N 2502/23 Gastro-intestinal tract cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: G01N 2333/485 Epidermal growth factor [EGF] (urogastrone)
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms

Abstract

An organotypic culture comprises an artificial stroma overlayed with epithelial cells isolated from a human colon or intestine. The stroma comprises a mixture of collagen and human fibroblasts isolated from a human colon or intestine. The culture contains a factor that binds the IGF-1 receptor, a factor that binds the EGF receptor, and a factor that binds the LIF receptor. These factors may be added exogenously to the culture via medium or may be expressed by various recombinantly engineered cell types in the culture. The organotypic culture can result in growth that is in situ-like or emphasizes other physiological or morphological states, depending on the balance of factors in the growth media. The organotypic culture may be used in methods for screening of therapeutic, carcinogenic, or growth enhancement factors, or for treating intestinal injuries by applying to the site of an injury the intact culture or the components thereof.

Claims

1. An organotypic culture comprising an artificial stroma comprising a mixture of collagen and human fibroblasts isolated from a human colon or intestine, said stroma overlayed with epithelial cells isolated from a human colon or intestine, wherein present in said culture are at least one growth factor that binds the insulin growth factor-1 (IGF-1) receptor, at least one growth factor that binds the epidermal growth factor (EGF) receptor, and at least one growth factor that binds the leukemia inhibitory factor (LIF) receptor.

2. The culture according to claim 1, wherein said collagen is selected from the group consisting of human collagen type I and bovine collagen type I.

3. The culture according to claim 1, wherein said fibroblasts are selected from the group consisting of adult human colon fibroblasts, fetal human colon fibroblasts, adult human small intestinal fibroblasts, fetal human small intestinal fibroblasts, and human fibroblast stem cells.

4. The culture according to claim 3, wherein said stem cells are derived from human bone marrow.

5. The culture according to claim 1, wherein said fibroblast cells are derived from the cell line FFC331.

6. The culture according to claim 1, wherein said epithelial cells are selected from the group consisting of adult human colon epithelial cells, fetal human colon epithelial cells, adult human small intestinal epithelial cells, fetal human small intestinal epithelial cells and human epithelial stem cells.

7. The culture according to claim 1, further comprising human smooth muscle cells embedded into said collagen.

8. The culture according to claim 1, further comprising a layer of endothelial cells underlying said collagen and fibroblast mixture, wherein said endothelial cells form a capillary network induced by fibroblasts in said mixture.

9. The culture according to claim 1, comprising at least one matrix protein selected from the group consisting of Laminin-1 and Laminin-2.

10. The culture according to claim 1, wherein said growth factor that binds the IGF-1 receptor is selected from the group consisting of insulin and IGF-1.

11. The culture according to claim 1, wherein said growth factor that binds the EGF receptor is selected from the group consisting of EGF and tumor growth factor-alpha (TGF-alpha).

12. The culture according to claim 1, wherein at least a portion of said fibroblasts are infected with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

13. The culture according to claim 12, wherein said vector is a virus.

14. The culture according to claim 13, wherein said virus is an adenovirus.

15. The culture according to claim 12, wherein said growth factor is selected from at least one of the group consisting of a growth factor that binds the IGF-1 receptor, a growth factor that binds the EGF receptor, a growth factor that binds the LIF receptor, a growth factor that binds the hepatocyte growth factor (HGF) receptor, a growth factor that binds the stem cell factor (SCF) receptor, a growth factor that binds the endothelin-3 (ET-3) receptor, a growth factor that binds the platelet derived growth factor (PDGF) receptor, Laminin-1 and Laminin-2.

16. The culture according to claim 1, wherein at least a portion of said epithelial cells are infected with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

17. The culture according to claim 16, wherein said vector is a virus.

18. The culture according to claim 17, wherein said vector is an adenovirus.

19. The culture according to claim 16, wherein said growth factor is selected from at least one of the group consisting of a growth factor that binds the IGF-1 receptor, a growth factor that binds the EGF receptor, a growth factor that binds the SCF receptor and a growth factor that binds the autocrine motility factor (AMF) receptor, a growth factor that binds the endothelin receptor A and a growth factor that binds the endothelin receptor B.

20. The culture according to claim 8, wherein at least a portion of said endothelial cells are infected with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

21. The culture according to claim 26 wherein said vector is an adenovirus.

24. A culture medium suitable for growth of an organotypic culture of claim 1 comprising a base media, at least one of insulin or IGF-1, at least one of EGF-1 or TGF-α

, and LIF.

25. The medium according to claim 24, further comprising one or more of a factor selected from the group consisting of ET-3, HGF, SCF and AMF.

26. The medium according to claim 25, further comprising an additional component selected from the group consisting of transferrin, fetal calf serum, and combinations thereof.

27. A method of preparing an organotypic culture of claim 1 comprising:

(a) assembling an artificial stroma by mixing collagen and fibroblasts;

(b) seeding said artificial stroma with epithelial cells in the presence of at least one growth factor that binds the IGF-1 receptor, at least one growth factor that binds the EGF receptor, and at least one growth factor that binds the LIF receptor.

28. The method according to claim 27, wherein said collagen is selected from the group consisting of human collagen type I and bovine collagen type I.

29. The method according to claim 27, wherein said fibroblasts are selected from the group consisting of adult human colon fibroblasts, fetal human colon fibroblasts, adult human small intestinal fibroblasts, fetal human small intestinal fibroblasts, and human fibroblast stem cells.

30. The method according to claim 29, wherein said stem cells are derived from human bone marrow.

31. The method according to claim 27, wherein said fibroblast cells are derived from the cell line FFC331.

32. The method according to claim 27, wherein said epithelial cells are selected from the group consisting of adult human colon epithelial cells, fetal human colon epithelial cells, adult human small intestinal epithelial cells, fetal human small intestinal epithelial cells and human epithelial stem cells.

33. The method according to claim 27, further comprising mixing human smooth muscle cells into said collagen mixture.

34. The method according to claim 27, further comprising layering said artificial stroma over a layer of endothelial cells.

35. The method according to claim 27, comprising introducing at least one matrix protein selected from the group consisting of Laminin-1 and Laminin-2 between said artificial stroma and said epithelial cells or overlaying said epithelial cells.

36. The method according to claim 37, wherein said growth factor that binds the IGF-1 receptor is selected from the group consisting of insulin and IGF-1.

37. The method according to claim 27, wherein said growth factor that binds the EGF receptor is selected from the group consisting of EGF and TGF-alpha.

38. The method according to claim 27, further comprising infecting prior to admixture with collagen, at least a portion of said fibroblasts with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

39. The method according to claim 38, wherein said vector is an adenovirus.

40. The method according to claim 38, wherein said growth factor is selected from at least one of the group consisting of a growth factor that binds the IGF-1 receptor, a growth factor that binds the EGF receptor, a growth factor that binds the LIF receptor, a growth factor that binds the hepatocyte growth factor (HGF) receptor, a growth factor that binds the stem cell factor (SCF) receptor, a growth factor that binds the endothelin-3 (ET-3) receptor, a growth factor that binds the platelet derived growth factor (PDGF) receptor, Laminin-1 and Laminin-2, and a combination of said factors.

41. The method according to claim 27, further comprising infecting prior to seeding, at least a portion of said epithelial cells with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

42. The method according to claim 41, wherein said vector is a recombinant adenovirus.

43. The method according to claim 41, wherein said growth factor is selected from at least one of the group consisting of a growth factor that binds the IGF-1 receptor, a growth factor that binds the EGF receptor, a growth factor that binds the SCF receptor and a growth factor that binds the autocrine motility factor (AMF) receptor, a growth factor that binds the endothelin receptor A and a growth factor that binds the endothelin receptor B and a combination of said factors.

44. The method according to claim 34, further comprising infecting at least a portion of said endothelial cells with a recombinant vector comprising a DNA sequence encoding a selected growth factor, under the control of regulatory sequences capable of expressing said factor in said fibroblast.

45. The method according to claim 44, wherein said vector is a recombinant adenovirus.

46. The method according to claim 27, comprising adding to said artificial stroma and said epithelial cells a medium comprising a base media, LIF, at least one of insulin or IGF-1, and at least one of EGF-1 or TGF-α

.

47. The method according to claim 46, wherein said medium further comprises one or more of a factor selected from the group consisting of ET-3, HGF, SCF and AMF.

48. The method according to claim 46, wherein said medium further comprises an additional component selected from the group consisting of transferrin, fetal calf serum and combinations thereof.

49. The method according to claim 27, further comprising seeding a malignant cell on the artificial stroma to create a model of tumor formation or tumor-stroma interaction.

50. A method of in vitro screening of an agent comprising contacting an organotypic culture of claim 1 with said agent in a vessel, and observing the effect of said agent upon said culture.

51. The method according to claim 50, wherein said agent is a drug candidate, and said screening comprises determining the absorption rate thereof, and wherein said observing step comprises observing the movement of said drug candidate through the epithelial cell layer at the top of said culture as a function of time.

52. The method according to claim 50, wherein said agent is screened for toxicity to human intestinal tissue, and wherein said observing step comprises observing the effects of said agent on the morphology and life span of said epithelial cells, whereby an agent which reduces the life span of the epithelial cells or has a negative impact on the morphology of said epithelial cells is toxic.

53. The method according to claim 50, wherein said agent is screened for its effects on hormone regulation in said culture.

54. The method according to claim 50, wherein said agent is screened for binding for a receptor in said culture.

55. A method for screening an agent for repairing effect on intestinal epithelial cell injury comprising:

disrupting the layer of epithelial cells on an organotypic culture of claim 1;

contacting said site of disruption with said agent; and

observing the effects of said agent on the repair of said epithelial cell layer, whereby an agent which promotes repair of the epithelial cell layer is capable of repairing said injury.

60. A method of treating an intestinal wound, comprising placing an organotypic culture of claim 1 on an intestinal wound in a patient.

61. The method according to claim 60, wherein at least one cell present in said organotypic culture is allogeneic to said patient receiving treatment.

62. The method according to claim 61, wherein said wound is caused by a condition selected from the group consisting of chronic inflammation, Crohn'"'"'s disease, ulcerative colitis, intestinal hemorrhage, hemorrhaging diarrhea, ulcer, post operative wound, a malabsorption syndrome, and post irradiation treatment.

22. An organotypic culture comprising an artificial stroma comprising a mixture of collagen and human fibroblasts isolated from a human colon or intestine, said stroma overlayed with epithelial cells isolated from a human colon or intestine, wherein present in said culture are at least one growth factor that binds the IGF—

I receptor, at least one growth factor that binds the EGF receptor, at least one growth factor that binds the LIF receptor, at least one growth factor that binds the ET-3 receptor, at least one growth factor that binds the HGF receptor, at least one growth factor that binds the SCF receptor, and at least one growth factor that binds the AMF receptor and a combination of two or more said factors.

23. The culture according to claim 22, further comprising an additional component selected from the group consisting of transferrin, fetal calf serum, and combinations thereof.

56. A method for enhancing epithelial cell repair at an in vivo site of intestinal or colonic injury comprising:

delivering to the site of said injury at least one of collagen, fibroblasts, at least one growth factor that binds the IGF-1 receptor, at least one growth factor that binds the EGF receptor, and LIF, and a combination thereof.

57. The method according to claim 56, wherein said delivery comprises administering to said site of said injury at least one recombinant vector comprising a polynucleotide molecule encoding at least one of a factor selected from the group consisting of a growth factor that binds the IGF-1 receptor, a growth factor that binds the EGF receptor, a growth factor that binds the LIF receptor, and a combination thereof.

58. The method according to claim 56, wherein said vector is present in a transfected or infected fibroblast delivered to the site of the injury.

59. The method according to claim 56, wherein said vector is present in transfected or infected intestinal or colonic epithelial cells delivered to the site of the injury.