Methods for forming normal regenerated tissues, the normal regenerated tissues, and method of calibrating sensitivity and so on

• US 20040241837A1
• Filed: 06/09/2004
• Published: 12/02/2004
• Est. Priority Date: 10/15/2001
• Status: Active Grant

• Current Assignee: Innovative Medicine and Any Peptide Pharmaceuticals Corp
• Original Assignee: Japan Science And Technology Corpration
• Inventors: Nakatsugawa, Shigekazu
• Granted Patent: US 8,592,176 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3813 Epithelial cells, e.g. keratinocytes, urothelial cells
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2502/088 neural stem cells
• CPC Class Codes: C12N 2502/095 mammary cells
• CPC Class Codes: C12N 2502/246 Cells of the male genital tract, non-germinal testis cells
• CPC Class Codes: C12N 2502/256 Renal cells
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2503/04 Screening or testing on artificial tissues
• CPC Class Codes: C12N 5/0625 Epidermal cells, skin cells; Cells of the oral mucosa
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

A normal regenerated tissue is formed by exposing to radiation a connective tissue or a supporting tissue originating in an organ to thereby form a feeder layer and then transplanting epithelial cells thereon to form a stratified structure. By conveniently and surely providing regenerated tissue by the 3-dimensional culture with the use of a human-origin normal tissue as a base, it is possible to construct systems for assessing effects and side effects of chemicals such as drugs or assessing sensitivities thereof with the use of regenerated tissues as models of corresponding tissues respectively.

Claims

1. A method for forming a normal regenerated tissue comprising irradiating an organ-derived connective tissue or its constituent cells or supporting tissue to form a feeder layer followed by transplanting epithelial cells to form a stratified structure.

2. The method for forming a normal regenerated tissue according to claim 1 wherein the connective tissue or its constituent cells or supporting tissue and the epithelial tissue are orthotopic with regard to the originating organ.

3. The method for forming a normal regenerated tissue according to claim 1 wherein the connective tissue or its constituent cells or supporting tissue are at least any of the organ-derived fibroblasts, endothelial cells or its constituent tissue.

4. The method for forming a normal regenerated tissue according to claim 1 wherein vascular endothelial cells are transplanted on organ-derived fibroblasts and then an irradiation is effected to form a feeder layer, and then the epithelial cells are transplanted to form a stratified structure.

5. The method for forming a normal regenerated tissue according to claim 1 wherein after transplanting the epithelial cells at least one extracellular matrix is added to form the epithelial cells in a stratified structure.

6. The method for forming a normal regenerated tissue according to claim 5 wherein the extracellular matrix is a structural component or adhesion molecule of an extracellular substrate.

7. The method for forming a normal regenerated tissue according to claim 5 wherein the extracellular matrix is at least one of collagen, elastin, proteoglycan, fibronectin, laminin and tenascin.

8. The method for forming a normal regenerated tissue according to claim 7 wherein after transplanting a collagen or collagen with fibronectin as well as laminin are added to form the epithelial cells in a stratified structure.

9. The method for forming a normal regenerated tissue according to claim 1 wherein the connective tissue or its constituent cells or supporting tissue is co-cultured with at least one of heterogenous constituent cells derived from an orthotopic organ or a culture supernatant thereof and thereafter irradiated to form a feeder layer.

10. The method for forming a normal regenerated tissue according to claim 1 wherein heterogenous constituent cells deriving from an orthotopic organ, epithelial cells and a culture supernatant thereof is co-cultured to form a stratified structure.

11. The method for forming a normal regenerated tissue according to claim 1 wherein the irradiation is an X-ray or γ

-ray irradiation.

12. The method for forming a normal regenerated tissue according to claim 1 wherein the stratified structure of the epithelial cells is formed by a culture at a carbon dioxide gas concentration of 5 to 15%, air concentration of 85 to 95% at a culture temperature of 20 to 40°

C.

13. A normal regenerated tissue comprising a stratified structure of epithelial cells on a feeder layer from an organ-derived connective tissue or constituent cells or supporting tissue.

14. The normal regenerated tissue according to claim 13 wherein the epithelial cells derives from nerve, oral mucosa, skin, bronchus, mammary gland, liver or kidney.

15. The normal regenerated tissue according to claim 13 wherein the connective tissue or its constituent cells or supporting tissue and the epithelial tissue are orthotopic with regard to the deriving organ.

16. The normal regenerated tissue according to claim 13 wherein the connective tissue or its constituent cell or supporting tissue are at least any of the organ-derived fibroblasts, endothelial cells or its constituent tissue.

18. The normal regenerated tissue according to claim 13 or 17 wherein the epithelial cells are in a stratified structure consisting of four or more layers.

19. The normal regenerated tissue comprising a normal regenerated tissue according to claim 13 or 17 placed on a basal plate.

20. The normal regenerated tissue according to claim 13 or 17 wherein a culture medium or culture fluid is allowed to flow in contact with a normal regenerated tissue.

21. The normal regenerated tissue wherein a plural of normal regenerated tissues according to claim 19 are allowed to pass through the channel of the culture medium or culture fluid.

22. The normal regenerated tissue which constitutes a regenerated organ model body of a normal regenerated tissue according to claim 13 or 17.

23. A method for assessing a sensitivity of cancer cells comprising inhibiting the proliferation of regenerated epithelial cells by irradiating a normal regenerated tissue according to claim 13 or 17 followed by transplanting the cancer cells on the epithelial cells in a stratified structure followed by adding a chemical substance or irradiation.

24. The method according to claim 23 wherein the assessment is effected at a low oxygen concentration.

25. The method according to claim 23 comprising supplementing at least one of collagen or other extracellular matrixes after transplanting cancer cells or transplanting cancer cells after supplementing at least one of collagen or other extracellular matrixes, followed by assessing the sensitivity.

26. A method for assessing an angiogenetic ability comprising inhibiting the proliferation of regenerated epithelial cells by irradiating a normal regenerated tissue according to claim 13 or 17 followed by transplanting the cancer cells on the epithelial cells in a stratified structure followed by supplementing at least one of collagen or other extracellular matrixes on which then vascular endothelial cells are transplanted followed by assessing the angiogenetic ability of the cancer cells in response to the addition of a chemical substance.

27. A method for assessing an angiogenetic ability comprising inhibiting the proliferation of regenerated epithelial cells by irradiating a normal regenerated tissue according to claim 13 or 17 followed by transplanting the cancer cells on the epithelial cells in a stratified structure followed by mounting at least one of collagen or other extracellular matrixes followed by inverting the entire structure whereby assessing a motility or invasion ability.

28. A method for assessing a sensitivity of a normal regenerated tissue according to claim 13 or 17 comprising an exposure of the tissue to a chemical substance or irradiation.

29. A method for assessing a gene transduction comprising assessing the efficiency of the gene transduction in a normal regenerated tissue according to claim 13 or 17.

30. The method according to claim 23 wherein the irradiation for inhibiting the proliferation of regenerated epithelial cells is an X-ray or γ

-ray irradiation.

17. A normal regenerated tissue comprising a feeder layer consisting of a lower layer of organ-derived fibroblasts and an upper layer of vascular endothelial cells and epithelial cells in a stratified structure placed on the feeder layer.