Method of producing three-dimensional tissue and method of producing extracellular matrix used in the same

Abstract

The present invention provides a novel method of producing a three-dimensional tissue by which cell lamination can be carried out easily. According to the method, a three-dimensional tissue in which cell layers are laminated with an extracellular matrix intervening between each pair of the adjacent cell layers is produced by: (A) forming a cell layer on a substrate; (B) bringing the cell layer formed on the substrate into contact with a solution containing a first substance and a solution containing a second substance alternately, thus forming, on the cell layer, an extracellular matrix in which the first substance and the second substance are laminated alternately; and (C) culturing a cell on the extracellular matrix to form a further cell layer. In the present invention, a combination of the first substance and the second substance is (a) a combination of a protein or polymer having an RGD sequence and a protein or polymer that interacts with the protein or polymer having the RGD sequence or (b) a combination of a protein or polymer that is positively charged and a protein or polymer that is negatively charged.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-I to 1-IV′ are schematic views illustrating an example of major steps included in a three-dimensional tissue production method according to the present invention.

FIG. 2 is a graph showing the relationship between a frequency shift and the number of dipping steps performed in one example of the present invention.

FIG. 3 shows a SEM image showing a three-dimensional tissue obtained in another example of the present invention.

FIG. 4 shows micrographs of the three-dimensional tissue obtained in the above example and a three-dimensional tissue obtained in a comparative example. In FIG. 4, the micrographs of the group (A) show the result obtained in Comparative Example 1 and the micrographs of the group (B) show the result obtained in Example 2. In each of the groups (A) and (B), the micrograph (1) is an image as viewed through a phase-contrast microscope and the micrograph (2) is an image as viewed through a confocal fluorescence microscope.

FIG. 5 shows confocal laser micrographs of a three-dimensional tissue in still another example of the present invention. In FIG. 5, the micrograph (A) shows the X-Y plane of the three-dimensional tissue, the micrograph (B) shows the X-Z plane of the three-dimensional tissue, and the micrograph (C) shows the Y-Z plane of the three-dimensional tissue.

FIG. 6 shows a 3-D image of the three-dimensional tissue obtained in the above example as viewed through a confocal laser microscope.

FIG. 7 shows SEM images of a three-dimensional tissue obtained in still another example of the present invention. In FIG. 7, the SEM image (A) shows a surface of the three-dimensional tissue and the SEM image (B) shows a cross section of the three-dimensional tissue.

FIG. 8 shows confocal micrographs of a three-dimensional tissue obtained in still another example of the present invention. In FIG. 8, the micrographs (A) and (B) both show the result obtained in Example 4.

FIG. 9 shows phase-contrast micrographs of a three-dimensional tissue obtained in still another example of the present invention. In FIG. 9, the micrograph (A) shows an image of a first cell layer, the micrograph (B) shows an image of a second cell layer, the micrograph (C) shows an image of a third cell layer, and the micrograph (D) shows an image of a fourth cell layer.

FIG. 10 is a graph showing the relationship between a frequency shift and the number of dipping steps performed in still another example of the present invention.

FIG. 11 is a graph showing the change in amount of a remaining ECM over time in still another example of the present invention.

Claims

1. A method of producing a three-dimensional tissue by cell lamination, the method comprising the steps of (A) forming a cell layer on a substrate;(B) bringing the cell layer formed on the substrate into contact with a solution containing a first substance and a solution containing a second substance alternately, thus forming, on the cell layer, an extracellular matrix in which the first substance and the second substance are laminated alternately; and(C) culturing a cell on the extracellular matrix to form a further cell layer,wherein a combination of the first substance and the second substance is (a) a combination of a protein or polymer having an RGD sequence and a protein or polymer that interacts with the protein or polymer having the RGD sequence or (b) a combination of a protein or polymer that is positively charged and a protein or polymer that is negatively charged.

2. The method according to claim 1, further comprising the step of repeating the step (B) and the step (C).

3. The method according to claim 1, wherein the extracellular matrix having a thickness of at least 1 nm is formed in the step (B).

4. The method according to claim 1, wherein the first substance is at least one selected from the group consisting of fibronectin, vitronectin, laminin, cadherin, collagen, chitin, chitosan, and polylysine, and the second substance is at least one selected from the group consisting of gelatin, albumin, globulin, heparin, heparan sulfate, dextran sulfate, polyglutamic acid, polyaspartic acid, and elastin.

5. The method according to claim 1, wherein the first substance is fibronectin or laminin.

6. The method according to claim 5, wherein the combination of the first substance and the second substance is a combination of fibronectin and gelatin or a combination of laminin and gelatin.

7. The method according to claim 1, wherein, in the step (B), the cell layer is brought into contact with the solution containing the first substance first to form a first substance layer as a lowermost layer of the extracellular matrix.

8. The method according to claim 1, wherein, in the step (B), the cell layer is brought into contact with the solution containing the first substance last to form a first substance layer as an uppermost layer of the extracellular matrix.

9. The method according to claim 1, further comprising, prior to the step (A), the step of bringing the substrate into contact with the solution containing the first substance and the solution containing the second substance alternately, thus forming, on the substrate, an extracellular matrix in which the first substance and the second substance are laminated alternately, wherein, in the step (A), the cell layer is formed on the extracellular matrix formed on the substrate.

10. The method according to claim 9, wherein the extracellular matrix having a thickness of at least 1 nm is formed on the substrate.

11. The method according to claim 9, wherein the substrate is brought into contact with the solution containing the first substance last to form a first substance layer as an uppermost layer of the extracellular matrix formed on the substrate.

12. The method according to claim 1, wherein the cell is at least one cell selected from the group consisting of a hepatocyte, a vascular endothelial cell, a fibroblast, an epidermic cell, an epithelial cell, a mammary glandular cell, a myocyte, a neurocyte, a tissue stem cell, an embryonic stem cell, a bone cell, and an immunocyte.

13. The method according to claim 1, wherein at least one of the solution containing the first substance and the solution containing the second substance further contains at least one substance selected from the group consisting of cell growth factors, cytokines, chemokines, hormones, and biologically active peptides.

14. The method according to claim 1, wherein at least one of the solution containing the first substance and the solution containing the second substance further contains at least one substance selected from the group consisting of therapeutic agents for treating diseases, prophylactic agents for preventing diseases, and inhibitors for inhibiting diseases.

15. A three-dimensional tissue obtained by the method according to claim 1.

16. A method of producing an extracellular matrix for adhering cell layers, the method comprising the step of bringing a cell layer into contact with a solution containing a first substance and a solution containing a second substance alternately, thus forming, on the cell layer, a thin film in which the first substance and the second substance are laminated alternately as an extracellular matrix,wherein a combination of the first substance and the second substance is (a) a combination of a protein or polymer having an RGD sequence and a protein or polymer that interacts with the protein or polymer having the RGD sequence or (b) a combination of a protein or polymer that is positively charged and a protein or polymer that is negatively charged.

17. The method according to claim 16, wherein the cell layer is formed on a substrate by culturing a cell on the substrate.

18. The method according to claim 16, wherein the thin film has a thickness of at least 1 nm.

19. The method according to claim 16, wherein the first substance is at least one selected from the group consisting of fibronectin, vitronectin, laminin, cadherin, collagen, chitin, chitosan, and polylysine, and the second substance is at least one selected from the group consisting of gelatin, albumin, globulin, heparin, heparan sulfate, dextran sulfate, polyglutamic acid, polyaspartic acid, and elastin.

20. An extracellular matrix obtained by the method according to claim 16.