REPRODUCTION OF A STEM CELL NICHE OF AN ORGANISM AND METHOD FOR THE GENERATION THEREOF

Abstract

The present invention relates firstly to a method for reproducing a stem cell niche of an organism. The invention further relates to a reproduction of a stem cell niche of an organism. According to the invention, an image of a tissue of an organism is generated, which tissue comprises at least one stem cell niche. The image is filtered in order to obtain a structural pattern of the imaged stem cell niche. In a further step, a lithographic mask is generated from the structural pattern. According to the invention, a starting material of a substrate is structured by means of indirect or direct application of the lithographic mask, whereby a structured substrate is obtained which represents the reproduction of the imaged stem cell niche of the organism. The reproduction can be characterised as biolithomorphic.

Claims

1. A method for reproducing a stem cell niche of an organism, comprising the following steps: creation of an image of a tissue comprising at least one stem cell niche of an organism; filtering of the image in order to produce a structural pattern of the reproduced stem cell niche; creation of a lithographic mask from the structural pattern; and structuring of a starting material through application of the lithographic mask, whereby a structured substrate is obtained which represents the reproduction of the imaged stem cell niche of the organism.

2. The method as set forth in claim 1, wherein the structural pattern comprises a substructural pattern for coarse structures of the imaged stem cell niche and a substructural pattern for fine structures of the imaged stem cell niche; wherein the lithographic mask comprises a submask for coarse structures that is produced from the substructural pattern for coarse structures; and wherein the lithographic mask comprises a submask for fine structures that is produced from the substructural pattern for fine structures.

3. The method as set forth in claim 2, wherein the fine structures have a maximum feature size of between 50 m and 75 m, and that the coarse structures have a minimum feature size of between 50 m and 75 m.

4. The method as set forth in claim 2, wherein the filtering of the image comprises different edge analyses with which the substructural pattern for the coarse structures and the substructural pattern for the fine structures are determined.

5. The method as set forth in any one of claim 1, wherein a tool is first created with the lithographic mask for deforming the starting material of the substrate, after which the starting material of the substrate is structured with the tool.

6. The method as set forth in claim 12, wherein the tool for deforming the starting material preferably includes a tool for creating coarse structures and a tool for creating fine structures, with the tool for creating coarse structures being created with the submask for coarse structures, and with the tool for creating fine structures being created with the submask for fine structures.

7. The method as set forth in claim 6, wherein the tool for creating fine structures is constituted by an embossing die for hot-embossing or by an embossing die for nanoimprint lithography, and that the tool for creating coarse structures is constituted by a thermoforming mold.

8. The method as set forth claim 1, wherein the structured substrate is populated with at least one stem cell.

9. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 1.

10. Reproduction of a stem cell niche of an organism, characterized in that it has geometric characteristics of the stem cell niche of the organism.

11. The method as set forth in claim 3, wherein filtering of the image comprises different edge analyses with which the substructural pattern for the coarse structures and the substructural pattern for the fine structures are determined.

12. The method as set forth in claim 2, wherein tool is first created with the lithographic mask for deforming the starting material of the substrate, after which the starting material of the substrate is structured with the tool.

13. The method as set forth in claim 3, wherein tool is first created with the lithographic mask for deforming the starting material of the substrate, after which the starting material of the substrate is structured with the tool.

14. The method as set forth in claim 4, wherein tool is first created with the lithographic mask for deforming the starting material of the substrate, after which the starting material of the substrate is structured with the tool.

15. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 2.

16. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 4.

17. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 5.

18. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 6.

19. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 7.

20. Reproduction of a stem cell niche of an organism, which can be achieved by means of a method as set forth in claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Additional advantages, details, and developments of the invention follow from the following description of preferred exemplary embodiments of the invention with reference to the drawing.

[0055] FIG. 1 shows cross-sectional representation of a structured substrate prepared according to the invention;

[0056] FIG. 2 shows an original image of a bone marrow section prepared according to the invention and structural patterns produced therefrom according to the invention;

[0057] FIG. 3 shows a comparison between the original image shown in FIG. 2 and a structural pattern prepared according to the invention;

[0058] FIG. 4 shows a flowchart of the inventive production of lithographic masks;

[0059] FIG. 5 shows polycarbonate films finely structured according to the invention;

[0060] FIG. 6 shows the polycarbonate films shown in FIG. 5 with supplementary coarse structures;

[0061] FIG. 7 shows a flowchart of a physical surface modification according to a preferred embodiment of the invention;

[0062] FIG. 8 shows a first preferred embodiment of an inventive reproduction of stem cell niches of a bone marrow;

[0063] FIG. 9 shows a second preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow;

[0064] FIG. 10 shows a detailed illustration of the reproduction shown in FIG. 9;

[0065] FIG. 11 shows a third preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow;

[0066] FIG. 12 shows a fourth preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow; and

[0067] FIG. 13 shows a fifth preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow in apposition.

DETAILED DESCRIPTION

[0068] FIG. 1 shows the objective of a preferred embodiment of the method according to the invention in a cross-sectional representation, namely the construction of a hierarchical structure in the form of a structured substrate that synthetically reproduces a plurality of stem cell niches of a biological tissue of an organism, consisting of a coarse mesostructure 01, a fine microstructure 02, and an even finer nanostructure 03. Thermoplastic films are preferably structured using various methods for this. This is followed by the culturing of haematopoietic stem cells (not shown) with the aim of maintaining their non-differentiated status.

[0069] FIG. 2 shows an original image A prepared according to the invention of a bone marrow section and binary images B, C, D, and E produced according to the invention after application of edge detection algorithms. The binary images B, C, D, E represent structural patterns that were obtained starting from the original image A. The binary images B, C, D, E exhibit edges, which represent walls of stem cell niches in the bone marrow. The edges were detected in the original image A, which can be a grayscale or RGB image, using various algorithms. The preferred edge-detection algorithms according to Canny B., Sobel C., Prewitt D., and Roberts E. are shown here.

[0070] FIG. 3 shows a comparison between the original image A already shown in FIG. 2 and a binary image prepared according to the invention after the application of the Canny detector. The original image of the bone marrow section A and the original image are shown with superimposed, extracted structures after edge detection with the Canny detector B.

[0071] FIG. 4 shows a flowchart of the creation of lithographic masks from data of an image of a tissue comprising at least one stem cell niche according to a preferred embodiment of the invention. The flowchart describes the procedure of the extraction of structures from an image file using different algorithms of the binary image creation and the use thereof in the designing of a photomask. After a selection 11 of an image file and reading 12 of the data of the image file into a computer program for solving mathematical problems, the image file is converted from the RGB color space or from a grayscale range into a binary image 13. Through the use of an edge-detection algorithm 14 and a local threshold calculation 16, structural patterns are obtained. A selection 17 is made of the most suitable structural patterns. The structural patterns are subjected to vectoring 18, so that vector data are obtained. Lithographic masks 19 are produced from the vector data.

[0072] FIG. 5 shows scanning electron microscope images of polycarbonate films hot-embossed according to the invention. By means of hot-embossing, 50 m-thick polycarbonate films were structured with fine structures using a lithographic mask that was prepared from the bone marrow structure obtained through imaging and filtering.

[0073] FIG. 6 shows additional scanning electron microscope images of the polycarbonate films shown in FIG. 5 after thermoforming. After hot-embossing with a bone marrow structure obtained through imaging and filtering, the 50 m-thick polycarbonate films were additionally structured by means of a microthermoforming process. Molding tools with cavities are used for this purpose. The cavities of these molding tools have a diameter of 300 m and a depth of also 300 m. Based on their size, these structures are very similar to those in the trabecular bone. The moldings that are marked come closest to the original.

[0074] FIG. 7 shows a flowchart of a physical surface modification according to a preferred embodiment of the invention. The flowchart shows the procedure for physical surface modification for producing a hierarchical architecture of a structured substrate to be produced according to the invention. After the preparation of a lithographic mask 21 as illustrated in FIG. 4, and after the processing of a wafer 22, a silicone case 23 of a master is produced, thus resulting in a film. This is followed by the modification of the film in a hot-embossing step 24 using a tool that was prepared through application of a lithographic mask which, in turn, was produced from a structure of a bone marrow that was obtained through imaging and filtering. Subsequently, in a microthermoforming process 25, structures of a trabecular bone are embodied, for which purpose an additional tool is used that was produced through application of the lithographic mask. In a final process step, the nanostructure 26 is applied through the dip-coating and incubation of the film in a solution or caustic.

[0075] FIG. 8 shows a scanning electron microscope image of a first preferred embodiment of an inventive reproduction of stem cell niches of a bone marrow. This embodiment is formed by a structured substrate made of polydimethylsiloxane, which was poured into a mold.

[0076] FIG. 9 shows a scanning electron microscope image of a second preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow. This embodiment is formed by a structured substrate made of silicon, which was structured using an embossing die (not shown).

[0077] FIG. 10 shows a detailed illustration of the reproduction shown in FIG. 9.

[0078] FIG. 11 shows a scanning electron microscope image of a third preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow. This embodiment is formed by a structured substrate made of silicon, which was structured by means of reactive ion etching.

[0079] FIG. 12 shows a scanning electron microscope image of a fourth preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow. This embodiment is formed by a structured substrate made of a borosilicate glass in which edges were rounded for structuring.

[0080] FIG. 13 shows a scanning electron microscope image of a fifth preferred embodiment of the inventive reproduction of stem cell niches of a bone marrow. In this embodiment, an extracted structure is multiply apposed for large-surface reproduction.

LIST OF REFERENCE SYMBOLS

[0081] 01 mesostructure [0082] 02 microstructure [0083] 03 nanostructure [0084] 04 [0085] 05 [0086] 06 [0087] 07 [0088] 08 [0089] 09 [0090] 10 [0091] 11 selection of an image file [0092] 12 reading of the data of the image file [0093] 13 binary image [0094] 14 edge-detection algorithm [0095] 15 [0096] 16 local threshold calculation [0097] 17 selection of the most suitable structural patterns [0098] 18 vectoring [0099] 19 lithographic masks [0100] 20 [0101] 21 creation of a lithographic mask [0102] 22 processing of a wafer [0103] 23 silicone cast [0104] 24 hot-embossing step [0105] 25 microthermoforming process [0106] 26 application of a nanostructure