NEURONALLY INDUCTIVE CULTIVATION MATRIX

Abstract

A matrix for the cultivation of biological cells and differentiation into neuronal cells consists of a polymer base body having a structured surface with a microstructure and a nanostructure embedded therein.

Claims

1. A matrix for cultivating biological cells and differentiating these cells into neuronal cells or neuronal tissue, made of a base body made of biocompatible or biological polymer having a structured surface, the matrix comprising: a first periodic microstructure, the first periodic microstructure made of parallel microdimensioned grooves; and, a second periodic nanostructure, the second periodic microstructure made of parallel nanodimensioned grooves, the second periodic microstructure extending parallel to the first periodic microstructure and embedded into the parallel microdimensioned grooves of the first periodic microstructure.

2. The matrix according to claim 1, wherein the polymer has a polymer rigidity on the structured surface equal to or not greater than a biological cell rigidity of the biological cells to be cultivated.

3. The matrix according to claim 1, wherein the parallel microdimensioned grooves of the first periodic microstructure have a depth of 0.7 to 1.5 m and a width of 1 to 2 m that have a center distance of 3 to 12 m between adjacent grooves.

4. The matrix according to claim 1, wherein the parallel nanodimensioned grooves of the second periodic nanostructure have a depth of 15 to 35 nm and a width of 450 to 550 nm that have a center distance of 500 to 550 nm between adjacent grooves.

5. A method for producing the matrix according to claim 1, the method comprising: forming the structured surface on the base body made of biocompatible or biological polymer by micro-milling, wherein the first periodic microstructure and the second periodic nanostructure embedded therein are formed.

6. A method for producing a matrix according to claim 1, the method comprising: forming a structured surface on a tool by machining, wherein the first periodic microstructure and the periodic nanostructure embedded therein are formed, and producing the structured surface on the base body made of biocompatible or biological polymer by: casting the polymer of the base body onto the tool, or, embossing of the polymer of the base body with the tool, and, optionally curing or drying the polymer of the base body on the tool and separating the cured, dry base body from the tool, such that the matrix is obtained as the base body having a structured surface.

7. The method according to claim 5, wherein the machining is performed with a machining tool having a cutting edge made of monocrystalline diamond with a tip radius of 10 m or less.

8. The method according to claim 5, wherein the polymer is selected from collagen, gelatin, agarose, derivatives and semi-synthetic modifications thereof.

9. Use of the matrix according to claim 1 for mechanotransductive differentiation of stem cells into neuronal tissue.

10. A method for producing neuronal cells or tissue from isolated biological cells, the method comprising: bringing isolated biological cells into contact with the matrix according to claim 1, cultivating the biological cells on and at the structured surface of this matrix, wherein mechanotransductive differentiation of the cells into neuronal cells and/or neuronal tissue takes place, and obtaining differentiated neuronal cells or tissues.

11. The method according to claim 10, wherein the isolated biological cell is a mesenchymal stem cell.

12. The method according to claim 6, wherein the machining is performed with a machining tool having a cutting edge made of monocrystalline diamond with a tip radius of 10 m or less.

13. The method according to claim 6, wherein the polymer is selected from collagen, gelatin, agarose, derivatives and semi-synthetic modifications thereof.

Description

[0028] The invention is described in greater detail below:

[0029] FIG. 1 is a detailed view of a schematic cross section through a neurotransductive cultivation matrix of the invention. The latter comprises a base body having a structured surface (20). The surface (20) is characterized by a microstructure (22) of parallel microdimensioned ridges (23) and a periodic nanostructure (26) embedded therebetween having parallel nanodimensioned grooves (27) running parallel to the microstructure (22).

[0030] FIG. 2 is a different magnification of a schematic cross section through another variant of a neurotransductive cultivation matrix of the invention. A structured surface (20) is embodied on one side on the base body (10) of the matrix. The structured surface is characterized by a periodic microstructure (22) having parallel microdimensioned grooves (23) and a periodic nanostructure (26) having parallel nanodimensioned grooves (27) running parallel to the microstructure (22), the periodic nanostructure (26) being embedded in the grooves (23) of the microstructure (22).

[0031] FIGS. 3A and 3B show a plan view of electron micrographs of inventively surface-structured cultivation matrices (scales added). FIG. 3A illustrates a cultivation matrix having the structure according to the schematic representation (not to scale) according to FIG. 1.

EXAMPLE

[0032] The entire process sequence for the mechanotransductive differentiation of stem cells into neuronal cells or neuronal tissue includes (a) production of a master tool having the negative of the inventive topography, (b) transfer of the topography onto a moldable material, (c) optionally further impressions from the master tool; and (d) cultivating stem cells on the molded, structured material.

[0033] To produce a surface-structured master tool, a cylindrical roller made of copper or nickel (or corresponding alloys) is clamped in a lathe and turned with an MDC cutting edge having a tip angle of 130 and a tip radius of 1 m at a speed of 150 min.sup.1 by means of a turning tool. The machining tool is guided such that ridges having a width of 1.2 m and spacing of 11.2 m are cut on the cylinder (topography 1). Alternatively, the advance is set such that ridges having a width of 1.24 m and spacing of 3.8 m are produced on the cylinder (topography 2). The ridges form the microstructure.

[0034] For machining, an advance of 0.5 mm per revolution (or tool stroke) is selected. Between the ridges of the microstructure, a nanostructure having channels of 32 nm, which are spaced 500 nm apart from one another (topography 1), or channels having a depth of 19.15 nm and set 40 nm apart from one another, is formed on the metal cylinder (topography 2).

[0035] The surface-structured master tool produced in this way is clamped as an embossing roller in an apparatus for embossing collagen films. A base body made of freshly poured lyophilized collagen is used as collagen film. The channel structure of the roller is embossed into the surface of the collagen film. Then the collagen film having a structured surface is dried by dehydration in a desiccator.

[0036] In an alternative approach, a correspondingly structured flat metal plate is produced by planing with a corresponding machining tool. The metal plate acts as a template. A freshly prepared liquid solution of lyophilized collagen is cast onto it and allowed to harden there. Then the collagen film produced is dried in the desiccator for about four days. The film shrinks by a few percent. It may be removed easily from the template due to shrinkage.

[0037] In a further alternative approach, a correspondingly structured flat casting mold made of silicone elastomer is produced by foil embossing or casting by means of a metal master tool. This mold is then used as a template accordingly.

[0038] The liquid solution of lyophilized collagen is cast onto the mold and allowed to harden there. Then the collagen film produced is dried in the desiccator for about four days. The film shrinks by a few percent. It may be removed easily from the template due to shrinkage. Alternatively, due to its elasticity, the casting mold is removed from the solidified or dried collagen gel.

[0039] It has been found that the embossed or cast collagen film can be stored for several weeks after drying. In the present case, the embossed film was moistened after a storage period of four weeks, so that the original shape, including the inventively structured surface, formed.

[0040] The freshly embossed or rehydrated collagen foil is used as a cultivation matrix. The surface structure of the cultivation matrix is shown in FIG. 3. FIGS. 3A and 3B illustrate SEM images of a surface-structured gelatin matrix (metacrylated gelatin) that was embossed with a tool having topography 1. The surface-structured gelatin matrix was produced in a comparable manner, as described here by way of example for a collagen matrix. FIG. 3A illustrates a gelatin matrix molded with a nickel master tool. FIG. 3B illustrates a gelatin matrix molded with a PDMS master tool.

[0041] Both the cast or embossed collagen film and the embossed gelatin matrix can be readily stored in the dried state for at least 3 weeks, so that the originally embossed surface structuring is retained after water or culture medium is added when swelling the gels.

[0042] For producing neuronal cells, suspended mesenchymal stem cells derived from adult tissue are seeded onto the embossed gelatin matrix or collagen film and cultivated for four weeks.

[0043] The cultivated cells develop an extension and parallel alignment of their morphology along the microstructuring of the surface of the cultivation matrix. The cells lose their original fibroblast-like shape rapidly, and the typical morphology of neuronal cells is recognizable after a few days. The cells develop very long dendritic or axonal extensions, this determining their good function as nerve cells.

[0044] The residual collagen of the collagen matrix is digested using collagenase after a four-week cultivation period so that the cells can be gently isolated without destroying their acquired morphology. Vitality tests demonstrate that there is no limitation to the vitality of these cells following collagenase digestion. In assays, neuronal markers were detected in the cells at the gene and protein levels.