Method for three-dimensional replication of a biological tissue

Abstract

A method for three-dimensional reproduction of a biological tissue in the framework of tissue engineering. A substrate is provided and primary cells of a first cell type are deposited on the substrate. A first precursor of a substance adhering the cells is arranged on at least a selection of the primary cells of the first cell type. A second precursor of the substance adhering the cells is arranged in a locally targeted manner on the selection of primary cells of the first cell type according to a structure formed by cells of the first cell type in the tissue to be reproduced. The first precursor and the second precursor react with each other to form the substance adhering the cells, which substance adheres the selection of the primary cells of the first cell type according to the structure formed by the cells of the first cell type in the tissue to be reproduced.

Claims

1. Method for the reproduction of biological tissue comprising the steps of: provision of a substrate (01); deposit of primary cells (08) of a first cell type on the substrate (01); arrangement of a first precursor of a substance (11) adhering the cells (08, 13) on at least a selection of the primary cells (08) of the first cell type; and arrangement in a locally targeted manner of a second precursor (12) of the substance (11) adhering the cells (08, 13) to the selection of primary cells (08) of the first cell type according to a structure formed by cells of the first cell type in the tissue to be reproduced, whereby the first precursor and the second precursor (12) react with each other to form the substance (11) adhering the cells (08, 13), which substance adheres the selection of primary cells (08) of the first cell type according to the structure formed by the cells of the first cell type in the tissue to be reproduced and forms a first layer of the primary cells (08) such that the selection of primary cells (08) of the first cell type constitutes a reproduction of at least a first section of the biological tissue to be reproduced.

2. The method according to claim 1, wherein the substrate (01) to be provided has microfluidic structural elements.

3. The method according to claim 1, wherein the primary cells (08) of the first cell type are deposited uniformly stochastically distributed on the substrate (01).

4. The method according to claim 1, wherein the arrangement of the first precursor of the substance (11) adhering the cells (08, 13) on the selection of the primary cells (08) of the first cell type is carried out with a locally metering dispensing system (09).

5. The method according to claim 4, wherein the locally metering dispensing system (09) is formed by a dispenser or pipetting system movable in the x-y-z axes.

6. The method according to claim 4, wherein the second precursor (12) is arranged in individual drops at or on the selection of primary cells (08) of the first cell type, wherein the smallest dispensable drops contain at most 5 picoliters of the second precursor (12).

7. The method according to claim 4, wherein during the arrangement in a locally targeted manner of the second precursor (12) with the locally metering dispensing system (09), travel paths are covered in all three spatial axes, wherein the travel paths are at least 10 mm.

8. The method according to claim 1, wherein the substance (11) adhering the cells (08, 13) is formed by a two-component hydrogel, wherein the first precursor constitutes a first component of the two-component hydrogel and the second precursor (12) constitutes a second component of the two-component hydrogel.

9. The method according to claim 1, wherein one of the two precursors (12) comprises an ionotropic polymer formed by alginate, pectin, poly(galacturonate), carrageenan, dextran, gellan, scleroglucan, chitosan, polyphosphazene, sodium polyacrylate and/or polyamino acid.

10. The method according to claim 1, wherein the deposit of the primary cells (08) of the first cell type takes place in a medium (04).

11. The method according to claim 10, wherein the medium (04) comprises at least one nutrient medium for the primary cells (08) of the first cell type.

12. The method according to claim 10, wherein the medium (04) further comprises the first precursor of the substance (11) adhering the cells (08, 13), or that the first precursor of the substance (11) adhering the cells (08, 13) is mixed into the medium (04) located on the substrate (01).

13. The method according to claim 1, wherein after the first precursor and the second precursor (12) have formed the substance (11) adhering the cells (08, 13), the primary cells (08) of the first cell type not belonging to the selection are removed from the substrate (01).

14. The method according to claim 1, wherein it comprises the following further steps: deposit of further primary cells (08) of the first cell type on the already formed reproduction of the first section of the biological tissue; arrangement of the first precursor of the substance (11) adhering the cells (08, 13) on at least a selection of the further primary cells (08) of the first cell type; and arrangement in a locally targeted manner of the second precursor (12) of the substance (11) adhering the cells (08, 13) to the selection of the further primary cells (08) of the first cell type according to the structure formed by cells of the first cell type in the tissue to be reproduced, whereby the first precursor and the second precursor (12) react with each other and form the substance (11) adhering the cells (08, 13), which substance adheres the selection of the further primary cells (08) of the first cell type according to the structure formed by the cells of the first cell type in the tissue to be reproduced, such that the selection of the further primary cells (08) of the first cell type constitutes a reproduction of a further section of the biological tissue to be reproduced.

15. The method according to claim 1, wherein it comprises the following further steps: deposit of primary cells (13) of a second cell type on the substrate (01) and/or on the already formed reproduction of the first section of the biological tissue; arrangement of the first precursor of the substance (11) adhering the cells (08, 13) on at least a selection of the primary cells (13) of the second cell type; and arrangement in a locally targeted manner of the second precursor (12) of the substance (11) adhering the cells (08, 13) to the selection of primary cells (13) of the second cell type according to a structure formed by cells of the second cell type in the tissue to be reproduced, whereby the first precursor and the second precursor (12) are amalgamated and form the substance (11) adhering the cells (08, 13), which substance adheres the selection of the primary cells (13) of the second cell type according to the structure formed by the cells of the second cell type in the tissue to be reproduced, such that the selection of the primary cells (13) of the second cell type constitutes a reproduction of a further section of the biological tissue to be reproduced.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Further advantages, details and further developments of the invention will be apparent from the following description of preferred embodiments of the invention, with reference to the drawing. Wherein:

[0045] FIG. 1: shows a first phase of a first preferred embodiment of a method according to the invention for reproduction of biological tissue;

[0046] FIG. 2: shows a second phase of the first preferred embodiment of the method according to the invention;

[0047] FIG. 3: shows a third phase of the first preferred embodiment of the method according to the invention;

[0048] FIG. 4: shows a fourth phase of the first preferred embodiment of the method according to the invention;

[0049] FIG. 5: shows a fifth phase of the first preferred embodiment of the method according to the invention;

[0050] FIG. 6: shows a sixth phase of the first preferred embodiment of the method according to the invention;

[0051] FIG. 7: shows a first phase of a second preferred embodiment of the method according to the invention for reproduction of biological tissue;

[0052] FIG. 8: shows a second phase of the second preferred embodiment of the method according to the invention;

[0053] FIG. 9: shows a third phase of the second preferred embodiment of the method according to the invention;

[0054] FIG. 10: shows a fourth phase of the second preferred embodiment of the method according to the invention;

[0055] FIG. 11: shows a fifth phase of the second preferred embodiment of the method according to the invention; and

[0056] FIG. 12: shows a sixth phase of the second preferred embodiment of the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0057] FIG. 1 shows a first phase of a first preferred embodiment of a method for the reproduction of biological tissue according to the invention. A structured substrate 01 in the form of a film with cavities 02 and elevations 03 is provided and arranged in a medium 04 in a machining tray 06. Using a pipette 07, primary cells 08 of a first cell type are deposited on the substrate 01. In this manner, some of the primary cells 08 of the first cell type do not remain on the substrate 01 but rather reach a bottom of the machining tray 06. The primary cells 08 of the first cell type are distributed in a locally untargeted manner over the substrate 01 using the pipette 07 such that the primary cells 08 are distributed in a largely uniform manner. When the primary cells 08 of the first cell type are deposited with the pipette 07, the primary cells 08 are not subjected to high compressive or shear stress. A dispensing system 09, which is movable in x-y axes and is not yet used in this first phase, is arranged above the machining tray 06. The dispensing system 09 can preferably also be configured in the method in the three x-y-z axes or can also be formed by a pipetting system.

[0058] The medium 04 comprises a first precursor for a later to be formed substance 11 to adhere the primary cells 08 (shown in FIG. 2). The medium 04 moreover comprises a nutrient medium for the primary cells 08 of the first cell type.

[0059] FIG. 2 shows a second phase of the first preferred embodiment of the method for the reproduction of biological tissue according to the invention. In this second phase, the dispensing system 09, which can be moved in the x-y axes, is used to selectively arrange a second precursor 12 of the substance 11 that adheres the cells 08 on a selection of the primary cells 08 of the first cell type. The second precursor 12 is arranged in a locally targeted manner according to a structure actually formed by the cells 08 of the first cell type in the tissue to be reproduced (not shown). The first precursor that is present in the medium 04 and the second precursor 12 immediately form the substance 11 in the form of a hydrogel adhering the cells 08. The adhered selection of primary cells 08 of the first cell type constitutes a reproduction of a first section of the biological tissue to be reproduced (not shown).

[0060] FIG. 3 shows a third phase of the first preferred embodiment of the method for the reproduction of biological tissue according to the invention. In this third phase, the primary cells 08 of the first cell type that are not adhered by the substance 11 are removed from the substrate 01 and from the medium 04 using the pipette 07.

[0061] FIG. 4 shows a fourth phase of the first preferred embodiment of the method for the reproduction of biological tissue according to the invention. The pipette 07 is now used to deposit primary cells 13 of a second cell type on the substrate 01. In this manner, some of the primary cells 13 of the second cell type do not remain on the substrate 01 but rather reach the bottom of the machining tray 06. The primary cells 13 of the second cell type are distributed in a locally untargeted manner over the substrate 01 with the pipette 07, such that the primary cells 13 are distributed in a largely uniform manner. When the primary cells 13 of the second cell type are deposited with the pipette 07, the primary cells 13 are not subjected to high compressive or shear stress.

[0062] FIG. 5 shows a fifth phase of the first preferred embodiment of the method for the reproduction of biological tissue according to the invention. In this fifth phase, the dispensing system 09, which can be moved in the x-y axes, is used to selectively arrange in a locally targeted manner further amounts of the second precursor 12 of the substance 11 adhering the cells 08, 13 on a selection of the primary cells 13 of the second cell type. The second precursor 12 is arranged in a locally targeted manner according to a structure actually formed by the cells 13 of the second cell type in the tissue to be reproduced (not shown). The first precursor that is present in the medium 04 and the second precursor 12, in turn, immediately form the substance 11, in the form of a hydrogel, adhering the cells 08, 13. The adhered selection of primary cells 13 of the second cell type constitutes a reproduction of a second section of the biological tissue to be reproduced (not shown).

[0063] FIG. 6 shows a sixth phase of the first preferred embodiment of the method for the reproduction of biological tissue according to the invention. In this sixth phase, the primary cells 13 of the second cell type that are not adhered by the substance 11 are removed from the substrate 01 and from the medium 04 using the pipette 07.

[0064] FIG. 7 shows a first phase of a second preferred embodiment of the method for the reproduction of biological tissue according to the invention. This second embodiment is initially similar to the first embodiment illustrated in FIGS. 1 to 6. In contrast to the first embodiment, in the second embodiment the substrate 01 is porous. The sequence of the first phase of the second embodiment is similar to the sequence of the first phase of the first embodiment shown in FIG. 1.

[0065] FIG. 8 shows a second phase of the second preferred embodiment of the method for the reproduction of biological tissue according to the invention. The sequence of the second phase of the second embodiment is similar to the sequence of the second phase of the first embodiment shown in FIG. 2.

[0066] FIG. 9 shows a third phase of the second preferred embodiment of the method for reproduction of biological tissue according to the invention. The sequence of the third phase of the second embodiment is similar to the sequence of the third phase of the first embodiment shown in FIG. 3.

[0067] FIG. 10 shows a fourth phase of the second preferred embodiment of the method for the reproduction of biological tissue according to the invention. The sequence of the fourth phase of the second embodiment is similar to the sequence of the fourth phase of the first embodiment shown in FIG. 4.

[0068] FIG. 11 shows a fifth phase of the second preferred embodiment of the method for the reproduction of biological tissue according to the invention. The sequence of the fifth phase of the second embodiment is similar to the sequence of the fifth phase of the first embodiment shown in FIG. 5.

[0069] FIG. 12 shows a sixth phase of the second preferred embodiment of the method for the reproduction of biological tissue according to the invention. The sequence of the sixth phase of the second embodiment is similar to the sequence of the sixth phase of the first embodiment shown in FIG. 6.

REFERENCE LIST

[0070] 01 Substrate [0071] 02 Cavity [0072] 03 Elevation [0073] 04 Medium [0074] 05 - [0075] 06 Machining tray [0076] 07 Pipette [0077] 08 Primary cells of a first cell type [0078] 09 Movable dispensing system [0079] 10 - [0080] 11 Substance adhering the primary cells [0081] 12 Second precursor [0082] 13 Primary cells of a second cell type