Autologous, prevascularized breast tissue constructs produced in a 3D printing method, and methods for producing same

Abstract

Autologous prevascularized breast tissue constructs created via 3D printing and methods for 3D printing autologous prevascularized breast tissue constructs. The method comprises steps of: (i) providing a triculture consisting of adipose mesenchymal stem cells, fibroblasts, and endothelial progenitor cells, (ii) mixing the triculture cells with a bioink composed of biopolymers, (iii) printing three-dimensional structures of the breast tissue construct using the triculture-added bioink from step (ii), where the cells of the triculture are pretreated with growth media before printing so that the endothelial progenitor cells differentiate into endothelial cells and the adipose mesenchymal stem cells differentiate into adipocytes. After 3D printing, the development of vascular-like structures is induced.

Claims

1. A method for 3D printing autologous prevascularized breast tissue constructs comprising the steps of: (i) Providing a triculture consisting of adipose mesenchymal stem cells, fibroblasts, and endothelial progenitor cells, (ii) Mixing the triculture cells with a bioink composed of biopolymers, (iii) Printing three-dimensional structures of the breast tissue construct using the triculture-added bioink from step (ii), wherein the cells of the triculture are pretreated with growth media prior to printing so that the endothelial progenitor cells differentiate into endothelial cells and the adipose mesenchymal stem cells differentiate into adipocytes, and wherein the development of vessel-like structures is induced after 3D printing.

2. The method according to claim 1, characterized in that the development of vessel-like structures after 3D printing is induced with collagen I.

3. The method according to claim 1, characterized in that the 3D printing is performed in a 1-channel system and/or a 2-channel system.

4. The method according to claim 1, characterized in that the vascular structures of the breast tissue construct are printed with vessel-forming cells.

5. The method according to claim 15, characterized in that the endothelial cells are differentiated endothelial cells or microvascular endothelial cells.

6. The method according to claim 1, characterized in that the bioink comprises cellulose, alginate, mannitol, gelatin methacrylate and/or collagen I.

7. The method according to claim 1, characterized in that the primary mesenchymal stem cells, fibroblasts and/or endothelial progenitor cells are derived from autologous cells of a patient.

8. The method according to claim 7, characterized in that the endothelial progenitor cells are late endothelial progenitor cells from the patient's blood.

9. The method according to claim 7, characterized in that the endothelial progenitor cells are obtainable by culturing the cells taken from the blood for several days and converting them to a gelatin-coated culture surface with a selection medium.

10. The method according to claim 1, characterized in that the bioink is contained in a composition comprising self-extracted extracellular matrix from adipose tissue (adECM).

11. The method of claim 7, characterized in that the adipose mesenchymal stem cells are selected from a present adipose tissue sample of the patient by anti-CD34 coupled magnetic beads.

12. The method according to claim 7, characterized in that the fibroblasts are selected from a present oral mucosa sample of the patient.

13. The method according to claim 1, characterized in that the endothelial cells are cultured as spheroids or on microcarriers prior to printing.

14. An autologous prevascularized breast tissue construct generated via a 3D printing process, comprising a three-dimensional structure of several different cell types, consisting of endothelial cells differentiated from endothelial progenitor cells, adipocytes differentiated from adipose-derived mesenchymal stem cells and fibroblasts, wherein the breast tissue construct is obtainable by a method according to claim 1.

15. The method according to claim 4, characterized in that the vessel-forming cells are endothelial cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Further features and advantages of the present invention will become apparent from the following description of preferred embodiments with reference to the accompanying figures, in which:

[0027] FIG. 1 shows the formation of vessel-like structures after 7 days of culture in a collagen I-containing bioink,

[0028] FIG. 2 shows the viability of an endothelial cell-fibroblast co-culture in a collagen I-containing bioink compared with a collagen I-free bioink,

[0029] FIG. 3 shows the cultivation of endothelial cells on microcarriers to increase cell viability and differentiation.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0030] The following embodiments serve to illustrate the invention. By no means is the invention limited to these embodiments. The invention also encompasses combinations of individual embodiments or any combinations of features of individual embodiments.

[0031] In the following embodiments, two different approaches are taken for the manufacturing of the prevascularized tissue constructs, which are based on either 1-channel, or 2-channel printing.

[0032] For the manufacturing of a finely branched network of capillary-like structures within smaller tissue constructs, for the first approach (1-channel) the detached cells are transferred together in equal parts into cell medium so that a triculture with a total cell concentration of 10.sup.6 cells/ml is achieved. The cell suspension is then mixed with the collagen-based bioink at a ratio of 1:10 using a Luer-lock syringe and Luer-lock adapter and transferred to a print cartridge. After inserting the cartridge: the 3D constructs are printed into a sterile well plate in a channel using a pressure of 9-15 kPa and a 25 G tip.

[0033] For the second approach, larger vessels in the form of a channel or tube system are printed into the tissue construct (1-channel and/or 2-channel). For this purpose, the cells are first separated after detachment from the culture vessels. In this process, mesenchymal stem cells or adipocytes and fibroblasts (biculture) are transferred together into a cell suspension and endothelial cells (monoculture) are transferred into another suspension, each with a total cell concentration of 10.sup.6 cells/ml. Subsequently, the different cell suspensions (monoculture and biculture) are mixed with the collagen-based bioink at a ratio of 1:10 as described above and divided into two print cartridges. For the procedure described here, 25 G pressure tips are also used. For 1-channel printing, a basic scaffold from the biculture is first printed to produce a connective tissue structure that has tubular recesses or porous structures, Subsequently, the endothelial cell-bioinks mixture is then used to print the vascular structures. In the 2-channel system, the basic scaffold is printed from the biculture and, simultaneously, using the second channel, vascular structures are printed with the endothelial cell monoculture. The results are summarized in the figures below.

[0034] FIG. 1 depicts the morphology of endothelial cells on a collagen I-based bioink visualized by CD31 staining (here black). Vessel-like structures (white arrows) are shown, which form after 7-days co-culture of endothelial cells with fibroblasts in a collagen I-containing bioink.

[0035] FIG. 2 shows the viability of an endothelial cell fibroblast co-culture in a collagen I-containing compared to a collagen I-free bioink, detected in an MTT assay. The collagen I content in the bioink is crucial for high viability.

[0036] FIG. 3 shows endothelial cells cultured on gelatin-coated microcarriers, recognizable as bright staining (CD31). Adherence to microcarriers allows endothelial cells to retain high cell and differentiation.