Mit biologischen Zellen besiedeltes 3D-Gerüst aus biokompatiblem Polymer und dessen Herstellung

Abstract

A 3D scaffold of a biocompatible polymer and colonized with biological cells is provided., The biological cells can be cultured to form a 3D cell culture construct that closely approximates a physiological architecture. A method for producing the 3D scaffold colonized with biological cells is also provided.

Claims

1-12. (canceled)

13. A method including: (a) constructing a 3D scaffold comprising a biocompatible polymer using a lithographic 3D printing method, the 3D scaffold having an at least partially covered cavity; and (b) filling the at least partially covered cavity with a suspension containing biological cells to colonize the 3D scaffold.

14. The method of claim 13 wherein the 3D scaffold is constructed using a stereolithographic 3D printing method.

15. The method claim 13 wherein constructing the 3D scaffold includes curing a first photopolymerizable or photocrosslinkable substance by focusing electromagnetic radiation in a first focal plane in which the first photopolymerizable or photocrosslinkable substance is present.

16. The method of claim 15 wherein constructing the 3D scaffold includes curing a second photopolymerizable or photocrosslinkable substance by focusing electromagnetic radiation in a second focal plane in which the second photopolymerizable or photocrosslinkable substance is present, the second focal plane being different from the first focal plane.

17. The method of claim 16 wherein the second photopolymerizable or photocrosslinkable substance is different from the first photopolymerizable or photocrosslinkable substance.

18. The method of claim 13 wherein filling the at least partially covered cavity with the suspension containing biological cells includes directing the suspension containing biological cells through a filling opening on the 3D scaffold.

19. The method of claim 13 further including drying the 3D scaffold prior to filling the at least partially covered cavity with the suspension containing biological cells.

20. The method of claim 13 further including, after filling the at least partially covered cavity with the suspension containing biological cells, culturing the biological cells to form a 3D cell culture construct.

21. The method of claim 20 wherein the 3D cell culture construct is penetrated by further cells, viruses, bacteria, enzymes or active substances.

22. A 3D scaffold comprising: (a) multiple layers of a biocompatible polymer, the multiple layers of the biocompatible polymer formed by a lithographic 3D printing method and defining an at least partially covered cavity; and (b) a suspension containing biological cells filling the at least partially covered cavity to colonize the 3D scaffold.

23. The 3D scaffold of claim 22 further including one or more outlet openings.

24. The 3D scaffold of claim 22 further including columns, grids, or crosspieces in the at least partially covered cavity.

25. The 3D scaffold of claim 22 wherein at least a portion of a first one of the multiple layers of the biocompatible polymer comprises a first photopolymerizable or photocrosslinkable substance cured by focusing electromagnetic radiation in a first focal plane in which the first photopolymerizable or photocrosslinkable substance is present.

26. The 3D scaffold of claim 25 wherein at least a portion of the first one of the multiple layers of the biocompatible polymer or a second one of the multiple layers of the biocompatible polymer comprises a second photopolymerizable or photocrosslinkable substance cured by focusing electromagnetic radiation in a second focal plane in which the second photopolymerizable or photocrosslinkable substance is present, the second focal plane being different from the first focal plane.

27. The 3D scaffold of claim 26 wherein the second photopolymerizable or photocrosslinkable substance is different from the first photopolymerizable or photocrosslinkable substance.

28. The 3D scaffold of claim 22 wherein the biological cells are cultured to form a 3D cell culture construct.

29. The 3D scaffold of claim 28 wherein the 3D cell culture construct is penetrated by further cells, viruses, bacteria, enzymes or active substances.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIG. 1 shows a view of an uncolonized 3D scaffold of a CAD file, having a centrally covered hollow body, a filling opening on the top side and a plurality of lateral outlet openings.

[0100] FIG. 2 shows a photograph of a top view of a 3D scaffold produced by lithographic 3D printing according to the CAD file of FIG. 1.

[0101] FIG. 3 shows a photograph of a top view of a 3D scaffold according to FIG. 2 colonized with tumor cells from a neuroblastoma.

[0102] FIG. 4 shows a view of an uncolonized 3D scaffold of a CAD file, having a central recess which is open at the top and also covered cavities in the form of vascular vessels, having two filling openings on the top side for the vascular vessels and two lateral outlet openings of the vascular vessels.

[0103] FIG. 5 shows a micrograph of a printed model according to the CAD file according to FIG. 4, from below.

DESCRIPTION OF REPRESENTATIVE EMBODIMENTS

[0104] FIG. 1 shows a model of a 3D scaffold 1, created in a CAD file, which can be used according to the invention. The 3D scaffold 1 preferably has a central cavity 2 which is predominantly covered. The cavity 2 is preferably accessible via a filling opening 3 designed as a filler neck. The area above the cavity 2 is preferably supported by columns 5 within the cavity 2. The 3D scaffold 1 preferably has a plurality of lateral outlet openings 4 in the form of outlet channels.

[0105] FIG. 2 shows a photograph of a 3D scaffold 1 which has been produced using a lithographic 3D printing method based on the CAD file according to FIG. 1. The top filler neck 3, the lateral channels 4 and the columns 5 in the cavity 2 can be clearly identified. The 3D scaffold shown in FIG. 2 is produced by the following method steps: [0106] 1.) creating the CAD file and calculating the master; [0107] 2.) equipping the printer with the photopolymerizable or photocrosslinkable liquids to be used; [0108] 3.) calibrating the printer, the axes and the printhead; [0109] 4.) carrying out the printing; the print platform lowers to the first printing plane for the first photopolymerizable or photocrosslinkable liquid; [0110] 5.) printing a first polymer from the first photopolymerizable or photocrosslinkable liquid for architectures consisting of polymer 1 for the first layer height of the construct to be printed; in the process, the calculated construction plan of the first polymer for the first layer height of the construct is projected onto the printing plane in which the printhead is located; here, one or more constructs can be produced simultaneously, depending on the user’s wishes and plans; the limiting factor here is the size of the print platform or the installation space; [0111] 6.) if necessary, step of washing the printer in order to prevent the first polymer spreading into a second photopolymerizable or photocrosslinkable liquid and vice versa - optional (if a second polymer is required); [0112] 7.) if necessary, printing the second polymer for architectures consisting of the second polymer for the first layer - optional (if a second polymer is used); [0113] 8.) repeating steps six and seven if a third polymer is used; [0114] 9.) changing the printing plane in order to be able to print the second layer height; [0115] 10.) printing the first polymer for architectures consisting of the first polymer for the second layer height of the construct to be printed; [0116] 11.) if necessary, step of washing the printer in order to prevent the first polymer spreading into the second photopolymerizable or photocrosslinkable liquid and vice versa - optional (if a second polymer is used); [0117] 12.) if necessary, printing the second polymer from a second photopolymerizable or photocrosslinkable liquid for architectures consisting of the second polymer for the second layer - optional (if a second polymer is used); [0118] 13.) repeating steps 11 and 12 if a third polymer is required; [0119] 14.) changing the printing plane in order to be able to print the third layer height; [0120] 15.) repeating steps five to eight until the complete architecture has been printed; [0121] 16.) after completion of the printing, the print platform is moved into the starting plane and the 3D scaffold obtained is removed; [0122] 17.) subsequently, the 3D scaffold can be dried or used immediately; [0123] 18.) if the 3D scaffold is dried, this takes place in a sterile atmosphere; here, all water is removed from the 3D scaffold, with the result that a dry polymer scaffold is formed; [0124] 19.) after drying, the 3D scaffold can be stored under sterile conditions.

[0125] In this example, the entire 3D scaffold is printed from the first photocrosslinkable liquid. Adding the photoblocker tartrazine to the photocrosslinkable liquid regulates the penetration depth of the light used for the polymerization, enabling the production of the covered cavity. Alternatively, for producing the covered cavity, use could be made of sacrificial inks (used in a further photocrosslinkable liquid, e.g. 15 g/kg hyaluronic acid dissolved in RPMI + 5 g/kg lithium phenyl-2,4,6-trimethylbenzoylphosphinate, printed and subsequently digested with hyaluronidase in order to produce the cavity), which are dissolved hydrolytically or by enzymatic digestion after completion of the printing.

[0126] FIG. 3 shows a photograph of a 3D scaffold 1 according to FIG. 2, which has been colonized with tumor cells from a neuroblastoma. To this end, the following further method steps are carried out: [0127] 20.) a cell suspension having a concentration set beforehand by the user is pipetted via the filler neck into a 3D scaffold produced according to 1.) to 19.); in the process, a cell suspension volume is used which corresponds at most to the volume of the cavity of the 3D scaffold; if the 3D scaffold is dried in step 18.), it must first be rehydrated by the user for reuse; a sterile medium such as water, PBS, cell culture medium or the like is suitable for this purpose; [0128] 21.) through the pipetting the suspension is distributed within the 3D scaffold and the cells can be cultured within the 3D scaffold;

[0129] To colonize the cavity with neuroblastoma cells (SK-N-BE(2)), a cell suspension is produced (solvent: DMEM high glucose + 10% FCS + 1% penicillin/streptomycin; cell concentration: 150 × 10.sup.6 cells/l). 6 .Math.l of this suspension are pipetted into the cavity of the 3D scaffold via the filler neck. The construct is subsequently cultured at 37° C. and 5% CO.sub.2.

[0130] FIG. 4 shows a model of a 3D scaffold 1, created in a CAD file, which can be used according to the invention. The 3D scaffold 1 has a central recess which is open at the top as a colonization region 6 for biological cells. Furthermore, it has cavities 2 which are closed at the top, i.e. covered, in the form of vascular vessels which preferably extend in the horizontal plane around the central colonization region 6. The 3D scaffold 1 shown in FIG. 4 further includes a plurality of lateral outlet openings 4 in the form of outlet channels.

[0131] FIG. 5 shows an actually printed 3D scaffold 1 under the microscope, from the underside thereof. Filler neck 3, cavities 2 in the form of vascular vessels and colonization region 6 can be clearly identified.

[0132] As used herein, whether in the above description or the following claims, the terms “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, that is, to mean including but not limited to.

[0133] The above-described preferred embodiments are intended to illustrate the principles of the invention, but not to limit the scope of the invention. Various other embodiments and modifications to these preferred embodiments may be made by those skilled in the art without departing from the scope of the present invention. For example, in some instances, one or more features disclosed in connection with one embodiment can be used alone or in combination with one or more features of one or more other embodiments. More generally, the various features described herein may be used in any working combination.

List of Reference Numbers

[0134] 1 3D scaffold [0135] 2 cavity [0136] 3 filling opening [0137] 4 outlet opening [0138] 5 columns [0139] 6 colonization region