WO2020190242A1

WO2020190242A1 - 3d bone tumor and metastasis model with scaffold structure

Info

Publication number: WO2020190242A1
Application number: PCT/TR2020/050218
Authority: WO
Inventors: Safiye AKTAŞ; Ömer BEKÇİOĞLU; Melek AYDIN; Buğra HÜSEMOĞLU
Original assignee: Dokuz Eylül Üni̇versi̇tesi̇ Rektörlüğü
Priority date: 2019-03-18
Filing date: 2020-03-18
Publication date: 2020-09-24
Also published as: TR201903959A2

Abstract

The invention is a 3D scaffold model (10) which is obtained by using biomaterials in the health sector, allows easy monitoring of in vivo bone tumor and bone metastasis by means of being implanted subdermally to the experimental animals, characterized in that; it has a hexagonal morphology (12) consisting of many hexagonal frames for carrying out the micro environment studies in relation with the metastatic bone tumor by means of mimicking the porous structure of a bone, comprises a tissue scaffold (11) where the cancer cells are cultured for observing the tumor formation.

Description

l

3D BONE TUMOR AND METASTASIS MODEL WITH SCAFFOLD STRUCTURE

Technological Field

The invention relates to a 3D scaffold model which is obtained by using biomaterials in the health sector, allows easy monitoring of in vivo bone tumor and bone metastasis by means of being implanted to the experimental animals subdermally.

State of the Art:

Oncology studies with 3D tissue scaffolds are found in the literature. The methods which are used in the in vitro cell culture studies generally consist of similar biomaterials. Generally hydrogel and scaffolds are used in the studies where 3D cell culture is performed. Filament selection in scaffold production takes an important place. Many different scaffold types using various biomaterials are developed; hydrogels, specific scaffolds, fibrous scaffolds, porous scaffolds, microspheres and natural tissue scaffolds. Particularly standardization of the pore structure in these scaffolds is very important. Ideal pore structure, pore size in used scaffolds is important for the morphological structure of the tissue scaffold. Cell involvement, growth and formation of microenvironment depend on the morphology of the tissue scaffold. Natural biomaterials used in 3D cell culture can be listed as silk, collagen, gelatin, chitosan, fibrinogen, hyaluronic acid and alginate. The synthetic biomaterials used in 3D cell culture can be listed as PEG, PGA, PMMA,

PLGA and PCL.

A model in which the trabecular morphological structure of the bone is considered and studied and different cancer cell lines are studied, are not found in prior art in

the literature. The models which will be compared with the developed model are only 3D cell culture models.

In the state of the art, 3D metastatik cell culture modeling is not taken into consideration sufficiently. Produced tissue scaffolds have not been standardized, a system which mimics the porous structure of the bone has not been used.

Cancer cells were not used in the tissue scaffolds as a xenograft model in the previous techniques. The model we developed is a new model, it is observed that the tissue scaffold was not used in a structure which can form xenograft tumor.

In the patent document No US2014051168 (A1) encountered in the literature research made, a three-dimensional cell culture scaffold composition including an absorbent rigid (AR) component is described. In said document, a scaffold in which a three-dimensional cell culture is formed is described. It can comprise different types of signal models in its structure. A statement which mentions study of different cancer, tumor (metastasis) cell lines and orientation to the hexagonal morphology has not been encountered.

The invention described in the patent document No US2015024967 (A1) encountered during another literature research made is a three dimensional scaffold combination comprising randomly oriented fibers, wherein the fibers comprise polyethylene glycol-polylactic acid block copolymer (PEG-PLA) and a poly (lactic-co-glycolic acid) (PLGA). In said document, a scaffold in which a three-dimensional cell culture is formed is disclosed. A statement which mentions study of different cancer, tumor (metastasis) cell lines and orientation to the hexagonal morphology has not been encountered.

The invention No W00121760 (A2) encountered in the literature research made, relates to culture devices which are compact, use a small amount of cell culture medium to form and sustain cell cultures and produce a large number of cells in a short time compared to other cell culture devices and methods. Any statement which mentions study of different cancer cell lines and orientation to the hexagonal bone structure has not been encountered.

Consequently, a new 3D scaffold model is required to be developed in which the state of the art is exceeded, the disadvantages are eliminated. Brief Description of the Invention

The invention is a new 3D scaffold models, in which the state of the art is exceeded, the disadvantages are eliminated, and additional advantages are brought.

The aim of the invention is to provide a new 3D scaffold model which intends to mimic the porous structure of the bone, has hexagonal morphology, forms a new xenograft tumor with the tissue scaffold.

Another aim of the invention is to provide a new 3D scaffold model in which the studies under in vivo conditions are configured such that they are pioneer and innovative to the metastatic tumor micro environment.

Another aim of the invention is to provide a new 3D scaffold model which enables tumor formation in experimental animals by means of culturing different types of cancer cells and placing them into the bodies of experimental animals, in order to make contribution to well- known literature studies.

Another aim of the invention is to provide a new 3D scaffold model which can be adapted to the cell culture materials containing bone tumor or bone metastasis model, is in the form of bone trabecular structure, is easy to manufacture and has low manufacturing cost.

In order to fulfill all abovementioned and below described aims in the description, the invention is a 3D scaffold model which is obtained by using biomaterials in the health sector, allows easy monitoring of in vivo bone tumor and bone metastasis by means of being implanted subdermally to the experimental animals, characterized in that; it has a hexagonal morphology consisting of many hexagonal frames for carrying out the micro environment studies in relation with the metastatic bone tumor by means of mimicking the porous structure of a bone, comprises a tissue scaffold where the cancer cells are cultured for observing the tumor formation.

Another preferred embodiment of the invention is characterized in that; it is configured with three-dimensional printers such that it is manufactured from PLA (polyactic acid) material.

Another preferred embodiment of the invention is characterized in that; the cell culture used in the tissue scaffold comprises; breast cancer cell lines (MCF-7 (HTB-22) incubated in 10 Fetal Bovine Serum (FBS), 1 % L-Glutamine, 1 % penicillin streptomycin, Dulbecco’s Modified Eagles Medium (DMEM), and 4T1 cell line incubated in 5 % carbon dioxide, at 370°C in RPMI -1640 medium comprising MDAMB-231 (HTB-26) and 10 % FBS.

Another preferred embodiment of the invention is characterized in that; polylactic acid (PLA) is used as a printing material in the three dimensional printer at a printing temperature of 250 ° C.

Another preferred embodiment of the invention is characterized in that; when the cell culture consisting of breast cancer cell lines (MCF-7 (HTB-22), MDA- MB-231 (HTB-26) and 4T1 cell line reaches to 80 % density, reproduction of thereof by means of passaging with 1/2 ratio after it is removed from the culture dishes by trypsin/EDTA solution, keeping 3D scaffold model (10) in a 5 % carbon dioxide incubator during 1 hour at 370°C within 2 ml medium (RPMI 1640-DMEM) after it is placed in a 12-well plate element in a sterile manner, culturing the cell culture such that it is within the pore on the tissue scaffold made of PLA material after it is removed from the culture dishes by trypsin/EDTA solution.

Description of the Figures:

The invention will be described with reference to the accompanying drawings, thus the characteristics of the invention will be understood clearly. However, the aim of this is not to limit the invention with such determined embodiments. On the contrary, it is aimed to cover all alternatives, amendments and equivalents which may be contained in the field defined by the accompanying claims. It is to be understood that the details shown are only shown for the sake of illustrating the preferred embodiments of the present invention and presented for both illustrating the methods and for providing description of the rules of the invention and the conceptual features of the invention to be easily understood. In these figures;

Figure 1 is an illustrative view of the inventive 3D scaffold.

The figures which enable to clarify this invention are enumerated as mentioned in the attached figure and they are given with their names herein below. Description of the References:

10.3D scaffold model

11. Tissue scaffold

12. Hexagonal morphology

Description of the Invention:

In this detailed description, the inventive 3D scaffold model (10) is described by means of examples only for clarifying the subject manner such that no limiting effect is created. In the specification, a 3D scaffold model which is obtained by using biomaterials in the health sector, allows easy monitoring of in vivo bone tumor and bone metastasis by means of being implanted subdermally to the experimental animals is disclosed.

Figure - 1 is an illustrative view of the inventive 3D scaffold (10). Accordingly, 3D scaffold model (10) consists of a tissue scaffold (11) has a hexagonal morphology (12) consisting of a plurality of hexagonal frame. It is in the form of a honeycomb with this structure. It is designed in this form in order to mimic the porous structure of the bone. The hexagonal structure within the porous structure in the bone; is from a morphology which is specific and known as a tight packing method. Cancer cells are used in the tissue scaffold (11). Breast cell cancer lines are cultured to a 3D scaffold model (10) which is produced from PLA (polylactic acid) and mimics the bone trabecular structure. Thus, 3D scaffold model (10) is implanted to the experimental animals, and tumor formation can be observed. It is possible to develop known literature in relation with the tumor and cancer disorders by means of observing tumor formation.

Production phase of 3D scaffold model (10) is realized as follows. A 3D scaffold model (10) production was realized according to the fused deposition modeling (FDM) in a single extruder 3D printer using a hot-tipped extruder in order to print the PLA filaments. This printer has 100-100-

200pm mechanical precision in X-Y-Z axis. Particularly, 3D scaffold model (10) was designed in CAD programs, then the distance between the columns and the critical parameters between the width of the columns and height was optimized, optimization of print parameters was made. In order to obtain a homogenous porosity in Z direction, the clearance speed in the sheer (Slic3r) program was optimized. 15 mm/s is determined as filling speed, 25 mm/s is determined for the clearance speed as the most appropriate. The motion speed of the extruder in the printer is set as 100 mm/s. The settings were a layer height of 0,3mm, a hexagonal fill pattern, a solid upper and lower value 0 and an environmental value 0. The print bearing was heated to 40 ° C for the first layer of the print in order to facilitate bonding. Printing was performed by a nozzle of 0,3 mm. The porosity of the scaffolds are adjusted to 70 %. Polylactic acid (PLA) was used a printing material at a printing temperature of 250 °C. After printing, 3D scaffold model (10) was removed carefully from the print bearing. After this process, 3D scaffold models (10) were obtained in the hexagonal morphology (12) in the internal structure of the bone.

Here the aim is to produce 3D scaffold models (10) in the same morphology to model the metastatic bone tumors.

The cell used in the tissue scaffold (11) of 3D scaffold model (10) was cultured by using 3 types of breast cancer cell line. In 3D scaffold model (10) MCF-7 (HTB-22), MDA- MB-231 (HTB-26), 4T1 cell lines are used. Breast cancer cell lines (MCF-7 (HTB-22), MDA- MB-231

(HTB-26) were incubated in 10 Fetal Bovine Serum (FBS), 1 % L-Glutamine, 1 % penicillin streptomycin, Dulbecco’s Modified Eagles Medium (DMEM), and 4T1 cell line was incubated in 5 % carbon dioxide, at 370°C in RPMI -1640 medium comprising 10 % FBS. When the cultures reach 80 % density, they were passaged with ratio of 1 :2 by means of removing thereof from the culture dishes with trypsin/EDTA solution. Cells are reproduced by means of passaging until sufficient number of cells are obtained for the experimental groups. 3D scaffold model (10) is placed in a 12-well plate element in a sterile manner. It is kept in a 5 % carbon dioxide incubator during 1 hour at 370°C within 2 ml medium (RPMI 1640-DMEM). The cells are cultured such that it is within the pore on the tissue scaffold (11) made of PLA material after it is removed from the culture dishes by trypsin/EDTA solution.

Claims

1 - 3D scaffold model (10) which is obtained by using biomaterials in the health sector, allows easy monitoring of in vivo bone tumor and bone metastasis by means of being implanted subdermally to the experimental animals, characterized in that; -it has a hexagonal morphology (12) consisting of many hexagonal frames for carrying out the micro environment studies in relation with the metastatic bone tumor by means of mimicking the porous structure of a bone,

-comprises a tissue scaffold (11) where the cancer cells are cultured for observing the tumor formation after it is implanted to the experimental animals.

2- 3D scaffold model (10) according to claim 1 , characterized in that; it is configured with three-dimensional printers such that it is manufactured from PLA (polylactic acid) material.

3- A 3D scaffold model (10) according to claim 1 , characterized in that; the cell culture used in the tissue scaffold (11) comprises; breast cancer cell lines (MCF-7 (HTB-22) incubated in 10 Fetal Bovine Serum (FBS), 1 % L-Glutamine, 1 % penicillin streptomycin, Dulbecco’s Modified Eagles Medium (DMEM), and 4T1 cell line incubated in 5 % carbon dioxide, at 370°C in RPMI -1640 medium comprising MDAMB-231 (HTB-26) and 10 % FBS.

4- A 3D scaffold model (10) according to claiml , characterized in that; polylactic acid (PLA) is used as a printing material in the three dimensional printer at a printing temperature of 250 ° C in its production.

5- A method for culturing cancer cell to a 3D scaffold model (10) according to claim 1 , characterized in that;

-when the cell culture consisting of breast cancer cell lines (MCF-7 (HTB-22), MDA- MB-231 (HTB-26) and 4T1 cell line reaches to 80 % density, reproduction of thereof by means of passaging with a ratio of 1/2 after it is removed from the culture dishes by trypsin/EDTA solution,

-keeping 3D scaffold model (10) in a 5 % carbon dioxide incubator during 1 hour at 370°C within 2 ml medium (RPMI 1640-DMEM) after it is placed in a 12-well plate element in a sterile manner, -culturing the cell culture such that it is placed within the pore and on the tissue scaffold (11) made of PLA material after it is removed from the culture dishes by trypsin/EDTA solution.