METHOD OF MANUFACTURING CELL SPHEROID USING THREE-DIMENSIONAL PRINTING METHOD

• US 20200131471A1
• Filed: 10/23/2019
• Published: 04/30/2020
• Est. Priority Date: 10/24/2018
• Status: Active Application

• Current Assignee: Postech Academy-Industry Foundation
• Original Assignee: Postech Academy-Industry Foundation
• Inventors: JANG, Jinah, PARK, Yejin, YONG, UiJung, DAS, Sanskrita, HWANG, Dong Gyu
• Application Number:
  US16/660,924
• Publication Number:
  US 20200131471A1
• Time in Patent Office: Days
• CPC Class Codes: B29C 64/106 using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
• CPC Class Codes: B29C 64/386 Data acquisition or data processing for additive manufacturing
• CPC Class Codes: B29K 2005/00 Use of polysaccharides or derivatives as moulding material
• CPC Class Codes: B29L 2031/40 Test specimens ; Models, e.g. model cars mannequins B29L2031/7028; Probes
• CPC Class Codes: B33Y 10/00 Processes of additive manufacturing
• CPC Class Codes: B33Y 50/00 Data acquisition or data processing for additive manufacturing
• CPC Class Codes: B33Y 70/00 Materials specially adapted for additive manufacturing
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture

Abstract

The present invention relates to a method of manufacturing a cell spheroid using three-dimensional bio-printing technology, and the cell spheroid may be used for preventing or treating vascular and endocrine diseases by including mesenchymal stem cells, induced pluripotent stem cells-derived cells, or the like as an active ingredient, or may be used as an in vitro drug testing model.

Claims

1. A method of manufacturing a cell spheroid using a three-dimensional printing method, comprising

preparing a bioink comprising cells, decellularized extracellular matrix and alginate;

and

manufacturing a cell spheroid by printing the bioink using the three-dimensional printing in a micro-extrusion manner.

2. The method of manufacturing according to claim 1, wherein manufacturing the spheroid comprises

inserting an nozzle to a mixed solution comprising a gelling agent of the alginate and hydrogel;

extruding the bioink by applying a pneumatic pressure; and

pulling out the injection nozzle from the mixed solution.

3. The method of manufacturing according to claim 2, further comprising applying high viscosity to the hydrogel by heating the mixed solution.

4. The method of manufacturing according to claim 2, wherein the mixed solution has enough high viscosity to form the cell spheroid by separating the extruded bioink from the injection nozzle with pulling out the injection nozzle.

5. The method of manufacturing according to claim 1, wherein the bioink is printed by being extruded at a pneumatic pressure of 30 kPa to 40 kPa.

6. The method of manufacturing according to claim 1, wherein the bioink comprises a decellularized extracellular matrix solution at a concentration of 0.5 to 5% by weight, and an alginate solution at a concentration of 0.5 to 5% by weight.

7. The method of manufacturing according to claim 1, wherein the alginate concentration of the bioink is equal to or more than the concentration of the decellularized extracellular matrix of the bioink.

8. The method of manufacturing according to claim 1, wherein the bioink comprises the decellularized extracellular matrix and the alginate at a concentration ratio of more than 1:

0.5 to 1;

5.

9. The method of manufacturing according to claim 1, wherein the bioink comprises the decellularized extracellular matrix solution and the alginate solution in a volume ratio of 1:

0.33 to 1;

3.

10. The method of manufacturing according to claim 1, wherein the cell spheroid has a diameter of 300 to 500 μ

m.

11. The method of manufacturing according to claim 1, wherein the bioink is extruded at a pneumatic-pressure time of 0.01 to 0.1 second using a 27 G nozzle, to form the spheroid having a diameter of 300 to 500 μ

m.

12. The method of manufacturing according to claim 1, wherein two or more of the cell spheroids are formed continuously as the bioink is sprayed with an injection nozzle and printed continuously, and located in circumstances of two or more concentric circles.

13. The method of manufacturing according to claim 1, wherein the manufacturing a cell spheroid by printing the bioink is performed by printing a cell spheroid with extruding the bioink by an injection nozzle moving along the spiral track.

14. The method of manufacturing according to claim 13, wherein the manufacturing a cell spheroid by printing the bioink comprises

determining information of a radius of a semicircle having the longest radius among the spiral tracks of the injection nozzle, the number of spheroids located in the circumference of the semicircle having the longest radius among the spiral tracks, a radius of a semicircle having the shortest radius among the spiral tracks, and an interval between circumferences of the spiral tracks;

determining the location at which the bioink is extruded by the injection nozzle using the determined information; and

manufacturing a plurality of cell spheroids continuously by printing the bioink on the determined locations.

15. The method of manufacturing according to claim 13, wherein the injection nozzle manufactures 100 or more of cell spheroids continuously.

16. The method of manufacturing according to claim 1, wherein the cell is at least one selected from the group consisting of cancer cell, stem cell, precursor cell, osteoblast, myoblast, tenocyte, neuroblast, fibroblast, glioblast, germ cell, hepatocyte, renal cell, Sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell, cardiocyte, cardiomyocyte, glial cell, endothelial cell, hormone-secreting cell, immunocyte, islet cell, pancreatic islet cell, neuron, thymocyte, adipocyte, alveolar cell, dental pulp cell, chondrocyte, oocyte and intestinal cell.

17. The method of manufacturing according to claim 16, wherein the stem cell is at least one selected from the group consisting of mesenchymal stem cell, induced pluripotent stem cell, induced pluripotent stem cell-based insulin producing cell, induced pluripotent stem cell-based cardiomyocyte, and induced pluripotent stem cell-based endothelial cell.

18. The method of manufacturing according to claim 1, wherein the cell spheroid is a drug testing model.

19. A composition for manufacturing a cell spheroid using a three-dimensional printing method, comprising cells, decellularized extracellular matrix and alginate as a gelated polymer.

20. The composition according to claim 19, wherein the concentration ratio of the decellularized extracellular matrix and the alginate is more than 1:

0.5 or more to 1;

5.