METHOD OF MANUFACTURING CELL SPHEROID USING THREE-DIMENSIONAL PRINTING METHOD

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 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 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0053] FIG. 1 is a schematic diagram of the process of manufacturing spheroids according to one embodiment of the present invention.

[0054] FIG. 2 is a schematic diagram showing the method of calculating the location where to produce spheroids according to one embodiment of the present invention.

[0055] FIG. 3a is schematic diagram showing the result of printing simulation using the CAMotics program which virtually confirms a G-code according to one embodiment of the present invention.

[0056] FIG. 3b is a drawing which shows the track of the spheroid manufactured by printing the bioink composition according to one embodiment of the present invention.

[0057] FIG. 4 is the experimental result showing the result of manufacturing a cell spheroid according to the mixing ratio of extracellular matrix and alginate which constitutes the bioink composition according to one embodiment of the present invention.

[0058] FIG. 5 is capsule photographs showing the result of printing under various nozzle and pneumatic pressure conditions according to one embodiment of the present invention.

[0059] FIG. 6a is a photograph showing the viability of cells in the cell spheroid manufactured according to one embodiment of the present invention over time, and FIG. 6b shows it divided by cell.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0060] The present invention will be described in more detail by the following examples, but the present invention is not intended to be limited by the following exemplary examples.

Example 1

Cell Spheroid Manufacture

(1) Bioink Manufacture

[0061] Bioink for manufacturing spheroids comprised cells, decellularized extracellular matrix and alginate. A sufficient number of cells should be secured to manufacture cell spheroids. The volume ratio of the cell suspension in which the decellularized extracellular matrix and cells comprised in bioink were mixed was 1:1. The decellularized extracellular matrix was a solution at a concentration of 2.6% by weight, and as the alginate solution, a solution at a concentration of 2% by weight was used.

[0062] As cells comprised in bioink, cardiac progenitor cells (CPC), endothelial progenitor cells (EPC), or cells mixing cardiac progenitor cells and endothelial progenitor cells at the same number ratio were used. The decellularized extracellular matrix obtained from swine heart tissue was used as the decellularized extracellular matrix of the bioink. The cell concentration used was all 10.sup.7 cells/mL, and in case of the mixed cells, cardiac progenitor cells (CPC) and endothelial progenitor cells (EPC) were mixed in equal numbers so that the total number of cells was 10.sup.7 cells/mL.

[0063] When printing once, cells were used in an amount of 10.sup.7 cells/mL, and decellularized extracellular matrix solution 200 ul, cell culture medium 60 ul, and alginate solution 260 ul were used, and thereby bioink in a volume of 520 ul in total was prepared. Then, the concentration of the decellularized extracellular matrix in the decellularized extracellular matrix solution and suspension of cell culture medium was 2.0% by weight, and it was same as the alginate use of the alginate solution of 2.0% by weight. Accordingly, the concentration of the decellularized extracellular matrix and the concentration of the alginate in the mixed solution in which the decellularized extracellular solution, cell culture medium and alginate solution were mixed were same as 1.0% by weight. After detaching cells from the dish using trypsin, they were mixed to the bioink in which alginate and decellularized extracellular matrix were mixed evenly. Then, care was taken to avoid bubbles, and since the decellularized extracellular matrix is not gelated during the experiment when being used under 4 C., ink was mixed in an ice-box.

(2) Bioink Printing

[0064] Bioink in which all substances were evenly mixed was put in a 3 ml syringe with a 27 G nozzle, and this was connected to the printer nozzle area. As a chamber, a petri dish was used, and the mixed solution of calcium chloride solution (0.1 M) and Pluronic F-127 solution (20% by weight) was added by 5 ml, and then to increase the viscosity of PF-127 solution, the temperature was set to 25 C.

[0065] When inputting the center of the water tank to the first part of the code, a cell spheroid is manufactured by drawing a circle around the position. The nozzle moved to the center of the bath, and when it reached the position corresponding to the same distance as the length of the radius from the center, the nozzle was lowered down so that the tip of the nozzle was submerged in the solution in the tank, and the pneumatic pressure was applied to form a spheroid at the tip of the nozzle. Then, when the nozzle was raised again, due to the viscosity of PF-127, the spheroid was not raised and was separated, being immersed in the chamber. The pneumatic pressure time was 0.032 seconds, and the pneumatic pressure was 30 kPa.

Example 2

Spheroid Manufacture According to Pneumatic Pressure Time Change

[0066] In order to investigate the effect according to the pneumatic pressure time, spheroids were manufactured under various conditions, and spheroids having various sizes according to the change of the pneumatic pressure time could be manufactured.

[0067] Specifically, bioink was manufactured by the same method as Example 1 to connect it to the printer nozzle. By increasing the time of applying the pneumatic pressure from the initial pneumatic pressure time 0.032 seconds to manufacture spheroids. To form spheroid at the end of the nozzle on the code, when applying the pneumatic pressure, the delay time was set to increase the pneumatic pressure time. By setting the pneumatic pressure time to 0.1 second, 0.3 seconds, 0.5 seconds and 0.7 seconds, spheroids were manufactured. The result was shown in Table 1.

TABLE-US-00001 TABLE 1 Pneumatic pressure time (sec) 0.032 0.1 0.3 0.5 0.7 Diameter of 372.24 476.67 530.34 734.83 1047.06 spheroid (um)

[0068] As a result, in the range of the pneumatic pressure time of 0.01 to 0.2 seconds (for example, 0.032 seconds), spheroid having a diameter of 300 to 500 m were manufactured, and when increasing the time of applying the pneumatic pressure to 0.7 seconds or more, spheroid having a size of 1000 m or more were manufactured.

Example 3

Consecutive Manufacture of Spheroids

[0069] To manufacture a large number of spheroids consecutively, bioink was distributed while the injection nozzle moved along the circumferences of concentric circles with a certain interval, and spheroids were consecutively manufactured in quantity.

[0070] Specifically, to manufacture a large number of spheroids at a certain interval, it was considered that it was the most effective that the injection nozzle moved along the track of circles, and accordingly, codes were produced so that the injection nozzle could move the circle track. In addition, it was not that the injection nozzle moved to one circle track and the spheroid manufacture was completed, but bioink was distributed along the concentric circles following the circle track with gradually decreasing radius, thereby allowing massive production of spheroids.

[0071] For example, after locating the nozzle on the position of the center of circles at first, the initial radius (23 mm) and the number of spheroids to be manufactured (90), the final radius (5.6 mm), and the interval between circumferences (0.3 mm) were substituted on codes, and then each point where spheroids were located was drawn using a trigonometric function. When the injection nozzle reached to each distributing point, after lowering the nozzle down as Example 1, the pneumatic pressure was distributed to manufacture spheroids. When distribution was completed, the nozzle rose up again, and immediately moved to the next position using the difference between the next position and the present position, and then bioink was distributed in the same manner to manufacture spheroids. When turning around all the track of the semicircle by repeating this process consecutively, by decreasing one of the number of spheroids on the circumference with the radius of 22.7 mm which was reduced by 0.3 mm from the initial radius, the point where the nozzle should be positioned was drawn using a trigonometrical function so as to manufacture 89 spheroids. When 89 spheroids were manufactured all while turning around the semicircle, the distribution position was drawn to produce 88 spheroids on the 22.4 mm circle with the radius reduced by 0.3 mm again. The above process was repeated until the smallest radius of the concentric circle reached 5.6 mm, and as a result, 3000 or more of spheroids could be automatically manufactured in one batch. FIG. 2 is a drawing which shows a spiral shape of track which can consecutively manufacture spheroids while decreasing the radius of the semicircle and the number of spheroids according to one example of the present invention. FIG. 3a is a schematic diagram showing the result of printing simulation using a program called CAMotics confirming G-code virtually according to one example of the present invention, and FIG. 3b is a photograph of the track of spheroids with actual printing.

Example 4

[0072] Spheroid Manufacture According to the Mixing Ratio of Decellularized Extracellular Matrix (dECM) and Alginate

[0073] To control the bioink composition, it was performed by the substantially same method as (1) of Example 1, but bioink was manufactured so as to comprising no cells and comprising decellularized extracellular matrix and alginate at various concentration ratios.

[0074] Specifically, cells were not used, and only the suspension comprising decellularized extracellular matrix and the alginate were used. For suspension manufacture, the decellularized extracellular matrix solution (concentration: 2.6%) of 400 ul was added to the cell culture medium of 120 ul, and they were mixed homogeneously to prevent bubbles. By the mixing, the concentration of the decellularized extracellular matrix of the suspension was 2.0% by weight. This was divided to 5 groups and 100 ul was put in an e-tube each.

[0075] The alginate solution (concentration: 2%) was mixed evenly in each tube by adding 300 ul, 200ul, 100 ul, 50 ul, or 33.33 ul, respectively, so that the concentration of the decellularized extracellular matrix and the concentration of the alginate were 1:3, 1:2, 1:1, 1:0.5, and 1:0.33, and the mixed volume ratio of the suspension and the alginate solution was 1:3, 1:2, 1:1, 1:0.5, and 1:0.33.

[0076] The bioink was moved to a syringe one by one in turn and was connected to the printer nozzle. Since the composition became heterogeneous over time when the bioink was moved to the syringe in advance, it was stored in the e-tube and was moved to a syringe just before printing. All was printed under the same printing condition, and thereby the shape of the spheroid found the homogeneous ratio. Eight spheroids were manufactured along the straight track by each experiment, and the average shape was examined The result was shown in FIG. 4.

[0077] FIG. 4 is the experimental result showing the result of cell spheroid manufacture according to the concentration ratio of decellularized extracellular matrix and alginate composing the bioink composition according to one example of the present invention. As shown in FIG. 4, when the concentration ratio of decellularized extracellular matrix and alginate was 1:3, 1:2, and 1:1, that is the concentration of alginate was more than the concentration of decellularized extracellular matrix concentration, the shape of spheroids was homogeneous. At this point, the cell affinity increased as the ratio of the alginate was low, and therefore it could be seen that the concentration ratio of the decellularized extracellular matrix and alginate of 1:1 to 1:2, for example, the concentration ratio of 1:1 was suitable for spheroid manufacture.

Example 5

Spheroid Manufacture According to Various Pneumatic Pressure Conditions

[0078] To investigate the change according to the pneumatic pressure during spheroid manufacture, it was performed by the substantially same method as Example 1, but bioink was manufactured and used so as to comprising no cells and comprising decellularized extracellular matrix and alginate.

[0079] Specifically, bioink having a ratio of dECM and alginate obtained in Example 4 (mixed concentration ratio 1:1) was manufactured. Alginate 260 ul at a concentration of 2% by weight and dECM 200 ul at a concentration of 2.6% by weight, and cell culture medium 60 ul were prepared and were injected to a syringe with a 27 G nozzle. The pneumatic pressure was provided equally for 0.032, and spheroids were manufactured while changing the pneumatic pressure size of 5 kPa, 10 kPa to 100 kPa in 10 kPa increments. One hundred spheroids were manufactured in each experiment and the average shape was examined. After adding the mixed solution of 2 ml each of the calcium chloride solution (0.1 M) and Pluronic F-127 solution (20% by weight) to a 6-well plate, 100 per one well were manufactured while varying the pressure, and all experiment groups were manufactured only in 2 batches in total.

[0080] When applying the pneumatic pressure of 5 kPa, the pressure was weak and therefore the tail from the nozzle was formed and thus the spheroid shape was not homogeneous, and when applying the pneumatic pressure of 100 kPa, the spheroid had a too big size. Accordingly, when applying the pneumatic pressure of 30 kPa to 40 kPa, homogeneous spheroids having a size of 300 to 350 m could be produced in quantity. FIG. 5 is a drawing which shows spheroids prepared by performing printing under various pneumatic pressure conditions according to one example of the present invention.

Example 6

Cell Viability Evaluation

[0081] The cell viability of cell spheroids manufactured in Example 1 was measured. Specifically, it was performed with 3 kinds of cells, that is, EPC only, CPC only, and mixed cells of mixing EPC and CPC at the same number ratio were used.

[0082] After preparing spheroids comprising each cell, a petri dish containing spheroids was transferred to a clean bench. To remove the mixed solution of calcium chloride (0.1 M) and Pluronic F-127 (20% by weight), the dish was cooled. When the viscosity of the mixed solution was lowered enough to flow, only the spheroids were filtered using a 100 um cell strainer. After two washes, the cell strainer comprising spheroids was placed on a 6-well plate. It was mixed to the cell culture medium so that the concentration of calcium chloride was 5 mM, and 5 ml each was put to each well so that the spheroids were sufficiently filled. Then, the cell culture medium was changed once a day. To see the viability, spheroids filtered with a sieve were added to a live/dead assay solution and were put in an incubator for 30 minutes, and then were observed with a fluorescent microscope.

[0083] FIG. 6a is a photograph showing the viability of cells in the cell spheroid manufactured according to one example of the present invention, and FIG. 6b shows the viability at Day 1 as divided by cell. The viability at Day 1 and Day 7 showed that most of the cells were alive. Through this, it could be seen that the environment in the spheroid was made into a cell friendly niche environment.