KR20180049998A - Method of manufacturing 3D cell spheroids using thermo-responsive hydrogel - Google Patents

Abstract

The present invention relates to a method for producing a three-dimensional cell spherical body using a thermosensitive hydrogel, and more particularly, to a method for producing a three-dimensional cell spherical body using a thermosensitive hydrogel, And a cell aggregate is formed and desorbed by volume expansion according to a decrease in the temperature of the thermosensitive hydrogel, thereby preparing a three-dimensional cell spherical body ≪ / RTI >
According to the present invention, it is possible to provide a novel three-dimensional cell culture system capable of easily controlling the size and distribution of cell spheroids by controlling the size of the cell adsorbing material pattern formed on the thermosensitive hydrogel. Therefore, the present invention can be widely applied to a medical cell cluster delivery and treatment system, a three-dimensional artificial tissue preparation for substituting an animal experiment and a clinical experiment, and a study model of tissue and tumor interaction.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for manufacturing a 3D cell spheroid using a thermosensitive hydrogel,

The present invention relates to a method for producing a three-dimensional cell spherical body using a thermosensitive hydrogel.

Body cells and tissues grow, differentiate, and develop while interacting within a very complex three-dimensional structure. However, most cell cultures are made on a two-dimensional impermeable flat plane. Therefore, the two-dimensional cell culture has a limitation that it can not accurately simulate the cell environmental conditions in our body.

Recently, a method of culturing a spheroid, which is a three-dimensional cell tissue having functions equivalent to those in vivo, has attracted attention. 3-dimensional cell spheres are used for cell aggregation induction to simulate cancer, transplantation of agglutinated cells for transplanting islet cells to induce normal secretion of insulin for diabetes treatment, and so on. Mass production . In addition, as the stem cell research has matured, various methods have been attempted to apply 3-D embryonic stem cells to studies of various differentiation mechanisms.

Conventional three-dimensional cell culture methods include a hanging-drop method, a rotary culture method using a bioreactor, a centrifugation method, and a micromolding method. For example, Japanese Patent Laid-Open Publication No. 6-327465 Discloses a method in which a plurality of single cells are seeded in a well having a funnel shape on the bottom surface and the single cells are aggregated and cleaved on the bottom surface to cultivate the spleoid. In Korean Patent No. 10-1341572, And a plurality of cell receiving portions extending from one surface of the substrate and including a hollow tube therein.

These three-dimensional cell culture methods can be used to evaluate the effects of regenerative medicine, hybrid artificial organs, production of bioactive materials, investigation and search of the functions of biological tissues or organs, screening of new drugs, An animal test substitution method, and a cell chip having a sensor function.

However, the above-described conventional three-dimensional cell culture methods are not only complicated in the culture method but also difficult to control the size of the cell spheroid and control the distribution of the cells, and it takes much labor and time to acquire a large amount of cell spheroids There are many limitations.

Disclosure of the Invention The present invention has been conceived in order to solve the above-mentioned problems. In the present invention, by using a microcontact printing method on a thermosensitive hydrogel reversibly exhibiting swelling and contraction according to temperature change, To form a cell aggregate by site-specific induction of cell adsorption, followed by volumetric expansion according to a decrease in the temperature of the thermosensitive hydrogel, thereby forming a three-dimensional cell spheroid.

In addition, the present invention provides a three-dimensional cell spherical body prepared according to the above-described method.

According to an aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising: (a) patterning a cell adsorbing material on a substrate; (b) contacting the substrate on which the cell adsorbing material is patterned with the surface of the thermosensitive hydrogel, and patterning the cell adsorbing material on the thermosensitive hydrogel; (c) dispensing and culturing cells on a thermosensitive hydrogel patterned with the cell adsorbing material; And (d) expanding the volume of the thermosensitive hydrogel to form and desorb the cell aggregate.

The substrate may be selected from the group consisting of polydimethylsiloxane (PDMS), glass, metal, and silicon.

In the step (a), the cell adsorbing material may be patterned on the substrate by a polymerization reaction performed under weakly basic conditions.

The cell adsorbing material may be a polycarate-based polymer, and the polycarate-based polymer may be selected from the group consisting of polydopamine, polyepinephrine, polynorepinephrine, and mixtures thereof.

The thermosensitive hydrogel may be bulky as the ambient temperature is lowered.

The thermosensitive hydrogel may be a copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide / thiamine conjugate hydrogel.

The cell adsorbing material pattern is formed in a circular or polygonal plate shape, and the diameter of the circular pattern or the length of one side of the polygonal pattern may be 100 to 500 mu m.

The cells may be selected from the group consisting of muscle cells, fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, osteoblasts, kidney cells, adipocytes, corneal epithelial cells, stem cells, neurons, keratinocytes, muscle cells, ≪ / RTI >

In addition, the present invention provides a three-dimensional cell spherical body produced according to the above production method.

According to the present invention, by controlling the size of the cell adsorbing material pattern formed on the thermosensitive hydrogel, a new three-dimensional cell culture which can easily control the size of the cell spheroid, the number of cells contained in the spheroid, System can be provided. Therefore, the present invention can be widely applied to a medical cell cluster delivery and treatment system, a three-dimensional artificial tissue preparation for substituting an animal experiment and a clinical experiment, and a study model of tissue and tumor interaction.

1 is a schematic view showing a process for producing a three-dimensional cell spherical body according to the present invention.
2 is a schematic view illustrating a process of patterning a cell adsorbing material on a thermosensitive hydrogel through a microcontact printing method according to an embodiment of the present invention.
FIG. 3 is a schematic view showing the principle of cell aggregation being formed and desorbed by the volume expansion of a hydrogel after cell culture on a thermosensitive hydrogel patterned with a cell adsorbing material according to an embodiment of the present invention.
FIG. 4 is an image of a wisdom micrograph of a surface of a thermosensitive hydrogel patterned with a cell adsorbing material before and after microcontact printing of a PDMS substrate on which a cell adsorbing material is patterned and a microcontact printing method.
5 is a graph showing a change in size of a pattern according to a temperature change of a thermosensitive hydrogel patterned with a cell adsorbing material.
6 is an image of a phase contrast microscope in which fibroblasts are coated on a thermosensitive hydrogel patterned with a cell adsorbing material, and the growth process is observed over time.
FIG. 7 is a graph showing cell counts according to image and pattern size visualized through fluorescent staining at 24 and 72 hours after the application of fibroblasts on a thermosensitive hydrogel patterned with a cell adsorbing material.
FIG. 8 is a graph showing the results of cell adhesion, cell desorption, and cell desorption obtained by applying the fibroblasts on a thermosensitive hydrogel patterned with a cell adsorbing material, culturing the cells for 24 hours, lowering the temperature of the thermosensitive hydrogel to 4 ° C for 20 minutes, Is a phase contrast microscope image of the surface of the post hydrogel.
FIG. 9 is a graph showing the results of cell adhesion, cell desorption, and cell desorption obtained by applying the fibroblasts on a thermosensitive hydrogel patterned with a cell adsorbing material, culturing the cells for 72 hours, lowering the temperature of the thermosensitive hydrogel to 4 DEG C for 20 minutes, Is a phase contrast microscope image of the surface of the post hydrogel.
FIG. 10 shows the survival rate and size distribution of the cell aggregate obtained after the culture of the fibroblasts on the thermosensitive hydrogel patterned with the cell adsorbing material for 24 hours, lowering the temperature of the thermosensitive hydrogel to 4 ° C, Dead assay and NIS-element.
Figure 11 shows the survival rate and size distribution of the cell aggregate obtained after the culture of the fibroblasts on the thermosensitive hydrogel with the cell adsorbing material patterned for 72 hours and lowering the temperature of the thermosensitive hydrogel to 4 ° C, Dead assay and NIS-element.
FIG. 12 shows the results of culturing the fibroblasts on a thermosensitive hydrogel patterned with the cell adsorbing material for 24 hours and 72 hours, lowering the temperature of the thermosensitive hydrogel to 4 ° C, and then determining the yield of the obtained cell aggregate Fig.
FIG. 13 is a graph showing the structure of the cell aggregate obtained after the culture of fibroblasts on a thermosensitive hydrogel patterned with a cell adsorbing material for 24 hours and 72 hours, lowering the temperature of the thermosensitive hydrogel to 4 ° C, actin staining and H & E staining.
FIG. 14 is an image of a phase contrast microscope obtained by culturing and observing for 24 hours after the application of the X-ray mesenchymal stem cells on the thermosensitive hydrogel patterned with the cell adsorbing material.
Fig. 15 is a graph showing the relationship between the cell aggregate obtained, the surface of the hydrogel after cell desorption, and the surface area of the cell aggregate obtained after the temperature was lowered to 4 < 0 > C for 10 minutes after culturing the tumoral stem cells on a thermosensitive hydrogel patterned with the cell- It is a phase contrast microscope image.
16 is a graph showing the survival rate and size distribution of the cell aggregate obtained after culturing the mesenchymal stem cells on a thermosensitive hydrogel patterned with a cell adsorbing material for 24 hours and lowering the temperature of the thermosensitive hydrogel to 4 ° C. / Dead assay and NIS-element, respectively.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method of forming a cell adsorbing material pattern having a predetermined region on a thermosensitive hydrogel reversibly exhibiting swelling and shrinkage according to a temperature change using a microcontact printing method, Dimensional cell spheres by forming and desorbing a cell aggregate by volume expansion due to a decrease in the temperature of the thermosensitive hydrogel.

A method for producing a three-dimensional cell spherical body according to the present invention includes the following steps.

(a) patterning a cell adsorbing material on a substrate; (b) contacting the substrate on which the cell adsorbing material is patterned with the surface of the thermosensitive hydrogel, and patterning the cell adsorbing material on the thermosensitive hydrogel; (c) dispensing and culturing cells on a thermosensitive hydrogel patterned with the cell adsorbing material; And (d) expanding the volume of the thermosensitive hydrogel to form and desorb the cell aggregate;

First, in step (a), the cell adsorbing material is patterned on one side of the substrate. At this time, the substrate may be selected from the group consisting of polydimethylsiloxane (PDMS), glass, metal, and silicon.

In addition, the cell adsorbing material is preferably a polycarate-based polymer. The catecholol polymer mimics a catechol group that is repeatedly found in adhesive proteins secreted from the mussel's footpath. Since the catecholate has bonding properties with various substrates, it is coated on the surface of various substrates, And have been attracting attention as materials capable of modifying the surface to have the same chemical properties. In the present invention, the cells are adsorbed along the polymer pattern of the polycarate based on the adhesive ability of the polymer, so that the adsorption of the cells can be region-specifically controlled.

At this time, the polycationic polymer as the cell adsorbing material may be patterned on the substrate by an autopolymerization reaction performed under weakly basic conditions.

The polycarate polymer may be selected from the group consisting of polydopamine, polyepinephrine, polynorepinephrine, and mixtures thereof, as long as it can induce adsorption of cells. Can

The shape of the pattern formed by the cell adsorbing material is not limited. For example, the pattern may be formed in the shape of a circular or polygonal plate. If the pattern is a circular pattern, the pattern is a diameter. If the pattern is a polygonal pattern And the length of one side may be 100 to 500 mu m.

Next, in the step (b), the substrate on which the cell adsorbing material is patterned is brought into contact with the surface of the thermosensitive hydrogel using a microcontact printing method, and the cell adsorbing material is patterned on the thermosensitive hydrogel. Through this process, the pattern of the cell adsorbing material formed on the substrate is transferred directly to the thermosensitive hydrogel, and the same pattern is formed on the thermosensitive hydrogel.

Next, in step (c), the cells are dispensed and cultured on the thermosensitive hydrogel patterned with the cell adsorbing material. The cells to be dispensed and cultured on the thermosensitive hydrogel are not particularly limited, and examples thereof include liver, kidney, spleen, bone, bone marrow, thymus, heart, muscle, lung, brain, testes, ovary, Cells capable of being separated or activated from the ear, skin, gallbladder tissue, prostate, bladder, embryo, immune system and hematopoietic system can be used, and it is preferable to use cells such as myofibroblasts, fibroblasts, vascular endothelial cells, cartilage cells, hepatocytes, Cells selected from the group consisting of cells, kidney cells, adipocytes, corneal epithelial cells, stem cells, nerve cells, keratinocytes, muscle cells, cardiac muscle cells and esophageal epithelial cells can be cultured.

Next, in step (d), the volume of the thermosensitive hydrogel is expanded to form an aggregate of cells cultured through the step (c), and desorbed from the thermosensitive hydrogel. At this time, an aggregation of cells is formed due to the volume expansion of the thermosensitive hydrogel, and when the thermosensitive hydrogel is inflated, the cells receive a force above a contractile force that the cells have when they adhere to the hydrogel Desorption occurs from the thermosensitive hydrogel.

The thermosensitive hydrogel reversibly exhibits shrinkage and swelling, rather than sol-gel behavior, depending on the temperature. Specifically, the thermosensitive hydrogel has a low critical solution temperature (LCST) Below the solution temperature, the water content increases and the water content decreases below the lower critical solution temperature. The thermosensitive hydrogel was prepared by adding a carnospermine peroxidase and hydrogen peroxide to a copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide / thiamine conjugate as a phenol analogue to form crosslinks through dehydrogen bonding between the conjugates, Oxide-polypropylene oxide-polyethylene oxide copolymer / thiamine conjugate hydrogel. At this time, as the copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a copolymer having a trade name of Pluronic, Poloxamer, Tetronic may be used. Tetronictyramine (Tet-TA) exhibits a low critical solution temperature at room temperature (15-25 DEG C) in the thermosensitive hydrogel used in the examples of the present invention.

In addition, the present invention provides a three-dimensional cell spherical body produced according to the above production method.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments and the like. It will be apparent to those skilled in the art, however, that these examples are provided for further illustrating the present invention and that the scope of the present invention is not limited thereto.

PDMS  Board Fabrication

First, a rectangular pattern with a depth of 30 μm and 50 μm was imprinted on a silicon substrate using a laser beam so that the lengths of the sides were 100, 200, and 300 μm. A PDMS substrate having a square pattern was prepared by curing polydimethylsiloxane (PDMS) (polymer: curing agent = 9: 1, 8: 2 95 ° C) on the patterned silicon substrate. The substrate was sterilized by ultraviolet (UV) irradiation for more than one day in 70% ethanol before coating with the cell adsorbing material, polydopamine, and then the surface was washed with distilled water and dried at room temperature.

Cell sorbents ( Polydopamine ) coating

First, the PDMS substrate was cut into a 6-well plate size (diameter = 35 mm) and prepared on the bottom. Next, dopamine hydrochloride (Sigma-Aldrich) was dissolved at a concentration of 2 mg / ml in 10 mM Tris HCl (37 DEG C, pH 8.5). 10 ml of the dissolved dopamine solution was applied onto the PDMS and self-polymerized and coated with polydodamine for 2-4 hours. After the polydopamine coating, the polydodamine solution was removed and the PDMS stamp was cut out using a circular punch of 10 mm after naturally dried for one day.

Temperature Sensitivity Hydrogel  making

First, the hydroxy functional group of the tetronic end is reacted with p-nitrophenyl chloroformate (PNC) and then reacted with tyramine to form a tetronic-tyramine conjugate (Tte-TA) according to the method disclosed in Japanese Patent Application Publication No. 2010-0076163. . Next, a 1: 1 solution of Tet-TA dissolved in 0.1% H ₂ O ₂ solution and 0.0025 mg / ml HRP solution at 15% was mixed in a ratio of 1: 1, . After crosslinking for 15 minutes, the hydrogel was cut into a circular shape using an 8 mm biopsy punch. The circular hydrogel was sterilized by irradiating ultraviolet rays (UV) in a 70% ethanol solution for 30 minutes or more, and washed with PBS at 37 ° C five times or more.

Temperature Sensitivity Hydrogel  On the surface, contact  Formation of micro-unit patterns using printing

The circular hydrogel was placed in the center of a 24-well tissue culture plate (diameter = 15 mm), and the PDMS stamp coated with the polydodamine was vertically lowered to contact the pattern surface and the surface of the hydrogel. After 3 minutes, the PDMS stamp was separated from the hydrogel to form a polydodamine pattern on the surface of the hydrogel, which was then stored in PBS at 37 ° C.

Micro-scale cell aggregation culture

Human dermal fibroblast was cultured in a cell culture dish (37 ° C, 5% CO ₂ ) with 10% fetal bovine serum (FBS) and 1% penicillin added high glucose Dulbecco's Modified Eagle's Medium. Human turbinate stem cells were cultured in a cell culture dish (37 ° C, 5% CO ₂ ) using Alpha Modified Eagle's Medium supplemented with 10% FBS and 1% penicillin. The fibroblasts were cultured for 3 minutes with trypsin-EDTA (0.05%) in a cell culture dish to obtain single cells. Then, the fibroblasts were cultured in a hydrogel phase at a density of 2 x 10 ⁴ cells / And cultured for 24 hours and 72 hours, respectively, to form microcell aggregates specifically adhered to the hydrogel pattern. The cells were incubated with trypsin-EDTA (0.05%) for 3 minutes to obtain single cells. The cells were coated on the hydrogel with the cell adsorbing material pattern at a density of 5 × 10 ⁴ cells / well, and cultured for 24 hours Thereby forming a microcell aggregate specifically adhered to the hydrogel pattern.

Obtain cell aggregate through temperature change

24, and 72 hours, respectively. The culture medium in which the cell aggregates formed at 37 ° C and 5% CO ₂ were formed was removed using a Pasteur pipette, and the 4 ° C culture solution stored in the refrigerator was treated with 500 μl of the culture solution on the hydrogel. The hydrogel was shaken in a 4 ° C refrigerator using a rocker shaker and kept for 20 minutes so that it could swell. Twenty minutes after the fibroblasts, 10 minutes after the transbronchial stem cells, the cell aggregates desorbed from the hydrogel were obtained using a pipette, and the cells were transferred to a three-dimensional cell culture dish for culturing.

Results and Discussion

Micro contact  Using printing Hydrogel  Result of surface patterning

FIG. 4 is an image of a wisdom micrograph of a surface of a thermosensitive hydrogel patterned with a cell adsorbing material before and after microcontact printing of a PDMS substrate on which a cell adsorbing material is patterned and a microcontact printing method. As a result, PDMS observed before microcontact printing showed that PDMS was coated with polydodamine, and PDMS after microcontact printing showed that polydopamine was reduced in pattern portion. In the case of the thermosensitive hydrogel, it was also confirmed that the polydopamine pattern was clearly formed on the surface which was clean before the microcontact printing, and that the polydopamine pattern was clearly formed after the printing process. According to the present invention, It was confirmed that the cell adsorbing material pattern was successfully formed.

Following the temperature change Hydrogel  Change of micro pattern

5 is a graph showing a change in size of a pattern according to a temperature change of a thermosensitive hydrogel patterned with a cell adsorbing material. Through this, we confirmed the change of the pattern of the hydrogel with pattern by microcontact printing according to the change of the temperature by the change of the pattern size (length of one side). Specifically, hydrogels of groups 100, 200 and 300 having 97.9 ± 5.4, 199.2 ± 3.6, and 307.5 ± 4.8 μm at 37 ° C were 134.5 ± 6.9 and 268.1 ± 9.0 when the temperature was changed to 4 ° C. 446.7 ± 4.4 ㎛, which is about 1.4 times larger than that of the others. In view of the similar increase in size regardless of the size of the pattern (length of one side) in all the three groups, it was confirmed that the patterning method using microcontact printing used in the present invention did not affect the temperature reduction property of the hydrogel Respectively.

Hydrogel  ≪ / RTI >

6 is an image of a phase contrast microscope in which fibroblasts are coated on a thermosensitive hydrogel patterned with a cell adsorbing material, and the growth process is observed over time.

Specifically, it was confirmed that the cells showed a specific growth pattern in all patterns ranging from 6 hours to 24 hours into the pattern. After 24 hours, the cells continued to grow in the pattern, It was confirmed that the cells appeared to aggregate and form aggregates.

FIG. 7 is a graph showing cell counts according to image and pattern size visualized through fluorescent staining at 24 and 72 hours after the application of fibroblasts on a thermosensitive hydrogel patterned with a cell adsorbing material. Cell nuclei were stained with Hoechst and cell number was counted by image analysis using NIS-element to confirm the cell number in the cell aggregate grown up to 72 hours.

As a result, it was confirmed that there was 13.8 ± 5.2 cells in the pattern after culturing for 24 hours at 100 μm, and the number increased to 29.0 ± 8.0 after 72 hours. Also, at 200 ㎛, the number of cells increased from 35.1 ± 8.0 at 24 hours to 60 ± 7.94 at 72 hours and increased from 66.6 ± 12.4 to 104.8 ± 13.2 at 300 ㎛ at 24 hours. As a result, it was confirmed that when the cell attachable portion in the pattern was gradually reduced in an environment in which cells could specifically adhere, the cells coagulated and continued to grow.

Obtain cell aggregate using temperature change

FIGS. 8 and 9 show the results of 24 hours after the application of the fibroblasts on the thermosensitive hydrogel patterned with the cell adsorbing material (FIG. 8). 72 hours (FIG. 9), lowered the temperature of the thermosensitive hydrogel to 4 占 폚, and left for 20 minutes, thereby obtaining the cell aggregate and the surface of the hydrogel after cell desorption.

First, the hydrogel at 37 ° C for 24 hours was treated with the culture at 4 ° C and maintained in a refrigerator at 4 ° C for 20 minutes so that cells expanded in the pattern formed an aggregate. After 20 minutes, it was confirmed that all the cells formed aggregates on the hydrogel. However, the pattern of 300 ㎛ did not form a complete cell aggregate as compared with other patterns, and it was confirmed that the cells were still held in a state in which they were attached to one pattern or one of them was attached.

Cells maintained in this state were obtained using a pipette and transferred to another cell culture dish to observe its shape and to confirm whether the cell aggregate remained in the hydrogel. At 24 hours of culture, it was confirmed that the cells aggregated at 100 and 200 ㎛, which are small enough in size, to form a cell aggregate. On the other hand, the aggregation of cells having a uniform shape .

 And the cell aggregate was desorbed by the same method as above for 24 hours even after 72 hours of culturing. After 20 minutes, it was confirmed that the cells aggregated on the hydrogel similarly to form a cell aggregate.

Cells maintained in this state were obtained using a pipette, transferred to another cell culture dish, and their morphology was observed. When the cell aggregate remained in the hydrogel, it was found that the cell aggregate having a complete shape and a uniform size . ≪ / RTI > This can be inferred from the presence of a sufficient amount of cells to form a cell aggregate on the hydrogel.

Fig. 12 is a diagram quantifying the degree of gain of the cell aggregate. Quantitative analysis of the degree of obtaining the cell aggregate revealed that it was possible to obtain cell aggregates of 90% or more in both 24 hours and 72 hours of culture.

Characterization of the obtained cell aggregate

FIGS. 10 and 11 show the results obtained when the fibroblasts were coated on the thermosensitive hydrogel patterned with the cell adsorbing material and cultured for 24 hours (FIG. 10) and 72 hours (FIG. 11), and the temperature of the thermosensitive hydrogel The survival rate and the size distribution of the cell aggregate obtained after lowering were confirmed by measuring the cell cluster diameter using the Live / Dead assay and the NIS-element.

As a result of observation, it was confirmed that most of the cell aggregates did not show red signal indicated by dead cells in all cell aggregates and survived. In addition, the cell aggregate cultured on the hydrogel for 24 hours had a diameter of about 40-60 mu m in the 100 mu m pattern, about 80% of the cells were in the same size range, and the 200 mu m pattern had a diameter of 60-80 mu m The cell aggregates were found to be close to 100%, while at 300 ㎛, various diameters from 70 ㎛ to 120 ㎛ appeared in a wide range. These results are similar to those obtained when the cells were first obtained, and it can be inferred that when the cells are specifically grown, a sufficient amount of cells are not present on the pattern to make the cell aggregate.

In the case of the cell aggregate cultured on the hydrogel for 72 hours, it was confirmed that most of the cells were alive as well as the cells cultured for 24 hours. The size of the cell aggregate was 50-70 ㎛, which was about 10 ㎛ larger than the cells cultured for 24 hours when it was obtained in the pattern of 100 ㎛, and it was confirmed that about 90% cell aggregate had a diameter within the same range. The pattern aggregates of 70-90 μm were present in 80% or more, and the aggregates obtained in the pattern of 300 μm were found to contain 60% or more of aggregates having a diameter of 90-120 μm.

13 is a diagram showing the structure of the cell aggregate through typical dyeing. The structure of the aggregate and the cell distribution inside the aggregate were confirmed by F-actin staining and H & E staining using a 200 ㎛ pattern having a general cell cluster size. The structure of the aggregate had an unstable structure, such as a nucleus emerging from the cultured cells for 24 hours, and it was confirmed that there was not enough cells in the aggregate when it was confirmed by the distribution of cells in the aggregate. On the contrary, in the cells cultured for 72 hours, it was confirmed that the cell aggregate was stably formed and the amount of cells in the aggregate was sufficient.

Stem cells  Fabrication and characterization of cell aggregates

FIGS. 14, 15, and 16 show the results of culturing osteomaic stem cells on a thermosensitive hydrogel patterned with a cell adsorbing material for 24 hours (FIG. 12), lowering the temperature of the thermosensitive hydrogel to 4 ° C (Fig. 13) and size distribution (Fig. 14) of a cell aggregate were determined by measuring the diameter of the cell cluster using Live / Dead assay and NIS-element.

As a result of observation, it was confirmed that most of the cell aggregates did not show red signal indicated by dead cells in all cell aggregates and survived. In addition, the cell aggregate cultured on the hydrogel for 24 hours had a diameter of about 70-110 mu m in the 200 mu m pattern and about 80% of the cells were in the same size range.

Claims (10)

(a) patterning a cell adsorbing material on a substrate;
(b) contacting the substrate on which the cell adsorbing material is patterned with the surface of the thermosensitive hydrogel, and patterning the cell adsorbing material on the thermosensitive hydrogel;
(c) dispensing and culturing cells on a thermosensitive hydrogel patterned with the cell adsorbing material; And
(d) expanding the volume of the thermosensitive hydrogel to form and desorb the cell aggregate. The method according to claim 1,
Wherein the substrate is selected from the group consisting of polydimethylsiloxane (PDMS), glass, metal, and silicon. The method according to claim 1,
Wherein the cell adsorbing material is patterned on a substrate by a polymerization reaction performed under weakly basic conditions in the step (a). The method according to claim 1,
Wherein the cell adsorbing material is a polycarate-based polymer. 5. The method of claim 4,
Wherein the polycarate-based polymer is selected from the group consisting of polydopamine, polyepinephrine, polynorepinephrine, and mixtures thereof. The method according to claim 1,
Wherein the thermosensitive hydrogel is bulky when the ambient temperature is lowered. The method according to claim 1,
Wherein the thermosensitive hydrogel is a copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide / thiamine conjugate hydrogel. The method according to claim 1,
The cell adsorbing material pattern is formed in a circular or polygonal plate shape,
Wherein the diameter of the circular pattern or the length of one side of the polygonal pattern is 100 to 500 mu m. The method according to claim 1,
The cells may be selected from the group consisting of muscle cells, fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, osteoblasts, kidney cells, adipocytes, corneal epithelial cells, stem cells, neurons, keratinocytes, muscle cells, ≪ RTI ID = 0.0 > 3. ≪ / RTI > A three-dimensional cell spherical body produced according to the manufacturing method of claim 1.

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