WO2018093069A1

WO2018093069A1 - Method for 3d-culturing cell

Info

Publication number: WO2018093069A1
Application number: PCT/KR2017/012196
Authority: WO
Inventors: 이재영; 장종욱; 박중건; 최고은
Original assignee: 광주과학기술원; 사회복지법인 삼성생명공익재단
Priority date: 2016-11-15
Filing date: 2017-10-31
Publication date: 2018-05-24

Abstract

The present invention relates to a microcapsule containing alginic acid therein, a preparation method therefor, a 3D culturing method of stem cells using the microcapsule, and use of the microcapsule in stem cell culture. In the present invention, micro-size capsule structures allowing stem cells to be cultured in a 3D manner are constructed with alginic acid or alginic acid modified with an extracellular matrix-derived peptide and can be used for efficient culturing which leads to enlarged spatial and temporal efficiency, cell activity, and mass production, compared to general 2D culture using a culture dish.

Description

Cell 3D Culture Method

The present invention relates to a microcapsule containing alginic acid, a method of preparing the microcapsules, a three-dimensional culture method of cells using the microcapsules, and a cell culture use of the microcapsules.

Stem cells have self-renewal and differentiation capacity, and are widely used in tissue engineering and regenerative medicine by secreting various bioactive factors. Active application research using various stem cells, such as embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells, is being conducted.

In particular, mesenchymal stem cells can be separated from bone marrow, adipose tissue, umbilical cord, umbilical cord blood, etc., can differentiate into adipocytes, bone cells, or chondrocytes, and have low immunogenicity and can play an immune mediating role. have. In addition, it expresses CD73, CD90, CD105 among surface antigens and does not express CD34, CD45, CD14, CD11b, CD19, CD79a.

In order for various stem cells to be clinically researched and commercialized as described above, the proliferation of stem cells is essential and must be proliferated through the culture of stem cells while maintaining appropriate differentiation and activity. However, if the conditions of proliferation are inadequate, there are problems such as slow growth rate of stem cells, initiation of spontaneous differentiation, and reduction of differentiation capacity.

In general, the stem cell culture method is used to culture the stem cells in a plastic culture dish or flask in a culture medium, a culture plate having a surface area of 25, 75, 150, 225 cm ² is mainly used. It consists of two-dimensional culture on plastics with high hardness. After cell proliferation, stem cells are generally detached and recovered by enzymes such as trypsin, and then passaged or used.

Clinical applications, including cell therapy and drug delivery, require large numbers of cells. When culturing cells in a general culture method, a large amount of culture dish or flask must be used.

However, when a large amount of flask is used, not only is it labor intensive but also variability for each flask and the possibility of contamination due to external factors increases, so the efficiency is very low. It is evaluated that it may be relatively unsuitable for proliferating cells to an appropriate level for mass production.

In addition, culturing in a culture dish or flask is different from the three-dimensional soft environment in which stem cells proliferate in a living body by culturing stem cells in a two-dimensional hardness environment, thereby inducing a risk of inducing stem cell degeneration. There is this.

Accordingly, it is urgent to develop a technique for efficiently and stably culturing stem cells, and the proliferation of stem cells in an industrial and clinical manner has a very important ripple effect.

The present inventors made diligent research efforts to develop a culture method capable of scale-up while culturing cells stably and with high efficiency.

As a result, when culturing cells using microcapsules containing alginic acid, the present invention was found to exhibit excellent effects in terms of high density culture, titer maintenance, culture stability, and scale-up efficiency compared to conventionally known culture methods. To complete.

It is therefore an object of the present invention to provide microcapsules comprising alginic acid.

Another object of the present invention is to provide a method for preparing microcapsules containing alginic acid.

Still another object of the present invention is to provide a three-dimensional culture method of cells using alginic acid microcapsules.

Yet another object of the present invention relates to the use of alginate microcapsules for culturing cells.

The present invention is to solve the problems such as inefficiency, slow growth rate, the risk of reducing the cell activity due to external harmful environment such as oxidative stress, according to the two-dimensional culture of the existing cell culture method, as a peptide derived from the extracellular matrix By supporting cells in biomimetic three-dimensional microcapsules using the converted natural polymers, a method of culturing stem cells with high density and high efficiency and easily recovering them was developed.

Accordingly, the present invention is to achieve a high density of the cells in the alginic acid three-dimensional microcapsules to cultivate the cells, thereby improving the growth of the supported cells and maintain the titer of the cells and can be easily recovered from the microcapsules By increasing the mass production of cells, the efficiency of the production process is increased.

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

One aspect of the invention relates to a microcapsule for cell culture comprising alginic acid.

The alginic acid may be substituted with an extracellular matrix-derived peptide.

The extracellular matrix-derived peptide may be an RGD motif, a DGEA motif, a REDV motif, an IKVAV motif, a VPGVG motif, a YIGSR motif, a KHIFSDDSSE motif, but may be, for example, an RGD motif, but is not limited thereto.

The average diameter of the microcapsules may be 30 um or more, 40 um or more, 50 um or 60 um or more. In addition, the average diameter of the microcapsules may be 1000 um or less, 900 um or less, 850 um or less, 800 um or less, 750 um or less, 700 um or less, 650 um or less, 600 um or less, 550 um or less or 500 um or less. have.

For example, the average diameter of the microcapsules is 30 to 1000 um, 30 to 900 um, 30 to 850 um, 30 to 800 um, 30 to 7500 um, 30 to 700 um, 30 to 650 um, 30 to 600 um , 30 to 550 um, 30 to 500 um, 40 to 1000 um, 40 to 900 um, 40 to 850 um, 40 to 800 um, 40 to 7500 um, 40 to 700 um, 40 to 650 um, 40 to 600 um , 40 to 550 um, 40 to 500 um, 50 to 1000 um, 50 to 900 um, 50 to 850 um, 50 to 800 um, 50 to 7500 um, 50 to 700 um, 50 to 650 um, 50 to 600 um , 50 to 550 um, 50 to 500 um, 60 to 1000 um, 60 to 900 um, 60 to 850 um, 60 to 800 um, 60 to 7500 um, 60 to 700 um, 60 to 650 um, 60 to 600 um , 60 to 550 um, may be 60 to 500 um, but is not limited thereto.

The cell may be a stem cell. The stem cells may be one or more selected from the group consisting of mesenchymal stem cells, blood stem cells, neural stem cells, muscle stem cells, myocardial stem cells, embryonic stem cells and induced pluripotent stem cells. For example, it may be mesenchymal stem cells.

Another aspect of the invention relates to a method for preparing microcapsules comprising alginic acid.

Alginic acid solution manufacturing step of preparing an alginic acid solution;

Mixing the cell dispersion solution and the alginic acid solution;

Forming step of forming microcapsules.

The alginic acid solution preparation step may be to prepare an alginic acid solution by adding an alginate substituted with alginate or a peptide derived from extracellular matrix to physiological saline or medium.

The alginic acid solution is 0.1 to 10% by weight, 0.1 to 8% by weight, 0.1 to 6% by weight, 0.1 to 4% by weight, 0.1 to 2% by weight, 0.5 to 10% by weight, 0.5 to 8% by weight, alginate To 6% by weight, 0.5 to 4% by weight, 0.5 to 2% by weight, 1 to 10% by weight, 1 to 8% by weight, 1 to 6% by weight, 1 to 4% by weight or 1 to 2% by weight, for example , 1.25% by weight may be included. Depending on the concentration of alginic acid can control the strength of the material in the same cross-linking solution, the solution of too high concentration in the range beyond the above range may cause high needle viscosity, there is a problem that the low viscosity in the case of too low concentration, Microsized capsules are prepared that are stable at the alginic acid solution concentrations above.

The microcapsule manufacturing method may further include filtering the alginic acid solution after the alginic acid solution preparation step.

The filtering may be filtering with a 0.2 um syringe filter, but is not limited thereto.

The mixing step may be to mix the cell dispersion solution and the alginic acid solution in a volume of 1: 0 to 1: 0, for example, 1: 4 volume, to adjust the final cell concentration and the concentration of alginic acid according to the mixing ratio Can be.

The concentration of cells contained in the mixed solution in which the cell dispersion and the alginic acid solution are mixed in the mixing step is 10 ³ to 10 ⁹ cell / mL, 10 ⁴ to 10 ⁹ cell / mL, 10 ⁵ to 10 ⁹ cell / mL, 10 ³ To 10 ⁸ cell / mL, 10 ⁴ to 10 ⁸ cell / mL, 10 ⁵ to 10 ⁸ cell / mL, 10 ³ to 10 ⁷ cell / mL, 10 ⁴ to 10 ⁷ cell / mL or 10 ⁵ to 10 ⁷ cell / It may be mL, for example, it may be 1.0 X 10 ⁶ cell / mL. At the cell concentration lower than the above range, the physiological activity of the cell is low and the growth is very slow after encapsulation, and if it is higher than the above range, it is difficult to concentrate the cells and it is difficult to supply nutrients to the encapsulated cells.

The cell dispersion solution may include cells recovered and / or concentrated from cultured cells, but is not limited thereto.

The forming step includes applying a voltage to the mixed solution; And adding a crosslinking solution.

The voltage is 1 to 16 kV, 1 to 14 kV, 1 to 12 kV, 1 to 10 kV, 2 to 16 kV, 2 to 14 kV, 2 to 12 kV, 2 to 10 kV, 4 to 16 kV, 4 to 14 kV, 4 to 12 kV, 4 to 10 kV, 6 to 16 kV, 6 to 14 kV, 6 to 12 kV, 6 to 10 kV, 8 to 16 kV, 8 to 14 kV, 8 to 12 kV or 8 to 10 kV, for example, may be 10 kV, but is not limited thereto.

The addition of the crosslinking solution is 1 to 10 mL / h, 1 to 9 mL / h, 1 to 8 mL / h, 1 to 7 mL / h, 1 to 6 mL / h, 2 to 10 mL / h, 2 To 9 mL / h, 2 to 8 mL / h, 2 to 7 mL / h, 2 to 6 mL / h, 3 to 10 mL / h, 3 to 9 mL / h, 3 to 8 mL / h, 3 to 7 mL / h, 3 to 6 mL / h, 4 to 10 mL / h, 4 to 9 mL / h, 4 to 8 mL / h, 4 to 7 mL / h, 4 to 6 mL / h, 5 to 10 It may be added at a flow rate of mL / h, 5 to 9 mL / h, 5 to 8 mL / h, 5 to 7 mL / h or 5 to 6 mL / h, for example, a flow rate of 6 mL / h It may be added as, but is not limited thereto.

The crosslinking solution may be an aqueous solution including sodium chloride (NaCl), and barium chloride (BaCl ₂ ), calcium chloride (CaCl ₂ ), or strontium chloride.

The sodium chloride may be a concentration of 0.05 to 0.3 M, 0.05 to 0.25 M, 0.05 to 0.2 M, 0.1 to 0.3 M, 0.1 to 0.25 M or 0.1 to 0.2 M, for example, may be 0.15 M concentration. . The concentration range is physiologically suitable for cell growth and activity, and the sodium chloride solution outside the concentration range lowers the activity of the cell and gradually leads to death.

The barium chloride, calcium chloride or strontium chloride is 0.01 M to 0.1 M, 0.01 M to 0.09 M, 0.01 M to 0.08 M, 0.01 M to 0.07 M, 0.01 M to 0.06 M, 0.01 M to 0.05 M, 0.01 M to 0.04 M Or it may be a concentration of 0.01 M to 0.03 M, for example, may be 0.02 M concentration. The strength of the capsule varies depending on the concentration of the crosslinking solution, and the concentration of the crosslinking solution can be varied depending on the direction of the desired differentiation. Capsules are difficult to produce at concentrations below this range, and cell viability is lower at concentrations above that range.

The forming step may be performed through electrospraying systems, emulsion dispersion, spray, or microfluidic systems, but is not limited thereto, and may be manufactured using any conventionally known method. Do.

The average diameter of the microcapsules containing alginic acid may be adjusted by adjusting the voltage and flow rate of the forming step.

Another aspect of the invention relates to a stem cell three-dimensional culture method comprising the following steps.

Preparing a microcapsule for stem cell culture comprising alginic acid;

A supporting step for supporting stem cells in the microcapsules; And

Culture step of culturing stem cells.

The microcapsule preparation step and the supporting step may be performed at the same time, but are not limited thereto.

The culturing step includes a washing step of washing the microcapsules with sodium chloride (NaCl) solution and a culture medium addition step of putting the cell culture solution; Transferring to a cell culture dish; And culturing in a cell incubator; but may not be limited thereto.

The cell culture medium may be added based on the basic cell culture medium FBS, antibiotic-antimycotic (antibiotics-antimycotic), for example, may be 10% FBS, 1% antibiotic-antibacterial to MEM-alpha cell culture medium It is not limited to this.

The effect of the present invention is that by culturing the cells on the microhydration gel attached to the extracellular matrix to mimic the living environment, high concentration culture of cells, induction of rapid proliferation rate, stable titer of cells, ease of recovery In this way, the proliferation of cells required for cell application research and clinical application can be efficiently and stably implemented.

By comparing the blood glucose consumption between the cells cultured using the microcapsules and the cells cultured by the general culture method, the number of cells, the growth rate of the cells is compared, and the expression and differentiation capacity of stem cell markers are compared. The titer retention of stem cells was tested, and it was found that mesenchymal stem cells can be cultured efficiently by the method of the present invention.

"Microcapsules containing alginic acid" of the present invention is a particle having an average diameter of several tens to hundreds of micrometers containing the alginic acid polymer as a main component, means a particle having a cell for culture therein, For convenience, it can be simply described as "alginic acid microcapsules" or "alginic acid capsules", and unless otherwise stated, all have the same meaning.

The present invention provides a method for preparing microcapsules for supporting stem cells in alginic acid modified with alginate or extracellular matrix-derived stem cells and high concentration culture of the stem cells, increase in stem cell growth rate, stable activity maintenance, and recovery. More specifically, a micro-sized capsule structure capable of culturing stem cells in three dimensions is formed of alginic acid or alginic acid modified with an peptide derived from an extracellular matrix, and more specifically than a two-dimensional culture using a culture dish. Efficient cultivation enables spatial and temporal efficiency enhancement, cell activity, and mass production.

1 is a process schematic diagram of high-efficiency bio simulation three-dimensional culture of stem cells according to an embodiment of the present invention.

Figure 2 is a photograph observing cells cultured in alginic acid capsules according to an embodiment of the present invention with an optical microscope.

Figure 3 is a photograph observing cells cultured in alginic acid capsules according to an embodiment of the present invention with an optical microscope.

Figure 4 is a photograph observed by Live / dead staining cells cultured in alginic acid capsules according to an embodiment of the present invention.

Figure 5 is a graph comparing the glucose changes and consumption of cells carried in alginic acid substituted with a peptide derived from the extracellular matrix and the general two-dimensional culture according to an embodiment of the present invention.

Figure 6 is a graph comparing the number of cells recovered in the two culture method according to an embodiment of the present invention.

Figure 7 is a photograph observed by Live / dead staining of the cells contained in the alginic acid capsules under the oxidative stress according to an embodiment of the present invention.

8 is a graph observed by Live / dead staining of cells supported in alginic acid capsules under an oxidative stress according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

실시예 1. 세포외기질 유래의 펩타이드의 알긴산 부착Example 1 Alginic Acid Adhesion of Peptides Derived from Extracellular Substrates

MES (2- (N-morpholino) ethanesulfonic acid) buffer was diluted to 0.2 M, then 0.3 M sodium chloride (NaCl) was added and the pH was titrated to 6.5. After dissolving alginic acid in the buffer at 1% by weight, NHS and EDC were added in a 1: 2 molar ratio to induce a reaction of EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysulfosuccinimide). Put it. Then, per 1 g of alginic acid, 22 umol of peptide was added and stirred for 24 hours, followed by dialysis for 4 days using a 3500 Da size membrane, and lyophilization. Peptides derived from extracellular matrix that can interact with integrins of cells were chemically attached to alginic acid through an EDC / NHS reaction.

실시예 2. 줄기세포 담지된 알긴산 캡슐 제작Example 2. Preparation of Stem Cell Supported Alginate Capsule

Alginate solution was prepared by adding alginate substituted with 1.25% by weight of alginate or an extracellular matrix-derived peptide (arginine-glycine-aspartic acid (RGD)) to distilled water, using a 0.2 μm syringe filter. Was filtered. Then, the cells were seeded at 4,000 to 5000 / cm ² in a 75 cm ² plastic cell dish, and when the cells proliferated in an area of about 90%, the cultured cells were treated with 0.05% trypsin-EDTA and then 1000 Centrifugation was performed for 10 minutes at rpm, and the mixed solution was prepared by mixing the alginic acid solution in a 1: 4 volume ratio so that the concentration of the last cells was 1.0 X 10 ⁶ cells / mL.

Then, 0.15 M sodium chloride (NaCl) and 0.02 M barium chloride (BaCl ₂ ) were added to the distilled water to prepare a crosslinking solution of the alginic acid solution.

Apply the mixed solution to a constant voltage of 1 to 16 kV according to the size of the capsule using the electrospinning equipment, ionic cross-linking occurs while falling at a fixed rate of 1 to 10 mL / h to the cross-linking solution By induction, a hydrogel capsule was prepared in which cells were loaded.

Depending on the supplied voltage and flow rate, the size of the capsule can be made differently. Using a 27 G nozzle, supply voltage is 10 kV and flow rate is 6 mL / h. Adjusted to. The formed capsules were sufficiently washed with a 0.15 M sodium chloride (NaCl) solution, and then the cell culture solution was placed in a cell culture dish and cultured in a cell incubator.

실시예 3. 배양액의 포도당 소모량 및 소모속도 비교Example 3. Comparison of glucose consumption and rate of culture

Cell growth was compared in the method of the present invention (Example) and two-dimensional culture (Comparative Example). Specifically, umbilical cord-derived human mesenchymal stem cells or capsules carrying the cells were cultured in a plastic plate of 75 cm ² , and 10 mL of the same medium was added to each other to compare the consumption and the rate of consumption for the first 48 hours.

Glucose and time-varying changes were observed immediately after the addition, and in two-dimensional culture, there was no significant change in glucose consumption, like the lag phase of the cell, and there was a tendency to decrease from about 24 hours later. Started. In contrast, the capsules carrying the cells were found to immediately start as the culture was consumed.

For effective culturing of stem cells, cells were microencapsulated using an electrospray system with alginic acid to which peptides derived from extracellular matrix were attached.

As can be seen in Figures 2 to 4, before and after encapsulation, the survival rate of the stem cells is live / dead fluorescence staining, and counts the number of living cells (green) and dead cells (red) therefrom and has activity when counting. It was confirmed that the percentage of cells is maintained more than 90%.

Unlike the two-dimensional culture method in the conventional culture flask, the three-dimensional support of the present invention can activate the metabolic process of the cells in a spatially high density, high efficiency culture. Thus, the concentration of glucose in the culture medium essential for cell growth was observed, and the metabolic processes of stem cells were compared, and the results are shown in FIG. 5 and Table 1. FIG.

	2D culture2D culture (비교예)(Comparative Example)	3D culture3D culture (실시예)(Example)
Total glucose consumption for 48 hours (mg/dL)Total glucose consumption for 48 hours (mg / dL)	12.5 mg/dL12.5 mg / dL	88.5 mg/dL88.5 mg / dL

As can be seen in FIG. 5, the comparative example consumed 12.5 mg / dL of glucose for the first 48 hours after inoculation culture, and the example consumed 88.5 mg / dL, and the example consumed glucose about 7 times faster than the comparative example. Confirmed to show. That is, the comparative example had to replace the medium at a 48-hour cycle, but the example had to change the culture medium at about 5-6 hour cycles due to the rapid decrease of glucose in the culture medium.

실시예 4: 알긴산 마이크로겔에 배양된 줄기세포 회수Example 4 Recovery of Stem Cells Cultured on Alginic Acid Microgel

50 mM EDTA was prepared based on physiological saline (pH 7) and used for decrosslinking of alginic acid.The EDTA solution was added to a capsule containing stem cells and incubated at 37 ° C. for 3 to 4 minutes. After the capsule was sufficiently decrosslinked, the supernatant was removed by centrifugation and the stem cell pellet was easily obtained. After several times washing with physiological saline, the stem cells were dispersed. Culture, storage, and application was carried out.

Stem cells were internally embedded in alginic acid, so it was possible to simply decrosslink alginic acid with EDTA solution and recover the cells without the use of enzymes such as trypsin. At this time, by washing with physiological saline it was able to remove alginic acid and EDTA components.

실시예 5. 세포 담지 및 생장 속도Example 5. Cell Support and Growth Rate

Unlike the conventional two-dimensional culture method in the culture flask, the three-dimensional support and culture in the hydrogel containing the extracellular matrix peptide of the present invention was spatially high density culture, it was possible to promote cell growth. The cell growth rate and the amplification ratio were compared by comparing the number of stem cells obtained during passage and the cells before supporting, and the results are shown in FIGS. 6 and 2.

	2D culture2D culture (비교예)(Comparative Example)	3D culture3D culture (( 실시예Example ))
Day 0(세포수 / ml)Day 0 (cell count / ml)	3.0 x 10⁵ 3.0 x 10 ⁵	3.0 x 10⁶ 3.0 x 10 ⁶
Day 5(세포수 / ml)Day 5 (cell count / ml)	2.4 x 10⁵ 2.4 x 10 ⁵	3.0 x 1073.0 x 107

As can be seen in Figure 6 and Table 2, two-dimensional culture in the existing culture flask was inoculated at the concentration of the initial 3 x 10 ⁵ cells / mL to obtain stem cells of about 3 x 10 ⁶ cells / mL after 5 days In comparison with the 2.2-day doubling time, in three-dimensional culture, the initial culture was possible at a concentration of 3 x 10 ⁶ cells / mL, and 2 days later, 7 x 10 ⁶ cells / mL stem cells were recovered. It was. It is possible to culture 10 times higher density based on the initial concentration, the culturing method of the present invention showed a growth rate of 2-3 times as fast as 0.81 days.

실시예 6. 산화스트레스 환경에서의 활성 시험Example 6 Activity Test in Oxidative Stress Environment

Stem cells are microencapsulated in alginic acid substituted with alginic acid or peptide derived from extracellular matrix, and then cultured in a normal culture solution for an initial day, and then hydrogen peroxide is added to H ₂ O such as 100 uM, 200 uM, 400 uM and 800 uM. Stem cells were exposed to hydrogen peroxide at ₂ concentrations in the culture medium, and live / dead staining and microscopic observation were performed 24 hours later to confirm cell viability. The results are shown in FIGS. 7 to 8 and Table 3 .

H2O2 conc. (uM)H2O2 conc. (uM)	00	100100	200200	400400	800800	10001000	15001500	20002000
Viability (%)Viability (%)	99.9499.94	99.5399.53	9.279.27	96.6196.61	77.577.5	78.5978.59	64.0564.05	53.5453.54

As can be seen in Figures 7 to 8 and Table 3, in the culture of stem cells, there is a risk that can adversely affect cell activity, including oxidative stress, such as reactive oxygen species according to changes in the external environment. Three-dimensional culture using alginic acid and the use of alginic acid with extracellular matrix peptides reinforce the signals that can reduce the autologous apoptosis of the cells, thereby improving the viability of stem cells during culture. It was confirmed that there is. When hydrogen peroxide was added to the culture medium to induce an oxidative stress environment, the activity of stem cells cultured by microencapsulation using alginic acid or alginic acid to which RGD peptides were attached was superior to those maintained in conventional flasks. In addition, it showed better results when incubated in the alginic acid to which the extracellular matrix peptide is attached. This may be due to the three-dimensional culture, the soft environment of the cell part by the hydrogel, the local high density stem cell culture, and the activation of the intracellular activation signaling system using the extracellular matrix peptide.

Claims

Microcapsules for cell culture comprising alginic acid.
The microcapsule for cell culture according to claim 1, wherein the alginic acid is substituted with a peptide derived from an extracellular matrix.
According to claim 2, The extracellular matrix-derived peptide is one or more selected from the group consisting of RGD motif, DGEA motif, REDV motif, IKVAV motif, VPGVG motif, YIGSR motif and KHIFSDDSSE motif, microcapsules for cell culture .
The microcapsules for cell culture according to claim 1, wherein the microcapsules have an average diameter of 30 to 1000 um.
The microcapsules for cell culture according to claim 1, wherein the cells are stem cells.
According to claim 5, The stem cells are mesenchymal stem cells, blood stem cells, neural stem cells, muscle stem cells, myocardial stem cells, embryonic stem cells and induced pluripotent stem cells that are one or more selected from the group consisting of stem cells. Microcapsules for cell culture.
The microcapsules for cell culture according to claim 1, wherein the microcapsules for cell culture are supporting cells.
Microcapsules comprising the alginic acid comprising the following steps:

Alginic acid solution manufacturing step of preparing an alginic acid solution;

A mixing step of preparing a mixed solution by mixing the cell dispersion solution and the alginic acid solution;

Forming step of forming microcapsules.
The method of claim 8, wherein the alginic acid is substituted with a peptide derived from an extracellular matrix.
The method of claim 9, wherein the extracellular matrix-derived peptide is at least one selected from the group consisting of an RGD motif, a DGEA motif, a REDV motif, an IKVAV motif, a VPGVG motif, a YIGSR motif, and a KHIFSDDSSE motif.
The method of claim 8, wherein the alginic acid solution comprises 0.1 to 10 wt% of alginate.
The method of claim 8, wherein the mixed solution is a cell dispersion solution and an alginic acid solution mixed at a volume of 1: 4.
The method of claim 8, wherein the concentration of the cells contained in the mixed solution is 1.0 X 10 6 cells / mL, microcapsules manufacturing method.
The method of claim 8, wherein the cells are stem cells.
The method of claim 14, wherein the stem cells are mesenchymal stem cells, blood stem cells, neural stem cells, muscle stem cells, myocardial stem cells, embryonic stem cells and induced pluripotent stem cells that are one or more selected from the group consisting of,
The method of claim 8, wherein the forming step is performed with electrospraying systems, emulsion dispersions, sprays or microfluidic systems.
The method of claim 8, wherein the microcapsules have an average diameter of 30 to 1000 um.
Cell three-dimensional culture method comprising the following steps:

Preparing a microcapsule for cell culture containing alginic acid;

A supporting step for supporting cells in the microcapsules; And

Cultivation step of culturing the cells.
The cell three-dimensional culture method of claim 18, wherein the microcapsule preparation step and the supporting step are performed simultaneously.
The method of claim 13, wherein the culturing step,

A washing step of washing the microcapsules with a sodium chloride (NaCl) solution;

Adding a culture medium to the cell culture;

Transferring to a cell culture dish; And

Culturing in a cell incubator;

That comprises, cell three-dimensional culture method.