KR100785049B1 - Method and apparatus for forming and growing embryoid body - Google Patents

Abstract

The present invention relates to a stem cell differentiation, and to a cell culture method and apparatus for generating embryoid bodies (EBs) of uniform size and shape, growth of generated EBs and subsequent cell differentiation.

The cell culture apparatus has a small diameter of 1.0 to 2.6 mm and uniformly and rapidly forms an EB (embryoid body) in a cylindrical vessel with an open upper and lower surface, and the bottom of the culture vessel for growing the formed embryoid body is fine. Since the micro-pore layer is bonded, the formed EB (embryoid body) does not pass to the bottom while the supply of the culture solution from the bottom is made smoothly and can be cultured for a long time. Using the present invention, embryonic body formation and cell differentiation can be rapidly and quantitatively progressed.

Embryonic Body, Embryonic Formation Layer, Culture Vessel Layer, Microporous Membrane Layer, Stem Cell, Differentiation

Description

Method and apparatus for forming and growing embryoid body

1A to 1H are a perspective view and a side view showing the stem cell differentiation apparatus and each component according to the present invention.

Figure 2 is a flow chart schematically showing a stem cell differentiation method according to the present invention.

Figure 3 is a perspective view showing the embryonic formation step of the stem cell differentiation method according to the present invention.

Figure 4 is a perspective view showing the embryonic growth stage of the stem cell differentiation method according to the present invention.

Figure 5 is a schematic diagram showing the manufacturing process of the microporous membrane of the stem cell differentiation apparatus according to the present invention.

Figure 6a is a picture of the cells differentiated according to the stem cell differentiation method according to the present invention Figure 6b is a cell differentiated by a conventional hanging drop method (hanging drop method).

Figure 7a is a result of measuring the stem cell markers of cells differentiated according to the stem cell differentiation method according to the present invention.

Figure 7b is compared with the result of measuring the differentiation of the differentiated cells (Device) according to the stem cell differentiation method according to the present invention (Device) and the result of measuring the differentiation of the cells differentiated by the conventional hanging drop method (HD) It is shown.

* Description of the main parts of the drawings *

11: Embryonic Formation Layer 12: Culture Vessel Layer

13: microporous membrane layer 14: bottom support layer

21: protrusion 22: groove

23: hole

31: cylindrical container 32: supplementary container

33: culture vessel 34: hemispherical droplets

35: embryonic body (EB)

41: pipette

The present invention relates to an effective stem cell differentiation, and to a cell culture method and apparatus capable of rapid and uniform production of an embryonic body (EB), growth and cell differentiation of the generated embryonic body (EB).

In conventional stem cell differentiation, most of the Hanging drop method (Hanging drop method) is used to form the EB. This is done by dropping a drop of culture medium containing cells on the inner side of the lid of the cell culture dish and inverting them so that the cells are collected at the tip of the drop by gravity and aggregated between the cells and the cells (cell mass) called EB. Make However, the size of the resulting droplets is 3mm in average diameter, which is too large for the cells and the shape of the droplets is not uniform. Therefore, it takes at least 3 days to form EBs. In order to obtain two EBs, it takes different time to form each EB, and the formation of an EB having a uniform size cannot be expected. In addition, EB is formed for more than 3 days during this process. During this time, it is not easy to exchange the droplets of the medium, and as time goes by, the state of the medium becomes poor for the cells to be cultured. Will receive. In general, EB is added to a bacterial dish so that the cells do not adhere to the bottom of the dish but remain floating after EB formation, and differentiate the desired cells from the stem cells. However, this also has some problems. Incubate in a dish that is too large for the size of the EB (about 200 ~ 300 um), resulting in the consumption of a large amount of unnecessary cell culture. In addition, many EBs in a wide dish transmit and receive many signals for differentiation. In such a large dish, differentiation-related signals such as in vivo microenvironment cannot be overloaded. There is a problem.

In order to solve the above problems, an object of the present invention is to modify an existing hanging drop method to form an EB quickly and uniformly.

It is another object of the present invention to efficiently perform cell differentiation by growing the formed EB under conditions that enable smooth signal transduction between cells.

Another object of the present invention is to prevent cells from degrading in quality over time so that cells grow in a healthier state.

Another object of the present invention is to allow the formation of EBs and the growth of the formed EBs efficiently in one system.

In order to achieve the above object, the method for forming an embryoid body from the stem cells according to the present invention, the cylindrical container is open at both ends vertically and vertically by filling the culture medium containing the stem cells in the cylindrical container vertically At the end of the cylindrical container, the culture medium is held by the surface tension while maintaining the hemispheric shape, and the stem cells are collected at the end of the drop of the hemispherical medium suspended by the gravity by the end of the cylindrical container (Embryoid Body, EB). It is characterized by forming a).

In the method for forming an embryonic body from the stem cells according to the present invention, the cylindrical container is characterized in that the diameter of 1.0 to 2.6mm.

In the method for forming an embryoid body from stem cells according to the present invention, the cylindrical container is characterized in that the size of the embryonic body formed by adjusting so that two or more and all cylindrical containers have the same diameter.

In the method for forming an embryoid body from the stem cells according to the present invention, the cylindrical container is two or more, the two or more cylindrical containers are adjusted to have a variety of diameters, to vary the size of the formed embryoid body It features.

In the method for forming an embryonic body from the stem cells according to the present invention, by supplementing the culture medium in the cylindrical container to prevent the culture medium from being evaporated and lost from the cylindrical container and to prevent degradation of the culture medium due to cell metabolism It features.

Stem cell differentiation method according to the present invention,

A first step of forming an embryoid body by a method of forming an embryoid body from the stem cells; And

The embryonic body formed in the first step is a cylindrical container in which the embryonic body is formed at the end of the drop of the culture medium through the first step, into the culture vessel containing the culture medium for embryo growth, the end of the drop of the culture medium to the embryo growth The embryos are dropped into the culture medium for embryonic growth by contact with the culture medium for embryonic growth by placing them at a height that is in contact with the culture medium for the embryo culture. And a second step of growing the embryonic body in a state of being attached to the culture vessel bottom.

In the stem cell differentiation method according to the present invention, the second step is characterized in that the growth of the embryo in a culture vessel of 2.0 to 4.0mm in diameter.

On the other hand, according to the present invention, a device for forming an embryoid body from stem cells is provided with a plurality of vertically open cylindrical vessels at both ends, so that the culture fluid is maintained on the surface tension while maintaining the hemispherical shape at the end of the vertically-shaped cylindrical vessel. By hanging by, the stem cells are collected at the end of the drop of the hemisphere-shaped culture solution suspended by the end of the cylindrical container by gravity to form a spherical embryo (Embryoid Body, EB).

In the above embryonic body forming apparatus according to the present invention, the cylindrical container is preferably in the range of 1.0 to 2.6mm in diameter.

In the embryonic body forming apparatus according to the present invention described above, it is preferable that the cylindrical containers all have the same diameter, or the size of the embryonic bodies formed by having various diameters.

The embryonic body forming apparatus according to the present invention is preferably a container surrounding the upper portion of the cylindrical container further comprises a replenishment container for replenishing and supplying the culture solution to the cylindrical container.

On the other hand, the apparatus for differentiating stem cells according to the present invention is characterized in that it comprises an embryonic body forming layer comprising the embryonic body forming apparatus described above.

The stem cell differentiation apparatus according to the present invention is stacked below the embryonic body forming layer, wherein the culture vessel layer for growth of the embryonic body formed in the embryonic body forming layer comprises the same number as the cylindrical container as the culture vessel layer The culture vessel is characterized in that it further comprises a culture vessel layer, characterized in that the upper and lower open tube form.

The stem cell differentiation apparatus according to the present invention is a microporous membrane layer composed of a microporous membrane which is stacked below the culture vessel layer and is in contact with the culture vessel. It characterized in that it further comprises a microporous membrane layer, characterized in that for passing the culture solution from.

In the stem cell differentiation apparatus according to the present invention, the culture vessel of the culture vessel layer is preferably 2.0 to 4.0mm in diameter.

In the stem cell differentiation apparatus according to the present invention, it is preferable that the embryonic body forming layer and the culture container layer are stacked such that the cylindrical container of the embryonic body forming layer and the culture container of the culture container layer are disposed at a height overlapping each other.

The stem cell differentiation apparatus according to the present invention, which is stacked below the microporous membrane layer, further comprises a bottom support layer that combines the culture vessel layer and the microporous membrane layer and protects the microporous membrane layer. It features.

In the stem cell differentiation apparatus according to the present invention, the microporous membrane of the microporous membrane layer is preferably ECM coated to improve the adhesion of the embryo.

Hereinafter, the method and apparatus according to the present invention will be described in detail.

Figure 1a to 1g is a diagram showing the stem cell differentiation device and each component according to the present invention. FIG. 1A shows an EB formation layer 11, FIG. 1B shows a culture well layer 12, FIG. 1C shows a micro-hole membrane 13, and FIG. 1D shows A bottom layer 14 is shown. According to FIG. 1E, the stem cell differentiation apparatus according to the present invention includes an embryonic body formation layer (EB formation layer) 11, a culture well layer 12, and a micro-hole membrane layer 13. ) Are stacked in order, and optionally, the bottom support layer 14 is further stacked below the microporous membrane layer. The protrusions 21 at the centers of both ends of the embryonic body forming layer 11 are inserted into and fixed in the grooves 22 at the centers of both ends of the culture chamber layer 12 for the bonding between the embryonic body forming layer 11 and the culture vessel layer 12. . In order to bond the culture vessel layer 12, the microporous membrane layer 13, and the bottom support layer 14, acrylic screws are inserted and fixed in four holes 23 existing at the corners of each layer.

1F is a perspective view of the embryonic body forming layer 11 (internal structure is indicated by a dotted line). The replenishment container 32 surrounds the upper circumference of the cylindrical container 31. At the center of both ends there is a projection 21 for bonding with the culture vessel layer.

Fig. 1G is a side view of the embryonic body forming layer 12 (internal structure is indicated by dotted lines). A plurality of cylindrical containers 31 are provided, and around the top thereof, the same number of replenishing containers 32 as the cylindrical containers are provided. In addition, in the cylindrical container 31, the embryonic body forming layer 11 and the culture container layer 12 are stacked so as to overlap with the culture container 33 of the culture container layer 12.

3 shows the embryo formation layer 11 in more detail. In the embryo forming layer of FIG. 3, a total of 25 cylindrical containers 31 for forming EBs are provided, and a supplementary container 32 surrounds each cylindrical container upper part. The replenishment container 32 serves to prevent the culture solution in the cylindrical container 31 from evaporating and to replenish the culture solution. The replenishment container 32 has a top diameter of 3 to 5 mm, preferably about 4 mm, and a depth of 1 to 3 mm, preferably about 2 mm. The replenishment vessel 32 prevents the culture medium from evaporating from the cylindrical container forming the EB to the outside and replenishes the culture solution to prevent the culture from deteriorating due to the very small amount of the culture medium of the EB forming cylinder. Play a role. The culture medium supplied from the replenishing container 32 enables EB to be cultured for a long time in the EB forming cylindrical container 31. The embryonic formation layer 11 and the replenishment vessel 32 may be integral with each other.

1H shows an example in which the cylindrical container 31 and the replenishment container 32 are combined. By filling the culture medium in the culture vessel 32 in addition to the cylindrical container 31 filled with the culture solution, it is possible to prevent the culture solution from evaporating from the cylindrical container 31 and to mitigate the deterioration of the culture medium as the cells grow.

It is preferable that the EB forming cylindrical container 31 is 1.0-2.6 mm in diameter. More preferably 1.2 to 2.0 mm. Most preferably about 1.6 mm. If the diameter is less than 1.0 mm, the amount of the culture solution in the cylindrical container may be small and evaporate in a short time. If the diameter is larger than 2.6 mm, the size of the hemispherical droplets hanging at the end of the cylindrical container may be too large, causing delayed EB formation. Occurs. On the other hand, the height of the cylindrical container is approximately 1 to 3 mm, preferably about 2 mm. If the height is less than 1mm, a small amount of medium enters to cause the medium to evaporate. If the height is more than 3mm, the weight of the medium in the container causes the droplet to overcome the surface tension of the wall and collapse, and the device becomes too high. Filling a culture vessel containing stem cells in a cylindrical container having the size as described above is a problem of the existing hanging drop method (hanging drop method), that is because the size of the drop is too large for the cells and the resulting drop form is also irregular The problem of non-uniformity and the problem of slow EB formation can be solved. In other words, cells can aggregate quickly by a narrow space of about 1.6 mm in diameter, and all the cylindrical vessels in the device have the same diameter, so that the droplets formed at the end also have the same shape and size. This allows uniform EB formation in all cylinders.

The culture vessel layer 12, the microporous membrane layer 13, and the bottom support layer 14 are places for attaching and culturing the EB formed in the embryonic body forming layer 11. The culture vessel 33 preferably has a diameter of 2 to 4 mm, more preferably about 3 mm. If the diameter is less than 2mm it is difficult to accommodate the cylindrical container, if the diameter exceeds 4mm it becomes too large compared to the size of the EB intercellular signal transmission is not smooth. The height of the culture vessel 33 is preferably 2 to 3 mm, more preferably 2.5 mm. If the height is less than 2mm, the culture capacity is reduced, and if the height exceeds 3mm, intercellular signal transmission is not smooth. Each of the EBs in the narrow culture vessel 33 as described above can smoothly receive differentiation signals that they send. The signal sent from the other EB may be received through the microporous membrane layer 14. Another role of the micro-hole membrane is to have a function of smoothing the movement of the culture solution from the bottom to the top without passing the formed EB. The diameter of the holes of the microporous membrane is preferably 25 to 35 mu m, most preferably about 30 mu m. If the diameter is less than 25 μm, medium exchange is not easy, and if the diameter is more than 35 μm, the diameter is too large, so that EB is not easy to attach. The distance between the holes is preferably 5 to 15 mu m, most preferably about 10 mu m. If the distance is less than 5µm, the distance between holes is too small for EB to attach, and if it exceeds 15µm, as mentioned above, the number of holes decreases, resulting in poor medium exchange. The microporous membrane is preferably coated with an extracellular matrix (ECM) on the top. The cell adhesion function is improved through the coating. The material of the microporous membrane may be selected from those that are transparent and easy for ECM to coat. The most preferred material for microporous membranes is negative photoresist SU-8 (Microchem Inc), which is easy to process, transparent and easy to coat.

The bottom support layer 14 serves to protect the microporous membrane to be maintained without breaking.

Hereinafter, a method for stem cell differentiation according to the present invention, that is, an EB formation method and a growth method of the formed EB will be described in detail. Stem cell differentiation method according to the present invention can be divided into EB formation step and EB growth step.

Figure 2 shows the whole cell culture process for cell differentiation. First, a culture solution containing stem cells is injected into an EB-forming cylindrical container [S21]. Thereafter, EB is formed within an average of one day [S22]. The cell differentiation device of the present invention is immersed in a general culture dish containing the culture solution [S23]. The culture medium enters into the culture vessel 33 through the microporous membrane layer 13 from the culture dish, and the embryos suspended at the end of the cylindrical vessel 31 fall off and adhere to the microporous membrane layer 13 by contact with the culture medium. [S24].

3 shows an EB formation step. A small pipette 41 is used to fill the cylindrical container by injecting a culture solution containing stem cells into each of the EB-forming cylindrical containers 31. At the bottom of the cylindrical container 31, the culture solution is suspended while maintaining the hemisphere shape without falling down by the surface tension. The culture medium in the EB-forming cylindrical container 31 is filled in the supplementary container 32 in order to keep the fresh state without evaporation. Spreading cells are collected by gravity into the tip of hemisphere droplet 34 and spherical EB is produced 35 within one day.

4 shows the EB growth stage. In order to grow the formed EB until differentiation, the apparatus of the present invention shown in FIG. 1 is immersed about half in a general culture dish containing the culture solution. Then, the culture solution in the culture dish enters the culture vessel 33 through the microporous membrane layer 13 and the culture solution goes up to the place where the EB is formed. When the culture solution of the culture vessel 33 comes into contact with the EB 35 suspended in the EB-forming cylindrical vessel, the EB instantly falls into the culture vessel 33. The fallen EB adheres and grows on the ECM-coated microporous membrane.

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

1. Preparation of Microporous Membrane

First, a microporous membrane was prepared according to the manufacturing process of the microporous membrane shown in FIG. 2. First, wafer cleaning was performed (1). A 4 inch Soralim glass wafer (in the form of a 4 inch circular disk) was cleaned with 300 ml DW (deionized water, sterile water) and dried for 1 minute with an N ₂ blower. Then SU-8 negative photoresist coating was performed (2). SU-8 100 series (Microchem. Inc.) was applied onto a glass wafer to make a 100 um thick SU-8 layer, followed by spin coating at 3000 rpm. The SU-8 coated wafer was baked for 10 minutes on a 65 ° C. hotplate and baked for 30 minutes on a 95 ° C. hotplate. Then, an ultraviolet exposure process was performed (3). Patterning was performed by exposure for 21.5 seconds using a lithography equipment (MA6). Then, the SU-8 development process was performed (4). Soaking the wafer for 10 minutes in a vessel containing the SU-8 developer (Microchem. Inc.) completes the development. SU-8 coated on the wafer must be separated from the wafer for use as a micro-pore layer. Therefore, a procedure was then followed to separate the SU-8 layer from the wafer (5). The wafers were immersed in 300 ml of DW (deionized water) and sonicated for 30 seconds in order to reduce the adhesion between the wafer and SU-8 and ensure complete separation. After dipping in 250 ml of IPA (2-propanol) for 10 seconds, the thin transparent SU-8 layer could be separated from the wafer.

The diameter of the holes of the prepared microporous membrane was 30 μm, and the distance between the holes and the holes was 10 μm.

2. Manufacture of stem cell differentiation device

The microporous membrane prepared as described above was used to manufacture the device of FIG. 1. The EB forming layer 11, the culture vessel layer 12, the microporous membrane layer 13, and the bottom support layer 14 were sequentially stacked to manufacture the apparatus of FIG. 1. The overall size of the device was 32 x 32 x 7.8 mm. In the apparatus used in this embodiment, the diameter of the cylindrical container 31 for forming the EB of the EB forming layer 11 was 1.6 mm and the height was 2 mm, and the diameter of the refill container 32 was 4 mm and the height was 2 mm. The total number of the cylindrical containers 31 and the refill containers 32 was 25 pieces. In addition, the diameter of the culture vessel 33 of the culture vessel layer 12 was 3 mm, and the height was 2.5 mm.

3. Perform differentiation process of stem cells

Stem cells were actually cultured using the stem cell differentiation device prepared as above.

P19 EC cells (ATCC number CRL-1825), a mouse embryonal carcinoma cell line derived from the C3H / He mouse strain, was prepared using 10% fetal bovine serum (FBS, Gibco). Dulbeco's modified Eagl's medium (DMEM, Gibco) (hereinafter referred to as EC medium) was cultured in cell culture dishes. Cultured P19EC cells were isolated from the cell culture dish by treatment with 0.05% Trypsin-EDTA (sigma) for 1 minute. 5000 stem cells were equally diluted in 7 μl of EC medium, and the culture solution containing the cells was injected into each cylindrical container. After 24 hours, it was confirmed that embryonic bodies of about 0.2 mm were formed in the hemisphere droplets suspended at the end of the cylindrical container 31.

At the same time, 500 stem cells were equally diluted in 10 µl of EC medium by the conventional hanging drop method, and then, the drop of the culture medium containing the cells was dropped on the lid of the cell culture dish. It was confirmed that embryonic bodies were formed after 2 to 3 days.

Each embryo formed above was further cultured for up to 6 days to observe the shape of the embryo.

When the stem cells were injected into the cell differentiation apparatus according to the present invention in the above, when embryos were formed after 24 hours, the formed embryoid bodies were further cultured in a suspended state for 1 day. Thereafter, the cell differentiation apparatus according to the present invention containing the cultured embryos was immersed in a cell culture dish dispensed with 8 ml of EC medium and further cultured for 5 days.

The embryos in the conventional hanging drop method were separated from the lid of the cell culture dish using a pipette, and then immersed in a 60 ° C bacterial culture dish in which 4 ml of EC medium was dispensed. The isolated embryos were further cultured in suspension for 3 days. The cultured embryos were then transferred to a cell culture dish in which 8 ml of EC medium was dispensed and further cultured for 5 days.

4. Observation of Embryonic Morphology

Each cell cultured as above was magnified 45 times in a zoom stereo microscope daily for 6 days of culture time to observe the shape. The results are shown in FIGS. 6A and 6B.

Figure 6a shows the change of the embryonic body formed using the stem cell differentiation apparatus according to the present invention, Figure 6b shows the change of the embryonic body formed by the conventional hanging drop method. As shown in FIG. 6a, when stem cells were cultured according to the present invention, it was confirmed that embryonic bodies were formed in a smooth, uniform and dense round shape at the end of one day. On the other hand, when stem cells were cultured according to the conventional hanging drop method, only three days passed, only embryonic bodies were formed, and even the formed embryoid bodies were uneven, uniform and low in density.

5. Changes in Stem Cell Markers

As described above, the change of the stem cell marker Oct3 / 4 was measured for cells cultured according to the method and apparatus according to the present invention. The results are shown in Figure 7a. Referring to FIG. 7A, it can be seen that over time, the stem cell marker Oct3 / 4 is gradually blurred. This means that differentiation is made from stem cells to other cells.

6. Differentiation into Cardiomyocytes and Neurons

RT-PCR was performed to confirm the differentiation of cells cultured according to the method according to the present invention and cells cultured by a conventional hanging drop method. In order to extract total RNA from the cells obtained during the experiment, 250 μl of Trizol reagent (invitrogen) was added to the cells from which the culture medium was completely removed to lyse the cells. Lysed cells were allowed to stand for 5 minutes at room temperature, 50 μl of chloroform was added, vortexed and allowed to stand at room temperature for 5 minutes. After centrifuging the sample at 12000 g, 4 degrees, only the supernatant was placed in a new container and 250 μl of isopropanol was added in the same amount, and left at room temperature for 5 minutes. The sample was centrifuged at 12000 g at room temperature, the supernatant was removed, 75% Ethanol was added, and 7500 g was centrifuged at room temperature. After removing the supernatant and drying completely, the sample was dissolved in DEPC treated water. In order to remove DNA from the sample, RQ1 DNase (Promega) 1U was treated and reacted. DNase-treated 2.5 μg of RNA was synthesized by cDNA through oligo dT and revese trascriptase provided by RT system (Promega). In order to confirm the differentiation pattern of the cells, the synthesized 2.5 μg cDNA was OCt3 / 4 (octamer-binding protein 4, 5'-GGCGTTCTCTTTGGAAAGGTTTC-3 ', 5), which is a stem cell specific primer in a PCR machine (BioRed). '-CTCGAACCACATCCTTCTCT-3'), Nkx2.5 (NK2 transcription factor related 5, 5'-AGCAACTTCGTGAACTTTG-3 ', 5'-CCGGTCCTAGTGTGGA-3'), a cardiomyocyte specific primer, MAP2 (neuronalal) microtubule-associated protein 2, 5'-TCAGACTTCCACCGAGCAG-3 ', 5'-AGTGCTGTACCCTGCTTCC-3'), GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 5'-TTCACCACCATGGAGAAGGC-3 ', 5') -GGCATGGACTGTGGTCATGA-3 ') and Taq polymerase (Genenmed) for 5 minutes pre-denaturation at 95 ℃, 30 seconds at 95 ℃, 30 seconds at 57.8 ℃, 40 seconds at 72 ℃ After amplifying 30 cycles, the final amplification was performed for 5 minutes at 72 ° C. Amplified DNA was confirmed by electrophoresis at 1.5% agarose.

The results are shown in Figure 7b. As shown in Figure 7b, it was confirmed that the cells (Device) cultured according to the method according to the invention is losing the propensity of stem cells from day 1, cells cultured by the conventional hanging drop method (HD) In comparison, the differentiation of cells into cardiomyocytes was observed from day 3 and the differentiation of neurons from day 6. As a result, it was confirmed that the differentiation in the cell differentiation apparatus according to the present invention proceeds faster than the differentiation indicated by the hanging drop method.

By using the present invention, EB can be formed quickly and uniformly, and cell formation can be efficiently performed by growing the formed EB in a state capable of smooth signal communication between cells. In addition, by preventing the quality of the cell culture solution to deteriorate over time, the cells can be grown in a healthier state, there is an advantage that the formation of EB and the growth of the formed EB can be efficiently performed in one system. .

Claims (19)

As a method for forming an embryoid body from stem cells, A cylindrical container with both ends opened upright and filled with a culture medium containing stem cells in the cylindrical container to hold the culture medium at the end of the vertically held cylindrical container by surface tension while maintaining a hemispherical shape, and the cylindrical container by gravity Method of forming an embryoid body from the stem cells, characterized in that to form a spherical embryo (Embryoid Body, EB) by causing the stem cells to collect at the end of the hemisphere-shaped culture droplets hanging on the end of. The method of claim 1, The diameter of the cylindrical container is a method for forming an embryonic body from stem cells, characterized in that 1.0 to 2.6mm. The method of claim 2, Said cylindrical container is two or more, all cylindrical containers are adjusted to have the same diameter, the method of forming an embryoid body from stem cells, characterized in that to uniformize the size of the embryoid body formed. The method of claim 2, The cylindrical container is two or more, the two or more cylindrical containers are adjusted to have a variety of diameters, the method for forming an embryoid body from stem cells, characterized in that varying the size of the embryonic body formed. The method of claim 1, wherein the culture medium is replenished in the cylindrical container to prevent the culture medium from being evaporated and lost from the cylindrical container and to prevent deterioration of the culture medium due to cell metabolism. . As a method of differentiating stem cells, A first step of forming an embryonic body by the method according to any one of claims 1 to 5; And The embryonic body formed in the first step is a cylindrical container in which the embryonic body is formed at the end of the drop of the culture medium through the first step, into the culture vessel containing the culture medium for embryo growth, the end of the drop of the culture medium to the embryo growth The embryos are dropped into the culture medium for embryonic growth by contact with the culture medium for embryonic growth by placing them at a height that is in contact with the culture medium for the embryo culture. Stem cell differentiation method comprising the step of growing an embryo in the state attached to the culture vessel bottom. The method of claim 6, wherein the second step is a stem cell differentiation method, characterized in that for growing the embryo in a culture vessel of 2.0 to 4.0mm in diameter. An apparatus for forming an embryoid body from stem cells, A plurality of cylindrical containers open at both ends which are vertically installed; And A container surrounding the upper portion of the cylindrical container includes a replenishment container for replenishing the culture solution to the cylindrical container, The plurality of cylindrical vessels allow the culture medium to be suspended by surface tension while maintaining the hemispherical shape at the end of the vertically cylindrical container, and stem cells are collected at the end of the droplets of the hemispherical medium suspended at the end of the cylindrical container by gravity. Form an embryonic body (EB) The cylindrical container is embryonic body forming apparatus, characterized in that the diameter of 1.0 to 2.6mm. delete delete delete delete As a device for differentiating stem cells, Stem cell differentiation apparatus comprising an embryonic body forming layer comprising an embryonic body forming apparatus according to claim 8. The method of claim 13, The culture vessel layer stacked below the embryonic body forming layer and including the same number of culture vessels as the cylindrical container for growth of the embryonic body formed in the embryonic body forming layer, characterized in that the culture vessel is in the form of a tube open up and down. Stem cell differentiation apparatus further comprises a culture vessel layer. The method of claim 14, A microporous membrane layer laminated under the culture vessel layer and composed of a microporous membrane in contact with the culture vessel, for attaching an embryonic body formed in the embryonic formation layer and passing the culture solution from the bottom thereof. Stem cell differentiation apparatus further comprises a sclera layer. The method of claim 14, Stem cell differentiation device, characterized in that the culture vessel of the culture vessel layer is 2.0 to 4.0mm in diameter. The method of claim 14, Stem cell differentiation apparatus, characterized in that the embryonic body forming layer and the culture vessel layer is laminated so that the cylindrical container of the embryonic body forming layer and the culture vessel of the culture vessel layer overlap each other. The method of claim 15, Stacked below the microporous membrane layer, the stem cell differentiation apparatus further comprises a bottom support layer for coupling the culture vessel layer and the microporous membrane layer and protects the microporous membrane layer. The method of claim 15, Stem cell differentiation apparatus, characterized in that the microporous membrane of the microporous membrane layer is ECM coated to improve the adhesion of the embryo.

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