A method for mass proliferation of stem cells without using hydrogel

Abstract

The present invention provides a method for mass proliferation of stem cells without using hydrogel.

Description

[Technical field]

[0001] The present invention relates to a method for mass-producing induced pluripotent stem cells without using a hydrogel, and more particularly to a method for producing and mass-producing induced pluripotent stem cells from somatic cells using a three-dimensional cell culture plate without using a hydrogel. [Background technology]

[0002] The process of returning differentiated somatic cells to undifferentiated cells (e.g., stem cells) is called reprogramming. Induced pluripotent stem cells (iPSCs), also known as reverse differentiation stem cells or reverse pluripotent stem cells, are produced by converting somatic cells into stem cells using reprogramming factors (Oct4, Klf4, Sox2, c-Myc, etc.) (Non-Patent Documents 1 and 2).

[0003] Induced pluripotent stem cells have a wide range of applications, including cell therapy, tissue engineering, new drug development, toxicology, and precision medicine. For these applications, it is necessary to culture induced pluripotent stem cells and embryonic stem cells with high efficiency and high quality, and stem cells with uniform size and efficiency are required, but in reality, research on such mass production technology is insufficient. In addition, the efficiency of reprogramming induced pluripotent stem cells is still very low. In recent years, much research has been conducted on methods for clinical application, i.e., methods that do not incorporate foreign genes into the inside, and among them, attempts have been made to produce induced pluripotent stem cells using methods such as episomal vectors, but these series of production methods have problems such as the low efficiency of 3D culture methods and the low reprogramming efficiency of somatic cells.

[0004] In addition, for induced pluripotent stem cell reprogramming, the bottom of a 2D cell culture plate is coated with hydrogel, but colonies are not formed well, and even if colonies are formed after cell reprogramming, they are not easy to separate, which is in fact an obstacle not only for research purposes but also for commercialization. In addition, with existing methods, it is almost impossible to produce a large number of clones, and it is difficult to stably secure clones in a state reprogrammed into induced pluripotent stem cells with existing methods. In addition, a platform that can easily screen induced pluripotent stem cells is also needed.

[0005] Meanwhile, Korean Patent Registration No. 10-1756901 (Patent Document 1) discloses a cell culture chip capable of culturing three-dimensional tissue cells. The cell culture chip in Patent Document 1 has a first culture section, a second culture section, and a third culture section formed in separate layers, and the degree of cell proliferation can be confirmed in each layer. However, the cell culture chip in Patent Document 1 has a problem in that spheroids and / or organoids cannot be obtained in high yield.

[0006] In addition, pipetting may be performed to exchange the culture medium during cell culture. However, in the case of Corning spheroid microplates, which are capable of 3D cell culture, pipetting can affect the spheroids or organoids being cultured, causing them to be sucked up or displaced during the process, which is undesirable for the cell culture environment.

[0007] Matrigel (BD Biosciences) is a protein complex extracted from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, and contains extracellular matrix (ECM) such as laminin, collagen, and heparan sulfate proteoglycan, and growth factors such as fibroblast growth factor (FGF), epidermal growth factor (EFG), insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), and platelet-derived growth factor (PDGF). The complex that constitutes Matrigel is used as a substrate for cell culture by providing the complex extracellular environment found in many tissues.

[0008] Matrigel is derived from mouse sarcoma, so there is a high risk of introducing immunogens and pathogens. Matrigel is used for cell growth and tissue formation, but it is a complex substance, so there is criticism that it has a major problem with cell reproducibility. It is unclear whether Matrigel simply functions as a passive 3D scaffold that provides physical support to growing spheroids, or whether it actively influences spheroid formation by providing biologically essential elements. Matrigel is also very expensive. Therefore, although Matrigel is a substance that has contributed to the development of cell culture technology, it is also true that Matrigel is a hindrance to the development of the technology field.

[0009] Therefore, the present inventors conducted extensive research into techniques for increasing the reprogramming efficiency of induced pluripotent stem cells without using hydrogel, and have now completed the present invention. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Republic of Korea Patent No. 10-1756901 [Non-patent literature]

[0011] [Non-Patent Document 1] Takahashi K, Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-676.

[0012] [Non-Patent Document 2] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell.2007;131:861-872. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a method for expanding stem cells.

[0014] However, the technical problem that the present invention aims to achieve is not limited to the above-mentioned problem, and other problems not mentioned can be clearly understood by a person of ordinary skill in the art from the following description. [Means for solving the problem]

[0015] In order to achieve the above object, the present invention provides a method for expanding stem cells, comprising:

[0016] i) culturing the cells; ii) preparing a hydrogel-free three-dimensional cell culture plate for producing stem cells; iii) reprogramming the cultured cells into stem cells in a 3D cell culture plate that does not contain the hydrogel; iv) forming spheroids of the reprogrammed stem cells; v) preparing a hydrogel-free three-dimensional cell culture plate for subculture; vi) isolating the spheroids and subculturing them one or more times in a three-dimensional cell culture plate not containing a hydrogel, The three-dimensional cell culture plate comprises: The present invention includes a well plate including a plurality of main wells and a plurality of sub wells formed below the main wells, into which a cell culture medium is injected and which include a recess on the bottom surface, and a connector for high content screening (HCS) that supports the well plate, The connector for high content screening (HCS) includes a base having a fixing means detachably attached to the lower end of a well plate, and a cover disposed on the upper part of the well plate and coupled to the base. The main well has a step formed so that a predetermined portion is tapered, and the step has an inclination angle (θ) of 10 to 60° with respect to the wall of the main well.

[0017] The cells may be somatic cells or stem cells.

[0018] The somatic cells may be, but are not limited to, fibroblasts, and any somatic cells known in the art may be used.

[0019] The stem cells may be one or more types selected from the group consisting of adult stem cells, embryonic stem cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, umbilical cord blood stem cells, and induced pluripotent stem cells, but are not necessarily limited thereto, and any stem cells known in the art can be used.

[0020] The cells can be cultured in common 2-dimensional well plates, 3-dimensional cell culture plates, or 3-dimensional plates according to the invention.

[0021] The hydrogel may be an extracellular matrix-based hydrogel.

[0022] The extracellular matrix-based hydrogel may be Matrigel (product name).

[0023] The subculture may be for 1 to 20 generations, but is not necessarily limited to this.

[0024] In the step vi), the spheroids can be separated into single cells and subcultured one or more times.

[0025] The subwells of the three-dimensional cell culture plate may have an inclined surface formed so as to taper toward the recesses, the upper end diameter of the subwell 120 may be in the range of 3.0 to 4.5 mm, the upper end diameter of the recess 121 may be in the range of 0.45 to 1.5 mm, the inclined surface (θ2) of the subwell and recess may be in the range of 40 to 50°, and the ratio of the length to the diameter of the subwell and the diameter of the recess may be in the range of 1:0.1 to 0.5.

[0026] The individual volumes of the main wells of the three-dimensional cell culture plate may be in the range of 100 to 300 μl, the individual volumes of the recesses may be in the range of 20 to 50 μl, and the individual volume ratio of the main wells to the recesses may be on average 1:0.1 to 0.5.

[0027] The main well may include a space between the step and the subwell, the height (ah) of the space being in the range of 2.0 to 3.0 mm on average, the height (bh) of the subwell being in the range of 1.0 to 2.0 mm on average, and the height ratio (ah:bh) of the space to the subwell being in the range of 1:0.3 to 1.

[0028] The somatic cells can be seeded into the subwells of the cell culture plate at 100 to 1000 cells / well.

[0029] The present invention will be described in detail below.

[0030] Since the present invention can be modified in various ways and can have various embodiments, specific embodiments are shown by way of example in the drawings and will be described in detail in the detailed description.

[0031] However, it should be understood that this is not intended to limit the present invention to a specific embodiment, and includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. In the description of the present invention, if it is determined that a detailed description of related publicly known technology may obscure the gist of the present invention, the detailed description will be omitted.

[0032] The terms used in this application are merely used to describe specific embodiments and are not intended to limit the present invention. The singular expressions include the plural expressions unless otherwise clearly defined in the context.

[0033] In the present invention, the terms "comprise" or "have" are intended to specify the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are to be understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0034] Generally, hydrogels have been used to play the role of extracellular matrix when culturing cells, spheroids, organoids, etc. Generally, when reprogramming induced pluripotent stem cells using 2D or 3D cell culture plates, extracellular matrix-based hydrogels (e.g., Matrigel) are used to coat the cell culture plates.

[0035] However, the present invention provides a method for producing induced pluripotent stem cells using a three-dimensional cell culture plate that does not contain a hydrogel. The three-dimensional cell culture plate of the present invention is specifically described as follows.

[0036] In one embodiment, the present invention uses a three-dimensional cell culture plate comprising:

[0037] The three-dimensional cell culture plate includes a well plate including a plurality of main wells and a plurality of sub wells formed below the main wells, into which a cell culture medium is injected and which include a recess on the bottom surface; A connector for high content screening (HCS) that supports a well plate; The connector for high content screening (HCS) includes a base having a fixing means detachably attached to a lower end of a well plate, and a cover disposed on an upper portion of the well plate and coupled to the base, The main well has a step formed at a predetermined portion so as to have a tapered shape, and the step has an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well.

[0038] In the case of conventional 96-well plates, it was necessary to perform experiments and analyses several times to evaluate the efficacy of high-yield drugs, which was time-consuming and costly. Furthermore, pipetting is often required to replace the culture medium during cell culture, but in the case of conventional Corning spheroid microplates, pipetting can affect the spheroids or organoids during cell culture, causing them to be sucked up or displaced during the pipetting process, which is unfavorable for the cell culture environment.

[0039] Therefore, the present invention has been made to solve the above-mentioned problems, and provides a cell culture plate that includes multiple subwells in multiple main wells formed in a well plate, which makes it possible to produce spheroids / organoids with high yields, includes a connector for high content screening (HCS) that supports the plate, reduces the tolerance during high-speed image capture of large volumes, and allows images in the well plate to be captured uniformly. Furthermore, the present invention provides a cell culture plate that minimizes the influence of pipetting work on the cultured cells during medium replacement due to the step of the main well.

[0040] A preferred embodiment of the present invention will be described in detail below with reference to the accompanying drawings. Prior to the description, the terms or words used in the present specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted as meanings or concepts that correspond to the technical ideas of the present invention, based on the principle that the inventor can appropriately define the concepts of terms in order to best describe his / her invention.

[0041] Therefore, it should be understood that the embodiments described in this specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not represent the entire technical idea of ​​the present invention, and therefore there may be various equivalents and modifications that can replace them at the time of this application.

[0042] FIG. 1(a) is a front view of a cell culture plate according to one embodiment of the present invention, FIG. 1(b) is a cross-sectional view of the cell culture plate according to one embodiment of the present invention, FIG. 2 is a detailed view of a main well formed in a cell culture plate according to one embodiment of the present invention, and FIG. 3 is a view showing a well plate, base and cover of a cell culture plate according to one embodiment of the present invention ((a) cover, (b) base, (c) fixing means for the microplate and base).

[0043] Hereinafter, a cell culture plate according to one embodiment of the present invention will be described in detail with reference to FIGS.

[0044] As shown in Figures 1 to 3, a cell culture plate 10 according to one embodiment of the present invention includes a well plate 100 including a plurality of main wells 110, a plurality of subwells 120 formed below each of the main wells 110, into which cell culture medium is injected, and including a recess 121 on the bottom surface, and a connector 200 for high content screening (HCS) that supports the well plate 100.

[0045] First, a well plate 100 according to an embodiment of the present invention will be described in detail with reference to FIGS.

[0046] The well plate 100 is manufactured in the form of a plastic injection molded plate through a mold. Such a plastic injection mold can be manufactured by having a repeating pattern of the main well 110 as a well structure, thereby reducing the production cost and facilitating the enlargement of the size through microfabrication. This makes it easy to mass-produce cells and can be modified to various sizes according to the needs of users.

[0047] A plurality of main wells 110 are formed in the well plate 100, and each main well 110 includes a step 101. The step 101 is formed at a predetermined portion of the main well 110, and more specifically, the step 101 may be formed at a position 1 / 3 to 1 / 2 of the total length of the main well 110, or the step 101 may be formed at a position 1 / 3 to 1 / 2 from the lower end of the main well 110.

[0048] Conventionally, when culturing cells on a microplate, a pipetting operation may be performed to replace the culture medium. In such cases, however, this may affect the spheroids or organoids being cultured, causing the spheroids or organoids to be sucked up or change position during the pipetting operation, which is undesirable for the cell culture environment. The step 101 is provided to prevent such problems.

[0049] The step 101 may be a space to which a pipette is applied, and specifically, may have an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well 110. Alternatively, it may have an inclination angle in the range of 20 to 50°, and preferably an inclination angle in the range of 30 to 45°. If the inclination angle of the step 101 is less than 10°, the inclination angle in the main well 110 is too small and there is not enough space to apply the pipette, so that when the culture solution in the main well 110 is aspirated, the pipette may slip inside the subwell 120, and the spheroids or organoids may be sucked up or changed position. In addition, if the inclination angle (θ) exceeds 60°, the space to which the pipette can be applied can be secured, but the inclination angle of the step 101 may be too large, making it difficult to aspirate the culture solution sufficiently, and when cells are seeded in the subwells 120, the cells may not fit into all the subwells 120 and may be seeded in the step 101. Therefore, it is preferable that the inclination angle is in the above-mentioned range.

[0050] In addition, the main well 110 according to an embodiment of the present invention may include a space 130 between the step 101 and a subwell 120 described below. Specifically, the space 130 is a space into which a culture medium is injected and allows cells inside the subwell 120 to share the same culture medium.

[0051] More specifically, the height (ah) of the space 130 may be in the range of 2.0 to 3.0 mm on average, or in the range of 2.2 to 2.8 mm on average, or in the range of 2.3 to 2.7 mm on average, and the height (bh) of the subwell 120 may be in the range of 1.0 to 2.0 mm on average, or in the range of 1.2 to 1.8 mm on average.

[0052] For example, the height (ah) of the space 130 may be 2.5 mm on average, and the height (bh) of the sub-well may be 1.5 mm on average.

[0053] At this time, the height ratio (ah:bh) of the space portion to the subwell 120 may be in the range of 1:0.3 to 1, and more specifically, the height ratio (ah:bh) of the space portion to the subwell 120 may be 1:0.4 to 0.9 or 1:0.5 to 0.8. If the height of the subwell 120 is less than 1:0.3 to the height of the space portion, cells being cultured inside the subwell 120 may come out even with a small force when replacing the medium in the subwell 120, and if the height of the subwell 120 is more than 1:1 to the height of the space portion, the culture medium required for the cells may not be sufficiently exchanged, and apoptosis may be induced. Therefore, it is preferable that the space portion 130 and the subwell 120 have the above-mentioned height range and height ratio.

[0054] Next, the sub-wells 120 are formed below each of the main wells 110 and include a recess 121 on the bottom surface. In a specific embodiment, the sub-wells 120 may be formed in a plurality of sub-wells 120 below the main well 110.

[0055] The subwells 120 contained below the main well 110 are of the same size and shape, which allows spheroids and organoids to be generated under uniform conditions.

[0056] The subwell 120 may have an inclined surface that tapers toward the recess 121. Specifically, the upper end of the subwell 120 may have a horizontal width that decreases as it descends in the vertical direction. For example, the upper end of the subwell 120 may have an inverted pyramid shape. In the illustrated embodiment, the upper end of the subwell 120 may have a shape such as a pyramid or funnel shape, in which the horizontal width decreases as it descends in the vertical direction.

[0057] In particular, by including a plurality of subwells 120 having the same size and shape, the cell culture plate can produce a large number of spheroids or organoids under uniform conditions.

[0058] In a specific embodiment, one main well 110 can contain 4 to 25 subwells 120 of the same size, and the entire microplate 100 can contain 96 to 1,728 subwells 120. This allows for uniform and precise size control.

[0059] In addition, the subwell 120 includes a recess 121, and a space is formed at the lower end of the recess 121 so that a 3D spheroid or organoid can be cultured in the recess 121. Specifically, the recess 121 may be "U-shaped", "V-shaped", or "Ц-shaped", and for example, the recess 121 may be "U-shaped".

[0060] The upper end diameter of the subwell 120 may be in the range of 3.0 to 4.5 mm, or 3.5 to 4.3 mm, or 4 mm on average. Furthermore, the upper end diameter of the recess 121 may be 0.45 to 1.5 mm, or 0.5 to 1.0 mm, or 0.5 mm on average.

[0061] In addition, the ratio of the diameter of the subwell 120 to the length of the diameter of the recess 121 may be in the range of 1:0.1 to 0.5, and preferably the ratio of the diameter of the subwell 120 to the length of the diameter of the recess 121 may be 1:0.12.

[0062] If the upper end diameter of recess 121 is less than 0.1 relative to the upper end diameter 1 of subwell 120, it is not possible to secure sufficient cell culture space in recess 121, and a problem may occur in which cells fall out even with a small force when changing the culture medium. If the upper end diameter of recess 121 is more than 0.5 relative to the upper end diameter 1 of subwell 120, it is not possible to change the culture medium required for the cells in sufficient quantity, which may make it difficult to culture them stably.

[0063] On the other hand, the inclined surfaces of the subwell 120 and the recess 121 based on the wall of the main well may have an inclination angle (θ2) of 40 to 50°, an inclination angle (θ2) in the range of 42 to 48°, an inclination angle (θ2) in the range of 43 to 47°, or an average inclination angle (θ2) of 45°.

[0064] The above-mentioned subwell 120 has an advantage that it is possible to culture cells at 100 to 1000 cells / well or less, and the size of the spheroids can be stably controlled.

[0065] Further, according to one embodiment of the present invention, the individual volumes of the main wells 110 are in the range of 100-300 μl, the individual volumes of the recesses 121 are in the range of 20-50 μl, and the individual volume ratio between the main wells 110 and the recesses 121 is 1:0.07-0.5 on average. Preferably, the individual volumes of the main wells 110 according to the embodiment may be in the range of 250-300 μl, the individual volumes of the recesses may be in the range of 25-35 μl, and the individual volume ratio between the main wells 110 and the recesses 121 may be 1:0.11 on average.

[0066] Specifically, if the volume of each main well 110 is less than 100 μl, there may be a problem of not being able to accommodate sufficient culture medium during cell culture, and if it exceeds 300 μl, the culture efficiency may decrease.

[0067] In addition, the recess 121 is a space in which cells are actually cultured, and if the volume is less than 20 μl, the cell culture space is insufficient, which may cause a problem of cells escaping, and if the volume exceeds 50 μl, there is a risk of a problem of it being difficult to stably culture cells, etc. Therefore, it is preferable that the main well 110 and the recess 121 have volumes in the above-mentioned ranges.

[0068] Due to the configuration of the cell culture plate of the present invention described above, reprogramming into induced pluripotent stem cells occurs with high efficiency even without containing a hydrogel, i.e., without coating the cell culture plate with a hydrogel, and spheroids are formed well after reprogramming.

[0069] Furthermore, when the spheroids are separated and subcultured in the cell culture plate of the present invention, the spheroids are formed very well. Specifically, the spheroids are separated into single cells and subcultured to produce hundreds to thousands of monoclonal induced pluripotent stem cells.

[0070] The cell culture plate 10 according to an embodiment of the present invention includes a connector 200 for high content screening (HCS) that supports a well plate 100. Here, the connector 200 for high content screening (HCS) refers to a connector 200 that is attached to an HCS (High Contents Screening) system, and specifically, the connector 200 may refer to a base 210 and a cover 220 in the present invention.

[0071] More specifically, the connector for high content screening (HCS) includes a base 210 having fastening means 140, 240 at the lower end of a well plate 100 so as to be detachable from each other, and a cover 220 disposed on the upper part of the well plate 100 and coupled to the base 210. The upper end of the base 210 and the lower end of the well plate 100 include fastening means 140, 240 which can be detachably fastened to each other.

[0072] At this time, the base includes a convex portion 240 for supporting the well plate 100, and the well plate 100 may include a recessed portion 140 facing the convex portion 240 of the base 210. The well plate 100 is fixed by the fixing means, and an image can be uniformly captured during screening.

[0073] The base may be made of, but is not necessarily limited to, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyester, polyvinyl chloride, polyurethane, polycarbonate, polyvinylidene chloride, polytetrafluoroethylene, polyether ether ketone, or polyetherimide materials.

[0074] The well plate may be made of, but is not necessarily limited to, polydimethyl silicone, high fat modified silicone, methylchlorophenyl silicone, alkyl modified silicone, methylphenyl silicone, silicone polyester, or amino modified silicone material.

[0075] On the other hand, it is not necessary to use Matrigel when forming induced pluripotent stem cells in the cell culture plate 100 of the present invention.

[0076] FIG. 4 shows a comparison of a method for producing induced pluripotent stem cells using a two-dimensional cell culture plate using Matrigel and a three-dimensional cell culture plate according to the present invention that does not require Matrigel. After culturing somatic cells (fibroblasts), an episomal vector is transfected into the fibroblasts by electroporation to induce reprogramming and produce induced pluripotent stem cells. In the case of two-dimensional Matrigel culture, the process of isolating induced pluripotent stem cell colonies is cumbersome, and the yield is low. However, when using the three-dimensional culture plate of the present invention, since there is no Matrigel, a large number of single cells reprogrammed into induced pluripotent stem cells gather together to form a spheroid, which is a three-dimensional spherical cell aggregate. This spheroid can be easily separated into a three-dimensional cell culture plate and can be subcultured (FIG. 7d). In other words, the reprogramming efficiency is very high.

[0077] As described above, the three-dimensional cell culture plate used in the present invention can include 4 to 25 subwells 120 of the same size in one main well 110, and the entire microplate 100 can include 96 to 1,728 subwells 120. This allows for mass production of identical induced pluripotent stem cells and their spheroids whose sizes can be precisely controlled.

[0078] Figure 10 is a schematic diagram showing the method for mass proliferation of induced pluripotent stem cell spheroids obtained in the induced pluripotent stem cell reprogramming process. It can be seen that the proliferation rate of the induced pluripotent stem cells of the present invention is very fast compared to two-dimensional cell culture plates (Matrigel coating). In addition, by separating the spheroids into single cells and replating them, and then continuing subculture, hundreds to thousands of monoclonal spheroids of uniform size are produced, so that an induced pluripotent stem cell spheroid bank can be created. Effect of the Invention

[0079] According to the production method of the present invention, stem cells with improved reprogramming efficiency can be produced and mass-produced without the need for hydrogel, and a monoclonal stem cell spheroid bank can be created. [Brief description of the drawings]

[0080] [Figure 1] FIG. 1(a) is a front view of a cell culture plate according to one embodiment of the present invention, and FIG. 1(b) is a cross-sectional view of the cell culture plate according to one embodiment of the present invention.

[0081] [Diagram 2] 2 is a detailed view of a main well formed in a cell culture plate according to an embodiment of the present invention; FIG.

[0082] [Diagram 3]1A to 1C show a well plate, a base and a cover of a cell culture plate according to one embodiment of the present invention ((a) cover, (b) base, (c) fixing means for a microplate and a base).

[0083] [Figure 4] FIG. 4(a) is a schematic diagram showing the process of producing induced pluripotent stem cells according to an embodiment of the present invention and a comparative example, and FIG. 4(b) is an image showing the generation of induced pluripotent stem cells according to an embodiment of the present invention and a comparative example (in all figures from FIG. 4 onwards, for the sake of convenience, the 3D cell culture plate of the present invention is not accurately depicted, but is depicted in a U-shape for the sake of convenience).

[0084] [Diagram 5] 1 shows images of an embodiment of the present invention (3D iPSC) and a comparative example (2D PSC).

[0085] [Figure 6] 1 shows AP (Alkaline Phosphatase) stained images of an example of the present invention (3Dsph-iPSC) and a comparative example (2D Matrigel).

[0086] [Figure 7] Figure 7(a) shows AP images over time (D4, D9, D15, and D21), Figure 7(b) shows a comparison of colony numbers, Figure 7(c) shows the E-cadherin expression results, and Figure 7(d) shows the process of spheroid formation of iPSCs.

[0087] [Figure 8] FIG. 8(a) shows the size distribution of spheroids (colonies) resulting from conventional 3D culture and culture according to one embodiment of the present invention, and FIG. 8(b) shows the results of expression of reprogramming factors (pluripotency markers).

[0088] [Figure 9] 1 shows the results of pluripotency marker expression in iPSCs according to one embodiment of the present invention.

[0089] [Figure 10] FIG. 1 is a schematic diagram showing a method for proliferating induced pluripotent stem cells according to an embodiment of the present invention and a comparative example.

[0090] [Figure 11] 1 shows images over time showing the proliferation process of induced pluripotent stem cells according to one embodiment of the present invention.

[0091] [Figure 12] 1 is a graph showing that the size distribution is uniform when induced pluripotent stem cells are subcultured according to one embodiment of the present invention.

[0092] [Figure 13] 1 is a graph comparing the proliferation efficiency according to an embodiment of the present invention (three-dimensional cell culture) and a comparative example (two-dimensional cell culture).

[0093] [Figure 14] 1 shows the results of pluripotency marker expression over the proliferation time of induced pluripotent stem cells.

[0094] [Figure 15] 1 shows the results of pluripotency marker expression over the proliferation time of induced pluripotent stem cells.

[0095] [Figure 16] FIG. 1 is a schematic diagram showing the production, proliferation and differentiation process of induced pluripotent stem cells according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] The present invention can be modified in various forms and can have various embodiments, and specific embodiments are illustrated in the drawings and described in detail below. However, it is not intended to limit the present invention to a specific embodiment, and it should be understood that it includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. In the description of the present invention, if a detailed description of related publicly known technology is deemed to make the gist of the present invention unclear, the detailed description will be omitted. EXAMPLES

[0097] Example 1. Experimental Method

[0098] 1-1: Fibroblast culture and generation of induced pluripotent stem cells

[0099] Human fibroblast cell line F134 (the german federal authorities / RKI: AZ 1710-79-1-4-41 E01) was cultured in DMEM containing 10% FBS (fetal bovine serum, Invitrogen, USA) and 1 mM L-glutamine (Invitrogen, USA) in 35 mm or 100 mm Petri dishes. Cultured fibroblasts were transfected with episomal iPSC reprogramming vectors (EP5™ kit: catalog number A16960. Invitrogen, Carlsbad, CA, USA) by electroporation (Neon™ transfection system) and reprogrammed. Electroporation was performed at 1,650 V, 10 ms, 3 pulses.

[0100] As shown in FIG. 4a, the transfected fibroblasts were seeded on the 3D cell culture plate of the present invention (without Matrigel, Example), a 2D 12-well plate (Matrigel-coated, Comparative Example 1), and a commercial product, Addgene (Comparative Example 2, Matrigel-coated, not shown in FIG. 4a), and cultured in N2B27 medium (containing bFGF). After culturing for 15 days, the medium was replaced with Essential 8TM medium. After 15 days, the 3D iPSCs of the Example (3D cell culture plate) were plated on a 2D 12-well plate, and the colony counts of the Example and Comparative Example were confirmed.

[0101] 1-2: Analysis of fibroblast reprogramming efficiency

[0102] According to the Alkaline Phosphatase Staining kit manual (System Biosciences, USA), the reprogrammed cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with Blue-color AP solution, washed twice with PBS, and observed under an optical microscope for the presence or absence of colony staining. The number of stained colonies was counted and quantified.

[0103] The cells cultured in the Example and Comparative Example were photographed to compare the size of the cell spheroids. The spheroids were imaged using an automatic plate machine, which automatically focused and advanced the image. The image size was analyzed using a macro program in the ImageJ program (related to Figures 5, 6, and 7).

[0104] 1-3: Optimization of 3D culture method for induced pluripotent stem cells

[0105] The 3D induced pluripotent stem cells cultured in the Example and Comparative Example were photographed, and the sizes of the cell balls were compared and measured (Figure 8(a)). To test the results of the images, an external testing company (Cell Bio CEFO, Korea) was commissioned, and the test was conducted as a blind test.

[0106] 1-4: Immunostaining

[0107] The reprogrammed cells were fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were incubated with 1% BSA and 0.5% Triton X-100 in PBS for 1 h at room temperature, then treated with primary antibodies Oct4 (1:100, SantaCruz, CA, USA), Sox2 (1:100, Cell Signalling, Danvers, MA, USA), Nanog (1:200, Cosmo Bio, Koto-Ku, Japan), and E-cadherin (1:200, Abcam), and reacted with FITC-conjugated goat anti-rabbit IgG or anti-mouse IgG (1:100, Invitrogen, Carlsbad, CA) as secondary antibodies. Fluorescent images were analyzed under a fluorescent microscope (Olympus, Shinjuku, Tokyo, Japan). DAPI was used as a nuclear staining solution.

[0108] 1-5: Verification of the efficiency of 3D mass culture of stem cells or induced pluripotent stem cells

[0109] Induced pluripotent stem cells were seeded at different cell numbers and compared by size after each day. Cell numbers of 0.1, 0.3, 0.5, 1, 2, and 4 x 105 were placed in the multi-wells according to the embodiment, and it was demonstrated that the cell size was maintained and the cell number increased steadily regardless of the cell number (see Figures 12 and 13).

[0110] Comparing this increase in cell number to the number of cells cultured in 2D over the same period, we confirmed an increase of 22.9 ± 4.33%.

[0111] 1-6:qPCR

[0112] Total RNA was extracted from fibroblasts and reprogrammed cells using an RNA mini kit (Qiagen, Inc.), and then converted to cDNA using Accupower RT mix reagent (Bioneer Corp., Seoul, Korea). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA). The primer sequences used in the present invention are shown in Table 1.

[0113] [Table 1]

[0114] Example 2. Confirmation of reprogramming efficiency

[0115] FIG. 4b shows that in the case of 2D culture, a small number of colonies begin to form only at D15. After inducing iPSC reprogramming up to D15, 3DiPSCs were plated on a 2D plate, and the number of colonies formed in Comparative Example 1 was compared with that in Example 1, showing a large difference in the number of colonies formed. It can be seen that the iPSC reprogramming yield in the Example is high because cells that have successfully differentiated from iPSCs form colonies. FIG. 5 and FIG. 7b show that there is a very large difference in the number of colonies between 2D culture (Comparative Example 1) and 3D culture (Example). FIG. 6 shows that the reprogramming efficiency is very high in 3D iPSC spheroids (Example, 3D sph-iPCs) as a result of AP (Alkaline phosphatase) staining. Furthermore, when looking at Figures 6 and 7a, when comparing 2D Matrigel (Comparative Example 1) with 3D iPSC spheroids (Example, 3D sph-iPCs), the images are uniform and clear, which indicates that the 3D cell culture plate of the present invention is capable of large-scale image analysis.

[0116] Figure 7c shows that the reprogramming efficiency is very good in the 3D cell culture plate. Also, Figure 7d shows that since the present invention does not use Matrigel, a large number of single cells reprogrammed by iPCs gather together to form spheroids, which are 3D spherical cell aggregates, and these spheroids can be easily separated from the 3D cell culture plate and replated. That is, the reprogramming efficiency is very high.

[0117] FIG. 8 compares the conventional 3D culture of Comparative Example 2 with the 3D culture of the embodiment of the present invention (Spheroid Film in FIG. 8b). The conventional 3D culture is not uniform in size and has a relatively low Oct4 expression level. However, the present invention is very uniform in size (99.45%) and has a very high reprogramming factor expression rate. That is, even when compared with the conventional 3D culture, the present invention is effective in stem cell culture and can increase the efficiency of reprogramming somatic cells into induced pluripotent stem cells. In addition, the uniform size means that standardized induced pluripotent stem cells and stem cells can be mass-produced in 3D in the form of spheroids.

[0118] Example 3. Analysis of the characteristics of induced pluripotent stem cells

[0119] FIG. 9 shows that the iPSCs produced according to the present invention have very high expression of pluripotency markers.

[0120] Example 4. Confirmation of Mass Proliferation of Stem Cells

[0121] 10 and 11 show that iPSCs produced according to the present invention can be mass-produced when subcultured. FIG. 13 is a graph comparing the proliferation efficiency of induced pluripotent stem cells reprogrammed in a 2D 12-well plate (Matrigel coating, Comparative Example 1) subcultured in a 2D Matrigel-coated plate and induced pluripotent stem cells reprogrammed according to an embodiment of the present invention subcultured in a 3D cell culture plate of the present invention. FIG. 13 shows that the proliferation efficiency of the present invention is increased by about 23 times. Considering the results of FIG. 12, mass proliferation of uniform size is possible. FIG. 14 and FIG. 15 show that the mass-produced iPSCs also have very high expression of pluripotency markers.

[0122] Figure 16 shows a schematic diagram of the present invention. Using the three-dimensional cell culture plate of the present invention, somatic cells can be reprogrammed into induced pluripotent stem cells with high efficiency without using hydrogel, and when the spheroids of the reprogrammed induced pluripotent stem cells are isolated and subcultured, the induced pluripotent stem cells can be mass-produced.

[0123] iPSCs are generated and proliferate in the wells of the cell culture plate of the present invention. They can be stored in this state, or the medium can be frozen at once. The size of the wells can be adjusted without using hydrogels such as Matrigel, so less medium is needed, making it economical. The large amounts of proliferated induced pluripotent stem cells can then be differentiated into various cells.

[0124] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. [Explanation of symbols]

[0125] 100: Well plate 101: Step 110: Main well 120: Subwell 121: Recess 130: Space part 140: Drop-in section 200: Connector for high content screening 210: Bass 220: Cover 240: Convex

Claims

1. A method for expanding stem cells, comprising: i) culturing the cells; ii) preparing a three-dimensional cell culture plate without hydrogel coating for producing stem cells; iii) reprogramming the cultured cells into stem cells in the three-dimensional cell culture plate without the hydrogel coating; iv) forming spheroids of the reprogrammed stem cells; v) preparing a three-dimensional cell culture plate without a hydrogel coating for subculture; vi) isolating the spheroids and subculturing them one or more times on a three-dimensional cell culture plate without the hydrogel coating; Each of the three-dimensional cell culture plates prepared in the steps ii) and iv) comprises: A well plate including a plurality of main wells and a plurality of sub wells each formed below the main wells, each sub well having a recess on a bottom surface into which a cell culture medium is injected; A connector for high content screening (HCS) that supports the well plate; The high content screening (HCS) connector comprises: a base provided with a fixing means at the lower end of the well plate such that the base can be detachably attached to the lower end of the well plate; a cover disposed on the top of the well plate and coupled to the base; The main well has a step formed so that a predetermined portion is tapered, The step has an inclination angle (θ1) in the range of 10 to 60° with respect to the wall of the main well, the main well includes a space between the step and the subwell, The subwell has an inclined surface (θ2) formed so as to be tapered toward the recess, The method for proliferating stem cells, wherein the inclined surface (θ2) between the subwell and the recess is in the range of between 40 and 50°.

2. The method of claim 1 , wherein the cells are somatic cells or stem cells.

3. The proliferation method according to claim 2, wherein the stem cells are one or more types selected from the group consisting of adult stem cells, embryonic stem cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, umbilical cord blood stem cells, and induced pluripotent stem cells.

4. The method according to claim 1 , wherein the subculture is for 1 to 20 generations.

5. The method of claim 1, wherein in step vi), the spheroids are separated into single cells and subcultured one or more times.

6. The upper end diameter of the subwell (120) is in the range of 3.0 to 4.5 mm; The upper end diameter of the recess (121) is in the range of 0.45 to 1.5 mm; 2. The method of claim 1, wherein the ratio of the diameter of the subwell to the length of the recess to the diameter of the recess is in the range of 1:0.1 to 0.

5.

7. The individual volumes of the main wells range from 100 to 300 μl; the individual volumes of said wells are in the range of 20-50 μl; The method of claim 1, wherein the individual volume ratio of the main well to the recess is on average 1:0.1-0.

5.

8. The height (ah) of the space portion is in the range of 2.0 to 3.0 mm on average, The height (bh) of the subwell is in the range of 1.0 to 2.0 mm on average; 2. The method according to claim 1, wherein the ratio of the height of the space to the height of the subwell (ah:bh) is in the range of 1:0.3 to 1:

1.

9. The method according to claim 1, wherein the cells are seeded into the subwells of the cell culture plate at 100 to 1000 cells / well.

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