Cell culture platform capable of simulating microgravity, and cell culture comprising cells prepared therefrom

Abstract

The present invention relates to: a cell culture platform in which cell culture is enabled in a state capable of simulating microgravity by mixing a density control solution into a cell culture medium to adjust density of the cell culture medium to a density range of cells; and a cell culture comprising cells prepared therefrom. Since the cell culture platform of the present invention can overcome limitations of existing microgravity platforms simulated on the ground, including excessive cost and side effects caused by application of additional external force, the cell culture platform is expected to be usefully applicable in the aspects of regenerative medicine, immunology, and pharmacology as well as in those of space biology and space medicine.

Description

A cell culture platform capable of simulating microgravity, and a cell culture comprising cells produced therefrom

[0001] The present invention relates to a cell culture platform capable of simulating microgravity using a cell culture medium (cell culture solution) having a density similar to that of cells, and a cell culture product containing cells produced therefrom. Specifically, the present invention relates to a cell culture platform capable of simulating a microgravity environment on Earth by adding a density-adjusting solution to the cell culture solution to adjust the density to be similar to that of cells, and a cell culture product containing cells produced therefrom.

[0002] All living things, including humans, are constantly subject to the Earth's gravity, and gravity plays a crucial role in maintaining biological homeostasis. With the advent of modern space exploration, the importance of gravity has been highlighted by the observation of numerous physiological changes in astronauts exposed to long-term, gravity-free space (microgravity). These changes include decreased muscle mass and bone density, impaired vision, decreased kidney function, decreased neural responses, and weakened immunity. These phenomena have been a key factor in spurring scientists to pursue research in astrobiology and space medicine.

[0003] Currently, research on microgravity is actively being conducted because it can be utilized in various aspects such as understanding the behavior of organisms in space, as well as regenerative medicine, immunology, and pharmacology.

[0004] However, these microgravity studies are conducted on the International Space Station orbiting the Earth, and thus accessibility to research is very limited due to the high cost, effort, and transportation constraints compared to the high research demand.

[0005] Accordingly, numerous researchers have attempted to replicate microgravity environments through Earth-based platforms. While parabolic flight systems, drop towers, and sounding rockets can temporarily simulate microgravity on Earth, these systems remain costly and, most importantly, remain too short to observe physiological changes in cells.

[0006] Non-patent literature 1 suggests the use of a microgravity simulator to investigate the effects of microgravity at the cellular level, including cell morphology, proliferation, and adhesion, as direct research in space is expensive.

[0007] Non-patent document 2 presents the results of evaluating the effects of microgravity on muscles to address the possibility of long-term human residence in space through an experimental device for culturing mouse muscle cells in a microgravity environment as a bioreactor for cell culture experiments on the International Space Station (ISS).

[0008] To address this, various platforms have been developed to simulate microgravity, including rotating-wall vessel bioreactors, 2D / 3D clinostats, random positioning machines, magnetic levitation, and sonic levitation, which counteract gravity with additional external forces to create sustained microgravity. However, these platforms are not similar to space-based microgravity because they also impose additional external forces on cells due to the shear stress of the liquid acting on the cells due to rotation, the shock from hitting the wall, and the magnetic field and sonic waves.

[0009] Accordingly, the purpose of the present invention is to provide a cell culture platform capable of providing neutral buoyancy to cells and simulating a new microgravity with minimal external force, overcoming the limitations of high costs and various external forces in cell-related research, such as cell culture, on a platform implementing an existing microgravity environment.

[0010] In addition, another object of the present invention is to provide a cell culture comprising cells cultured using the cell culture platform.

[0011] A cell culture platform capable of providing neutral buoyancy to cells and implementing microgravity to achieve the purpose of the present invention is characterized in that it is possible to culture cells in an environment simulating microgravity by mixing a density control solution into a cell culture solution to control the density of the cell culture solution to a density range that cells have.

[0012] According to one embodiment of the present invention, the density control solution may be any one selected from a first material comprising iodine as a component; a second material based on polysaccharides; a third material based on silica particles; and a mixture of two or more selected from the first material, the second material, and the third material.

[0013] Among the density-controlling solutions of the present invention, the first material containing iodine as a component may preferably be any one selected from the group consisting of Optiprep, Visipaque, Cardiolek, RosetteSep, Omnipaque, NycoDenz, and HistoDenz.

[0014] Among the density-controlling solutions of the present invention, the second material based on the polysaccharide may preferably be any one selected from the group consisting of Ficoll, Ficoll-Paque, PolymorphPrep, LymphoPrep, Histopaque, sucrose, and dextran.

[0015] Among the density control solutions of the present invention, the third material based on the silica particles may preferably be Percoll or GM501 (Gynemed).

[0016]

[0017] In addition, when the density control solution of the present invention is a first material containing iodine as a component, it is preferable that the density of the cell culture solution has a range of 1.05 to 1.10 g / mL.

[0018] In another embodiment of the present invention, when the density control solution is a second material based on polysaccharides, the density of the cell culture medium is preferably in the range of 1.06 to 1.08 g / mL.

[0019] In another embodiment of the present invention, when the density control solution is a third material based on silica particles, it is preferable that the density of the cell culture medium has a range of 1.05 to 1.06 g / mL.

[0020] The cell culture medium according to the present invention may include a basic medium, serum, and antibiotics.

[0021] The basic medium constituting the cell culture medium of the present invention may preferably be any one serum-free medium selected from the group consisting of BME (basal medium Eagle's), MEM (minimal essential medium), α-MEM (α-minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM / F-12, Ham's F-10, Ham's F-12, Leibovitz's L-15 Medium, RPMI 1640, M199 medium, IMDM (Iscove's modified Dulbecco's medium), McCoy's 5A (modified) medium, William's medium E, and MesenCult-ACF Plus medium.

[0022] In addition, the serum, which is a component of the cell culture medium, may be one or two or more selected from fetal bovine serum (FBS), bovine calf serum (BCS), newborn calf serum (NCS), horse serum, and normal human serum.

[0023] It is preferable that the above serum be included in an amount of 1 to 40% of the weight of the basic medium.

[0024] The antibiotics that constitute the cell culture medium of the present invention may be one or two or more selected from penicillin, streptomycin, amphotericin B, ampicillin, gentamicin, neomycin, kanamycin, and polymyxin B.

[0025] It is preferable that the above antibiotics be included in an amount of 0.1 to 10% relative to the weight of the basic medium.

[0026] According to one embodiment of the present invention, it is preferable that the density of the cell culture medium has a range of 1.05 to 1.10 g / mL.

[0027] In addition, the present invention is characterized in that the density of the cell culture solution can be continuously maintained during cell culture, at the time when cell culture is completed, and even after cell culture is completed.

[0028]

[0029] Additionally, the present invention can provide a cell culture comprising cells produced from the cell culture platform.

[0030] The cells manufactured on the cell culture platform according to the present invention are characterized in that they are buoyant within the culture medium and remain suspended in the middle or on the surface of the liquid of the cell culture medium, so that the cells do not sink and the cells remain continuously suspended.

[0031] According to the present invention, a cell culture platform capable of simulating microgravity with neutral buoyancy, which offsets the gravity exerted on cells by utilizing buoyancy within the liquid, can be manufactured by making the densities of cells and cell culture medium (cell culture solution) similar, and such a cell culture platform has the effect of providing a microgravity environment similar to space on the ground by overcoming the limitations of existing microgravity simulating devices or platforms that simulate a weightless environment.

[0032] Furthermore, the novel platform for studying microgravity effects on the ground according to the present invention has potential applications in astrobiology, space medicine, and various other fields utilizing the same.

[0033] Figure 1 shows the in vitro location of bone marrow-derived stem cell spheroids according to Examples 1 to 7 and Comparative Examples 1 to 8 of the present invention.

[0034] Figure 2 shows the morphology of cell spheroids cultured in Example 1, which can impart neutral buoyancy in Figure 1, over time. (A) Microscope images of cell spheroids cultured in Example 1 for 1, 3, 7, and 14 days, and (B) a graph of the change in diameter of cell spheroids measured based on the microscope images. Cell spheroids cultured only in cell culture medium were used as a negative control, and the data are expressed as the mean ± standard error.

[0035] Figure 3 shows the results of changes in the height of cell spheroids cultured in Example 1 over 14 days within the medium. (A) A graph recording the position within the medium over time in Example 1, and (B) images capturing the position of cell spheroids at specified time intervals are shown. Data are expressed as mean ± standard error.

[0036] Figure 4 is a graph showing the cytotoxicity of cell spheroids for the components of Example 1 over time. Cell spheroids cultured in a normal cell culture medium were used as a negative control, and the data are expressed as the mean ± standard error.

[0037] Figure 5 is a graph confirming the expression of pluripotency-specific genes OCT4, SOX2, and NANOG in cell spheroids cultured in Example 1 over time at the RNA level. Bone marrow-derived stem cells cultured two-dimensionally (2D culture) were used as a negative control group, and cell spheroids cultured in a general cell culture medium (3D culture) were used as a positive control group. Data are expressed as mean ± standard error, and statistically significant data are indicated by superscripts (*p<0.05, **p<0.01).

[0038] Figure 6 shows the expression of pluripotency-specific proteins in cell spheroids cultured in Example 1 over time using immunoblotting. Pluripotency-specific proteins were analyzed using antibodies to OCT4, SOX2, and NANOG. Bone marrow-derived stem cells cultured in two dimensions (2D culture) served as a negative control, and cell spheroids cultured in a general cell culture medium (3D culture) served as a positive control.

[0039] Figure 7 is a graph measuring the expression of RUNX2 and OCN, osteogenic differentiation-specific factors, in cell spheroids cultured in Example 1 over time at the RNA level. Bone marrow-derived stem cells cultured two-dimensionally (2D culture) served as a negative control, and cell spheroids cultured in a general cell culture medium (3D culture) served as a positive control. Data are expressed as mean ± standard error, and statistically significant data are indicated by superscripts (*p<0.05, **p<0.01).

[0040] The present invention is described in more detail below.

[0041] The terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the invention.

[0042] As used herein, the singular forms include the plural forms unless the context clearly dictates otherwise. Furthermore, as used herein, the words "comprise" and / or "comprising" specify the presence of stated features, numbers, steps, operations, elements, components and / or groups thereof, but do not preclude the presence or addition of one or more other features, numbers, operations, elements, components and / or groups thereof.

[0043]

[0044] The present invention relates to a cell culture platform capable of simulating microgravity, and a cell culture comprising cells produced therefrom.

[0045] The 'cell culture platform' used in the present invention is used to mean a cell culture medium or cell culture solution that satisfies conditions for culturing cells.

[0046] The present invention is characterized in that a cell culture platform capable of culturing cells in an environment simulating microgravity mixes a density control solution into a cell culture solution to control the density of the cell culture solution to a density range possessed by cells, thereby enabling cell culture while maintaining microgravity.

[0047] The cell culture medium according to the present invention may be composed of a basic medium, serum, and antibiotics.

[0048] The above basic medium may preferably be any one serum-free medium selected from the group consisting of BME (basal medium Eagle's), MEM (minimal essential medium), α-MEM (α-minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM / F-12, Ham's F-10, Ham's F-12, Leibovitz's L-15 Medium, RPMI 1640, M199 medium, IMDM (Iscove's modified Dulbecco's medium), McCoy's 5A (modified) medium, William's medium E, and MesenCult-ACF Plus medium.

[0049] In addition, the serum, which is a component of the cell culture medium, may be one or more selected from among fetal bovine serum (FBS), bovine calf serum (BCS), newborn calf serum (NCS), horse serum, and normal human serum, and the serum may be included in an amount of 1 to 40% relative to the weight of the basic medium.

[0050] In addition, it is preferable that the antibiotics that constitute the cell culture solution of the present invention are one or more selected from penicillin, streptomycin, amphotericin B, ampicillin, gentamicin, neomycin, kanamycin, and polymyxin B, and the content thereof may be included at 0.1 to 10% relative to the weight of the basic medium.

[0051] The present invention is characterized in that a density control solution is added to the cell culture medium to control the density to a level similar to the density range of the cells. The density control solution may be any one selected from a first material including iodine as a component; a second material based on polysaccharides; a third material based on silica particles; and a mixture of two or more selected from the first material, the second material, and the third material.

[0052] Among the density-adjusting solutions, the first material containing iodine as a component may preferably be any one selected from the group consisting of Optiprep, Visipaque, Cardiolek, RosetteSep, Omnipaque, NycoDenz, and HistoDenz.

[0053] According to one embodiment of the present invention, when a first material containing iodine as a component is added to the density control solution, the density of the cell culture solution can be controlled to a range of 1.05 to 1.10 g / mL.

[0054] In addition, according to another embodiment of the present invention, the second material based on polysaccharide in the density control solution may preferably be any one selected from the group consisting of Ficoll, Ficoll-Paque, PolymorphPrep, LymphoPrep, Histopaque, sucrose, and dextran.

[0055] When a second material based on polysaccharides is used as the density control solution, the density of the cell culture medium is controlled to a range of 1.06 to 1.08 g / mL.

[0056] In addition, according to another embodiment of the present invention, the density-controlling solution may be a third material based on silica particles, such as Percoll or GM501 (Gynemed). When the third material based on silica particles is used as the density-controlling solution, the density of the cell culture medium may be controlled to a range of 1.05 to 1.06 g / mL.

[0057] In the present invention, it is preferable to add each of the density control solutions to the cell culture medium so that the density range of the cell culture medium is controlled within 1.05 to 1.10 g / mL, and the density range can be controlled by controlling the content according to the characteristics of each density control solution.

[0058]

[0059] Therefore, according to the present invention, a density-controlled cell culture platform can be manufactured in an environment (neutral buoyancy) where various cells are buoyant by being lifted off the bottom, and the density of the cell culture solution of the cell culture platform is characterized by being maintained without sinking until the end of the culture, even when the cells begin to grow and become heavier. In addition, it has the effect of maintaining the buoyancy for about 14 days after the end of the cell culture.

[0060] Accordingly, various cell cultures including cells produced from the cell culture platform according to the present invention can be provided.

[0061] In addition, the cells manufactured on the above cell culture platform have the characteristic of being buoyant in the cell culture medium and being suspended in the middle or on the surface of the liquid of the cell culture medium, so that the cells do not sink and the cells are continuously maintained in suspension.

[0062] The cells according to the present invention are preferably, but not limited to, various stem cells such as adult mesenchymal stem cells (ASCs), hematopoietic stem cells (HSCs), umbilical cord blood stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs).

[0063] In addition, the cells according to the present invention can be both two-dimensional cells and three-dimensional spheroid cells, and their shapes are not particularly limited.

[0064]

[0065] Additionally, the density of the cell culture medium in the cell culture platform of the present invention can be maintained constant by periodically replacing it with new medium.

[0066] Preferred embodiments of the present invention will be described in detail below. The following examples are intended solely to illustrate the present invention and should not be construed as limiting the scope of the present invention. Furthermore, while specific compounds are used in the examples below, it will be apparent to those skilled in the art that equivalent compounds of these compounds can also produce similar or equivalent effects.

[0067]

[0068] Examples 1-7: Manufacturing of cell culture platforms with various densities

[0069] 1. Preparation of cells

[0070] Bone marrow-derived mesenchymal stem cells (BM-MSCs) were purchased from StemCell Technologies. Bone marrow-derived mesenchymal stem cells were cultured in DMEM (Welgene) supplemented with 20% fetal bovine serum (Welgene) and 1% penicillin-streptomycin (Welgene) based on the weight of the medium. The cell culture medium thus prepared had a density of 1.02 g / mL.

[0071]

[0072] 2. Production of cell spheroids

[0073] Cell suspension 5*10 5 100 μL of cells / mL was dispensed into each well of an ultralow attached plate (Perkin Elmer) and centrifuged at 700 g for 1 minute to induce cell aggregation. Cell spheroids were then produced by culturing the cells in a 37°C, 5% CO2 incubator for one day.

[0074]

[0075] 3. Preparation of cell culture medium with controlled density

[0076] Optiprep (density: 1.32 g / mL, Axis-shield), which is composed of iodixanol, which is widely used for cell separation and X-ray contrast agents, Percoll (density: 1.13 g / mL, Cytiva), which is based on silica particles used for density gradient cell separation, and Ficoll-Paque (density: 1.08 g / mL, Cytiva), which is used for lymphocyte separation, were added to the cell culture medium to control the density as shown in Table 1 below.

[0077] Example 1234567 Density control solution OptiPrepColpicoll-Fake Addition amount (vol%) 202520305080100 Density after addition (g / mL) 1.081.101.051.061.081.071.08

[0078]

[0079] 4. Cell culture on a cell culture platform containing cell culture medium with controlled density.

[0080] After carefully removing the cell culture medium of the cell spheroids manufactured in the above 2, 100 μL of cell culture medium having a density adjusted according to Table 1 was dispensed and the cells were cultured under those conditions.

[0081]

[0082] Comparative Examples 1 to 8: Cell culture on a cell culture platform containing cell culture medium using lower and higher densities than cells.

[0083] In the above example, steps 1 and 2 were carried out in the same manner, and the process for manufacturing a cell culture medium with an adjusted density of step 3 was carried out as follows.

[0084] Optiprep (density: 1.32 g / mL, Axis-shield), which contains iodixanol, widely used in cell separation, X-ray contrast agents, etc., Percoll (density: 1.13 g / mL, Cytiva), which is a silica particle-based material used for density gradient cell separation, and Ficoll-Paque (density: 1.08 g / mL, Cytiva), which is used for lymphocyte separation, were added to the cell culture medium to adjust the density as shown in Table 2 below.

[0085]

[0086] Comparative Example 12345678 Density Control Solution OptiPreppercolpicol-Fake Addition Amount (vol%) 1301158012050 Density after addition (g / mL) 1.0261.1101.0241.0391.1091.0241.0341.044

[0087] Next, cells were cultured in the same manner as in Example 4 except that a medium of cell culture medium having a density adjusted according to Table 2 was used as the cell culture medium.

[0088]

[0089] Experimental Example 1: Identification of the optimal medium for maintaining neutral buoyancy of cell spheroids.

[0090] When culturing cells using the cell culture platform according to the above examples and comparative examples, the appearance of the cells was observed to determine whether neutral buoyancy was maintained according to each adjusted density, and the results are shown in the following Figure 1.

[0091] Referring to the following Figure 1, in Comparative Examples 1, 3, 4, and 6 to 8, the cell spheroids (white round shapes) with densities less than 1.05 g / mL were shown to sink due to gravity. In addition, in Comparative Examples 2 and 5, where the density of the mixed medium exceeded 1.10 g / mL, the buoyancy was so high that the cell spheroids floated exposed on the liquid surface.

[0092] However, in Examples 1 to 7, where the density of the cell culture medium was adjusted to between 1.05 and 1.10 g / mL, cell spheroids were observed to be buoyant and suspended in the liquid. From these results, it was confirmed that when the density adjustment solution is added in the present invention to maintain the density of the cell culture medium at a level similar to that of the cells, cell culture is possible under conditions where the cells maintain their neutral buoyancy until the culture is completed.

[0093]

[0094] Among these, the following experiment was conducted using Example 1 having a density of 1.08 g / mL.

[0095]

[0096] Experimental Example 2: Morphological Analysis of Cell Spheroids in Neutral Buoyancy Medium

[0097] After removing all the medium from the wells containing cell spheroids, 100 μL of Example 1 was dispensed. As a negative control, cell spheroids cultured in cell culture medium (cell culture medium; density, 1.023 g / mL) containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin based on the weight of the DMEM medium were added. The morphology and diameter of the cell spheroids observed under a microscope for 14 days are shown in Figure 2.

[0098] As can be seen in Figures 2A and 2B, the morphological changes in the spherical shape of the cell spheroids did not differ significantly compared to the negative control cell culture medium. The decrease in the size of the cell spheroids over time is thought to be due to the strengthening of the cohesion between the cells of the cell spheroids and the limited supply of oxygen and nutrients inside the cell spheroids, which reduces cell survival. In addition, Example 1 showed less decrease in diameter than the negative control group until day 7, which is thought to be a result of the reduced force on the cells due to microgravity. However, it did not show statistical significance.

[0099]

[0100] Experimental Example 3: Confirmation of the Sustained Floating Ability of Cell Spheroids in Neutral Buoyancy Medium

[0101] The floating persistence of cell spheroids in the medium of Example 1 was observed for 14 days. Half of the total medium volume was replaced every two days, and the height of cell spheroids was observed by observing the side of the well plate, as shown in Figures 3A and 3B.

[0102] As can be seen in Figures 3A to 3B, the height of the cell spheroids cultured in Example 1 was found to remain constant for 14 days. From these results, it was determined that the cell spheroids cultured in Example 1 could simulate a microgravity environment through continuous suspension. In addition, it was confirmed that microgravity was maintained during cell culture, at the time when cell culture was completed (usually taking about 24 hours), and for about 14 days after culture was completed.

[0103]

[0104] Experimental Example 4: Confirmation of Cytotoxicity of Neutral Buoyancy Medium

[0105] The cell spheroids of Example 1 were cultured for 14 days (Experimental Example 3), and the cell spheroids cultured in cell culture medium (Experimental Example 2) were set as a control group. After 1, 3, 7, and 14 days, a general cell culture medium containing 10% cell counting kit (Dojindo) solution was dispensed to all groups, and the cells were cultured for 3 hours in a 37°C, 5% CO2 incubator. The absorbance was measured at 450 nm using a UV / VIS Spectrophotometer, and the results are shown in Fig. 4.

[0106] Referring to the following Figure 4, the cell viability of Example 1 was found to be no significant difference from the control group, and thus it was determined that Example 1 had no cytotoxicity. Nevertheless, it was determined that the reason why the viability of the cell spheroids decreased over time was due to the characteristics of the cell spheroids themselves, as explained above.

[0107]

[0108] Experimental Example 5: Measurement of pluripotency-specific gene expression levels

[0109] RNA of cell spheroids cultured in Example 1 was extracted at a given time using a phenol-chloroform-based RNA extraction technique. Two-dimensional monolayer-cultured cells (2D culture) and three-dimensional cell spheroids (3D culture) were set as controls. The extracted RNA was synthesized into complementary DNA using Prime Script RT Master Mix (Takara). The synthesized complementary DNA was subjected to real-time PCR using the following pluripotency-specific gene TaqMan probes (ThermoFisher Scientific): OCT4 (Hs04260367_gH), SOX2 (Hs04234836_s1), and NANOG (Hs02387400_g1). The gene expression levels were plotted in Figure 5 using the ddCt method with GAPDH (Hs02786624_g1) as a housekeeping gene.

[0110] Under conditions of microgravity, various stem cells are known to remain undifferentiated rather than differentiated, and markers of terminal differentiation are suppressed. In the absence of external constraints like gravity, the expression of pluripotency-specific genes increases due to simultaneous changes, including cytoskeletal rearrangement, altered cellular metabolism, and a decrease in aryl hydrocarbon receptors (AHR), which detect external stimuli like gravity.

[0111] Looking at the gene expression results of Figure 5 below, representative pluripotency-related genes, OCT4, SOX2, and NANOG, all show higher expression in Example 1, a microgravity environment (μg), than in an actual gravity (1g) environment over time. Therefore, it is judged that the simulated microgravity environment utilizing neutral buoyancy of Example 1 maintains a high undifferentiated capacity of stem cells, rather than an environment with gravity, due to the results of Example 1 of the present invention.

[0112]

[0113] Experimental Example 6: Measurement of pluripotency-specific protein expression levels using immunoblotting.

[0114] The protein expression of the cell spheroids of Example 1 was analyzed using the same experimental group composition as Experimental Example 5. 2D culture, 3D culture, and the cell spheroids of Example 1 were each lysed with RIPA buffer to isolate proteins. The separated proteins were classified by size using polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane. The membrane with attached proteins was blocked with 5% bovine serum albumin and bound to the target proteins with anti-OCT4 (Cell Signaling Technology), anti-SOX2 (Abcam), and anti-NANOG (Cell Signaling Technology) primary antibodies and HRP-conjugated secondary antibodies, respectively. Afterwards, the target protein bands were confirmed by luminol emission by treating with ECL reagent. The observation results are shown in Figure 6.

[0115] As a result of observing the bands of the target proteins in Fig. 6, a graph showing that the expression levels of SOX2 and NANOG increased over time in Example 1 was observed. High expression of OCT4 was also observed in Example 1, but no significant difference in expression was observed compared to 3D culture. In the 3D culture group, the expression levels of SOX2 and NANOG were observed to be significantly lower than in Example 1, but the expression of OCT4 increased similarly to Example 1. This can be judged to mean that the shape of the cell spheroid contributes to the pluripotency of stem cells to some extent, although it is lower than in a microgravity environment.

[0116] In summary, when cells are cultured in a spheroid form, the expression of some pluripotency proteins increases, but when cultured as cell spheroids in the environment of Example 1, high expression of representative pluripotency-specific proteins such as OCT4, SOX2, and NANOG were observed to increase over time. In other words, it can be seen that the neutral buoyancy environment of Example 1 significantly increased the pluripotency of stem cells by simulating microgravity.

[0117]

[0118] Experimental Example 7: Analysis of osteogenic differentiation potential of cell spheroids in a simulated microgravity environment.

[0119] The protein expression of the cell spheroids of Example 1 was analyzed using the same experimental group composition as Experimental Example 5. An osteogenic differentiation medium was prepared by adding 100 nM dexamethasone (Sigma-Aldrich), 50 μM ascorbic acid (Sigma-Aldrich), and 10 mM beta-glycerophosphate (Sigma-Aldrich) to the cell culture medium. Based on the osteogenic differentiation medium prepared in this way, the cell spheroids cultured in Example 1 and Comparative Example 5 were measured for the expression of RUNX2 and OCN, which are osteogenic differentiation-related genes, using the same method as Experimental Example 5.

[0120] It is known that differentiation of stem cells into bone lineage cells is inhibited in a microgravity environment. As shown in Figure 7, both RUNX2, an early osteogenic differentiation factor, and OCN, a late osteogenic differentiation factor, were significantly reduced compared to the 2D and 3D culture controls in Example 1.

[0121] These results suggest that the neutral buoyancy of Example 1 mimicked the microgravity environment, inhibiting the early to late osteogenic differentiation of stem cells.

[0122]

[0123] This confirms that the neutral buoyancy environment of Example 1 can sufficiently simulate microgravity by maintaining the differentiation ability of cell spheroids and suppressing osteogenic differentiation, and suggests that it can overcome the limitations of existing microgravity simulation devices that cannot sufficiently simulate microgravity on the ground by significantly applying rotational force, magnetic force, sound waves, etc. to cells.

Claims

1. A cell culture platform characterized in that cell culture is possible in an environment that simulates microgravity by mixing a density control solution into the cell culture medium to control the density of the cell culture medium to a density range that cells have.

2. In paragraph 1, The above density control solution comprises a first substance comprising iodine as a component; Second material based on polysaccharides; A third material based on silica particles; and A cell culture platform, wherein the cell culture platform is one selected from a mixture of two or more materials selected from the first material, the second material, and the third material.

3. In paragraph 2, A cell culture platform wherein the first material comprising iodine as a component is any one selected from the group consisting of Optiprep, Visipaque, Cardiolek, RosetteSep, Omnipaque, NycoDenz, and HistoDenz.

4. In paragraph 2, A cell culture platform wherein the second material based on the above polysaccharide is any one selected from the group consisting of Ficoll, Ficoll-Paque, PolymorphPrep, LymphoPrep, Histopaque, sucrose, and dextran.

5. In paragraph 2, A cell culture platform wherein the third material based on the above silica particles is Percoll or GM501 (Gynemed).

6. In paragraph 2, A cell culture platform in which the density of the cell culture solution has a range of 1.05 to 1.10 g / mL when the density control solution is a first substance containing iodine as a component.

7. In paragraph 2, A cell culture platform wherein the density of the cell culture solution has a range of 1.06 to 1.08 g / mL when the density control solution is a second material based on polysaccharides.

8. In paragraph 2, A cell culture platform wherein the density of the cell culture solution has a range of 1.05 to 1.06 g / mL when the density control solution is a third material based on silica particles.

9. In paragraph 1, A cell culture platform wherein the above cell culture medium comprises a basic medium, serum, and antibiotics.

10. In paragraph 9, A cell culture platform wherein the above basic medium is any one serum-free medium selected from the group consisting of BME (basal medium Eagle's), MEM (minimal essential medium), α-MEM (α-minimal essential medium), DMEM (Dulbecco's modified Eagle's medium), DMEM / F-12, Ham's F-10, Ham's F-12, Leibovitz's L-15 Medium, RPMI 1640, M199 medium, IMDM (Iscove's modified Dulbecco's medium), McCoy's 5A (modified) medium, William's medium E, and MesenCult-ACF Plus medium.

11. In paragraph 9, A cell culture platform wherein the serum, which is a component of the above cell culture medium, is one or more types selected from among fetal bovine serum (FBS), bovine calf serum (BCS), newborn calf serum (NCS), horse serum, and normal human serum.

12. In paragraph 9, A cell culture platform wherein the serum is included in an amount of 1 to 40% of the basic medium weight.

13. In paragraph 9, A cell culture platform in which the antibiotics that are a component of the cell culture medium are one or more selected from penicillin, streptomycin, amphotericin B, ampicillin, gentamicin, neomycin, kanamycin, and polymyxin B.

14. In paragraph 9, A cell culture platform wherein the above antibiotic is included at 0.1 to 10% of the weight of the basic medium.

15. In paragraph 1, A cell culture platform wherein the density of the above cell culture medium is 1.05 to 1.10 g / mL.

16. In paragraph 1, A cell culture platform in which the density of the above cell culture medium can be continuously maintained during cell culture, at the time when cell culture is completed, and after cell culture is completed.

17. A cell culture comprising cells prepared from a cell culture platform according to paragraph 1.

18. In paragraph 17, A cell culture characterized in that cells produced on the above cell culture platform are buoyant within the culture medium and remain suspended in the middle or on the surface of the liquid of the cell culture medium, so that the cells do not sink and the cells remain continuously suspended.

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