Method for producing differentiated cells or differentiated tissues

Abstract

To provide a technology that more efficiently promotes cell differentiation without depending on the initial conditions of a stimulus when promoting cell differentiation by applying the stimulus.SOLUTION: Provided is a method for producing differentiated cells or differentiated tissues, the method comprising: a culture step of culturing cells while applying a stimulus under conditions where a plurality of strain fields with different combinations of strains in a plurality of directions occur; a measuring step of non-invasively measuring the expression level of a gene related to differentiation of the cells in each strain field, in the culturing step; and an adjusted culture step of adjusting stimulation and culturing based on the results of the measurement step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing differentiated cells or differentiated tissues, etc. [Background technology]

[0002] Articular cartilage covers the joint surface and possesses excellent mechanical functions such as load support, shock absorption, and lubrication. Furthermore, joint movements such as walking subject chondrocytes to complex mechanical stimuli, including compressive and shear forces, influencing the formation of articular cartilage and the maintenance of cellular characteristics. However, because articular cartilage lacks lymphatic vessels, blood vessels, and nerves, its self-repair capacity is poor, making self-repair difficult for damaged articular cartilage. A representative disease affecting articular cartilage is osteoarthritis of the knee, and autologous cultured cartilage transplantation using culture media is attracting attention. However, maintaining the characteristics of cultured cartilage for extended periods is difficult, and its mechanical properties are inferior to those of articular cartilage in vivo. In fact, it has been reported that when chondrocytes are cultured in vitro, the expression level of Col2a1, a chondrocyte differentiation marker, decreases, while the expression level of Col1a1, a dedifferentiation marker, increases, leading to fibrochondrosis. Therefore, in the production of cultured cartilage, increasing Col2a1 expression and suppressing Col1a1 expression are considered crucial.

[0003] Numerous studies have reported that physiological mechanical stimuli affect the activity and morphological maintenance of chondrocytes, and that mechanical stimulation during the culture process improves the matrix production and mechanical function of cultured cartilage tissue. For example, Non-Patent Documents 1 and 2 report that periodic hydrostatic pressure and mechanical compression stimulation of cultured cartilage tissue promote the production of extracellular matrix proteins such as type II collagen and aggrecan. Non-Patent Documents 3 and 4 report that excessive strain on chondrocytes decreases Col2a1 expression and increases Col1a1 expression. In addition to these, research is being conducted on regenerative medicine for bone and cartilage using ES / iPS cells and mesenchymal stem cells to address the problem of cell sources. Mesenchymal stem cells have inferior proliferative and differentiation capabilities compared to ES / iPS cells, but they have a lower risk of tumorigenesis and can be used relatively easily as autologous cells, making them less of a barrier to clinical application. Mechanical stimulation is also effective for chondrogenic differentiation of mesenchymal stem cells, and it has been reported that shear stimulation and compression stimulation promote chondrogenic differentiation. Living tissues achieve structural construction and functional expression by reflecting cellular responses to stimuli transmitted via the mechanical field surrounding the cells. Therefore, it is believed that if appropriate stimuli can be applied to cells according to the tissue state, efficient production of cultured cartilage will be possible.

[0004] As mentioned above, the mechanical field surrounding cells also changes in accordance with culture, so in order to set appropriate mechanical stimuli, it is necessary to monitor the gene expression dynamics in response to the mechanical field. However, in many studies to date, the amount of stimulation and the timing of stimulation loading are set in feedforward, and the amount of gene expression and protein production are evaluated after a certain period of culture. As a result, information on cell differentiation is obtained as a quantity for the entire tissue, and the relationship with spatial information such as the minute "mechanical field" surrounding the cells and temporal information such as real-time information is lost. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Buschmann, MD, Kim, YJ, Wong, M, Frank, E, Hunziker, EB, and Grodzinsky, J, Archives of Biochemistry and Biophysics, Vol. 366 , ( 1999 ), pp. 1-7

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[0006] An object of the present invention is to provide a technique for more efficiently promoting cell differentiation promoted by application of stimulus, regardless of the initial conditions of the stimulus. Means for Solving the Problem

[0007] As a result of intensive research conducted by the present inventors in view of the above problem, it has been found that the above problem can be solved by a method for producing a differentiated cell or a differentiated tissue, the method comprising: a culture step of culturing cells while applying a stimulus thereto under conditions where a plurality of strain fields with mutually different combinations of strains in a plurality of directions are generated; a measurement step of non-invasively measuring the expression level of a gene associated with differentiation of the cells in each strain field in the culture step; and an adjusted culture step of adjusting the stimulus based on the result of the measurement step and continuing the culture. The present inventors have further advanced research based on this finding, and as a result completed the present invention. That is, the present invention includes the following embodiments.

[0008] Item 1. A method for producing a differentiated cell or a differentiated tissue, comprising: a culture step of culturing cells while applying a stimulus thereto under conditions where a plurality of strain fields with mutually different combinations of strains in a plurality of directions are generated; a measurement step of non-invasively measuring the expression level of a gene associated with differentiation of the cells in each strain field in the culture step; an adjusted culture step of adjusting the stimulus based on the result of the measurement step and culturing the cells; a method comprising the above steps.

[0009] Item 2. The method according to Item 1, wherein the stimulus is a mechanical stimulus, an electrical stimulus, or a drug stimulus.

[0010] Item 3. The method according to Item 1, wherein the stimulus is a mechanical stimulus.

[0011] Item 4. The method according to Item 3, wherein the mechanical stimulus is at least one selected from the group consisting of tensile stimulation, compressive stimulation, shear stimulation, hydrostatic pressure, sliding stimulation, and torsion.

[0012] Item 5. The method according to item 1, wherein there are two directions.

[0013] Item 6. The method according to item 5, wherein the two aforementioned directions intersect at an angle of 60 to 120 degrees.

[0014] Item 7. The method according to item 1, wherein the culture step is two-dimensional culture or three-dimensional culture.

[0015] Item 8. The method according to Item 1, wherein the cells are at least one selected from the group consisting of chondrocytes, chondrocytes, myoprogenitor cells, myocytes, osteoblasts, osteocytes, and stem cells.

[0016] Item 9. The method according to Item 1, wherein the gene is at least one selected from the group consisting of type I collagen gene, type II collagen gene, type X collagen gene, PRG4 gene, aggrecan gene, MMP13 gene, and SOX9 gene.

[0017] Item 10. The method according to Item 1, wherein the cells include an expression cassette comprising a promoter of the gene and a reporter gene located downstream thereof.

[0018] Item 11. The method according to item 10, wherein the measurement step is performed by measuring the reporter signal intensity of the cell.

[0019] Item 12. The method according to Item 1, wherein in the preparation culture step, the stimulation is adjusted based on the results of the measurement step to promote cell differentiation and then cultured. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a technique that promotes cell differentiation more efficiently when stimulating cells, regardless of the initial conditions of the stimulus. [Brief explanation of the drawing]

[0021] [Figure 1] The mechanical stimulation culture apparatus used in the example is shown. [Figure 2] The sheet shape and dimensions of the culture sheet used in the example are shown. [Figure 3] This shows a two-dimensional strain field contour map for the strain εxx generated when displacement (sheet displacement: (a) 1.520 mm, (b) 1.380 mm, (c) 1.240 mm, (d) 1.100 mm, (e) 0.960 mm, (f) 0.820 mm, (g) 0.680 mm, (h) 0.540 mm) is applied by a stepping motor. [Figure 4] This shows a two-dimensional strain field contour map for the strain εyy generated when displacement (sheet displacement: (a) 1.520 mm, (b) 1.380 mm, (c) 1.240 mm, (d) 1.100 mm, (e) 0.960 mm, (f) 0.820 mm, (g) 0.680 mm, (h) 0.540 mm) is applied by a stepping motor. [Figure 5] This shows the relationship between the average fluorescence intensity values ​​calculated from fluorescence observation images and the gene expression levels measured by RT-qPCR (N=4, mean±SD). [Figure 6] This section shows a comparison of experimental and analytical values ​​using the sigmoid function. [Figure 7]The following shows the results of measuring Col2a1 and Col1a1 expression levels on day 6 of culture by RT-qPCR when mechanical stimulation was controlled with an initial displacement of 0.960 mm (N=4, mean±SD, * indicates p<0.05 (Student's t-test)). [Figure 8] The results of measuring Col2a1 and Col1a1 expression levels by RT-qPCR on day 6 of culture under constant mechanical stimulation at 0.540 mm are shown (N=4, mean±SD, * indicates p<0.05 (Student's t-test)). [Figure 9] The following shows the results of measuring Col2a1 and Col1a1 expression levels on day 6 of culture by RT-qPCR when mechanical stimulation was controlled with an initial displacement of 0.540 mm (N=4, mean±SD, * indicates p<0.05 (Student's t-test)). [Figure 10] The response surfaces for each day ((a): day1, (b): day2, (c): day3, (d): day4, (e): day5) when the initial displacement was set to 0.960 mm and mechanical stimulation was controlled are shown. [Figure 11] The response surfaces for each day ((a): day1, (b): day2, (c): day3, (d): day4, (e): day5) when the initial displacement was set to 0.540 mm and mechanical stimulation was controlled are shown. [Modes for carrying out the invention]

[0022] In this specification, the expressions “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consistent only of.” In one embodiment, the present invention relates to a method for producing differentiated cells or differentiated tissue, comprising: a culture step of culturing cells while applying stimuli under conditions that generate a plurality of strain fields in which combinations of strains in a plurality of directions are mutually different; a measurement step of non-invasively measuring the expression levels of genes related to the differentiation of the cells in each strain field during the culture step; and an adjusted culture step of culturing the cells after adjusting the stimuli based on the results of the measurement step (which may also be referred to as “the method of the present invention” in this specification). This will be described below.

[0023] The cells used in the culture process are not particularly limited, as long as they are cells whose differentiation can be promoted by the application of stimuli. Examples of such cells include chondrocytes, chondrocytes, muscle (e.g., cardiac muscle, skeletal muscle) progenitor cells, muscle (e.g., cardiac muscle, skeletal muscle) cells, osteoblasts, osteocytes, and stem cells (e.g., mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, etc.).

[0024] Cells can be cell lines or primary cultured cells. Furthermore, cells may be isolated individual cells or form planar or three-dimensional tissues.

[0025] The culture process is carried out under a stimulating condition. The stimulus is not particularly limited as long as it can promote cell differentiation, and examples include mechanical stimulation, electrical stimulation, and drug stimulation. From the viewpoint of suitably applying the method of the present invention and easily obtaining the desired effect, mechanical stimulation is particularly preferred. Examples of mechanical stimulation include tensile stimulation, compressive stimulation, shear stimulation, hydrostatic pressure, sliding stimulation, and torsion.

[0026] The stimulus is applied to the cells under conditions that generate multiple strain fields, each with a different combination of strains in multiple directions. One of the features of the method of the present invention is that it takes into account strains in multiple directions.

[0027] A strain field is a region with a certain area or volume that divides a cell culture surface or a three-dimensional structure composed of cells. The cell culture surface or the three-dimensional structure composed of cells is divided into multiple strain fields, each with a different combination of strains in multiple directions.

[0028] "Multiple strain fields with different combinations of strain in multiple directions" refers to multiple strain fields where the combination of the numerical value of strain in one direction (direction 1) and the numerical value of strain in a direction other than direction 1 (direction 2) are different from each other. More specifically, this can include multiple strain fields consisting of, for example, a strain field where the strain in direction 1 is X1% and the strain in direction 2 is Y1%, a strain field where the strain in direction 1 is X2% and the strain in direction 2 is Y1%, a strain field where the strain in direction 1 is X3% and the strain in direction 2 is Y2%, and so on (where X1, X2, X3, ... represent different numerical ranges, and Y1, Y2, ... represent different numerical ranges).

[0029] The number of strain directions to consider is, for example, 2 to 4, preferably 2 to 3, and particularly preferably 2.

[0030] The angle at which one direction intersects with another is, for example, 30 to 150 degrees, preferably 60 to 120 degrees, and more preferably 80 to 100 degrees.

[0031] One of the multiple directions may be the direction in which the stimulus is applied (for example, the tensile direction in the case of a tensile stimulus), and the other may be a direction that intersects that direction at, for example, 30 to 150 degrees, preferably 60 to 120 degrees, and more preferably 80 to 100 degrees.

[0032] The strain range is preferably 0 to 11%. More specifically, for example, in the case of tensile stimulation, the strain in the tensile direction can be 0 to 11%, and the strain in the direction intersecting the tensile direction can be 0 to 5%.

[0033] The width of the strain range that divides the strain field (the width of the numerical range of X1, X2, X3, Y1, Y2, ... in the above description) is not particularly limited, but can be, for example, 0.1 to 3%, preferably 0.3 to 2%, more preferably 0.6 to 1.5%, and even more preferably 0.8 to 1.2%.

[0034] Cell culture is preferably carried out on a substrate made of a stretchable material so that strain can be applied to the cells. Such materials are not particularly limited, but examples include silicone sheets, agarose gel, collagen gel, etc. In a preferred embodiment of the present invention, the shape of the culture substrate and the intensity of the stimulus applied to the culture substrate are predetermined so that the above-mentioned multiple strain fields are generated, and the stimulus is applied to the culture substrate under these predetermined conditions, thereby applying the same stimulus to the cells.

[0035] The shape of the culture substrate is not particularly limited, but it is usually in the form of a sheet.

[0036] Culture media can be prepared based on basal media used for culturing animal cells. Examples of basal media include Glasgow's Minimal Essential Medium (GMEM), IMDM, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's, Neurobasal Medium (Life Technologies), and mixtures thereof. The media may or may not contain serum. If necessary, the culture medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol, as well as one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts.

[0037] Regarding the culture conditions, the culture temperature is not particularly limited, but is approximately 30-40°C, preferably approximately 37°C. The culture is carried out in an atmosphere of CO2-containing air, and the CO2 concentration is preferably approximately 2-5%.

[0038] The culture time is not particularly limited, as long as it is long enough for differentiation-related genes to be expressed in at least one strain field. The culture time can be, for example, 12 hours to 7 days, preferably 15 hours to 2 days.

[0039] The genes related to differentiation are not particularly limited, as long as they are genes that promote differentiation, inhibit differentiation, or promote dedifferentiation. Here, differentiation may differ depending on the type of cell, but can be, for example, cartilage differentiation, bone differentiation, muscle differentiation, etc. Specific examples of such genes in the case of cartilage include, for example, type I collagen gene, type II collagen gene, type X collagen gene, aggrecan gene, PRG4 gene, MMP13 gene, and SOX9 gene. Among these, type I collagen gene and type II collagen gene are preferred, and a combination of type I collagen gene and type II collagen gene is particularly preferred.

[0040] The organism from which the genes related to differentiation originate is not particularly limited and can be any animal, such as various mammals including humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Among these, humans are particularly preferred.

[0041] The base sequences of genes related to differentiation are publicly known and can be obtained using various databases such as NCBI.

[0042] The method for measuring the expression levels of differentiation-related genes is not particularly limited, as long as it is non-invasive, i.e., can be performed without destroying cultured cells. One such method involves using cells containing an expression cassette that includes a promoter of differentiation-related genes and a reporter gene located downstream thereof, as the cells to be cultured, and further measuring the reporter signal intensity of these cells.

[0043] A promoter is a region required for the expression of a gene related to differentiation, and is not particularly limited in this respect. Typically, a promoter may include the transcription start site of a gene related to differentiation, its upstream (5') sequence, and optionally its downstream (3') sequence. The base length of the upstream sequence can be, for example, 100 or more, 200 or more, 300 or more, 500 or more, 1000 or more, 2000 or more, or 3000 or more, and can be, for example, 10000 or less, 7000 or less, or 5000 or less.

[0044] Examples of reporter genes include fluorescent protein genes such as GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, and mPlum.

[0045] The reporter signal intensity measured by the above method represents the amount of reporter protein expressed by the promoter of differentiation-related genes; therefore, the signal intensity reflects the expression level of differentiation-related genes. The signal intensity or its relative value can be directly treated as the expression level of differentiation-related genes, or the correlation between the signal intensity and the expression level of differentiation-related genes can be measured in advance, and the signal intensity can be converted to the expression level of differentiation-related genes based on that correlation.

[0046] In the measurement step, the expression levels of differentiation-related genes are measured in each of the multiple strain fields. This provides expression level information for each strain field. In the method of the present invention, the stimulation in the culture step is adjusted based on the obtained expression level information, and the culture is continued.

[0047] Expression level information can be used directly as an indicator for adjusting stimuli, or it can be processed before use. For example, if expression level information for multiple genes is obtained, each expression level can be subjected to arithmetic operations such as addition, subtraction, multiplication, and division, and the resulting value can be used as an indicator for adjusting stimuli. In a preferred example, the value obtained by subtracting the expression level of type II collagen gene from the expression level of type II collagen gene can be used as an indicator.

[0048] In the adjusted culture step, preferably, the stimulation is adjusted based on the results of the measurement step to promote cell differentiation and then cultured. A specific example is as follows: For each strain field, the value X is calculated by multiplying the area of ​​the strain field by the value of the above index. Next, the total value Y is calculated by adding up all the values ​​X obtained for each strain field. Subsequently, based on the information on the change in the area of ​​each strain field when the stimulation is changed, the mechanical stimulation is changed and cultured so that the total value Y changes in a direction that further promotes differentiation.

[0049] The method of the present invention makes it possible to obtain differentiated cells or tissues that are more differentiated than the cells at the time of the culture process. The method of the present invention can promote differentiation more efficiently regardless of the initial stimulation conditions. Furthermore, according to a preferred embodiment of the present invention, it is possible to promote differentiation more efficiently than by continuously applying a constant stimulus.

[0050] "Advanced differentiation" encompasses not only the progression of cell differentiation, but also the fact that the substances produced by the cells (such as extracellular matrix components like collagen) and the tissues containing the cells become closer in composition to the body's internal tissues. [Examples]

[0051] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0052] Test Example 1. Production of Differentiated Cells <1. Overview> When culturing cells while applying mechanical stimuli, the expression levels of type I collagen gene (Col1a1) and type II collagen gene (Col2a1) in multiple strain fields with different combinations of tensile and compressive strains were measured using reporter assays based on the promoters of these genes. Based on the obtained measurement results, the mechanical stimuli were adjusted to further increase Col2a1 expression and / or further suppress Col1a1 expression, and the cells were further cultured to produce differentiated cells. Specifically, the procedure was as follows.

[0053] <2. Test Method> <2.1 Overview of the Mechanical Stimulation Stressing Culture System> In this study, to evaluate the transcriptional response of genes associated with cartilage differentiation in situ under mechanical stimulation culture, the mechanical stimulation culture apparatus shown in Figure 1 was used. The total length of the mechanical stimulation culture apparatus is 156 × 230 mm, which is less than the total length of the automatic positioning stage of the fluorescence microscope (ECLIPSE TE2000-U Nikon). Furthermore, by providing holes in the substrate of the mechanical stimulation culture apparatus for fixing to the automatic positioning stage, fluorescence observation at the same coordinate position is possible. In this study, cells were cultured on four silicon sheets (Sanshin Enterprise Co., Ltd.) measuring 10 mm wide, 20 mm long, and 200 μm thick, placed in the center of the mechanical stimulation culture apparatus, and mechanical stimulation was applied. In this apparatus, a uniaxial stepping motor (DRL28, Oriental motor) is driven to apply forced displacement from both sides of the silicon sheets, resulting in a mechanism where the silicon sheets experience symmetrical displacement. The uniaxial stepping motor was programmed to control its direction, drive speed, and drive amount via a controller (EMP400, Oriental motor) on a PC using the software "Hyper terminal". A linear motion guide rail (SSEB6-25, MISUMI) was installed to ensure that the jig fixing the silicone sheet moved in a straight line without lateral movement when forced displacement was applied from both sides of the silicone sheet. Furthermore, to prevent the silicone sheet from twisting when fixing both ends, fixing fixtures made of SUS316 were used and screwed in. A glass slide was placed on the bottom of the observation area, allowing fluorescence observation with the lid attached, thus enabling continued cell culture after fluorescence observation. Using this culture apparatus, cell responses under mechanical stimulation were observed in situ. In this study, cells were cultured using the mechanical stimulation culture apparatus in an incubator at 37°C and a CO2 concentration of 5%.

[0054] <2.2 Method for culturing ATDC5 cells> In this study, we used ATDC5 cells, a mouse embryonic carcinoma-derived clonal cell line used as a model cell for chondrogenesis in the early stages of cartilage development. It has been confirmed that ATDC5 cells show increased expression of the chondrogenesis markers Col2a1 and Acan, and that differentiation progresses in the presence of insulin. In the differentiation culture, Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham (D8062-500ML, SIGMA-ALDRICH) culture medium containing 5% FBS and 50 μg / ml kanamycin was used. Transferrin (10652202001, SIGMA-ALDRICH) was added to the maintenance culture medium at a final concentration of 50 μg / ml, and 3 × 10⁶ cells were added. -8 A culture medium supplemented with M-grade sodium selenite (S5261-10G, SIGMA-ALDRICH) and 10 μg / ml insulin (11376497001, SIGMA-ALDRICH) was used. During differentiation, the culture medium was 2.4 × 10⁶. 4 cells / cm 2 ATDC5 cells were seeded at a density and cultured in an incubator at 37°C with a CO2 concentration of 5%. The culture medium was changed every 48 hours for both maintenance and differentiation cultures.

[0055] <2.3 Design of substrate sheet shape using finite element analysis> This study first focused on the heterogeneous two-dimensional strain fields generated around cells when mechanical stimuli are applied. After creating a sheet shape that generates these strains, the aim was to establish a stimulus control method that enables the desired gene expression state across the entire sheet. The study investigated the effects of mechanical stimuli on chondrocyte substrate production and gene expression levels. Numerous previous studies have shown that, under the two-dimensional culture conditions of this study, periodic tensile stimulation at 0.17 Hz, 3% strain, and 0-2 hours / day has little effect on the expression of substrate-related genes. Furthermore, it has been reported that periodic tensile stimulation at 0.17-0.5 Hz, 3-10% strain, and 2-12 hours / day promotes chondrocyte anabolic reactions, while periodic tensile stimulation at 0.5 Hz, 10% or more strain, and 12 hours / day enhances chondrocyte catabolic reactions. Based on this information, the sheet shape and dimensions determined by finite element analysis are shown in Figure 2. Furthermore, Figure 3 shows the contour map of the two-dimensional strain field generated when cells are seeded on this sheet and displacement is applied by a stepping motor. As shown in Figure 3, the strain ε in the x-direction (tensile direction) xx Based on the aforementioned prior research, the conditions for promoting increased gene expression in chondrocytes were set to 1-11%. Furthermore, the sheet shape design specifications were set so that (1) the set strain amount could be distributed within the sheet, and (2) the set strain ε was a minimum of 1% and a maximum of 11%. xx The change in fluorescence intensity value in the culture device must be observable via fluorescence in the observation area of ​​the mechanical stimulation-loaded culture apparatus. The sheet shape shown in Figure 2 was designed to satisfy these specifications, with a sheet length of 30 mm and a shape obtained by cutting a circle symmetrically along the x-axis, passing through the three points (x, y) = (±10 mm, ±5 mm) and (x, y) = (0 mm, ±2 mm) with the sheet center as the origin. For the analysis, the physical properties of the silicon sheet were determined to be Young's modulus 5.7 MPa, Poisson's ratio 0.49, and density 1040 kg / cm³. 3 The same strain field was defined as being within ±0.5% of the reference value. Figure 3(h) shows the minimum strain ε set in this study. xxIn order to perform fluorescence observation of 1% strain ε in the sheet using a mechanical stimulation loading culture device, a sheet displacement that generates a maximum strain ε of 4% in the sheet xx was required. Therefore, finite element analysis was used to search for the sheet displacement required to increase from 4% by 1% increments until the maximum value of strain ε generated in the sheet xx reached 11%. As a result, by using eight types of sheet displacement as shown in Figure 3, the set strain ε ranging from 1% to 11% xx was distributed in the sheet, and changes in fluorescence luminance values at each strain level became observable via fluorescence using the mechanical stimulation loading culture device. In this study, by using this sheet shape, a mechanism that applies eight types of set mechanical stimulation according to cell state was obtained.

[0056] <2.4.1 Construction of ZsGreen1 Expression Vector Having Col2a1-Specific Promoter> This study utilized the function of promoter regions in the gene transcription process of organisms. To evaluate the promotion of ATDC5 cell differentiation, the promoter and enhancer regions of the Col2a1 gene, which encodes the genetic information of type II collagen, the major substrate of mouse articular cartilage, were used. Based on previous studies, the Col2a1 promoter region was selected using the region containing the reaction region (309 bp) of the promoter region (687 bp) and the enhancer sequence (182 bp) in the intron regions of exon 1 and exon 2, which have been identified as functioning specifically on cartilage during mouse development. Furthermore, transcriptional activity was improved by tandem-repeating the enhancer region twice. Previous studies have confirmed that these regions function specifically on cartilage in the shoulder joint and costal cartilage in mouse fetuses. ZsGreen1 was used as the reporter protein. In this study, pZsGreen1-1 (Clontech Laboratories, Inc.), a promoter-less vector containing the ZsGreen1 sequence, was used. We prepared and used Col2a1 promoter (687 bp)-Col2a1 intron (182×2 bp)-pZsGreen1-1 by inserting a selected promoter region and a double-tandem-replicated intron region into the multi-cloning site (MCS) of the pZsGreen1-1 vector. Hereafter in this study, this vector will be referred to as Col2a1 promoter (687 bp)-i182×2-pZsGreen1.

[0057] <2.4.2 Method for introducing vectors into ATDC5 cells> For gene transfer into ATDC5 cells, the transfection reagents Lipofectamine LTX Reagent (100014470, Thermo Fisher Scientific) and Lipofectamine Plus Reagent (100014473, Thermo Fisher Scientific) were used. The amount of reporter vector introduced was 1.2 × 10⁶. 4For ATDC5 cells from cells, the Col2a1 promoter (687 bp)-i182×2-pZsGreen1 : CMV-pDsRed-Express2 was administered in a ratio of 600 ng : 400 ng. 250 μl of Opti-MEM (31985-070, Thermo Fisher Scientific) was mixed with 7.5 μl of Lipofectamine LTX Reagent, 2.5 μl of Lipofectamine Plus Reagent, and the reporter vector prepared in the above ratio. The mixture was incubated at room temperature for 15 minutes. During incubation, the entire cell culture medium was replaced with Opti-MEM, and the mixture was added dropwise after incubation was complete. Since the total cell count differed between static culture using wells or plates and under mechanical stimulation, the amount of transgene and reagents for each condition were adjusted according to the manufacturer protocols for Lipofectamine LTX Reagent and Lipofectamine Plus Reagent.

[0058] <2.5.1 Composition of a DsRed-Express2 expression vector with a Col1a1-specific promoter> Previous studies have reported many conditions for mechanical stimulation that increase Col2a1 expression in chondrocytes. ,However, it is possible that not only differentiation-promoting marker genes such as Col2a1, but also cartilage dedifferentiation marker genes, may have altered expression levels due to mechanical stimulation. Therefore, in this study, we will simultaneously evaluate the transcriptional response of Col1a1, a cartilage dedifferentiation marker, in addition to Col2a1, a cartilage differentiation-promoting marker. To this end, we first created a fluorescent protein expression vector containing the Col1a1 promoter. Based on previous studies, we selected a 3.2 kbp promoter region that has been confirmed to exhibit specific expression in dermal fibroblasts, osteoblasts, odontoblasts, tendons, and fascial fibroblasts in in vivo mice. Furthermore, we used a promoter-less vector, pDsRed-Express2-1, containing the gene sequence of the reporter protein DsRed-Express2, as the vector. First, we cloned the DNA fragment of the Col1a1 promoter region from BAC clone (Thermo Fisher Scientific).

[0059] <2.5.2 Expression evaluation of DsRed-Express2 expression vectors with Col1a1-specific promoters> The responsiveness of Col1a1 promoter (3.2bp)-pDsRed-Express2-1, prepared in section 2.5.1, was evaluated. The vector, prepared in NIH3T3 cells (mouse-derived fibroblasts known to produce type I collagen), was introduced using the method described in section 2.4.2, and its function was evaluated by fluorescence observation. Furthermore, to confirm the expression of the fluorescent protein in ATDC5 cells used in this study, the vector, prepared in undifferentiated ATDC5 cells known to produce type I collagen, was introduced using the method described in section 2.4.2, and fluorescence observation was performed. Gene introduction was performed 18 hours after seeding of each cell type, and fluorescence observation was performed 5 days after gene introduction. In addition to Col1a1 promoter (3.2bp)-pDsRed-Express2-1, CMV-pZsGreen1-1 was also introduced simultaneously. The amount of reporter vector introduced was 1.2 × 10⁻⁶. 4For cells, the Col1a1 promoter (3.2bp)-pDsRed-Express2-1 : CMV-pZsGreen1 = 600 ng : 400 ng was used.

[0060] <2.5.3 Evaluation of the relationship between Col2a1 and Col1a1 expression levels and their respective fluorescence intensity values> Changes in fluorescence intensity in response to changes in gene expression levels were evaluated. ATDC5 cells were maintained for 4 days and then differentiated for 7 days, followed by 2.4 × 10⁶ 4 cells / cm 2 Seeds were sown at the following density. Eighteen hours after sowing, Col2a1 promoter (687 bp)-i182×2-pZsGreen1 and Col1a1 promoter (3.2 kbp)-pDsRed-Express2 were simultaneously introduced. The amount of reporter vector introduced was 1.2 × 10⁻⁶. 4 For cells from the Cells group, the following mixtures were used: Col2a1 promoter (687 bp)-i182×2-pZsGreen1 : Col1a1 promoter (3.2 kbp)-pDsRed-Express2 = 500 ng : 500 ng. Fluorescence observation and gene extraction were performed on days 1, 3, 5, and 7 of culture, and the relationship between the expression level of each gene and the fluorescence intensity value was evaluated. mRNA extraction and RT-qPCR were performed according to standard procedures. The target genes for RT-qPCR were Col2a1, Col1a1, and G3pdh as an endogenous control, and primers with the nucleotide sequences shown in Table 2.2 were used for each gene during measurement.

[0061] <2.5.4 Strain analysis in the compression direction using finite element analysis> In this study, strain ε is identified as a strain that can affect gene expression levels. xx In addition, strain ε in the tensile and perpendicular directions yy We focused on the possibility that this is also the case. In particular, in a two-dimensional plane stress state, tensile strain ε xx against ε yy This indicates compression. Therefore, in this study, when evaluating the expression levels of Col2a1 and Col1a1 associated with dynamic field fluctuations across the entire sheet, strain ε xx In addition, strain εyy The expression levels of each gene are evaluated considering the value of ε. The strain ε generated when using the eight types of sheet displacement shown in Figure 3 of Section 2.3 yy The strain ε was obtained by finite element analysis. Figure 4 shows the strain ε at each of the eight obtained sheet displacement values. yy The analysis results are shown. In this study, the strain ε is used in Figures 3 and 4. xx and strain ε yy The expression levels of each gene in this region were evaluated.

[0062] <2.5.5 Algorithm for controlling mechanical stimuli using Col2a1 and Col1a1 promoter activity> To increase Col2a1 expression and decrease Col1a1 expression, mechanical stimulation culture was performed by varying the amount of mechanical stimulation based on Col2a1 and Col1a1 promoter activity. Eight types of strain ε obtained in sections 2.3 and 2.5.4 were used. xx and strain ε yy Based on the distribution patterns, the area of ​​each strain field occurring within each sheet was calculated using ImageJ, and the results are shown in Tables 1 and 2. The sheet displacements are as follows: (a) 1.520 mm, (b) 1.380 mm, (c) 1.240 mm, (d) 1.100 mm, (e) 0.960 mm, (f) 0.820 mm, (g) 0.680 mm, (h) 0.540 mm.

[0063] [Table 1]

[0064] [Table 2]

[0065] ATDC5 cells were maintained in culture for 4 days, then differentiated in culture for 7 days, and then 2.4 × 10⁶ cells were placed across the entire area of ​​the silicone sheet. 4 cells / cm 2Seeds were sown at the following density. Eighteen hours after sowing, Col2a1 promoter (687 bp)-i182×2-pZsGreen1 and Col1a1 promoter (3.2 kbp)-pDsRed-Express2 were introduced using the method described in section 2.4.3. The amount of reporter vector introduced was 1.2 × 10⁻⁶. 4 For cells, the following strains were used: Col2a1 promoter (687 bp)-i182×2-pZsGreen1 : Col1a1promoter (3.2 kbp)-pDsRed-Express2 = 500 ng : 500 ng. After acquiring the fluorescence intensity values ​​for each strain field on the first day of mechanical stimulation culture, the gene expression levels for each strain field were estimated based on the relationship between gene expression levels and fluorescence intensity values ​​obtained in section 2.5.3. Note that the fluorescence intensity values ​​were obtained by subtracting the background of an empty vector from the acquired images. The difference in expression levels in each strain field, i.e., "average value of Col2a1 mRNA - average value of Col1a1 mRNA", was used as the target for strain ε xx : 0~11%, strain ε yy : Between 0 and 5%, the response surface z = β0 + β1x + β2y + β3x 2 +β4y 2 +β5xy was created. Here, z is Col2a1 mRNA expression level - Col1a1 mRNA expression level, β i (i=1~5) are constants, and x is the strain ε xx The value of y is the strain ε. yy This is the value. When creating the response surface diagram, only the condition that satisfies "mean value of Col2a1 mRNA in the strain field of the stimulus group > mean value of Col2a1 mRNA in the control group" was adopted. The z value of each strain field obtained from the response surface diagram was multiplied by the area of ​​each strain field calculated to weight the z value in each strain field. Then, the sum of "area of ​​each strain field" × "z value" was calculated to obtain the total z value for the entire region of each of the 8 types of sheets. After that, the total z values ​​of the 8 z values ​​were compared and the stimulus amount that maximized was set as the next stimulus condition. Control according to this determination method was performed daily for 6 days. As an initial condition, a strain of 1-4% ε was placed in the sheet. xxUsing a sheet displacement of 0.540 mm that results in this condition, periodic tensile stimulation is applied at 0.5 Hz for 5 hours a day, and a strain of 1-7% ε is applied within the sheet. xx Mechanical stimulation control was performed under two conditions: periodic tensile stimulation at 0.5 Hz for 5 hours a day, using a sheet displacement of 0.960 mm that causes a certain amount of stress. For comparison, constant mechanical stimulation culture was performed under two sheet displacement conditions: 0.540 mm and 0.960 mm. For each unstimulated group, cells seeded on a silicon sheet were cultured statically on a 6-well plate. mRNA extraction was performed on day 6 of culture under two conditions with different initial conditions: mechanical stimulation culture based on Col2a1 and Col1a1 promoter activity, and constant mechanical stimulation culture. After extraction, the Col2a1 and Col1a1 expression levels for each condition, measured using RT-qPCR, were evaluated by dividing by the unstimulated group. mRNA extraction and RT-qPCR were performed using the same methods as described in section 2.4.5. The target genes were Col2a1, Col1a1, and G3pdh as an endogenous control, and the expression levels of these three genes were quantitatively evaluated.

[0066] <3.Results> <3.1 Col2a1 and Col1a1 expression in response to mechanical stimulation based on Col2a1 and Col1a1 promoter activity> Quantitative evaluation <3.1.1 Evaluation of DsRed-Express2 expression vectors with Col1a1-specific promoters> In NIH3T3 cells introduced with CMV-pZsGreen1-1 and Col1a1 promoter (3.2bp)-pDsRed-Express2, we confirmed the expression of green and red fluorescent proteins. This confirmed that the constructed vector functioned normally. Furthermore, since we confirmed the expression of green and red fluorescent proteins in undifferentiated ATDC5 cells introduced in the same manner, it is suggested that introducing the constructed vector into ATDC5 cells used in this study allows for the observation of Col1a1 expression dynamics over time.

[0067] <3.1.2 Evaluation of the relationship between Col2a1 and Col1a1 expression levels and their respective fluorescence intensity values> From fixed-point fluorescence imaging images of ATDC5 cells introduced with Col2a1 promoter (687 bp)-i182×2-pZsGreen1 and Col1a1 promoter (3.2 kbp)-pDsRed-Express2 as culture days progressed, a qualitative increase in the average fluorescence intensity values ​​of ZsGreen1 and DsRed-Express2 was observed as culture days progressed, indicating that the transcription of Col2a1 and Col1a1 was activated at the same observation site. Figure 5 shows the relationship between the average fluorescence intensity values ​​calculated from the fluorescence imaging images and the expression levels of each gene measured by RT-qPCR. From Figure 5, it was confirmed that the average fluorescence intensity values ​​increased with the increase in the expression levels of each gene. Therefore, in order to determine the relationship between the average fluorescence intensity values ​​and mRNA expression levels, curve approximation was performed based on the graph in Figure 5, which plots each value. In this study, we used the sigmoid function as a function that approaches a constant value for fluorescence intensity, referencing the fact that expression levels and luminescence levels are approximated by the sigmoid function in reporter assays, one of the transcriptional activity evaluation methods using luciferase. The sigmoid function is an S-shaped curve with the inflection point at the coordinate point (0,0.5), and equation (1) using the sigmoid function used in this study is shown below.

[0068]

number

[0069] Here, x represents gene expression level, y represents average fluorescence intensity, and a represents gain, with b being the value shifted in the x-axis direction. In equation (1), a and b were identified such that the sum of squared residuals between the experimental and analytical values ​​was minimized. The condition (x, y) = (0, 0) was added to the experimental values. Figure 6 shows a comparison of the analytical results calculated based on the identified values ​​with the experimental values. From Figure 6, the gain values ​​a for Col2a1 and Col1a1 were 10.15 and 2.04, respectively, and the value b was 1.12 and 1.95, respectively. The correlation coefficient between the analytical and experimental values ​​was 0.99 for both Col2a1 and Col1a1.

[0070] <3.1.3 Control and Evaluation of Dynamical Stimuli Based on Col2a1 and Col1a1 Promoter Activity> Figure 7 shows the results of measuring Col2a1 and Col1a1 expression levels by RT-qPCR on day 6 of culture when mechanical stimulation control was performed with an initial displacement of 0.960 mm. From Figure 7, Col1a1 expression levels were significantly decreased in the stimulated group compared to the unstimulated group, and Col2a1 expression levels showed an increasing trend, although the difference was not statistically significant. Table 3 shows the daily sheet displacement for each condition. The response surface is shown in Figure 10. From Table 3, under the condition using the mechanical control method, the sheet displacement increased to the maximum value of this system, 1.52 mm, on day 2 of culture, and the sheet displacement was 0.68 mm on day 6.

[0071] [Table 3]

[0072] Figure 8 shows the results of measuring Col2a1 and Col1a1 expression levels by RT-qPCR on day 6 of culture under constant mechanical stimulation with an initial displacement of 0.540 mm, where no increase in Col2a1 expression was observed in the validation experiment. From Figure 8, there was no significant difference in Col2a1 expression levels between the stimulated group and the unstimulated group, and although Col1a1 expression levels showed a decreasing trend, the difference was not statistically significant. From the above, it was confirmed that constant mechanical stimulation using a sheet displacement of 0.540 mm had no effect not only on promoting differentiation, as confirmed in the prior validation experiment, but also on reducing dedifferentiation. On the other hand, Figure 9 shows the results of measuring Col2a1 and Col1a1 expression levels by RT-qPCR on day 6 of culture when mechanical stimulation was controlled by setting the initial displacement to 0.540 mm. From Figure 9, Col1a1 expression levels were significantly decreased in the stimulated group compared to the unstimulated group, and although there was no statistically significant difference in Col2a1 expression levels, there was an increasing trend. Table 4 shows the daily sheet displacement levels for each condition. (The response surface is shown in Figure 11). (a) shows the case of constant mechanical stimulation, and (b) shows the case of mechanical stimulation control.

[0073] [Table 4]

[0074] Table 4 shows that under the conditions using the mechanical control method, the sheet displacement increased on day 2 of culture, decreased on day 3, and then reached a maximum value of 1.52 mm from day 4 onwards, which was equal to the displacement from day 2 to day 5 under the control with an initial displacement of 0.96 mm. Next, the area ratio of each strain under each condition was calculated. The group with constant mechanical stimulation had a strain of 2-3% ε xx , 0-2% strain ε yy This range accounted for 77.9% of the total. On the other hand, when this control method was used, the 7% strain ε xx Furthermore, a strain of 3% ε yy This was the most common pattern, accounting for 11.3% of all patterns over the 6 days, and strain ε xx is 1-11%, strain ε yy The cells were subjected to a wide range of stresses from 0-5%. In the mechanically stimulated culture shown in Figure 9, the stimulated group showed an increasing trend in Col2a1 expression and a decreasing trend in Col1a1 expression compared to the stimulated group in the constant mechanically stimulated culture shown in Figure 8. This suggests that even when mechanical stimulation is controlled using a sheet displacement of 0.540 mm, which did not change Col2a1 expression, differentiation promotion and dedifferentiation reduction are possible. These findings suggest that the mechanical stimulation control method used in this study can increase Col2a1 expression and decrease Col1a1 expression compared to the unstimulated group, regardless of the initial displacement. Through these experiments, it was found that strains of 5% or more (ε) may increase Col2a1 expression and decrease Col1a1 expression compared to the unstimulated group. xx , strain ε of 1% or more yy We confirmed the existence of ε with a strain of 1-4%. xx , 0-2% strain ε yyEven under conditions where this occurs, the system was suggested to be effective in reducing dedifferentiation compared to the unstimulated group. In the mechanical stimulation method using this system, the effect on reducing dedifferentiation was stronger than the effect on promoting differentiation for a constant stimulus. This is thought to be influenced by the setting of the response variable (z in this study) and the strain distribution which depends on the sheet shape.

[0075] In the preliminary study of this research, strain in one direction (strain ε) xx When only ) was considered, the Col2a1 expression level tended to show a low rate of increase. From these preliminary study results and the results above, in a stress field, strain in multiple directions (strain ε) xx and ε yy The study suggested that evaluating gene expression levels over time and estimating them, taking these factors into consideration, could more efficiently promote cell differentiation.

Claims

1. A method for producing cartilage cells or cartilage tissue, A cell culture process in which cells are cultured while mechanical stimuli are applied under conditions that generate multiple strain fields, each with a different combination of strains in multiple directions. In the culture process, a measurement step is performed to non-invasively measure the expression levels of type I collagen genes and type II collagen genes in each strain field. A culture adjustment step in which the mechanical stimulus is adjusted and culture is performed based on the results of the measurement step, Methods that include...

2. The method according to claim 1, wherein the mechanical stimulus is at least one selected from the group consisting of tensile stimulation, compressive stimulation, shear stimulation, hydrostatic pressure, sliding stimulation, and torsion.

3. The method according to claim 1, wherein there are two directions.

4. The method according to claim 3, wherein the two aforementioned directions intersect at an angle of 60 to 120 degrees.

5. The method according to claim 1, wherein the culture step is two-dimensional culture or three-dimensional culture.

6. The method according to claim 1, wherein the cells are chondrocytes and / or cartilage cells.

7. The method according to claim 1, wherein the cell comprises an expression cassette containing a promoter of the gene and a reporter gene located downstream thereof.

8. The method according to claim 7, wherein the measurement step is performed by measuring the reporter signal intensity of the cell.

9. The method according to claim 1, wherein in the preparation culture step, the stimulation is adjusted based on the results of the measurement step to promote cell differentiation and then cultured.

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