Method for producing culture base material, cell culture method using culture base material, and culture base material

Abstract

To provide a method for producing a culture base material that enables suitable production of a culture base material with excellent oxygen permeability.SOLUTION: There is provided a method for producing a culture base material comprising a film or a sheet provided with a plurality of recesses for culturing cells, the method being characterized in that, when the average film thickness of the recesses is T (μm), the aperture ratio of the recesses is A (%), and the oxygen transmission coefficient of the culture base material is P0 (ml μm / m2 24Hr atm), the average film thickness T and the aperture ratio A of the recesses are defined so that the oxygen permeability P (ml / m2 24Hr atm) of the culture base material fulfills the following formulae, and a plurality of recesses are formed on one surface of the film or sheet according to the defined average film thickness T and aperture ratio A of the recesses: P=P0×(1 / T)×(A / 100), A≥75, P>9000.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] The present invention relates to a cell culture technique, and more particularly to a technique for culturing cells in the form of spheres. [Background technology]

[0002] In sphere (cell aggregate) culture, it is known that the lower the initial seeding cell number (cells / well), the higher the cell proliferation rate. Specifically, in an experiment using a well bag (a culture bag with many wells formed on the culture surface), 500 single cells were seeded in each well, and when culture was started with a total seeded cell count of approximately 300,000, approximately 10% of the cells died without forming spheres after one day, but after seven days the cells had proliferated to approximately six times the number of cells seeded.

[0003] In contrast, in an experiment using the same well bag, when 10 single cells were seeded in each well, for a total seeded cell count of approximately 5,000, and culture was initiated, approximately 30-40% of the cells died without forming spheres after one day, but after seven days the cells had proliferated to approximately 150 times the number of cells seeded. Thus, when cells are mass-cultured in the form of spheres, it is efficient to increase the number of cells with high proliferation efficiency based on a small number of seeded cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7348586 Summary of the Invention [Problem to be solved by the invention]

[0005] In recent years, culturing cells in the form of spheres (hereinafter, this may be referred to as sphere culture) has become widespread using a bioreactor or the like. Specifically, sphere culture of iPS cells and the like is carried out using, for example, a 30 mL bioreactor. However, such sphere culture using a bioreactor has the problem that it is difficult to control the size of the spheres, making it difficult to obtain spheres of a uniform size.

[0006] For this reason, when culturing spheres, it is advantageous to use a well bag that allows control of the size of the spheres, as this is more convenient. In this situation, it would be desirable to provide a culture substrate with excellent oxygen permeability that is suitable for culturing spheres in large quantities at high density using well bags. Furthermore, if it becomes possible to provide culture vessels such as well bags using such culture substrates, it will be possible to increase cells with higher proliferation efficiency in sphere cultures using a smaller number of seeded cells.

[0007] Therefore, the present inventors conducted extensive research and succeeded in producing a culture substrate with excellent oxygen permeability, thereby completing the present invention. Specifically, the average thickness of the recesses of a culture substrate made of a film or sheet with multiple recesses for culturing cells is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The average film thickness of the recesses and the opening ratio of the recesses were set so that the film thickness (24Hr·atm) satisfied the following formula: Then, based on the set average film thickness and opening ratio of the recesses, a plurality of recesses were formed on one surface of the film or sheet to obtain a culture substrate. P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0008] At the time of filing this application, no commercially available culture substrates that satisfy these conditions were available. Furthermore, Patent Document 1, a patent application filed by the present applicant, discloses a bag-shaped culture vessel made of a soft packaging material and having a culture section with multiple recesses for accommodating spheres, the culture vessel being equipped with at least one port, the depth of the recess being 50 to 500 μm, at least a portion of the side wall of the recess being vertical, and the vertical length of the vertical portion of the side wall being longer than 25 μm. However, this document neither describes nor suggests how to set the average film thickness and opening ratio of the recesses in the present invention, nor does it disclose a method for manufacturing such a culture substrate.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing a culture substrate that can suitably obtain a culture substrate with excellent oxygen permeability, a cell culture method using the culture substrate, and a culture substrate. [Means for solving the problem]

[0010] In order to achieve the above object, the method for producing a culture substrate of the present invention is a method for producing a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, in which the average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The method is to set the average film thickness of the recesses and the opening ratio of the recesses so that the thickness (atm) of the recesses (atm) satisfies the following formula, and form a plurality of recesses on one side of the film or sheet based on the set average film thickness of the recesses and the opening ratio of the recesses. P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0011] Furthermore, the method for producing a culture substrate of the present invention is preferably a method in which the distance from the surface opposite to the one surface of the film or sheet to the lowest part of the recess is greater than 0 μm and equal to or less than 40 μm. In addition, the method for producing a culture substrate of the present invention is preferably a method in which the internal inclination angle of the recess is greater than 0° and not greater than 30°. Furthermore, the method for producing a culture substrate of the present invention is preferably a method in which the maximum length of the opening of the recess, parallel to the surface of the film or sheet, is 20 μm or more and 2000 μm or less.

[0012] Furthermore, in the method for producing a culture substrate of the present invention, the film or sheet is preferably made of a thermoplastic resin, and the thermoplastic resin is preferably an olefin. Furthermore, the method for producing a culture substrate of the present invention is also preferably a method in which the above-mentioned configurations for producing a culture substrate are combined in various ways.

[0013] The cell culture method using the culture substrate of the present invention is a culture method using a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, wherein the average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm, the oxygen permeability P (ml / m 2 This method involves culturing spheres in a culture vessel using a culture substrate in which the average thickness of the wells, T, and the opening ratio, A, of the wells are set so that the thickness (μm / cm²) of the wells (μm / cm²) satisfies the following equation: P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0014] Furthermore, the cell culture method using the culture substrate of the present invention is preferably a method in which the size of the spheres is 20 μm to 200 μm.

[0015] The culture substrate of the present invention is a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, and the average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2·24H·atm, the oxygen permeability P (ml / m 2 It is configured with a temperature of 24 hours (24 hours atm). P = P0 × (1 / T) × (A / 100) A≧75 P>9000 [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a method for producing a culture substrate that can suitably obtain a culture substrate with excellent oxygen permeability, a cell culture method using the culture substrate, and the culture substrate. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is an explanatory view showing a plurality of recesses surrounded by a wall portion of a culture substrate in a method for producing a culture substrate according to an embodiment of the present invention. FIG. [Figure 2] 1A to 1C are explanatory views showing various recesses of a culture substrate in a method for producing a culture substrate according to an embodiment of the present invention. [Figure 3] FIG. 2 is an explanatory diagram showing a method for calculating the average film thickness of recesses in a culture substrate in a method for producing a culture substrate according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the set conditions of the culture substrate used in Test 1. [Figure 5] FIG. 1 is an explanatory diagram showing the culture substrate and other materials used in Test 1. [Figure 6] FIG. 1 shows the results of Test 1. [Figure 7] FIG. 1 shows the set conditions of the culture substrate used in Test 2. [Figure 8] FIG. 1 is a graph showing the relationship between the calculated and measured oxygen transmission rates of the culture substrate used in Test 2. [Figure 9] FIG. 1 shows the results of Test 3. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, a method for producing a culture substrate, a cell culture method using the culture substrate, and embodiments of the culture substrate of the present invention will be described, although the present invention is not limited to the specific details of the following embodiments and examples described later.

[0019] The method for producing a culture substrate of this embodiment is a method for producing a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, in which the average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The method is characterized in that the average thickness T of the recesses and the opening ratio A of the recesses are set so that the thickness (T / 24Hr / atm) of the recesses satisfies the following formula, and a plurality of recesses are formed on one side of the film or sheet based on the set average thickness T of the recesses and the opening ratio A of the recesses. P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0020] The multiple recesses of the culture substrate in this embodiment are portions that form wells of a well bag or the like. Specifically, the recess in this embodiment can be formed, for example, in the shape of a rectangular parallelepiped and an inverted square pyramid, as shown in FIG.

[0021] 1(A) is a top view of a recess 10 of a culture substrate 1, in which the recess 10 is surrounded on all four sides by walls 11. The opening of the recess 10 surrounded by the walls 11 has a square shape. Figure 1(B) is a diagram showing the AA cross section in Figure 1(A). The bottom of the recess 10 is shaped like an inverted square pyramid, and has a sloping bottom 12. The angle between the sloping bottom 12 and the horizontal plane (the internal inclination angle of the recess) is indicated by θ (°).

[0022] In Figure 1(B), the angle between the horizontal plane and the line connecting the rising portion 14, which is the boundary between the inclined bottom portion 12 and the wall portion 11, and the lowest portion 13 of the inclined bottom portion 12 (the lowest portion of the recess), is shown as θ (°). The distance between the lowest portion 13 and the bottom surface of the culture substrate 1 (the surface of the culture substrate 1 opposite to the surface on which the recesses 10 are formed) is indicated by D (μm). One side of the square that forms the opening is indicated by L (μm). Furthermore, the width of the wall portion 11 between the recesses 10 is indicated by M (μm).

[0023] In this embodiment, the shape of the recess 10 is not limited to that shown in Fig. 1 and may be an inverted quadrangular pyramid only. The recess 10 may also be a cylinder and an inverted cone, or may be an inverted cone only, or may have any other shape as long as it has the inclined bottom 12.

[0024] Specifically, for example, as shown in FIG. 2(A), the recess 10 may be formed in the shape of a rectangular parallelepiped and an inverted pyramid, or a cylinder and an inverted cone. As shown in FIG. 2(B), the recess 10a may be provided with a wall 11a, and the inclined bottom 12a may be configured to have a rounded shape.

[0025] Furthermore, as shown in FIG. 2(C), the recess 10b may have no wall portion, but may have an intermediate portion 11b between the recesses, and may be formed of only an inverted pyramid or only an inverted cone. As shown in FIG. 2(D), the recess 10c may not have walls, but may have a recessed portion 11c between the recesses, and the inclined bottom 12c may be configured in a rounded shape.

[0026] In the method for producing a culture substrate of this embodiment, the oxygen permeability P (ml / m 2 ·24Hr·atm) is preferably greater than 9000, more preferably greater than 9400, and even more preferably greater than 10000. According to this embodiment, the oxygen permeability P of the culture substrate is calculated by P = P0 × (1 / T) × (A / 100), and the average film thickness T of the recesses of the culture substrate and the opening ratio A of the recesses are set so that the oxygen permeability P of the culture substrate satisfies the above conditions, thereby making it possible to suitably manufacture a culture substrate with such excellent oxygen permeability.

[0027] Next, in the method for producing a culture substrate of this embodiment, the average thickness of the recesses 10 of the culture substrate 1 is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The reason for setting the average film thickness T of the recesses 10 and the opening ratio A of the recesses so that the thickness (T / 24Hr / atm) satisfies the following formula will be explained. P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0028] In general, for a flat film-shaped culture substrate, when the thickness of the culture substrate increases by n times, the oxygen permeability of the culture substrate decreases by 1 / n times. In other words, the thickness of the culture substrate and oxygen permeability are inversely related. On the other hand, the culture substrate 1 of this embodiment has an uneven shape (plurality of recesses 10) due to processing of its surface, and can therefore be divided into a wall portion 11 (or the area between recesses that does not have a wall portion), which is a portion that is difficult for oxygen to permeate, and a sloping bottom portion 12, which is a portion that is easy for oxygen to permeate. Since the wall portion 11 makes a small contribution to the oxygen permeability of the culture substrate 1, the oxygen permeability P of the culture substrate 1 can be estimated based on the thickness of the inclined bottom portion 12.

[0029] The oxygen permeability P of a culture substrate 1 having a plurality of recesses 10 increases as the distance D between the lowest portion 13 and the bottom surface of the culture substrate 1 (hereinafter, sometimes referred to as the substrate bottom thickness) decreases. Furthermore, the oxygen permeability P increases as the angle θ between the inclined bottom portion 12 and the horizontal plane (hereinafter, sometimes referred to as the bottom inclination angle) decreases within a range of 0° or more. Furthermore, the oxygen permeability P increases as the maximum length of the opening of the recesses 10 decreases.

[0030] The maximum length of the opening of recess 10 is the length of the longest line segment connecting within the horizontal region surrounded by wall 11. Recess 10 has inclined bottom 12, and as the maximum length of the opening of recess 10 increases, the thickness of the substrate increases. Therefore, the oxygen permeability P of culture substrate 1 increases when the maximum length of the opening of recess 10 is shorter.

[0031] Based on the above considerations, the inventors expressed the oxygen permeability P of the culture substrate 1 as the above formula, and confirmed in the examples described below whether a culture substrate 1 that satisfies the conditions of this formula has excellent oxygen permeability. Furthermore, in the examples described below, the oxygen permeability P (ml / m 2 The validity of the formula for calculating the temperature (H2O 24Hr atm) was verified. In the method for manufacturing the culture substrate of this embodiment, the longest length of the opening of the recess 10 is not used directly, but rather the average thickness of the inclined bottom 12 (average film thickness of the recess 10 of the culture substrate 1) and the opening ratio of the recess 10 are used, as described below.

[0032] Here, since the thickness of the inclined bottom portion 12 is not constant from the lowest portion 13 to the rising portion 14, it is preferable to use the average thickness of the inclined bottom portion 12 as described above. The average thickness of the inclined bottom portion 12 can be calculated by dividing the "volume of the inclined bottom portion 12" by the "bottom area of ​​the inclined bottom portion 12".

[0033] Furthermore, the "volume V of the inclined bottom 12" can be calculated by subtracting the "volume V2 of the inverted square pyramid" from the "volume V1 of the rectangular parallelepiped" as shown in Fig. 3. Note that the shape of the opening of the recess 10 in Fig. 3 (the horizontal shape of the upper end of the opening when viewed from above) is square. In Figure 3, the angle between the inclined bottom 12 and the horizontal plane is θ (°), the distance between the lowest part 13 and the bottom surface of the culture substrate 1 is D (μm), and one side of the square that forms the opening is L (μm).

[0034] In this case, the volume V1 of the rectangular parallelepiped can be calculated using the formula [D+(L / 2)×tan(θ)]×L×L. In addition, the "volume V2 of the inverted square pyramid" can be calculated using the formula (1 / 3) × L × L × (L / 2) × tan(θ).

[0035] Therefore, the average thickness of the inclined bottom 12 is (volume V1 - volume V2) / L 2 It can be expressed using the following formula: In the method for producing a culture substrate of this embodiment, the average thickness of the inclined bottom 12, ie, the average film thickness of the recesses 10 of the culture substrate 1, is defined as T (μm).

[0036] In the method for producing the culture substrate of this embodiment, the opening ratio of the recesses 10 is the percentage of the area of ​​the openings of the recesses 10 relative to the surface area of ​​the culture substrate 1, and is represented as A (%). The opening of the recess 10 means a horizontal area surrounded by the wall 11, and the area of ​​the opening of the recess 10 is equal to the "bottom area of ​​the inclined bottom 12", and in FIG. 2 The opening of the recess 10 is shown as region S in FIG.

[0037] The opening ratio of the recess 10 is calculated by using the length L (μm) of one side of the square constituting the opening shown in FIG. 1(B) and the width M (μm) of the wall 11 between the recesses 10. 2 / (L+M) 2 It can be expressed as x100. Furthermore, in the method for producing a culture substrate of this embodiment, the oxygen permeability coefficient of the culture substrate 1 is set to P0 (ml μm / m 2 ·24Hr·atm).

[0038] Therefore, the oxygen permeability (ml / m 2 The formula for calculating P=P0×(1 / T)×(A / 100) for calculating the temperature (T·24Hr·atm)P can be expressed as follows when the culture substrate 1 shown in FIG. 1 is used: P=P0×L 2 / (Volume V1 - Volume V2) x (A / 100) =P0×3 / (3D+L×tan(θ))×(A / 100)

[0039] Here, as shown in the examples described later, it is preferable that the opening ratio A of the recesses 10 is 75% or more. That is, when the opening ratio A of the recesses 10 of the culture substrates in Examples 1 to 5 is 75.1% in all cases, if the opening ratio A is 75% or more in the formula for calculating P, the oxygen permeability P of the culture substrate can be made approximately the same or larger.

[0040] According to this embodiment, the oxygen permeability P of the culture substrate 1 is 9000 (ml / m 2 By setting the distance D (μm) between the lowest part 13 and the bottom surface of the culture substrate 1, the length L (μm) of one side of the square that forms the opening, and the angle θ (°) between the inclined bottom part 12 and the horizontal plane so that the oxygen permeability is greater than 1 / 24 Hr·atm, it is possible to suitably manufacture a culture substrate 1 with such excellent oxygen permeability.

[0041] In the above calculation example, for ease of explanation, the case where the culture substrate 1 shown in FIG. 1 is used has been described. However, the method for producing the culture substrate of this embodiment is based on the average film thickness of the recesses 10 of the culture substrate 1, the opening ratio of the recesses, and the oxygen permeability (ml / m 2 1.·24Hr·atm), it goes without saying that the present invention is not limited to the case where the culture substrate 1 having the recesses 10 shown in FIG. 1 is used.

[0042] In addition, in the method for manufacturing the culture substrate of this embodiment, it is preferable to set the distance D (μm) from the surface opposite to one side of the film or sheet on which multiple recesses are formed to the lowest part 13 of the recesses (the distance between the lowest part 13 and the bottom surface of the culture substrate 1) to be greater than 0 μm and less than 40 μm. Furthermore, the method for producing the culture substrate of this embodiment is preferably a method for setting the internal inclination angle θ (°) of the recess (the angle between the inclined bottom 12 and the horizontal plane) to greater than 0° and equal to or less than 30°. Furthermore, the method for producing the culture substrate of this embodiment is preferably a method in which the longest length (μm) of the opening of the well parallel to the surface of the film or sheet is set to 20 μm or more and 2000 μm or less.

[0043] By setting the distance D between the lowest part 13 and the bottom surface of the culture substrate 1, the angle θ between the inclined bottom part 12 and the horizontal plane, and the longest length of the opening of the recess 10 as described above, it becomes easier to meet the setting conditions for the average film thickness T of the recess of the culture substrate and the opening ratio A of the recess, and it becomes possible to more suitably manufacture a culture substrate with oxygen permeability that can improve cell growth efficiency in sphere culture.

[0044] Furthermore, in the method for producing a culture substrate of this embodiment, it is preferable that the film or sheet having a plurality of recesses for culturing cells is made of a thermoplastic resin, and it is more preferable that the thermoplastic resin is an olefin. Specifically, for example, polyolefin resins such as polyethylene and polypropylene can be used as the material for the film or sheet. For example, polyethylene, a copolymer of ethylene and an α-olefin, a copolymer of ethylene and vinyl acetate, an ionomer using a copolymer of ethylene and acrylic acid or methacrylic acid and a metal ion, etc. can be suitably used.

[0045] Other materials that can be used for the film or sheet include styrene-based elastomers, polyester-based thermoplastic elastomers, etc. Furthermore, soft vinyl chloride resin, polybutadiene resin, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), and fluorine-based resins may also be used.

[0046] The cell culture method using the culture substrate of this embodiment is a culture method using a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, in which the average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The spheres are cultured in a culture vessel using a culture substrate in which the average thickness T of the wells and the opening ratio A of the wells are set so that the thickness (μm / cm²) of the wells (μm / cm²) satisfies the following formula: P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0047] In the cell culture method using the culture substrate of this embodiment, the size of the spheres to be cultured is preferably 20 μm to 200 μm.

[0048] Furthermore, the culture substrate of this embodiment is a culture substrate made of a film or sheet having a plurality of recesses for culturing cells, The average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P0 (ml μm / m 2 ·24H·atm, the oxygen permeability P (ml / m 2 A culture substrate characterized by having a temperature of 24Hr·atm. P = P0 × (1 / T) × (A / 100) A≧75 P>9000

[0049] The cell culture method using the culture substrate of this embodiment and each configuration of the culture substrate can be the same as the above-described method for producing a culture substrate of this embodiment.

[0050] According to the method for manufacturing a culture substrate of this embodiment, as will be explained in the examples below, by satisfying the setting conditions of the average film thickness T of the recesses of the culture substrate and the opening ratio A of the recesses, it is possible to suitably manufacture a culture substrate having an oxygen permeability that can improve the cell growth efficiency in sphere culture. Furthermore, according to the method for producing a culture substrate, the cell culture method using the culture substrate, and the culture substrate of this embodiment, it is possible to improve the cell growth efficiency in sphere culture. [Example]

[0051] Hereinafter, a method for producing a culture substrate according to an embodiment of the present invention, a cell culture method using the culture substrate, and tests carried out to confirm the effects of the culture substrate will be described. [Test 1] A culture substrate was manufactured using the culture substrate manufacturing method of this embodiment, and its oxygen permeability was confirmed. In addition, sphere culture was performed in a culture vessel using the culture substrate, and the cell proliferation efficiency was confirmed.

[0052] First, the average thickness of the recesses in the culture substrate is defined as T (μm), the opening ratio of the recesses in the culture substrate is defined as A (%), and the oxygen permeability coefficient of the culture substrate is defined as P0 (ml μm / m 2 24Hr·atm), the oxygen permeability P (ml / m 2 The average thickness T of the recesses of the culture substrate and the opening ratio A of the recesses were set so that the thickness (atm) of the recesses (atm) satisfies the following formula, and multiple recesses were formed on one side of the film or sheet based on the set average thickness T of the recesses and the opening ratio A of the recesses to produce the culture substrate. P = P0 × (1 / T) × (A / 100) A≧75 P>10000

[0053] Then, iPS cells were cultured as spheres using the culture substrate, and the size of the spheres was measured, and the number of cultured cells was counted to calculate the cell density, thereby confirming the cell proliferation efficiency when sphere culture was performed in a culture vessel using a culture substrate manufactured by the method for manufacturing a culture substrate of this embodiment.

[0054] Specifically, culture substrates were manufactured for Examples 1 and 2, in which the recesses 10 shown in Fig. 1 were rectangular parallelepiped and inverted pyramid shapes, respectively. The material of these culture substrates was LLDPE (Linear Low-Density Polyethylene). As shown in FIG. 4, the number of wells / cm of the culture substrates in Examples 1 and 2 2 The total number of samples was 1,111. The side length L (μm) of the square forming the openings in the culture substrates of Examples 1 and 2 was 260 μm in both cases, and the width M (μm) of the wall between the openings was 40 μm in both cases, although not shown.

[0055] Furthermore, the distance D (μm) between the lowest part of the culture substrate and the bottom surface of the culture substrate in Examples 1 and 2 was 15 μm and 35 μm, respectively. Furthermore, the angles θ (°) formed between the inclined bottoms and the horizontal plane of the culture substrates of Examples 1 and 2 were 10° and 30°, respectively.

[0056] The average film thickness T (μm) of the recesses of the culture substrates of Examples 1 and 2 calculated from these values ​​was 30.3 μm and 85 μm, respectively. The opening ratio A (%) of the recesses of the culture substrates of Examples 1 and 2 was 75.1%.

[0057] Furthermore, the oxygen permeability coefficient P0 (ml μm / m 2 24Hr atm) are 1.14×10 6 ml·μm / m 2 ·24Hr·atm. Therefore, the oxygen permeability P (ml / m 2 The calculated values ​​for (24Hr·atm) are as follows: P = 1.14 × 10 6 ×(1 / 30.3)×(75.1 / 100) ≒28255

[0058] In addition, the oxygen permeability P (ml / m 2 The calculated values ​​for (24Hr·atm) are as follows: P = 1.14 × 10 6 ×(1 / 85)×(75.1 / 100) ≒10072 Therefore, the culture substrates of Examples 1 and 2 both satisfy the condition of P>10,000.

[0059] Furthermore, a culture substrate of Reference Example 1 was produced in which the calculated oxygen permeability P did not satisfy the condition of P > 10,000. Then, iPS cell sphere culture was performed using a culture vessel using the culture substrate of Reference Example 1 produced, and the sphere size was measured, and the number of cultured cells was counted to calculate the cell density.

[0060] Specifically, a culture substrate of Reference Example 1 was produced in which the recesses 10 shown in Fig. 1 were rectangular parallelepipeds and inverted square pyramids. The material of these culture substrates was LLDPE (Linear Low-Density Polyethylene). The culture substrate of Reference Example 1 was produced by attaching a 110 μm flat film to the bottom surface (the surface opposite to the surface on which the recesses 10 were formed) of the culture substrate of Example 2. The material of this flat film was the same as that of the culture substrate. As shown in FIG. 4, the number of wells / cm of the culture substrate of Reference Example 1 2 The number was 1,111. The length L (μm) of one side of the square constituting the opening of the culture substrate of Reference Example 1 was 260 μm. The width M (μm) of the wall between the openings was 40 μm, although not shown.

[0061] Furthermore, the distance D (μm) between the lowest part of the culture substrate and the bottom surface of the culture substrate in Reference Example 1 was 145 μm. The angle θ (°) formed between the inclined bottom of the culture substrate of Reference Example 1 and the horizontal plane was 30°.

[0062] The average thickness T (μm) of the recesses of the culture substrate of Reference Example 1 calculated from these values ​​was 195 μm. The opening ratio A (%) of the recesses of the culture substrate of Reference Example 1 was 75.1%.

[0063] Furthermore, the oxygen permeability coefficient P0 (ml μm / m 2 ·24Hr·atm) is 1.14×10 6 ml·μm / m 2 ·24Hr·atm. Therefore, the oxygen permeability P (ml / m 2 The calculated values ​​for (24Hr·atm) are as follows: P = 1.14 × 10 6 ×(1 / 195)×(75.1 / 100) ≒4390 Therefore, the culture substrate of Reference Example 1 does not satisfy the condition of P>10,000.

[0064] In addition, the actual oxygen permeability of each culture substrate in Example 1, Example 2, and Reference Example 1 was measured by a conventional method using a flow-type gas / water vapor permeability measuring device (GTR-20XFTSK, manufactured by GTR Tech Co., Ltd.).

[0065] Next, as shown in Figure 5, a polyethylene tube 2 was heat-sealed to the surface of the culture substrate 1 in each of Examples 1, 2, and Reference Example 1, on which multiple recesses were formed, to prepare a culture vessel for sphere culture. The polyethylene tube 2 had an inner diameter of 30 mm, and the culture area of ​​the culture vessel was 7 cm. 2 It was. 2.0 × 10 iPS cells (1231A3 strain) were cultured in each of these culture vessels. 6 cells / cm 2 The number of cells seeded per well was 1800 cells / well. StemFit medium (Ajinomoto Co., Inc.) was used as the medium, and 10 mL of the medium was filled into each culture vessel.

[0066] Then, spheres were cultured for 2 days at 37°C using the culture vessels using the culture substrates of Example 1, Example 2, and Reference Example 1. During this time, half of the medium was replaced every half day (5 mL of medium was discarded and 5 mL of new medium was added). 48 hours after cell seeding, the spheres were collected and the average size of the spheres was measured.

[0067] Next, the spheres were dissociated into single cells using TrypLE (Thermo Fisher Scientific), and the cell number was counted to calculate the cell density. Cell counting was performed by standard methods using a cell counter (NC-200, ChemoMetec) and a counting cassette (Via1-Cassette, ChemoMetec). The results are shown in Figure 6.

[0068] As shown in FIG. 6, the measured oxygen permeability of the culture substrate of Example 1 was 22,600 ml / m 2 24Hr·atm, the calculated value is 28255ml / m 2 ·24Hr·atm was roughly the same. The average size of the spheres obtained by sphere culture using the culture substrate of Example 1 was 245.8 μm, and the number of cells was 1.37E×10 7 cells, and the cell density was 1.96 × 10 6 cells / cm 2 It was.

[0069] The measured oxygen permeability of the culture substrate of Example 2 was 12,400 ml / m 2 24Hr atm, the calculated value is 10072ml / m 2 ·24Hr·atm was roughly the same. The average size of the spheres obtained by sphere culture using the culture substrate of Example 2 was 226.2 μm, and the number of cells was 1.12 × 10 7 cells, and the cell density was 1.60 × 10 6 cells / cm 2 It was.

[0070] Furthermore, the measured oxygen permeability of the culture substrate of Reference Example 1 was 4960 ml / m 2 24Hr·atm, the calculated value is 4390ml / m 2 ·24Hr·atm was roughly the same. The average size of the spheres obtained by sphere culture using the culture substrate of Reference Example 1 was 217.2 μm, and the number of cells was 7.27 × 10 6 cells, and the cell density was 1.04 × 10 6 cells / cm 2 It was.

[0071] As described above, the culture substrates of Examples 1 and 2, which were manufactured based on the culture substrate manufacturing method of this embodiment, showed a larger average size of the cultured spheres, and also a larger number of cells and cell density, compared to the culture substrate of Reference Example 1, which does not satisfy the setting conditions for the average film thickness T of the recesses of the culture substrate and the opening rate A of the recesses in the culture substrate manufacturing method of this embodiment. Therefore, it has become clear that the method for producing a culture substrate of this embodiment can suitably provide a culture substrate that can improve cell proliferation efficiency in sphere culture.

[0072] [Test 2] Various culture substrates were manufactured with different substrate thicknesses D (μm) at the bottom, square side lengths L (μm) at the opening, and inclination angles θ (°) at the inclined bottom. The calculated and measured oxygen permeabilities of these culture substrates were compared to determine the oxygen permeability P (ml / m 2 The validity of the formula for calculating the temperature (H2O 24Hr atm) was verified.

[0073] Specifically, the oxygen permeability coefficient P0 (ml μm / m 2 ·24Hr·atm) is 7.21×10 5 ml·μm / m 2A culture substrate was prepared using LLDPE (Linear Low-Density Polyethylene) with a 24 Hr atm oxygen permeability, and its oxygen permeability was measured by standard methods using a flow-type gas and water vapor permeability measuring device (GTR-20XFTSK, manufactured by GTR Tech Co., Ltd.).

[0074] In addition, the average film thickness T (μm) of the recesses was calculated based on the substrate thickness D (μm) at the lowest part of each culture substrate, the length L (μm) of one side of the square opening, and the inclination angle θ (°) of the inclined bottom.The oxygen permeability P of the culture substrate was calculated based on the obtained average film thickness T (μm) of the recesses, the opening ratio A (%) of the recesses, and the oxygen permeability coefficient P0. A graph was then created showing the oxygen permeability P (calculated value) of each culture substrate on the horizontal axis and the actually measured oxygen permeability Pm (measured value) on the vertical axis.

[0075] Figure 7 shows the substrate thickness D (μm) at the lowest part of each culture substrate (No. 1-11), the length of one side of the square opening L (μm), the inclination angle θ (°) of the inclined bottom, the average film thickness T (μm) of the recess, the opening ratio A (%) of the recess, the oxygen permeability P (calculated value), and the oxygen permeability Pm (measured value). FIG. 8 shows a graph showing the oxygen permeability P and oxygen permeability Pm of each culture substrate.

[0076] As shown in Figure 7, the substrate thickness D (μm) at the lowest part of substrate No. 1, the length of one side of the square opening L (μm), the inclination angle θ (°) of the inclined bottom, the average film thickness T (μm) of the recess, the opening ratio A (%) of the recess, the oxygen permeability P (calculated value), and the oxygen permeability Pm (measured value) were 22 μm, 260 μm, 10°, 37.3 μm, 75.1%, and 14526 ml / m, respectively. 2 24Hr atm, 18095ml / m 2 ·24Hr·atm.

[0077] Those of substrate No. 2 are 38 μm, 260 μm, 10°, 53.3 μm, 75.1%, 10164 ml / m 2 24Hr atm, 11081ml / m 2·24Hr·atm. Those of substrate No. 3 are 62 μm, 260 μm, 10°, 77.3 μm, 75.1%, 7007 ml / m 2 24Hr atm, 7361ml / m 2 ·24Hr·atm.

[0078] Those of substrate No. 4 are 83 μm, 260 μm, 10°, 98.3 μm, 75.1%, 5510 ml / m 2 24Hr atm, 5653ml / m 2 ·24Hr·atm. Those of substrate No. 5 are 113 μm, 260 μm, 10°, 128.3 μm, 75.1%, 4222 ml / m 2 24Hr atm, 4362ml / m 2 ·24Hr·atm.

[0079] Those of substrate No. 6 are 116 μm, 260 μm, 10°, 131.3 μm, 75.1%, 4125 ml / m 2 24Hr atm, 4257ml / m 2 ·24Hr·atm. Those of substrate No. 7 are 7 μm, 260 μm, 30°, 57.0 μm, 75.1%, 9495 ml / m 2 24Hr atm, 10812ml / m 2 ·24Hr·atm. Those of substrate No. 8 are 28 μm, 260 μm, 30°, 78.0 μm, 75.1%, 6940 ml / m 2 24Hr atm, 6144ml / m 2 ·24Hr·atm. Those of substrate No. 9 are 52 μm, 260 μm, 30°, 102.0 μm, 75.1%, 5307 ml / m 2 24Hr atm, 4893ml / m 2 ·24Hr·atm.

[0080] Those of substrate No. 10 are 77 μm, 260 μm, 30°, 127.0 μm, 75.1%, 4263 ml / m2 24Hr atm, 4096ml / m 2 ·24Hr·atm. Those of substrate No. 11 are 90 μm, 260 μm, 30°, 140.0 μm, 75.1%, 3867 ml / m 2 24Hr atm, 4007ml / m 2 ·24Hr·atm.

[0081] As shown in Figure 8, the oxygen permeability P (calculated value) and the oxygen permeability Pm (measured value) of these culture substrates show a linear relationship with a slope of approximately 1 (y = 1.3027X - 1590.3, R 2 =0.9813). Therefore, it was found that there is a general correlation between the calculated value calculated using the formula P = P0 × (1 / T) × (A / 100) for calculating the oxygen permeability P of the culture substrate in the culture substrate manufacturing method of this embodiment and the actually measured oxygen permeability Pm, and it was confirmed that there is validity in calculating the oxygen permeability P of the culture substrate using this formula.

[0082] [Test 3] Sphere culture was performed using culture vessels using the culture substrates of various Examples manufactured using the culture substrate manufacturing method of this embodiment and the culture substrates of various Reference Examples, and the cell proliferation efficiency was confirmed.

[0083] Specifically, the culture substrates Nos. 1, 2, and 7 used in Test 2 were used in this test as Examples 3, 4, and 5, respectively. In addition, the culture substrates Nos. 3, 8, and 9 used in Test 2 were used in this test as Reference Examples 2, 3, and 4, respectively.

[0084] Furthermore, the substrate thickness D (μm) at the lowest part, the length of one side of the square opening L (μm), the inclination angle θ (°) of the inclined bottom, the average film thickness T (μm) of the recess, the opening ratio A (%) of the recess, the oxygen permeability P (calculated value), and the oxygen permeability Pm (measured value) were newly calculated as 9 μm, 260 μm, 65°, 194.5 μm, 75.1%, and 2784 ml / m, respectively. 2 24Hr atm, 5670ml / m 2This was designated as Reference Example 5.

[0085] Using these culture substrates, similar to Test 1, a polyethylene tube 2 was heat-sealed to the surface of each culture substrate 1 on which multiple recesses were formed, as shown in Figure 5, to create a culture vessel for sphere culture. The polyethylene tube 2 had an inner diameter of 30 mm, and the culture area of ​​the culture vessel was 7 cm. 2 It was.

[0086] In each of these culture vessels, 1.5 × 10 iPS cells (1231A3 strain) were cultured. 6 cells / cm 2 The number of cells seeded per well was 1,350 cells / well. StemFit medium (Ajinomoto Co., Inc.) was used as the medium, and 10 mL of the medium was filled into each culture vessel.

[0087] Here, the seeding cell density is 1.5 x 10 6 cells / cm 2 is a fairly high density for cell culture, and a large amount of medium and oxygen is required to maintain that density. Therefore, in Test 3, in order to confirm whether the culture substrate is capable of supplying enough oxygen to maintain the initial seeded cell number when a sufficient amount of medium (10 mL initially, half of which was replaced every half day) is provided, the cell density used to judge the cell count results described below was set to the same value as the seeded cell density.

[0088] Then, spheres were cultured in each culture vessel at 37°C for 2 days, with half of the medium replaced every half day (5 mL of medium was discarded and 5 mL of new medium was added), and the spheres were collected 48 hours after cell seeding. Next, the spheres were dissociated into single cells using TrypLE (Thermo Fisher Scientific), and the number of cells was counted. The number of cells was counted by a conventional method using a cell counter (NC-200, ChemoMetec) and a counting cassette (Via1-Cassette, ChemoMetec).

[0089] When iPS cells are cultured on a flat surface, the cell density is generally 0.3 to 0.4 × 10 even when they are in a confluent state (a state in which the cell density is high in the later stage of culture). 6 cells / cm 2 It is known that they only grow to a certain extent. In contrast, when static sphere culture is performed using a culture vessel with a culture substrate having multiple recesses, it is possible to increase the cell density per unit area compared to planar adhesion culture.

[0090] Therefore, in this study, the cell density was increased to 1.5 × 10 cells, which is 4 to 5 times higher than that of flat adherent culture. 6 cells / cm 2 Cultures with cell densities above this level were determined to be high-density cultures. That is, the cell count was 1.5 x 10 6 cells / cm 2 When the cell density was above this level, it was judged that high-density culture was possible (◯), and when it was below this level, it was judged that high-density culture was not possible (×). The oxygen permeability at which high-density culture was possible was confirmed. The results are shown in Figure 9.

[0091] As shown in FIG. 9, the number of wells / cm of the culture vessel in Example 3 2 , the thickness of the substrate at the lowest part D (μm), the length of one side of the square opening L (μm), the inclination angle θ (°) of the inclined bottom part, the oxygen permeability P (calculated value), and the oxygen permeability Pm (measured value), the cell density after 48 hours (cells / cm 2 ), and the possibility of high density culture is 1111, 22 μm, 260 μm, 10°, 14526 ml / m 2 24Hr atm, 18100ml / m 2 24Hr atm, 2.10×10 6 cells / cm 2, it was ○.

[0092] Those of the culture vessel of Example 4 are 1111 pieces, 38 μm, 260 μm, 10°, 10164 ml / m 2 24Hr atm, 11081ml / m 2 24Hr atm, 1.70×10 6 cells / cm 2 , it was ○. Those of the culture vessel of Example 5 are 1111 pieces, 7 μm, 260 μm, 30°, 9495 ml / m 2 24Hr atm, 10812ml / m 2 24Hr atm, 1.88×10 6 cells / cm 2 , it was ○.

[0093] Those of the culture vessel of Reference Example 2 are 1111 pieces, 62 μm, 260 μm, 10°, 7007 ml / m 2 24Hr atm, 7361ml / m 2 24Hr atm, 1.22×10 6 cells / cm 2 , was ×. Those of the culture vessel of Reference Example 3 are 1111 pieces, 28 μm, 260 μm, 30°, 6940 ml / m 2 24Hr atm, 6144ml / m 2 24Hr atm, 1.01×10 6 cells / cm 2 , was ×.

[0094] Those of the culture vessel of Reference Example 4 are 1111 pieces, 52 μm, 260 μm, 30°, 5307 ml / m 2 24Hr atm, 4893ml / m 2 24Hr atm, 0.92×10 6 cells / cm 2 , was ×. Those of the culture vessel of Reference Example 5 are 1479 pieces, 9 μm, 260 μm, 65°, 2784 ml / m 2 24Hr atm, 5670ml / m 2 24Hr atm, 1.39×106 cells / cm 2 , was ×.

[0095] As described above, the culture substrates of Examples 3 to 5, which were manufactured based on the culture substrate manufacturing method of this embodiment, had higher cell densities than the culture substrates of Reference Examples 2 to 5, which did not satisfy the setting conditions for the average film thickness T of the recesses of the culture substrate and the opening ratio A of the recesses in the culture substrate manufacturing method of this embodiment. Therefore, it has been revealed that according to the method for manufacturing a culture substrate of this embodiment, by satisfying the setting conditions of the average film thickness T of the recesses of the culture substrate and the opening ratio A of the recesses, a culture substrate having an oxygen permeability that can improve the cell growth efficiency in sphere culture can be suitably manufactured.

[0096] The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention. For example, although iPS cells are used in the examples, the method may also be applied to the culture of other undifferentiated cells such as ES cells. Furthermore, it can also be suitably used when producing a culture substrate for use in a culture bag. [Industrial Applicability]

[0097] The present invention can be suitably used when producing a culture substrate suitable for the expansion and culture of iPS cells and the like. [Explanation of symbols]

[0098] 1 Culture substrate 10 well 11 Wall (between recesses) 12 Slanted bottom 13 lowest part 14 Rising section 2 Cylinder part L length of one side of the square opening θ The angle of inclination of the inclined bottom D: Base material thickness at the lowest point

Claims

1. A method for producing a culture substrate comprising a film or sheet having a plurality of recesses for culturing cells, comprising: The average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P ０ (ml μm / m 2 ・24Hr・atm), the oxygen permeability P (ml / m 2 an average film thickness T of the recessed portion and an opening ratio A of the recessed portion are set so that the film thickness (at 24 Hr atm) satisfies the following formula: The plurality of recesses are formed on one surface of the film or sheet based on the set average film thickness T of the recesses and the set aperture ratio A of the recesses. A method for producing a culture substrate comprising: P=P ０ ×(1 / T)×(A / 100) A≧75 P>9000

2. 2. The method for producing a culture substrate according to claim 1, wherein the distance from the surface opposite to the one surface of the film or sheet to the lowest point of the recess is greater than 0 μm and not more than 40 μm.

3. 3. The method for producing a culture substrate according to claim 1, wherein the internal inclination angle of the recess is greater than 0° and not greater than 30°.

4. 3. The method for producing a culture substrate according to claim 1, wherein the maximum length of the opening of the recess, parallel to the surface of the film or sheet, is 20 μm or more and 2000 μm or less.

5. 3. The method for producing a culture substrate according to claim 1, wherein the film or sheet is made of a thermoplastic resin.

6. 6. The method for producing a culture substrate according to claim 5, wherein the thermoplastic resin is an olefin.

7. A cell culture method using a culture substrate made of a film or sheet having a plurality of wells for culturing cells, The average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P ０ (ml μm / m 2 ・24Hr・atm), the oxygen permeability P (ml / m 2 Spheres are cultured in a culture vessel using a culture substrate in which the average thickness T of the wells and the opening ratio A of the wells are set so that the thickness (24 Hr atm) satisfies the following formula: A cell culture method using a culture substrate characterized by: P=P ０ ×(1 / T)×(A / 100) A≧75 P>9000

8. 8. The cell culture method using a culture substrate according to claim 7, wherein the size of the spheres is 20 μm to 200 μm.

9. A culture substrate consisting of a film or sheet having a plurality of recesses for culturing cells, The average thickness of the recesses is T (μm), the opening ratio of the recesses is A (%), and the oxygen permeability coefficient of the culture substrate is P ０ (ml μm / m 2 ・24Hr・atm), the oxygen permeability P (ml / m 2 A culture substrate characterized by having a temperature of 24 hours atm. P=P ０ ×(1 / T)×(A / 100) A≧75 P>9000

Images