Adhesive cell culture equipment, culture container, cell detachment method, and method for producing adhesive cell culture equipment

Abstract

A large number of adherent cells can be cultured and the adherent cells can be easily peeled off from the culture container. A material for culturing adherent cells is a sheet-like material for manufacturing a culture container for adherent cells, and a groove including a mountain-shaped portion and a valley-shaped portion is provided on one surface side of the material, and the distance from the top of the mountain-shaped portion to the other surface of the material is less than 1 mm. In addition, it is preferred that the cross section perpendicular to the direction in which the groove extends is set to a roughly V-shaped shape, and the inclination angle of the side of the roughly V-shaped shape in the groove is set to less than 80 degrees. In addition, it is preferred that a plurality of grooves are provided in parallel in a straight line, and the top of the mountain-shaped portion is set to a straight line.

Description

Technical Field

[0001] The present invention relates to cell culture technology, and in particular to equipment used in culture containers for adherent cells. Background Art

[0002] In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., there has been a demand for efficient mass culture of cells, tissues, etc. under an artificial environment.

[0003] When iPS cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells and other adherent cells are attached to the surface (culture part) for culturing cells in a culture container and cultured in large quantities, the yield is limited by the surface area of ​​the culture part. Therefore, it would be beneficial if the yield of adherent cells could be increased by manufacturing a culture container with a large surface area of ​​the culture part.

[0004] As such a culture container with a large surface area of ​​the culture part, for example, a multi-stage culture container (Corning(R) CellSTACK, etc.) is commercially available in which a plurality of culture parts are stacked in multiple stages to form a culture container, and a culture medium can be passed between the culture parts. If such a culture container is used, a large number of adherent cells can be cultured.

[0005] However, when the culture of adherent cells is completed and the cells are recovered, since the adherent cells adhere to the culture part and the cells also adhere to each other, it is not easy to peel off from the culture part even if the culture container is directly hit, and the cells cannot be recovered. Therefore, a peeling agent (cell dissociation enzyme, TrypLE TM After the cells are separated by using a flow cytometer (Select, etc.), the cells are recovered by pipetting, etc.

[0006] However, when using the multi-stage culture container as described above, there is a problem that it becomes very complicated to pipette the culture parts of each stage. In addition, in the case of such a rigid (hard) container, after using the stripping agent, even if an impact such as knocking is applied from the outside of the container, it is impossible to fully peel off the adhered cells.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 6-38734

[0010] Patent Document 2: Japanese Utility Model Application Laid-Open No. 1-98599 Summary of the invention

[0011] Problems to be solved by the invention

[0012] On the other hand, it has been proposed to form grooves or recessed portions of a special shape on the culture surface of the culture container to obtain a culture container having a large surface area of ​​the culture portion.

[0013] Specifically, the culture container described in Patent Document 1 has a V-shaped line formed in the culture portion, thereby obtaining a larger culture area than a conventional culture container of the same size. In addition, the culture container described in Patent Document 2 has a conical or hemispherical concave portion formed in addition to the V-shaped line, thereby increasing the surface area of ​​the culture portion.

[0014] However, when culturing adherent cells using a culture container having a culture area increased by forming a V-shaped line or the like in such a culture portion, there is a problem that it becomes very difficult to detach the adherent cells from the culture container when collecting the cells.

[0015] That is, since grooves and recesses of special shapes are formed in the culture part, even if the liquid is pipetted after using a peeling agent, the adhered cells are not easily peeled off from the culture container, and there is a problem that the recovery of cells is extremely difficult.

[0016] Furthermore, these culture containers are all rigid containers and have a large thickness. Therefore, even if an impact such as a knock is applied from the outside of the container, the adhered cells cannot be sufficiently detached.

[0017] Therefore, the present inventors conducted intensive studies to obtain a device that can increase the surface area of ​​the culture portion in a culture container and can easily remove adherent cells from the culture container after culturing them using the culture container, and have successfully developed an adherent cell culture device that can achieve this.

[0018] Specifically, the following conditions were found: as the structure of the adherent cell culture equipment, V-shaped grooves such as lines are formed, and after culturing adherent cells on the surface of the equipment, after using a peeling agent, the adherent cells can be easily peeled off from the culture surface by applying impact such as hitting from the outside.

[0019] Furthermore, by manufacturing a culture container using such an adherent cell culture device, a culture container that can culture adherent cells in large quantities and can be easily peeled off can be obtained.

[0020] In particular, by manufacturing a bag-shaped culture container (culture bag) using such an adhesion cell culture device, it is possible to obtain a culture container in which cells can be efficiently recovered by tapping from the outside.

[0021] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an adherent cell culture device, a culture container, a cell detachment method, and a method for producing an adherent cell culture device that can culture adherent cells in large quantities and can easily detach adherent cells from the culture container.

[0022] Means for solving problems

[0023] In order to achieve the above-mentioned purpose, the adhesion cell culture device of the present invention is a sheet-like device for manufacturing a culture container for adhesion cells, and its structure is as follows: a groove including a mountain-shaped portion and a valley-shaped portion is provided on one surface side of the device, and the distance from the top of the mountain-shaped portion to the other surface of the device is less than 1 mm.

[0024] In the adhesive cell culture device of the present invention, the cross section perpendicular to the direction in which the groove extends is substantially V-shaped, and the inclination angle of the side surface of the substantially V-shaped groove is preferably 80 degrees or less.

[0025] Furthermore, a culture container of the present invention is manufactured using the above-mentioned adhesion cell culture device, and the culture container is configured to have a side of the adhesion cell culture device having the above-mentioned groove as a culture portion.

[0026] In addition, the cell detachment method of the present invention is a cell detachment method using the above-mentioned culture container, wherein the above-mentioned culture container is filled with adherent cells and a culture medium, the adherent cells are cultured on the surface of the above-mentioned groove, the culture medium is discharged after the culture is completed, and the above-mentioned culture container is filled with a detachment liquid. After a certain period of time, the detachment liquid is discharged from the above-mentioned culture container, the culture medium is filled into the above-mentioned culture container, and an impact is applied to the above-mentioned culture container from the outside, thereby detaching the adherent cells from the surface of the above-mentioned groove.

[0027] In addition, the method for producing a device for culturing adherent cells of the present invention is a method as follows: a surface treatment for producing the device for culturing adherent cells is performed on the surface of a sheet material for producing a culture container for adherent cells, and then a groove including a mountain-shaped portion and a valley-shaped portion is processed on one surface side of the material so as to form a cross section perpendicular to the direction in which the groove extends and having a substantially V-shaped shape, and an inclination angle of a side surface of the substantially V-shaped portion in the groove is 80 degrees or less, thereby forming the device for culturing adherent cells, and before the surface treatment, the material is selected to have a thickness that allows the distance from the top end of the mountain-shaped portion to the other surface of the material to be 1 mm or less.

[0028] Effects of the Invention

[0029] According to the present invention, it is possible to provide an adherent cell culture device, a culture container, a cell detachment method, and a method for producing an adherent cell culture device that can culture adherent cells in large quantities and can easily detach adherent cells from the culture container. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an explanatory diagram regarding the unique property (directionality of proliferation relative to the culture surface) exhibited by adherent cells during their proliferation.

[0031] Figure 2 The figure shows a photograph showing a unique property (directionality of proliferation with respect to the culture surface) exhibited when adherent cells proliferate.

[0032] Figure 3 It is a cross-sectional explanatory view showing the structure of a groove etc. included in the adhesive cell culture device according to one embodiment of the present invention.

[0033] Figure 4 It is an explanatory diagram regarding a V-shaped groove (line shape) formed in an adhesion cell culture material and a quadrangular pyramid-shaped recess (box shape) formed in a spheroid cell culture material according to one embodiment of the present invention.

[0034] Figure 5 The calculated value of the number of cells increased in a culture container formed using the adhesion cell culture material of one embodiment of the present invention and a culture container formed using the control material (cell density at the time of seeding: 1500 cells / cm 2 )’s picture.

[0035] Figure 6 The calculated values ​​are as follows (cell density at the time of seeding: 2250 cells / cm2) showing the number of cells increased in a culture container formed using the adhesion cell culture material of one embodiment of the present invention and a culture container formed using the control material. 2 )’s picture.

[0036] Figure 7 This is a graph showing the processing performance of the adhesive cell culture device at various V-angles in Test 1 (whether a processing mold can be prepared and whether a workpiece can be processed).

[0037] Figure 8 The diagram shows microscopic photographs showing the state of culturing adherent cells using the adherent cell culture material (material having a 75-degree V-shaped groove) in Experiment 2 and a culture container formed using the material.

[0038] Fig. 9The cell proliferation rate, etc. of the culture container formed by using the adhesion cell culture material (material with 60-degree V-shaped groove) in Experiment 2 and the culture container formed by using the control material (cell density at the time of seeding: 1500 cells / cm 2 )’s picture.

[0039] Fig.10 The cell proliferation rate of the culture container formed by using the adhesion cell culture material (material with 60-degree V-shaped groove) in Experiment 2 and the culture container formed by using the control material (cell density at the time of seeding 2250 cells / cm 2 )’s picture.

[0040] Fig.11 The figure shows a microscope photograph (a cross section perpendicular to the direction in which the V-shaped groove extends) of the adhesive cell culture device formed to various thicknesses (0.42 mm, 0.6 mm, 0.9 mm, and 1.1 mm) in Test 3.

[0041] Fig.12 This is a graph showing the results (peeling rates) of adherent cells detaching from the adherent cell culture material of various thicknesses when adherent cells were cultured using a culture container formed using the adherent cell culture material in Test 3.

[0042] Fig.13 The diagram shows microscopic photographs showing the results of detachment of adherent cells when adherent cells were cultured in Experiment 3 using culture containers formed into adherent cell culture materials having various thicknesses (0.6 mm, 1.1 mm). DETAILED DESCRIPTION

[0043] Embodiments of the adherent cell culture device, culture container, cell detachment method, and method for producing the adherent cell culture device of the present invention are described in detail below. However, the present invention is not limited to the specific contents of the following embodiments and examples.

[0044] First, use Figure 1 and Figure 2 The discovery made by the present inventors that led to the present invention will be described. Figure 1 This is an illustration of the unique properties of adherent cells during proliferation (the directionality of proliferation relative to the culture surface). Figure 2 The figure shows a photograph showing the same.

[0045] The present inventors cultured adherent cells using a culture container having a culture surface with height differences and observed their proliferation. As a result, they noticed that there was regularity in the directionality of the proliferation.

[0046] That is, Figure 1As shown, when the adherent cells were present on the upper side of the step (upper stage) and proliferated, they were not observed to spread to the slope of the step and proliferate on the lower side of the step (lower stage).

[0047] On the other hand, when the adhered cells were present on the lower side of the step and proliferated, they did not spread to the upper side of the step and proliferate, but were observed to spread to the slope of the step and proliferate.

[0048] Figure 2 The tilt angle of the height difference of the culture surface in the left side photo is 60 degrees, and the tilt angle of the height difference of the culture surface in the right side photo is 30 degrees. In addition, the section on the lower side of the photo is the upper section, and the section on the upper side is the lower section, which are all taken from the upper side of the paper surface. The part colored in black is the part of cell proliferation. In any photo, the adherent cells all extend from the lower section to the inclined surface and proliferate, but do not extend from the upper section to the inclined surface and proliferate. In addition, although not shown, it is also the same when the tilt angle of the height difference is 90 degrees, the adherent cells climb the vertical surface from the lower section and proliferate, but do not proliferate downward along the vertical surface from the upper section.

[0049] This revealed that when an upwardly inclined surface is formed on the culture surface (when the upwardly inclined angle is at least 30 degrees), the adherent cells can spread on the inclined surface and proliferate.

[0050] In contrast, it was found that when a downwardly inclined surface was formed on the culture surface (when the downwardly inclined angle was at least 30 degrees), the adherent cells did not spread on the inclined surface and proliferate.

[0051] Thus, adherent cells are able to climb a hill that exists in the direction of travel, but are unable to descend over it.

[0052] Based on such results, the present inventors have come to the following conclusion: In order to effectively increase the culture area of ​​adherent cells, it is preferable to consider the directionality of adherent cell proliferation. In addition, a device for culturing adherent cells has been invented, which reduces the downward slope (relative to the direction of proliferation) in which adherent cells do not expand and proliferate, forms a culture portion extending in a manner that allows adherent cells to easily expand and proliferate, and can easily peel adherent cells from the culture container after culture.

[0053] That is, the adherent cell culture material of this embodiment is a sheet material for manufacturing a culture container for adherent cells, characterized in that it has a groove including a mountain-shaped portion and a valley-shaped portion on one surface side of the material, and the distance from the top of the mountain-shaped portion to the other surface of the material is less than 1 mm.

[0054] In addition, in the adhesion cell culture device, the cross section perpendicular to the direction in which the groove extends is preferably substantially V-shaped. Hereinafter, a groove of such a shape may be referred to as a V-shaped groove.

[0055] In addition, in the substantially V-shape, the inclination angle of the side surface of the substantially V-shape of the groove (hereinafter sometimes referred to as V-angle) is preferably 80 degrees or less.

[0056] Here, when the inclination angle of the substantially V-shaped side surface of the groove is 60 degrees, the surface area of ​​the groove is twice the surface area of ​​the corresponding region in the absence of the groove.

[0057] Similarly, when the inclination angle is 65 degrees, the surface area of ​​the groove is 2.37 times, when the inclination angle is 70 degrees, the surface area of ​​the groove is 2.92 times, when the inclination angle is 75 degrees, the surface area of ​​the groove is 3.86 times, and when the inclination angle is 80 degrees, the surface area of ​​the groove is 5.67 times.

[0058] Therefore, from the viewpoint of the culture area, the larger the inclination angle, the more preferred. On the other hand, the larger the inclination angle, the greater the chippings generated at the front end of the processing mold, or the front end of the processed object deformed, etc., and the processing performance is reduced. When the inclination angle is 83 degrees, the surface area of ​​the groove becomes 8.21 times, but at this inclination angle, the processing mold cannot be made and the processed object cannot be processed. Making a processing mold with an inclination angle of 80 degrees and using it to make a processed object is roughly the limit.

[0059] Next, refer to Figure 3 The structure of such a V-shaped groove will be described. This figure is a cross-sectional explanatory diagram showing the structure of the groove included in the adhesive cell culture device of the present embodiment.

[0060] like Figure 3 As shown, the adhesion cell culture device of this embodiment is preferably provided on one surface side of the device ( Figure 3 The device has a structure with multiple V-shaped grooves in parallel).

[0061] In the figure, angle θ is the inclination angle (V-angle) of the V-shaped groove, which represents the angle formed by the side surface of the V-shaped groove and a plane parallel to the other surface of the device (the lower surface of the device) and passing through the lowest point of the groove.

[0062] The distance a is the horizontal distance from the top of the mountain-shaped portion to the center of the groove (= the horizontal distance from the lowest point of the groove to the center of the mountain-shaped portion). The distance (pitch) between the tops of the mountain-shaped portions is 2a.

[0063] The distance b is the depth of the groove, and represents the vertical distance from the top of the mountain-shaped portion to the plane passing through the lowest point of the groove. That is, the depth of the groove is calculated by b=tanθ×a.

[0064] The distance c is the thickness of the bottom of the device, and is the distance obtained by subtracting the depth of the groove from the thickness of the device.

[0065] The distance h is the thickness of the device and is calculated by h=b+c=tanθ×a+c.

[0066] The distance H is the thickness of the raw material used in the manufacture of the adherent cell culture device of the present embodiment, and is calculated by H=b / 2+c=tanθ×a / 2+c.

[0067] Here, the adherent cell culture device of the present embodiment can be suitably manufactured by placing a molding die on a workpiece and performing heat transfer.

[0068] At this time, the convex part of the forming die corresponding to the groove of the equipment is pressed by the material to form a groove, and the material existing in the groove part is pushed away to form a mountain-shaped part, so the thickness of the equipment is greater than the thickness of the original material. Figure 3 The paper surface of the figure extends in the vertical direction. In this figure, the relationship a×(Hc)=a×b / 2 holds. Therefore, the thickness H of the original material is calculated by the above formula.

[0069] In addition, the adhesion cell culture device of the present embodiment preferably includes a plurality of grooves arranged in parallel in a straight line, and the top end of the mountain-shaped portion is preferably in a straight line.

[0070] Specifically, if Figure 4 As shown in the left photograph of , it is preferably configured to have a V-shaped groove in a linear shape.

[0071] When the adherent cell culture material of the present embodiment is configured in this manner, the adherent cells can be proliferated along the linear grooves, and thus the culture area can be effectively increased.

[0072] In addition, in the adhesion cell culture device of the present embodiment, the top end of the mountain-shaped portion may be flat.

[0073] On the other hand, Figure 4 As shown in the right photograph of , in the case of the culture surface having a box-shaped recess, the culture area is substantially the same as that of the culture surface having a linear V-shaped groove in the left photograph.

[0074] However, since adherent cells do not spread and proliferate on the downwardly sloping surface, adherent cells cultured in a certain concave portion do not spread and proliferate in the adjacent concave portion. In addition, from the viewpoint of proliferation efficiency, it is not desirable to seed cells in all concave portions.

[0075] Therefore, when culturing adherent cells, a culture surface having a box shape and recessed portions is not suitable.

[0076] In addition, the adhesion cell culture device of this embodiment can have multiple grooves in concentric circles or concentric ellipses, and the top of the mountain-shaped part is circular or elliptical. In addition, the adhesion cell culture device of this embodiment can have one or more grooves in a disc shape, and the top of the mountain-shaped part is disc-shaped.

[0077] As the material of the adhesion cell culture equipment of the present embodiment, polyolefin resins such as polyethylene and polypropylene can be suitably used. For example, copolymers of polyethylene, ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers of ethylene and acrylic acid, methacrylic acid copolymers and metal ions, etc. can be cited. In addition, polyolefins, styrene elastomers, polyester thermoplastic elastomers, silicone thermoplastic elastomers, silicone resins, etc. can also be used. In addition, silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, such as SBS (styrene butadiene styrene), SIS (styrene isoprene styrene), SEBS (styrene ethylene butylene styrene), SEPS (styrene ethylene propylene styrene), polyolefin resins, fluorine resins, etc. can also be used.

[0078] The adherent cell culture device of the present embodiment can be suitably produced by using these materials, placing a molding die on a workpiece, and performing heat transfer as described above.

[0079] Specifically, for example, a polyethylene sheet or other workpiece is placed on a rubber stand, and a silicon-made forming mold with a desired pattern is overlapped on the surface of the silicon wafer. It should be noted that the processing of the silicon wafer can usually be performed by cutting and etching. Next, a pressure plate is configured and heated to 150°C. Then, a pressure of 100 kPa or the like is applied, and the forming mold is pressed against the workpiece for about a few seconds, thereby transferring the mold and enabling manufacturing.

[0080] The culture container of the present embodiment is manufactured using the adhesion cell culture device of the present embodiment, and is characterized in that the culture container has a side of the device having a groove as a culture portion.

[0081] In addition, the culture container of this embodiment is preferably configured as follows: the culture container is formed into a bag shape using an upper surface side member and a lower surface side member, at least one of the upper surface side member and the lower surface side member is formed by the adhesion cell culture device of this embodiment, and a culture part is provided in the culture container.

[0082] The culture container of this embodiment is preferably configured such that the upper surface member and the lower surface member are formed of the adhesion cell culture device of this embodiment, and the culture portion is provided on the upper surface side and the lower surface side in the culture container.

[0083] In addition, in the culture container, it is preferably configured such that the direction in which the grooves in the upper surface member extend is not parallel to the direction in which the grooves in the lower surface member extend, and it is also preferably configured such that the angle between these directions is approximately 90°.

[0084] If the culture container of this embodiment is configured as such, the adhered cells can be proliferated along the grooves formed in the device, thereby effectively increasing the culture area. In addition, the thickness of the device is 1 mm or less, so after using the stripping solution, the adhered cells can be easily stripped by tapping from the outside of the culture container. In addition, by making the thickness of the device 1 mm or less, the air permeability of the culture surface can also be improved.

[0085] Furthermore, by forming the culture container in a bag shape, adhered cells can be more effectively detached by tapping the culture container from the outside.

[0086] In addition, by providing culture sections on both the upper and lower sides of the culture container, the culture area can be further increased, and by staggering the directions in which the grooves on the upper and lower sides extend, it is possible to prevent the grooves on the upper and lower sides from overlapping and hindering cell culture.

[0087] Here, refer to Figure 5 and Figure 6 The culture performance of the culture container of this embodiment will be described. These figures show how the culture performance can be improved by increasing the culture area.

[0088] The numerical values ​​corresponding to the "V-angle (θ)" of 60 degrees, 65 degrees, 70 degrees, and 75 degrees indicate the culture performance of the culture container having a culture portion with grooves formed at the respective V-angles. In addition, the culture performance of the culture container having a flat culture portion without grooves formed at the V-angle is shown in comparison.

[0089] "The ratio of the surface area of ​​the culture part" represents the ratio of the surface area of ​​the culture part in the culture container of this embodiment relative to the surface area of ​​the culture part in the control, and the ratios of V-angles of 60 degrees, 65 degrees, 70 degrees, and 75 degrees are expressed as 2 times, 2.37 times, 2.92 times, and 3.86 times, respectively.

[0090] "Cell density at inoculation relative to the surface area of ​​the culture part" is the assumed cell density at inoculation. Figure 5 In the above, it is assumed that for all culture containers, the surface area of ​​the culture part is 1500 cells / cm 2 The density of inoculation was Figure 6 For all culture containers, it is assumed that the surface area of ​​the culture part is 2250 cells / cm 2 Inoculate at a density of .

[0091] "Cell density at the time of inoculation relative to the bottom area of ​​the container" is an indicator for measuring the density of cells relative to the bottom area of ​​the container, and is calculated by "multiple of the surface area of ​​the culture part" × "cell density at the time of inoculation relative to the surface area of ​​the culture part".

[0092] The number of cells inoculated is the number of cells inoculated when the bottom area of ​​the culture unit is 50 cm 2 The calculated value at the time of inoculation was calculated by multiplying the “cell density at the time of inoculation relative to the bottom area of ​​the container” by 50.

[0093] The “cell number at the end of culture” was calculated by “inoculated cell number”דproliferation factor after 7 days of culture” using the “proliferation factor after 7 days of culture” obtained in Experiment 2 of Examples described below.

[0094] The "cell increase number" was calculated by subtracting the "cell number at the end of culture" from the "inoculated cell number".

[0095] exist Figure 5 In the figure, the "proliferation rate after 7 days of culture" of the control was 202 times, and the "proliferation rate after 7 days of culture" of the culture container of the present embodiment was 143 times. Figure 5 The cell density at the time of inoculation was set to 1.5 times the value when Figure 6 The "proliferation factor after 7 days of culture" of the control was 157 times, and the "proliferation factor after 7 days of culture" of the culture container of the present embodiment was 145 times.

[0096] The reason why the proliferation rate based on the control is greater than the proliferation rate based on the culture container of this embodiment is that, as described above, the adherent cells can climb up the hill existing in the traveling direction, but cannot go over the hill and descend. That is, the adherent cells cannot go over the top of the hill-shaped portion in the culture portion having the V-shaped groove, and thus the proliferation rate becomes lower than that of the control.

[0097] In the control, when the cell density at the time of seeding was set to 1.5 times, the proliferation factor decreased from 202 times to 157 times, but in the culture container of this embodiment, the proliferation factor was 143 times and 145 times, which was almost unchanged.

[0098] This is because, as shown in the results of Experiment 2 of the embodiment described later, the area of ​​the culture portion of the control is smaller than that of the culture container of the present embodiment, so if the number of cells inoculated increases above a certain level, the space for cell growth disappears, and the colonies (aggregates of adherent cells) are connected, making it difficult for them to proliferate further. The culture of adherent cells is preferably carried out at 350,000 / cm 2 In the culture of adherent cells, it is usually desirable to limit the number of cells seeded to allow for a margin of proliferation.

[0099] In the culture container of this embodiment, although the proliferation multiple is lower than that of the control, the number of cells to be seeded can be increased.

[0100] Therefore, in Figure 5 In the case of , the number of cells increased based on the control was about 15.1 million, while the number of cells increased based on the culture container of this embodiment was about 21.3 million at 60 degrees, about 25.2 million at 65 degrees, about 31.1 million at 70 degrees, and about 41.1 million at 75 degrees. Figure 6 In the case of the control, the number of cells increased was about 17.6 million, while the number of cells increased in the culture container based on the present embodiment was about 32.4 million at 60 degrees, about 38.4 million at 65 degrees, about 47.3 million at 70 degrees, and about 62.5 million at 75 degrees.

[0101] As described above, according to the culture container of this embodiment, cells can be grown much more efficiently compared to the control.

[0102] In addition, the cell detachment method of the present embodiment is characterized in that the culture container of the present embodiment is filled with adherent cells and a culture medium, the adherent cells are cultured on the surface of the groove, the culture medium is discharged after the culture is completed, and the culture container is filled with a detachment liquid. After a certain period of time, the detachment liquid is discharged from the culture container, the culture container is filled with a culture medium, and an impact is applied to the culture container from the outside, thereby detaching the adherent cells from the surface of the groove.

[0103] The culture container of this embodiment has grooves formed on the culture surface, but since it is formed using a material with a thickness of less than 1 mm, it is not difficult to peel the adhered cells from the surface of the grooves according to the cell peeling method of this embodiment. After culturing the adhered cells, they can be easily peeled off by knocking from the outside of the culture container using a peeling solution.

[0104] In addition, the method for manufacturing an adherent cell culture device according to the present embodiment is characterized in that a surface treatment for generating adhesion of the device to adherent cells is performed on the surface of a sheet material for manufacturing a culture container for adherent cells, and then a groove including a mountain-shaped portion and a valley-shaped portion is processed on one surface side of the material so as to form a shape in which a cross section perpendicular to a continuous direction of the groove is substantially V-shaped, and an inclination angle of a side surface of the substantially V-shaped portion in the groove is 80 degrees or less, thereby forming the adherent cell culture device, and before the surface treatment, a material having a thickness that can be formed into a shape in which the distance from the top end of the mountain-shaped portion to the other surface is 1 mm or less is selected.

[0105] That is, the adhesion cell culture device of the present embodiment can be appropriately manufactured by heat transfer or the like, but needs to be formed into a form having the above-mentioned special-shaped grooves and a thickness of 1 mm or less.

[0106] Therefore, as described above, this can be achieved by selecting a material having a thickness calculated according to tanθ×a / 2+c based on the inclination angle (θ) of the roughly V-shaped side surface in the groove, the horizontal distance (a) from the top of the mountain-shaped portion to the center of the groove, and the thickness (c) of the bottom of the device.

[0107] In addition, the adherent cell culture device of this embodiment needs to be surface treated (corona treatment, excimer treatment, etc.) in order to allow adherent cells to adhere to the surface. However, after the grooves are formed on the surface of the device, if the grooves are thin and deep, it is difficult for the light beam or light to enter the inside of the grooves, and sometimes the surface treatment cannot be fully performed.

[0108] Therefore, in the method for producing the adhesive cell culture device of the present embodiment, the surface treatment is performed before processing the grooves.

[0109] Therefore, according to the method for producing an adhesive cell culture device of the present embodiment, the device can be suitably produced.

[0110] Example

[0111] Hereinafter, experiments conducted to confirm the effects of the adherent cell culture device, the culture container, the cell detachment method, and the method for producing the adherent cell culture device according to the embodiments of the present invention will be described in detail.

[0112] [Test 1]

[0113] First, various molding dies for molding the adhesive cell culture device of the present embodiment are prepared.

[0114] Specifically, a silicon mold was produced in which a plurality of V-shaped grooves were arranged in parallel in a straight line and the top of the mountain-shaped part was in a straight line. At this time, V-angles (θ) of 60 degrees, 65 degrees, 70 degrees, and 75 degrees were produced. It should be noted that a mold with a V-angle (θ) of 83 degrees could not be produced.

[0115] Next, a polyethylene sheet (UMERIT 125FN, UBE-MARUZEN POLYETHYLENE CO., LTD.) as a workpiece was placed on a rubber stand, and the above-mentioned silicon molding mold was overlapped, and a press plate was placed and heated to 150°C. Next, the silicon molding mold was pressed at 100 kPa for 10 seconds to transfer the mold. At this time, the surface of the polyethylene sheet reached about 110°C. The results are shown in Figure 7 .

[0116] like Figure 7 As shown, processing molds with V-angles (θ) of 60 degrees, 65 degrees, 70 degrees, and 75 degrees can be produced, and these processing molds can be used to process the adhesion cell culture equipment.

[0117] [Test 2]

[0118] A culture bag was prepared using the adherent cell culture device of the present embodiment, and a culture test of adherent cells was performed.

[0119] Specifically, first, a polyethylene sheet (UMERIT 125FN, UBE-MARUZEN POLYETHYLENE CO., LTD.) as a workpiece was subjected to surface treatment. The surface treatment was hydrophilized using an excimer irradiation device (manufactured by MDCOM CO., LTD.). In this test, the test was carried out under the conditions of a voltage of 12 V, an irradiation distance of 4 mm, and an irradiation speed of 2 mm / s × 3 times or less.

[0120] Next, the workpiece was subjected to heat transfer in the same manner as in Experiment 1 to produce adhesive cell culture equipment having a V-angle (θ) of 60 degrees and 75 degrees. Then, the equipment was used to produce a culture bag as follows.

[0121] First, the produced adhesive cell culture device was used on the bottom surface side, and the above-mentioned polyethylene sheet without grooves was used on the top surface side to prepare a rectangular film with a long side of 15 cm and a short side of 8 cm.

[0122] Next, the bottom side film is overlapped with the upper side film, and the peripheral part is bonded by heat sealing to form a culture bag. At this time, a port is sandwiched on one side of the culture bag and bonded to form a culture bag with one port. The area of ​​the culture part of the culture bag excluding the peripheral sealing part and the port setting part is about 50cm 2 .

[0123] Six culture bags using an adhesion cell culture device having a V-angle (θ) of 60 degrees were prepared, and one culture bag using an adhesion cell culture device having a V-angle (θ) of 75 degrees was prepared.

[0124] In addition, as a control, six culture bags using the above-mentioned polyethylene sheets for both the bottom membrane and the upper surface membrane were prepared.

[0125] Then, StemFit(R) (AK-02N, AJINOMOTO HEALTHY SUPPLY Co., Ltd.) was sealed in each culture bag as a culture medium, and adherent cells (iPS cell line 1231A3, iPS Cell Research Institute, Kyoto University) were seeded and cultured for 7 days.

[0126] At this time, 3 control culture bags (samples 1 to 3), 3 culture bags using an adhesion cell culture device with a V-angle (θ) of 60 degrees (samples 4 to 6), and a culture bag using an adhesion cell culture device with a V-angle (θ) of 75 degrees were subjected to a 1500 cell / cm 2 In addition, for three control culture bags (samples 7 to 9) and three culture bags using an adhesion cell culture apparatus with a V-angle (θ) of 60 degrees (samples 10 to 12), the cells were inoculated at a density of 2250 cells / cm 2 The cells were seeded at a density of .

[0127] After 7 days of culture, cells were counted in the culture bags of samples 1 to 12. Cell counts were performed using TrypLE TM The detached cells were suspended in culture medium using ELISA Select (Thermo Fisher Scientific) and counted using a hemacytometer.

[0128] exist Figure 8 , a microscope photograph of a manufactured device having a 75-degree V-shaped groove (left) and a culture bag made using the device for culturing adherent cells (right). The figure shows the proliferation of adherent cells on the side of the V-shaped groove of the culture bag using the device.

[0129] exist Fig. 9 The cell density at the time of inoculation is 1500 cells / cm 2The cell proliferation rate at Fig.10 The cell density at the time of inoculation is 2250 cells / cm 2 The cell proliferation rate at that time, etc.

[0130] The cell density at the time of inoculation was 1500 cells / cm 2 In the case of , the proliferation rates of the control culture bag were 183 times, 220 times, and 204 times, respectively, and the average proliferation rate was 202 times. On the other hand, the proliferation rates of the culture bag using the adhesion cell culture equipment with a V-shaped angle of 60 degrees were 151 times, 132 times, and 145 times, respectively, and the average proliferation rate was 143 times.

[0131] In addition, the cell density of the control culture bag on the 7th day was 275,000 cells / cm 2 , 330,000 / cm 2 , 306,000 / cm 2 The cell density on the 7th day of the culture bag using the adhesion cell culture equipment with a V-shaped angle of 60 degrees was 226,000 cells / cm 2 , 198,000 / cm 2 , 218,000 / cm 2 .

[0132] The cell density at the time of inoculation was 2250 cells / cm 2 In the case of , the proliferation rates of the control culture bag were 161 times, 152 times, and 159 times, respectively, and the average proliferation rate was 157 times. On the other hand, the proliferation rates of the culture bag using the adhesion cell culture equipment with a V-angle of 60 degrees were 144 times, 143 times, and 146 times, respectively, and the average proliferation rate was 145 times.

[0133] In addition, the cell density of the control culture bag on the 7th day was 362,000 cells / cm 2 , 342,000 / cm 2 、358,000 / cm 2 The cell density on the 7th day of the culture bag using the adhesion cell culture equipment with a V-shaped angle of 60 degrees was 325,000 cells / cm 2 , 321,000 / cm 2 , 329,000 / cm 2 .

[0134] The cell density at the time of inoculation was 2250 cells / cm 2 In the case of , the proliferation rate obtained using the control culture bag is the same as the cell density at the time of inoculation of 1500 cells / cm 2The reason why the cell density was lower than that of the control is presumably because the cell density increased, resulting in a decrease in the space required for the proliferation of adherent cells. It is believed that in the control culture bag, even if the number of cells at the time of inoculation was increased, it was difficult to further increase the number of cells appropriately.

[0135] In contrast, in the culture bag using the adhesion cell culture equipment with a V-angle of 60 degrees, the cell density at the time of seeding was 1500 cells / cm 2 Case and 2250 pieces / cm 2 Under the same conditions, the proliferation rate was maintained at about 145 times.

[0136] In addition, regarding the number of cells at the end of the culture, the cell density at the time of inoculation was 1500 cells / cm 2 In the case of , the control culture bag had 13.75 million to 16.5 million cells, while the culture bag using the adhesion cell culture equipment with a V-angle of 60 degrees had 19.8 million to 22.6 million cells. In addition, the cell density at the time of inoculation was 2250 cells / cm 2 In the case of , the number of cells in the control culture bag was 17.1 million to 18.1 million, while the number of cells in the culture bag using the adhesion cell culture equipment with a V-angle of 60 degrees was 32.18 million to 32.9 million.

[0137] As described above, it is clear that by using the culture container of this embodiment, cells can be cultured efficiently in large quantities.

[0138] [Test 3]

[0139] A test was conducted in which a culture bag was prepared using an adherent cell culture device having a 75-degree V-shaped groove, and the adherent cells were cultured and then peeled off.

[0140] Specifically, the culture bag was manufactured using the adhesive cell culture materials formed so that the material thickness (H) was 0.42 mm, 0.6 mm, 0.9 mm, and 1.1 mm, respectively. The thickness (c) of the bottom corresponding to these material thicknesses was 0.03 mm, 0.41 mm, 0.51 mm, and 0.71 mm, respectively.

[0141] Microscopic photographs of each of the adhesion cell culture devices (a cross section perpendicular to the direction in which the V-shaped grooves extend) are shown in FIG. Fig.11 .

[0142] At this time, the surface treatment of the material of the adhesion cell culture equipment, the processing of the grooves by heat transfer, and the preparation of the culture bag were performed in the same manner as in Experiment 2.

[0143] Then, StemFit(R) (AK-02N, AJINOMOTO HEALTHY SUPPLY Co., Ltd.) was sealed in each culture bag as a culture medium, and adherent cells (iPS cell line 1231A3, iPS Cell Research Institute, Kyoto University) were seeded and cultured for 7 days.

[0144] Then, the culture medium was drained, the culture bag was washed with PBS (phosphate buffered saline), and the PBS was drained. Next, a cell dissociation enzyme (TrypLE TM Select, Thermo Fisher Scientific) was diluted 2-fold with PBS and placed at 37°C for 5 minutes. Then, the cell dissociation enzyme was removed, the cells were washed with PBS, and the culture medium was added.

[0145] Then, each culture bag was knocked from the outside to peel off the adhered cells. In addition, the peeling rate of the adhered cells after peeling was confirmed. The peeling rate was confirmed by taking pictures of the surface area of ​​the culture container before and after peeling and calculating the occupancy rate of the cell adhesion area. The results are shown in Fig.12 and Fig.13 .

[0146] like Fig.12 As shown, the peeling rate of the culture bag with a thickness (H) of 0.42 mm and 0.6 mm was more than 95%. In addition, the peeling rate of the culture bag with a thickness (H) of 0.9 mm was about 90%. On the other hand, the peeling rate of the culture bag with a thickness (H) of 1.1 mm was about 50%.

[0147] Microscopic photographs of the peeled surface of a culture bag having a material thickness (H) of 0.6 mm (left side) and the peeled surface of a culture bag having a material thickness (H) of 1.1 mm (right side) are shown in FIG. Fig.13 .

[0148] There are almost no residual cells in the culture bag on the left, whereas there are residual cells in the culture bag on the right.

[0149] From these results, it is understood that when the thickness of the adhesive cell culture device is 1 mm or less, excellent detachability of the adherent cells can be obtained.

[0150] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications can be made within the scope of the present invention. For example, the arrangement of the grooves of the adhesion cell culture device can be appropriately changed to a curved shape instead of a straight line.

[0151] Industrial Applicability

[0152] The present invention can be suitably used when, for example, a large amount of adherent cells are to be produced efficiently.

[0153] The contents of the documents mentioned in this specification and the specification of the Japanese application based on the Paris priority of this application are incorporated herein in their entirety by reference.

Claims

1. An adhesive cell culture device, characterized in that: The invention relates to a sheet-like device for manufacturing a culture container for adherent cells, wherein a groove including a mountain-shaped portion and a valley-shaped portion is provided on one surface side of the device, and the distance from the top of the mountain-shaped portion to the other surface of the device is less than 1 mm. The cross section perpendicular to the direction in which the groove extends is V-shaped, and the inclination angle of the side surface of the V-shaped groove is less than 80 degrees. The plurality of grooves are provided in parallel in a straight line rather than in a box shape, and the top end of the mountain-shaped portion is in a straight line shape.

2. A culture container, characterized in that: The culture container is manufactured using the adhesive cell culture material according to claim 1, wherein the culture container has a side of the adhesive cell culture material having the groove as a culture portion.

3. The culture container according to claim 2, characterized in that: The bag is formed using an upper surface side member and a lower surface side member, at least one of the upper surface side member and the lower surface side member is formed by the adhesion cell culture device, and the culture part is provided in the culture container.

4. The culture container according to claim 3, characterized in that: The upper surface side member and the lower surface side member are formed by the adhesion cell culture device, and the culture portion is provided on the upper surface side and the lower surface side in the culture container.

5. The culture container according to claim 4, characterized in that: It is formed in a form in which the direction in which the groove in the upper surface side member extends is not parallel to the direction in which the groove in the lower surface side member extends.

6. The culture container according to claim 5, characterized in that: It is formed in a form in which the angle formed by the extending direction of the groove in the upper surface side member and the extending direction of the groove in the lower surface side member is 90 degrees.

7. A method for detaching cells, characterized in that: This is a method for removing cells using the culture container according to any one of claims 2 to 6, The culture container is filled with adherent cells and a culture medium, and the adherent cells are cultured on the surface of the groove. After the culture is completed, the culture medium is discharged and the culture container is filled with a stripping solution. After a certain period of time, the stripping solution is discharged from the culture container, and the culture container is filled with a culture medium. By applying an impact to the culture container from the outside, the adhered cells are peeled off from the surface of the groove.

8. A method for manufacturing the adhesive cell culture device according to claim 1, characterized in that: The surface of a sheet material used for manufacturing a culture container for adherent cells is subjected to a surface treatment for improving the adhesion of the adherent cells to the device. Next, a groove including a mountain-shaped portion and a valley-shaped portion is processed on one surface side of the material to form a V-shaped cross section perpendicular to the direction in which the groove extends, and the inclination angle of the side surface of the V-shaped groove is 80 degrees or less, thereby forming an adhesive cell culture device, and, Before the surface treatment, the material is selected to have a thickness that allows the material to be formed into a shape in which the distance from the top end of the mountain-shaped portion to the other surface of the material is 1 mm or less.

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