JPWO2014156455A1 - Cell culture tool - Google Patents

Abstract

The cells are cultured without moving around the culture surface. The well (10) includes a container body (11) and a culture carrier (12). The culture carrier (12) has a plurality of holes on the culture surface (12a) for culturing cells, and has irregularities. The volume ratio of the culture carrier (12) in the range from the culture surface (12a) to the depth of 0.1 μm is in the range of 5% to 80%, and the opening ratio in the culture surface (12a) is 30% to 95%. Range. The container body is composed of a cylindrical cylindrical member (15) and a low affinity part (16). The low affinity part (16) is formed as a coating covering the inner surface of the side wall of the cylindrical member (15). The low affinity part (16) is formed of PEG and has a lower affinity for cells than the culture surface (12a).

Description

  The present invention relates to a cell culture device for culturing cells.

  A honeycomb structure film having a honeycomb structure by forming a plurality of minute holes side by side along the film surface is used as a culture carrier for culturing cells. This honeycomb structure film is manufactured by, for example, a dew condensation method. The dew condensation method is a method for producing a film by casting a solution for forming a film to form a cast film, and condensing the cast film to evaporate a solvent and water droplets. According to this dew condensation method, extremely small pores are formed, and the resulting film is more susceptible to erosion of cells with reduced elongation compared to films with flat culture surfaces and cell culture dishes. (Cell aggregates) are easily formed.

  Since spheroids exhibit behavior similar to that of a living body, spheroids are very useful when performing experiments that mimic cell behavior in vivo outside the body. As a culture carrier for forming spheroids in this way, there is one produced using a nanoimprint method in addition to a honeycomb structure film formed by a dew condensation method. For example, the culture carrier of Patent Document 1 has a cell-to-cell width of 3 μm or less and a depth of 10 nm or more on the culture surface by a nanoimprint method, and the cell shape viewed from the direction perpendicular to the culture surface is a polygon. As described above, the culture carrier of Patent Document 1 also has a minute recess on the culture surface, like the honeycomb structure film formed by the dew condensation method.

  Moreover, the culture container of patent document 2 is equipped with a culture bed and a hydrophilic layer. The culture bed is provided in a portion where the cells on the inner surface of the culture vessel are in contact, and the hydrophilic layer is provided in a portion where the culture solution is in contact with the inner surface of the culture vessel. As a result, the culture container of Patent Document 2 prevents the physiologically active substance from adhering to a portion other than the culture bed and also prevents cell adhesion.

Japanese Patent No. 4159103 JP 2004-290111 A

  However, when a honeycomb structure film obtained by the dew condensation method is used as a culture carrier, and the culture carrier is placed in a culture vessel and cells are cultured, the cells move around the culture carrier, and the desired number of cells are cultured. Does not remain on the carrier. This phenomenon is similarly confirmed when the culture carrier of Patent Document 1 is used. As described above, the phenomenon that the cells move to the surroundings is particularly prominent when irregularities are formed on the culture surface and the size of either the concave portion or the convex portion is in the range of 50 nm to 50 μm. is there. Moreover, the culture container of patent document 2 is equipped with the culture support | carrier which adheres a cell positively, a cell does not migrate to the first place, and does not move to the circumference | surroundings of a culture support | carrier. Therefore, the invention described in Patent Document 2 is not a solution when using a culture carrier in which cells move like a honeycomb structure film.

  Accordingly, an object of the present invention is to provide a cell culture device that includes a culture carrier having a culture surface on which irregularities are formed, and that cultivates cells while suppressing the movement of cells to the periphery of the culture surface.

  In order to solve the above problems, the cell culture instrument of the present invention includes a culture carrier and a low affinity part. The culture carrier has irregularities formed on the culture surface for culturing cells, the volume ratio in the range from the culture surface to a depth of 0.1 μm is in the range of 5% to 80%, and the open area ratio in the culture surface Is in the range of 30% to 95%. The low affinity part is provided so as to surround the periphery of the culture surface, and has a lower affinity with the cell than the culture surface.

  It is preferable that the low affinity part is provided in an upright posture with respect to the culture surface. It is preferable that the container body contains a culture medium and has an inner surface formed by a low-affinity part, and the culture carrier is provided at the bottom of the container body, and the culture surface is separated from the inner side surface of the container body. It is preferable that it is formed.

  The culture carrier is a film having a honeycomb structure when the culture surface is viewed from the vertical direction by forming a plurality of concave portions having a certain size along the culture surface, or a certain height. It is preferable that it is a film in which a plurality of convex portions having a thickness and a shape are formed side by side along the culture surface.

  According to the present invention, since the low affinity portion is provided around the culture surface on which the irregularities are formed, the cells can be cultured while suppressing the movement of the cells to the periphery of the culture surface.

It is the schematic of the plane and cross section of the well which are 1st Embodiment. It is a plane schematic diagram of a culture carrier. It is sectional drawing which follows the III-III line of FIG. It is explanatory drawing explaining how to obtain | require a volume ratio. It is explanatory drawing explaining how to obtain | require an aperture ratio. It is the schematic of the plane and cross section of the well which is 2nd Embodiment. It is the cross-sectional schematic of the well which is 3rd Embodiment. It is the cross-sectional schematic of the well which is 4th Embodiment. It is the cross-sectional schematic of the well which is 5th Embodiment. It is the cross-sectional schematic of the well which is 6th Embodiment. It is the cross-sectional schematic of the well which is 7th Embodiment. It is the cross-sectional schematic of the well which is 8th Embodiment. It is the schematic of the plane of the chip | tip which is 9th Embodiment, and a cross section. It is the schematic of the multiwell plate which is 10th Embodiment. It is sectional drawing of a multiwell plate. It is the schematic of the multichip plate which is 11th Embodiment. It is sectional drawing of a multichip plate. It is sectional drawing which shows the outline of a culture support | carrier. It is sectional drawing which shows the outline of a culture support | carrier. It is sectional drawing which shows the outline of a culture support | carrier. It is sectional drawing which shows the outline of a culture support | carrier. It is a top view which shows the outline of a culture support | carrier. It is sectional drawing which follows the XXIII-XXIII line | wire of FIG. It is sectional drawing which follows the XXIV-XXIV line | wire of FIG. It is a top view which shows the outline of a culture support | carrier. It is sectional drawing which follows the XXVI-XXVI line of FIG. It is a top view which shows the outline of a culture support | carrier. It is sectional drawing which follows the XXVIII-XXVIII line of FIG. It is a top view which shows the outline of a culture support | carrier. It is sectional drawing which follows the XXX-XXX line of FIG.

  With reference to FIG. 1 and FIG. 2, a cell culture tool according to a first embodiment of the present invention will be described. A well (also called a microflask) 10 as a cell culture tool is composed of a container body 11 for containing a culture solution and a culture bed for culturing cells, that is, a culture carrier 12 as a culture substrate. The container body 11 has a cylindrical shape with an outer diameter D11 of approximately 16 mm and a depth DE11 of approximately 10 mm. However, the size of the container body 11 is not limited to this, and may be set as appropriate according to the usage form after cell culture, the purpose of use of the cultured cells, and the like. For example, when drug discovery screening is planned in a state where cultured cells are in the well 10, the outer diameter is preferably in the range of 1 mm to 16 mm. Moreover, when it is planned to observe the cultured cells in the state of being in the well 10, the outer diameter is preferably in the range of 1 mm to 150 mm.

  The culture carrier 12 is formed in a film shape, is provided on the lower end surface of the container body 11, and constitutes the bottom of the well 10. The upper surface forming the inner surface of the well 10 of the culture carrier 12 is a culture surface 12a for culturing cells. The culture carrier 12 has a plurality of holes 13. Each hole 13 opens to the culture surface 12 a and forms a recess of the culture carrier 12. The plurality of holes 13 have a constant size and are densely arranged. As a result, the culture carrier 12 has a honeycomb structure when the culture surface 12a is viewed from the vertical direction, that is, a so-called honeycomb structure.

  In the present specification, the honeycomb structure means a structure in which holes having a fixed shape and a fixed size are arranged along the culture surface 12a and regularly arranged as described above. The basic structure forming the honeycomb structure is such that a plurality of (for example, six) holes are arranged so as to surround any one hole on the culture surface 12a. The number of holes surrounding any one hole is not limited to six, but may be three to five or seven or more.

  And the dimension and formation density of the hole 13 differ with manufacturing conditions mentioned later. The pore formation density is the number of pores per unit area of the culture surface 12a. The form of the culture carrier 12 is not particularly limited. For example, as shown in FIG. 3, the thickness TH12 is preferably in the range of 0.05 μm to 20 μm, and in the range of 0.1 μm to 20 μm. It is more preferable that it is within the range of 0.1 μm or more and 10 μm or less. The diameter D13 of the hole 13 is preferably in the range of 0.05 μm to 30 μm, more preferably in the range of 0.05 μm to 20 μm, and in the range of 0.1 μm to 20 μm. It is particularly preferred. The pitch P13 of the holes 13 is preferably in the range of 0.05 μm to 50 μm, more preferably in the range of 0.05 μm to 40 μm, and preferably in the range of 0.1 μm to 40 μm. Particularly preferred.

  When the depth from the culture surface 12a at the top of the convex portion to the bottom 13a of the hole 13 is DE13, the value of DE13 / D13 is preferably 0.05 or more and 1.2 or less, and 0.2 or more and 1 More preferably, it is 0.0 or less.

  The volume ratio of the culture carrier 12 will be described with reference to FIG. In FIG. 4, a symbol DEa is attached to a range corresponding to the culture surface 12 a to a depth of 0.1 μm. Here, when the hole 13 is not formed on the culture surface 12a, that is, when the culture surface is flat, the volume in the range of a depth of 0.1 μm is defined as VS, and the hole 13 at the depth DEa of 0.1 μm from the culture surface 12a. The volume is V13. VS is the sum of the volumes of the hatched portion and the cross-hatched portion in FIG. 4, and V13 is the volume of the cross-hatched portion. The volume ratio (hereinafter simply referred to as volume ratio) VR (unit:%) in the range from the culture surface 12a of the culture carrier 12 to a depth of 0.1 μm is obtained by {(VS−V13) / VS} × 100. .

  Since the culture carrier 12 has the holes 13 formed in the culture surface 12a as described above, the volume ratio VR is not 100% but is in the range of 5% to 80%. When the volume ratio VR is 80% or less, spheroids are easily formed as compared with a case where the volume ratio VR is larger than 80%. In addition, when the volume ratio VR is 5% or more, the cells are more reliably cultured than when the volume ratio VR is less than 5%. When the volume ratio VR is less than 5%, there are problems such as cells entering the pores 13 or dying (apoptosis) during culture. The volume ratio VR is more preferably in the range of 35% or more and 55% or less from the viewpoint of easy formation of spheroids and reliable cell culture.

  The aperture ratio on the culture surface 12a will be described with reference to FIG. Here, when the culture surface 12a is viewed from the vertical direction, the area of the opening of the hole 13 in the culture surface 12a is S13, and the area of the culture surface 12a is S12a. The area S13 of the opening of the hole 13 is the area of the portion indicated by cross hatching in FIG. 5, and the area S12a of the culture surface 12a is the area of the hatched portion indicated by hatching. The aperture ratio SR (unit:%) on the culture surface 12a is determined by {S13 / (S12a + S13)} × 100.

  The culture carrier 12 has an opening ratio SR of 30% or more and 95% or less on the culture surface 12a. When the aperture ratio SR is 95% or less, the cells are more reliably cultured as compared with a case where the aperture ratio SR is greater than 95%. If it is larger than 95%, there are problems such as cells entering into the pores 13 or dying (apoptosis) during culture. In addition, when the aperture ratio SR is 30% or more, the cells are more likely to migrate during culturing than in the case where the aperture ratio SR is less than 30%, so that the cells are likely to gather together, and thus spheroids are easily formed. The aperture ratio SR is more preferably in the range of 40% or more and 70% or less from the viewpoint of more reliably culturing cells and ease of spheroid formation.

  In this embodiment, the culture carrier 12 is made of polystyrene (PS), but is not limited thereto. For example, the culture carrier 12 may be composed of any one polymer such as polybutadiene, polycarbonate, polylactic acid, polycaprolactone, and polyglycolic acid instead of polystyrene.

  The container body 11 includes a cylindrical member 15 and a low affinity portion 16. The cylindrical member 15 is for blocking the accommodated culture solution and is made of polystyrene. The cylindrical member 15 also has a function of reducing the influence (for example, evaporation of the culture solution) due to disturbance during the culture and maintaining the culture environment constant.

  In the present embodiment, the cylindrical member 15 is made of polystyrene, but is not limited thereto. For example, instead of polystyrene, the cylindrical member 15 is made of a polymer such as polycarbonate (PC) or polyethylene terephthalate (PET), a metal such as stainless steel (for example, SUS) or aluminum, glass, or at least two of these. You may comprise from the comprised composite.

  The low affinity portion 16 has a lower affinity for cells than the culture carrier 12, specifically the culture surface 12a. The low affinity portion 16 has a water contact angle of 40 ° or less. In contrast, the contact angle of the culture surface 12a is approximately 120 °. Thus, the function as the low affinity part 16 is expressed by making the contact angle smaller than the culture surface 12a. In order to express the function as the low affinity part 16, the difference in the contact angle with the culture surface 12a is preferably in the range of approximately 20 ° to 130 °. The low affinity portion 16 is a coating that covers the inner wall surface of the cylindrical member 15, thereby forming the inner surface of the side wall of the container body 11. Thus, the low affinity part 16 is provided with the attitude | position standing with respect to the culture surface 12a.

  The low affinity part 16 is formed of solid polyethylene glycol. Since the inner wall surface of the cylindrical cylindrical member 15 is covered with the low affinity portion 16, the cells do not move from the culture surface 12a toward the periphery, and the cells increase on the culture surface 12a. Since the pores 13 having a diameter D13 in the range of 0.05 μm or more and 30 μm or less are formed on the culture surface 12a, the cells easily form spheroids as they increase. Therefore, the cells obtained by culturing in the well 10 often exhibit a behavior similar to that in the living body in vitro, and can be expected to be used for drug discovery screening and cosmetic safety tests.

  The thickness T16 of the low affinity portion 16 is approximately 100 nm, but is not limited thereto. In addition, there exists a tendency for affinity with a cell to rise, so that thickness T16 is large. The reason is that as the thickness T16 is increased, the low affinity portion contains free water, and the protein adheres to the surface of the low affinity portion 16 together with the free water. It is thought that cells adhere through the protein. Therefore, it is preferable to form the low affinity portion 16 so that the thickness T16 is as small as possible, thereby preventing the attachment of proteins or cells more reliably. In addition, the said protein is originally contained in a culture solution, or exists in a culture solution by a cell producing and releasing.

  The low affinity portion 16 is formed by applying a solution obtained by dissolving polyethylene glycol (PEG) in a solvent to the inner surface of the cylindrical member 15, drying the formed coating film, and evaporating the solvent component. The thickness T16 can be adjusted by adjusting the concentration of PEG in the solution, the type of solvent, and the drying conditions. The material forming the low affinity part 16 by coating is not limited to PEG, and for example, albumin, cytochrome, chitin, chitosan, hyaluronic acid, chondroid sulfate, heparin sulfate, deoxyribonucleic acid, ribonucleic acid, poly (2-methoxyethyl) Acrylate) (PMEA), polyethylene oxide, polyacrylamide, polysiloxane, 2-methacryloyloxyethyl phosphorylcholine polymer (MPC polymer), polyhydroxyalkyl methacrylate, polyvinyl pyrrolidone, polymethyl vinyl ether, polytetrahydrofurfuryl acrylate, poly (2-hydroxy) Alkyl methacrylate), polysulfopetine, silicone, and polyvinyl alcohol. Even if the low-affinity portion 16 is constituted by these coatings, the contact angle of water is 40 ° or less, and the affinity with cells is surely lower than that of the culture surface 12a. In the case of polyvinyl alcohol, for example, a solution in which polyvinyl alcohol is dissolved in a solvent is applied to the inner surface of the cylindrical member 15, and the coating film is dried to form a coating film.

  The low-affinity portion 16 is not limited to being formed as a coating on the inner wall surface of the cylindrical member 15, but may be formed as a charging portion. For example, a charged part having a charge is formed by applying a negative charge or a positive charge to the inner wall surface of the cylindrical member 15 by performing a discharge process or an electrostatic process. This charging part functions as a low affinity part. This ensures that the affinity with the cells is lower than that of the culture surface 12a. The potential of the culture surface 12a made of polystyrene is approximately −80 mV, and in this case, for example, by forming a charged portion in which the potential is in the range of −30 mV to less than 0 mV, the charged portion becomes a low-affinity portion. As a function.

  Thus, the charged portion having a potential different from that of the culture surface 12a functions as a low affinity portion. In addition, the function as a low affinity part is expressed when there is a potential difference from the culture surface 12a. Therefore, according to the electric potentials of the inner wall surface of the cylindrical member 15 and the culture surface 12a, both of them may be subjected to a predetermined charging process, or only one of them may be subjected to a predetermined charging process. However, the cell has a lower affinity when the potential is 0 (zero) than when it is positive or negative, and the closer the potential is to 0 in each of the positive potential region and the negative potential region, the lower the affinity. Tend. Therefore, it is preferable to perform a charging process corresponding to each potential between the inner wall surface of the cylindrical member 15 and the culture surface 12a in consideration of the affinity tendency at such a potential. In addition, as a method for forming a charged portion having a negative charge, there is a sulfonation treatment. The sulfonation treatment is performed, for example, by placing concentrated sulfuric acid at 30 ° C. for 1 minute in a cylindrical member having a positive potential. The entire surface of the cylindrical member may be sulfonated by immersing the cylindrical member in concentrated sulfuric acid at 30 ° C. for 1 minute. In addition, it is preferable to wash | clean with water etc. after a sulfonation process.

  In the present embodiment, the container body 11 has a cylindrical shape, but is not limited thereto, and may be, for example, a rectangular or polygonal cylinder. However, from the viewpoint of forming spheroids, the culture unit is preferably circular as in this embodiment.

  In the present embodiment, the culture carrier 12 is fixed to the lower end surface of the container body 11 with an adhesive, but the fixing method is not limited to this. For example, a double-sided adhesive tape may be used instead of the adhesive, or the culture carrier 12 may be fixed to, for example, an annular holder, and this holder may be fitted to the container body 11. Both the adhesive and the double-sided pressure-sensitive adhesive tape are non-toxic to cells. Alternatively, the culture carrier 12 may be fixed to the lower end surface of the container body 11 by heat welding or ultrasonic welding. Further, a casting film is formed by putting a solution for forming the culture carrier 12 in the container main body 11 or the cylindrical member 15, and the culture carrier 12 is formed on the container main body 11 or the cylindrical member 15 by the dew condensation method described later. May be.

  Alternatively, the culture carrier 12 may be detachable by applying an electric charge to the container body 11 and the culture carrier 12 or making it magnetic. By making the culture carrier 12 detachable, the cultured cells can be detached from the container body 11 together with the culture carrier 12. When a charge is applied, one of the container body 11 and the culture carrier 12 is charged with a minus (−) charge and the other is charged with a plus (+) charge. When magnetism is applied, the container body 11 and the culture carrier 12 have different magnetic poles, one being the N pole and the other being the S pole.

  In the well 10, the culture surface 12a is formed on the entire bottom inner surface of the container body 11, but the region where the culture surface 12a is formed is not limited thereto. As shown in FIG. 6, the well 20 according to the second embodiment includes a container body 21 for containing a culture solution and a culture carrier 12 as a culture substrate. In FIG. 6, the same members as those in FIG. 1 are denoted by the same reference numerals as those in FIG.

  The container main body 21 includes a cylindrical member 25 formed of the same material as the cylindrical member 15 and a low affinity portion 16 provided on the inner surface of the cylindrical member 25, and has a shape in which a lower end is bent inward. Since the film-shaped culture carrier 12 is provided inside the bent lower end, the diameter D12a of the culture surface 12a is smaller than the inner diameter d21 of the side wall of the container body 21.

  Thus, the culture carrier 12 constitutes a part of the bottom of the well 20, specifically, the center of the bottom, and the culture surface 12 a is separated from the inner surface of the side wall of the container body 21. The culture solution may be sown during cell culture, and if it stagnates, cell growth may be inhibited. The stagnation is particularly remarkable in the vicinity of the side wall at the bottom of the well 20. However, since the periphery of the culture surface 12 a is separated from the side wall of the well 20, the entire culture surface 12 a effectively acts on cell culture, and spheroids are formed earlier on the culture surface 12 a than the well 10. Or formed larger.

  In the first and second embodiments, the culture carrier 12 constitutes the bottom of the wells 10 and 20, but is not limited to this mode. For example, as in the following third and fourth embodiments, the culture carrier 12 may be accommodated in the container bodies 31 and 41 having the bottom formed in advance.

  As shown in FIG. 7, the well 30 according to the third embodiment includes a container body 31 for containing a culture solution and a culture carrier 12. In FIG. 7, the same members as those in FIG. 1 are denoted by the same reference numerals as those in FIG. The container body 31 includes a cylindrical container (hereinafter referred to as a cylindrical container) 35 whose one end is a bottom, and a low affinity part 16 formed on the inner surface of the cylindrical container 35. The culture carrier 12 is fixed on the bottom. In this embodiment, this fixing is performed using an adhesive in the same manner as the method of fixing the container body 11 and the culture carrier 12 in the well 10, but may be fixed by the other methods described above. The culture carrier 12 may be detachably installed without being fixed to the container body 11.

  Since the cylindrical container 35 is made of polystyrene like the cylindrical member 15 of the well 10 and the inner wall surface of the cylindrical container 35 is covered with the low affinity portion 16, the cells move from the culture surface 12a toward the periphery. Without increasing, it increases on the culture surface 12a and forms a spheroid.

  As shown in FIG. 8, the well 40 according to the fourth embodiment includes a container body 31 and a culture carrier 12. In FIG. 8, the same members as those in FIG. 7 are denoted by the same reference numerals as those in FIG. The culture carrier 12 is placed on the bottom but is not fixed. When the culture solution is put in the well 40, the culture carrier 12 may float during the culture or the like. In order to prevent this, the pressing member 42 is placed on the culture carrier 12.

  The holding member 42 has a ring shape that covers the peripheral edge of the culture carrier 12 and opens the center of the culture carrier 12. The surface of the culture carrier 12 exposed by this opening functions as a culture surface 12a for culturing cells. The pressing member 42 is a so-called weight that suppresses the lifting of the culture carrier 12, and is made of glass in this embodiment, and the surface is covered with a coating 42a whose affinity for cells is lower than that of the culture surface 12a. . As the material of the coating 42a, the same material as that of the low affinity portion 16 can be used. Thus, also in this embodiment, the cells do not move from the culture surface 12a toward the periphery or the side surface of the culture carrier 12, but increase on the culture surface 12a to form spheroids. Even when the culture carrier 12 is installed in the well 30 shown in FIG. 7 without being fixed on the container body 11, it is possible to prevent the culture carrier 12 from being lifted by placing the pressing member 42 on the culture carrier 12. preferable.

  The pressing member 42 may be configured such that the pressing member main body on which the coating 42 a is formed is made of metal, a solid polymer, or a composite of at least two of glass, metal, and solid polymer instead of glass. . The pressing member 42 may be formed by forming a negatively charged layer by providing the above-described sulfonation treatment or polyvinyl alcohol coating on the surface of a stainless pressing member main body, for example, instead of the coating 42a.

  The culture carrier 12 may be fixed to the container body 31 in the same manner as the culture carrier 12 of the first to third embodiments 10, 20, and 30. The fixation may be performed by an adhesive as in the fixing method of the container body 11 and the culture carrier 12 in the well 10, or may be fixed by the other method described above.

  The culture carrier 12 may constitute a well together with a support that supports the culture carrier 12. For example, as shown in FIG. 9, the well 50 of the fifth embodiment includes the same container body 11 as the well 10 of FIG. 1, a culture carrier 12, and a support body 52 that supports the culture carrier 12. The support body 52 supports the culture carrier 12 from below, that is, from the surface opposite to the culture surface 12a. In the present embodiment, the support 52 is made of glass, but the support 52 may be made of a polymer such as PET instead of glass. The culture carrier 12 and the support body 52 may be integrated and then fixed to the container body 11, or the culture carrier 12 and the container body 11 may be fixed and then the culture carrier 12 may be integrated.

  The culture carrier 12 is fixed to the support 52. The culture carrier 12 is fixed to the support body 52 with an adhesive in the same manner as the container body 11 and the culture carrier 12 in the well 10 are fixed. However, the method of fixing the culture carrier 12 to the support 52 may be the above-described other method of the container body 11 of the well 10 and the culture carrier 12. Also, a solution for forming the culture carrier 12 was cast on the support 52 to form a cast film, and the culture carrier 12 was formed by the dew condensation method described later, and the culture carrier 12 was fixed on the support 52. A culture carrier 12 is obtained.

  Also in this example, since the inner wall surface of the cylindrical member 15 is covered with the low affinity portion 16, the cells do not move from the culture surface 12a toward the periphery, but increase on the culture surface 12a and on the culture surface 12a. Spheroids are formed.

  As shown in FIG. 10, the well 60 of the sixth embodiment includes the same container body 21 as the well 20 of FIG. 6, the culture carrier 12, and a support 52. Also in this example, the cells do not move from the culture surface 12a toward the periphery, but increase on the culture surface 12a, and spheroids are formed on the culture surface 12a. Furthermore, in this example, the culture surface 12a is separated from the inner surface of the side wall of the container main body 21 as in the well 20 of FIG. Therefore, spheroids are formed earlier or larger on the culture surface 12a than the well 50 of FIG.

  By making the support 52 and the culture carrier 12 integrated with each other detachable from the container main body 21, the culture carrier 12 after the culture can be detached from the container main body 21 together with the support 52. In the case of observing the cultured cells, it is better to remove the support 52 and the culture carrier 12 from the container body 21 after culturing and observe the cells in such a detached state.

  As shown in FIG. 11, the well 70 of the seventh embodiment includes the same container body 31 as the well 30 of FIG. 7, the culture carrier 12, and a support 52. The culture carrier 12 is integrated with the support body 52 and installed on the bottom of the container body 31. The culture carrier 12 and the support 52 have the same area as the upper surface of the bottom of the container body 31 and cover the upper surface of the bottom. Also in this example, the cells do not move from the culture surface 12a toward the periphery, but increase on the culture surface 12a, and spheroids are formed on the culture surface 12a.

  As shown in FIG. 12, the well 80 of the eighth embodiment includes a container main body 31, a culture carrier 12, and a support body 52, similar to the well 70 of FIG. 11. It is united and installed on the bottom of the container body 31. The culture carrier 12 and the support 52 are smaller than the top surface of the bottom of the container body 31 and are provided apart from the side surfaces. Also in this example, the cells do not move from the culture surface 12a toward the periphery, but increase on the culture surface 12a, and spheroids are formed on the culture surface 12a. Furthermore, spheroids are formed earlier or larger on the culture surface 12a than the well 70 of FIG.

  The well 80 preferably includes a pressing member 42 similar to that shown in FIG. 8 in order to prevent the culture carrier 12 from being lifted during the culture. Similarly, the well 70 of FIG. 11 is provided with the pressing member 42 to prevent the culture carrier 12 from being lifted during the culture.

  Although all the above embodiments are wells, the cell culture device of the present invention may be a chip. As shown in FIG. 13, the chip 90 according to the ninth embodiment includes a circular culture carrier 12 and a low affinity portion 16. The culture carrier 12 is circular, and the low-affinity portion 16 is formed in an annular shape and covers the periphery of the culture carrier 12. The low-affinity portion 16 is provided on the surface on which the culture carrier hole 13 (see FIGS. 2 and 3) is formed. A portion exposed from the opening of the annular low affinity portion 16, that is, a portion surrounded by the low affinity portion 16 is the culture surface 12a. The culture solution is accommodated in a portion surrounded by the low affinity portion 16.

  The diameter D90 of the chip 90 is approximately 25 mm, the diameter D12a of the culture surface 12a is approximately 15 mm, the width W16 of the low affinity portion 16 is approximately 5 mm, and the thickness TH16 of the low affinity portion 16 is approximately 100 μm. However, the diameters D90 and D12a of the chip 90 and the culture surface 12a and the width W16 of the low affinity portion 16 are not limited to this, and the usage form after cell culture is the same as the size of the container body 11 shown in FIG. And may be appropriately set according to the purpose of use of the cultured cells. The thickness TH16 of the low-affinity portion 16 is preferably 10 μm or more, so that the culture solution is reliably accommodated.

  Also in this example, the inner peripheral surface of the low-affinity portion 16 is formed in a posture standing up from the culture surface 12a. For this reason, cells increase on the culture surface 12a without moving from the culture surface 12a to the periphery, and spheroids are formed on the culture surface 12a.

  Each of the above examples is a cell culture tool having one culture surface 12a, but may be a cell culture tool having a plurality of culture surfaces 12a. As shown in FIGS. 14 and 15, the multiwell plate 100 that is the tenth embodiment of the cell culture instrument has a plurality of well portions 102 formed on the upper surface of a plate body 101 as a container body. In the present embodiment, the well portion 102 has a plurality of well portions 102 formed in a matrix on the plate body 101, but this arrangement is not limited to a matrix. In addition, the multiwell plate 100 of the present embodiment includes 24 well portions 102, but the number of well portions 102 is not limited to this, and may be any number such as 2 to 23 and 25 or more.

  The plate body 101 is made of the same material as the container body 11 of the well 10, and the inside of the well portion 102 has the same configuration as the inside of the well 10 of FIG. 1. That is, the plate body 101 includes a polystyrene plate member 103 in which a plurality of circular holes for forming each well portion 102 are formed, and a low affinity portion formed on the side surface of each hole of the plate member 103. 16. The culture carrier 12 is arranged at the lower part of the plate body 101 and constitutes a flat bottom portion of the well portion 102. The part surrounded by the low affinity part 16 is the culture surface 12a.

  Also in this example, the culture surface 12 a is surrounded by the low affinity portion 16. For this reason, the cells increase on the culture surface 12a without moving from the culture surface 12a to the surroundings, and spheroids are formed on the culture surface 12a.

  In addition, although the inside of the well part 102 in this embodiment has the same structure as the inside of the well 10 of FIG. 1, it is not limited to this. For example, like the inside of the well 20 shown in FIG. 6, the center of the bottom of the well portion 102 is the culture surface 12 a, and the periphery of the culture surface 12 a is separated from the side surface formed by the low affinity portion 16. May be. Further, the plate body 101 may have a bottom portion made of polystyrene or the like covered with the low affinity portion 16 like the container body 31 shown in FIGS. 7 and 6, and the culture carrier 12 is installed on the bottom portion. Good. Further, the culture carrier 12 may be supported by the support body 52 in the same manner as the wells 50, 60, 70, and 80 shown in FIGS. Furthermore, the culture carrier 12 of the well portion 102 may be detachably provided on the plate body 101 or may be fixed.

  As shown in FIGS. 16 and 17, the multichip plate 110 that is the eleventh embodiment of the cell culture device has a plurality of chip portions 112 formed on the upper surface of the plate main body 111 as a container main body. In the present embodiment, the chip portion 112 has a plurality of chip portions 112 formed in a matrix on the plate body 111, but this arrangement is not limited to a matrix. In addition, the multichip plate 110 of the present embodiment includes 24 chip portions 112, but the number of chip portions 112 is not limited to this, and may be any number such as 2 to 23 and 25 or more.

  The plate body 111 is made of the same material as the container body 11 of the well 10, and the inside of the chip portion 112 has the same structure as the chip 90 of FIG. 13. That is, the plate body 111 includes a polystyrene plate member 113 in which a plurality of circular holes for forming each chip portion 112 is formed, and the low affinity portion 16 formed on the side surface of each hole. The culture carrier 12 is disposed at the lower part of the plate body 111 and constitutes a flat bottom portion of the chip portion 112. The part surrounded by the low affinity part 16 is the culture surface 12a.

  Also in this example, the culture surface 12 a is surrounded by the low affinity portion 16. For this reason, the cells increase on the culture surface 12a without moving from the culture surface 12a to the surroundings, and spheroids are formed on the culture surface 12a.

  The multi-well plate 100 and the multi-chip plate 110 described above are plate bodies 101 and 111 having a well portion 102 and a chip portion 112 formed therein. However, the multiwell plate and the multichip plate are not limited to these embodiments. For example, a well 10, 20, 30, 40, 50, 60, 70, 80, a chip is formed in each of the through hole or non-through hole of a plate body (not shown) in which a plurality of through holes or non-through holes are formed. 90 may be fitted.

  A method for producing the culture carrier 12 will be described. The culture carrier production process for producing the culture carrier 12 includes a solution preparation process, a cast film formation process, and a film formation process. The solution preparation step is a step of preparing a solution for forming the culture carrier 12. Specifically, for example, a hydrophobic polymer forming the culture carrier 12 is dissolved in a solvent to form a solution.

  The cast film forming step is a step of forming the cast film by flowing and spreading the solution obtained in the solution preparing step on the support. It is preferable that the temperature of the support is adjusted in advance, and the temperature is also adjusted while the casting film is formed.

  The film forming process includes a water droplet forming process and an evaporation process. The water droplet forming step is a step of forming water droplets by condensation on the film surface of the casting film. The water droplets are formed by cooling the support so that the temperature is lower than the temperature of the atmosphere around the casting membrane. Here, from the viewpoint of aligning the timing of the generation of a plurality of water droplets or uniforming the size of the formed water droplets, humidifying while adjusting the temperature of the support so that the support is maintained at a predetermined temperature. It is preferable to supply the gas (for example, air) to the casting film.

  The evaporation step is a step of evaporating the solvent and the water droplets formed in the water droplet forming step. Since the holes 13 (see FIGS. 2 and 3) are formed with water droplets as a mold, the solvent evaporates faster than the water droplets so that the water droplets sink into the casting film. For this purpose, it is preferable to use a solvent having a higher evaporation rate than water. However, the timing at which the water droplets start to evaporate may not be after all of the solvent has been evaporated. Further, as long as the formed hole 13 is maintained, some solvent may remain in the casting film after the evaporation of the water droplet is completed, and the remaining solvent is after the evaporation of the water droplet is completed. It may evaporate. Thus, a film formation process is a process of the dew condensation method known as a manufacturing method of a porous film.

  Instead of the culture carrier 12, another culture carrier may be used. Other culture carriers will be described. The culture carrier 120 shown in FIG. 18 has a plurality of holes 121 formed deeper than the holes 13 of the culture carrier 12. Thereby, in the culture carrier 120, the hole 121 is more spherical than the hole 13 of the culture carrier 12. Further, the culture carrier 125 shown in FIG. 19 has a hole 126 penetrating in the thickness direction, and the hole 126 is opened on the opposite surface in addition to the culture surface 120a. The plurality of holes 126 arranged along the culture surface 120a are independent of each other.

  In the culture carrier 130 shown in FIG. 20, the holes 131 arranged along the culture surface 130a communicate with each other. The diameter D131 of the hole 131 is in the range of 10 nm to 100 μm. The culture carrier 135 shown in FIG. 21 has a hole 136 penetrating in the thickness direction, and the hole 136 is opened on the opposite surface in addition to the culture surface 135a. The holes 136 arranged along the culture surface 135a communicate with each other. The diameter D136 of the hole 136 is in the range of 10 nm to 100 μm. The plan views of the above culture carriers 120, 125, 130, and 135 are the same as those in FIG. As described above, the culture carriers 120, 125, 130, 135 are all formed in the culture surfaces 120 a, 125 a, 130 a, 135 a with the holes 121, 126, 131, 136 having a constant size as recesses. , 126, 131, 136 are formed side by side at a constant pitch.

  The film-shaped culture carrier 140 shown in FIGS. 22 to 24 is a so-called pillar structure film in which a plurality of columnar convex portions 141 are formed on one film surface. The plurality of convex portions 141 have a substantially constant height and shape. The plurality of convex portions 141 are arranged at a constant pitch and regularly arranged, so that the tip surface 141a constitutes the culture surface 140a. As described above, the culture carrier is not limited to a honeycomb structure in which pores are formed, and any culture carrier having a certain pattern of irregularities on the surface may be used.

  When the culture carrier 140 is viewed from the direction perpendicular to the culture surface 140a, the tip surface 141a of the convex portion 141 has, for example, a shape surrounded by three arcs that are convex inward as shown in FIG. . The distance L1 between the adjacent convex portion 141 and the convex portion 141 is fixed in the range of 50 nm to 250 μm. The thickness TA is in the range of 50 nm to 50 μm. In this case, the size of the convex portion 141 is in the range of 50 nm to 50 μm. In addition, when the convex part 141 is seen from the perpendicular direction of the culture surface 140a, the longest dimension part D141 of the convex part 141 is regarded as the size of the convex part 141. In addition, although illustration is abbreviate | omitted, as for the film of the pillar structure as a culture support | carrier, the front-end | tip of a convex part differs from the convex part 141 of FIGS. 22-24, and there is also a thing with the sharp shape, ie, a taper, Something like that.

  Any of the above culture carriers 12, 120, 125, 130, 135, and 140 can be produced by the above-described dew condensation method. Examples of other culture carriers that can be used in place of the culture carrier 12 include porous materials obtained by nanoimprinting, borogen leaching, phase separation, and fiber welding. As a porous material obtained by the fiber welding method, there is a so-called knit mesh shape.

  The culture carrier 150 shown in FIGS. 25 and 26 is an example of a porous material obtained by nanoimprinting. The culture carrier 150 has a plurality of convex portions 151 formed in a lattice shape, and a culture surface 150 a is formed by the convex portions 151. The concave portion 152 surrounded by the convex portion 151 is a substantially square rectangle. In this case, the size of the recess 152 is in the range of 50 nm to 50 μm. When a circle 153 having the same area as the opening of the recess 152 on the culture surface 150a is drawn, the diameter D151 of the circle is regarded as the size of the recess. Although the recessed part 151 of this embodiment is substantially square, it is not restricted to this. For example, it may be a rectangle such as a rectangle, trapezoid or parallelogram, a polygon such as a triangle or a pentagon, or a circle such as a perfect circle or an ellipse.

  The culture carrier 160 shown in FIGS. 27 and 28 is an example of a porous material obtained by nanoimprinting. In the culture carrier 160, a plurality of frustoconical convex portions 161 obtained by cutting out the top of the cone at a certain height are formed in a matrix, and the culture surface 160a is formed by the convex portions 161. In this case, the size of the convex portion is in the range of 50 nm to 50 μm. In addition, let the diameter D161 of the circle | round | yen at the front-end | tip of the convex part 161 be the magnitude | size of a convex part. Although the convex part 161 of this embodiment is made into the truncated cone shape, it is not restricted to this. For example, it may be a truncated pyramid cut out at the height of the top.

  The culture carrier 170 shown in FIGS. 29 and 30 is an example of a porous material obtained by the phase separation method. The culture carrier 170 has a convex portion 171 formed in an irregular shape, and a culture surface 170 a is formed by the convex portion 171. The concave portion 172 is formed as a portion surrounded by the convex portion 171 and is indefinite like the convex portion 171. In this example, the convex portion 171 and the concave portion 172 are formed as a sea-island structure (the convex portion 171 is a sea portion and the concave portion 172 is an island portion), and the convex portion 171 is formed in a sea shape and the concave portion 172 is formed in an island shape. . In this case, the size of the recess 172 is in the range of 50 nm to 50 μm. When a circle 172 having the same area as the opening of the recess 172 on the culture surface 170a is drawn, the diameter D172 of the circle 172 is regarded as the size of the recess 172. In the present embodiment, each recess 172 is an independent island, but the sea-island structure may be reversed. That is, a convex part may be an island part and a recessed part may be a sea part, and each convex part may be independent.

  The culture carrier 120, 125, 130, 135, 140, 150, 160, 170 is the same as the culture carrier 12, from the culture surface 120a, 125a, 130a, 135a, 140a, 150a, 160a, 170a to a depth of 0.1 μm. The volume ratio VR is in the range of 20% to 80%, and the opening ratio SR on the culture surface is in the range of 30% to 90%.

  Further, the culture carriers 12, 120, 125, 130, 135, 140, 150, 160, and 170 are all film-like, but may be block-like. In the case of a block-shaped culture carrier, a culture surface on which a concave portion or a convex portion within a range of 50 nm to 50 μm is formed.

  Examples of the present invention will be described below. Details will be described in Example 1, and Comparative Example 1 for the present invention will be described only for conditions different from those of Example 1.

  A polystyrene film was cast on a glass plate as the support 52 to form a cast film. The cast film was dried while supplying humidified air to the cast film, whereby water droplets were formed by condensation on the surface of the cast film, and finally the solvent and the water droplets were evaporated. In order to evaporate the water droplets, the air to be blown was switched from humidified to dry. By this film forming step, a culture carrier 12 having a honeycomb structure made of polystyrene and having holes 13 arranged along the culture surface 12a was formed. Thus, in this example, the culture carrier 12 was formed by the condensation method. The obtained culture carrier 12 had a uniform pore diameter, and the average pore diameter (average pore diameter) was about 5 μm. The volume ratio VR was about 35%, and the opening ratio SR on the culture surface 12a was about 50%.

  A container body 31 in which the inner surface of a polystyrene cylindrical container 35 was covered with the low affinity portion 16 was prepared. First, a platinum (Pt) film having a thickness of 9 nm was formed on the inner surface of the cylindrical container 35 using a sputtering apparatus (E-1030, manufactured by Hitachi, Ltd.). Next, the low affinity part 16 was formed on this Pt film. The low affinity part 16 was formed by applying a solution of a synthetic polymer (manufactured by NOF Corporation) having a polyethylene glycol (PEG) chain having a weight average molecular weight of 30000 and a thiol group and evaporating the solvent. A well 40 shown in FIG. 8 was created using the container body 31. That is, the culture carrier 12 integral with the support 52 was placed on the bottom of the container body 31 and the pressing member 42 was placed on the culture carrier 12 to prevent the culture carrier 12 from being lifted. A glass pressing member 42 covered with a PEG coating 42a was used.

  Inside the well 40, cells to be cultured and a culture solution (liquid medium) were placed. The cells were human liver-derived cells (HepG2), and the culture broth was 18.8 g / L of Williams E medium (Williams's Medium E, Sigma-Aldrich Japan LLC), 58.8 mg / L of penicillin (MeijiSeika). Pharma Co., Ltd.), 100 mg / L streptomycin (Meiji Seika Pharma Co., Ltd.), 2.2 g / L sodium bicarbonate (Wako Pure Chemical Industries, Ltd.), 10% fetal bovine serum (FBS, Invitrogen Corp.), a serum-added culture solution. The number of cells charged is 5000. The amount of the culture solution was such that the liquid level reached the middle position of the depth of the well 40 (fifth minute). The culture was carried out at an oxygen dioxide concentration of 5% and a temperature of 37 ° C. for 24 hours.

  24 hours after the start of the culture, the number of cells in the well 40 was counted and found to be 10,000, of which the number of cells on the culture surface 12a was 9000 or more. In addition, spheroids were formed on the culture surface 12a.

[Comparative Example 1]
The same cylindrical container 35 as in Example 1 was prepared, and the low affinity portion 16 was not formed on the inner surface of the cylindrical container 35. The same culture carrier 12 as in Example 1 was installed together with the support 52 on the bottom of the cylindrical container 35. Moreover, the peripheral part of the culture support | carrier 12 was hold | suppressed with the press member formed in the ring shape similarly to the press member 42. FIG. However, the used pressing member is one in which the coating 42a is not formed. Other conditions were the same as in Example 1, and the same cells as in Example 1 were cultured.

  Although spheroids were formed on the culture surface 12 after 24 hours from the start of the culture, the number of cells in the well was counted to be 10,000, of which the number of cells on the culture surface 12a was 1000. It was the following.

10, 20, 30, 40, 50, 60, 70, 80 Well 12, 120, 125, 130, 135, 140 Culture carrier 90 chip 100 multiwell plate 110 multichip plate 13, 121, 126, 131, 136, 141 Hole 141 Convex

In order to solve the above problems, the cell culture instrument of the present invention includes a culture carrier and a low affinity part. The culture carrier has irregularities formed on the culture surface for culturing cells, the volume ratio in the range from the culture surface to a depth of 0.1 μm is in the range of 5% to 80%, and the open area ratio in the culture surface Is in the range of 30% to 95%. The low affinity part is provided so as to surround the periphery of the culture surface, and has a lower affinity with the cell than the culture surface. The contact angle of the surface of the low affinity part is smaller than the contact angle of the culture surface, and the difference in contact angle between the surface of the low affinity part and the culture surface is 20 ° or more and 130 ° or less. Or the potential of the low affinity part as the charging part is in the range of −30 mV to less than 0 mV.

Claims (8)

Concavities and convexities are formed on the culture surface for culturing the cells, the volume ratio in the range from the culture surface to a depth of 0.1 μm is in the range of 5% to 80%, and the opening ratio in the culture surface is 30% or more. A culture carrier within a range of 95% or less;
A low-affinity portion that is provided around the culture surface and has a lower affinity for the cells than the culture surface;
A cell culture tool comprising:   The cell culture instrument according to claim 1, wherein the low affinity part is provided in an upright posture with respect to the culture surface. Containing a culture medium, comprising a container body whose inner surface is formed by the low affinity part,
The cell culture instrument according to claim 1 or 2, wherein the culture carrier is provided on the bottom of the container body.   The cell culture instrument according to claim 3, wherein the culture surface is formed so as to be separated from an inner side surface of the container body.   3. The film according to claim 1 or 2, wherein the culture carrier is a film having a honeycomb structure when the culture surface is viewed from a vertical direction by forming a plurality of concave portions having a certain size along the culture surface. The cell culture device described.   4. The film according to claim 3, wherein the culture carrier is a film having a honeycomb structure when the culture surface is viewed from a vertical direction by forming a plurality of concave portions having a certain size along the culture surface. The cell culture device described.   The cell culture tool according to claim 1 or 2, wherein the culture carrier is a film in which a plurality of convex portions having a certain height and shape are formed side by side along the culture surface.   The cell culture device according to claim 3, wherein the culture carrier is a film in which a plurality of convex portions having a certain height and shape are formed side by side along the culture surface.

Images