Cell culture method and cell culture apparatus used therein

Abstract

The present invention provides: a cell culture method that enables mass production of a BBB model which has a three-layer structure comprising vascular endothelial cells, pericytes, and astrocytes; a cell culture device that is used for the cell culture method; and a cell culture method that enables an increase in mass productivity of the BBB model.　A cell culture method according to the present invention comprises: a step for dispensing a culture solution that is in a suspension state onto the outer surface of a porous film of a cell culture insert; a step for seeding astrocyte cells in the culture solution and culturing astrocytes; a step for seeding pericyte cells inside the cell culture insert that has been turned upside down, and culturing pericytes; a step for seeding thermosensitive vascular endothelial cells that are embedded in thermosensitive gel, and culturing vascular endothelial cells; and a step for dissolving the thermosensitive gel.

Description

Technical Field

[0001] The present invention relates to a technique for culturing cells constituting a blood-brain barrier (BBB) in vitro model (hereinafter referred to as a BBB model) outside the body, and particularly to a cell culture method enabling mass production of a three-layer-structured BBB model composed of vascular endothelial cells, pericytes, and astrocytes, and a cell culture apparatus used therefor. Note that "in vitro" is a Latin word meaning "in a glass container", and generally represents a state in which a part of a living body is removed or separated "outside the body".

Background Art

[0002] Anatomically, the blood-brain barrier (BBB) is cerebral capillaries, which are composed of vascular endothelial cells, pericytes, astrocytes, etc. originating from the brain. Pericytes existing so as to surround vascular endothelial cells have a role of controlling the differentiation and proliferation of vascular endothelial cells, and astrocytes have a role of supplying nutrients to nerve cells and protecting nerve cells by quickly removing excessive ions and neurotransmitters. The blood-brain barrier (BBB) has an important function of restricting the exchange of substances between blood and brain tissue fluid, and this function maintains the homeostasis of nerve cells. However, this function has become a major obstacle in developing preventive and therapeutic drugs used for central nervous system diseases. For example, a drug intended to act on the central nervous system may actually be blocked from migrating into the brain by the BBB, and the expected effect may not be obtained. In addition, a drug that was not originally expected to migrate into the brain may penetrate the BBB contrary to expectations, and as a result, it may have an adverse effect on the central nervous system.

[0003] Regarding what substances penetrate the BBB, there is no fixed law, so when developing preventive and therapeutic drugs used for central nervous system diseases, it is necessary to perform screening using a BBB model. The inventors of this invention have previously succeeded in developing a model that accurately reproduces the anatomical structure of the blood-brain barrier (BBB), which has dramatically improved the level of research in drug screening. This BBB model has a structure in which astrocytes and pericytes are formed in layers with a porous membrane in between, and layered vascular endothelial cells are arranged in direct contact with the pericytes. Traditionally, this BBB model has been prepared manually by skilled researchers, and because its preparation requires specialized techniques, it has been difficult to mass-produce.

[0004] As a technology related to the creation of the above BBB model, for example, Patent Document 1 discloses an invention relating to a device for co-culturing cells on both sides of a porous membrane, under the name "cell culture insert." The cell culture apparatus disclosed in Patent Document 1 comprises an outer tubular body with a porous membrane attached to its lower end, a complementary tubular body that fits into the outer diameter of the lower end of the outer tubular body, and a suspension component having at least one flange extending laterally from the upper end, the entire circumference or part of the outer diameter of which fits into the inner diameter of the outer tubular body. The apparatus is characterized in that, in a first step, the outer tubular body is connected to the complementary tubular body to form a self-supporting insert for a first cell culture, and in a second step, the outer tubular body is disengaged from the complementary tubular body and attached to the suspension component for a second cell culture.

[0005] In this cell culture apparatus, a cell culture chamber is formed by connecting a complementary tubular body to the lower end of an outer tubular body and then inverting the two. In this cell culture chamber, cells are seeded on the outer surface of the membrane protruding into the complementary tubular body (first cell seeding). Next, after an appropriate culture period has elapsed, the combination of the outer tubular body and the complementary tubular body is turned inside out, the suspension component is connected to the outer tubular body, and then the complementary tubular body is removed. The resulting suspension insert is placed in the wells of a cell culture plate, and a second cell seeding is performed on the inner surface of the membrane that is protruding upward. Thus, with the cell culture apparatus described above, cells can be co-cultured on both sides of the membrane by performing cell seeding in two stages on the outer and inner surfaces of the membrane. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 5674953 [Overview of the project] [Problems that the invention aims to solve]

[0007] Although the invention disclosed in Patent Document 1 allows for the creation of a cell culture model in which different types of cell layers are formed on both sides of a porous membrane, it is unsuitable for mass production of cell culture models because the operations for forming the cell culture chamber and suspension insert must be performed manually one by one. Therefore, even if the cell culture apparatus disclosed in Patent Document 1 is applied to the creation of a BBB model, it is considered that mass production of BBB models is not possible. The first object of the present invention is to address the aforementioned conventional circumstances and to provide a cell culture method and a cell culture apparatus used therefor that can mass-produce a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes. The second object of the present invention is to provide a cell culture method that can enhance the mass production of a BBB model consisting of these cell layers by maintaining the stacked state of the three cells for a long period of time. [Means for solving the problem]

[0008] To achieve the above objective, the first invention is characterized by comprising a cell culture chamber cover that simultaneously closes at least one of the divided and integrated cell culture chambers, each comprising a pair of cell culture chambers formed in a cylindrical shape by a porous membrane on both sides capable of fixing biomaterials, with the inner wall of each chamber being divided into upper and lower sections, and a plurality of these chambers being connected together to integrate the upper and lower sections. In cell culture apparatuses with this structure, a cell culture chamber cover is provided to conceal the open end of the cell culture chamber, thus preventing the cell culture medium injected into the cell culture chamber or the seeded cells from leaking out during handling. The reason for configuring the cell culture chambers so that at least one of the pair is closed off by a cell culture chamber cover is that if one cell culture chamber containing the biomaterial initially fixed to the upper side (first surface) of the porous membrane is closed off, then that cell culture chamber is inverted so that the other cell culture chamber faces upwards, and the biomaterial is fixed to the back side (second surface) of the porous membrane. If the membrane is not then inverted, the biomaterial can be cultured on both sides of the porous membrane without closing off both sides. Furthermore, "simultaneously blocking at least one of the fractionated and integrated cell culture chambers" means that in addition to simultaneously blocking the entire integrated cell culture chamber, it also includes simultaneously blocking each individual cell culture chamber that makes up the integrated cell culture chamber.

[0009] The second invention is characterized in that, in the first invention, a cell culture plate having a plurality of first through-holes used as an inner wall and a sheet made of a porous membrane placed on the bottom surface so as to cover the first through-holes, a first bottom cover having an open top and a plurality of second through-holes provided in the bottom plate which are used as an inner wall together with the first through-holes and placed on the bottom surface of the cell culture plate, and a second bottom cover as a cell culture chamber cover which is placed on the bottom plate of the first bottom cover, the second through-holes are provided at locations that correspond to the first through-holes when the first bottom cover is viewed from above in its installed state on the bottom surface of the cell culture plate. In the second invention, in addition to the effects of the first invention, when the cell culture plate is inverted and the first bottom cover is placed on the bottom surface, and then astrocytes are seeded into the second through-holes along with the cell culture medium, the leakage of the cell culture medium and astrocytes is prevented by the inner wall of the second through-holes, thus allowing the astrocytes to be easily cultured on the outer surface of the porous membrane. Furthermore, when the second bottom cover is placed on the first bottom cover, and the cell culture plate is inverted along with the first and second bottom covers, and then pericytes and vascular endothelial cells are seeded into the first through-holes of the cell culture plate along with the cell culture medium, the leakage of the cell culture medium, pericytes, and vascular endothelial cells is prevented by the inner wall of the first through-holes, thus allowing the pericytes and vascular endothelial cells to be easily cultured on the inner surface of the porous membrane. Moreover, the cell culture plate with the first and second bottom covers installed functions as a microplate.

[0010] The third invention is characterized in that, in the first invention, it comprises a first inner wall of a cell culture insert, one end of which is closed by a porous membrane and used as the inner wall of a cell culture chamber; a cell culture plate into which the cell culture insert is inserted and which has a plurality of through holes and is used as the inner wall of a cell culture chamber; and a cell culture chamber cover comprising a first plate cover and a second plate cover, which are flat plates installed on both sides of the cell culture plate and cover the through holes in which the cell culture insert is inserted. In the third invention, in addition to the effects of the first invention, if a first plate cover is placed on the upper surface of a cell culture plate in which a cell culture insert is installed in the through-holes with the bottom facing downwards, and then the cell culture plate is inverted together with the first plate cover, and astrocytes are seeded into the through-holes of the cell culture plate together with the cell culture medium, the leakage of the cell culture medium and astrocytes is prevented by the inner wall of the through-holes of the cell culture plate, thus allowing the astrocytes to be easily cultured on the outer surface of the porous membrane. Furthermore, if a second plate cover is placed on the side of the cell culture plate where the first plate cover is not installed, and the cell culture plate is inverted together with the first and second plate covers, the cell culture insert is held in a bottom-down position by the through-holes of the cell culture plate, thus facilitating the process of seeding pericytes and vascular endothelial cells into the cell culture insert together with the cell culture medium, and the process of culturing vascular endothelial cells and pericytes inside the cell culture insert. In addition, the cell culture plate with the second plate cover installed functions as a microplate.

[0011] The fourth invention is a membrane holder used as a cell culture chamber, having one end closed by a porous membrane and a second inner wall used as an inner wall; a plate on one side of a flat plate-shaped first base having a plurality of first through-holes formed thereon, communicating with the first through-holes, with the membrane holder installed inside and a first cylindrical body erected vertically on the plate, which is used as an inner wall together with the first through-holes; a plate cover that covers the first through-holes with the membrane holder installed inside, having a plurality of first protrusions provided on one side of the flat plate-shaped second base that simultaneously fit onto the plurality of first cylindrical bodies; and a plate cover that simultaneously fits onto the plurality of first cylindrical bodies. The present invention is characterized by comprising a spacer cover that covers a first through-hole with a membrane holder installed inside, with a plurality of second protrusions provided on one side of a flat third base, and a spacer that forms a cell culture chamber together with a first cylinder, with a plurality of second through-holes used as an inner wall, on one side of a flat fourth base, with the same number of second through-holes as the first through-hole, and a plurality of second cylinders that are simultaneously inserted into each of the plurality of first cylinders and used as an inner wall together with the second through-hole, erected vertically so as to communicate with the second through-hole, and an extruder that has a plurality of extrusions erected vertically on one side of a flat extruder plate, with a plurality of extrusions that are simultaneously inserted into each of the plurality of first through-holes.

[0012] In the fourth invention, in addition to the effects of the first invention, a membrane holder is installed on the upper end of the second cylindrical body of the spacer, the first cylindrical body of the plate is fitted onto the second cylindrical body of the spacer, and the spacer cover is attached to the spacer by fitting the second protrusion onto the first cylindrical body. This prevents leakage of the cell culture medium injected into the second cylindrical body of the spacer through the first through-hole of the plate, and the membrane holder is held in place by the second cylindrical body of the spacer, thus facilitating the cultivation of astrocytes on the inner surface of the porous membrane. Furthermore, after discarding the cell culture medium inside the second cylindrical body of the spacer, attaching the spacer cover to the spacer by fitting the first projection onto the first cylindrical body, inverting the spacer together with the plate cover and spacer cover, and then removing the spacer cover, seeding perisites together with the cell culture medium inside the second cylindrical body of the spacer, the leakage of the cell culture medium and perisites is prevented by the inner wall of the second cylindrical body of the spacer, thus facilitating the cultivation of perisites on the outer surface of the porous membrane. Then, after removing the plate cover, spacer cover, and spacer from the plate, and inserting the extrusion part of the extruder into the first through hole of the plate, the extruder is brought closer to the plate so that the extrusion plate contacts the first base of the plate, and the extrusion part pushes the membrane holder out of the first cylindrical body. Furthermore, after laying a cell sheet of vascular endothelial cells on the upper surface of the membrane holder extruded to the outside of the first cylindrical body of the plate, the cell sheet of vascular endothelial cells is punched out from above with a sleeve, and the sleeve is then pushed into the membrane holder, thereby attaching the membrane holder to the tip of the sleeve. This process integrates the membrane holder and the sleeve, resulting in a cell culture insert with three types of cell layers formed at the bottom: a cell sheet of vascular endothelial cells, pericytes, and astrocytes.

[0013] The fifth invention is characterized in that, in the first invention, it comprises a membrane holder used as a cell culture chamber, having a third inner wall that is closed at one end by a porous membrane and used as an inner wall; a plate as a cell culture chamber having a plurality of through holes, with the membrane holder installed inside a flat base and used as an inner wall of the cell culture chamber; two flat plate covers that are installed on the plate as a cell culture chamber cover, covering the through holes with the membrane holder installed inside; and an extruder having a plurality of vertically erected extruders on one side of a flat extruder plate, each of which is simultaneously inserted into the plurality of through holes. In the fifth invention, in addition to the effects of the first invention, when a plate cover is attached to the lower surface of the plate, and a membrane holder is placed inside the through-holes with the outer surface of the porous membrane facing downwards, and then cell culture medium is injected into the through-holes of the plate, the inner wall of the through-holes of the plate prevents leakage of the cell culture medium, thus facilitating the cultivation of astrocytes on the inner surface of the porous membrane.

[0014] Furthermore, if another plate cover is attached to the top surface of the plate, and the plate is inverted, and then the first plate cover is removed from the plate, the membrane holder will be inverted inside the plate's through-holes, and the outer surface of the porous membrane will face upwards. This allows perisites to be easily cultured on the inner surface of the porous membrane by seeding them inside the plate's through-holes. Furthermore, by removing the other plate cover from the plate and inserting the extrusion part of the extruder into the through hole in the plate, the extruder is brought closer to the plate so that the extrusion plate contacts the base of the plate, and the extrusion part pushes the membrane holder out of the cylindrical body. Then, after laying a cell sheet of vascular endothelial cells on the upper surface of the membrane holder that has been pushed out to the outside of the cylindrical body of the plate, the cell sheet of vascular endothelial cells is punched out from above with the insert body, and the insert body is then pushed into the membrane holder, thereby attaching the membrane holder to the tip of the insert body. As a result, the membrane holder and the insert body become one, and a cell culture insert is obtained in which three types of cell layers consisting of a cell sheet of vascular endothelial cells, pericytes, and astrocytes are formed at the bottom.

[0015] The sixth invention is characterized in that, in the fifth invention, the plate is provided with first and second protrusions on both sides of the base, which communicate with the through hole, and which surround the through hole when the base is viewed from above; the plate cover is provided with a plurality of first cylindrical bodies on one side, which fit simultaneously with the plurality of first and second protrusions; and the extruder is shorter than the extruder and is provided on one side of the extruder plate with a plurality of second cylindrical bodies on which fit simultaneously with the plurality of first and second protrusions, respectively, surrounding the extruder. In the sixth invention, the "first and second protrusions of the plate," the "first cylindrical body of the plate cover," and the "second cylindrical body of the extruder" correspond to the higher-level concepts of the "first annular protrusion 14b and second annular protrusion 14c of the plate 14," the "short cylindrical body constituting the holding portion 15b of the plate cover 15," and the "short cylindrical body constituting the holding portion 17b of the extruder 17," respectively, which will be described later using Figures 11(a), 11(c), and 11(f) in the embodiment. In the sixth invention, in addition to the effects of the fifth invention, the first cylindrical body of the plate cover is fitted onto the first or second protrusion of the plate, thereby increasing the adhesion of the plate cover to the plate, and the second cylindrical body of the extruder is fitted onto the first or second protrusion of the plate, thereby ensuring accurate alignment of the extruder with respect to the plate.

[0016] The seventh invention is characterized in that, in the first invention, it comprises a cell culture insert used as a cell culture chamber, having a fourth inner wall that is closed at one end by a porous membrane and used as an inner wall; a cell culture case used as a cell culture chamber, having an open top and a plurality of through holes in the bottom plate that are used as an inner wall and into which the cell culture insert is inserted; an insert holding means for holding a plurality of cell culture inserts in a state that allows them to be simultaneously inserted into the plurality of through holes of the cell culture case; and a case lid that is placed over the top of the cell culture case as a cell culture chamber cover. In the seventh invention, in addition to the operation of the first invention, after placing the plurality of cell culture inserts held by the insert holding means in a lying state and installing the cell culture case so that the bottom of each of the plurality of cell culture inserts protrudes from each through-hole, when a cell culture solution is injected into the cell culture case, the cell culture case has a function of storing the cell culture solution inside so that it does not leak. Therefore, it has the effect that astrocytes are easily cultured on the outer surface of the porous membrane.

[0017] Also, after attaching a lid for the case to the upper surface of the cell culture case, when the cell culture case is inverted together with the insert holding means, leakage of the cell culture solution from the cell culture case is prevented by the lid for the case, and the plurality of cell culture inserts are inverted simultaneously and face upward. Therefore, by seeding pericytes inside the cell culture insert, it has the effect that pericytes are easily cultured on the inner surface of the porous membrane. Furthermore, when a cell culture insert having three cell layers consisting of vascular endothelial cells, pericytes, and astrocytes formed at the bottom is obtained by seeding and culturing vascular endothelial cells inside the cell culture insert, since the plurality of cell culture inserts are integrated by the insert holding means, the operation of transferring them to the wells of the microplate is easy.

[0018] The eighth invention is a cell culture method for producing a three-layer structure BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using a plurality of pairs of cell culture chambers in which the inner wall formed in a cylindrical shape is vertically partitioned by a porous membrane on both sides of which a biomaterial can be fixedly formed. The method includes a step of seeding astrocytes on the first surface facing upward in the porous membrane, a step of culturing astrocytes, a step of simultaneously closing the upper end openings of the plurality of pairs of cell culture chambers, a step of simultaneously inverting the plurality of pairs of cell culture chambers and seeding pericytes on the second surface of the porous membrane, a step of culturing pericytes, a step of laying a sheet-like vascular endothelial cell on the second surface of the porous membrane, and a step of culturing vascular endothelial cells. In the embodiments of the invention, the steps of culturing astrocytes, pericytes, and vascular endothelial cells and the step of injecting a cell culture solution into a cell culture insert are described as separate steps. However, depending on the structure of the cell culture device, when culturing pericytes or vascular endothelial cells, there may be a situation where the cell culture insert used for culturing astrocytes can be utilized, and it may not be necessary to newly inject the cell culture solution into the cell culture insert. Therefore, in the eighth and ninth inventions, the step of culturing astrocytes, pericytes, and vascular endothelial cells includes either a step of utilizing the cell culture solution already injected into the cell culture insert or a step of newly injecting the cell culture solution into the cell culture insert. In the eighth invention, since it includes a step of simultaneously inverting the cell culture chambers with the upper openings of a plurality of pairs of cell culture chambers closed simultaneously, it has the effect that the cell culture solution injected into the cell culture chambers and the seeded cells are unlikely to leak from the cell culture chambers during handling.

[0019] The ninth invention is characterized in that, in the eighth invention, the vascular endothelial cells have temperature sensitivity and cell growth stops at a desired temperature, and instead of the step of laying a sheet-like vascular endothelial cell on the second surface of the porous membrane, it includes a step of seeding on the second surface of the porous membrane in a state where the vascular endothelial cells are embedded in a temperature-sensitive gel that dissolves at the above-mentioned temperature, and also includes a step of dissolving the temperature-sensitive gel after the step of culturing the vascular endothelial cells. In the ninth invention, in addition to the effect of the eighth invention, due to the dissolution of the temperature-sensitive gel, a state where astrocytes, pericytes, and vascular endothelial cells are neatly laminated is completed, and the state where the above three cells are laminated is maintained until their temperature is lowered to the temperature at which cell growth starts.

Advantages of the Invention

[0020] According to the first invention, at least one of the cell culture chambers, which are formed by linking multiple pairs of cell culture chambers separated by a porous membrane, is closed off by a cell culture chamber cover. This prevents the cell culture medium injected into the cell culture chamber or the seeded cells from leaking out during inversion in handling, allowing handling to proceed simultaneously. Therefore, by using a handling device, it is possible to efficiently perform tasks such as inverting the porous membrane and transferring the prepared BBB model to the wells of a microplate. Furthermore, since it is possible to perform handling on multiple cell culture chambers without any time differences, it is possible to carry out homogeneous cell culture between cell culture chambers.

[0021] According to the second invention, in addition to the effects of the first invention, it eliminates the need for cell culture inserts and allows the cell culture plate to be used directly as a microplate. This results in increased mass production efficiency of the three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes compared to cases where cell culture inserts or microplates are used.

[0022] According to the third invention, since the cell culture medium and astrocytes do not spill from the outer surface of the porous membrane, in addition to the effects of the first invention, it has the effect of efficiently forming an astrocyte cell layer on the outer surface of the porous membrane. Furthermore, when a cell culture plate with cell culture inserts placed inside multiple through-holes is inverted, the inversion of the porous membrane is performed simultaneously for all cell culture inserts, so that astrocyte and pericyte cell layers are formed on both sides of the porous membrane in a short time. Therefore, according to the third invention, it is possible to efficiently create a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes.

[0023] According to the fourth invention, since the operations of inverting the porous membrane, removing the membrane retainer from inside the plate's through-holes, and positioning vascular endothelial cells on top of the perisite are performed simultaneously for multiple porous membranes and membrane retainers, in addition to the effects of the first invention, it has the effect of being able to produce multiple BBB models in a short time.

[0024] According to the fifth invention, the operations of inverting the porous membrane necessary for culturing astrocytes and pericytes on both sides of the porous membrane, removing the membrane holder from inside the through-holes of the plate, and placing vascular endothelial cells on top of the pericytes are performed simultaneously for multiple porous membranes and membrane holders. Therefore, in addition to the effects of the first invention, it has the effect of being able to produce multiple BBB models in a short time.

[0025] According to the sixth invention, the first cylindrical body enhances the adhesion of the plate cover to the plate, thus, in addition to the effects of the fifth invention, it reliably prevents the cell culture medium injected into the through-holes from leaking out between the plate and the plate cover. Furthermore, according to the sixth invention, the second cylindrical body ensures accurate alignment of the extruder with respect to the plate, thus reducing the likelihood of malfunction of the plate and extruder.

[0026] According to the seventh invention, since multiple cell culture inserts can be inverted or transferred to microplate wells while remaining integrated, in addition to the effects of the first invention, it has the effect of being able to efficiently produce multiple BBB models in a short time.

[0027] In the eighth invention, there is no risk of leakage of the cell culture medium or seeded cells injected into the cell culture chamber during handling, so multiple pairs of cell culture chambers can be efficiently inverted simultaneously using a handling device or the like. This increases the mass production capability of the BBB model with a three-layer structure consisting of vascular endothelial cells, pericytes, and astrocytes.

[0028] According to the ninth invention, in addition to the effects of the eighth invention, it is not necessary to prepare a cell sheet of vascular endothelial cells in advance, and the process of creating the cell sheet can be omitted, thereby increasing the mass-producibility of the BBB model consisting of the three cell layers described above. [Brief explanation of the drawing]

[0029] [Figure 1] (a) and (b) are external perspective views and longitudinal section views of the cell culture insert, respectively; (c) is an external perspective view of the microplate; and (d) is a cross-sectional view taken along the line AA in figure (c). [Figure 2] This is a process diagram showing the procedure for a cell culture method according to the first embodiment of the present invention. [Figure 3] (a) and (b) are diagrams showing how cell culture medium is injected into the outer surface of the porous membrane of a cell culture insert, and (c) is a diagram showing how cells are seeded into a cell culture insert placed in the wells of a microplate. [Figure 4] (a) is a cross-sectional view taken along the line BB in Figure 3(c), and (b) is an enlarged view of the area enclosed by the dashed line in Figure 3(a). [Figure 5] (a) and (b) are perspective views of the insert holding plate and cell culture case, respectively. [Figure 6] (a) and (b) are perspective views of the insert retainer and the case lid, respectively. [Figure 7] This is a process diagram showing the procedure for a cell culture method according to a second embodiment of the present invention. [Figure 8] (a) is a perspective view showing the cell culture insert placed on the insert holding plate, and (b) is a cross-sectional view taken along the CC line in figure (a). [Figure 9] (a) is a perspective view showing the cell culture case placed above the insert holding plate in Figure 8(a), and (b) is a perspective view showing the cell culture case in Figure 8(a) with the case lid attached and inverted together with the insert holding plate. [Figure 10] (a) is a cross-sectional view taken along the DD line in Figure 9(b), and (b) is a perspective view showing a cell culture insert with an insert holding plate and insert retainer attached placed in a well of a microplate. [Figure 11] (a) is a side view of the plate cover and plate, (b) and (c) are side views of the spacer and extruder, respectively, and (d) through (f) are external perspective views of the porous membrane, membrane holder and insert body, respectively. [Figure 12] (a) and (c) are plan views of the plate and plate cover shown in Figure 11(a), respectively, and (b) and (d) are cross-sectional views taken along the EE line in Figure 11(a) and along the FF line in Figure 11(c), respectively. [Figure 13] (a) is a plan view of the spacer and extruder shown in Figures 11(b) and 11(c), and (b) and (c) are cross-sectional views of the spacer and extruder, respectively. [Figure 14] (a) and (c) are plan views of the insert body and film holder shown in Figure 11(f) and Figure 11(e), respectively, and (b) and (d) are cross-sectional views taken along the line HH in Figure (a) and along the line II in Figure (c), respectively. [Figure 15] This is a process diagram showing the procedure for a cell culture method according to a third embodiment of the present invention. [Figure 16] (a) is a cross-sectional view showing a state in which astrocyte cells are seeded inside a plate with the bottom surface covered by a plate cover and a membrane holder installed inside, and (b) is a cross-sectional view showing a state in which the top of the plate is covered by a plate cover. [Figure 17] (a) is a cross-sectional view showing the state after the plate cover that was installed on the bottom surface has been removed and pericyte cells have been seeded inside the plate, after the plate has been inverted vertically in Figure 16(b). (b) is a cross-sectional view showing the state after the plate cover that was installed on the bottom surface in Figure 16(a) has been removed and the extruder shown in Figure 11(c) has been installed. [Figure 18](a) is a cross-sectional view showing the state in which sheet-like vascular endothelial cells are laid on the outer surface of the porous membrane in Figure 17(b), (b) is a cross-sectional view showing the state in which the membrane holder is attached to the lower end of the insert body in Figure 17(a), and (c) is a cross-sectional view of the cell culture insert consisting of the membrane holder and the insert body. [Figure 19] (a) is a cross-sectional view showing a state in which astrocyte cells are seeded inside a plate in which the bottom surface is covered with the spacer shown in Figure 11(b) and a membrane retainer is installed inside, and (b) is a cross-sectional view showing the state in which the top and bottom of the plate are covered with spacers. [Figure 20] Figure 19(b) is a cross-sectional view showing the plate after it has been inverted vertically, with the spacer that was installed on the bottom surface removed and pericyte cells seeded inside the plate. [Figure 21] (a) and (b) are perspective views of the plate, and (c) and (d) are perspective views of the plate cover (spacer cover) and spacer, respectively. [Figure 22] (a) to (c) are perspective views of the external appearance of the extruder, film holder, and sleeve, respectively, and (d) and (e) are cross-sectional views taken along the line JJ in Figure (b) and along the line KK in Figure (c), respectively. [Figure 23] (a) and (b) are longitudinal cross-sectional views showing the cylindrical spacer body inserted into the cylindrical body of the plate and the extrusion part of the extruder inserted into the cylindrical body of the plate, respectively. [Figure 24] This is a process diagram showing the procedure for a cell culture method according to a fourth embodiment of the present invention. [Figure 25] (a) is a diagram showing the state in which the membrane holder is installed on the upper end of the cylindrical body of the spacer shown in Figure 21(d), (b) is a diagram showing the state in which the spacer cover and plate are attached to the spacer in Figure 21(a) and the top and bottom are reversed, and (c) is a longitudinal cross-sectional view of the cylindrical body of the spacer and plate and the membrane holder in Figure 21(b). [Figure 26](a) is a diagram showing the plate in Figure 25(b) with the plate cover attached and the plate inverted, and (b) is a diagram showing the same figure (a) with the spacer cover removed. [Figure 27] (a) is a longitudinal cross-sectional view of the cylindrical spacer and plate and the membrane holder in Figure 26(b), (b) shows the spacer cover reattached to the spacer in Figure 26(b), and (c) shows the plate cover and spacer cover removed in Figure 26(b) and the extruder attached to the plate. [Figure 28] (a) is a longitudinal cross-sectional view of the cylindrical plate, the extruder section, and the membrane holder in Figure 27(c); (b) is a diagram showing the state in which a cell sheet of vascular endothelial cells is laid on the upper surface of the membrane holder that has been extruded upward from the cylindrical plate in Figure 27(c); and (c) is a longitudinal cross-sectional view of the cylindrical plate, the extruder section, and the membrane holder in Figure 27(b). [Figure 29] (a) is a diagram showing how the cell sheet of vascular endothelial cells shown in Figure 28(b) is punched out by a sleeve, and (b) is a diagram showing the state in which a membrane retainer is attached to the sleeve from which the cell sheet of vascular endothelial cells was punched out in Figure 28(a). [Figure 30] (a) is a longitudinal cross-sectional view of a cell culture insert consisting of a sleeve and a membrane holder in Figure 29(b), and (b) is a diagram showing how the cell culture insert shown in Figure 29(a) is placed in a well of a microplate. [Figure 31] This figure shows a modified version of the process diagram shown in Figure 24. [Figure 32] (a) is a diagram showing the state in which the sleeve is attached to the membrane holder in Figure 27(c), and (b) is a longitudinal cross-sectional view of the cell culture insert consisting of the cylindrical plate, the extrusion part of the extruder, the sleeve and membrane holder in Figure 27(a). [Figure 33] (a) to (c) are perspective views of the cell culture plate, top cover (bottom cover), and cell culture insert, respectively. [Figure 34]This is a process diagram showing the procedure for a cell culture method according to a fifth embodiment of the present invention. [Figure 35] (a) is a diagram showing how the cell culture insert shown in Figure 33(c) is installed in the through-hole of the cell culture plate, and (b) and (c) are cross-sectional views taken along the LL line and MM line in Figure 33(a), respectively. [Figure 36] (a) is a diagram showing how cells are seeded into cell culture inserts placed in the through-holes of a cell culture plate, (b) is a cross-sectional view taken along the line NN in figure (a), and (c) is a magnified view of the area enclosed by the dashed line in figure (b). [Figure 37] (a) is a diagram showing how cells are seeded into cell culture inserts placed in the through-holes of a cell culture plate, with the state of Figure 36(a) inverted vertically; (b) is a cross-sectional view taken along the PP line in Figure 36(a); and (c) is an enlarged view of the area enclosed by the dashed line in Figure 36(b). [Figure 38] (a) and (c) are perspective views of the cell culture plate, and (b) is a cross-sectional view taken along the QQ line in figure (a). [Figure 39] (a) and (c) are perspective views of the first and second bottom covers, respectively, and (b) is a cross-sectional view taken along the RR line in Figure (a). [Figure 40] This is a process diagram showing the procedure for a cell culture method according to the sixth embodiment of the present invention. [Figure 41] (a) is a diagram showing a cell culture plate inverted with the first bottom cover attached, (b) is a cross-sectional view taken along the SS line in (a), and (c) is an enlarged view of the area enclosed by the dashed line in (b). [Figure 42] (a) is a diagram showing the cell culture plate in the same state as in Figure 41(a), with the second bottom cover attached to the first bottom cover and the top and bottom of the plate inverted; (b) is a cross-sectional view taken along the TT line in Figure 41(a); and (c) is an enlarged view of the area enclosed by the dashed line in Figure 41(b). [Modes for carrying out the invention]

[0030] The cell culture method and cell culture apparatus used therein of the present invention will be described with reference to Figures 1 to 42. All components of the cell culture apparatus of the present invention are made of plastic. Furthermore, components that have already been described will be denoted by the same reference numerals, and their descriptions will be omitted as appropriate. In addition, although the microplate in the description of the embodiment has a structure with 12 wells, the number of wells is not limited to this and can be changed as appropriate. For example, even if the microplate has 384 wells, the operation and effects of the present invention described below will be similarly exhibited. Furthermore, although a temperature-sensitive gel that dissolves at 37°C is used as an example in the following description, the dissolution temperature is not limited to 37°C, as any temperature-sensitive gel that dissolves at a temperature at which cell proliferation of vascular endothelial cells stops is acceptable. [Examples]

[0031] A cell culture apparatus according to the first embodiment of the present invention will be described with reference to Figure 1. Figures 1(a) and 1(b) are external perspective views and longitudinal cross-section views of a cell culture insert, respectively, Figure 1(c) is an external perspective view of a microplate, and Figure 1(d) is a cross-sectional view taken along the line AA in Figure 1(c). To avoid cluttering the figures, only one well is labeled in Figures 1(c) and 1(d). As shown in Figures 1(a) to 1(d), the cell culture apparatus of this embodiment is installed in the wells 2b of a microplate 2 having circular bottomed holes when performing biochemical analysis or clinical tests, and is equipped with a cell culture insert 1 whose side 1a is trumpet-shaped. The cell culture insert 1 has a pair of flanges 1d, 1d facing each other on the outer surface of the end on the side of the large-diameter opening 1b, and a porous polycarbonate membrane 3, formed on both sides to allow the attachment of biomaterials, is provided on the inner surface of the bottom so as to close the small-diameter opening 1c. The microplate 2 is rectangular in shape, and 12 wells 2b (3 rows vertically x 4 rows horizontally) are provided on the upper surface 2a. The porous membrane 3 has pores with a diameter of approximately 1 to 3 μm, allowing the cell culture medium described later to pass through, and is fixed to the cell culture insert 1 by heat fusion. Furthermore, the inner diameter s1 of well 2b (see Figure 1(d)) is longer than the outer diameter s2 of side surface 1a where the pair of flanges 1d, 1d are provided (see Figure 1(b)), and shorter than the distance s3 between the outermost surfaces of the pair of flanges 1d, 1d (see Figure 1(b)). In addition, the depth h1 of well 2b (see Figure 1(d)) is longer than the distance h2 along the central axis X from the end face on the small diameter opening 1c side of side surface 1a to where the pair of flanges 1d, 1d are provided (see Figure 1(b)). In other words, the microplate 2 is structured so that, with the pair of flanges 1d, 1d locked to the edge of well 2b, the outer surface of the porous membrane 3 can be placed inside well 2b without contacting the bottom surface 2c of well 2b (see Figure 1(d)) (see Figure 4(a)). Furthermore, the cell culture insert 1 equipped with a fourth inner wall 1e, which is used as the inner wall of the cell culture chamber, is used as a cell culture chamber as described later.

[0032] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 2 to 4. Figure 2 is a process diagram showing the procedure of a cell culture method according to the first embodiment of the present invention. Figures 3(a) and 3(b) show how cell culture medium is injected into the outer surface of the porous membrane of a cell culture insert, and Figure 3(c) shows how cells are seeded into a cell culture insert placed in the wells of a microplate. Furthermore, Figure 4(a) is a cross-sectional view taken along the line BB in Figure 3(c), and Figure 4(b) is an enlarged view of the area enclosed by the dashed line in Figure 4(a). To avoid making the diagrams cluttered, Figures 3(c) and 4(a) only label one well, and Figure 3(c) shows the state in which cell culture inserts are placed in only 4 of the 12 wells provided in the microplate.

[0033] As shown in Figure 3(a), first, the cell culture insert 1 is placed upside down (with the bottom facing upwards), and the suspended cell culture medium 5 is injected onto the outer surface of the porous membrane 3 using a pipette 4 (step S1 in Figure 2). Then, as shown in Figure 3(b), astrocytes are seeded into the cell culture medium 5, which has risen in a roughly hemispherical shape on the outer surface of the porous membrane 3 without permeating through it due to surface tension (step S2 in Figure 2). After that, the cell culture medium 5 is cultured for several days while maintaining its temperature at, for example, 33°C, until the astrocytes form layers (step S3 in Figure 2). As shown in Figure 3(c) and Figure 4(a), the cell culture insert 1 is inverted and placed in well 2b of a microplate 2 filled with cell culture medium 5 (Step S4 in Figure 2). Then, using a pipette 4, pericyte cells are seeded into the cell culture insert 1 (Step S5 in Figure 2), and the pericytes are cultured for several days while maintaining the temperature inside the cell culture insert 1 at, for example, 33°C until they form layers (Step S6 in Figure 2). After the pericytes have grown in layers, the cell culture medium 5 is discarded. In this way, the astrocytes that have already formed in layers on the outer surface of the porous membrane 3 have legs extending from the cells through the pores of the porous membrane 3 to the vicinity of the perisites formed on the inner surface of the porous membrane 3. Next, a temperature-sensitive gel 6 (e.g., gelatin LS-250 manufactured by Nitta Gelatin Co., Ltd.) that dissolves at a temperature above which vascular endothelial cell proliferation stops (e.g., 37°C) is applied to the pericyte cell layer (step S7 in Figure 2). Furthermore, the vascular endothelial cells are embedded in this temperature-sensitive gel 6 (step S8 in Figure 2), and the vascular endothelial cells are cultured for several days while maintaining the temperature inside the cell culture insert 1 at, for example, 33°C (step S9 in Figure 2). As a result, as shown in Figure 4(b), three cell layers consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 embedded in the temperature-sensitive gel 6 are formed at the bottom of the cell culture insert 1. Pericytes 8 have the function of controlling the differentiation and proliferation of vascular endothelial cells 7, but in this state, the vascular endothelial cells 7 embedded in the temperature-sensitive gel 6 are not in contact with the pericytes 8, so this function is not exercised. Therefore, when the internal temperature of the cell culture insert 1 is raised to 37°C, the cell proliferation of vascular endothelial cells 7 stops and the temperature-sensitive gel 6 dissolves (step S10 in Figure 2). This brings the vascular endothelial cells 7 into contact with the pericytes 8. By maintaining the internal temperature of the cell culture insert 1 at 33°C in this state and restarting the culture of vascular endothelial cells 7, a three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 is completed.

[0034] Thus, in the cell culture apparatus of this embodiment, the injection of cell culture medium 5 onto the outer surface of the porous membrane 3 of the cell culture insert 1, the seeding of astrocytes 9, or the transfer of the cell culture insert 1 with the BBB model at the bottom to the well 2b of the microplate 2 are simple operations, and a simple handling device can be used for each operation. Furthermore, by using such a handling device, it becomes possible to mass-produce a three-layer BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9. Furthermore, the cell culture method of this embodiment includes a step of seeding vascular endothelial cells 7 embedded in a temperature-sensitive gel 6 onto the cell layer of pericytes 8. In this case, since the vascular endothelial cells 7 do not come into contact with the pericytes 8 until the temperature-sensitive gel 6 dissolves, the function of pericytes 8 in controlling the differentiation and proliferation of vascular endothelial cells 7 is not exercised. In other words, the cell culture method of this embodiment maintains the temperature inside the cell culture insert 1 at a temperature that does not cause the temperature-sensitive gel 6 to dissolve, thereby maintaining the layered state of vascular endothelial cells 7, pericytes 8, and astrocytes 9 formed at the bottom of the cell culture insert 1 by steps S1 to S9 shown in Figure 2 for a long period of time. In addition, when using the temperature-sensitive gel 6, it is not necessary to prepare a cell sheet of vascular endothelial cells in advance, so the step of producing the cell sheet can be omitted. Therefore, according to the cell culture method described above, it is possible to increase the mass production capacity of the BBB model consisting of the three cell layers described above. Furthermore, the cell culture insert 1 equipped with a fourth inner wall 1e, which is used as the inner wall of the cell culture chamber, is used as a cell culture chamber as described later. [Examples]

[0035] A cell culture apparatus according to a second embodiment of the present invention will be described with reference to Figures 5 and 6. Figures 5(a) and 5(b) are external perspective views of the insert holding plate and cell culture case, respectively, and Figures 6(a) and 6(b) are external perspective views of the insert retainer and case lid, respectively. To avoid making the diagrams too complex, only some of the square and round holes are labeled with reference numerals in Figures 5(a) and 5(b).

[0036] The cell culture apparatus of this embodiment comprises an insert holding plate 10 (see Figure 5(a)) and a cell culture case 11 (see Figure 5(b)) that form a rectangular shape of the same size when viewed from above, an insert holder 12 (see Figure 6(a)), and a case lid 13 (see Figure 6(b)). As shown in Figure 5(a), the insert holding plate 10 has a pair of parallel side plates 10a, 10a, each with five rectangular holes 10c, and twelve circular holes 10d (3 rows vertically x 4 rows horizontally) in the bottom plate 10b. The cell culture case 11, as shown in Figure 5(b), has an open top and twelve circular through-holes 11b (3 rows vertically x 4 rows horizontally) in the bottom plate 11a. Furthermore, in the bottom plate 10b of the insert holding plate 10, the three vertically aligned circular holes 10d are formed so that their central axes are located on the same plane perpendicular to the side plate 10a and the bottom plate 10b, while the four horizontally aligned circular holes 10d are formed so that their central axes are located on the same plane parallel to the side plate 10a. In addition, the distance s5 between the centerlines of two circular holes 10d, 10d (see Figure 5(a)) is longer than the distance s3 between the outermost surfaces of a pair of flanges 1d, 1d in the cell culture insert 1 (see Figure 1(b)). In other words, when multiple cell culture inserts 1 are placed on the bottom plate 10b of the insert holding plate 10 such that their respective central axes X (see Figure 1(b)) coincide with the central axes of the circular holes 10d, adjacent cell culture inserts 1 do not come into contact with each other. Furthermore, when the cell culture case 11 is positioned above the insert holding plate 10, parallel to the insert holding plate 10, and in a state where the two completely overlap when viewed from above, the central axes of the 12 through holes 11b coincide with the central axes of the 12 circular holes 10d of the insert holding plate 10. The insert retainer 12 consists of five square bars 12a arranged parallel to each other and simultaneously insertable into five square holes 10c, as shown in Figure 6(a), and a connecting plate 12b that connects one end of these five square bars 12a. The square bars 12a are formed to be longer than the distance between the outer surfaces of the pair of side plates 10a, 10a of the insert holding plate 10. Furthermore, the case lid 13, which has a rectangular shape in plan view and is used as a cell culture room cover, consists of a rectangular recess 13a and a frame-shaped projection 13b provided around the recess 13a, as shown in Figure 6(b). When the recess 13a is installed on top of the cell culture case 11, a pair of side plates 10a, 10a can be placed inside the projection 13b. Furthermore, the cell culture case 11, which is equipped with through-holes 11b used as the inner wall of the cell culture chamber, is used as a cell culture chamber, as will be described later.

[0037] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 7 to 10. Figure 7 is a process diagram showing the procedure of a cell culture method according to a second embodiment of the present invention; Figure 8(a) is a perspective view showing the state in which the cell culture insert is fixed to the insert holding plate by an insert holder; and Figure 8(b) is a cross-sectional view taken along the line CC in Figure 8(a). Figure 9(a) shows the state in which the cell culture case is placed above the insert holding plate in Figure 8(a); and Figure 9(b) shows the state in which the cell culture case with the case lid attached is inverted together with the insert holding plate in Figure 9(a). Figure 10(a) is a cross-sectional view taken along the line DD in Figure 9(b); and Figure 10(b) is a perspective view showing the state in which the cell culture insert with the insert holding plate and insert holder attached is placed in the wells of a microplate. To avoid making the diagrams cluttered, in Figures 9(b) and 10(a), only some of the rectangular and circular holes are labeled with reference numerals, and in Figures 8(a), 9(a), and 9(b), only one cell culture insert is labeled with a reference numeral. Also, in Figure 10(a), the cell culture case and its lid are not shown.

[0038] As shown in Figure 8(a), the 12 cell culture inserts 1 are placed face down on the bottom plate 10b of the insert holding plate 10 so that each central axis X (see Figure 1(b)) aligns with the central axis of the circular hole 10d (step S1 in Figure 7). Next, the five square bars 12a of the insert holder 12 are simultaneously connected to all of the square holes 10c, 10c provided in the pair of side plates 10a, 10a of the insert holding plate 10. Furthermore, the distance s4 between the two rectangular holes 10c, 10c of the insert holding plate 10 (see Figure 5(a)) is longer than the outer diameter s2 of the side surface 1a where the pair of flanges 1d, 1d are provided in the cell culture insert 1 (see Figure 1(b)), and shorter than the distance s3 between the outermost surfaces of the pair of flanges 1d, 1d (see Figure 1(b)). Also, the height h3 of the rectangular holes 10c from the bottom plate 10b (see Figure 5(a)) is slightly longer than the thickness h4 of the flange 1d in the cell culture insert 1 (see Figure 1(b)). The rectangular holes 10c are formed at opposing positions on the two side plates 10a, 10a, respectively, so that the rectangular bars 12a of the insert holder 12 (see Figure 6(a)) can be connected. Therefore, as shown in Figure 8(a), the 12 cell culture inserts 1 placed on the bottom plate 10b of the insert holding plate 10 are arranged in groups of three between the two square bars 12a, 12a of the insert holder 12, and a pair of flanges 1d, 1d are positioned between the two square bars 12a, 12a and the bottom plate 10b of the insert holding plate 10, respectively (see Figure 8(b)). As a result, the cell culture inserts 1 become unable to detach from the insert holding plate 10. In this way, the insert holder 12 connects the square bar 12a to a pair of square holes 10c, 10c formed at opposing positions in the pair of side plates 10a, 10a of the insert holding plate 10, thereby fixing the cell culture insert 1 to the insert holding plate 10 (step S2 in Figure 7).

[0039] Next, as shown in Figure 9(a), the cell culture case 11 is placed above the insert holding plate 10 in the state shown in Figure 8(a), parallel to it, and so that they completely overlap when viewed from above. The bottom of the cell culture insert 1 is made to protrude from the through-hole 11b, so that the outer surface of the porous membrane 3 is exposed to the inside of the cell culture case 11 (step S3 in Figure 7). Then, a suspended cell culture medium (not shown) is injected into the cell culture case 11 and astrocytes 9 are seeded (steps S4 and S5 in Figure 7). The astrocytes 9 are then cultured over several days while maintaining the temperature of the cell culture medium at, for example, 33°C, until they form layers (step S6 in Figure 7). After attaching the case lid 13 to the top surface of the cell culture case 11, the cell culture case 11 is inverted along with the insert holding plate 10 and insert retainer 12, taking care not to spill the cell culture medium and astrocytes 9, as shown in Figure 9(b) (Step S7 in Figure 7). Then, using a pipette 4, perisites 8 are seeded into the cell culture insert 1 through the round holes 10d of the insert holding plate 10 (Step S8 in Figure 7), and the perisites 8 are cultured for several days while maintaining the temperature inside the cell culture insert 1 at, for example, 33°C, until they form layers (Step S9 in Figure 7). The cell culture medium is then discarded when the perisites have grown in layers. As a result, the astrocytes 9 that have already formed in layers on the outer surface of the porous membrane 3 will have their legs extending through the small pores of the porous membrane 3 to the vicinity of the perisites 8 formed on the inner surface of the porous membrane 3. Thus, when the bottom of the cell culture insert 1 protrudes from the through-hole 11b, the inside of the cell culture case 11 can be used as a cell culture chamber in the same way as the cell culture insert 1, and the inside of the cell culture insert 1 and the inside of the cell culture case 11 are separated by the porous membrane 3. In other words, when multiple cell culture inserts 1 are placed in the cell culture case 11 so that their bottoms protrude from the through-hole 11b, the inside of the cell culture case 11 is separated from each of the multiple cell culture inserts 1 by the porous membrane 3. Therefore, the cell culture apparatus of this embodiment can be said to have a structure in which the fourth inner wall 1e of the cylindrical cell culture insert 1 (see Figure 1(b)) and the inner wall of the through-hole 11b provided in the bottom plate 11a of the cell culture case 11 constitute a pair of cell culture chambers divided vertically by the porous membrane 3, and multiple of these cell culture chambers are connected and each of the vertically divided parts is integrated. Next, a temperature-sensitive gel 6 that dissolves at a temperature above which vascular endothelial cell proliferation stops (e.g., 37°C) is spread inside the cell culture insert 1 on top of the pericyte cell layer (step S10 in Figure 7), and then the vascular endothelial cells 7 are embedded in this temperature-sensitive gel 6 (step S11 in Figure 7). Subsequently, the vascular endothelial cells 7 are cultured for several days while maintaining the temperature inside the cell culture insert 1 at, for example, 33°C (step S12 in Figure 7). In this way, as shown in Figure 10(a), three types of cell layers consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 embedded in the temperature-sensitive gel 6 are formed at the bottom of the cell culture insert 1. Then, the 12 cell culture inserts 1, which are fixed to the insert holding plate 10 by the insert holder 12, are removed from the through-hole 11b of the cell culture case 11 and moved together with the insert holding plate 10 and the insert holder 12 to the microplate 2 (step S13 in Figure 7).

[0040] With the bottoms of all cell culture inserts 1 positioned inside the wells 2b of the microplate 2, when all the square bars 12a of the insert holder 12 are simultaneously removed from the square holes 10c of the insert holding plate 10, the constraint on the cell culture inserts 1 from the insert holding plate 10 is released, and all cell culture inserts 1 are simultaneously positioned inside the wells 2b of the microplate 2. Then, when the insert holding plate 10 and the insert holder 12 are removed from the cell culture inserts 1, the process of placing the cell culture inserts 1 into the wells 2b of the microplate 2 is completed, resulting in the state shown in Figure 4(a) above (step S14 in Figure 7). Furthermore, when the internal temperature of the cell culture inserts 1 is raised to 37°C, cell proliferation of vascular endothelial cells 7 stops, the temperature-sensitive gel 6 dissolves (step S15 in Figure 7), and the vascular endothelial cells 7 come into contact with the pericytes 8. By maintaining the internal temperature of cell culture insert 1 at 33°C in this state and restarting the culture of vascular endothelial cells 7, a three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 is completed.

[0041] Thus, in the cell culture apparatus of this embodiment, the insert holding plate 10 and the insert holder 12 function as insert holding means that hold multiple cell culture inserts 1 in a state that allows them to be simultaneously inserted into the through-holes 11b of the cell culture case 11. As a result, multiple cell culture inserts 1 can be inverted as a single unit or transferred to the wells 2b of the microplate 2. Therefore, with the cell culture apparatus of this embodiment, it is possible to efficiently produce multiple BBB models in a short time. Furthermore, the cell culture method of this embodiment includes a step of applying a temperature-sensitive gel 6 onto the cell layer of pericytes 8. In this case, since it is not necessary to prepare a cell sheet of vascular endothelial cells 7 in advance, the step of preparing the cell sheet can be omitted. The through-holes provided in the bottom plate 11a of the cell culture case 11 are not limited to through-holes 11b, but may be rectangular holes or other shapes. However, in order to prevent leakage of the cell culture medium 5 injected into the cell culture case 11, the cell culture insert 1 must have a structure that fits into these through-holes. [Examples]

[0042] A cell culture apparatus according to a third embodiment of the present invention will be described with reference to Figures 11 to 24. Figure 11(a) is a side view of the plate cover and plate, Figures 11(b) and 11(c) are side views of the spacer and extruder, respectively, and Figures 11(d) to 11(f) are external perspective views of the porous membrane, membrane holder and insert body, respectively. Figures 12(a) and 12(c) are plan views of the plate and plate cover shown in Figure 11(a), respectively, and Figures 12(b) and 12(d) are cross-sectional views taken along the line EE in Figure 12(a) and along the line FF in Figure 12(c), respectively. Figure 13(a) is a plan view of the spacer and extruder shown in Figures 11(b) and 11(c), respectively. Figures 13(b) and 13(c) are cross-sectional views of the spacer and extruder, respectively, both corresponding to the cross-sectional view taken along the GG line in Figure 13(a). Figures 14(a) and 14(c) are plan views of the insert body and film holder shown in Figures 11(f) and 11(e), respectively. Figures 14(b) and 14(d) are cross-sectional views taken along the HH line in Figure 14(a) and along the II line in Figure 14(c), respectively. Note that in Figures 13(a) to 13(c), the spacer and extruder are shown in a more magnified view than the plate and plate cover shown in Figures 12(a) to 12(d). Also, since the spacer and extruder have the same shape when viewed from above, their reference numerals are shown together in Figure 13(a).

[0043] As shown in Figures 11(a) to 11(f), the cell culture apparatus of this embodiment comprises a plate 14 having a flat base 14a with a first annular projection 14b and a second annular projection 14c having the same outer diameter on both sides, a plate cover 15 having a flat base 15a with a holding portion 15b on one side, and a spacer 16 and an extruder 17 having a base 16a and an extruder plate 17a made of flat material with a cylindrical body 16c and a cylindrical extruder 17c formed to be sized to fit into the holding portions 16b, 17b and the first annular projection 14b and the second annular projection 14c on one side. Furthermore, this cell culture apparatus comprises a membrane holder 18 that is short cylindrical in shape and sized to fit into the second annular projection 14c, a porous polycarbonate membrane 3 that is attached to the membrane holder 18 so as to close one end and is formed so as to be able to fix biomaterials on both sides, and an insert body 19 that is cylindrical in shape, with the membrane holder 18 attached to one end and a pair of flanges 19a, 19a provided at the other end, and together with the membrane holder 18 it constitutes a cell culture insert. The plate cover 15, spacer 16, and extruder 17 are formed to a size that can close the openings of the first annular projection 14b and the second annular projection 14c of the plate 14. The porous membrane 3 is fixed to the membrane holder 18 by heat fusion.

[0044] As shown in Figures 12(a) and 12(b), the base 14a of the plate 14 has a plurality of through-holes 14d which are used as the inner wall of the cell culture chamber. On both sides of the base 14a, a first annular projection 14b and a second annular projection 14c are provided symmetrically on either side of the base 14a, with the same inner diameter as the through-holes 14d, and are in communication with each other via the through-holes 14d. As shown in Figures 12(c) and 12(d), the retaining portion 15b provided on one side of the plate cover 15 is composed of a plurality of short cylindrical bodies, each formed to a size that allows the open ends of the first annular projection 14b and the second annular projection 14c of the plate 14 to be fitted. In other words, the plate cover 15 used as a cell culture room cover is structured to be detachable from the top and bottom of the plate 14 by fitting the open ends of the first annular projection 14b and the second annular projection 14c into the retaining portion 15b (see Figure 16(b)). As shown in Figures 13(a) to 13(c), the retaining portions 16b and 17b provided on one side of the spacer 16 and the extruder 17 are composed of multiple short cylindrical bodies that simultaneously fit onto the open ends of the multiple first annular projections 14b provided on the plate 14. In other words, the spacer 16 and the extruder 17 are detachably attached to the top and bottom of the plate 14 by fitting the open ends of the first annular projections 14b onto the retaining portions 16b and 17b, respectively (see Figures 17(b) and 20).

[0045] The base 16a of the spacer 16 and the extrusion plate 17a of the extruder 17 have multiple cylindrical bodies 16c and extrusion parts 17c on the side where the holding parts 16b and 17b are provided. These are arranged so that their cylindrical axes coincide with the cylindrical bodies that make up the holding parts 16b and 17b, and when the base 16a and the extrusion plate 17a are arranged parallel to the base 14a of the plate 14, the cylindrical bodies 16c and extrusion parts 17c are located at positions that correspond to the first annular projection 14b and the second annular projection 14c when viewed in a direction perpendicular to either the base 16a or the extrusion plate 17a and both the base 14a. In other words, the spacer 16 and the extrusion plate 17a are formed such that when either the base 16a or the extrusion plate 17a is arranged parallel to the base 14a of the plate 14, the cylindrical axes of all the cylindrical bodies 16c and extrusion parts 17c coincide with the cylindrical axes of all the first annular projections 14b and the second annular projections 14c. Furthermore, the cylindrical body 16c and the extruded portion 17c have an outer diameter smaller than the inner diameter of the first annular projection 14b, and are formed to be fitted into the first annular projection 14b, the second annular projection 14c, and the through hole 14d. In other words, the spacer 16 and the extruder 17 are structured so that all the cylindrical bodies 16c and extruded portions 17c can be simultaneously fitted into all the first annular projections 14b, the second annular projections 14c, and the through hole 14d, respectively, while the base portion 16a and the extruded plate 17a are parallel to the base portion 14a, and the cylindrical body 16c and the extruded portion 17c can move inside the through hole 14d while maintaining the parallel state of the base portion 16a and the extruded plate 17a to the base portion 14a. The sum of twice the length of the cylindrical body 16c of the spacer 16 and the length of the membrane holder 18 is equal to the sum of the lengths of the first annular projection 14b, the second annular projection 14c, and the through hole 14d. In other words, the spacer 16 is constructed such that when the cylindrical body 16c of the spacer 16 is inserted into the through hole 14d of the plate 14 and the base 16a is brought into contact with the end face of the second annular projection 14c, and the cylindrical body 16c of another spacer 16 is inserted into the through hole 14d of the plate 14 and the base 16a is brought into contact with the end face of the first annular projection 14b, the membrane holder 18 is sandwiched from above and below by the cylindrical bodies 16c of the two spacers 16, as will be described later (see Figure 19(b)). Furthermore, the combined length of the extrusion portion 17c and the membrane holder 18 of the extruder 17 is longer than the combined length of the first annular projection 14b, the second annular projection 14c, and the through hole 14d. In other words, the extruder 17 is structured such that when the extrusion portion 17c is inserted into the through hole 14d of the plate 14 and the extrusion plate 17a is brought into contact with the end face of the second annular projection 14c, the porous membrane 3 attached to the membrane holder 18 installed on the tip surface 17d of the extrusion portion 17c is pushed out to the outside of the first annular projection 14b together with the membrane holder 18, as will be described later (see Figure 17(b)).

[0046] The inner and outer diameters of the insert body 19 are equal to the inner and outer diameters of the membrane holder 18, respectively, but the outer diameter of the membrane holder 18 is smaller than the inner diameter of the through hole 14d of the plate 14. Furthermore, as shown in Figure 14(a), the ends of the insert body 19 on the side without flanges 19a are provided with sharp portions 19b, and the end of the membrane holder 18 on the side to which the porous membrane 3 is attached is provided with an annular recess (engagement portion 18a) that can engage with the sharp portions 19b of the insert body 19. Furthermore, the inner diameter of the cylindrical body 16c of the spacer 16 and the extrusion portion 17c of the extruder 17 is equal to the inner diameter of the membrane holder 18. Therefore, when the porous membrane 3 is installed inside the through-hole 14d of the plate 14 so as to be parallel to the base 14a of the plate 14, as will be described later using Figure 17(a), and the cylindrical body 16c of the spacer 16 or the extrusion portion 17c of the extruder 17 is inserted into the through-hole 14d of the plate 14, the tip surfaces 16d, 17d of the cylindrical body 16c or the extrusion portion 17c (see Figures 13(b) and 13(c)) do not come into contact with the porous membrane 3 (see Figures 17(b) and 20). In other words, the cylindrical body 16c of the spacer 16 and the extrusion portion 17c of the extruder 17 are structured to allow the membrane holder 18 to be pushed and moved without affecting the cell layer formed on the outer surface of the porous membrane 3, by bringing their tip surfaces 16d and 17d into contact with the end face on the side of the membrane holder 18 to which the porous membrane 3 is attached. Furthermore, the membrane holder 18, which is equipped with a third inner wall 18b (see Figure 14(d)) used as the inner wall of the cell culture chamber, is used as a cell culture chamber together with a plate 14 having multiple through-holes 14d in its base 14a, which is also used as the inner wall of the cell culture chamber, as will be described later.

[0047] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 15 to 20. Figure 15 is a process diagram showing the procedure of a cell culture method according to a third embodiment of the present invention. Figure 16(a) is a cross-sectional view showing a state in which astrocyte cells are seeded inside a plate, with the bottom surface covered by a plate cover and a membrane holder installed inside. Figure 16(b) is a cross-sectional view showing a state in which the top of the plate is covered by a plate cover. Figure 17(a) is a cross-sectional view showing a state in which, after the top and bottom of Figure 16(b) have been inverted, the plate cover that was installed on the bottom surface has been removed and pericyte cells have been seeded inside the plate. Figure 17(b) is a cross-sectional view showing a state in which the plate cover installed on the bottom surface in Figure 17(a) has been removed and the extruder shown in Figure 11(c) has been installed. Figure 18(a) is a cross-sectional view showing the state in Figure 17(b) where a sheet of vascular endothelial cells is laid on the outer surface of the porous membrane, Figure 18(b) is a cross-sectional view showing the state in Figure 18(a) where the membrane holder is attached to the lower end of the insert body, and Figure 18(c) is a cross-sectional view of a cell culture insert consisting of the membrane holder and the insert body. Furthermore, Figure 19(a) is a cross-sectional view showing a state in which astrocyte cells are seeded inside a plate with the bottom surface covered by the spacer shown in Figure 11(b) and the membrane holder installed inside, and Figure 19(b) is a cross-sectional view showing the state in which the top and bottom of the plate are covered by spacers.In addition, Figure 20 is a cross-sectional view showing the state in which the top and bottom of Figure 19(b) have been inverted, the spacer that was installed on the bottom surface has been removed, and pericyte cells have been seeded inside the plate.

[0048] First, a plate cover 15 is attached to the lower surface of a plate 14 which has been set up so that its base 14a is horizontal and the first annular projection 14b is open upward. Then, a membrane holder 18 is placed inside the through-hole 14d with the outer surface of the porous membrane 3 facing downward (step S1 in Figure 15). Next, as shown in Figure 16(a), a cell culture medium (not shown) is injected into the through-hole 14d of the plate 14, and astrocytes 9 are seeded (steps S2 and S3 in Figure 15). In this state, the temperature inside the through-hole 14d is maintained at, for example, 33°C, and the astrocytes 9 are cultured for several days until they form layers (step S4 in Figure 15). As shown in Figure 16(b), after attaching the plate cover 15 to the top surface of the plate 14, the plate 14 is inverted, taking care not to spill the cell culture medium and astrocytes 9 (step S5 in Figure 15). As shown in Figure 17(a), after removing the plate cover 15 that was attached to the side of the second annular projection 14c, perisites 8 are seeded inside the through-holes 14d of the plate 14 (step S6 in Figure 15). Then, while maintaining the temperature inside the through-holes 14d at, for example, 33°C, the perisites 8 are cultured for several days until they form layers, and the cell culture medium is discarded when the perisites 8 have grown in layers (step S7 in Figure 15). As a result, the astrocytes 9 that have already formed in layers on the inner surface of the porous membrane 3 have legs extending from the cells through the small pores of the porous membrane 3 to the vicinity of the perisites 8 formed on the outer surface of the porous membrane 3. Thus, the interior of the through-hole 14d of the plate 14 is used as a cell culture chamber, and when the membrane holder 18 is installed inside, the cell culture chamber is divided into two by the porous membrane 3. Therefore, the cell culture apparatus of this embodiment can be said to have a structure in which the third inner wall 18b of the cylindrical membrane holder 18 (see Figure 14(d)) and the inner wall of the through-hole 14d provided in the base 14a of the plate 14 constitute a pair of cell culture chambers divided vertically by the porous membrane 3, and multiple such cell culture chambers are connected and each of the vertically divided sections is integrated.

[0049] In Figure 17(a), the plate cover 15 is removed from the first annular projection 14b of the plate 14, and as shown in Figure 17(b), the extrusion part 17c of the extruder 17 is inserted from below into the through hole 14d of the plate 14, and the membrane holder 18 is pushed out toward the second annular projection 14c by this extrusion part 17c, as shown in Figure 17(b) (step S8 in Figure 15). As shown in Figure 18(a), a cell sheet 20 of vascular endothelial cells 7 is laid on top of the pericytes 8 exposed to the outside of plate 14 in Figure 17(b) (step S9 in Figure 15). Furthermore, the cell sheet 20 of vascular endothelial cells 7 is punched out from above with the insert body 19, and the insert body 19 is then pushed into the membrane holder 18 to attach the membrane holder 18 to the tip of the insert body 19 (see Figure 18(b)). As a result, three types of cell layers consisting of the cell sheet 20 of vascular endothelial cells 7, pericytes 8, and astrocytes 9 are arranged inside the insert body 19 (step S10 in Figure 15). In this state, the temperature inside the insert body 19 is maintained at, for example, 33°C, and the vascular endothelial cells 7 are cultured for several days (step S11 in Figure 15). Then, once the culture of vascular endothelial cells 7 is complete, the cell culture insert 21, which consists of the insert body 19 and the membrane holder 18, is removed from the plate 14 as shown in Figure 18(c) (step S12 in Figure 5). Furthermore, when inverting the plate 14 in step S5, a spacer 16 may be used instead of the plate cover 15 to prevent the membrane holder 18 from moving vertically inside the through hole 14d. Specifically, in step S1, instead of the plate cover 15, a spacer 16 is attached to the lower surface of the plate 14 as shown in Figure 19(a), and in step S5, instead of the plate cover 15, a spacer 16 is attached to the upper surface of the through-hole 14d as shown in Figure 19(b) to invert the plate 14. Furthermore, after removing the spacer 16 that was attached to the side of the second annular projection 14c as shown in Figure 20, perisites 8 are seeded inside the through-hole 14d of the plate 14 in step S6. In this case, when the plate 14 is inverted, the membrane holder 18 is held between the extruded portions 17c of the two spacers 16 and does not move vertically inside the through-hole 14d, which has the advantage that the cell culture medium and astrocytes 9 inside the membrane holder 18 are less likely to spill.

[0050] Thus, with the cell culture apparatus of this embodiment, the operations necessary for culturing astrocytes 9 and pericytes 8 on both sides of the porous membrane 3, the operation of inverting the porous membrane 3, the operation of removing the membrane holder 18 from inside the through-hole 14d of the plate 14, and the operation of placing vascular endothelial cells 7 on top of the pericytes 8 are performed simultaneously for multiple porous membranes 3 and membrane holders 18, making it possible to create multiple BBB models in a short time. Furthermore, in the cell culture apparatus of this embodiment, by fitting the holding portion 15b of the plate cover 15 onto the first annular projection 14b or the second annular projection 14c of the plate 14, the adhesion of the plate cover 15 to the plate 14 is increased, and by fitting the holding portion 17b of the extruder 17 onto the first annular projection 14b or the second annular projection 14c of the plate 14, the positioning of the extruder 17 to the plate 14 is accurately achieved. Therefore, according to the cell culture apparatus of this embodiment, the holding part 15b increases the adhesion of the plate cover 15 to the plate 14, thereby reliably preventing the cell culture medium injected into the through-hole 14d from leaking out between the plate 14 and the plate cover 15. In addition, the holding part 17b ensures accurate alignment of the extruder 17 with respect to the plate 14, making the plate 14 and the extruder 17 less prone to malfunction. The cell culture apparatus of the present invention is not limited to the structure shown in this embodiment. For example, a watertight film made of paraffin, polyethylene, or the like may be installed between the holding portion 15b of the plate cover 15 and the first annular projection 14b or second annular projection 14c of the plate 14, or between the holding portion 16b of the spacer 16 or the holding portion 17b of the extruder 17 and the first annular projection 14b or second annular projection 14c of the plate 14. In this case, the watertightness between the plate 14 and the plate cover 15, or between the plate 14 and the spacer 16 or extruder 17 is improved, which has the advantage that the cell culture medium 5 injected into the through-hole 14d is less likely to leak out from between the plate cover 15 and the extruder 17. [Examples]

[0051] A cell culture apparatus according to a fourth embodiment of the present invention will be described with reference to Figures 21 to 23. Figures 21(a) and 21(b) are external perspective views of the plate, and Figures 21(c) and 21(d) are external perspective views of the plate cover (spacer cover) and spacer, respectively. Figures 22(a) to 22(c) are external perspective views of the extruder, membrane holder, and sleeve, respectively, and Figures 22(d) and 22(e) are cross-sectional views taken along the line JJ in Figure 22(b) and along the line KK in Figure 22(c), respectively. Figures 23(a) and 23(b) are longitudinal cross-sectional views showing the cylindrical body of the spacer inserted into the cylindrical body of the plate and the extruder part of the extruder inserted into the cylindrical body of the plate, respectively. Note that the plate cover and the spacer cover differ only in the outer diameter of the circular projection, and their external shapes are substantially identical; therefore, both are indicated by their respective reference numerals in Figure 21(c). Also, to avoid making the diagrams cluttered, in Figures 21(a) to 21(d) and Figure 22(a), only one cylindrical body, a circular projection, and a through hole are indicated by reference numerals. Furthermore, Figure 23(a) shows the cylindrical body of the plate and a portion of the periphery around its base end, the circular projections of the plate cover and spacer cover and a portion of the periphery around their base ends, the cylindrical body of the spacer and a portion of the periphery around its base end, and the membrane holder installed on the cylindrical body of the spacer, all cut across a plane containing the cylindrical axis of the cylindrical body of the plate. Furthermore, Figure 23(b) shows the extrusion part of the extruder and a portion of the periphery around its base end, the membrane holder extruded by the extruder, and the cylindrical body of the plate and a portion of the periphery around its base end, all cut across a plane containing the cylindrical axis of the cylindrical body of the plate.

[0052] As shown in Figures 21(a) to 21(d) and Figures 22(a) to 22(e), the cell culture apparatus of this embodiment comprises a plate 22 having a flat base 22a with multiple through-holes 22b formed therein, on one side of which cylindrical bodies 22c used as the inner wall of the cell culture chamber are provided in the same number as the through-holes 22b, and a plate cover 23 and a spacer cover 24 having the same number of circular protrusions 23b and 24b as the cylindrical bodies 22c of the plate 22 on one side of the flat bases 23a and 24a, respectively, and used as cell culture chamber covers, The cell culture chamber includes a spacer 25 having the same number of through-holes 25b (see Figure 23(a)) as the through-holes 22b formed on one side of a flat plate-shaped base 25a, and the same number of cylindrical bodies 25c as the through-holes 25b provided on one side; an extruder 26 having the same number of cylindrical extrusion portions 26b as the cylindrical bodies 22c of the plate 22 on one side of a flat plate-shaped extruder 26a; a membrane holder 27 that is roughly cylindrical and can be inserted into the cylindrical bodies 22c of the plate 22; and a roughly cylindrical sleeve 28 to which the membrane holder 27 is attached at one end. As shown in Figures 22(b) and 22(d), the membrane holder 27 has a flange 27a at one end, and the porous membrane 3 is fixed to the inner surface of the other end by heat fusion.

[0053] The cylindrical body 22c of the plate 22 has an inner diameter equal to the diameter of the through hole 22b, and is positioned so that each cylindrical axis coincides with the central axis of all the through holes 22b. The circular projections 23b of the plate cover 23 are fitted onto the cylindrical body 22c of the plate 22, and are positioned so that, when the bases 22a and 23a are parallel, the central axis of each circular projection 23b coincides with the central axis of all the through holes 22b. In other words, the plate cover 23 is structured so that when the base 23a is brought closer to the base 22a of the plate 22 while maintaining the parallel state of the base 23a to the base 22a, all the circular projections 23b fit simultaneously onto all the cylindrical body 22c (see Figure 23(a)). On the other hand, the circular projections 24b of the spacer cover 24 are fitted onto the cylindrical bodies 25c of the spacer 25, and are positioned so that, when the bases 25a and 24a are parallel, the central axis of each circular projection 24b coincides with the cylindrical axis of all the cylindrical bodies 25c. In other words, the spacer cover 24 is structured so that when the base 24a is brought closer to the base 25a of the spacer 25 while maintaining the parallel state of the base 24a to the base 25a, all the circular projections 24b fit simultaneously onto all the cylindrical bodies 25c (see Figure 23(a)). The cylindrical body 25c of the spacer 25 has an outer diameter smaller than the inner diameter of the cylindrical body 22c of the plate 22, is fitted into the cylindrical body 22c, and is positioned so that, when the base 22a and base 25a are parallel, each cylindrical axis coincides with the central axis of all the through holes 22b. In other words, the spacer 25 is structured so that when the base 25a is brought closer to the base 22a of the plate 22 while maintaining the parallel state of the base 25a to the base 22a, all the cylindrical bodies 25c fit into all the cylindrical bodies 22c simultaneously (see Figure 23(a)). The extrusion portion 26b of the extruder 26 has an outer diameter smaller than the inner diameter of the cylindrical body 22c of the plate 22, is fitted into the cylindrical body 22c, and is positioned so that, when the base 22a and the extrusion plate 26a are parallel, each cylindrical axis coincides with the central axis of all the through holes 22b. In other words, the extruder 26 is structured so that when the extrusion plate 26a is brought closer to the base 22a of the plate 22 while maintaining the parallel state of the extrusion plate 26a to the base 22a of the plate 22, all the extrusion portions 26b fit into all the cylindrical bodies 22c simultaneously (see Figure 23(b)). Furthermore, the combined length of the extrusion portion 26b of the extruder 26 and the film holder 27 is longer than the combined length of the base 22a and the cylindrical body 22c. In other words, the extruder 26 is structured such that when the extrusion portion 26b is inserted into the through hole 22b of the plate 22 and the extrusion plate 26a is brought into contact with the base portion 22a, the porous membrane 3 attached to the membrane holder 27 installed on the tip surface 26c of the extrusion portion 26b is pushed out to the outside of the cylindrical body 22c together with the membrane holder 27 (see Figure 23(b)). Furthermore, the membrane holder 27, which is equipped with a second inner wall 27b (see Figure 22(d)) used as the inner wall of the cell culture chamber, is used as a cell culture chamber, as described later, together with a plate 22 having multiple through holes 22b and cylindrical bodies 22c provided at its base 22a and a spacer 25 having multiple through holes 25b and cylindrical bodies 25c provided at its base 25a, both of which are used as the inner wall of the cell culture chamber.

[0054] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 24 to 30. Figure 24 is a process diagram showing the procedure for a cell culture method according to the fourth embodiment of the present invention. Figure 25(a) shows the state in which a membrane holder is installed on the upper end of the cylindrical body of the spacer shown in Figure 21(d), and Figure 25(b) shows the state in which the spacer cover and plate are attached to the spacer in Figure 25(a) and the top and bottom are reversed. Figure 25(c) is a longitudinal cross-sectional view of the cylindrical body of the spacer and plate and the membrane holder in Figure 25(b). Figure 26(a) shows the state in which the plate cover is attached to the plate in Figure 25(b) and the top and bottom are reversed, and Figure 26(b) shows the state in Figure 26(a) with the spacer cover removed. Figure 27(a) is a longitudinal cross-sectional view of the cylindrical spacer and plate and the membrane holder in Figure 26(b), and Figure 27(b) shows the spacer cover reattached to the spacer in Figure 26(b). Figure 27(c) shows the state in Figure 27(b) where the plate cover and spacer cover have been removed and the extruder has been attached to the plate. Figure 28(a) is a longitudinal cross-sectional view of the cylindrical plate, the extrusion section of the extruder, and the membrane holder in Figure 27(c). Figure 28(b) shows the state in which a cell sheet of vascular endothelial cells is laid on the upper surface of the membrane holder extruded upward from the cylindrical plate in Figure 27(c). Figure 28(c) is a longitudinal cross-sectional view of the cylindrical plate, the extrusion section of the extruder, and the membrane holder in Figure 28(b). Figure 29(a) shows the process of punching out the cell sheet of vascular endothelial cells shown in Figure 28(b) with a sleeve, and Figure 29(b) shows the state in which the membrane holder is attached to the sleeve from which the cell sheet of vascular endothelial cells was punched out in Figure 29(a). Furthermore, Figure 30(a) is a longitudinal cross-sectional view of the cell culture insert consisting of a sleeve and a membrane holder in Figure 29(b), and Figure 30(b) shows the state in which the cell culture insert shown in Figure 30(a) is placed in the wells of a cell culture plate. To avoid making the diagrams cluttered, Figure 25(a) shows a state in which the membrane holder 27 is installed on only some of the cylindrical bodies. Also, in Figures 25(a), 25(b), 26(a), 26(b), 27(b), 27(c), 28(b), 29(a), and 29(b), only one cylindrical body, membrane holder, and through-hole are labeled, and in Figure 30(b), only one well is labeled, showing how a cell culture insert is installed in only one of the 12 wells provided in the cell culture plate. Furthermore, Figure 25(c) shows the cylindrical body of the spacer and a portion of the periphery around its base end, the circular projection of the spacer cover and a portion of the periphery around its base end, the cylindrical body of the plate and a portion of the periphery around its base end, and the membrane holder installed on the cylindrical body of the spacer, all cut along a plane containing the cylindrical axis of the cylindrical body of the plate. Also, Figure 27(a) shows the cylindrical body of the spacer and a portion of the periphery around its base end, the circular projection of the plate cover and a portion of the periphery around its base end, the cylindrical body of the plate and a portion of the periphery around its base end, and the membrane holder installed on the cylindrical body of the spacer, all cut along a plane containing the cylindrical axis of the cylindrical body of the plate. And in Figures 28(a) and 28(c), the extrusion part of the extruder and a portion of the periphery around its base end, the membrane holder extruded by the extruder, and the cylindrical body of the plate and a portion of the periphery around its base end are all cut along a plane containing the cylindrical axis of the cylindrical body of the plate. Furthermore, Figure 30(a) shows the cell culture insert cut along a plane containing the cylindrical axis of the sleeve.

[0055] First, as shown in Figure 25(a), a membrane holder 27 is placed on the upper end of the cylindrical body 25c of the spacer 25 (step S1 in Figure 24). Next, the plate 22 is positioned with the cylindrical body 22c facing downwards so that the base 25a is parallel to the base 22a, and all the cylindrical bodies 22c are fitted onto all the cylindrical bodies 25c, and the spacer cover 24 is attached to the spacer 25 by fitting the circular projection 24b onto the cylindrical body 25c from the lower side of the base 25a (step S2 in Figure 24). After that, as shown in Figure 25(b), a pipette 4 is used to inject a suspended cell culture medium (not shown) into the interior of the cylindrical body 25c of the spacer 25 through the through-hole 22b of the plate 22, and astrocyte 9 cells (see Figure 25(c)) are seeded (steps S3 and S4 in Figure 24). Then, while maintaining the cell culture medium temperature at, for example, 33°C, the astrocytes 9 are cultured over several days until they form layers (Step S5 in Figure 24). From the state shown in Figure 25(b), discard the cell culture medium and attach the plate cover 23 to the plate 22 by fitting the circular projection 23b into the through hole 22b from the lower side of the base 22a. Then, as shown in Figure 26(a), invert the plate 22 together with the plate cover 23, spacer cover 24, and spacer 25 (step S6 in Figure 24). Furthermore, as shown in Figure 26(b), remove the spacer cover 24 and use the pipette 4 to inject the aforementioned cell culture medium into the cylindrical body 25c of the spacer 25 and seed the pericyte 8 cells (see Figure 27(a)) (steps S7 and S8 in Figure 24). Subsequently, as shown in Figure 27(b), the spacer cover 24 is reattached to the spacer 25, and the perisites 8 are cultured for several days while maintaining the temperature inside the cylindrical body 25c of the spacer 25 at, for example, 33°C, until they form layers. The cell culture medium is then discarded when the perisites 8 have grown in layers (steps S9 and S10 in Figure 24). As a result, the astrocytes 9 that have already formed in layers on the inner surface of the porous membrane 3 have their legs extending through the pores of the porous membrane 3 to the vicinity of the perisites 8 formed on the outer surface of the porous membrane 3, as shown in Figure 28(a). Thus, the through-hole 22b of the plate 22 and the inside of the cylindrical body 25c of the spacer 25 are used as cell culture chambers, and when the membrane holder 27 is installed inside, the cell culture chamber is divided into two by the porous membrane 3. Therefore, the cell culture apparatus of this embodiment can be said to have a structure in which the second inner wall 27b of the cylindrical membrane holder 27 (see Figure 22(d)) and the inner wall of the through-hole 25b and cylindrical body 25c provided in the base 25a of the spacer 25, or the second inner wall 27b of the membrane holder 27 and the inner wall of the through-hole 22b and cylindrical body 22c provided in the base 22a of the plate 22, constitute a pair of cell culture chambers divided vertically by the porous membrane 3, and multiple such cell culture chambers are connected and each of the vertically divided sections is integrated.

[0056] As shown in Figure 27(c), the plate cover 23, spacer cover 24, and spacer 25 are removed, and the extrusion portion 26b of the extruder 26 is inserted into the through hole 22b of the plate 22. Then, the extruder 26 is brought closer to the plate 22 so that the extrusion plate 26a contacts the base 22a. This causes the membrane holder 27 attached to the tip surface 26c of the extrusion portion 26b (see Figure 28(a)) to be pushed out of the cylindrical body 22c (step S11 in Figure 24). As shown in Figure 27(c), a cell sheet 20 of vascular endothelial cells 7 is laid on the upper surface of the membrane holder 27 that is extruded to the outside of the cylindrical body 22c of the plate 22, as shown in Figures 28(b) and 28(c) (step S12 in Figure 24). Then, as shown in Figure 29(a), the cell sheet 20 of vascular endothelial cells 7 shown in Figure 28(b) is punched out from above by the sleeve 28, and the remaining punched-out cell sheet 20 is removed (step S13 in Figure 24). Then, the membrane holder 27 is attached to the tip of the sleeve 28 by pushing the sleeve 28 into the membrane holder 27, as shown in Figure 29(b) (step S14 in Figure 24). As a result, the membrane holder 27 and the sleeve 28 become integrated, and as shown in Figure 30(a), three types of cell layers consisting of the cell sheet 20 of vascular endothelial cells 7, pericytes 8 and astrocytes 9 are formed at the bottom of the cell culture insert 21. Subsequently, the cell culture insert 21 is removed from the extrusion section 26b of the extruder 26 and placed in the well 2b of the microplate 2 as shown in Figure 30(b) (step S15 in Figure 24). Then, the internal temperature of the cell culture insert 21 is maintained at, for example, 33°C, and the vascular endothelial cells 7 are cultured for several days (step S16 in Figure 24).

[0057] Thus, with the cell culture apparatus of this embodiment, the operations of inverting the porous membrane 3, removing the membrane holder 27 from inside the through-hole 22b of the plate 22, and placing vascular endothelial cells 7 on top of the perisite 8 are performed simultaneously for multiple porous membranes 3 and membrane holders 27, making it possible to produce multiple BBB models in a short time.

[0058] Modified examples of the cell culture method of this embodiment will be described with reference to Figures 31 and 32. Figure 31 shows a modified example of the process diagram shown in Figure 24. Figure 32(a) shows the state in Figure 27(c) where the sleeve is attached to the membrane holder, and Figure 32(b) is a longitudinal cross-sectional view of the cell culture insert consisting of the cylindrical plate, the extrusion part of the extruder, the sleeve, and the membrane holder in Figure 32(a). To avoid making the figure too complex, only one cylinder and one sleeve are labeled in Figure 32(a). Figure 32(b) shows the state in which the extrusion part of the extruder and a part of the area around its base end, the membrane holder extruded by the extruder, and the cylindrical plate and a part of the area around its base end are cut in a plane containing the cylindrical axis of the cylindrical plate. Furthermore, of the steps shown in Figure 31, steps S1 to S11 are the same as steps S1 to S11 in Figure 24, so the explanation of those steps will be omitted. Specifically, after steps S1 to S11, the sleeve 28 is pressed into the membrane holder 27 in the state shown in Figure 27(c), and the membrane holder 27 is attached to the tip of the sleeve 28 as shown in Figure 32(a) (step S12 in Figure 31). Next, a temperature-sensitive gel 6 that dissolves at a temperature above which vascular endothelial cell proliferation stops (e.g., 37°C) is applied to the cell layer of pericytes 8 inside the cell culture insert 21 (step S13 in Figure 31), and then vascular endothelial cells 7 are embedded in this temperature-sensitive gel 6 (step S14 in Figure 32). After that, the vascular endothelial cells 7 are cultured for several days while maintaining the temperature inside the cell culture insert 21 at, for example, 33°C (step S15 in Figure 31). As a result, as shown in Figure 32(b), three cell layers consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 embedded in the temperature-sensitive gel 6 are formed at the bottom of the cell culture insert 21. Then, the cell culture insert 21 is removed from the extrusion section 26b of the extruder 26 and placed in the well 2b of the microplate 2 as shown in Figure 30(b) (step S16 in Figure 31). Furthermore, when the internal temperature of the cell culture insert 21 is raised to 37°C, cell proliferation of the vascular endothelial cells 7 stops and the temperature-sensitive gel 6 dissolves (step S17 in Figure 31), and the vascular endothelial cells 7 come into contact with the pericytes 8. In this state, the internal temperature of the cell culture insert 21 is maintained at 33°C and the culture of the vascular endothelial cells 7 is restarted, completing a three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9.

[0059] Pericytes 8 have the function of controlling the differentiation and proliferation of vascular endothelial cells 7, but when vascular endothelial cells 7 are embedded in the temperature-sensitive gel 6, the vascular endothelial cells 7 are not in contact with pericytes 8, and therefore this function is not exercised. In other words, in the cell culture method shown in Figure 31, by maintaining the temperature inside the cell culture insert 21 at a temperature that does not cause the temperature-sensitive gel 6 to dissolve, the layered state of vascular endothelial cells 7, pericytes 8, and astrocytes 9 formed at the bottom of the cell culture insert 21 by steps S1 to S15 shown in Figure 31 is maintained for a long period of time. Furthermore, when using the temperature-sensitive gel 6, there is no need to prepare a cell sheet of vascular endothelial cells in advance, so the step of producing the cell sheet can be omitted. Therefore, according to the cell culture method described above, it is possible to increase the mass production capacity of the BBB model consisting of the three cell layers described above. [Examples]

[0060] A cell culture apparatus according to a fifth embodiment of the present invention will be described with reference to Figure 33. Figures 33(a) to 33(c) are external perspective views of the cell culture plate, the first plate cover (second plate cover), and the cell culture insert, respectively. To avoid clutter in the figures, only one well is labeled in Figure 33(a). Also, since the first plate cover and the second plate cover have the same shape when viewed from above, their labels are shown together in Figure 33(b). As shown in Figures 33(a) to 33(c), the cell culture apparatus of this embodiment comprises a cell culture plate 29 having circular through-holes 29e used as the inner wall of the cell culture chamber, a first plate cover 30 and a second plate cover 31 installed above and below the cell culture plate 29, respectively, and used as cell culture chamber covers, and a cell culture insert 32. As shown in Figure 33(a), the cell culture plate 29 is roughly rectangular in shape, and has 12 through-holes 29e (3 rows vertically x 4 rows horizontally) that function as wells on its upper surface 29a, and a first rectangular projection 29c and a second rectangular projection 29d (see Figure 35(b)) are provided on the upper surface 29a and the lower surface 29b (see Figure 35(b)). As shown in Figure 33(b), the first plate cover 30 and the second plate cover 31 consist of rectangular recesses 30a and 31a and frame-shaped protrusions 30b and 31b provided around the recesses 30a and 31a. When the recesses 30a and 31a are placed on the upper surface 29a of the cell culture plate 29, the structure allows the first rectangular protrusion 29c and the second rectangular protrusion 29d to be positioned inside the protrusions 30b and 31b. As shown in Figure 33(c), the cell culture insert 32 consists of a cylindrical body with a flange 32a at one end, and a porous membrane 3 is fixed to the inner surface of the other end of this cylindrical body by heat fusion. The through-hole 29e of the cell culture plate 29 has a stepped structure with a large-diameter section 29f and a small-diameter section 29g. The large-diameter section 29f has an inner diameter larger than the outer diameter of the flange 32a and has a depth that allows the flange 32a to be placed inside. The small-diameter section 29g has an inner diameter smaller than the outer diameter of the flange 32a and allows the portion of the cell culture insert 32 other than the flange 32a to be placed inside, and has a depth that prevents the end face on the side of the cell culture insert 32 to which the porous membrane 3 is attached from contacting the bottom surface when the flange 32a is locked to the large-diameter section 29f. Furthermore, the cell culture insert 32, which is equipped with a first inner wall 32b (see Figure 33(c)) used as the inner wall of the cell culture chamber, is used as a cell culture chamber together with a cell culture plate 29, which is provided with multiple through-holes 29e and is also used as the inner wall of the cell culture chamber, as will be described later.

[0061] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 34 to 37. Figure 34 is a process diagram showing the procedure of a cell culture method according to the fifth embodiment of the present invention. Figure 35(a) shows how the cell culture insert shown in Figure 33(c) is installed in the through-holes of a cell culture plate, and Figures 35(b) and 35(c) are cross-sectional views taken along the LL line and MM line in Figure 35(a), respectively. Figure 36(a) shows how cells are seeded into the cell culture insert installed in the through-holes of a cell culture plate, and Figure 36(b) is a cross-sectional view taken along the NN line in Figure 36(a), and Figure 36(c) is an enlarged view of the area enclosed by the dashed line in Figure 36(b). Figure 37(a) shows how cells are seeded into the cell culture insert installed in the through-holes of a cell culture plate, with the state inverted vertically from Figure 36(a), and Figure 37(b) is a cross-sectional view taken along the PP line in Figure 37(a), and Figure 37(c) is an enlarged view of the area enclosed by the dashed line in Figure 37(b).

[0062] First, as shown in Figures 35(a) and 35(c), the cell culture insert 32 is placed in the through-hole 29e by positioning the flange 32a on the large diameter portion 29f of the cell culture plate 29 (step S1 in Figure 34). Then, the first plate cover 30 is placed on the upper surface 29a of the cell culture plate 29, and then, as shown in Figure 36(b), the cell culture plate 29, which is integrated with the first plate cover 30, is inverted (steps S2 and S3 in Figure 34). Next, as shown in Figure 36(a), a cell culture medium (not shown) is injected into the through-hole 29e of the cell culture plate 29 using a pipette 4, and astrocytes 9 are seeded (steps S4 and S5 in Figure 34). Furthermore, while maintaining the temperature inside the through-hole 29e at, for example, 33°C, the astrocytes 9 are cultured over several days until they form layers as shown in Figure 36(c) (step S6 in Figure 34). Then, the second plate cover 31 is placed on the lower surface 29b of the cell culture plate 29, and the cell culture plate 29, which is integrated with the first plate cover 30 and the second plate cover 31, is inverted (steps S7 and S8 in Figure 34). Furthermore, as shown in Figure 37(b), after removing the first plate cover 30 from the upper surface 29a of the cell culture plate 29, as shown in Figure 37(a), a cell culture medium (not shown) is injected into the cell culture insert 32 installed in the through-hole 29e of the cell culture plate 29 using a pipette 4, and pericytes 8 are seeded (steps S9 to S11 in Figure 34). Then, while maintaining the temperature inside the cell culture insert 32 at, for example, 33°C, the pericytes 8 are cultured for several days until they form layers, and the cell culture medium is discarded when the pericytes 8 have grown in layers (step S12 in Figure 34). As a result, the astrocytes 9 that have already formed in layers on the outer surface of the porous membrane 3 have legs extending from the cells through the small pores of the porous membrane 3 to the vicinity of the pericytes 8 formed on the inner surface of the porous membrane 3. Thus, the interior of the through-hole 29e of the cell culture plate 29 is used as a cell culture chamber, and when a cell culture insert 32 is installed inside it, the cell culture chamber is divided into two by the porous membrane 3. Therefore, the cell culture apparatus of this embodiment can be said to have a structure in which the first inner wall 32b of the cylindrical cell culture insert 32 (see Figure 33(c)) and the inner wall of the through-hole 29e provided in the cell culture plate 29 constitute a pair of cell culture chambers divided vertically by the porous membrane 3, and multiple such cell culture chambers are connected and each of the vertically divided sections is integrated.

[0063] Next, a temperature-sensitive gel 6 that dissolves at a temperature above which vascular endothelial cell proliferation stops (e.g., 37°C) is applied to the cell layer of pericytes 8 (step S13 in Figure 34), and then vascular endothelial cells 7 are embedded in this temperature-sensitive gel 6 (step S14 in Figure 34). Subsequently, the vascular endothelial cells 7 are cultured for several days while maintaining the temperature inside the cell culture insert 32 at, for example, 33°C (step S15 in Figure 34). As a result, as shown in Figure 37(c), three cell layers consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 embedded in the temperature-sensitive gel 6 are formed at the bottom of the cell culture insert 32. Then, the cell culture insert 32 is removed from the through-hole 29e of the cell culture plate 29 and placed in well 2b of the microplate 2 (see Figure 1(c)). Furthermore, when the internal temperature of the cell culture insert 32 is raised to 37°C, the cell proliferation of vascular endothelial cells 7 stops, and the temperature-sensitive gel 6 dissolves (step S16 in Figure 34), causing the vascular endothelial cells 7 to come into contact with the pericytes 8. At this point, the internal temperature of the cell culture insert 21 is maintained at 33°C, and the culture of vascular endothelial cells 7 is restarted. This completes the three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9.

[0064] Thus, in the cell culture apparatus of this embodiment, after placing the first plate cover 30 on the upper surface 29a of the cell culture plate 29 in which the cell culture insert 32 is placed with the through-hole 29e facing downwards, the cell culture plate 29 is inverted together with the first plate cover 30, and astrocytes 9 are seeded into the through-hole 29e together with the cell culture medium 5. The inner wall of the through-hole 29e prevents leakage of the cell culture medium 5 and astrocytes 9, thus allowing the astrocytes 9 to be easily cultured on the outer surface of the porous membrane 3. Furthermore, if the second plate cover 31 is placed on the lower surface 29b of the cell culture plate 29, and the cell culture plate 29 is inverted together with the first plate cover 30 and the second plate cover 31, the through-hole 29e will hold the cell culture insert 32 in a position with the bottom facing downwards. Therefore, the process of seeding pericytes 8 and vascular endothelial cells 7 together with the cell culture medium 5 inside the cell culture insert 32, and the process of culturing the vascular endothelial cells 7 and pericytes 8 inside the cell culture insert 32, becomes easier. Furthermore, the cell culture plate 29 on which the second plate cover 31 is installed functions as a microplate 2. Thus, with the cell culture apparatus of this embodiment, it is possible to efficiently produce multiple BBB models. Furthermore, the cell method of this embodiment includes a step of applying a temperature-sensitive gel 6 onto the cell layer of pericytes 8. In this case, since it is not necessary to prepare a cell sheet 20 of vascular endothelial cells 7 in advance, the step of preparing the cell sheet can be omitted. [Examples]

[0065] A cell culture apparatus according to a sixth embodiment of the present invention will be described with reference to Figures 38 and 39. Figures 38(a) and 38(c) are external perspective views of a cell culture plate, and Figure 38(b) is a cross-sectional view taken along the QQ line in Figure 38(a). Figures 39(a) and 39(c) are external perspective views of a first bottom cover and a second bottom cover, respectively, and Figure 39(b) is a cross-sectional view taken along the RR line in Figure 39(a). As shown in Figures 38(a) to 38(c) and Figures 39(a) to 39(c), the cell culture apparatus of this embodiment comprises a cell culture plate 33 having circular through-holes 33d used as the inner wall of the cell culture chamber, a first bottom cover 35 installed on the bottom surface 33b of the cell culture plate 33, and a second bottom cover 36 installed on the bottom plate 35a of the first bottom cover 35 and used as a cell culture chamber cover. As shown in Figures 38(a) to 38(c), the cell culture plate 33 is roughly rectangular in shape, with 12 through-holes 33d (3 rows vertically x 4 rows horizontally) that function as wells provided on the upper surface 33a, and a sheet 34 made of a porous membrane 3 attached or heat-sealed to a rectangular projection 33c provided on the bottom surface 33b. As shown in Figures 39(a) to 39(c), the first bottom cover 35 and the second bottom cover 36 are provided with rectangular bottom plates 35a and 36a, and frame-shaped protrusions surrounding these bottom plates 35a and 36a. The first bottom cover 35 is structured so that when it is placed on the bottom surface 33b of the cell culture plate 33, the rectangular protrusions 33c can be positioned inside the protrusions, and the second bottom cover 36 is structured so that when it is placed on the first bottom cover 35, the rectangular protrusions 35b can be positioned inside the protrusions. Furthermore, when the first bottom cover 35 is placed on the bottom surface 33b of the cell culture plate 33, two circular through-holes 35c (3 rows vertically x 4 rows horizontally) are provided on the bottom plate 35a at locations corresponding to the 12 through-holes 33d, and their inner diameter is equal to that of the through-holes 33d, and these are used as the inner wall of the cell culture chamber. Furthermore, the cell culture plate 33, which has through-holes 33d used as the inner wall of the cell culture chamber, is used as a cell culture chamber together with the first bottom cover 35, which has multiple through-holes 35c used as the inner wall of the cell culture chamber, as will be described later.

[0066] Here, the cell culture method of the present invention for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes using the cell culture apparatus of this embodiment will be explained with reference to Figures 40 to 42. Figure 40 is a process diagram showing the procedure for a cell culture method according to the sixth embodiment of the present invention. Figure 41(a) shows a cell culture plate inverted upside down with the first bottom cover attached, Figure 41(b) is a cross-sectional view taken along the SS line in Figure 41(a), and Figure 41(c) is an enlarged view of the area enclosed by the dashed line in Figure 41(b). Furthermore, Figure 42(a) shows the cell culture plate inverted upside down with the second bottom cover attached to the first bottom cover in Figure 41(a), Figure 42(b) is a cross-sectional view taken along the TT line in Figure 42(a), and Figure 42(c) is an enlarged view of the area enclosed by the dashed line in Figure 42(b).

[0067] First, as shown in Figures 41(a) and 41(b), the first bottom cover 35 is placed on the bottom surface 33b of the inverted cell culture plate 33 (step S1 in Figure 40). Next, as shown in Figure 41(a), a cell culture medium (not shown) is injected into the through-hole 35c of the first bottom cover 35 using a pipette 4, and astrocytes 9 are seeded (steps S2 and S3 in Figure 40). Furthermore, while maintaining the temperature inside the through-hole 35c at, for example, 33°C, the astrocytes 9 are cultured over several days until they form layers as shown in Figure 41(c) (step S4 in Figure 40). Then, after placing the second bottom cover 36 on the first bottom cover 35, the cell culture plate 33, which is integrated with the first bottom cover 35 and the second bottom cover 36, is inverted (steps S5 and S6 in Figure 40). Furthermore, as shown in Figure 42(a), a cell culture medium (not shown) is injected into the through-holes 33d of the cell culture plate 33 using a pipette 4, and pericytes 8 are seeded (steps S7 and S8 in Figure 40). Subsequently, the temperature inside the through-holes 33d of the cell culture plate 33 is maintained at, for example, 33°C, and the perisites 8 are cultured over several days until they form layers. Once the perisites 8 have grown in layers, the cell culture medium is discarded (step S9 in Figure 40). As a result, the astrocytes 9 that have already formed in layers on the outer surface of the porous membrane 3 have their legs extending through the pores of the porous membrane 3 to the vicinity of the perisites 8 formed on the inner surface of the porous membrane 3. Thus, the interiors of the through-holes 33d in the cell culture plate 33 and the through-holes 35c in the first bottom cover 35 are used as cell culture chambers. When the first bottom cover 35 is installed on the cell culture plate 33, it is as if one cell culture chamber is divided into two by the porous membrane 3. Therefore, the cell culture apparatus of this embodiment can be said to have a structure in which the inner walls of the through-holes 33d in the cell culture plate 33 and the through-holes 35c in the first bottom cover 35 constitute a pair of cell culture chambers divided vertically by the porous membrane 3, and multiple such cell culture chambers are connected and each of the vertically divided chambers is integrated.

[0068] Next, a temperature-sensitive gel 6 that dissolves at a temperature above which vascular endothelial cell proliferation stops (e.g., 37°C) is spread onto the cell layer of pericytes 8 (step S10 in Figure 40), and then the vascular endothelial cells 7 are embedded in this temperature-sensitive gel 6 (step S11 in Figure 40). Subsequently, the vascular endothelial cells 7 are cultured for several days while maintaining the temperature inside the through-holes 33d of the cell culture plate 33 at, for example, 33°C (step S12 in Figure 40). As a result, as shown in Figure 42(c), three cell layers consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 embedded in the temperature-sensitive gel 6 are formed at the bottom of the cell culture insert 32. Subsequently, when the temperature inside the through-holes 33d of the cell culture plate 33 is raised to 37°C, the cell proliferation of vascular endothelial cells 7 stops, the temperature-sensitive gel 6 dissolves (step S12 in Figure 40), and the vascular endothelial cells 7 come into contact with the pericytes 8. In this state, when the temperature inside the through-holes 33d of the cell culture plate 33 is maintained at 33°C and the culture of vascular endothelial cells 7 is restarted, a three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9 is completed.

[0069] Thus, in the cell culture apparatus of this embodiment, when the cell culture plate 33 is inverted and the first bottom cover 35 is placed on the bottom surface 33b, and then astrocytes 9 are seeded inside the through-holes 35c together with the cell culture medium 5, the leakage of the cell culture medium 5 and astrocytes 9 is prevented by the inner wall of the through-holes 35c, thus allowing the astrocytes 9 to be easily cultured on the outer surface of the porous membrane 3. Furthermore, when the second bottom cover 36 is placed on the first bottom cover 35, and the cell culture plate 33 is inverted together with the first bottom cover 35 and the second bottom cover 36, and then pericytes 8 and vascular endothelial cells 7 are seeded inside the through-holes 33d of the cell culture plate 33 together with the cell culture medium 5, the leakage of the cell culture medium 5, pericytes 8 and vascular endothelial cells 7 is prevented by the inner wall of the through-holes 33d, thus allowing the pericytes 8 and vascular endothelial cells 7 to be easily cultured on the inner surface of the porous membrane 3. Furthermore, the cell culture plate 33 on which the first bottom cover 35 and the second bottom cover 36 are installed functions as a microplate 2. Therefore, the cell culture apparatus of this embodiment does not require a cell culture insert, and the cell culture plate 33 can be used directly as a microplate 2. Compared to the case where a cell culture insert or a microplate 2 is used, it is possible to increase the mass production capacity of the three-layered BBB model consisting of vascular endothelial cells 7, pericytes 8, and astrocytes 9. Furthermore, the cell method of this embodiment includes a step of coating a temperature-sensitive gel 6 onto the cell layer of pericytes 8. In this case, since it is not necessary to prepare the cell sheet 20 of vascular endothelial cells 7 in advance, the step of producing the cell sheet can be omitted. Therefore, the mass production capability of the BBB model described above is further enhanced. [Industrial applicability]

[0070] The cell culture method and cell culture apparatus used therein according to the present invention are applicable to the mass production of a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes. [Explanation of Symbols]

[0071] 1 Cell culture insert 1a side 1b Large diameter opening 1c Small diameter opening 1d flange 1e Fourth Inner Wall 2 microplates 2a Top side 2b well 2c Bottom 3 Porous membrane 4 pipettes 5 Cell culture medium 6. Temperature-sensitive gel 7 Vascular endothelial cells 8 Perisite 9 Astrocytes 10 Insert holding plate 10a Side plate 10b Bottom plate 10c square hole 10d round hole 11 Cell culture cases 11a Bottom plate 11b Through hole 12 Insert retainer 12a square bar 12b Connecting plate 13 Case Lid 13a Recess 13b Projection 14 plates 14a Base 14b First annular projection 14c Second annular projection 14d through hole 15 Plate Cover 15a base 15b Holding part 16 Spacers 16a base 16b Holding part 16th century cylindrical body 16d Tip surface 17 Extrusion tool 17a Extruded sheet 17b Holding part 17c Extrusion section 17d Tip surface 18 Membrane holder 18a Engagement part 18b 3rd inner wall 19 Insert body 19a Flange 19b Sharp section 20 cell sheets 21 Cell culture inserts 22 plates 22a base 22b Through hole 22c Cylindrical body 23 Plate Cover 23a base 23b Circular protrusion 24 Spacer Cover 24a base 24b Circular protrusion 25 Spacers 25a base 25b Through hole 25c Cylindrical body 26 Extrusion tool 26a Extruded sheet 26b Extrusion section 26c Tip surface 27 Membrane holder 27a Flange 28 sleeves 28a Flange 29 Cell culture plates 29a Top side 29b Bottom side 29c First rectangular projection 29d Second rectangular projection 29e Through hole 29f Large diameter section 29g small diameter part 30. First plate cover 30a recess 30b Protrusion 31. Second plate cover 31a Recess 31b Protrusion 32 Cell culture inserts 32a Flange 32b 1st inner wall 33 Cell culture plates 33a Top side 33b Bottom 33c Rectangular protrusion 33d through hole 34 seats 35. First bottom cover 35a bottom plate 35b Rectangular protrusion 35c through hole 36. Second bottom cover 36a bottom plate

Claims

1. A porous membrane (3) formed on both sides so that biomaterials can be fixed to it, Multiple cell culture chambers having a pair of upper and lower partitioned spaces by dividing the inside of the cylindrical inner wall into two by the porous membrane (3) which is positioned to extend in a direction intersecting its central axis, Equipped with a cell culture room cover, Multiple of the aforementioned cell culture chambers are connected to each other and integrated into one unit. The cell culture chamber cover simultaneously closes at least one of the upper or lower sides of the multiple cell culture chambers. A membrane holder (27) used as the cell culture chamber, having one end closed by the porous membrane (3) and equipped with a second inner wall (27b) used as the inner wall, A plate (22) is provided on one side of a flat plate-shaped first base (22a) having a plurality of first through holes (22b) formed therein, with a first cylindrical body (22c) erected vertically on one side that communicates with the first through holes (22b), and the membrane holder (27) is installed inside, and which is used as the inner wall together with the first through holes (22b), Multiple first protrusions (23b) that simultaneously fit onto each of the multiple first cylindrical bodies (22c) are provided on one side of a flat second base (23a), and the cell culture chamber cover is a plate cover (23) that covers the first through-hole (22b) with the membrane holder (27) installed inside, Multiple second protrusions (24b) that simultaneously fit onto each of the multiple first cylindrical bodies (22c) are provided on one side of a flat third base (24a), and the cell culture chamber cover includes a spacer cover (24) that covers the first through-hole (22b) with the membrane holder (27) installed inside, On one side of a flat plate-shaped fourth base (25a) having the same number of second through-holes (25b) as the first through-holes (22b) used as the inner wall, there is a spacer (25) erected vertically so that the multiple second cylindrical bodies (25c), which together constitute the cell culture chamber and are simultaneously inserted into each of the multiple first cylindrical bodies (22c), and which together constitute the inner wall with the second through-holes (25b), communicate with the second through-holes (25b). The extruder (26) comprises a flat extruder plate (26a) with a plurality of extruders (26b) vertically erected on one side, each of which is simultaneously inserted into a plurality of first through holes (22b), A cell culture apparatus characterized in that the extrusion portion (26b) of the extruder (26) is inserted into the first through hole (22b) of the plate (22), thereby extruding the porous membrane (3) together with the membrane holder (27) to the outside of the first cylindrical body (22c).

2. A porous membrane (3) formed on both sides so that a biomaterial can be fixed to it, Multiple cell culture chambers having a pair of upper and lower partitioned spaces by dividing the inside of the cylindrical inner wall into two by the porous membrane (3) which is positioned to extend in a direction intersecting its central axis, Equipped with a cell culture room cover, Multiple of the aforementioned cell culture chambers are connected to each other and integrated into one unit. The cell culture chamber cover simultaneously closes at least one of the upper or lower sides of the multiple cell culture chambers. A membrane holder (18) used as the cell culture chamber, having one end closed by the porous membrane (3) and equipped with a third inner wall (18b) used as the inner wall, A plate (14) serving as a cell culture chamber, having a flat base (14a) on which the membrane holder (18) is installed internally and which is provided with a plurality of through holes (14d) used as the inner wall of the cell culture chamber, The cell culture chamber cover consists of two flat plate covers (15) that are placed on the plate (14) so ​​as to cover the through-hole (14d) in which the membrane holder (18) is installed inside, The extruder (17) comprises a flat extruder plate (17a) with multiple extruder sections (17c) vertically erected on one side, each of which is simultaneously inserted into a plurality of through holes (14d). A cell culture apparatus characterized in that the extrusion portion (17c) of the extruder (17) is inserted into the through-hole (14d) of the plate (14), thereby causing the porous membrane (3) to be extruded from the through-hole (14d) together with the membrane holder (18).

3. The plate (14) is provided with a first projection (14b) and a second projection (14c) on both sides of the base (14a) that communicate with the through hole (14d), respectively, so as to surround the through hole (14d) when viewed from above. The plate cover (15) has a plurality of first cylindrical bodies (15b) on one side which fit simultaneously with the plurality of first protrusions (14b) and the second protrusions (14c), respectively. The cell culture apparatus according to claim 2, characterized in that the extruder (17) is shorter than the extrusion section (17c), and a plurality of second cylindrical bodies (17b) that simultaneously fit with the plurality of first protrusions (14b) and the second protrusions (14c), respectively, are provided on one side of the extrusion plate (17a) such that they surround the extrusion section (17c).

4. A cell culture method for creating a three-layered BBB model consisting of vascular endothelial cells, pericytes, and astrocytes, using multiple pairs of cell culture chambers in which the inner wall of a cylindrical structure formed by a porous membrane (3) formed on both sides to allow the attachment of biomaterials, is divided into upper and lower sections. The process involves sowing astrocytes on the first surface of the porous membrane (3) that is facing upward, The process of culturing the astrocytes, A step of simultaneously closing the upper openings of multiple pairs of the cell culture chambers, The process involves simultaneously inverting multiple pairs of the cell culture chambers and seeding the perisites onto the second surface of the porous membrane (3), The process of culturing the perisite, A step of seeding the vascular endothelial cells embedded in the temperature-sensitive gel onto the second surface of the porous membrane (3), The process of culturing the vascular endothelial cells, The process includes a step of dissolving the temperature-sensitive gel, A cell culture method characterized in that the vascular endothelial cells are temperature-sensitive and cell proliferation stops at a desired temperature.

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