Cell culture method and cell culture membrane

Abstract

To provide cell culture methods that can create a cell state closer to a state in vivo.SOLUTION: Provided is a cell culture method comprising: a preparation step of preparing a cell culture membrane comprising (i) a membrane body made of a thermosetting resin and having a first surface and a second surface facing the first surface, and (ii) a plurality of through holes formed in the membrane body and penetrating from the first surface to the second surface; and a culture step of seeding and culturing cells on each of the first and second surfaces of the prepared cell culture membrane, the plurality of through holes having a first average hoe diameter on the first surface smaller than a second average hole diameter on the second surface, and the hole density of the plurality of through holes being 2.0×105 / cm2 or less.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a cell culture method and a cell culture membrane. [Background technology]

[0002] Conventionally, there is a technique using a cell culture membrane to culture adherent cells three-dimensionally (for example, Patent Document 1). By culturing cells on both sides of the cell culture membrane, three-dimensional cell culture becomes possible. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-29092 A Summary of the Invention [Problem to be solved by the invention]

[0004] Further improvements in cell culture methods and cell culture membranes are needed to create cellular conditions that more closely resemble those in vivo. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms. According to one embodiment of the present disclosure, a cell culture method is provided, which includes: (i) a preparation step of preparing a cell culture membrane including a membrane body formed of a thermosetting resin and having a first surface and a second surface opposed to the first surface, and (ii) a plurality of through-holes formed in the membrane body and penetrating from the first surface to the second surface, and a culture step of seeding and culturing cells on each of the first surface and the second surface of the prepared cell culture membrane, wherein a first average pore size of the plurality of through-holes on the first surface is smaller than a second average pore size on the second surface, and a pore density of the plurality of through-holes is 2.0×10 5 pieces / cm 2 or less, wherein in the culturing step, the types of cells seeded and cultured on the first surface and the types of cells seeded and cultured on the second surface are different, and the first average pore size is 7 μm or less.

[0006] (1) According to one embodiment of the present disclosure, there is provided a cell culture method, comprising: (i) a preparation step of preparing a cell culture membrane including a membrane body formed of a thermosetting resin and having a first surface and a second surface opposed to the first surface, and (ii) a plurality of through-holes formed in the membrane body and penetrating from the first surface to the second surface; and a culture step of seeding and culturing cells on each of the first surface and the second surface of the prepared cell culture membrane, wherein a first average pore size of the plurality of through-holes on the first surface is smaller than a second average pore size on the second surface, and a pore density of the plurality of through-holes is 2.0×10 5 pieces / cm 2 According to this embodiment, the cells seeded on the second surface can contact the cells cultured on the first surface through the through-holes while suppressing the cells from wrapping around. Here, wrapping around means that the cells seeded on either the first surface or the second surface move through the through-holes to the other surface. Therefore, when different types of cells are cultured on the first surface and the second surface, a state close to the in vivo state where different types of cells interact with each other can be created. This makes it possible to create an organ model with good transport capacity and barrier properties. (2) In the cell culture method of the above aspect, in the culture step, the cells seeded and cultured on the first surface and the cells seeded and cultured on the second surface may be of different types. According to this aspect, by culturing different types of cells on the first surface and the second surface, it is possible to create a state close to the in vivo state in which different types of cells interact with each other. (3) In the cell culture method of the above aspect, the culture step may include a first step of seeding and culturing cells on the first surface, and a second step of seeding and culturing cells on the second surface after the first step. According to this aspect, the culture on the first surface and the culture on the second surface can each be performed well. (4) In the cell culture method of the above aspect, the first cells seeded and cultured on the first surface may be intestinal epithelial cells, and the second cells seeded and cultured on the second surface may be vascular endothelial cells. According to this aspect, an organ model can be produced. (5) In the cell culture method according to the above aspect, the first average pore size may be 7 μm or less. According to this aspect, when cells having a size of about 10 μm are cultured, the intrusion of the cells can be further suppressed. (6) In the cell culture method of the above embodiment, the first average pore size may be 3 μm or more and 5 μm or less. According to this embodiment, when cells having a size of about 5 μm are seeded, a pore size of 5 μm or less can further suppress cell wraparound, while a pore size of 3 μm or more can facilitate contact between the cells seeded on the second surface and the cells cultured on the first surface. (7) In the cell culture method of the above aspect, the pore density is 1.5×10 5 pieces / cm 2 According to this embodiment, the intrusion of cells can be further suppressed. (8) According to one embodiment of the present disclosure, there is provided a cell culture membrane comprising: a membrane body made of a thermosetting resin and having a first surface and a second surface opposite to the first surface; and a plurality of through-holes formed in the membrane body and penetrating from the first surface to the second surface, the plurality of through-holes having a first average pore size in the first surface that is smaller than a second average pore size in the second surface, and a pore density of the plurality of through-holes being 2.0×10 5 pieces / cm 2 The first surface is used for seeding and culturing a first type of cells, and the second surface is used for seeding and culturing a second type of cells different from the first type of cells. According to this embodiment, the cells seeded on the second surface can enter the through-holes and approach the cells cultured on the first surface while suppressing the cells from wrapping around. Therefore, by using a cell culture membrane, when different types of cells are cultured on the first surface and the second surface, a state close to the in vivo state where different types of cells interact with each other can be created. The present disclosure can be realized in various forms, and in addition to the forms described above, can be realized in the form of a method for producing a cell culture membrane, a method for producing a cell culture device, etc. [Brief description of the drawings]

[0007] [Figure 1] 1 is a flow chart showing steps of a cell culture method. [Diagram 2] FIG. 2 is a perspective view of the insert with the cell culture membrane attached. [Diagram 3] Cross-section of a cell culture membrane. [Figure 4] FIG. 2 is a diagram illustrating each step of the cell culture process. [Diagram 5] FIG. 4 is a diagram illustrating the attachment of a first jig. [Figure 6] FIG. 11 is a diagram illustrating the attachment of a second jig. [Figure 7] FIG. 13 is a diagram showing the relationship between pore density and the presence or absence of cell wraparound. [Figure 8] FIG. 1 shows the results of transepithelial electrical resistance. [Figure 9] FIG. 1 shows the results of a Lucifer Yellow permeation test. [Figure 10] FIG. 1 is a diagram explaining the method of transportation testing. [Figure 11] FIG. 1 shows the results of a digoxin transport study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] A. Embodiment: FIG. 1 is a flow chart showing the steps of the cell culture method. FIG. 2 is a perspective view of an insert 10 to which a cell culture membrane 20 is attached. FIG. 3 is a cross-sectional view of the cell culture membrane 20. FIG. 4 is a diagram for explaining each step of the cell culture process. In this embodiment, different types of cells are cultured on each of the two sides of the cell culture membrane 20, which is a porous membrane. The cells that are seeded and cultured first are called first cells CA, and the cells that are seeded and cultured next are called second cells CB. In this embodiment, the first cells CA as the first type of cells are Caco-2 (a cell line derived from human colon cancer), and the second cells CB as the second type of cells are vascular endothelial cells.

[0009] As shown in FIG. 1, the cell culture process includes a preparation step P10 and culture steps from step P20 to step P90. In the preparation step P10, a cell culture membrane 20 is prepared. As shown in FIG. 2, in this embodiment, the cell culture membrane 20 is used in a state where it is attached to an insert 10. In FIG. 2, an X-axis, a Y-axis, and a Z-axis that are mutually perpendicular are shown. The direction in which the arrow of the Z-axis points is the upward direction, and the opposite direction is the downward direction. The same applies to the following figures and explanations. The insert 10 is a hanging insert and is appropriately selected from TninCert manufactured by Greiner, Transwell manufactured by Corning, and the like. The insert 10 has a cylindrical portion 11 and a flange portion 12. The diameter of the cylindrical portion 11 is 6.5 mm. In addition, the diameter of the cylindrical portion 11 is, in detail, the inner diameter of the other end of the cylindrical portion 11 described later. The flange portion 12 has a disk-like shape and extends in the outer diameter direction from one end of the cylindrical portion 11. A circular cell culture membrane 20 is attached to cover the other end of the cylindrical portion 11. In this embodiment, the cell culture membrane 20 is attached so that a first surface 22 described later faces the inside of the cylindrical portion 11.

[0010] The cell culture membrane 20 is made of a thermosetting resin. Specifically, polyurethane, polyurethane, polyurea, silicone resin, phenol resin, epoxy resin, unsaturated polyester, polyimide, etc. can be used, and polyurethane is used in this embodiment. The thickness of the cell culture membrane 20 is 3 μm or more and 7 μm or less.

[0011] As shown in FIG. 3, the cell culture membrane 20 has a membrane body 21 having a first surface 22 and a second surface 23 opposite to the first surface 22, and a plurality of through holes 24 formed in the membrane body 21. The plurality of through holes 24 are holes penetrating from the first surface 22 to the second surface 23. The hole diameter of the through hole 24 on the first surface 22 is different from the hole diameter on the second surface 23. The hole diameter of the through hole 24 on the first surface 22 is referred to as a first hole diameter Da, and the hole diameter of the through hole 24 on the second surface 23 is referred to as a second hole diameter Db. The first hole diameter Da is smaller than the second hole diameter Db. In addition, a first average hole diameter, which is the average value of the first hole diameters Da of the plurality of through holes 24, is different from a second average hole diameter, which is the average value of the second hole diameters Db on the second surface 23. The first average hole diameter is smaller than the second average hole diameter. The first average hole diameter is preferably 7 μm or less. As a result, after the first cells CA are seeded on the cell culture membrane 20, the migration of the first cells CA through the through-holes 24 to the surface opposite to the surface on which they were seeded can be suppressed. In the following description, the migration of the first cells CA through the through-holes 24 to the surface opposite to the surface on which they were seeded may be referred to as "wandering". It is more preferable that the first average pore size is 3 μm or more and 5 μm or less. As a result, when the size of the first cells CA at the time of seeding is about 5 μm, the first average pore size is 5 μm or less to suppress the wandering of the first cells CA, while the first average pore size is 3 μm or more to facilitate contact between the first cells CA and the second cells CB. The case where the first average pore size is 3 μm or more means the case where the second average pore size is larger than 3 μm. In this case, the second cells CB can easily enter the through-holes 24 from the second surface 23 side, and the first cells CA can easily contact the second cells CB.

[0012] Here, the first average hole diameter is a value obtained by shining light on the first surface 22 facing upward with the first surface 22 facing upward and observing with a microscope in a state where the first surface 22 is facing upward and the second surface 23 is facing downward. Since light is not reflected from the through-holes 24, the through-holes 24 are visually recognized as black. Specifically, the circle-equivalent diameters of all the through-holes 24 observed in a specific field of view are measured, and the average of the measured values ​​is obtained. The circle-equivalent diameter refers to the diameter of a perfect circle equivalent to the area of ​​the through-holes 24. The second average hole diameter is also a value obtained in the same manner. In other words, the value obtained by shining light on the second surface 23 facing upward with the first surface 22 facing downward and observing with a microscope in a state where the second surface 23 is facing upward.

[0013] The hole density of the plurality of through holes 24 is 2.0×10 5 pieces / cm 2 This makes it possible to suppress the wraparound of the first cells CA. The hole density of the multiple through-holes 24 is 1.5×10 5 pieces / cm 2 The following is preferable. This can further suppress the intrusion of the first cells CA. There are cases where the second cells CB are difficult to settle on the membrane surface of the cell culture membrane 20 on which the first cells CA are settled. Therefore, by suppressing the intrusion of the first cells CA, the first cells CA and the second cells CB can be cultured well on each of the two sides of the cell culture membrane 20. This can create a state closer to that of a living organism.

[0014] The cell culture membrane 20 can be manufactured by supplying water vapor to an uncured polyurethane raw material formed in a thin plate on a film formation substrate, and foaming and curing the uncured polyurethane raw material. According to this manufacturing method, the through-holes 24 are formed by foaming, so that the cell culture membrane 20 having the first hole diameter Da and the second hole diameter Db different from each other can be manufactured.

[0015] In this embodiment, the shape of the through-holes 24 in the first surface 22 is close to a perfect circle. Specifically, in the above-mentioned observation with the microscope, when a rectangle circumscribing the through-holes 24 is drawn, the average value of the ratio of the long side to the short side is about 1.1, but is not limited to this. Also, in this embodiment, the second average hole diameter which is the average value of the second hole diameters Db of the multiple through-holes 24 is 4 μm or more and 7 μm or less, but is not limited to this.

[0016] In step P20 of FIG. 1, the cell culture membrane 20 is immersed in phosphate-buffered saline (PBS) and incubated overnight. The incubation conditions are a temperature of 37° C. and a CO2 concentration of 5%. Specifically, as shown in step P20 of FIG. 4, the insert 10 is placed in one well 30 of a 24-well plate. Since the outer diameter of the flange 12 is larger than the diameter of the well 30, the flange 12 abuts against the peripheral surface of the well 30, and the cell culture membrane 20 is held apart from the bottom surface of the well 30. By incubating overnight in step P20, air bubbles that have adhered to the membrane surface of the cell culture membrane 20 when the cell culture membrane 20 is immersed in phosphate-buffered saline can be removed.

[0017] 1, the first surface 22 and the second surface 23 of the cell culture membrane 20 are coated with fibronectin. This can improve the adhesion rate of the second cells CB, which are vascular endothelial cells, to be seeded second.

[0018] As shown in FIG. 1 and FIG. 4, in steps P40 and P50 as the first step, the first cells CA are seeded and cultured on the first surface 22. In step P40, Caco-2 cells CA are seeded inside the insert 10, i.e., on the first surface 22 of the cell culture membrane 20. As shown in FIG. 1 and FIG. 4, in step P50, the first cells CA are cultured for three days. In this embodiment, 50,000 first cells CA are seeded. The incubation conditions are a temperature of 37° C. and a CO2 concentration of 5%. The medium used is serum-free D-MEM supplemented with bovine serum albumin (BSA) at a concentration of 5 μM.

[0019] In step P60 shown in FIG. 1, the first jig 40 and the second jig 50 are attached to the insert 10. FIG. 5 is a diagram for explaining the attachment of the first jig 40 to the insert 10. FIG. 6 is a diagram for explaining the attachment of the second jig 50 to the insert 10. Specifically, first, as shown in FIG. 5, the first jig 40 is inserted into the insert 10. The first jig 40 has flexibility and a cylindrical shape with a diameter that decreases toward the tip 41. The first jig 40 has a size such that the outer circumferential surface of the first jig 40 is in close contact with the inner circumferential surface of the cylindrical portion 11 during insertion into the cylindrical portion 11. Therefore, before the tip 41 of the first jig 40 abuts against the cell culture membrane 20, the first jig 40 is held in the cylindrical portion 11 with the outer circumferential surface of the first jig 40 in close contact with the inner circumferential surface of the cylindrical portion 11. Then, with the culture medium put in, the inside of the insert 10 is sealed by the first jig 40. Next, the insert 10 with the first jig 40 attached is turned upside down, and the second jig 50 is attached to the outer periphery of the insert 10 so as to cover the cell culture membrane 20, as shown in FIG. 6. The second jig 50 has flexibility and a cylindrical shape. In the attached state, the end of the second jig 50 farther from the flange portion 12, i.e., the upper end, is attached in a state separated from the cell culture membrane 20. As a result, a space in which the culture medium is accommodated is formed by the cell culture membrane 20 and the inner circumferential surface of the second jig 50.

[0020] As shown in FIG. 1 and FIG. 4, in step P70 and step P80 as the second step, the second cells CB are seeded and cultured on the second surface 23. First, in step P70 shown in FIG. 1, the second cells CB are seeded on the inside of the second jig 50, that is, on the second surface 23 of the cell culture membrane 20. As shown in FIG. 1 and FIG. 4, in step P80, the first cells CA and the second cells CB are cultured for 4 hours. In this embodiment, the number of second cells CB seeded is 100,000. The same vascular endothelial cell medium is used for the first cells CA and the second cells CB. As shown in FIG. 1 and FIG. 4, in step P90, the first jig 40 and the second jig 50 are removed from the insert 10. Thereafter, the insert 10 is placed in the well 30, the medium is replaced every day, and the insert 10 is cultured for three days. In addition, in the culture, a medium suitable for each of the first cells CA and the second cells CB is used. The above-described cell culture method realizes a bilayer culture of a Caco-2 cell layer and a vascular endothelial cell layer. The cell culture method according to this embodiment can be used to create an organ model, specifically a small intestine model.

[0021] According to the embodiment described above, in the preparation step P10, the cell culture membrane 20 is prepared. The cell culture membrane 20 is made of a thermosetting resin, and includes a membrane body 21 having a first surface 22 and a second surface 23, and a plurality of through-holes 24 formed in the membrane body 21. The plurality of through-holes 24 have a first average pore size on the first surface 22 that is smaller than a second average pore size on the second surface 23, and a pore density of 2.0×10 5 pieces / cm 2 In the culture step, cells are seeded and cultured on each of the first surface 22 and the second surface 23 of the cell culture membrane 20. The pore density is 2.0×10 5 pieces / cm 2By setting the above, it is possible to suppress the movement of the first cells CA seeded on the first surface 22 through the through-holes 24 to the second surface 23, which is the wraparound. Furthermore, since the second average pore size is larger than the first average pore size, the second cells CB seeded on the second surface 23 can enter the through-holes 24 and approach the first cells CA. Therefore, when the first cells CA and the second cells CB, which are different from each other, are cultured on the first surface 22 and the second surface 23, the distance between the second cells CB and the first cells CA is reduced, and a state close to the state in the living body where different types of cells interact with each other can be created. Therefore, an organ model with good transport ability and barrier properties can be created. By using this organ model, it is possible to shorten the time for drug discovery and development, and reduce the development cost of drug discovery.

[0022] The culture process includes steps P40 and P50 of seeding and culturing the first cells CA on the first surface 22, and steps P70 and P80 of seeding and culturing the second cells CB on the second surface 23. This allows the culture on the first surface 22 and the culture on the second surface 23 to be performed satisfactorily.

[0023] Furthermore, the first cells CA are intestinal epithelial cells, and the second cells CB are vascular endothelial cells. This allows the cultured first cells CA and second cells CB to be used as an organ model.

[0024] B. Other embodiments: (B1) In the above embodiment, first cells CA and second cells CB, which are different types from each other, are cultured on the first surface 22 and the second surface 23 of the cell culture membrane 20. This is not limiting, and the same type of cells may be cultured on the first surface 22 and the second surface 23, or two or more types of cells may be cultured on each of the first surface 22 and the second surface 23. Also, the first cells CA may be cultured on the second surface 23, and the second cells CB may be cultured on the first surface 22. Also, the first cells CA, i.e., the cells seeded and cultured first, may be vascular endothelial cells, and the second cells CB, which are seeded and cultured next, may be Caco-2 cells.

[0025] (B2) In the above embodiment, the first cell CA is Caco-2, and the second cell CB is vascular endothelial cell. The first cell CA is not limited to Caco-2, and may be other intestinal epithelial cells. As other intestinal epithelial cells, HT-29 cells, primary small intestinal epithelial cells, iPS-derived small intestinal epithelial cells, stem cells, Paneth cells, crypt cells, mucus-secreting cells, etc. can be used. In addition, in the above embodiment, vascular endothelial cells are used as the second cell CB to prepare a small intestine model. The second cell CB may be selected according to the organ model to be prepared. As other organ models, a cerebrovascular model, a liver model, a kidney model, and a lung model can be mentioned.

[0026] The present disclosure is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features of the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention can be appropriately replaced or combined to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

[0027] C. Examples and Comparative Examples: C1. Evaluation of wraparound: FIG. 7 is a diagram showing the relationship between pore density and the presence or absence of cell wraparound. Various polyurethane cell culture membranes with a first average pore size and pore density were prepared. The membrane thickness was 3 μm or more and 7 μm or less. The second average pore size was 4 μm or more and 7 μm or less. Using the prepared cell culture membrane, the same process as the cell culture process of the above embodiment was performed to perform cell culture. Caco-2 was cultured on the first surface 22, and vascular endothelial cells were cultured on the second surface 23. Then, Caco-2 and vascular endothelial cells were stained, and the presence or absence of Caco-2 wraparound to the second surface 23 was evaluated by observation with a fluorescent inverted microscope. The size of Caco-2 at the time of seeding is about 5 μm, which is the same as the cell nucleus. The size of Caco-2 at the time of settlement is about 10 μm. Therefore, by using a cell culture membrane with a first average pore size of 5 μm or less, it is possible to suppress the seeded Caco-2 from passing through the through holes 24 and wrapping around from the first surface 22 to the second surface 23. In addition, by using a cell culture membrane having a first average pore size of 3 μm or more, it is possible to facilitate contact between Caco-2 cells and vascular endothelial cells. Therefore, the inventors conducted experiments using cell culture membranes having a first average pore size of 3 μm or more and 5 μm or less.

[0028] As shown in FIG. 7, the first average pore size is 3 μm or more and 5 μm or less, and the pore density is 2.0×10 5 pieces / cm 2 It can be seen that the use of the following cell culture membranes can suppress the infiltration of Caco-2. Furthermore, the pore density is 1.5×10 5 pieces / cm 2 It can be seen that the use of the following cell culture membranes can further suppress the penetration of Caco-2 cells.

[0029] C2. Barrier evaluation: Fig. 8 is a diagram showing the results of transepithelial electrical resistance. Fig. 9 is a diagram showing the results of a Lucifer Yellow permeation test. In the example, the first average pore size is 3 μm or more and 5 μm or less, and the pore density is 2.0 × 10 5 pieces / cm 2The results are as follows: the results were obtained using a cell culture membrane in which the above cell culture was performed and no wraparound was observed. The number of samples was two, and average values ​​are shown in Figures 8 and 9. The comparative example was a membrane made of PET (Polyethylene terephthalate) with a pore diameter of 3 μm and a pore density of 2.0 × 10 6 pieces / cm 2 The results are from a cell culture membrane with a thickness of 10 μm. In the comparative example, the through-holes are formed by a laser, so the pore size on the first surface of the cell culture membrane is approximately the same as that on the second surface. In the Lucifer Yellow permeation test, Lucifer Yellow was added to the inside of the insert 10, and after incubation, the solution between the well 30 and the insert 10 was collected and measured, and the permeability coefficient was calculated.

[0030] As shown in FIG. 8, the transepithelial electrical resistance of the Example is higher than that of the Comparative Example. Also, as shown in FIG. 9, the permeability coefficient of Lucifer Yellow of the Example is smaller than that of Lucifer Yellow of the Comparative Example. Therefore, it can be seen that the Example has higher barrier properties than the Comparative Example. This is considered to be because, in the Example, Caco-2 and vascular endothelial cells are well cultured, and in the cell culture membrane of the Example, Caco-2 and vascular endothelial cells come into contact with each other, creating a state closer to that of a living body than in the Comparative Example. In contrast, in the cell culture membrane of the Comparative Example, the pore size on the first surface and the pore size on the second surface are approximately the same, so that the vascular endothelial cells cannot enter the through holes, and there is no or little opportunity for Caco-2 and vascular endothelial cells to come into contact with each other.

[0031] C3. Evaluation of selective transport: FIG. 10 is a diagram for explaining the method of the transport test. FIG. 11 is a diagram showing the results of the digoxin transport test. The examples and comparative examples shown in FIG. 11 are the same samples as those used in the evaluation of barrier properties. FIG. 10 is a diagram showing, in detail, the procedure of the transport test (A to B) from the Caco-2 cell side (apical side) to the vascular endothelial cell membrane side (basal side). In step 1 of FIG. 10, the medium is replaced with Hank's balanced salt solution (HBSS) and incubated for 20 minutes. The incubation conditions are a temperature of 37° C. and a CO2 concentration of 5%. In step 2, a digoxin solution is added to the insert 10. The concentration of digoxin is adjusted to 5 μM. In step 3, the culture is performed for 2 hours. The incubation conditions are a temperature of 37° C. and a CO2 concentration of 5%. In step 4, 100 μL of solution is collected twice from between the well 30 and the insert 10. In step 5, the digoxin concentration is measured by a liquid chromatograph mass spectrometer (LC / MS / MS) and the transport rate is calculated. A transport test from the vascular endothelial cell side to the Caco-2 side (B to A) is also performed in the same manner. That is, in step 2, a digoxin solution is added between the well 30 and the insert 10, and in step 4, the digoxin concentration of the solution collected from inside the well 30 is measured and the transport rate is calculated. The transport rate B to A is divided by the transport rate A to B to calculate the transport rate ratio.

[0032] 11, the transport rate ratio is larger in the Example than in the Comparative Example, and the selective transport ability of digoxin is higher than in the Comparative Example. As with the above-mentioned barrier property results, this is thought to be because the cell culture membrane used in the Example creates a state closer to that of a living body, as Caco-2 and vascular endothelial cells come into contact with each other in the cell culture membrane used in the Example than in the cell culture membrane used in the Comparative Example. [Explanation of symbols]

[0033] 10...insert, 11...cylindrical portion, 12...flange portion, 20...cell culture membrane, 21...membrane body, 22...first surface, 23...second surface, 24...through hole, 30...well, 40...first fixture, 41...tip, 50...second fixture, CA...first cell, CB...second cell, Da...first hole diameter, Db...second hole diameter, P10...preparation step, P20 to P90...steps

Claims

1. 1. A cell culture method comprising: (i) a membrane body formed of a thermosetting resin and having a first surface and a second surface opposite to the first surface; and (ii) a plurality of through holes formed in the membrane body and penetrating from the first surface to the second surface; A culture step of seeding and culturing cells on each of the first surface and the second surface of the prepared cell culture membrane, The plurality of through holes have a first average hole diameter in the first surface that is smaller than a second average hole diameter in the second surface, The hole density of the plurality of through holes is 2.0×10 ５ pieces / cm ２ is as follows: In the culturing step, the types of cells seeded and cultured on the first surface and the types of cells seeded and cultured on the second surface are different from each other; The cell culture method, wherein the first average pore size is 7 μm or less.

2. The cell culture method according to claim 1, The culture step comprises: a first step of seeding and culturing cells on the first surface; A cell culture method comprising: a second step of seeding and culturing cells on the second surface after the first step.

3. The cell culture method according to claim 1 or 2, the first cells seeded and cultured on the first surface are intestinal epithelial cells; A cell culture method, wherein the second cells seeded and cultured on the second surface are vascular endothelial cells.

4. The cell culture method according to claim 1, The cell culture method, wherein the first average pore size is 3 μm or more and 5 μm or less.

5. The cell culture method according to any one of claims 1 to 4, The pore density is 1.5×10 ５ pieces / cm ２ The cell culture method is as follows:

6. 1. A cell culture membrane comprising: A membrane body formed of a thermosetting resin and having a first surface and a second surface opposite to the first surface; A plurality of through holes are formed in the film body and extend from the first surface to the second surface, The plurality of through holes have a first average hole diameter in the first surface that is smaller than a second average hole diameter in the second surface, The hole density of the plurality of through holes is 2.0×10 ５ pieces / cm ２ is as follows: The first average pore size is 7 μm or less; the first surface is used for seeding and culturing a first type of cell; The second surface is a cell culture membrane used for seeding and culturing a second type of cells that is different from the first type of cells.

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