WO2012032646A1 - Cell culture device and cell culture method - Google Patents

Abstract

A cell culture device, comprising: an intermediate layer consisting of two porous membranes (14, 24); a first culture chamber (11a) and a second culture chamber (21a), said culture chambers being partitioned by the intermediate layer; and limiting layers (13, 23) which are positioned on both sides of the intermediate layer and provided with multiple openings (13a, 23a) for exposing the intermediate layer to a liquid flowing between the culture chambers (11a, 21a). Since the intermediate layer consists of the two porous membranes (14, 24), different scaffolds, which are suitable for the type of cells to be cultured, can be coated on both sides of the intermediate layer. Since areas to which cells can be adhered are limited by the limiting layers (13, 23), the cells can be fixed to the intermediate layer in a desired pattern that is suitable for the expression of the function thereof. Thus, an environment closer to the in vivo environment can be constructed in the device and cells can be cultured over a long period of time while sustaining the cell function.

Description

Cell culture device and cell culture method

The present invention relates to a cell culture device and a cell culture method using the same.

Cell culture is generally performed in a state where cells and a liquid medium are contained in a container such as a petri dish. However, in recent years, with the progress of microfabrication technology in the semiconductor manufacturing field, the application of microdevices manufactured by microfabrication technology has been promoted in the medical and biotechnology research fields, and cell culture using such microdevices has been promoted. (For example, refer to Patent Document 1).

A microdevice for cell culture (cell culture device) is formed by forming a culture chamber and a microchannel inside a flat substrate, and performs cell culture by accommodating cells and a medium in the culture chamber. The medium is exchanged using the microchannel.

By the way, in recent years, there has been an expectation for the development of an artificial organ, and many studies have been made on the development of an artificial liver, which is a part of the development. However, since only the liver is known, it performs over 500 kinds of metabolic reactions, and it is extremely difficult to completely supplement such liver functions with only an artificial device. Therefore, a so-called hybrid type artificial liver combining an artificial device and a living hepatocyte has been devised, and its development is underway.

In the liver of a living body, hepatic parenchymal cells constituting 70-80% of the liver and non-parenchymal cells such as sinusoidal endothelial cells are regularly arranged. Signal transduction and fluid circulation between these cells are It plays an important role in maintaining normal liver function.

The hepatic parenchymal cells and sinusoidal endothelial cells are arranged side by side, but an extracellular matrix called a dyssel cavity exists between them, and the cell line of hepatic parenchymal cells and the cell line of sinusoidal endothelial cells There is no direct contact. Further, outside the respective cell rows, the hepatic parenchymal cell side is a bile duct and the sinusoidal endothelial cell side is a blood vessel, and drugs and nutrients are supplied from the blood vessel side and discharged to the bile duct side.

In order to establish an artificial liver system with liver function, regular arrangement of these cell types and construction of an extracellular matrix are important. However, hepatocytes are difficult to culture for a long period of time, and moreover, it is more difficult to form a tissue close to the liver in vivo. Therefore, an artificial system that can maintain a stable liver function over a long period of time has not yet been constructed.

International Publication WO2009 / 099066 Pamphlet

In connection with the construction of such an artificial liver system, the present inventors have proposed a biodevice using two types of living cells (Patent Document 1). This uses a porous membrane as an alternative to the extracellular matrix, and by fixing and culturing different cells on both sides of the membrane, signal transmission and substance exchange between the cells on both sides via the porous membrane Or it is made to move.

Such a culture device is epoch-making in that it can realize a configuration close to that of a living body as compared with the case of culturing one kind of cell alone, but there is room for further improvement in terms of long-term maintenance of cell functions. was there.

The present invention has been made in view of the above points. The object of the present invention is to use a cell culture device capable of maintaining a stable function over a long period of time using two types of biological cells, and the same. It is to provide a cell culture method.

The cell culture device according to the present invention, which has been made to solve the above problems,
a) an intermediate layer consisting of a porous membrane and coated with a scaffold for cell culture;
b) a first culture chamber and a second culture chamber formed by partitioning the space inside the device with the intermediate layer;
c) a first limiting layer that covers a part of one surface of the intermediate layer and limits a region where the intermediate layer is in contact with the liquid flowing in the first culture chamber;
d) a second limiting layer that covers a part of the other surface of the intermediate layer and limits the region where the intermediate layer is in contact with the liquid flowing in the second culture chamber;
It is characterized by having.

The cell culture device according to the present invention has two culture chambers partitioned by an intermediate layer, and can culture different types of cells simultaneously in each culture chamber. The heterogeneous cells are fixed to both sides of the intermediate layer coated with the scaffold, and signal transmission between the cells and exchange of substances can be performed through the porous membrane constituting the intermediate layer. Conventionally, when cells are introduced into such a cell culture device, the cells are suspended in a medium and the suspension is injected into the culture chamber by a syringe or the like. However, in this case, the injected cells are biased to adhere to the vicinity of the entrance of the culture chamber, making it difficult to spread the cells throughout the culture chamber. In particular, depending on the type of cell, there are those that can not function unless they form a tight junction with the surrounding cells, and if there are many areas with low cell density in the culture chamber, stable function may not be obtained. there were. On the other hand, according to the cell culture device of the present invention having the above-described configuration, the cell suspension and the intermediate layer are introduced when cells are introduced into the device by providing the first limited layer and the second limited layer. Can be limited, so that a region to which cells adhere can be optimally designed in the culture chamber. As a result, an environment closer to the living body can be easily realized, and a stable cell function can be obtained over a long period of time.

As the porous film, one made of polycarbonate can be suitably used, but is not limited to this, and is made of polytetrafluoroethylene, mixed cellulose, polyethylene terephthalate (PET), glass fiber, polyether, or fluorine resin. And various types such as cellulose-based, nylon-based, and ceramic-based porous membranes can be used. In addition, the porous membrane must be such that it does not pass cells and does not hinder the efficiency of substance exchange and signal transmission between cells. Therefore, it is desirable to use a porous membrane having a thickness of 1 mm or less and an average pore diameter of 0.1 μm to 10 μm.

The first limited layer and the second limited layer have a plurality of openings that expose the intermediate layer to the liquid flowing through the culture chambers, and the opening area per one opening is 0. It is desirable that the thickness be 0.01 mm ² to 10 mm ² .

If the opening area is smaller than this, the number of cells that can be fixed to the intermediate layer with one opening is reduced, and if it is larger than this, it is difficult to obtain the effect of limiting the adhesion region. The thickness of the limiting layer is suitably 1 mm or less. When larger than this, since the said opening part becomes deep, the efficiency of the liquid exchange around a cell in culture | cultivation falls.

In the cell culture device according to the present invention, it is preferable that the intermediate layer is formed by stacking a plurality of porous membranes.

This makes it possible to coat different scaffolds according to the type of cultured cells on the porous membrane on the first culture chamber side and the porous membrane on the second culture chamber side, realizing an environment closer to the living body. It becomes possible to do.

As the scaffold, E-cad-Fc (a fusion protein of an extracellular region of E-cadherin and an Fc fragment of an antibody IgG molecule) can be preferably used, but in addition, it is generally used as a scaffold. And polymers containing collagen, fibronectin, laminin, PVLA (poly-Np-vinylbenzyl-D-lactone amide) and the like can be used.

The cell culture method according to the present invention is a cell culture method using the cell culture device according to the present invention, wherein different types of cells are fixed to one surface and the other surface of the intermediate layer and cultured. It is characterized by doing.

Here, the different cells are typically hepatic parenchymal cells and sinusoidal endothelial cells, but the cell device according to the present invention is not limited to these and can be used for culturing various cells.

As described above, according to the cell culture device and the cell culture method according to the present invention, it is possible to easily realize an environment close to the living body in the cell culture using two types of living cells, and stable cells. The function can be obtained over a long period of time.

The top view of the cell culture device which concerns on one Example of this invention. FIG. 2 is a cross-sectional view taken along arrow XX in FIG. 1. The disassembled perspective view of the cell culture device which concerns on the Example. The schematic block diagram of the cell culture system containing the cell culture device of the Example. The graph which shows the evaluation result of the testosterone hydroxylation ability of the cultured cell in Experimental example 1. FIG. The graph which shows the hydroxylation pattern of the cultured cell and liver microsome in Experimental example 1. FIG. The graph which shows the evaluation result of the testosterone hydroxylation ability of the cultured cell in Experimental example 2. FIG. The graph which shows the evaluation result of the urea synthetic ability of the cultured cell in Experimental example 2. FIG.

Hereinafter, modes for carrying out the present invention will be described using examples. 1 is a plan view of the cell culture device according to the present embodiment, FIG. 2 is a cross-sectional view taken along the line XX of FIG. 1, and FIG. 3 is an exploded perspective view of the cell culture device according to the present embodiment. In FIG. 1, for the sake of explanation, a part of the internal structure is shown through.

The cell culture device 10 according to the present embodiment has a configuration in which two porous films 14 and 24, limited layers 13 and 23, and seal layers 12 and 22 are sandwiched between two substrates 11 and 21, respectively. It has become. In addition, the dimension and material of each part described below are an example to the last, and this invention is not limited to this.

The substrates 11 and 21 are made of PDMS (manufactured by Toray Dow Corning, SILPOT184). On one surface of the substrate 11, there are a first culture chamber 11a composed of a rectangular recess having a length of 16 mm and a width of 2 mm, and groove-shaped channels 11b and 11c extending from the vicinity of both ends of the first culture chamber 11a. Is formed. The first culture chamber 11a and the channels 11b and 11c all have a depth of 0.1 mm and can be formed by molding. The ends of the two flow paths 11b and 11c communicate with a liquid introduction port 11d or a liquid discharge port 11e each formed of a through hole extending in the thickness direction of the substrate 11. Further, a second culture chamber 21a, flow paths 21b and 21c, a liquid inlet 21d, and a liquid outlet 21e are formed in the substrate 21 in the same manner.

The limiting layers 13 and 23 are made of a metal mask made of SUS having a thickness of 0.1 mm, and the central portion thereof has a diameter of 0.5 mm in a region corresponding to the first culture chamber 11a or the second culture chamber 21a, respectively. 30 through- holes 13a and 23a are formed. The through holes 13a and 23a correspond to the openings in the present invention. The shape and number of the openings are not limited to the above, and can be various depending on the type of cells to be cultured and the purpose of the culture. The limiting layers 13 and 23 are made of a material that is difficult to adhere to cells to be cultured (at least those having lower cell adhesion than the porous membranes 14 and 24 coated with a scaffold). desirable. As such a material, in addition to the metal such as SUS, various resins can be used.

The seal layers 12 and 22 are made of silicon rubber having a thickness of 0.1 mm, and the length and width are almost the same as those of the substrates 11 and 21. A rectangular hole having substantially the same length and width as the limiting layers 13 and 23 is formed in the center of the sealing layers 12 and 22, and the limiting layer 13 or the limiting layer 23 is fitted into the rectangular holes.

The porous membranes 14 and 24 are punched into a rectangle having a width of 2 mm and a length of 14 mm. In this example, polycarbonate membrane filters (Millipore Corp., Isopore HTTP04700, filter pore diameter 0.4 μm, thickness 7-22 μm) were used as the porous membranes 14, 24. In addition, what overlap | superposed these porous membranes 14 and 24 is equivalent to the intermediate | middle layer in this invention.

The porous membranes 14 and 24 are each coated with a scaffold according to the type of cells to be cultured. In this example, the porous membrane 14 on the first culture chamber 11a side was coated with E-cad-Fc, and the porous membrane 24 on the second culture chamber 21a side was coated with type I collagen. The method of coating the scaffold material is not particularly limited. For example, after the porous films 14 and 24 are dipped and taken out from the liquefied scaffold material, the scaffold material adhered to the porous films 14 and 24 is solidified. Can be considered. Such coating with a scaffold may be performed at the manufacturing stage of the cell culture device, or may be performed by a user who has purchased the cell culture device.

When assembling the cell culture device 10 of the present embodiment, each of the above layers is sterilized and the porous membranes 14 and 24 are coated with a scaffold, and then the substrate 21, the seal layer 22, the limiting layer 23, and the porous membrane 24. The porous film 14, the limiting layer 13, the seal layer 12, and the substrate 11 are superposed in this order. At this time, the substrates 11 and 21 are configured such that the surface on which the first culture chamber 11a is formed faces the surface on which the second culture chamber 21a is formed. As described above, since the limiting layers 13 and 23 are fitted into the holes in the center of the sealing layers 12 and 22, the sealing layer 12 and the limiting layer 13, and the sealing layer 22 and the limiting layer 23 respectively. It will be arranged at the same position in the thickness direction.

When the layers are overlapped as described above, the substrate 11 and the seal layer 12, the substrate 21 and the seal layer 22, and the seal layers 12 and 22 are adsorbed by the self-adsorption property of the silicon rubber constituting the seal layers 12 and 22. The Therefore, the substrate 11 and the substrate 21 can be fixed to each other with the porous films 14 and 24 and the limiting layers 13 and 23 sandwiched therebetween.

When cell culture is performed by the cell culture device 10 described above, hepatic parenchymal cells are introduced from the liquid inlet 11d into the first culture chamber 11a, and sinusoidal endothelial cells are introduced from the liquid inlet 21d into the second culture chamber 21a. Each cell is fixed to the surface of the porous membranes 14 and 24, respectively. Specifically, the cell culture device 10 is first placed so that the first culture chamber 11a side is facing upward, the liver parenchymal cells are suspended in the medium, and the suspension is injected into the first culture chamber 11a with a syringe or the like. To do. The injected cells sink by gravity and adhere to the porous membrane 14. Thereafter, the cell culture device 10 is placed so that the second culture chamber 21a side is up, and sinusoidal endothelial cells are injected into the second culture chamber 21a in the same manner and adhered to the porous membrane 24.

Part of the porous membranes 14 and 24 is masked by the limiting layers 13 and 23 so that the liquid in the first culture chamber 11a or the second culture chamber 21a can be contacted only at the through holes 13a and 23a. It has become. For this reason, some of the cells introduced together with the medium from the liquid inlets 11d and 21d as described above enter the through holes 13a and 23a near the upstream of the culture chamber and adhere to the porous membranes 14 and 24. The remaining cells float in the medium or fall on the surfaces of the limiting layers 13 and 23. Since the limiting layers 13 and 23 are less likely to adhere cells than the porous membranes 14 and 24, these cells ride on the flow of the medium and flow downstream of the culture chambers 11a and 21a. Then, in the process, one of the through- holes 13a and 23a enters, and adheres to the porous films 14 and 24 there. As described above, in the culture device according to the present embodiment, since the portions where the cells can contact the porous membranes 14 and 24 are limited by the limiting layers 13 and 23, the cells injected into the device are cultured as in the past. It is possible to prevent the cells from being biased to adhere to the vicinity of the entrance of the chamber and to spread the cells throughout the culture chamber. Further, since the cells gather inside the through holes 13a and 23a, stable cell functions can be exhibited even when culturing cells that form a tight junction with the surrounding cells.

FIG. 4 shows an outline of a cell culture system using the cell culture device 10 according to the present example. This is a combination of the cell culture device 10 and a liquid feeding mechanism for continuously feeding a medium to the cell culture device 10. The liquid feeding mechanism is configured to operate the medium storage units 31 and 41, the medium supply pipes 32 and 42, the medium discharge pipes 33 and 43, the waste liquid storage units 34 and 44, the liquid feed pumps 35 and 45, and the liquid feed pumps 35 and 45. The control part 50 which controls is provided. One end of each of the medium supply pipes 32 and 42 is inserted into the medium reservoirs 31 and 41, and the other end is inserted into the liquid introduction ports 11d and 21d of the cell culture device 10, respectively. One ends of the medium discharge pipes 33 and 43 are respectively inserted into the liquid discharge ports 11e and 21e of the cell culture device 10, and the other ends are respectively inserted into the waste liquid storage portions 34 and 44.

A medium suitable for culturing hepatocytes is stored in the medium reservoir 31, and a medium suitable for culturing sinusoidal endothelial cells is stored in the medium reservoir 41. The medium stored in the medium storage unit 31 is sucked by the liquid feed pump 35 and sent to the cell culture device 10 through the medium supply pipe 32. The medium supplied to the cell culture device 10 passes through the first culture chamber 11a, and is discharged to the waste liquid storage part 34 through the medium discharge pipe 33 connected to the liquid discharge port 11e. Similarly, the culture medium stored in the culture medium storage unit 41 is sucked by the liquid feed pump 45, passes through the culture medium supply pipe 42, passes through the second culture chamber 21 a, and is discharged to the waste liquid storage part 44 through the culture medium discharge pipe 43. Is done.

In addition, since the first culture chamber 11a and the second culture chamber 21a are sealed by the self-adsorbing sealing layers 12 and 22, the culture medium is exposed to the outside from the liquid inlets 11d and 21d or the liquid outlets 11e and 21e. Although it does not leak out, in order to prevent liquid leakage more reliably, it is desirable to fix the cell culture device 10 by sandwiching it from above and below with an appropriate jig.

According to the cell culture device 10 according to the above-described embodiment, the porous membranes 14 and 24 can realize an ordered arrangement of cells instead of the extracellular matrix, and signal transmission and substance exchange between cells can be realized. It can be secured. Furthermore, the culture media in the first culture chamber 11a and the second culture chamber 21a play roles similar to bile and blood, respectively, and can exhibit functions close to the living body such as metabolism and drug uptake / discharge.

In addition, as described above, according to the cell culture device 10 according to the present example, by restricting the region to which the cells can be adhered by the limiting layers 13 and 23, the cells are biased and adhered near the entrances of the culture chambers 11a and 21a. The cells can be adhered to the culture chambers 11a and 21a in a desired pattern suitable for function expression. Furthermore, by providing two porous membranes 14 and 24, it becomes possible to use different scaffolding materials according to the types of cells cultured in the first culture chamber 11a and the second culture chamber 21a. As a result, an environment closer to the living body can be established inside the device, and cell culture can be performed in a state where expression of liver function is maintained for a long period of time.

As mentioned above, although the form for implementing this invention using the Example was demonstrated, this invention is not limited to this, A change is suitably accept | permitted in the range of the meaning of this invention. . For example, in the above example, the substrates 11 and 21 are fixed to each other by the seal layers 12 and 22. However, the seal layers 12 and 22 are not provided and the self-adsorption property of the PDMS constituting the substrates 11 and 21 is not provided. The substrates 11 and 21 may be fixed to each other. In this case, in order to obtain stronger adhesion, it is desirable to activate and bond the bonding surfaces of the substrates 11 and 21 with oxygen plasma or ultraviolet rays.

In addition, the cell culture device according to the present invention can be used as an artificial organ such as the above-described hybrid type artificial liver, and can also be used as a reaction container when performing a drug metabolism test using cells. In the above example, the case where hepatocytes and sinusoidal endothelial cells are co-cultured has been described. However, the cell culture device of the present invention can also be used for culturing other cells.

Hereinafter, a cell culture experiment using the cell culture device according to the present invention will be described.

[Experimental Example 1]
In the cell culture device 10 as shown in FIGS. 1 to 3, cell culture was performed by changing the material of the porous membranes 14 and 24, and the difference in liver function was evaluated. As the porous membranes 14 and 24, three types of porous membranes each made of polycarbonate, polytetrafluoroethylene, or mixed cellulose were used, and each was coated with type I collagen and used in the experiment.

In this experimental example, the liver function on the first day of culture was evaluated by examining the hydroxylation ability of testosterone by CYP (cytochrome P450). Specifically, first, about 3 × 10 ³ liver parenchymal cells were seeded in the first culture chamber, and about 3 × 10 ³ sinusoidal endothelial cells were seeded in the second culture chamber. Then, a culture medium containing testosterone at a concentration of 0.25 mM is poured into the first culture chamber, a culture medium containing no testosterone is poured into the second culture chamber, and the culture is conducted to the medium (waste liquid) discharged from the first culture chamber. The concentration of testosterone hydroxide contained was measured by HPLC. Each medium was continuously fed to each culture chamber at a flow rate of 40 μL / hr.

As a result, as shown in FIG. 5, it was found that the highest testosterone hydroxylation ability was obtained when a porous film made of polycarbonate was used as the porous films 14 and 24.

Furthermore, the hydroxylation pattern (that is, the abundance ratio of various hydroxylated testosterone in the waste liquid) in the case of using the above three kinds of porous membranes was compared with the hydroxylation pattern by liver microsomes. The hydroxylation pattern by liver microsomes was determined by adding the same medium as that introduced into the first culture chamber to the liver microsome fraction, incubating for 24 hours, collecting the supernatant, and containing various hydroxides by HPLC. Determined by measuring the amount.

The comparison result of the above hydroxylation pattern is shown in FIG. In the figure, 16β-OHT, 2α-OHT, 16α-OHT, 6β-OHT, and 7α-OHT mean testosterone in which the 16β, 2α, 16α, 6β, and 7α positions are hydroxylated, respectively. As is apparent from the figure, it was found that when a porous membrane made of polycarbonate was used, a hydroxylation pattern closest to liver microsomes was obtained.

[Experiment 2]
In a separate preliminary experiment, it was found that type I collagen is suitable as a scaffold for culturing sinusoidal endothelial cells. Therefore, only the scaffold on the liver parenchymal cell side was changed, and cell culture was performed. The liver function was evaluated.

The cell culture device used in this example uses a polycarbonate porous membrane as the porous membranes 14 and 24, and a scaffold material to be coated on the porous membrane 14 on the liver parenchymal cell side (that is, the first culture chamber 11a side) is used. Except for the changed points, the second embodiment is the same as the first embodiment. As the liver parenchymal cell side coating, six types of type I collagen, type IV collagen, fibronectin, laminin, PVLA, and E-cad-Fc were used.

Similar to Experimental Example 1, hepatocellular parenchymal cells were seeded on the first culture chamber side and about 3 × 10 ³ sinusoidal endothelial cells were seeded on the second culture chamber side, and cell culture was performed. Hydroxylation ability and urea synthesis ability (capability of decomposing ammonia to synthesize urea) on the first day, the fourth day, and the seventh day of the culture were evaluated.

The testosterone hydroxylation ability was discharged from the first culture chamber by culturing by flowing a medium containing testosterone in the first culture chamber and a medium not containing testosterone in the second culture chamber, as in Experimental Example 1 above. The concentration of testosterone hydroxide contained in the medium was evaluated by measuring with HPLC. The ability to synthesize urea was obtained by flowing a medium containing NH ₄ Cl at a concentration of 2.0 mM in the first culture chamber, and a medium not containing NH ₄ Cl flowing in the second culture chamber, On day 7, the urea concentration in the medium discharged from the first culture chamber was evaluated by measuring by HPLC. In each case, each medium was continuously fed to each culture chamber at a flow rate of 40 μL / hr.

FIG. 7 shows the evaluation results of testosterone hydroxylation ability and FIG. 8 shows the evaluation results of urea synthesis ability. As shown in FIG. 7, when the porous membrane on the liver parenchymal cell side was coated with PVLA and E-cad-Fc, it was coated with type I collagen (that is, the same as the sinusoidal endothelial cell side). Testosterone hydroxylating ability was higher than when the coating was applied.

In addition, as shown in FIG. 8, the porous membrane is made of type IV collagen, fibronectin, laminin, PVLA, or E-cad-Fc rather than the case where the porous membrane on the hepatocyte side is coated with type I collagen. In the case of coating, it was confirmed that the urea synthesis ability was maintained for a longer period of time (that is, the urea synthesis ability on the seventh day of culture increased).

Thus, different results were obtained for both testosterone hydroxylation ability and urea synthesis ability depending on the type of coating. This indicates the effectiveness of coating an appropriate scaffold according to the cell type in each culture chamber.

DESCRIPTION OF SYMBOLS 10 ... Cell culture device 11, 21 ... Board | substrate 11a ... 1st culture chamber 21a ... 2nd culture chamber 11d, 21d ... Liquid inlet 11e, 21e ... Liquid outlet 12, 22 ... Seal layer 13, 23 ... Limitation layer 13a, 23a ... through holes 14, 24 ... porous membranes 31, 41 ... medium reservoirs 32, 42 ... medium supply pipes 33, 43 ... medium discharge pipes 34, 44 ... waste liquid storage parts 35, 45 ... feed pump 50 ... control part

Claims (6)

1. a) an intermediate layer consisting of a porous membrane and coated with a scaffold for cell culture;
   b) a first culture chamber and a second culture chamber formed by partitioning the space inside the device with the intermediate layer;
   c) a first limiting layer that covers a part of one surface of the intermediate layer and limits a region where the intermediate layer is in contact with the liquid flowing in the first culture chamber;
   d) a second limiting layer that covers a part of the other surface of the intermediate layer and limits the region where the intermediate layer is in contact with the liquid flowing in the second culture chamber;
   A cell culture device comprising:
2. The first limited layer and the second limited layer have a plurality of openings for exposing the intermediate layer to the liquid flowing through the culture chambers, and the opening area per one opening is 0. The cell culture device according to claim 1, wherein the cell culture device is 0.01 mm ² to 10 mm ² .
3. The cell culture device according to claim 1 or 2, wherein the intermediate layer is formed by stacking a plurality of porous membranes.
4. The porous membrane on the first culture chamber side and the porous membrane on the second culture chamber side among the plurality of porous membranes are coated with different scaffolds, respectively. Cell culture device.
5. A cell culture method using the cell culture device according to any one of claims 1 to 4,
   A cell culturing method, wherein different types of cells are fixed and cultured on one side and the other side of the intermediate layer.
6. The cell culture method according to claim 5, wherein the different cells are liver parenchymal cells and endothelial cells.

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