Cell culture apparatus and cell culture method

Abstract

This cell culture apparatus is equipped with a hydrogel chamber, a culture vessel and a pneumatic device. The hydrogel chamber holds a hydrogel for culturing a cell. The culture vessel is equipped with a first culture solution storage unit which is connected to a first surface of the hydrogel chamber, and a second culture solution storage unit which is connected to a second surface of the hydrogel chamber which differs from the first surface. The pneumatic device is connected to the culture vessel, produces a pressure difference based on air pressure between the first culture solution storage unit and the second culture solution storage unit, and causes pressurized osmosis of the culture solution stored in the first culture solution storage unit and / or the second culture solution storage unit into the hydrogel in the hydrogel chamber.

Description

Technical Field

[0001] The present invention relates to a cell culture device and a cell culture method. This application claims priority based on Japanese Patent Application No. 2022-110291 filed in Japan on July 8, 2022, and incorporates its content herein by reference.

Background Art

[0002] Patent Document 1 below discloses a microchannel device. This microchannel device includes a base material made of an optically transparent material, and further includes one or more fluid channels, one or more fluid channel inlets, one or more fluid channel outlets, one or more gel cage regions, and a plurality of struts. All or part of each gel cage region has all or part of one or more fluid channels disposed on the side, thereby creating one or more gel cage region-fluid channel interface regions.

[0003] Each gel cage region includes at least one row of struts forming the gel cage region, and in at least one row of struts, each strut is formed in a triangle, a trapezoid including an interior angle of less than 90°, or a combination thereof. Also, the sum of the interior angle of each strut's corner and the contact angle of the gel on the base material surface in at least one row of struts is 180°, and the distance between each pair of adjacent struts in at least one row of struts is 50 micrometers to 300 micrometers.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Conventionally, a cell culture method has been known that uses the above-mentioned microfluidic devices to construct microvessels and other structures in vitro by placing vascular endothelial cells inside or near a hydrogel and inducing spontaneous vascular tissue formation. However, this method has limitations in the size of the vascular tissue that can be constructed due to restrictions on the supply of oxygen and nutrients to the hydrogel.

[0006] This invention has been made in view of the above-mentioned problems, and aims to provide a cell culture apparatus and a cell culture method that can efficiently construct large cell tissues in vitro. [Means for solving the problem]

[0007] A cell culture apparatus according to one aspect of the present invention comprises a hydrogel chamber for holding a hydrogel for culturing cells, a culture vessel having a first culture medium storage section communicating with a first surface of the hydrogel chamber, and a second culture medium storage section communicating with a second surface of the hydrogel chamber different from the first surface, and a pneumatic device connected to the culture vessel, wherein the pneumatic device generates a pressure difference based on air pressure between the first culture medium storage section and the second culture medium storage section, thereby pressurizing and permeating the culture medium stored in at least one of the first culture medium storage section and the second culture medium storage section into the hydrogel of the hydrogel chamber.

[0008] Furthermore, in a cell culture apparatus according to one aspect of the present invention, at least one of the first culture medium storage section and the second culture medium storage section may include a culture medium storage tank for storing culture medium and a microchannel for connecting the culture medium storage tank and the hydrogel chamber.

[0009] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the microchannel may be provided with one or more resistive channels having a smaller cross-sectional area than the microchannel, arranged in parallel.

[0010] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the flow resistance (R) of the resistive channel MC) is defined as the viscosity of the culture medium (μ), the length from the first surface to the second surface of the hydrogel chamber (L), the height of the hydrogel chamber (d), and the value obtained by dividing the width of the first or second surface by the height of the hydrogel chamber (N), where R MC ≥8 μL / πd 4 The relationship N may also be satisfied.

[0011] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the microchannels may be provided in pairs.

[0012] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the first culture medium storage unit comprises a first culture medium storage tank as the culture medium storage tank and a first microchannel as the microchannel, the second culture medium storage unit comprises a second culture medium storage tank as the culture medium storage tank and a second microchannel as the microchannel, and the return channel for the culture medium may be provided with a culture medium return channel connecting the first culture medium storage tank and the second culture medium storage tank, at least one of the first microchannel and the second microchannel, and a mechanism to prevent backflow of the culture medium.

[0013] Furthermore, in a cell culture apparatus according to one aspect of the present invention, a passive valve that prevents the entry of air bubbles may be provided as a mechanism to prevent backflow.

[0014] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the pressure resistance of the passive valve may be in the range of 0.1 [kPa] to 10 [kPa].

[0015] Furthermore, in a cell culture apparatus according to one aspect of the present invention, an opening may be formed directly above the hydrogel chamber.

[0016] Furthermore, in a cell culture apparatus according to one aspect of the present invention, a third culture medium storage section is provided that communicates with the opening, and the second opening may be provided with a permeable membrane having one side facing the hydrogel and the other side facing the culture medium.

[0017] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the pneumatic device may be connected to the third culture medium reservoir.

[0018] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the pneumatic device includes pneumatic piping connected to the culture vessel, and a filter may be provided in the pneumatic piping.

[0019] Furthermore, in a cell culture apparatus according to one aspect of the present invention, the hydrogel chamber may be provided with a mechanism for holding the hydrogel in a state where a pressure difference based on the air pressure is generated.

[0020] A cell culture method according to one aspect of the present invention involves culturing cells in the hydrogel chamber using the cell culture apparatus described above.

[0021] Furthermore, in a cell culture method according to one aspect of the present invention, the cells may be cultured and vascular tissue may be formed in the hydrogel chamber. [Effects of the Invention]

[0022] According to one aspect of the present invention described above, large cell tissues can be efficiently constructed in vitro. [Brief explanation of the drawing]

[0023] [Figure 1] This is a perspective view showing a cell culture apparatus according to the first embodiment. [Figure 2] This is a cross-sectional view taken along the line II-II shown in Figure 1. [Figure 3] This is a cross-sectional view taken along the line III-III shown in Figure 2. [Figure 4] This is an enlarged view of region A shown in Figure 3. [Figure 5] This is a perspective view showing a cell culture apparatus according to the second embodiment. [Figure 6] This is a cross-sectional view taken along the line VI-VI shown in Figure 5. [Figure 7] This is a cross-sectional view taken along the line VII-VII shown in Figure 6. [Figure 8] This is a perspective view showing a cell culture apparatus according to the third embodiment. [Figure 9] This is a plan cross-sectional view showing the main part of a cell culture apparatus according to the third embodiment. [Figure 10] This is a plan cross-sectional view showing the main parts of a cell culture apparatus according to the fourth embodiment. [Figure 11] This is a magnified view of region B shown in Figure 10. [Figure 12] This is a perspective view showing a cell culture apparatus according to the fifth embodiment. [Figure 13] Figure 12 shows a cross-sectional view taken along the line XIII-XIII. [Figure 14] Figure 13 shows a cross-sectional view taken along the line XIV-XIV. [Figure 15] This is a perspective view showing a cell culture apparatus according to the sixth embodiment. [Figure 16] Figure 15 shows a cross-sectional view taken along the line XVI-XVI. [Figure 17] This is a cross-sectional view taken along the line XVII-XVII shown in Figure 16. [Figure 18] This is a schematic plan view showing a cell culture apparatus according to the seventh embodiment. [Figure 19] This is a schematic plan view showing a cell culture apparatus according to the eighth embodiment. [Figure 20] This is a schematic plan view showing a cell culture apparatus according to the ninth embodiment. [Figure 21] This graph shows the relationship between blood vessel diameter, shear stress, and flow rate according to the first embodiment. [Figure 22] This is a fluorescence microscope image of vascular tissue in the case of "no resistance channel" according to the first embodiment. [Figure 23] This is a fluorescence microscope image of vascular tissue in the case of "with resistance channel" according to the first embodiment. [Figure 24] This is a fluorescence microscope image of the entire vascular tissue in the case of "no resistance channel" according to the first embodiment. [Figure 25] This is a fluorescence microscope image of the entire vascular tissue in the case of "with resistance channel" according to the first embodiment. [Figure 26]This graph shows the relationship between the sequential flow rate of the culture medium and the culture period in the first embodiment. [Figure 27] This is a schematic plan view showing a cell culture apparatus according to the second embodiment. [Figure 28] These are fluorescence microscope images of Calcein immediately after pressurizing the culture vessel according to the second example to 2.0 [kPa] and 50 seconds later. [Figure 29] This graph shows the results of measuring the migration of the Rhodamine-Dextran fluorescence interface over time when the culture vessel according to the second example was pressurized at 0.5 [kPa], 1.0 [kPa], 2.0 [kPa], and 3.0 [kPa]. [Figure 30] This graph shows the results of measuring the interface migration of calcein fluorescence over time when the culture vessel according to the second example was pressurized at 0.5 [kPa], 1.0 [kPa], 2.0 [kPa], and 3.0 [kPa]. [Figure 31] These are fluorescence microscope images showing the blood vessel formation behavior with and without mesenchymal stem cells according to the second embodiment. [Figure 32] This graph shows the changes in blood vessel flow with and without mesenchymal stem cells according to the second embodiment. [Figure 33] This is a schematic plan view showing a cell culture apparatus according to a modified example of the second embodiment. [Figure 34] These are fluorescence microscope images showing the formation behavior of vascular tissue under unidirectional and reciprocating flow environments in the second embodiment and one of its modified examples. [Figure 35] This graph shows the flow rate changes in vascular tissue under unidirectional and reciprocating flow conditions in the second embodiment and one of its modified examples. [Figure 36] This is an image analysis diagram used to determine the permeability coefficient of a fluorescent substance through the blood vessel wall in order to evaluate the barrier function of vascular tissue formed using the cell culture apparatus according to the second embodiment and one of its modified forms. [Figure 37] The permeability coefficients of Calcein obtained under three conditions for the second embodiment and one of its modifications: (i) without mesenchymal stem cells and unidirectional flow, (ii) with mesenchymal stem cells and unidirectional flow, and (iii) with mesenchymal stem cells and reciprocating flow. [Figure 38] The permeability coefficients of Rhodamine-Dextran obtained under three conditions for the second embodiment and one of its modifications: (i) without mesenchymal stem cells and unidirectional flow, (ii) with mesenchymal stem cells and unidirectional flow, and (iii) with mesenchymal stem cells and reciprocating flow. [Figure 39] This figure shows images of vascular endothelial cells formed in the cell culture apparatus according to the second embodiment and one of its modifications, stained with the marker CD31 and stained with NG2, a marker for mesenchymal stem cells. [Figure 40] This figure shows images of vascular endothelial cells formed in a cell culture apparatus according to the second embodiment and one of its modifications, stained with ZO-1, which constitutes the tight junctions in the blood vessel wall. [Modes for carrying out the invention]

[0024] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Furthermore, this invention can be applied to vascular permeability testing of drug candidate compounds in drug discovery, organoid culture for regenerative medicine and cell assays, and cancer invasion evaluation. In drug discovery, the permeability of a drug to the vascular wall is sometimes evaluated when assessing the pharmacokinetics of a drug. The cell culture apparatus and cell culture method of the present invention can be used as a test apparatus and test method for evaluating vascular permeability. In particular, it can be used as an apparatus and test method that can accurately evaluate permeability across the blood-brain barrier. Furthermore, organoid culture methods have recently attracted attention in regenerative medicine, and the cell culture apparatus and cell culture method of the present invention can be used to culture angioplastic organoids in order to further mature the organoids. Furthermore, since distant metastasis of cancer involves the movement of cancer cells via blood vessels, the cell culture apparatus and cell culture method of the present invention can be used in drug discovery and clinical testing as a model for evaluating cancer cell metastasis.

[0025] (First Embodiment) Figure 1 is a perspective view showing a cell culture apparatus 1 according to the first embodiment. Figure 2 is a cross-sectional view taken along line II-II in Figure 1. Figure 3 is a cross-sectional view taken along line III-III in Figure 2. Figure 4 is an enlarged view of region A shown in Figure 3. As shown in Figure 1, the cell culture apparatus 1 includes a culture vessel 2 that holds a hydrogel 100 for culturing cells and stores a culture medium 101 that supplies oxygen, nutrients, etc., to the cells.

[0026] In Figure 1, the external shape of the culture vessel 2 of the cell culture apparatus 1 is shown with a dashed line to improve the visibility of its internal structure, allowing the internal structure to be seen through it. The same applies to Figures 5, 8, 12, and 15, which will be described later. The culture vessel 2 is preferably made of a transparent material with high light transmittance for the purpose of observing cells, but it may also be made of a material with low or no light transmittance. In that case, a glass window or the like may be provided in a position where the cells can be seen.

[0027] The culture vessel 2 comprises a hydrogel chamber 10, a first culture medium storage section 20, and a second culture medium storage section 30. The hydrogel chamber 10 is positioned between the first culture medium storage section 20 and the second culture medium storage section 30. In the example shown in Figure 1, the hydrogel chamber 10 is positioned horizontally adjacent to the first culture medium storage section 20 and the second culture medium storage section 30.

[0028] In the following explanation, an XYZ Cartesian coordinate system will be established, and the positional relationships of each component will be described with reference to this XYZ Cartesian coordinate system. The X-axis direction is the first horizontal direction in which the first culture medium reservoir 20, the hydrogel chamber 10, and the second culture medium reservoir 30 are aligned. The Y-axis direction is the second horizontal direction perpendicular to the X-axis direction. The Z-axis direction is the vertical direction perpendicular to both the X-axis and Y-axis directions.

[0029] As shown in Figure 2, the hydrogel chamber 10 has a flattened internal space 11. The height (dimension in the Z-axis direction) of the internal space 11 of the hydrogel chamber 10 is, for example, in the range of 100 [μm] to 500 [μm]. The internal space 11 of the hydrogel chamber 10 is filled with hydrogel 100. The hydrogel 100 has a three-dimensional network structure of polymers that retains moisture, embeds or adheres cells, and allows for cell culture.

[0030] As shown in Figure 3, the hydrogel chamber 10 is formed in a hexagonal shape in plan view. While the plan view shape of the hydrogel chamber 10 is not particularly limited, the length adjacent to the first culture medium reservoir 20 and the second culture medium reservoir 30 (dimension in the Y-axis direction) should preferably be greater than the width (dimension in the X-axis direction). The Y-axis dimension of the hydrogel chamber 10 is, for example, within the range of 1 mm to 20 mm. The X-axis dimension of the hydrogel chamber 10 is, for example, within the range of 1 mm to 10 mm.

[0031] The hydrogel chamber 10 has a first surface 12 that communicates with the first culture medium storage section 20 and a second surface 13 that communicates with the second culture medium storage section 30. The first surface 12 and the second surface 13 face each other in the X-axis direction and extend parallel to each other in the Y-axis direction. Both ends of the hydrogel chamber 10 in the Y-axis direction communicate with introduction holes 14 and 15 via introduction passages 14a and 15a. A hydrogel storage tank 16 for introducing hydrogel 100 is formed directly above the hydrogel chamber 10.

[0032] The inlet hole 14 connects the top surface of the hydrogel chamber 10 to the bottom surface of the hydrogel storage tank 16 at one end of the hydrogel chamber 10 in the Y-axis direction (-Y side). The inlet hole 15 connects the top surface of the hydrogel chamber 10 to the bottom surface of the hydrogel storage tank 16 at the other end of the hydrogel chamber 10 in the Y-axis direction (+Y side). The two inlet holes 14 and 15 make it easier to remove air from the hydrogel chamber 10 and to fill the hydrogel chamber 10 with hydrogel 100 from the hydrogel storage tank 16.

[0033] The first culture medium storage section 20 includes a first culture medium storage tank 21 for storing the culture medium 101. The first culture medium storage tank 21 is formed in a long slit or elliptical shape extending in the Y-axis direction in a plan view. The plan view shape of the first culture medium storage tank 21 is not particularly limited. It is preferable that the internal space (volume) of the first culture medium storage tank 21 is larger than that of the hydrogel chamber 10. The side surface of the bottom of the first culture medium storage tank 21 on the hydrogel chamber 10 side (+X side) is in communication with the first surface 12 of the hydrogel chamber 10.

[0034] The second culture medium storage section 30 includes a second culture medium storage tank 31 for storing the culture medium 101. The second culture medium storage tank 31 is formed in a long slit or elliptical shape extending in the Y-axis direction in a plan view. The planar shape of the second culture medium storage tank 31 is not particularly limited. It is preferable that the internal space (volume) of the second culture medium storage tank 31 is larger than that of the hydrogel chamber 10. The side surface of the bottom of the second culture medium storage tank 31 on the hydrogel chamber 10 side (-X side) is in communication with the second surface 13 of the hydrogel chamber 10.

[0035] As shown in Figure 3, the culture vessel 2 with the above configuration is connected to a pneumatic device 5. The pneumatic device 5 includes a pneumatic pump 50 connected to the first culture medium storage tank 21 and a pneumatic pump 60 connected to the second culture medium storage tank 31. The pneumatic pumps 50 and 60 can continuously pressurize the inside of the culture vessel 2, for example, while a motor (not shown) is being driven. The pneumatic device 5 creates a pressure difference based on air pressure between the first culture medium storage tank 21 and the second culture medium storage tank 31, thereby pressurizing and permeating the culture medium 101 stored in at least one of the first culture medium storage tank 21 and the second culture medium storage tank 31 into the hydrogel 100 of the hydrogel chamber 10.

[0036] Furthermore, if the direction in which the culture medium 101 is supplied to the hydrogel chamber 10 is limited to one direction, from either the first culture medium storage tank 21 or the second culture medium storage tank 31 to the other, then one of the pneumatic pumps 50 or 60 may be omitted. Alternatively, pneumatic piping may be branched from a single pneumatic pump and connected to the first culture medium storage tank 21 and the second culture medium storage tank 31, respectively, and a switching valve may be provided on the branch pipe of the pneumatic piping to switch which culture medium storage tank is pressurized. The pneumatic device 5 is equipped with, for example, a pressure regulator (not shown) and adjusts the pressure within the range of 0.1 [kPa] to 10 [kPa] to pressurize at least one of the first culture medium storage tank 21 and the second culture medium storage tank 31.

[0037] The culture vessel 2 is equipped with column rows 22 and 32 as a mechanism to hold the hydrogel 100 in the hydrogel chamber 10 even when a pressure difference based on the above-mentioned pneumatic pressure is generated. Furthermore, the hydrogel chamber 10 may be subjected to a surface treatment (such as a coating treatment to adjust the wettability of the surface) to hold the hydrogel 100.

[0038] The column row 22 is provided along the first surface 12 of the hydrogel chamber 10, which is in communication with the first culture medium storage tank 21. The height of the column row 22 is the same as the height of the internal space 11 of the hydrogel chamber 10. The distance between each column in the column row 22 is, for example, in the range of 50 [μm] to 300 [μm]. The detailed structure of the column row 22 will be described below with reference to Figure 4.

[0039] As shown in Figure 4, the column row 22 comprises a plurality of columns 40 that form the interface region between the hydrogel chamber 10 and the first culture medium storage tank 21. The columns 40 are formed at equal intervals with gaps in the Y-axis direction. Fluid channels are formed between adjacent columns 40 in the Y-axis direction, allowing communication between the hydrogel chamber 10 and the first culture medium storage tank 21.

[0040] The support column 40 is formed in a substantially pentagonal shape in a plan view. The side surface of the support column 40 includes a pair of inclined surfaces 41 arranged on the side of the hydrogel chamber 10, a flat surface 42 arranged on the side of the first culture solution reservoir 21, and a pair of curved surfaces 43 connecting between the pair of inclined surfaces 41 and the flat surface 42.

[0041] The ends of the pair of inclined surfaces 41 facing the inside of the hydrogel chamber 10 are connected to each other. The connection angle of the pair of inclined surfaces 41 is an acute angle. Also, the angles of the pair of inclined surfaces 41 with respect to the X-Z plane are equal. The flat surface 42 forms a plane parallel to the Y-Z plane. The pair of curved surfaces 43 smoothly connect the end portion on the +Y side of the flat surface 42 and the end portion of the inclined surface 41 arranged on the +Y side, and also smoothly connect the end portion on the -Y side of the flat surface 42 and the end portion of the inclined surface 41 arranged on the -Y side.

[0042] The gap (dimension in the Y-axis direction) between adjacent support columns 40 is width w1 in the flat surface 42 portion, the minimum width w2 in the curved surface 43 portion, and the maximum w3 in the end connection portion of the pair of inclined surfaces 41. The widths w1, w2, and w3 have the relationship of w2 < w1 < w3. That is, the gap between adjacent support columns 40 decreases from the first culture solution reservoir 21 toward the hydrogel chamber 10 and then turns to increase. Thereby, it is easy to supply the culture solution 101 from the first culture solution reservoir 21 to the hydrogel chamber 10, and the wedge effect can make it difficult for the hydrogel 100 to flow out from the hydrogel chamber 10 to the first culture solution reservoir 21.

[0043] The support column row 32 is provided along the second surface 13 of the hydrogel chamber 10 communicating with the second culture solution reservoir 31. The support column row 32 includes a plurality of support columns 40 forming an interface region between the hydrogel chamber 10 and the second culture solution reservoir 31. Since the configuration of the support columns 40 in the support column row 32 is symmetric to the support columns 40 in the support column row 22 arranged on the opposite side (the side of the first culture solution reservoir 21) across the hydrogel chamber 10, the description thereof is omitted. The shape of the support column 40 is preferably the above shape, but it may be formed in a known triangle, trapezoid, etc.

[0044] According to the first embodiment of the above configuration, culture medium 101 is stored in the first culture medium storage section 20 and the second culture medium storage section 30 adjacent to the hydrogel chamber 10, and by applying pneumatic pressure to the stored culture medium 101, the culture medium 101 can be permeated into the hydrogel 100 and cells can be cultured. In this way, by connecting the pneumatic device 5 to the culture vessel 2 and culturing cells (for example, vascular endothelial cells) while permeating the culture medium 101 into the hydrogel 100 with pneumatic pressure, the formation of vascular tissue in the hydrogel 100 can be promoted.

[0045] By applying pneumatic pressure to the culture medium 101 and permeating it into the hydrogel 100, it becomes possible to form larger vascular tissue. Furthermore, by adjusting the pneumatic pressure, it is possible to perfuse the culture medium 101 into the vascular tissue at an appropriate flow rate as the vascular tissue matures. In addition, by adjusting the pneumatic pressure and perfusing the culture medium 101, it is possible to avoid the problem of the flow rate decreasing as the fluid is delivered, as is the case when delivering fluid using conventional water column pressure.

[0046] As described above, the cell culture apparatus 1 according to the first embodiment comprises a culture vessel 2 having a hydrogel chamber 10 for holding a hydrogel 100 for culturing cells, a first culture medium storage section 20 communicating with a first surface 12 of the hydrogel chamber 10, and a second culture medium storage section 30 communicating with a second surface 13 different from the first surface 12 of the hydrogel chamber 10, and a pneumatic device 5 connected to the culture vessel 2. The pneumatic device 5 generates a pressure difference based on air pressure between the first culture medium storage section 20 and the second culture medium storage section 30, thereby pressurizing and permeating the culture medium 101 stored in at least one of the first culture medium storage section 20 and the second culture medium storage section 30 into the hydrogel 100 of the hydrogel chamber 10. With this configuration, large cell tissues can be efficiently constructed in vitro.

[0047] Furthermore, in the first embodiment, the hydrogel chamber 10 is provided with column rows 22 and 32 as a mechanism for holding the hydrogel 100 while creating a pressure difference based on pneumatic pressure. With this configuration, it is easy to supply the culture medium 101 from the first culture medium storage section 20 and the second culture medium storage section 30 to the hydrogel chamber 10, and even when pneumatic pressure is applied, it is difficult for the hydrogel 100 to flow out of the hydrogel chamber 10 to the first culture medium storage section 20 or the second culture medium storage section 30. In addition, it is preferable to apply a surface treatment to the hydrogel chamber 10 to hold the hydrogel 100.

[0048] The cell culture method according to the first embodiment involves culturing cells in a hydrogel chamber 10 using the cell culture apparatus 1 described above. With this configuration, large cell tissues can be efficiently constructed in vitro.

[0049] Furthermore, in the first embodiment, cells are cultured and vascular tissue is formed within the hydrogel chamber 10. With this configuration, large vascular tissue can be efficiently constructed in vitro.

[0050] (Second Embodiment) Next, a second embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the above-described embodiment will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0051] Figure 5 is a perspective view showing the cell culture apparatus 1 according to the second embodiment. Figure 6 is a cross-sectional view taken along the line VI-VI in Figure 5. Figure 7 is a cross-sectional view taken along the line VII-VII in Figure 6. As shown in Figure 5, the second embodiment differs from the above embodiment in that a lid 3 to which pneumatic pipes 51 and 61 are connected is fixed to the upper part of the culture vessel 2.

[0052] As shown in Figure 6, the lower surface of the lid 3 has a first recess 23 that is concave upward and communicates with the first culture medium storage tank 21, and a second recess 33 that is concave upward and communicates with the second culture medium storage tank 31. The upper part of the hydrogel storage tank 16 is closed off by the lower surface of the lid 3. A pneumatic pipe 51 is connected to the lid 3 so as to communicate with the first recess 23. A pneumatic pipe 61 is also connected to the lid 3 so as to communicate with the second recess 33. The direction in which the pneumatic pipes 51 and 61 are connected to the lid 3 is not limited to the Y-axis direction, but may also be the X-axis direction or the Z-axis direction.

[0053] As shown in Figure 7, the pneumatic piping 51 is connected to the pneumatic pump 50. The pneumatic piping 61 is also connected to the pneumatic pump 60. This allows the first culture medium storage tank 21 to be pressurized from the pneumatic pump 50 via the pneumatic piping 51. Similarly, the second culture medium storage tank 31 can be pressurized from the pneumatic pump 60 via the pneumatic piping 61. It is preferable to provide filters in parts of the pneumatic piping 51 and 61 to prevent foreign matter such as dust from entering the first culture medium storage tank 21 or the second culture medium storage tank 31. For example, it is preferable to use a filter made of a hydrophobic material with a pore size of 0.22 [μm] or less.

[0054] O-rings may be placed on the opening edges of the first culture medium storage tank 21 and the second culture medium storage tank 31 of the culture vessel 2, and sandwiched between them and the lid 3. This can suppress pressure leakage from the gap in the lid 3. Alternatively, the lid 3 may be fixed to the culture vessel 2 with bolts or the like so as to compress the O-rings in the vertical direction. This can improve the sealing performance between the container body of the culture vessel 2 and the lid 3.

[0055] As described above, according to the second embodiment, the culture container 2 is equipped with a lid 3. Pneumatic pipes 51 and 61 are connected to the lid 3. With this configuration, the connection between the culture container 2 and the pneumatic device 5 can be easily released by removing the pneumatic pipes 51 and 61 from the lid 3. This allows the culture container 2 to be transported to another location, such as a clean bench, with the pneumatic pipes 51 and 61 removed from the lid 3, and then the lid 3 can be removed to easily supply hydrogel 100 and culture medium 101 to the culture container 2. In addition, filters are provided on the pneumatic pipes 51 and 61. With this configuration, it is possible to suppress the ingress of foreign matter such as dust into the first culture medium storage tank 21 or the second culture medium storage tank 31 when applying air pressure.

[0056] (Third embodiment) Next, a third embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the above-described embodiments will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0057] Figure 8 is a perspective view showing the cell culture apparatus 1 according to the third embodiment. Figure 9 is a plan cross-sectional view showing the main part of the cell culture apparatus 1 according to the third embodiment. Figure 9, like Figures 3 and 7 described above, shows a plan cross-sectional view along the hydrogel chamber 10. As shown in Figure 8, the third embodiment differs from the above embodiment in that the first culture medium storage unit 20 includes a first microchannel 24 that connects the first culture medium storage tank 21 and the hydrogel chamber 10. Furthermore, the third embodiment differs from the above embodiment in that the second culture medium storage unit 30 includes a second microchannel 34 that connects the second culture medium storage tank 31 and the hydrogel chamber 10.

[0058] The first microchannel 24 has a flattened internal space, similar to the hydrogel chamber 10, and extends in a strip shape in the X-axis direction. The height of the internal space of the first microchannel 24 (dimension in the Z-axis direction) is, for example, the same as that of the hydrogel chamber 10. Also, as shown in Figure 9, the width of the first microchannel 24 (dimension in the Y-axis direction) is, for example, the same as the width of the first surface 12 (dimension in the Y-axis direction).

[0059] Furthermore, the second microchannel 34 has a flattened internal space, similar to the hydrogel chamber 10, and extends in a strip shape in the X-axis direction. The height of the internal space of the second microchannel 34 (dimension in the Z-axis direction) is, for example, the same as that of the hydrogel chamber 10. Also, the width of the second microchannel 34 (dimension in the Y-axis direction) is, for example, the same as the width of the second surface 13 (dimension in the Y-axis direction). Note that the dimensions of the first microchannel 24 and the second microchannel 34 are not limited to the above dimensions.

[0060] According to the third embodiment of the above configuration, the hydrogel chamber 10 and the first culture medium storage tank 21 can be connected in the first culture medium storage section 20 by the first microchannel 24. With this configuration, space can be secured between the first culture medium storage tank 21 and the hydrogel chamber 10. Therefore, it is possible to secure design space for the culture vessel 2 for connecting to the pneumatic device 5, and design space for the culture vessel 2 to widen the opening for introducing the hydrogel 100 into the hydrogel storage tank 16 of the hydrogel chamber 10, thereby improving the degree of design freedom.

[0061] Furthermore, in the second culture medium storage section 30, connecting the hydrogel chamber 10 and the second culture medium storage tank 31 by the second microchannel 34 makes it easier to secure design space for the culture vessel 2. Note that if sufficient design space for the culture vessel 2 can be secured by providing a microchannel in either the first culture medium storage section 20 or the second culture medium storage section 30, the other of the first culture medium storage section 20 or the second culture medium storage section 30 may not be equipped with a microchannel.

[0062] (Fourth Embodiment) Next, a fourth embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the above-described embodiments will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0063] Figure 10 is a plan cross-sectional view showing the main part of the cell culture apparatus 1 according to the fourth embodiment. Figure 11 is an enlarged view of region B shown in Figure 10. Note that Figure 10, like Figures 3, 7, and 9 described above, shows a plan cross-sectional view along the hydrogel chamber 10. As shown in Figure 10, the fourth embodiment differs from the above embodiment in that the first microchannel 24 includes a resistive channel 25. The resistive channel 25 may also be provided in the second microchannel 34.

[0064] When culturing vascular tissue formed in hydrogel 100 while perfusing it with culture medium 101 using pneumatic drive, the cross-sectional area of ​​the vascular tissue increases as it matures, and the flow rate of the culture medium 101 increases. On the other hand, it is difficult to predict changes in the cross-sectional area of ​​the vascular tissue, and it is also difficult to predict the flow rate of the culture medium 101. Therefore, for example, when pressurizing the first culture medium storage tank 21 (second culture medium storage tank 31) to pressurize and permeate the culture medium 101 stored in the first culture medium storage section 20 (second culture medium storage section 30) into the hydrogel 100 in the hydrogel chamber 10, it is difficult to predict when to replenish the culture medium 101 in the first culture medium storage section 20 (second culture medium storage section 30), and it is cumbersome to frequently replenish the culture medium 101 when the flow rate of the culture medium 101 becomes too high. For this reason, in the fourth embodiment, a resistance channel 25 with a small cross-sectional area of ​​the channel is provided in a part of the first microchannel 24. By providing the resistive channel 25, it is possible to control the increase in the flow rate of the culture medium 101 even when the blood vessels become wider as the vascular tissue matures.

[0065] As shown in Figure 11, the first microchannel 24 has multiple resistance channels 25 arranged in parallel, each having a smaller cross-sectional area than the first microchannel 24. To avoid clogging of the resistance channels 25 due to bubbles or dust, it is preferable to have multiple resistance channels 25 arranged in parallel as shown in Figure 11, preferably 10 or more resistance channels 25 arranged in parallel. However, if there is no concern about clogging due to bubbles or dust, one resistance channel 25 (one channel) is sufficient.

[0066] By making the flow path resistance of the resistance flow path 25 larger than the flow path resistance of the blood vessels formed in the hydrogel 100, it becomes possible to more accurately control the flow rate of the culture solution 101 even when the blood vessel diameter changes with the maturation of the blood vessels. Here, the flow path resistance (R MC ) of the resistance flow path 25 is related to the viscosity (μ) of the culture solution 101, the length (L MC ) of the flow path, the cross-sectional diameter (d MC ) of the flow path, and the number (N MC ) of the flow paths arranged in parallel, and is defined as in the following formula (1). R MC = 8μL MC / πd MC 4 N MC …(1)

[0067] Here, d MC indicates the diameter when the flow path cross-section is circular, but when the flow path cross-section is non-circular, it can be substituted with the equivalent diameter. The equivalent diameter (d eq ) is related to the flow path cross-sectional area (S) and the flow path cross-sectional perimeter (L P ), and is defined as in the following formula (2). d eq = 4S / L P …(2)

[0068] The flow path resistance (R V ) of the blood vessels formed in the hydrogel 100 can be estimated by the following formula (3) from the viscosity (μ) of the culture solution 101, the length (L V ) of the blood vessels, the cross-sectional diameter (d V ) of the blood vessels, and the number (N V ) of the blood vessels formed in parallel. R V = 8μL V / πd V 4 N V …(3)

[0069] The blood vessel diameter and number of the blood vessels formed in the hydrogel 100 vary depending on the shape of the hydrogel chamber 10 and change with maturation, but the length (L V) is the length of the hydrogel chamber 10 in the direction of blood vessel extension (dimension in the X-axis direction), and the cross-sectional diameter of the blood vessel (d V ) is the height of the hydrogel chamber 10 (dimension in the Z-axis direction), and the number of blood vessels formed in parallel (N V ) is the width of the hydrogel chamber 10 (the dimension in the Y-axis direction of the first surface 12 or the second surface 13) and the cross-sectional diameter of the blood vessel (d V The value obtained by dividing by (R) can be used as a representative value to estimate the flow resistance of blood vessels formed in the hydrogel 100. This also allows for the estimation of the flow rate of the culture medium 101, as described later. For example, if 50 blood vessels with a diameter of 300 [μm] and a length of 2000 [μm] are arranged in parallel, the flow resistance of the blood vessels (R) can be estimated. V ) is approximately 2 × 10 -10 [Pa·s / μm 3 ]

[0070] In this case, the flow resistance of the resistive flow path 25 is 2 × 10 -10 [Pa·s / μm 3 By setting it to ] or higher, it can function as a flow channel resistance that is practical for use as a culture vessel 2. For example, if 18 resistive flow channels 25 with a width of 200 [μm], a depth of 200 [μm], and a length of 200 [μm] are arranged in parallel, the flow channel resistance (R) of the resistive flow channels 25 will be MC ) is 2 × 10 -10 [Pa·s / μm 3 ]

[0071] Resistance of the resistive flow path 25 (R MC To make this value larger, according to equation (1), the width and depth of the flow path may be made smaller, the length of the flow path may be made larger, or the number of resistive flow paths 25 may be reduced by adjusting the gap w4 of the resistive flow path 25 as shown in Figure 11.

[0072] In other words, the flow resistance (R) of the resistive flow path MCThe hydrogel chamber 10 should satisfy the following relationship (5), where (μ) is the viscosity of the culture medium, (L) is the length from the first surface 12 to the second surface 13 of the hydrogel chamber 10, (d) is the height of the hydrogel chamber 10, and (N) is the value obtained by dividing the width of the first surface 12 or the second surface 13 by the height of the hydrogel chamber 10. Note that N should be a natural number. R MC ≥ 8 μL / πd 4 N …(5)

[0073] The flow rate (Q) of the culture medium 101 is determined by the pressure (P) applied to the first culture medium reservoir 20 (second culture medium reservoir 30) and the flow resistance (R) of the resistive channel 25. MC ), the flow resistance of blood vessels that may be formed in hydrogel 100 (R V ) can be estimated using the following equation (6). Q = P / (R MC +R V ) …(6)

[0074] As described above, when culturing vascular tissue formed in hydrogel 100 while perfusing it with culture medium 101 using pneumatic drive, the flow rate of culture medium 101 increases as the cross-sectional area of ​​the vascular tissue increases with maturation. However, the maximum flow rate of culture medium 101 when the cross-sectional area of ​​the vascular tissue becomes very large can be estimated by the following equation (7). Q = P / R MC …(7)

[0075] By estimating the maximum flow rate of the culture medium 101 in this way, it is possible to estimate the shortest interval between replenishment of the culture medium 101, thereby streamlining the process of performing perfusion culture over a long period.

[0076] According to the fourth embodiment described above, the first microchannel 24 is provided with a resistive channel 25 having a smaller cross-sectional area than the first microchannel 24. The flow resistance (R) of the resistive channel 25 MCThe above equation (5) satisfies the relationship when the viscosity of the culture medium is (μ), the length from the first surface 12 to the second surface 13 of the hydrogel chamber 10 is (L), the height of the hydrogel chamber 10 is (d), and (N) is the value obtained by dividing the width of the first surface 12 or the second surface 13 by the height of the hydrogel chamber 10. With this configuration, even when the blood vessels become wider as the vascular tissue matures, the increase in the flow rate of the culture medium 101 can be controlled and the maximum flow rate of the culture medium 101 can be estimated, making the work of performing perfusion culture over a long period of time more efficient.

[0077] (Fifth embodiment) Next, a fifth embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the above-described embodiments will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0078] Figure 12 is a perspective view showing the cell culture apparatus 1 according to the fifth embodiment. Figure 13 is a cross-sectional view taken along the line XIII-XIII shown in Figure 12. Figure 14 is a cross-sectional view taken along the line XIV-XIV shown in Figure 13. As shown in Figure 12, the fifth embodiment differs from the above embodiment in that a third culture medium storage section 80 is provided directly above the hydrogel chamber 10.

[0079] The third culture medium storage section 80 includes a third culture medium storage tank 81 for storing the culture medium 101. In the example shown in the figure, the third culture medium storage tank 81 also serves as a hydrogel storage tank 16 for introducing the hydrogel 100 into the hydrogel chamber 10. The third culture medium storage tank 81 is formed in a long-slot or elliptical shape extending in the Y-axis direction in a plan view. The plan view shape of the third culture medium storage tank 81 is not particularly limited. It is preferable that the internal space (volume) of the third culture medium storage tank 81 is larger than that of the hydrogel chamber 10.

[0080] As shown in Figure 13, a large opening 82 is formed in the center of the bottom surface of the third culture medium storage tank 81, communicating with the top surface of the hydrogel chamber 10. A permeable membrane 82a may be provided in the opening 82. The permeable membrane 82a has one side facing the hydrogel 100 and the other side facing the culture medium 101. The permeable membrane 82a should, for example, be impermeable to the hydrogel 100 but permeable to the culture medium 101. Furthermore, the permeable membrane 82a should be attached to the opening 82 in a detachable manner.

[0081] As shown in Figure 14, the pneumatic device 5 includes a pneumatic pump 90 connected to the third culture medium storage tank 81. Returning to Figure 12, the top of the third culture medium storage tank 81 is closed by a lid 3. A third recess 83, which is concave upwards and communicates with the third culture medium storage tank 81, is formed on the lower surface of the lid 3. A pneumatic pipe 91 is connected to the lid 3 so as to communicate with the third recess 83. The pneumatic pipe 91 is connected to the pneumatic pump 90. This allows the third culture medium storage tank 81 to be pressurized from the pneumatic pump 90 via the pneumatic pipe 91.

[0082] In the fifth embodiment described above, an opening 82 is formed directly above the hydrogel chamber 10. In organoid culture for regenerative medicine and drug discovery, it is necessary to anastomose microvessels with organoids, which are cell aggregates, and to perfuse the culture medium 101 through the microvessels. As shown in Figures 13 and 14, by providing one large opening 82 directly above the hydrogel chamber 10, the introduction of the hydrogel 100 becomes easier. Furthermore, the opening 82 can be used to place cell aggregates on top of the hydrogel 100, and the culture medium 101 can be perfused to the cell aggregates via the microvessel network.

[0083] Furthermore, in the fifth embodiment, a permeable membrane 82a can be provided at the opening 82, with one side facing the hydrogel 100 and the other side facing the culture medium 101. In pharmacokinetic studies of drug candidate compounds, the permeation rate of the drug candidate compound from microvessels such as the blood-brain barrier is sometimes evaluated. To evaluate the permeation rate, it is necessary to collect the compound that has leaked out to the outside of the microvessels, i.e., into the hydrogel 100, and evaluate it with analytical instruments such as mass spectrometry. To make this possible, as shown in Figure 13, a permeable membrane 82a is provided at the opening 82 located directly above the hydrogel chamber 10, with one side of the permeable membrane 82a facing the hydrogel 100 and the other side facing the third culture medium storage tank 81 that holds the culture medium 101. This makes it possible to collect and analyze the compound exposed in the hydrogel 100 through the permeable membrane 82a.

[0084] Furthermore, as in the fifth embodiment, when an opening 82 is provided directly above the hydrogel chamber 10, when pressurizing at least one of the first culture medium storage section 20 and the second culture medium storage section 30 to pressurize and permeate the culture medium 101 into the hydrogel 100, the culture medium 101 may leak out from the opening 82 of the hydrogel chamber 10. To prevent this, a pneumatic device 5 is connected to the third culture medium storage section 80. By applying a similar amount of pressure to the third culture medium storage section 80 when pressurizing at least one of the first culture medium storage section 20 and the second culture medium storage section 30, leakage of the culture medium 101 from the opening 82 directly above the hydrogel chamber 10 can be prevented.

[0085] (Sixth Embodiment) Next, a sixth embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the embodiments described above will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0086] Figure 15 is a perspective view showing the cell culture apparatus 1 according to the sixth embodiment. Figure 16 is a cross-sectional view taken along the line XVI-XVI shown in Figure 15. Figure 17 is a cross-sectional view taken along the line XVII-XVII shown in Figure 16. As shown in Figure 15, the sixth embodiment differs from the above embodiment in that hydrogel storage tanks 16A and 16B are provided separately from the third culture medium storage tank 81.

[0087] The hydrogel storage tanks 16A and 16B are provided as a pair, sandwiching the third culture medium storage tank 81 in the Y-axis direction. The hydrogel storage tanks 16A and 16B are circular in shape when viewed from above. However, the shape of the hydrogel storage tanks 16A and 16B in terms of their shape when viewed from above is not particularly limited. From the viewpoint of, for example, installation space, it is preferable that the internal space (volume) of the hydrogel storage tanks 16A and 16B is smaller than that of the third culture medium storage tank 81.

[0088] As shown in Figure 16, the bottom surface of the hydrogel storage tank 16A is connected to the hydrogel chamber 10 via an inlet hole 14. Similarly, the bottom surface of the hydrogel storage tank 16B is connected to the hydrogel chamber 10 via an inlet hole 15. As shown in Figure 17, since the hydrogel storage tanks 16A and 16B are connected to the hydrogel chamber 10 via inlet holes 14 and 15 which are sufficiently smaller than the opening 82, there is little problem of leakage of culture medium 101 from the hydrogel chamber 10. Therefore, they are not connected to the pneumatic device 5 like the third culture medium storage tank 81, but they may be connected to the pneumatic device 5 in the same way as the third culture medium storage tank 81.

[0089] According to the sixth embodiment described above, the introduction holes 14 and 15 for introducing the hydrogel 100 into the hydrogel chamber 10 are located at positions separate from the opening 82 directly above the hydrogel chamber 10. This makes it easier to perform tasks such as collecting and analyzing compounds exposed in the hydrogel 100 via a permeable membrane 82a.

[0090] (Seventh Embodiment) Next, a seventh embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the embodiments described above will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0091] Figure 18 is a schematic plan view showing the cell culture apparatus 1 according to the seventh embodiment. As shown in Figure 18, the seventh embodiment differs from the above embodiment in that the first microchannel 24 and the second microchannel 34 are each provided in a pair.

[0092] The first microchannel 24 comprises a pair of microchannels 24A and 24B. In other words, the first culture medium storage tank 21 and the hydrogel chamber 10 are connected by two microchannels 24A and 24B. The first microchannel 24 is formed in a C-shape or U-shape when viewed from above. The first microchannel 24 communicates with the first culture medium storage tank 21 at both ends of the C-shape or U-shape, and communicates with the first surface 12 of the hydrogel chamber 10 at a location other than both ends (the middle portion).

[0093] The second microchannel 34 comprises a pair of microchannels 34A and 34B. In other words, the second culture medium reservoir 31 and the hydrogel chamber 10 are connected by two microchannels 34A and 34B. The second microchannel 34 is formed in a C-shape or U-shape when viewed from above. The second microchannel 34 communicates with the second culture medium reservoir 31 at both ends of the C-shape or U-shape, and communicates with the second surface 13 of the hydrogel chamber 10 at a location other than both ends (the middle section).

[0094] According to the seventh embodiment described above, a pair of first microchannels 24 and second microchannels 34 are provided. For example, in the first microchannel 24, air bubbles in the first microchannel 24 can be easily removed by introducing the culture medium 101 from the first culture medium storage tank 21 into either microchannel 24A or 24B. Similarly, in the second microchannel 34, air bubbles in the second microchannel 34 can be easily removed by introducing the culture medium 101 from the second culture medium storage tank 31 into either microchannel 34A or 34B.

[0095] (Eighth embodiment) Next, an eighth embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the embodiments described above will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0096] Figure 19 is a schematic plan view showing the cell culture apparatus 1 according to the eighth embodiment. As shown in Figure 19, the eighth embodiment differs from the above embodiment in that a return channel 110 is provided that connects the first culture medium storage tank 21 and the second culture medium storage tank 31.

[0097] In Figure 19, the white arrows indicate the flow of the culture medium 101. In other words, in the example shown in Figure 19, the culture medium 101 is supplied from the first culture medium storage tank 21 to the hydrogel chamber 10 via the first microchannel 24, flows out from the hydrogel chamber 10 to the second culture medium storage tank 31 via the second microchannel 34, and returns from the second culture medium storage tank 31 to the first culture medium storage tank 21 via the return channel 110.

[0098] The return channel 110 is provided with a passive valve 111 as a mechanism to prevent backflow of the culture medium 101 from the first culture medium storage tank 21 to the second culture medium storage tank 31. The passive valve 111 has, for example, a minute fluid channel, and by adjusting the cross-sectional area, shape, length, and wettability of the fluid channel, it is possible to prevent air bubbles from entering from the first culture medium storage tank 21 while allowing the culture medium 101 to flow in one direction from the second culture medium storage tank 31 to the first culture medium storage tank 21.

[0099] The pressure resistance of the passive valve 111 should be within the range of 0.1 [kPa] to 10 [kPa]. This allows for culture operations within a range that can be controlled by a commercially available pressure regulator and within a physiologically acceptable range for the culture vessel 2. Note that the pressure resistance of the passive valve 111 (ΔP Lap ) can be estimated using the interfacial tension (γ) and the channel cross-sectional diameter (d) as shown in equation (8) below. ΔP Lap = 4γ / d …(8)

[0100] Furthermore, check valves 36 are provided at both ends of the second microchannel 34 on the second culture medium storage tank 31 side as a mechanism to prevent backflow of the culture medium 101. This prevents backflow of the culture medium 101 to the hydrogel chamber 10 side when the second culture medium storage tank 31 is pressurized and the culture medium 101 is returned to the first culture medium storage tank 21.

[0101] The pneumatic device 5 switches between supplying the culture medium 101 and returning the culture medium 101 at predetermined intervals. It is preferable to set the supply time of the culture medium 101 to be longer than the return time of the culture medium 101. When supplying the culture medium 101, the pneumatic device 5 pressurizes the first culture medium storage tank 21 and supplies the culture medium 101 to the hydrogel chamber 10 by the pressure difference between it and the second culture medium storage tank 31.

[0102] When returning the culture medium 101, the pneumatic device 5 pressurizes the second culture medium storage tank 31 where the culture medium is stored, and the differential pressure between it and the first culture medium storage tank 21 causes the culture medium 101 to be returned to the first culture medium storage tank 21 via the return channel 110. It is preferable that the end of the return channel 110 on the first culture medium storage tank 21 side be higher than the end of the return channel 110 on the second culture medium storage tank 31 side. This suppresses the backflow of the culture medium 101 from the first culture medium storage tank 21 to the second culture medium storage tank 31.

[0103] According to the eighth embodiment described above, a return channel 110 for the culture medium 101 connects the first culture medium storage tank 21 and the second culture medium storage tank 31, and the second microchannel 34 and the return channel 110 are equipped with a mechanism to prevent backflow of the culture medium 101. With this configuration, depletion of the culture medium 101 in the first culture medium storage tank 21 can be prevented, and cells can be cultured for a long period of time.

[0104] Furthermore, in the eighth embodiment, a passive valve 111 is provided as a mechanism to prevent backflow, which prevents the entry of air bubbles. With this configuration, when the culture medium 101 is returned and circulated by pneumatic pressure, the introduction of air into the return channel 110 can be prevented at the position of the passive valve 111, and the generation of air bubbles in the first culture medium storage tank 21 and the second culture medium storage tank 31 due to air introduced into the return channel 110 can be prevented, thereby stabilizing the culture operation.

[0105] Furthermore, in the eighth embodiment, the pressure resistance of the passive valve 111 is within the range of 0.1 [kPa] to 10 [kPa]. This configuration allows for culture operations within a range controllable by a commercially available pressure regulator, and within a physiologically appropriate range.

[0106] (Ninth Embodiment) Next, a ninth embodiment of the present invention will be described. In the following description, components identical or equivalent to those in the embodiments described above will be denoted by the same reference numerals, and their descriptions will be simplified or omitted.

[0107] Figure 20 is a schematic plan view showing the cell culture apparatus 1 according to the ninth embodiment. As shown in Figure 20, the ninth embodiment differs from the above embodiment in that a resistive channel 35 is provided in each of the pair of microchannels 34A and 34B of the second microchannel 34.

[0108] The resistance channel 35 provided in the second microchannel 34 has the same configuration as the resistance channel 25 provided in the first microchannel 24 shown in Figure 11 above. In other words, the second microchannel 34 is provided with one or more resistance channels 35 arranged in parallel, each having a smaller cross-sectional area than the second microchannel 34. By making the flow resistance of the resistance channel 25 greater than the flow resistance of the blood vessels formed in the hydrogel 100, it becomes possible to control the flow rate of the culture medium 101 more accurately even if the blood vessel diameter changes as the blood vessels mature.

[0109] In other words, the flow resistance (R) of the resistive flow path 35 MC) is defined as follows: the viscosity of the culture medium (μ), the length from the first surface 12 to the second surface 13 of the hydrogel chamber 10 (L), the height of the hydrogel chamber 10 (d), and the value obtained by dividing the width of the first surface 12 or the second surface 13 by the height of the hydrogel chamber 10 (N), R MC ≥8 μL / πd 4 It is desirable to satisfy the relationship N. With this configuration, even if the blood vessels become thicker as the vascular tissue matures, the increase in the flow rate of the culture medium 101 can be controlled and the maximum flow rate of the culture medium 101 can be estimated, making the process of performing perfusion culture over a long period of time more efficient. Furthermore, by designing the flow resistance of the passive valve 111 to be greater than the flow resistance of the blood vessel, it can also serve as a resistive flow path 35.

[0110] The effects of the present invention will be further clarified by the following examples. However, the present invention is not limited to the following examples and can be implemented with appropriate modifications without altering its essence.

[0111] [First Embodiment] In the first embodiment shown below, the channels for angiogenesis (hydrogel chambers 10, etc.) in the culture vessel 2 shown in Figures 19 and 20 were fabricated by photolithography and PDMS (polydimethylsiloxane) molding. The dimensions of the hydrogel chamber 10 are 5.6 mm in the length direction of the blood vessel (dimension in the X-axis direction), 9.7 mm in width (dimension in the Y-axis direction), and 0.3 mm in depth (dimension in the Z-axis direction).

[0112] Figure 21 is a graph showing the relationship between blood vessel diameter, shear stress, and flow rate according to the first embodiment. In Figure 21, the relationship between blood vessel diameter, shear stress, and flow rate is estimated using equation (6). In Figure 21, "no resistance channel" refers to the calculation result when vascular tissue is formed in culture vessel 2 without the resistance channel 35, as shown in Figure 19. Also, in Figure 21, "with resistance channel" refers to the calculation result when vascular tissue is formed in culture vessel 2 with the resistance channel 35, as shown in Figure 20. In the first embodiment, the culture vessel 2 was pressurized to 1 [kPa], and the flow resistance (R) of the resistive flow channel 25 was set. MC ) is 2 × 10 -8 [Pa·s / μm 3 The viscosity (μ) of the culture medium 101 is set to 0.001 [Pa·s], and the length of the formed blood vessels (L V The diameter is set to 5600 [μm], and the number of blood vessels formed in parallel (N V The result shown is the calculation result when the value is set to 16. In culture vessel 2 without a resistive channel, it can be seen that the flow rate of the culture medium 101 increases as the blood vessel diameter increases. On the other hand, in culture vessel 2 with a resistive channel, it can be seen that the flow rate of the culture medium does not increase above 230 [μL / min] even when the blood vessel diameter increases.

[0113] In addition, in the first example, to coat the channel in the culture vessel 2, poly-L-lysine hydrobromide (molecular weight 30,000-70,000: manufactured by SIGMA-ALDRICH), which had been pre-adjusted to 614 [μg / ml] with water, was injected and left to stand at 37 [°C] for 1 hour, after which it was washed twice with phosphate-buffered saline.

[0114] Furthermore, to prepare hydrogel 100, fibrinogen, derived from human plasma (Wako), was dissolved in D-PBS(-) (Wako) culture medium 101 at 37°C to a concentration of 6 mg / ml, and then filtered and sterilized using a 0.22 filter Milliex-GV (Millipore) and a Terumo syringe (TERMO).

[0115] Furthermore, in order to observe the process of angiogenesis, cultured human vascular endothelial cells (HUVECs) were used in CellTracker. TM The cells were fluorescently stained with Red CMTPX Dye (Invitrogen). Afterward, they were detached by trypsin treatment, centrifuged, and resuspended in resuspension buffer (a cell suspension containing thrombin at a final concentration of 4 [U / mL]). The cell concentration was then counted. The cell concentration was 12 × 10⁶. 6The mixture was diluted again with resuspension buffer to achieve the desired cell count ([cells / mL]).

[0116] The culture vessels 2 shown in Figures 19 and 20 were pre-cooled to 4°C. A pregel solution was prepared by mixing fibrinogen solution and a cell suspension containing HUVEC in a 1:1 ratio, and the pregel solution was injected into the hydrogel chamber 10 of each culture vessel 2. The vessels were then placed in a 37°C, 5% CO2 incubator and kept warm for 15 minutes to solidify the gel, after which the dedicated culture medium EGM was added to the culture medium channel. TM BulletKit TM EGM TM Endothelial Cell Growth Medium BulletKit TM The Lonza product was introduced through the openings of the first microchannel 24 and the second microchannel 34, and the mixture was placed in a 37°C, 5% CO2 incubator and kept warm for 60 minutes. After removing the bubbles that formed at the boundary between the gel and the culture medium by pipetting, a check valve 36 was installed at the opening of the second microchannel 34 located in the second culture medium storage tank 31.

[0117] Then, 1 mL of culture medium was added to the first culture medium storage tank 21, and pressurized circulating culture was started. Specifically, the first culture medium storage tank 21 was pressurized to 1 kPa for 240 seconds to deliver the culture medium to the cells, and the second culture medium storage tank 31 was pressurized to 1 kPa for 60 seconds to return the culture medium. This cycle was repeated for 7 days.

[0118] The day the cells were introduced was designated as day 0, and fluorescence microscopy observation and progressive flow rate measurements were performed from days 3 to 7. The culture medium was changed on days 3 and 6. From days 4 to 7, 10 μl of 70 kDa FITC dextran (SIGMA) was flowed under hydrostatic pressure for 5 minutes, followed by fluorescence microscopy observation to analyze the shape and conductivity of the blood vessels.

[0119] Figure 22 is a fluorescence microscopy image of vascular tissue in the case of "no resistance channel" according to the first embodiment. Figure 23 is a fluorescence microscopy image of vascular tissue in the case of "with resistance channel" according to the first embodiment. Figures 22 and 23 show fluorescence microscopy images observed on day 0, day 3, day 5, and day 7 of the above results. On day 3, in both culture vessels 2, "without resistance channel" and "with resistance channel," blood vessel elongation and expansion had progressed, and the cells were oriented along the flow of the culture medium. On day 5, blood vessel expansion had progressed further, and stronger cell orientation was observed than on day 3. On day 7, HUVECs had proliferated to the point where the gel portion between blood vessels was almost invisible, and cell orientation was lost.

[0120] Figure 24 is a fluorescence microscope image of the entire vascular tissue in the case of "no resistance channel" according to the first embodiment. Figure 25 is a fluorescence microscope image of the entire vascular tissue in the case of "with resistance channel" according to the first embodiment. Figures 24 and 25 show the results of observing the overall state of the blood vessels after flowing 70 kDa FITC dextran for 3 days after vascular omission. As shown in Figure 24, vascular omission occurred on day 5 in the "no resistance channel" case and on day 4 in the "with resistance channel" case. For the following 3 days, the blood vessels expanded in both channels, and no difference was observed in the rate of expansion. In both cases, it was shown that it is possible to construct vascular tissue with a size of 5.6 mm in the longitudinal direction of the blood vessel within the hydrogel 100. This demonstrates that the method of the present invention can form larger vascular tissue than previously reported.

[0121] Figure 26 is a graph showing the relationship between the sequential flow rate of the culture medium 101 and the culture period according to the first embodiment. As shown in Figure 26, in the "no resistance channel" case, the blood vessels became partially conductive on day 4, and the flow rate was 81.8 [μl / min]. Subsequently, the flow rate increased rapidly as the blood vessels expanded, reaching 330.2 [μl / min] on day 5 and 839.2 [μl / min] on day 7. In the "with resistance channel" case, the blood vessels became conductive on day 4, and the flow rate was 288.5 [μl / min]. In the "with resistance channel" case, the flow rate did not increase from the time the blood vessels became conductive until day 7, and was maintained at approximately 230 [μl / min]. In both culture vessels 2, it was shown that it is possible to pressurize and permeate the culture medium 101 into the hydrogel 100 when blood vessels have not yet formed, and then perfuse the culture medium 101 into the vascular tissue as the blood vessels mature.

[0122] Conventionally, examples of constructing vascular tissue with a length of 2 mm or less have been reported, but there are no reports of constructing larger vascular tissue. In the first embodiment, it was confirmed that vascular tissue up to 5.6 mm in length can be constructed by applying the present invention. When using vascular tissue for vascular permeability testing, using larger vascular tissue allows for the testing of compounds using vascular tissue with a larger surface area. Furthermore, using vascular tissue with a larger surface area has the advantage of increasing the amount of compound recovered after permeating the vascular wall, thereby improving the sensitivity of the permeability test.

[0123] Thus, according to the present invention, by forming microvascular tissue while continuously applying pneumatic pressure, it becomes possible to perfuse the culture medium 101 in accordance with the formation of the microvascular tissue. According to the present invention, the flow rate of the culture medium 101 can be reported over time, and the microvascular tissue can be stably maintained over a long period of time. Such perfusion in accordance with the formation of microvascular tissue and the construction of stable vascular tissue over a long period of time can improve the stability of compound permeability tests and, when applied to organoid culture bound to vascular tissue, enable the formation of more mature organoids.

[0124] [Second Example] Figure 27 is a schematic plan view showing the cell culture apparatus 1 according to the second embodiment. In the second embodiment, the cell culture apparatus 1 shown in Figure 27 was used. This cell culture apparatus 1 differs from the configuration shown in Figure 20 (first embodiment) in that the second culture medium storage tank 31 of the second culture medium storage section 30 is divided into second culture medium storage tanks 31A and 31B, and a resistance channel 25 is provided in the first microchannel 24.

[0125] The pneumatic pump 60 comprises pneumatic pump 60A connected to the second culture medium storage tank 31A and pneumatic pump 60B connected to the second culture medium storage tank 31B. The first culture medium storage tank 21 is connected to pneumatic pump 50. The third culture medium storage tank 81 is connected to pneumatic pump 90. The second culture medium storage tanks 31A and 31B are connected to each other by a connecting channel 37. The connecting channel 37 equalizes the liquid level of the culture medium 101 in the second culture medium storage tanks 31A and 31B.

[0126] The return channel 110 is connected between the second culture medium storage tank 31A and the first culture medium storage tank 21, but it may also be connected between the second culture medium storage tank 31B and the first culture medium storage tank 21. The resistance channel 25 is provided in each of the microchannels 24A and 24B of the first microchannel 24.

[0127] In the second example, each channel for angiogenesis in the culture vessel 2 (hydrogel chamber 10, etc.) was fabricated by photolithography and PDMS (polydimethylsiloxane) molding. The dimensions of the hydrogel chamber 10 were 5.6 mm in length (X-axis direction), 9.7 mm in width (Y-axis direction), and 0.3 mm in depth (Z-axis direction).

[0128] Furthermore, the resistive channel 25 of the first microchannel 24 and the resistive channel 35 of the second microchannel 34 have a length of 1.2 [mm], a width of 0.08 [mm], and a height of 0.06 [mm]. Assuming that the interfacial tension between the culture medium and air is [60 mN / m], the pressure resistance of the first microchannel 24 and the second microchannel 34 is estimated to be 3.5 [kPa] from equation (8). The resistive channel 25 also functions as a passive valve.

[0129] The culture vessel 2 shown in Figure 27 has a resistance channel 25 in the first microchannel 24, compared to the culture vessel 2 shown in Figures 19 and 20. Also, the opening of the second microchannel 34 is approximately 1 cm higher than the bottom surface of the second culture medium storage tanks 31A and 31B. The opening of the first microchannel 24 is located at the bottom surface of the first culture medium storage tank 21. This configuration restricts the flow of the culture medium only from the first culture medium storage tank 21 to the second culture medium storage tanks 31A and 31B (in the direction of the arrows in the figure), thereby preventing backflow.

[0130] In other words, when the first culture medium storage tank 21 is pressurized by the pneumatic pump 50, the culture medium in the first culture medium storage tank 21 flows through the opening of the first microchannel 24, the first microchannel 24, the hydrogel chamber 10, and the second microchannel 34 to the opening of the second microchannel 34. On the other hand, when the pneumatic pumps 60A and 60B are pressurized, the opening of the second microchannel 34 is higher than the liquid level of the culture medium in the second culture medium storage tanks 31A and 31B. As a result, air flows into the second microchannel 34 from its opening, and the resistance channel 35 acts as a passive valve, stopping the inflow of air and thus stopping the flow of the culture medium. Therefore, by alternately pressurizing the pneumatic pump 50 and the pneumatic pumps 60 (60A and 60B), a unidirectional flow from the opening of the first microchannel 24 to the opening of the second microchannel 34 can be created.

[0131] Furthermore, the dimensions of the passive valve 111 of the return channel 110 are 0.24 mm in length, 0.08 mm in width, and 0.06 mm in height. Assuming that the interfacial tension between the culture medium and air is 60 mN / m, the pressure resistance of the passive valve is estimated to be 3.5 kPa from equation (8). The opening of the return channel 110 on the first culture medium storage tank 21 side is approximately 1 cm higher than the bottom surface of the first culture medium storage tank 21, and the opening of the return channel 110 on the second culture medium storage tank 31A side is located at the bottom surface of the second culture medium storage tank 31A. This restricts the flow of the culture medium 101 only from the second culture medium storage tanks 31A and 31B towards the first culture medium storage tank 21 (in the direction of the arrow in the figure), thereby preventing backflow.

[0132] In other words, when the second culture medium storage tanks 31A and 31B are pressurized by the pneumatic pumps 60A and 60B, the culture medium 101 in the second culture medium storage tank 31A flows through the return channel 110 to the opening on the second culture medium storage tank 31A side of the return channel 110, and then through the return channel 110 to the opening on the first culture medium storage tank 21 side of the return channel 110. On the other hand, when the pneumatic pump 50 is pressurized, the opening on the first culture medium storage tank 21 side of the return channel 110 is higher than the liquid level of the culture medium in the first culture medium storage tank 21. As a result, air flows into the return channel 110 from the opening on the first culture medium storage tank 21 side of the return channel 110, and the passive valve 111 stops the inflow of air, thus stopping the flow of the culture medium 101. Therefore, by alternately pressurizing the pneumatic pump 50 and the pneumatic pump 60, a unidirectional flow can be generated from the opening of the return channel 110 on the second culture medium storage tank 31A side toward the opening of the return channel 110 on the first culture medium storage tank 21 side.

[0133] In the second example, to coat the channel in culture vessel 2, poly-L-lysine hydrobromide (molecular weight 30,000-70,000: manufactured by SIGMA-ALDRICH), which had been pre-adjusted to 500 [μg / ml] with water, was injected and left to stand at 37 [°C] for 1 hour. After that, it was washed at least three times with a sterile 50% ethanol aqueous solution and then completely dried.

[0134] In the second embodiment, the following tests were conducted using the cell culture apparatus 1 shown in Figure 27 to confirm how the culture medium components permeate into the hydrogel 100 by pressurized perfusion. To prepare hydrogel 100, fibrinogen, derived from bovine plasma (Wako), was dissolved in D-PBS(-) (Wako) culture medium 101 at 37°C to a concentration of 5 mg / ml, and filtered sterilization was performed using a 0.22 filter Milliex-GV (Millipore) and a Terumo syringe (TERMO). A culture medium containing 4 U / mL of thrombin (Wako) (EGM-2MV, Lonza) was prepared.

[0135] Culture vessel 2 was pre-cooled to 4°C, and a pregel solution was prepared by mixing fibrinogen solution and a culture medium containing thrombin in a 1:1 ratio. The pregel solution was then poured into the hydrogel chamber 10 of culture vessel 2. The culture vessel was then placed in a 37°C, 5% CO2 incubator and kept warm for 35 minutes to solidify the gel. After that, the culture medium EGM-2MV was introduced through the openings of the second microchannel 34 and the first microchannel 24, and the culture vessel was placed in a 37°C, 5% CO2 incubator and kept warm for 60 minutes. The culture medium was then introduced into the return channel 110 from the opening on the first culture medium storage tank 21 side of the return channel 110.

[0136] Next, 1 mL of culture medium was added to the first culture medium storage tank 21, and after connecting pneumatic pumps 50, 60, and 90 to the culture vessel 2, pressurized perfusion was started under the following conditions. Pressurized perfusion was performed in an incubator at 37°C and 5% CO2. Pressurization was performed by pressurizing the first culture medium storage tank 21 and the third culture medium storage tank 81 at 0.5 kPa for 1440 seconds to sequentially move the culture medium, and then pressurizing the second culture medium storage tanks 31A and 31B for 360 seconds to return the culture medium 101. This cycle was repeated.

[0137] After pressurized perfusion overnight, the first microchannel 24 was replaced with culture medium 101 containing 20 [μM] Calcein (DOJINDO LABORATORIES) and 20 [μM] Rhodamine-Dextran (molecular weight 70,000 Da: Thermo Fisher Scientific). Then, 500 [μL] of culture medium 101 was added to the first culture medium storage tank 21. The first culture medium storage tank 21 was then pressurized at 0.5 [kPa], 1.0 [kPa], 2.0 [kPa], and 3.0 [kPa], and fluorescence microscope images were acquired at predetermined intervals.

[0138] Figure 28 shows fluorescence microscope images of Calcein immediately after pressurizing culture vessel 2 according to the second embodiment to 2.0 [kPa] and 50 seconds later. As shown in Figure 28, the fluorescence interface of Calcein shifts upward by approximately 2.0 to 2.5 mm after 50 seconds compared to immediately after pressurization.

[0139] Figure 29 is a graph showing the results of measuring the migration of the Rhodamine-Dextran fluorescence interface over time when the culture vessel 2 according to the second example was pressurized at 0.5 [kPa], 1.0 [kPa], 2.0 [kPa], and 3.0 [kPa]. Figure 30 is a graph showing the results of measuring the migration of the Calcein fluorescence interface over time when the culture vessel 2 according to the second example was pressurized at 0.5 [kPa], 1.0 [kPa], 2.0 [kPa], and 3.0 [kPa]. As shown in Figures 29 and 30, the migration velocities of the fluorescence interfaces of Calcein and Rhodamine-Dextran were similar, and it was observed that the migration velocities of the fluorescence interfaces increased in proportion to the pressure.

[0140] It is generally known that the molecular weights of calcein and rhodamine-dextran differ significantly, at 623 [Da] and 70,000 [Da] respectively, resulting in vastly different diffusion rates. Therefore, the results shown in Figures 29 and 30 strongly suggest that calcein and rhodamine-dextran penetrate the hydrogel 100 not by diffusion, but by pressurized osmosis. This confirms that the pressurized osmosis method of the present invention is effective in allowing nutrients contained in the culture medium 101 to penetrate the hydrogel 100.

[0141] Next, using the cell culture apparatus 1 shown in Figure 27, the following tests were conducted to confirm angiogenesis by pressurized perfusion. During this process, the effect of culturing a mixture of mesenchymal stem cells and human umbilical vein endothelial cells (HUVECs) was also investigated. To prepare hydrogel 100, fibrinogen derived from bovine plasma (Wako) was dissolved in D-PBS(-) (Wako) culture medium 101 at 37°C to a concentration of 5 mg / ml, and then filtered and sterilized using a 0.22 filter Milliex-GV (Millipore) and a Terumo syringe (TERMO).

[0142] Furthermore, in order to observe the process of angiogenesis, cultured HUVECs were placed in CellTracker TM The cells were fluorescently stained with Red CMTPX Dye (Invitrogen). Subsequently, HUVEC and mesenchymal stem cells (UE7T-13, JCRB cell bank) exfoliated with 0.05% or 0.25% Trypsin-EDTA were suspended in EGM-2MV containing 4 U / mL of Thrombin (FUJIFILM Wako Pure Chemical Corporation) to concentrations of 12 × 10⁶ [cells / mL] and 4 × 10⁶ [cells / mL], respectively.

[0143] The culture vessels 2 shown in Figure 27 were pre-cooled to 4°C. A pregel solution was prepared by mixing fibrinogen solution and a cell suspension containing HUVEC and MSCs in a 1:1 ratio, and the pregel solution was injected into the hydrogel chamber 10 of each culture vessel 2. The vessels were then placed in a 37°C, 5% CO2 incubator and incubated for 35 minutes to solidify the gel. After that, the culture medium EGM-2MV was introduced through the openings of the first microchannel 24 and the second microchannel 34, and the vessels were placed in a 37°C, 5% CO2 incubator and incubated for 60 minutes. The culture medium 101 was introduced into the return channel 110 from the opening on the first culture medium storage tank 21 side of the return channel 110.

[0144] Next, 1.2 mL of culture medium was added to the first culture medium reservoir 21, and after connecting the device to the pressurized perfusion apparatus, pressurized perfusion was started under the following conditions. Pressurized perfusion was performed in an incubator at 37°C and 5% CO2. Pressurization was performed at 2.0 kPa, and the first culture medium reservoir 21 and the third culture medium reservoir 81 were pressurized for 360 seconds and the medium was transferred sequentially. Then, the second culture medium reservoirs 31A and 31B were pressurized for 90 seconds and the medium was returned. This cycle was repeated for 12 days. The day on which the cells were introduced was designated as day 0, and fluorescence microscopy observation and sequential flow rate measurements were performed on days 1 to 12. The culture medium was changed each time the flow rate was measured.

[0145] Figure 31 is a fluorescence microscope image showing the blood vessel formation behavior with and without mesenchymal stem cells according to the second embodiment. Figure 32 is a graph showing the change in blood vessel flow with and without mesenchymal stem cells according to the second embodiment. As shown in Figure 31, microvascular formation was observed on day 4, regardless of the presence or absence of mesenchymal stem cells. During this period, an increase in the flow rate of culture medium 101 was observed in conjunction with the formation of vascular tissue (see Figure 32).

[0146] As shown in Figure 31, in the system without mesenchymal stem cells, HUVECs proliferated to such an extent that the intervascular gel portion was almost completely obscured between day 6 and day 8. On the other hand, in the system containing mesenchymal stem cells, the tubular structure of the vascular tissue was still confirmed on day 8, and as shown in Figure 32, although a decrease in the culture medium flow rate was observed with the proliferation of mesenchymal stem cells, peaking on day 6, it was confirmed that a constant flow rate of culture medium to the vascular tissue could be maintained. Based on the above, it was confirmed that by culturing mesenchymal stem cells mixed with HUVEC during the formation of vascular tissue, the vascular tissue can be stably maintained for 12 days.

[0147] The culture vessel 2 shown in Figure 27 has a backflow prevention mechanism and a return channel 110 that control the direction of flow of the culture medium 101, and is configured so that the culture medium flows in one direction from the first culture medium storage tank 21 to the second culture medium storage tanks 31A and 31B into the hydrogel chamber 10. To verify the effect of the backflow prevention mechanism, that is, the effect of unidirectional flow in vascular tissue formation, the culture vessel shown in Figure 33 was fabricated.

[0148] Figure 33 is a schematic plan view showing a cell culture apparatus 1 according to a modified example of the second embodiment. The culture vessel 2 shown in Figure 33 does not have a return channel 110. Also, the opening of the second microchannel 34 is provided on the bottom surface of the second culture medium storage tanks 31A and 31B. With this configuration, by alternately pressurizing the pneumatic pump 50 and pneumatic pumps 60A and 60B, the culture medium 101 can be moved back and forth between the first culture medium storage tank 21 and the second culture medium storage tanks 31A and 31B via the first microchannel 24, the hydrogel chamber 10, and the second microchannel 34.

[0149] In the formation of vascular tissue, the behavior of vascular tissue formation under unidirectional and reciprocating flow conditions was compared using culture vessel 2 shown in Figure 27 and culture vessel 2 shown in Figure 33. Similar to the above experiment (conditions for culturing a system containing mesenchymal stem cells using culture vessel 2 shown in Figure 27), HUVEC and mesenchymal cells were introduced into hydrogel 100, and pressurized perfusion was started. For pressurized perfusion conditions to create a unidirectional flow, the first culture medium reservoir 21 and the third culture medium reservoir 81 were pressurized at 2.0 [kPa] for 360 seconds and moved sequentially, and the second culture medium reservoirs 31A and 31B were pressurized for 90 seconds and returned, and this cycle was repeated for 12 days. Furthermore, to generate a reciprocating flow, the first culture medium storage tank 21 and the third culture medium storage tank 81 were pressurized at 2.0 [kPa] for 360 seconds, and then the second culture medium storage tanks 31A and 31B were pressurized for 360 seconds. This cycle was repeated for 12 days.

[0150] Figure 34 is a fluorescence microscope image showing the formation behavior of vascular tissue under unidirectional and reciprocating flow environments in the second embodiment and one of its modified examples. In both unidirectional and reciprocating flow conditions, vascular tissue formation was observed around day 4 after cell introduction. Figure 34 shows an image of vascular tissue on day 6 after cell introduction. Vascular tissue formation was observed in both unidirectional and reciprocating flow conditions, but in the unidirectional flow condition, the vascular tissue appeared to be more oriented in the direction of the flow compared to the reciprocating flow condition.

[0151] Figure 35 is a graph showing the flow rate changes in vascular tissue under unidirectional and reciprocating flow environments in the second embodiment and one of its modified examples. As shown in Figure 35, in both unidirectional and reciprocating flow cases, an increase in flow rate was observed from day 4 to day 6 after cell introduction, due to the formation of vascular tissue, and the flow of culture medium 101 through vascular tissue was confirmed until day 12.

[0152] Next, the effects of the presence or absence of mesenchymal stem cells, and the effects of unidirectional and reciprocating flow on the expression of proteins involved in the barrier function and vascular function of the vascular tissue formed in cell culture apparatus 1 were evaluated. For this purpose, vascular tissue was formed under three conditions using the culture vessels shown in Figure 27 and Figure 33: (i) no mesenchymal stem cells and unidirectional flow, (ii) mesenchymal stem cells present and unidirectional flow, and (iii) mesenchymal stem cells present and reciprocating flow.

[0153] Figure 36 is an image analysis diagram showing the process of determining the permeability coefficient of a fluorescent substance through the blood vessel wall in order to evaluate the barrier function of vascular tissue formed in the cell culture apparatus 1 according to the second embodiment and one of its modified examples. To evaluate the barrier function of vascular tissue, the permeability coefficient of fluorescent substances through the vascular wall was determined by image analysis. For systems without mesenchymal stem cells, the permeability coefficient of the fluorescent substance was determined on day 4 of culture, and for systems co-cultured with mesenchymal stem cells, it was determined on day 6 of culture, using the following method.

[0154] A culture medium containing 20 μM of calcein and 20 μM of rhodamine-dextran (molecular weight 70,000 Da) was added to the first culture medium reservoir 21 in a volume of 500 μL. A fluorescence microscope, IX71 (Olympus Corporation), was used to acquire fluorescence microscope images for 3 minutes every 20 seconds. The images shown in Figure 36 were obtained by acquiring two fields of view for each sample, and for each field of view, a region surrounded by 25 blood vessels was randomly selected as the analysis region. The transmission coefficients of calcein and rhodamine-dextran from vascular tissue were calculated using the following formula (9).

[0155]

number

[0156] Here, P [cm / sec] is the transmission coefficient. s [mol / sec] is the amount of substance that permeates per unit time. w [cm 2 ] is the area of ​​the blood vessel wall through which the substance permeates. L [mol / cm 3 ] represents the concentration difference between the inside and outside of the blood vessel wall. ΔI ex,mean [-] represents the time change in the average fluorescence intensity of the region surrounded by blood vessels. A i [cm 2 ] is the area of ​​the region surrounded by blood vessels. Δt[sec] is the rate of change over time. w [cm] represents the circumference of the area surrounded by blood vessels. i,mean [-] represents the average fluorescence intensity on the vascular side near the region surrounded by blood vessels. b,mean [-] represents the average fluorescence intensity of the region surrounded by blood vessels. Image data was analyzed using ImageJ (1.53c, NIH).

[0157] Figure 37 shows the permeability coefficients of Calcein obtained under three conditions for the second embodiment and one of its modifications: (i) without mesenchymal stem cells and unidirectional flow, (ii) with mesenchymal stem cells and unidirectional flow, and (iii) with mesenchymal stem cells and reciprocating flow. Figure 38 shows the permeability coefficients of Rhodamine-Dextran obtained under three conditions for the second embodiment and one of its modifications: (i) without mesenchymal stem cells and unidirectional flow, (ii) with mesenchymal stem cells and unidirectional flow, and (iii) with mesenchymal stem cells and reciprocating flow. In both the case of Calcein and Rhodamine-Dextran, the permeability coefficient was lower in the system containing mesenchymal stem cells. Furthermore, a tendency for the permeability coefficient to be lower was observed for unidirectional flow compared to reciprocating flow. These results suggest that the culture vessel 2 configured in Figure 27, which has a backflow prevention mechanism to control the direction of flow of the culture medium 101 and a return channel 110, is useful for forming vascular tissue with high barrier function.

[0158] Furthermore, immunohistochemical staining was performed using the following method to confirm the expression of proteins involved in vascular function. Vascular tissue formed under HUVEC conditions alone was fixed with 4% paraformaldehyde on day 4, and vascular tissue formed by mixing with mesenchymal stem cells was fixed with 4% paraformaldehyde on day 6.

[0159] The samples were then washed twice with phosphate buffer and permeabilized with 0.1% Triton-X aqueous solution for 15 minutes. After washing twice with phosphate buffer, blocking was performed at room temperature for 2 hours using Blocking One Hist (Nacalai Tesque). After washing three times with phosphate buffer (PBST) containing Tween20, the primary antibody used was reacted overnight at 4°C using the following antibody. Subsequently, the samples were washed three times with PBST and reacted at room temperature for 2 hours with a secondary antibody to which Phalloidin-iFluor or 4',6-diamidino-2-phenylindole (DAPI) had been added. The samples were washed three times with PBST before observation.

[0160] CD31 was labeled with a 200-fold dilution of rabbit monoclonal antibody (28364 or 32457: abcam). NG2 was labeled with a 200-fold dilution of mouse monoclonal antibody (MAB2585: R&D Systems). Vascular endothelial cadherin (VE-cadherin), laminin, and Invitrogen33-9100 were labeled with a 100-fold dilution of rabbit polyclonal antibody (CST2158S, abcam11575, and Invitrogen33-9100, respectively).

[0161] Fluorescently labeled secondary antibodies (Invitrogen A11011, A11034, or abcam150113) were used at 200-400-fold dilutions. Cell nuclei were stained with Cellstain-DAPI solution (Dojin Chemical Laboratories) at a 1:1000 dilution. F-actin filaments were stained with Phalloidin-iFluor 488 (Cayman Chemical Company) at a 1:1000 dilution.

[0162] Figure 39 shows images of vascular endothelial cells formed in the cell culture apparatus 1 according to the second embodiment and one of its modified forms, stained with the marker CD31 and stained with the mesenchymal stem cell marker NG2. As shown in Figure 39, in the system cultured with mesenchymal stem cells, the presence of mesenchymal stem cells around the vascular tissue formed by vascular endothelial cells was confirmed under both unidirectional and reciprocating flow conditions.

[0163] Figure 40 shows images of vascular endothelial cells formed in the cell culture apparatus 1 according to the second embodiment and one of its modifications, stained with ZO-1, which constitutes the tight junctions of the blood vessel wall. As shown in Figure 41, under the conditions of mesenchymal stem cells and unidirectional flow, ZO-1 is expressed throughout the entire vascular wall compared to other conditions. This suggests that the high vascular barrier function observed in Figure 36, i.e., the low permeability coefficient, is achieved by the expression of tight junction molecules.

[0164] The second embodiment and one of its modifications described above demonstrate that the culture vessel 2 configured as shown in Figure 27, which has a backflow prevention mechanism to control the direction of flow of the culture medium 101 and a return channel 110, is useful for forming vascular tissue with high barrier function.

[0165] While preferred embodiments and examples of the present invention have been described and explained above, it should be understood that these are illustrative examples of the present invention and should not be considered as limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the scope of the invention. Therefore, the present invention should not be considered limited by the foregoing description, but rather limited by the claims. [Explanation of Symbols]

[0166] 1...Cell culture device 2...Culture container 3…Lid part 5... Pneumatic equipment 10…Hydrogel Chamber 11…Interior space 12...Side 1 13…Second side 14…Inlet hole 14a... Introduction path 15…Inlet hole 15a...Introduction path 16…Hydrogel storage tank 16A... Hydrogel storage tank 16B... Hydrogel storage tank 20...First culture medium storage section 21…First culture medium storage tank 22...Strut row 23…First recess 24…First microchannel 24A... Microfluidic 24B... Microfluidic 25… Resistive channel 30...Second culture medium storage section 31…Second culture medium storage tank 32…Strut row 33...Second recess 34…Second microchannel 34A... Microfluidic 34B... Microfluidic 35… Resistive channel 36... Check valve 40…post 41…Slope 42…Plane 43...Curved surface 50…Pneumatic pump 51... Pneumatic piping 60... Pneumatic pump 60A... Pneumatic pump 60B... Pneumatic pump 61... Pneumatic piping 80...Third culture medium storage section 81...Third culture medium storage tank 82…Opening 82a...Membrane 83...Third recess 90... Pneumatic pump 91... Pneumatic piping 100... Hydrogel 101…Culture solution 110... Return channel 111... Passive valve

Claims

1. A hydrogel chamber that holds a hydrogel for culturing cells, A first culture medium storage section communicating with the first surface of the hydrogel chamber, A culture vessel comprising a second culture medium storage section communicating with a second surface of the hydrogel chamber that is different from the first surface, The culture vessel is connected to a pneumatic device, The pneumatic device generates a pressure difference based on air pressure between the first culture medium storage section and the second culture medium storage section, thereby pressurizing and permeating the culture medium stored in at least one of the first culture medium storage section and the second culture medium storage section into the hydrogel of the hydrogel chamber. At least one of the first culture medium storage section and the second culture medium storage section is A culture medium storage tank for storing the culture medium, The system includes a microchannel that connects the culture medium storage tank and the hydrogel chamber, The microchannel is provided with one or more resistive channels having a smaller cross-sectional area than the microchannel, arranged in parallel. The flow resistance (R MC) of the resistive channel is given by the viscosity of the culture medium (μ), the length from the first surface to the second surface of the hydrogel chamber (L), the height of the hydrogel chamber (d), and the value obtained by dividing the width of the first or second surface by the height of the hydrogel chamber (N), R MC ≧ 8 μL / πd 4 N Satisfying the relationship, Cell culture equipment.

2. The aforementioned microchannels are provided in pairs. The cell culture apparatus according to claim 1.

3. The first culture medium storage unit comprises a first culture medium storage tank as the culture medium storage tank and a first microchannel as the microchannel, The second culture medium storage unit comprises a second culture medium storage tank as the culture medium storage tank and a second microchannel as the microchannel, A return channel for the culture medium that connects the first culture medium storage tank and the second culture medium storage tank, The first microchannel and the second microchannel, and the return channel, are provided with a mechanism to prevent backflow of the culture medium. The cell culture apparatus according to claim 1.

4. As a mechanism to prevent backflow, it is equipped with a passive valve to prevent the entry of air bubbles. The cell culture apparatus according to claim 3.

5. The pressure resistance of the passive valve is within the range of 0.1 [kPa] to 10 [kPa]. The cell culture apparatus according to claim 4.

6. An opening is formed directly above the hydrogel chamber. The cell culture apparatus according to claim 1.

7. A third culture medium storage section is provided directly above the hydrogel chamber, communicating with it through the opening. The opening is provided with a permeable membrane, one side of which faces the hydrogel and the other side of which faces the culture medium. The cell culture apparatus according to claim 6.

8. The pneumatic device is connected to the third culture medium storage unit. The cell culture apparatus according to claim 7.

9. The pneumatic device includes pneumatic piping connected to the culture vessel, The aforementioned pneumatic piping is equipped with a filter. The cell culture apparatus according to claim 1.

10. The hydrogel chamber is provided with a row of support columns along the first and second surfaces of the hydrogel chamber as a mechanism for holding the hydrogel in a state where a pressure difference based on the air pressure is generated. In the row of support columns, the gaps between adjacent support columns change in the order w1, w2, w3 toward the hydrogel chamber, and the relationship w2 < w1 < w3 is maintained. The cell culture apparatus according to claim 1.

11. A cell culture apparatus according to any one of claims 1 to 10 is used to culture cells in the hydrogel chamber. Cell culture method.

12. The cells are cultured and vascular tissue is formed in the hydrogel chamber. The cell culture method according to claim 11.

Images