Cell culture chip and cell culture device

Abstract

The present invention addresses the problem of providing a cell culture chip in which unevenness of flow velocity distribution in a flow channel width direction is reduced in a curved flow channel in order to prevent bubble entrapment in the cell culture chip and to uniformly seed cells. A cell culture chip (1) according to an embodiment of the present invention includes a laminate (100, 200) having a flow channel structure therein, wherein: the laminate (100, 200) includes a flow channel substrate (L2, L4) in which a flow channel (F2, F4) leading to a port (P1, P2, P3, P4) is formed; the flow channel (P1, P2, P3, P4) includes a curved flow channel (FC) formed in a curved shape in plan view of the flow channel substrate (L2, L4) and having a cross-sectional area that gradually changes in the flow direction of a fluid, and a first connection flow channel (FJ1) connecting the port and the curved flow channel (FC) and having a constant cross-sectional area in the flow direction of the fluid; the cross-sectional area of the first connection flow channel (FJ1) is smaller than the minimum value of each of the cross-sectional area of the port (P1, P2, P3, P4) and the cross-sectional area of the curved flow channel (FC); and in plan view, the flow channel width of the curved flow channel (FC) gradually changes in the flow direction of the fluid.

Description

Cell culture chips and cell culture devices

[0001] This invention relates to cell culture chips and cell culture devices.

[0002] Conventional cell culture methods using culture dishes, as employed in traditional cell assays, create an environment significantly different from that in vivo. Therefore, a common problem with conventional cell culture methods is the loss of many of the functions expressed by cells in vivo. To address this issue, culture devices incorporating microchannels for culturing cells while delivering culture medium have been developed. Patent Document 1 discloses a method for manufacturing channel shapes using laser processing. However, with channel shapes where the channel width and bend angle are constant, bubbles may be generated during culture medium introduction, or cells may not be uniformly seeded due to differences in flow distribution within the channel.

[0003] International Publication No. 2023 / 027147

[0004] In cell culture chips with a flow channel structure, increasing the size of the opening at the end of the flow channel, which connects to the inlet (port) for test solutions (drug solutions), makes it easier for "bubble trapping" to occur during the liquid introduction operation into the flow channel. To prevent bubble trapping, it is necessary to limit the dimensions (height and width) of the opening at the end of the flow channel to a predetermined range. On the other hand, cell culture chips are instruments for evaluating cell function. Therefore, they are required to (1) be able to culture the number of cells necessary for the evaluation test, (2) have sufficient culture area for that purpose, (3) be able to hold the volume of liquid necessary for analyzing the evaluation compounds contained in the test solution (drug solution), and (4) be able to uniformly seed cells in the cell culture area. Consequently, the width dimension of the main cell culture area may fall outside the appropriate size range for the opening at the end of the flow channel. To satisfy these two objectives ("prevention of bubble trapping" and "uniform seeding of cells"), it becomes necessary to expand the flow channel width between the opening at the end of the flow channel and the main cell culture area. Incidentally, two-layered channels separated by a porous membrane include I-shaped channels, where the channel shape is I-shaped in plan view, and X-shaped channels, where the channel shape is X-shaped in plan view. An I-shaped channel is formed, for example, by making the submembrane channel longer than the supramembrane channel and shifting the positions of the channel inlet and outlet. In an I-shaped channel, unlike an X-shaped channel, the inlet and outlet of the supramembrane channel are located above the submembrane channel, so there is a possibility that the membrane at the inlet and outlet of the supramembrane channel may be damaged by the tip of the pipette. The X-shaped channel is a widely used design as a two-layered channel separated by a porous membrane. In a typical X-shaped channel, the flow velocity distribution may be uneven at the bends in the channel, making it difficult to uniformly seed cells. Furthermore, uneven flow velocity distribution can cause cell sheets to detach easily, impair measurements and their accuracy, and have an impact on the cells. For example, if you try to secure a culture area while narrowing the inlet width, the flow velocity distribution will be uneven depending on the shape of the bends. Therefore, it is necessary to curve the shape of the channel that connects from the port to the main cell culture area. An X-shaped channel, including a curved channel formed in a curved shape in plan view, does not suffer from the problems of the I-shaped channel described above or the problems of a normal X-shaped channel. Therefore, in order to prevent foam buildup and uniformly seed cells, it is necessary to design an X-shaped channel (curved channel) to reduce the bias in the flow velocity distribution in the channel width direction.

[0005] Therefore, the present invention aims to reduce the bias in the flow velocity distribution in the width direction of a curved channel in order to prevent foam buildup and to uniformly seed cells.

[0006] (1) A cell culture chip according to one aspect of the present invention (first aspect) is a cell culture chip comprising a laminate having a flow channel structure inside, wherein the laminate includes a flow channel substrate having a flow channel leading to a port, the flow channel includes a curved flow channel formed in a curved shape in a plan view of the flow channel substrate and having a cross-sectional area that gradually changes in the direction of fluid flow, and a first connecting flow channel connecting the port and the curved flow channel and having a constant cross-sectional area in the direction of fluid flow, wherein the cross-sectional area of ​​the first connecting flow channel is smaller than the minimum value of the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved flow channel, respectively, and in the plan view, the flow channel width of the curved flow channel gradually changes in the direction of fluid flow.

[0007] With this configuration, the cross-sectional area of ​​the first connecting channel is smaller than the minimum values ​​of the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved channel, thereby reducing the risk of foam contamination compared to the case where the cross-sectional area of ​​the first connecting channel is equal to or greater than the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved channel. Furthermore, by providing a curved channel between the main cell culture area and the port, the bias in the flow velocity distribution can be reduced, and cells can be uniformly seeded in the main cell culture area. In addition, in a plan view, the channel width of the curved channel changes gradually in the direction of fluid flow, thereby reducing the bias in the flow velocity distribution in the direction of the channel width in the curved channel. Therefore, in order to prevent foam contamination and uniformly seed cells, the bias in the flow velocity distribution in the direction of the channel width can be reduced in the curved channel.

[0008] (2) In the cell culture chip described in (1) above, the length of the inner circumference of the curved channel in the plan view is L １ The length of the outer circumference of the curved channel is L. ２ In that case, 1.3 < L ２ / L １ < May satisfy 5.0.

[0009] According to this configuration, the velocity difference of the fluid flow between the inner peripheral side and the outer peripheral side of the curved flow path can be made as small as possible. Therefore, sedimentation of cells and substances in the curved flow path can be reduced.

[0010] (3) In the cell culture chip according to the above (1) or (2), the flow path further includes a second connection flow path connecting the first connection flow path and the curved flow path, and the cross-sectional area of the second connection flow path may gradually change in the fluid flow direction.

[0011] According to this configuration, the velocity difference of the fluid flow in the second connection flow path can be gradually changed. Therefore, the risk of bubble entrainment in the curved flow path can be further reduced.

[0012] (4) In the cell culture chip according to the above (3), the flow path further includes a third connection flow path connected to an end portion of the curved flow path on the side opposite to the second connection flow path and having a constant cross-sectional area in the fluid flow direction, and the cross-sectional area of the third connection flow path may be the same as the maximum value of the cross-sectional area of the curved flow path.

[0013] According to this configuration, by providing a third connection flow path between the main cell culture region and the curved flow path, cells can be seeded more uniformly.

[0014] (5) In the cell culture chip according to any one of the above (1) to (4), in a side view of the flow path substrate, the flow path height of the curved flow path may be constant in the fluid flow direction.

[0015] According to this configuration, the height (thickness) of the flow path substrate can be maintained.

[0016] (6) In the cell culture chip according to any one of the above (1) to (5), the cross-sectional shape of the curved flow path may be formed in a substantially rectangular shape.

[0017] According to this configuration, when processing the flow path substrate by laser processing, the curved flow path can be more easily formed compared to the case where the cross-sectional shape of the curved flow path is formed in a circular shape.

[0018] (7) In the cell culture chip according to any one of (1) to (6) above, the laminate includes a bottom plate substrate, a first flow path substrate in which a first flow path is formed, a porous membrane, a second flow path substrate in which a second flow path is formed, and a top plate substrate in this order, and the curved flow path may be formed in each of the first flow path and the second flow path.

[0019] According to this configuration, in each of the first flow path and the second flow path, foaming can be prevented and cells can be seeded uniformly, so that the deviation of the flow velocity distribution in the flow path width direction in the curved flow path can be reduced.

[0020] (8) In the cell culture chip according to (7) above, the cell culture chip is provided with a first introduction port for introducing a liquid for cell culture into the first flow path, a first discharge port for discharging the liquid from the first flow path, a second introduction port for introducing the liquid into the second flow path, and a second discharge port for discharging the liquid from the second flow path. The first flow path includes a first central flow path, a first introduction flow path connecting the first introduction port and the first central flow path, and a first discharge flow path connecting the first discharge port and the first central flow path. The second flow path includes a second central flow path that at least partially overlaps the first central flow path in plan view, a second introduction flow path connecting the second introduction port and the second central flow path, and a second discharge flow path connecting the second discharge port and the second central flow path. The curved flow path may be formed in each of the first introduction flow path, the first discharge flow path, the second introduction flow path, and the second discharge flow path.

[0021] According to this configuration, the deviation of the flow velocity distribution in the flow path width direction in each of the curved flow paths of the first introduction flow path, the first discharge flow path, the second introduction flow path, and the second discharge flow path can be reduced.

[0022] (9) A cell culture device according to an aspect (second aspect) of the present invention includes the cell culture chip according to any one of (1) to (8) above.

[0023] This configuration, which includes the cell culture chip described above, provides a cell culture device that can prevent foam buildup and uniformly seed cells, thereby reducing the bias in the flow velocity distribution in the width direction of the channel in a curved channel.

[0024] According to the cell culture chip and cell culture device of this embodiment, it is possible to prevent foam buildup and uniformly seed cells by reducing the bias in the flow velocity distribution in the width direction of the channel in a curved channel.

[0025] Perspective view of the cell culture chip of the embodiment. Exploded view of the cell culture chip of Figure 1. Top view of the cell culture chip of the embodiment. Cross-sectional view along line IV-IV of Figure 3. Cross-sectional view along line V-V of Figure 3. Diagram showing an example of the layer configuration of the cell culture chip of the embodiment. Diagram showing another example of the layer configuration of the cell culture chip of the embodiment. Diagram showing an example of cell culture using the cell culture chip of the embodiment. Perspective view of the flow path of the embodiment. Top view illustrating the curved flow path of the embodiment. Top view of the flow path of Example 1. Top view of the flow path of Example 2. Top view of the flow path of Example 3. Top view of the flow path of Example 4. Top view of the flow path of Example 5. Top view of the flow path of Comparative Example 1. Top view of the flow path of Comparative Example 2. Diagram showing the evaluation results of foam inclusion and liquid uniformity. Top view of the flow path of Comparative Example 1. Top view of the flow path of Example 3. Top view of the flow path of Example 5. Diagram showing the evaluation results of the flow velocity distribution. Top view showing the flow in the curved flow path of Comparative Example 1. A top view showing the flow in the curved channel of Example 3. A diagram showing the flow after 4.5 hours in the curved channel of Comparative Example 1. A diagram showing the flow after 4.5 hours in the curved channel of Example 3.

[0026] Embodiments of the present invention will be described below with reference to the drawings. The drawings show an XYZ coordinate system as necessary. In this specification, each direction will be defined and described along the XYZ coordinate system as necessary. In this embodiment, the X direction is an example of a first direction and is a direction along the horizontal plane. The Y direction is an example of a second direction perpendicular to the first direction in the horizontal plane. The Z direction is an example of a third direction perpendicular to both the first and second directions and is a direction along the vertical direction. The +Z direction corresponds to the upper side of the vertical direction. The -Z direction corresponds to the lower side of the vertical direction. In the drawings used in the following description, characteristic parts may be shown enlarged in order to make the features of the present invention easier to understand. In the drawings used in the following description, the dimensional ratios of each component may not be the same as in reality.

[0027] <Cell Culture Chip> Figure 1 is a perspective view of the cell culture chip 1 of the embodiment. Figure 2 is an exploded view of the cell culture chip 1 of Figure 1. Figure 3 is a top view of the cell culture chip 1 of the embodiment. Figure 4 is a cross-sectional view taken along line IV-IV in Figure 3. Figure 5 is a cross-sectional view taken along line V-V in Figure 3. As shown in Figures 1 to 5, the cell culture chip 1 includes a laminate having a channel structure inside.

[0028] <Laminate> The laminate includes channel substrates L2 and L4, which have channels leading to ports P1 to P4. The laminate includes a bottom substrate L1, a first channel substrate L2, a porous membrane L3, a second channel substrate L4, and a top substrate L5 in this order. The laminate may further include a cover substrate L6. In the example shown in the figure, the bottom substrate L1, the first channel substrate L2, the porous membrane L3, the second channel substrate L4, the top substrate L5, and the cover substrate L6 are laminated in this order. Reservoirs may be provided in the parts of the cell culture chip 1 corresponding to each of ports P1 to P4.

[0029] A first channel F2 is formed in the first channel substrate L2. A second channel F4 is formed in the second channel substrate L4. By stacking the substrates as described above, a channel structure consisting of the first channel F2 and the second channel F4 is formed inside the cell culture chip 1.

[0030] The cell culture chip 1 is provided with port P1 as a drug solution introduction port to the first channel F2, port P2 as a drug solution discharge port from the first channel F2, port P3 as a drug solution introduction port to the second channel F4, and port P4 as a drug solution discharge port from the second channel F4.

[0031] The first channel F2 is formed from a first central channel F2a, a first introduction channel F2b connecting port P1 and the first central channel F2a, and a first discharge channel F2c connecting port P2 and the first central channel F2a. The chemical solution introduced from port P1 is discharged from port P2 after passing through the first introduction channel F2b, the first central channel F2a, and the first discharge channel F2c. The second channel F4 is formed from a second central channel F4a, a second introduction channel F4b connecting port P3 and the second central channel F4a, and a second discharge channel F4c connecting port P4 and the second central channel F4a. The chemical solution introduced from port P3 is discharged from port P4 after passing through the second introduction channel F4b, the second central channel F4a, and the second discharge channel F4c.

[0032] The first central channel F2a and the second central channel F4a are arranged such that at least a portion of each channel overlaps. The first central channel F2a and the second central channel F4a are separated by a porous membrane M. In the cell culture chip 1, the first central channel F2a is a lower channel located below the porous membrane M. The second central channel F4a is an upper channel located above the porous membrane M.

[0033] The channel widths of the first central channel F2a and the second central channel F4a can be appropriately set according to the purpose of the cell culture chip 1. Examples of channel widths include 1 to 2000 μm, 10 to 1500 μm, 50 to 1000 μm, or 100 to 500 μm.

[0034] The cover substrate L6 is provided with an opening W so that the first central channel F2a and the second central channel F4a can be observed.

[0035] <Resin contained in the channel substrate> In the cell culture chip, the channel substrate (first channel substrate L2, second channel substrate L4) has a glass transition temperature (hereinafter also referred to as "Tg") of 37°C or higher, and an elastic modulus of 1 × 10 at 25°C. ９It is Pa or higher, and its modulus of elasticity at 150°C is 1 × 10⁻⁶. ７ The material contains a resin (hereinafter referred to as "resin A") with a pH of Pa or less. Channels are formed in the channel substrate by laser processing. In the cell culture chip, the channel substrate forms partitions for the channels.

[0036] ≪Resin (A)≫ Resin (A) has a Tg of 37°C or higher, and an elastic modulus of 1 × 10 at 25°C. ９ It is Pa or higher, and its modulus of elasticity at 150°C is 1 × 10⁻⁶. ７ It is less than or equal to Pa.

[0037] In this specification, "glass transition temperature (Tg)" means the temperature at which the tangents intersect at the inflection point of the measured temperature obtained by differential scanning calorimetry (DSC) under a heating rate of 20°C / min. In this specification, "elastic modulus" is the complex elastic modulus measured using a dynamic viscoelasticity measuring device. Specifically, the elastic modulus can be obtained by preparing a resin sheet as a test piece with a size of 5 mm × 40 mm and measuring the dynamic elastic modulus of the test piece using a dynamic viscoelasticity measuring device. The measurement conditions should be tensile conditions at a frequency of 1 Hz, with the temperature raised from a starting temperature of 25°C to 150°C at a heating rate of 2°C / min. As a dynamic viscoelasticity measuring device, for example, Rheogel-E4000 (manufactured by UBM) can be used. The elastic modulus at 25°C is the complex elastic modulus at 25°C measured by the dynamic viscoelasticity measuring device. The elastic modulus at 150°C is the complex elastic modulus at 150°C measured by the dynamic viscoelasticity measuring device.

[0038] Examples of resin (A) include polyester resins, acrylic resins, cycloolefin copolymer resins, urethane resins, polyolefin resins, fluororesins, silicone resins, or mixtures or modified resins of these resins. Among these, polyester resins, acrylic resins, and cycloolefin copolymer resins are preferred as resin (A).

[0039] When the flow channel substrate has a multilayer structure, it is preferable that the flow channel substrate has at least one layer containing resin (A) (hereinafter also referred to as "layer A"). The content of resin (A) in layer A is preferably 70% by mass or more, more preferably 80% by mass or more, even more preferably 90% by mass or more, and particularly preferably 95% by mass or more, based on the total mass (100% by mass) of the resin contained in layer A. The content of resin (A) in layer A may be 100% by mass, based on the total mass (100% by mass) of the resin contained in layer A.

[0040] <<Resin with Tg less than 37°C: Resin (B)>> When the channel substrate has a multilayer structure, the channel substrate may include other layers in addition to layer A. Examples of the other layers include a layer containing a resin with a Tg of less than 37°C (hereinafter also referred to as "resin (B)") (hereinafter also referred to as "layer B"). Including layer B in the channel substrate makes it easier to bond with other components.

[0041] Examples of resin (B) include polyester resins with a Tg of less than 37°C. Examples of polyester resins include polyester resins and polyester urethane resins.

[0042] <Example of Laminate Structure> Figure 6 shows an example of the layer structure of the cell culture chip according to the embodiment. The laminate 100 shown in Figure 6 is composed of, from bottom to top, a bottom plate substrate L1, a first channel substrate L2, a porous membrane L3, a second channel substrate L4, a top plate substrate L5, and a cover substrate L6. In Figure 6, A represents the A layer and B represents the B layer.

[0043] <<Bottom Plate Substrate L1>> The bottom plate substrate L1 forms the bottom of the first channel F2. In the laminate 100, the bottom plate substrate L1 is composed of an S1 layer. The material of the S1 layer is not particularly limited, but a material with high biocompatibility is preferred. Since observation may be performed using a phase-contrast microscope or the like while culturing cells in the cell culture chip 1, the S1 layer is preferably made of a transparent material, and a material with low autofluorescence is more preferred. To enhance transparency, the S1 layer is preferably free of fillers (antiblocking agents).

[0044] The thickness of the S1 layer is not particularly limited, but for example, it can be set to 50 to 300 μm. Examples of the thickness of the substrate S1 include 100 μm to 250 μm, or 150 μm to 200 μm, etc.

[0045] <<First flow path substrate L2>> A first flow path F2 is formed in the first flow path substrate L2. In the cell culture chip, the first flow path substrate L2 forms the partition wall of the first flow path F2. In the laminate 100, the first flow path substrate L2 has a multilayer structure.

[0046] In the laminate 100, the first flow path substrate L2 is composed of a laminate in which a B layer, an A layer, and a B layer are laminated in this order. By providing B layers on both sides of the A layer, it becomes easier to perform adhesion with the bottom plate substrate L1 and the porous membrane L3. The B layer functions as an adhesive layer.

[0047] The thickness of the first flow path substrate L2 defines the height (vertical width) of the first flow path F2. Therefore, the thickness of the first flow path substrate L2 can be appropriately set according to the height of the target first flow path F2. The thickness of the first flow path substrate L2 can be appropriately adjusted, for example, by changing the thickness of the A layer. Examples of the thickness of the first flow path substrate L2 include 300 to 2000 μm. Examples of the thickness of the first flow path substrate L2 include 300 to 2000 μm, 400 to 1800 μm, 500 to 1500 μm, 600 to 1200 μm, 600 to 1000 μm, 600 to 9000 μm, 600 to 800 μm, or 700 to 800 μm, etc.

[0048] <<Porous membrane L3>> The porous membrane L3 separates the first flow path F2 and the second flow path F4. The porous membrane L3 is composed of a porous membrane M. The porous membrane M is not particularly limited as long as it has a pore diameter through which the cells to be cultured do not pass and the chemical solution can pass. Examples of the average pore size of the porous membrane M include 0.05 to 10 μm. Examples of the pore density of the porous membrane M include 10 ５ to 10 ９ per cm ２ or so.

[0049] The porous membrane M is preferably made of a material with high biocompatibility. Examples of materials for the porous membrane M include polycarbonate, polyester, polyethylene terephthalate, and polytetrafluoroethylene.

[0050] The thickness of the porous membrane M is not particularly limited. Examples of the thickness of the porous membrane M include 0.1 to 100 μm, 0.5 to 50 μm, 1 to 30 μm, 5 to 20 μm, or 5 to 15 μm.

[0051] <<Second channel substrate L4>> A second channel F4 is formed on the second channel substrate L4. In the cell culture chip, the second channel substrate L4 forms a partition wall for the second channel F4. In the laminate 100, the second channel substrate L4 has a multilayer structure.

[0052] In the laminate 100, the layer configuration of the second channel substrate L4 is the same as that of the first channel substrate L2. That is, it is composed of a laminate in which layers B, A, and B are stacked in this order. By providing layers B on both sides of layer A, adhesion between the porous film L3 and the second channel substrate L4 is facilitated. Layer B functions as an adhesive layer.

[0053] The thickness of the second channel substrate L4 defines the height (vertical width) of the second channel F4. Therefore, the thickness of the second channel substrate L4 can be appropriately set to match the desired height of the second channel F4. The thickness of the second channel substrate L4 may also be adjusted as appropriate, for example, by changing the thickness of layer A. Examples of the thickness of the second channel substrate L4 include 300 to 2000 μm. Examples of the thickness of the first channel substrate L2 include 300 to 2000 μm, 400 to 1800 μm, 500 to 1500 μm, 600 to 1200 μm, 600 to 1000 μm, 600 to 9000 μm, 600 to 800 μm, or 700 to 800 μm.

[0054] ≪Top Substrate L5≫ The top substrate L5 forms the ceiling of the second flow channel F4. In the laminate 100, the top substrate L5 is composed of an S1 layer. The S1 layer is the same as the S1 layer in the bottom substrate L1 described above.

[0055] The thickness of the top substrate L5 is not particularly limited, but can be, for example, 50 to 600 μm. Examples of the thickness of the top substrate L5 include 100 μm to 500 μm, or 150 μm to 400 μm.

[0056] ≪Cover Substrate L6≫ The cover substrate L6 is provided on the top layer of the cell culture chip. In the laminate 100, the cover substrate L6 is composed of a laminate in which the B layer and the S2 layer are stacked in that order. The B layer functions as an adhesive layer.

[0057] Since the S2 layer may be observed using a phase-contrast microscope or the like while cells are being cultured on the cell culture chip 1, a transparent material is preferred, and a low autofluorescence material is even more preferred. To enhance transparency, the S2 layer is preferably free of fillers (antiblocking agents).

[0058] The thickness of the S2 layer is not particularly limited, but can be, for example, 500 to 5000 μm. Examples of S2 layer thicknesses include 800 μm to 3000 μm, 1000 μm to 2500 μm, or 1500 to 2500 μm.

[0059] The thickness of the cover substrate L6 is not particularly limited, but can be, for example, 500 to 5000 μm. Examples of the thickness of the substrate S2 include 800 μm to 3000 μm, 1000 μm to 2500 μm, or 1500 to 2500 μm.

[0060] <Other Examples of Laminate Configurations> Figure 7 shows another example of the layer configuration of the cell culture chip of the embodiment. The laminate 200 shown in Figure 7 is composed of, from bottom to top, a bottom plate substrate L1, a first channel substrate L2, a porous membrane L3, a second channel substrate L4, and a top plate substrate L5. In Figure 7, A is the A layer and B is the B layer.

[0061] The laminate 200 is an example configuration that does not have a cover substrate L6. In the laminate 200, the bottom substrate L1, the first channel substrate L2, the porous film L3, and the second channel substrate L4 are the same as those in the laminate 100.

[0062] In the laminate 200, the top substrate L5 is composed of an S1 layer. In the laminate 200, the thickness of the top substrate L5 is not particularly limited, but examples include 50 to 300 μm, 100 to 200 μm, or 150 to 200 μm.

[0063] The relationships between the multiple S1 layers, multiple A layers, and multiple B layers included in the laminate 200 are the same as those for the laminate 100 described above.

[0064] According to the cell culture chip of this embodiment, the channel substrate has a glass transition temperature of 37°C or higher, and an elastic modulus of 1 × 10 at 25°C. ９ It is Pa or higher, and its modulus of elasticity at 150°C is 1 × 10⁻⁶. ７ The material contains resin A, which has a pressure of Pa or less. This suppresses the sorption and adsorption of the drug onto the partitions of the channel when the drug solution is perfused or retained in the channel. As a result, the decrease in the drug solution is suppressed. Therefore, a cell culture chip with suppressed drug reduction can be obtained.

[0065] The cell culture chip of this embodiment suppresses drug depletion when the drug solution is perfused or retained in the flow path, and is therefore suitable for use in cell culture in the presence of a drug. The cell culture chip of this embodiment can be applied, for example, to drug evaluation tests using cells (efficacy evaluation tests, safety evaluation tests, pharmacokinetic evaluation tests, etc.).

[0066] (Cell culture device) A second aspect of the present invention is a cell culture device comprising the cell culture chip of the first aspect.

[0067] The cell culture device comprises a first cell culture chip and a mechanism for performing cell culture using the cell culture chip. The mechanism of the cell culture device can be modified as appropriate depending on the purpose of cell culture.

[0068] Mechanisms included in cell culture devices include, for example, a liquid delivery mechanism for supplying drug solution to the flow channel of the cell culture chip (drug solution tank, delivery tube, delivery pump, etc.), a drainage mechanism for discharging drug solution from the flow channel of the cell culture chip (drainage tank, drainage tube, discharge pump, etc.), a temperature maintenance mechanism for maintaining the culture temperature of the cell culture chip (thermostat, etc.), and a cleaning mechanism for cleaning the flow channel of the cell culture chip (cleaning solution tank, tube, pump, etc.).

[0069] The cell culture device of this embodiment includes the cell culture chip of the first embodiment, and therefore can perform cell culture while suppressing the reduction of the drug in the drug solution. For this reason, it can be suitably used for cell culture in the presence of a drug. The cell culture device of this embodiment can be applied, for example, to drug evaluation tests using cells (efficacy evaluation tests, safety evaluation tests, pharmacokinetic evaluation tests, etc.).

[0070] (Method for manufacturing cell culture chips) A third aspect of the present invention is a method for manufacturing cell culture chips including a laminate having a channel structure inside. The manufacturing method according to this aspect includes step A of laminating a bottom plate substrate, a channel substrate having channels formed by laser processing, and a top plate substrate in this order, and step B of joining the laminated substrates. The channel substrate has a glass transition temperature of 37°C or higher and an elastic modulus of 1 × 10 at 25°C. ９ It is Pa or higher, and its modulus of elasticity at 150°C is 1 × 10⁻⁶. ７ It contains resin and has a pressure of Pa or less.

[0071] <Process A> Process A is a process of stacking a bottom plate substrate, a channel substrate in which a channel is formed by laser processing, and a top plate substrate in this order.

[0072] The channel substrate may include a first channel substrate L2 and a second channel substrate L4. The cell culture chip may further include a porous membrane L3 and a cover substrate L6.

[0073] ≪Bottom Plate Substrate L1≫ The bottom plate substrate L1 can be the same as described above. The bottom plate substrate L1 can be manufactured by processing the S1 layer to the size of a cell culture chip using laser processing or the like. For laser processing, for example, a carbon dioxide laser can be used.

[0074] <<First channel substrate L2>> The first channel substrate L2 can be the same as described above. Preferably, the first channel substrate L2 has a multilayer structure. If the first channel substrate L2 has a multilayer structure, a laminate for the first channel substrate L2 can be obtained by manufacturing each layer constituting the first channel substrate L2, and then laminating and pressing the layers together. The layers can be pressed together using a laminator and a press machine. For example, after laminating each layer, pressing them together with a laminator and then applying pressure with a press machine can be used to obtain a laminate for the first channel substrate L2 in which each layer is joined together.

[0075] The laminate for the first channel substrate L2 is processed to the size of a cell culture chip by laser processing. Furthermore, channels are formed by laser processing. For example, a carbon dioxide laser can be used for laser processing.

[0076] ≪Porous membrane L3≫ The porous membrane L3 can be the same as described above. The porous membrane L3 is processed to the size of the cell culture chip by laser processing or the like. Furthermore, necessary processing such as forming openings for port P3 and port P4 is performed by laser processing or the like.

[0077] <<Second channel substrate L4>> The second channel substrate L4 can be the same as the one described above. The second channel substrate L4 preferably has a multilayer structure. The second channel substrate L4 can be manufactured in the same way as the first channel substrate L2.

[0078] ≪Top Plate Substrate L5≫ The top plate substrate L5 can be the same as described above. The top plate substrate L5 may or may not have a multilayer structure. If the top plate substrate L5 has a multilayer structure, the laminate for the top plate substrate L5 can be manufactured in the same manner as the first channel substrate L2. The laminate for the top plate substrate L5 or the substrate for the top plate substrate L5 is processed to the size of the cell culture chip by laser processing or the like. Furthermore, the top plate substrate L5 can be manufactured by performing necessary processing such as forming openings for ports P1 to P4 by laser processing or the like.

[0079] ≪Cover Substrate L6≫ The same type of cover substrate L6 as described above can be used. The cover substrate L6 is processed to the size of the cell culture chip by laser processing or the like. Furthermore, the top substrate L5 can be manufactured by performing necessary processing such as forming openings for ports P1 to P4 and opening W by laser processing or the like.

[0080] The components prepared as described above are stacked in the following order: bottom plate substrate L1, first channel substrate L2, second channel substrate L4, second channel substrate L4, top plate substrate L5, and cover substrate L6. When stacking the components, ensure that the openings for ports P1 to P4 are aligned. If the cell culture chip does not include a cover substrate L6, the cover substrate L6 is not stacked.

[0081] <Process B> Process B is the process of joining the stacked substrates together.

[0082] The substrates can be joined using a laminator and a press. For example, by laminating each substrate of the laminate with a laminator and then applying pressure with a press, a cell culture chip with the substrates of the laminate joined together can be obtained.

[0083] The cell culture chip manufactured according to this embodiment is a cell culture chip according to the first embodiment. Therefore, the manufacturing method according to this embodiment can be applied to the manufacture of a cell culture chip according to the first embodiment.

[0084] (Method for culturing cells) A fourth aspect of the present invention is a method for culturing cells. The method for culturing cells according to this aspect includes the step of culturing cells in the flow channel of the cell culture chip of the first aspect in the presence of a drug.

[0085] <Culture Process> Figure 8 shows an example of cell culture using the cell culture chip of the embodiment. In the cell culture chip 1, the first central channel F2a and the second central channel F4a are separated by a porous membrane L3. Cells C are cultured on the porous membrane L3 facing the second central channel F4a.

[0086] Cell C is not particularly limited, and any cell can be used. Cell C can be an animal cell. Examples of animal cells include human cells and cells from non-human animals (such as monkeys, mice, rats, guinea pigs, marmosets, dogs, cats, and insects). The type of cell is not particularly limited and can be appropriately selected according to the purpose. Examples of cells include, but are not limited to, immune cells, germ cells, nerve cells, fibroblasts, mesenchymal stem cells, hormone-secreting cells, various organ cells, cancer cells, various disease cells, and pluripotent stem cells.

[0087] Any drug can be used. The drug may be a candidate drug planned for development as a treatment for any disease. Examples of drugs include, but are not limited to, small molecule drugs (molecular weight 500 or less), medium molecule drugs (molecular weight 500 to 2000), and high molecular weight drugs (nucleic acid drugs, protein drugs, polymers, etc.).

[0088] The culture medium used is not particularly limited and can be appropriately selected depending on the type of cell. The medium may be a basal medium for animal culture to which evaluation agents and other necessary components may be added as appropriate. Known basal media for animal culture can be used. Examples of basal media include Doublebecko's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these. The basal medium may contain serum (such as fetal bovine serum (FBS)) or a serum substitute as needed. Examples of serum substitutes include albumin, transferrin, sodium selenite, ITS-X (Invitrogen), knockout serum substitute (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may contain, as needed, components such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts. These can be used in appropriate combinations.

[0089] The culture conditions can be those commonly used for culturing animal cells. The culture temperature can be, for example, 32-40°C, with 35-38°C (e.g., 37°C) being preferred. ２ The concentration can be, for example, about 2-5% (for example, 5%).

[0090] The culture procedure can be carried out as follows, for example: First, culture medium is injected from port P1 to introduce the medium into the first central channel F2a. Next, cell culture medium is injected from port P3 to introduce the cell culture medium into the second central channel F4a. The cell culture medium is left to stagnate in the second central channel F4a and incubated for a desired amount of time to allow the cells to settle on the porous membrane L3. Next, a drug solution is injected from port P3 to introduce the drug solution into the second central channel F4a and perform cell culture. During cell settlement and culture, the drug solution may be perfused through the second channel F4 or left to stagnate within the second channel F4. During culture, the culture medium may be perfused through the first channel F2 or left to stagnate within the first channel F2. By analyzing the state of the cells during or after culture, the effects of the drug on the cells can be evaluated.

[0091] In the culture method of this embodiment, since the culture is performed using the cell culture chip according to the first embodiment, the reduction of the drug due to sorption and / or adsorption to the channel partition of the cell culture chip can be suppressed. Therefore, the effect of the drug on cells can be accurately evaluated.

[0092] <Flow Channel> Figure 9 is a perspective view of the flow channel of the embodiment. Figure 10 is a top view illustrating the curved flow channel FC of the embodiment. Referring together to Figures 9 and 10, the flow channel includes a curved flow channel FC formed in a curved shape in a plan view of the flow channel substrate. The cross-sectional area of ​​the curved flow channel FC gradually changes in the direction of fluid flow.

[0093] Referring together to Figures 1 and 2, the laminate includes, in this order, a bottom substrate L1, a first channel substrate L2 on which the first channel F2 is formed, a porous film L3, a second channel substrate L4 on which the second channel F4 is formed, and a top substrate L5. The curved channel FC is formed in the first channel F2 and the second channel F4, respectively.

[0094] The cell culture chip 1 is provided with a first introduction port P1 for introducing cell culture liquid into a first channel F2, a first discharge port P2 for discharging liquid from the first channel F2, a second introduction port P3 for introducing liquid into a second channel F4, and a second discharge port P4 for discharging liquid from the second channel F4.

[0095] The first flow path F2 includes a first central flow path F2a, a first inlet flow path F2b connecting the first inlet port P1 and the first central flow path F2a, and a first discharge flow path F2c connecting the first discharge port P2 and the first central flow path F2a. The second flow path F4 includes a second central flow path F4a which overlaps with the first central flow path F2a in at least a portion in a plan view, a second inlet flow path F4b connecting the second inlet port P3 and the second central flow path F4a, and a second discharge flow path F4c connecting the second discharge port P4 and the second central flow path F4a. The curved flow path FC is formed in each of the first inlet flow path F2b, the first discharge flow path F2c, the second inlet flow path F4b, and the second discharge flow path F4c.

[0096] In Figure 9, a portion of the second flow path F4 (the second inlet flow path F4b) is shown with a solid line, while the rest is shown with a dashed line. In the following explanation, the curved flow path FC formed in the second inlet flow path F4b will be described in detail. The curved flow paths FC formed in the other flow paths (first inlet flow path F2b, first discharge flow path F2c, and second discharge flow path F4c) are similar to the curved flow path FC formed in the second inlet flow path F4b, except for the direction of curvature and location, so their detailed explanation will be omitted.

[0097] In Figure 10, a portion of the second channel F4 is shown with a solid (thin) line, and the curved channel FC formed in the second inlet channel F4b is shown with a thick line. In plan view, the length of the inner circumference of the curved channel FC is L. １ The length of the outer circumference of the curved channel FC is L ２ In that case, 1.3 < L ２ / L １ <5.0 is satisfied. For example, 1.3 < L ２ / L １ It is preferable that the condition <1.7 is satisfied, and 1.3 < L ２ / L １ It is more preferable that the condition <1.5 be met.

[0098] Furthermore, in a curved channel FC where the channel width is constant in plan view, the centers of the outer and inner circular arcs are the same. In this case, L １ = 2R １ π(θ / 360), L ２ = 2R ２ It is given by π(θ / 360). From these equations, computationally R ２ / R １= L ２ / L １ This is the case. When the distance from the outer side wall to the inner side wall (flow channel width) and the distance from the inner side wall to the center of the arc are the same in a plan view, R ２ / R １ = L ２ / L １ = 2. When the latter is greater than the former, R ２ / R １ = L ２ / L １ < 2

[0099] The following description will explain an example configuration of the second inlet channel F4b. The configuration examples of the other channels (first inlet channel F2b, first discharge channel F2c, and second discharge channel F4c) are the same as the configuration example of the second inlet channel F4b, except for the orientation and location of the channels, so a detailed explanation of them will be omitted. As shown in Figure 9, the second inlet channel F4b may further include a first connecting channel FJ1 that connects port P3 and curved channel FC and has a constant cross-sectional area in the direction of fluid flow. The first connecting channel FJ1 is a channel that connects the end of port P3 on the curved channel FC side and the end of curved channel FC on the port P3 side in the direction of fluid flow, and has a constant cross-sectional area between them. The cross-sectional area of ​​the first connecting channel FJ1 is smaller than the minimum value of the cross-sectional area of ​​port P3 and the cross-sectional area of ​​curved channel FC, respectively. The first connecting channel FJ1 extends from port P3 toward the -Y side with a constant cross-sectional area.

[0100] The second inlet channel F4b may further include a second connecting channel FJ2 that connects the first connecting channel FJ1 and the curved channel FC. The second connecting channel FJ2 is a channel that connects the end of the first connecting channel FJ1 on the curved channel FC side and the end of the curved channel FC on the first connecting channel FJ1 side in the direction of fluid flow. The cross-sectional area of ​​the second connecting channel FJ2 gradually changes in the direction of fluid flow. The second connecting channel FJ2 extends from downstream of the first connecting channel FJ1 toward the -Y side such that its cross-sectional area gradually increases.

[0101] The second inlet channel F4b may further include a third connecting channel FJ3, which is connected to the end of the curved channel FC opposite to the second connecting channel FJ2 and has a constant cross-sectional area in the direction of fluid flow. The third connecting channel FJ3 is a channel that connects the end of the curved channel FC opposite to the second connecting channel FJ2 side and the end of the second central channel F4a on the curved channel FC side in the direction of fluid flow, and has a constant cross-sectional area between them. The cross-sectional area of ​​the third connecting channel FJ3 is the same as the maximum value of the cross-sectional area of ​​the curved channel FC. The third connecting channel FJ3 extends downstream of the curved channel FC toward the +X side with a constant cross-sectional area. In the example in Figure 9, the cross-sectional area SA of the third connecting channel FJ3 is shown by a cross-sectional hatch.

[0102] The second inlet channel F4b includes, in the direction of fluid flow, the first connecting channel FJ1, the second connecting channel FJ2, the curved channel FC, and the third connecting channel FJ3 in that order. The curved channel FC is formed in a curved shape in plan view (specifically, a shape that curves toward the -X and -Y sides in plan view) such that its cross-sectional area gradually increases from downstream of the second connecting channel FJ2 to upstream of the third connecting channel FJ3.

[0103] In a side view of the flow channel substrate, the flow channel height of the curved flow channel FC is constant in the direction of fluid flow (see Figures 4 and 9). In a plan view, the flow channel width of the curved flow channel FC gradually changes in the direction of fluid flow (see Figures 3 and 9).

[0104] The cross-sectional shape of the curved channel FC is formed to be approximately rectangular (see Figure 9). For example, the cross-sectional shape of the curved channel FC is formed to be rectangular (specifically, a rectangle with a shorter side in the Z direction).

[0105] In addition to the rectangle described above, the generally rectangular shape includes the biconcave lens type, plano-concave lens type, concave meniscus lens type, biconvex lens type, plano-convex lens type, and convex meniscus lens type. The cross-sectional shape of the curved channel FC may be formed in any of the following shapes: biconcave lens type, plano-concave lens type, concave meniscus lens type, biconvex lens type, plano-convex lens type, or convex meniscus lens type.

[0106] <Effects> As described above, the cell culture chip 1 according to this embodiment is a cell culture chip that includes a laminate having a flow channel structure inside. The laminate includes flow channel substrates L2 and L4 on which flow channels F2 and F4 leading to ports P1 to P4 are formed. Flow channels F2 and F4 are formed in a curved shape in plan view of the flow channel substrates L2 and L4 and include a curved flow channel FC having a cross-sectional area that gradually changes in the direction of fluid flow, and a first connecting flow channel FJ1 that connects ports P1 to P4 and the curved flow channel FC and has a constant cross-sectional area in the direction of fluid flow. The cross-sectional area of ​​the first connecting flow channel FJ1 is smaller than the minimum value of the cross-sectional area of ​​ports P1 to P4 and the cross-sectional area of ​​the curved flow channel FC, respectively. In plan view, the flow channel width of the curved flow channel FC gradually changes in the direction of fluid flow. With this configuration, the cross-sectional area of ​​the first connecting channel FJ1 is smaller than the minimum cross-sectional area of ​​ports P1 to P4 and the cross-sectional area of ​​the curved channel FC, thereby reducing the risk of foam contamination compared to the case where the cross-sectional area of ​​the first connecting channel FJ1 is equal to or greater than the cross-sectional area of ​​ports P1 to P4 and the cross-sectional area of ​​the curved channel FC. Furthermore, by providing the curved channel FC between the main cell culture area and ports P1 to P4, the bias in the flow velocity distribution can be reduced, and cells can be uniformly seeded in the main cell culture area. In addition, in a plan view, the channel width of the curved channel FC changes gradually in the direction of fluid flow, thereby reducing the bias in the flow velocity distribution in the channel width direction in the curved channel FC. Therefore, in order to prevent foam contamination and uniformly seed cells, the bias in the flow velocity distribution in the channel width direction can be reduced in the curved channel FC. For example, by reducing the bias in the flow velocity distribution in the channel width direction in the curved channel FC, mass transfer and mechanical effects associated with the fluid flow can be uniformly applied to the cultured cells. For example, seeded cells will adhere uniformly to the curved channel FC.

[0107] In the cell culture chip 1 according to this embodiment, the length of the inner circumference of the curved channel FC in a plan view is L. １ The length of the outer circumference of the curved channel FC is L ２ In that case, 1.3 < L ２ / L １ <5.0 is satisfied. For example, 1.3 < L ２ / L １It is preferable that the condition <1.7 is satisfied, and 1.3 < L ２ / L １ It is more preferable to satisfy <1.5. With this configuration, the difference in fluid flow velocity between the inner and outer sides of the curved channel FC can be minimized. Therefore, stagnation of cells and substances in the curved channel FC can be reduced.

[0108] The flow path according to this embodiment further includes a second connecting flow path FJ2 that connects the first connecting flow path FJ1 and the curved flow path FC. The cross-sectional area of ​​the second connecting flow path FJ2 gradually changes in the direction of fluid flow. With this configuration, the velocity difference of the fluid flow in the second connecting flow path FJ2 can be gradually changed. Therefore, the risk of foam entrapment in the curved flow path FC can be further reduced.

[0109] The flow path according to this embodiment further includes a third connecting flow path FJ3 which is connected to the end of the curved flow path FC opposite to the second connecting flow path FJ2 and has a constant cross-sectional area in the direction of fluid flow. The cross-sectional area of ​​the third connecting flow path FJ3 is the same as the maximum value of the cross-sectional area of ​​the curved flow path FC.

[0110] With this configuration, by providing a third connecting channel FJ3 between the main cell culture region and the curved channel FC, cells can be seeded more uniformly.

[0111] In the cell culture chip 1 according to this embodiment, the channel height of the curved channel FC is constant in the direction of fluid flow when viewed from the side of the channel substrates L2 and L4. With this configuration, the height (thickness) of the channel substrates L2 and L4 can be maintained.

[0112] The cross-sectional shape of the curved channel FC according to this embodiment is formed to be substantially rectangular. With this configuration, when processing the channel substrates L2 and L4 by laser processing, the curved channel FC can be formed more easily compared to the case where the cross-sectional shape of the curved channel FC is formed to be circular.

[0113] The laminate according to this embodiment includes, in this order, a bottom substrate L1, a first channel substrate L2 on which a first channel F2 is formed, a porous membrane L3, a second channel substrate L4 on which a second channel F4 is formed, and a top substrate L5. The curved channel FC is formed in each of the first channel F2 and the second channel F4. With this configuration, in each of the first channel F2 and the second channel F4, it is possible to prevent foam from getting trapped and to uniformly seed cells, thereby reducing the bias in the flow velocity distribution in the channel width direction in the curved channel FC.

[0114] The cell culture chip 1 according to this embodiment is provided with a first introduction port P1 for introducing a cell culture liquid into a first channel F2, a first discharge port P2 for discharging the liquid from the first channel F2, a second introduction port P3 for introducing the liquid into a second channel F4, and a second discharge port P4 for discharging the liquid from the second channel F4. The first channel F2 includes a first central channel F2a, a first introduction channel F2b connecting the first introduction port P1 and the first central channel F2a, and a first discharge channel F2c connecting the first discharge port P2 and the first central channel F2a. The second channel F4 includes a second central channel F4a which overlaps with the first central channel F2a in at least a portion in a plan view, a second introduction channel F4b connecting the second introduction port P3 and the second central channel F4a, and a second discharge channel F4c connecting the second discharge port P4 and the second central channel F4a. The curved flow channels FC are formed in each of the first inlet channel F2b, the first discharge channel F2c, the second inlet channel F4b, and the second discharge channel F4c. With this configuration, the bias in the flow velocity distribution in the width direction of each of the curved flow channels FC in the first inlet channel F2b, the first discharge channel F2c, the second inlet channel F4b, and the second discharge channel F4c can be reduced.

[0115] The cell culture device according to this embodiment includes the cell culture chip 1 described above. With this configuration, because it includes the cell culture chip 1 described above, it is possible to provide a cell culture device that can prevent foam from getting trapped and reduce the bias in the flow velocity distribution in the width direction of the channel in a curved channel in order to uniformly seed cells.

[0116] <Modification> In the above embodiment, in a plan view, the length of the inner circumference of the curved channel is L. １The length of the outer circumference of the curved channel is L. ２ In that case, 1.3 < L ２ / L １ <I have given an example that satisfies 5.0, but it is not limited to this. For example, 1.3 ≥ L ２ / L １ It may also be L ２ / L １ It may be ≥ 5.0. L ２ / L １ The scope can be changed according to the design specifications.

[0117] In the embodiments described above, the flow path further includes a second connecting flow path that connects the first connecting flow path and the curved flow path, and the cross-sectional area of ​​the second connecting flow path is described as gradually changing in the direction of fluid flow, but the invention is not limited to this. For example, the cross-sectional area of ​​the second connecting flow path may have a constant cross-sectional area in the direction of fluid flow. The cross-sectional area of ​​the second connecting flow path can be changed according to the design specifications.

[0118] In the embodiments described above, the flow path further includes a third connecting flow path connected to the end opposite to the second connecting flow path in the curved flow path and having a constant cross-sectional area in the direction of fluid flow, and the example given is that the cross-sectional area of ​​the third connecting flow path is the same as the maximum value of the cross-sectional area of ​​the curved flow path, but the invention is not limited to this. For example, the cross-sectional area of ​​the third connecting flow path may be different from the maximum value of the cross-sectional area of ​​the curved flow path. The cross-sectional area of ​​the third connecting flow path can be changed according to the design specifications.

[0119] In the embodiments described above, an example was given in which the channel height of the curved channel is constant in the direction of fluid flow when viewed from the side of the channel substrate, but this is not limited to this example. For example, the channel height of the curved channel may vary in the direction of fluid flow when viewed from the side of the channel substrate. The channel height of the curved channel can be changed according to the design specifications when viewed from the side of the channel substrate.

[0120] In the embodiments described above, the cross-sectional shape of the curved channel was explained as being formed in a substantially rectangular shape, but it is not limited to this. For example, the cross-sectional shape of the curved channel may be formed in a circular shape (e.g., a perfect circle, an ellipse, an oblong shape, etc.). The cross-sectional shape of the curved channel can be changed according to the design specifications.

[0121] In the embodiments described above, the laminate includes a bottom plate substrate, a first channel substrate with a first channel formed thereon, a porous film, a second channel substrate with a second channel formed thereon, and a top plate substrate, in this order, and the curved channel is formed in each of the first and second channels, as described in the example, but it is not limited to this. For example, the curved channel may be formed in either the first channel or the second channel. The laminate may be configured to include a bottom plate substrate, a channel substrate with a channel formed thereon, and a top plate substrate, in this order. The configuration of the laminate can be changed according to the design specifications.

[0122] In the embodiments described above, the cell culture chip is provided with a first introduction port for introducing a cell culture liquid into a first channel, a first discharge port for discharging the liquid from the first channel, a second introduction port for introducing the liquid into a second channel, and a second discharge port for discharging the liquid from the second channel. The first channel includes a first central channel, a first introduction channel connecting the first introduction port and the first central channel, and a first discharge channel connecting the first discharge port and the first central channel. The second channel includes a second central channel that overlaps with the first central channel in at least a portion in a plan view, a second introduction channel connecting the second introduction port and the second central channel, and a second discharge channel connecting the second discharge port and the second central channel. The curved channels have been described with examples of being formed in each of the first introduction channel, the first discharge channel, the second introduction channel, and the second discharge channel, but are not limited to these examples. For example, the curved channel may be formed in at least one of the first inlet channel, the first discharge channel, the second inlet channel, and the second discharge channel. The configuration of the curved channel in relation to the first inlet channel, the first discharge channel, the second inlet channel, and the second discharge channel can be changed according to the design specifications.

[0123] Furthermore, it is possible to replace the components in the above-described embodiments with well-known components without departing from the spirit of the present invention. Also, the above-described modifications may be combined.

[0124] Next, the cell culture chip according to the above embodiment of the present invention will be described in detail with reference to examples. Note that the following examples are specific examples to which the present invention is applied and do not limit the present invention.

[0125] <Examples of Flow Channel Construction for Evaluation of Foam Inclusion and Liquid Uniformity> Figure 11 is a top view of the flow channel of Example 1. Figure 12 is a top view of the flow channel of Example 2. Figure 13 is a top view of the flow channel of Example 3. Figure 14 is a top view of the flow channel of Example 4. Figure 15 is a top view of the flow channel of Example 5. Figure 16 is a top view of the flow channel of Comparative Example 1. Figure 17 is a top view of the flow channel of Comparative Example 2. The top view of the flow channel of Comparative Example 1 is shown in Figure 19, which will be described later. The top view of the flow channel of Example 3 corresponds to Figure 20, which will be described later. Referring to Figures 11 to 15, in Examples 1 to 5, the flow path includes a curved flow path formed in a curved shape in a plan view of the flow path substrate and having a cross-sectional area that gradually changes in the direction of fluid flow, and a first connecting flow path that connects the port and the curved flow path and has a constant cross-sectional area in the direction of fluid flow, wherein the cross-sectional area of ​​the first connecting flow path is smaller than the minimum values ​​of the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved flow path, and the flow path width of the curved flow path gradually changes in the direction of fluid flow in a plan view. In Examples 3 and 4, the flow path further includes a second connecting flow path that connects the first connecting flow path and the curved flow path, and the cross-sectional area of ​​the second connecting flow path gradually changes in the direction of fluid flow (see Figures 13 and 14). As shown in Figures 16 and 19, in Comparative Example 1, the flow path includes a curved flow path formed in a curved shape in a plan view of the flow path substrate, and the cross-sectional area of ​​the curved flow path is constant in the direction of fluid flow. In Comparative Example 1, the cross-sectional area of ​​the first connecting channel was the same as the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved channel. As shown in Figure 17, in Comparative Example 2, the channel included a curved channel formed in a curved shape in a plan view of the channel substrate, and the cross-sectional area of ​​the curved channel was the largest at the center in the direction of fluid flow. In Comparative Example 2, the cross-sectional area of ​​the first connecting channel was the same as the cross-sectional area of ​​the port and the same as the minimum value of the cross-sectional area of ​​the curved channel. The respective channel shape designs (plan view shapes) are shown in Figures 11 to 17 and Figures 19 to 21, and the unit [mm] is omitted for each dimension.

[0126] In each of the above examples, a cell suspension was flowed through the channel, and foam formation and liquid homogeneity were evaluated.

[0127] <Evaluation Results of Foam Inclusion and Liquid Uniformity> Figure 18 shows the evaluation results of foam inclusion and liquid uniformity. In Figure 18, regarding foam inclusion, "A" means that foam inclusion does not occur even once, and "B" means that foam inclusion occurs at least once out of 10 times. On the other hand, regarding liquid uniformity, "A" means that the velocity of the liquid on the outer and inner circumferences of the flow path is closer (the velocity distribution on the outer and inner circumferences is similar), "B" means that the velocity of the liquid on the outer and inner circumferences of the flow path is close (the velocity difference is less than a predetermined value), and "C" means that the velocity of the liquid on the outer and inner circumferences of the flow path is different (the velocity difference is greater than a predetermined value). As shown in Figure 18, according to Comparative Example 2, it was confirmed that the risk of foam inclusion can be reduced by making the cross-sectional area of ​​the curved flow path maximum in the center in the direction of fluid flow. According to Examples 1 to 5, it was confirmed that the risk of foam inclusion can be reduced by making the inlet narrower than in Comparative Example 1. Furthermore, Examples 1 to 5 confirmed that liquid uniformity can be improved by reducing the difference in radius (R) between the outer and inner circumferences of the flow path compared to Comparative Examples 1 and 2. Examples 1, 3, and 5 were found to have superior liquid uniformity compared to Examples 2 and 4 by minimizing the difference in radius (R) between the outer and inner circumferences of the flow path.

[0128] <Examples of Flow Path Fabrication for Evaluation of Flow Velocity Distribution> Figure 19 is a top view of the flow path of Comparative Example 1. Figure 20 is a top view of the flow path of Example 3. Figure 21 is a top view of the flow path of Example 5. As shown in Figure 19, in Comparative Example 1, the flow path included a curved flow path formed in a curved shape in a plan view of the flow path substrate, and the cross-sectional area of ​​the curved flow path was constant in the direction of fluid flow. Referring to Figures 20 and 21 as well, in Examples 3 and 5, the flow path included a curved flow path formed in a curved shape in a plan view of the flow path substrate and having a cross-sectional area that gradually changes in the direction of fluid flow, and a first connecting flow path that connects the port and the curved flow path and has a constant cross-sectional area in the direction of fluid flow, wherein the cross-sectional area of ​​the first connecting flow path was smaller than the minimum value of the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved flow path, and the flow path width of the curved flow path gradually changed in the direction of fluid flow in a plan view. In Example 5, a modified version of the flow path shape of Example 3 (a willow-shaped flow path) was used.

[0129] In each of the above examples, the flow path volume was introduced into the flow path at a rate of 1 s (0.479 m / s), and the flow velocity distribution was evaluated.

[0130] <Evaluation Results of Flow Velocity Distribution> Figure 22 shows the evaluation results of the flow velocity distribution. Figure 22 corresponds to the normalized graph of the simulation results when the flow channel volume is filled in 1 s for each channel type.

[0131] As shown in Figure 22, it was confirmed that, according to Examples 3 and 5, the bias in the flow velocity distribution in the width direction of the flow channel can be reduced in a curved flow channel compared to Comparative Example 1.

[0132] Figure 23 is a top view showing the flow in the curved channel of Comparative Example 1. Figure 24 is a top view showing the flow in the curved channel of Example 3. Referring to both Figures 23 and 24, it was confirmed that in Example 3, there was less stagnant area at the outer corner compared to Comparative Example 1, and the flow was relatively uniform.

[0133] Figure 25 shows the flow in the curved channel of Comparative Example 1 after 4.5 hours (before improvement, after 4.5 hours). Figure 26 shows the flow in the curved channel of Example 3 after 4.5 hours (after improvement, after 4.5 hours). Referring to Figures 25 and 26 together, it was confirmed that in Example 3, the flow was more uniform at the corner than in Comparative Example 1. It was also found that by reducing the flow velocity over time, the difference in flow velocity between the outer and inner sides could be reduced.

[0134] By utilizing the cell culture chip and cell culture device of the present invention, it is possible to provide a cell culture chip and cell culture device that can prevent foam buildup and reduce the bias in the flow velocity distribution in the width direction of the channel in a curved channel, thereby enabling uniform cell seeding.

[0135] 1...Cell culture chip, 100, 200...Laminate, F2...First channel, F2a...First central channel, F2b...First introduction channel, F2c...First discharge channel, F4...Second channel, F4a...Second central channel, F4b...Second introduction channel, F4c...Second discharge channel, FC...Curved channel, FJ1...First connecting channel, FJ2...Second connecting channel, FJ3...Third connecting channel, L1...Bottom plate substrate, L2...First channel substrate, F3...Porous membrane, L4...Second channel substrate, L5...Top plate substrate, P1...First introduction port, P2...First discharge port, P3...Second introduction port, P4...Second discharge port, SA...Cross-sectional area

Claims

1. A cell culture chip comprising a laminate having a flow channel structure inside, wherein the laminate includes a flow channel substrate having a flow channel formed therein leading to a port, the flow channel includes a curved flow channel formed in a curved shape in a plan view of the flow channel substrate and having a cross-sectional area that gradually changes in the direction of fluid flow, and a first connecting flow channel connecting the port and the curved flow channel and having a constant cross-sectional area in the direction of fluid flow, wherein the cross-sectional area of ​​the first connecting flow channel is smaller than the minimum value of the cross-sectional area of ​​the port and the cross-sectional area of ​​the curved flow channel, and in the plan view, the flow channel width of the curved flow channel gradually changes in the direction of fluid flow.

2. In the plan view, the length of the inner circumference of the curved channel is L. １ The length of the outer circumference of the curved channel is L. ２ In that case, 1.3 < L ２ / L １ A cell culture chip according to claim 1, satisfying <5.

0.

3. The cell culture chip according to claim 1 or 2, wherein the flow path further includes a second connecting flow path connecting the first connecting flow path and the curved flow path, and the cross-sectional area of ​​the second connecting flow path gradually changes in the direction of fluid flow.

4. The cell culture chip according to claim 3, wherein the flow path further includes a third connecting flow path connected to the end of the curved flow path opposite to the second connecting flow path and having a constant cross-sectional area in the direction of fluid flow, the cross-sectional area of ​​the third connecting flow path being the same as the maximum value of the cross-sectional area of ​​the curved flow path.

5. In a side view of the flow channel substrate, the flow channel height of the curved flow channel is constant in the direction of fluid flow, as described in claim 1 or 2.

6. The cell culture chip according to claim 1 or 2, wherein the cross-sectional shape of the curved channel is formed to be substantially rectangular.

7. The laminate comprises, in this order, a bottom plate substrate, a first channel substrate in which a first channel is formed, a porous membrane, a second channel substrate in which a second channel is formed, and a top plate substrate, and the curved channel is formed in each of the first channel and the second channel, the cell culture chip according to claim 1 or 2.

8. The cell culture chip is provided with a first introduction port for introducing a cell culture liquid into the first channel, a first discharge port for discharging the liquid from the first channel, a second introduction port for introducing the liquid into the second channel, and a second discharge port for discharging the liquid from the second channel, wherein the first channel includes a first central channel, a first introduction channel connecting the first introduction port and the first central channel, and a first discharge channel connecting the first discharge port and the first central channel, wherein the second channel includes a second central channel that overlaps with the first central channel in at least a portion in a plan view, a second introduction channel connecting the second introduction port and the second central channel, and a second discharge channel connecting the second discharge port and the second central channel, wherein the curved channel is formed in each of the first introduction channel, the first discharge channel, the second introduction channel and the second discharge channel, the cell culture chip according to claim 7.

9. A cell culture device comprising the cell culture chip according to claim 1 or 2.

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