Cell culture container

Abstract

To provide a cell culture vessel capable of efficiently culturing nerve cells.SOLUTION: The cell culture vessel has an upper surface, a lower surface opposite to the upper surface, a recess opened in the upper surface, and a flow path communicating with the recess. The recess has a side surface including an inclined portion inclined with respect to the upper surface, and a bottom surface substantially parallel to the upper surface or the lower surface. The flow path has a first portion extending in a direction parallel to the upper surface or the lower surface and located on a lower surface side of the bottom surface, and a second portion connecting the first portion and an opening provided in the bottom surface.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture vessel. [Background technology]

[0002] In recent years, there has been a demand for in vitro culture of various cells for purposes such as drug discovery research. Neurons, in particular, have a distinctive morphology, consisting of a cell body and processes extending from the cell body: an axon and dendrites. The axon is the longest process extending from the cell body of a neuron. To improve biological compatibility with in vivo neural tissue, a method has been developed for culturing axons by extending the axons away from the cell body and isolating the cell body from the axon.

[0003] Patent Documents 1 and 2 disclose cell culture devices used for culturing neurons. The cell culture devices of Patent Documents 1 and 2 are configured so that the cell bodies of neurons and the axons extending from the cell bodies are received in separate portions. The cell culture device of Patent Document 1 has a portion for receiving a liquid medium and a channel connected to the portion for receiving the liquid medium, the channel having a portion for receiving the cell bodies and a portion for receiving the axons. The portion for receiving the axons is located on the opposite side of the portion for receiving the cell bodies from the portion for receiving the liquid medium, and is configured so that the axons extend away from the portion for receiving the liquid medium. The cell culture device of Patent Document 2 has a culture module having two recesses (chambers) and a channel connecting them. The culture module of the cell culture device of Patent Document 2 is configured so that the cell bodies of neurons are placed in one of the two recesses, and axons extending from the cell bodies are formed in the channel. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 261775 [Patent Document 2] Patent No. 6430680 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for more efficient cultivation of nerve cells.

[0006] An object of the present invention is to provide a cell culture vessel capable of efficiently culturing nerve cells. [Means for solving the problem]

[0007] According to an embodiment of the present invention, the following solutions are provided:

[0008] [Item 1] an upper surface and a lower surface opposite the upper surface; a recessed portion that opens to the upper surface and has a bottom surface that is substantially parallel to the upper surface or the lower surface and a side surface that includes an inclined portion that is inclined with respect to the bottom surface; a flow path communicating with the recess; and The flow path is a first portion extending in a direction parallel to the bottom surface and located on the lower surface side of the bottom surface; a second portion connecting the first portion and an opening provided in the bottom surface; A cell culture vessel comprising: [Item 2] Item 2. The cell culture vessel according to item 1, wherein the second portion extends in a direction intersecting the bottom surface. [Item 3] 3. The cell culture vessel according to item 1 or 2, wherein the second portion is arranged to overlap the bottom surface in a top view. [Item 4] 4. The cell culture vessel according to any one of items 1 to 3, wherein the side surface has one or more protrusions that protrude into the recess. [Item 5] 5. The cell culture vessel according to item 4, wherein the protrusion extends from the side surface in a direction parallel to the bottom surface or toward the top surface. [Item 6] the one or more protrusions are a plurality of protrusions, Item 6. The cell culture vessel according to item 4 or 5, wherein the plurality of protrusions are arranged at equal intervals at an intersection between a predetermined plane parallel to the bottom surface and the side surface. [Item 7] Item 7. The cell culture vessel according to item 6, wherein the plurality of protrusions include a pair of protrusions symmetrical with respect to the center of the recess in a top view. [Item 8] 8. The cell culture vessel according to any one of items 4 to 7, wherein the protrusion is integrally formed with the side surface. [Item 9] The recess further includes a support portion that is disposed in contact with the side surface of the recess and matches the side surface, 8. The cell culture vessel according to any one of items 4 to 7, wherein the protrusion is supported by the support. [Item 10] the side surface has a first portion and a second portion located on the upper surface side of the first portion of the side surface, 10. The cell culture vessel according to any one of items 1 to 9, wherein the second portion of the side surface has lower cell adhesiveness than the first portion of the side surface. [Item 11] the second portion of the side surface has a fine uneven structure on the surface, Item 11. The cell culture vessel according to item 10, wherein the first portion of the side surface does not have a micro-relief structure on the surface. [Item 12] the first portion of the side surface has a cell adhesion layer having cell adhesive properties on its surface, Item 12. The cell culture vessel according to item 10 or 11, wherein the second portion of the side surface does not have the cell adhesion layer on its surface. [Item 13] Item 11. The cell culture vessel according to item 10, wherein the portion of the side surface having the second portion is formed from a material with lower cell adhesiveness than the portion of the side surface having the first portion. [Item 14] 14. The cell culture vessel of any one of items 1 to 13, further comprising one or more electrodes disposed within the first portion of the flow path. [Item 15] a substrate having a top surface; a well member supported on the upper surface of the substrate, the well member having the recess and the channel; and 15. The cell culture vessel according to any one of items 1 to 14, wherein the upper surface of the substrate constitutes a bottom of the first portion of the channel. [Effects of the Invention]

[0009] According to an embodiment of the present invention, a cell culture vessel capable of efficiently culturing nerve cells is provided. [Brief explanation of the drawings]

[0010] [Figure 1] 1A and 1B are diagrams schematically showing a top view and a cross-sectional view of a cell culture vessel 50A according to an embodiment of the present invention. [Figure 2A] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 50A. FIG. [Figure 2B] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 50A. FIG. [Figure 2C] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 50A. FIG. [Figure 2D] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 50A. FIG. [Figure 3A] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 950A according to a comparative example. FIG. [Figure 3B] 10 is a schematic cross-sectional view illustrating a method for culturing nerve cells using a cell culture vessel 950A according to a comparative example. FIG. [Figure 4] FIG. 10 is a schematic cross-sectional view for explaining a method for culturing nerve cells using a cell culture vessel 950B according to another comparative example. [Figure 5A] FIG. 2 is a schematic cross-sectional view of a cell culture vessel 50A. [Figure 5B] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50A1 according to a modified example of the embodiment of the present invention. [Figure 6A] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50B according to another embodiment of the present invention. [Figure 6B] 10 is a schematic top view of a recess 32 of a cell culture vessel 50B. FIG. [Figure 6C] 10 is a schematic top view of a recess 32 showing another example of a protrusion 44 of a cell culture vessel 50B. FIG. [Figure 6D] 10 is a schematic top view of a recess 32 showing another example of a protrusion 44 of a cell culture vessel 50B. FIG. [Figure 6E] 10 is a schematic cross-sectional view of the cell culture vessel 50B showing another example of the protrusion 44 of the cell culture vessel 50B. FIG. [Figure 7] 10A and 10B are schematic cross-sectional views showing an example of a process for culturing nerve cells using a cell culture vessel 50A. [Figure 8A] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50B1 according to a modified example of another embodiment of the present invention. [Figure 8B] 10 is a schematic top view of a recess 32 of a cell culture vessel 50B1. FIG. [Figure 9] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50C according to yet another embodiment of the present invention. [Figure 10] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50C1 according to a modification of still another embodiment of the present invention. [Figure 11] FIG. 10 is a schematic cross-sectional view of a cell culture vessel 50D according to yet another embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, a cell culture vessel according to an embodiment of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the embodiments exemplified below. In the following drawings, components having substantially the same functions are designated by common reference numerals, and their description may be omitted.

[0012] [Embodiment 1] 1 shows a schematic top view and a cross-sectional view of a cell culture vessel 50A according to an embodiment of the present invention. The top view of the cell culture vessel 50A is shown in the upper part of Fig. 1, and the cross-sectional view taken along line A-A' in the upper part of Fig. 1 is shown in the lower part of Fig. 1.

[0013] The cell culture vessel 50A has an upper surface 51, a lower surface 52 opposite to the upper surface 51, a recess 32 opening to the upper surface 51, and a flow path 34 communicating with the recess 32. In this example, the cell culture vessel 50A has a substrate 11 and a well member 30 supported by the substrate 11.

[0014] The substrate 11 has two opposing main surfaces, an upper surface 11a and a lower surface 11b. The upper surface 11a and the lower surface 11b of the substrate 11 are, for example, substantially parallel to each other. The upper surface 11a and the lower surface 11b of the substrate 11 are, for example, surfaces substantially parallel to the xy plane. The well member 30 is supported on the upper surface 11a of the substrate 11. In this example, the lower surface 52 of the cell culture vessel 50A includes the lower surface 11b of the substrate 11.

[0015] The substrate 11 is, for example, a glass substrate or a substrate formed from a resin material. At least the upper surface 11a of the substrate 11 is required to be formed from a material with low cytotoxicity. The substrate 11 may be formed from a material with high visible light transmittance. Examples of materials for the substrate 11 with low cytotoxicity and high visible light transmittance include glass, polycarbonate, PDMS (polydimethylsiloxane), COP (cycloolefin polymer), and PET (polyethylene terephthalate). When the substrate 11 with high visible light transmittance is used, cells in the cell culture vessel 50A (e.g., cells being cultured) can be observed using an inverted microscope. The visible light transmittance of the substrate 11 may be, for example, 80% or more, or 90% or more.

[0016] The well member 30 has a lower surface 30b on the substrate 11 side and an upper surface 30a opposite to the lower surface 30b. The lower surface 30b of the well member 30 is, for example, a surface that is approximately parallel to the upper surface 11a of the substrate 11. The lower surface 30b of the well member 30 is, for example, a surface that is approximately parallel to the xy plane. The upper surface 30a of the well member 30 may be a surface that is approximately parallel to the lower surface 30b of the well member 30, or may be a surface that intersects with the lower surface 30b of the well member 30. In this example, the upper surface 51 of the cell culture vessel 50A includes the upper surface 30a of the well member 30.

[0017] The well member 30 may be formed from a material with low cytotoxicity (e.g., a resin material, a resist, etc.). The well member 30 may also be formed from a material with high transmittance to visible light. The well member 30 may be formed from the same material as the resin material of the substrate 11 described above. For example, by using known injection molding or photolithography techniques, the well member 30 having the structure and shape described below can be formed.

[0018] The substrate 11 and the well member 30 may be bonded to each other via an adhesive member. Examples of the adhesive member that can be used include ultraviolet curable resin, epoxy resin, and double-sided tape. A highly biocompatible material is preferably used as the adhesive member. A highly water-resistant material is also preferably used as the adhesive member. A highly biocompatible and highly water-resistant material is even more preferably used as the adhesive member. Alternatively, the substrate 11 and the well member 30 may be bonded to each other without using an adhesive by performing a surface treatment using plasma treatment on the upper surface 11a of the substrate 11 and the lower surface 30b of the well member 30. Examples of materials for the substrate 11 and the well member 30 that can be subjected to plasma treatment include glass, PDMS, and COP.

[0019] The well member 30 has a recess 32 that opens to the upper surface 30a (i.e., the upper surface 51 of the cell culture vessel 50A) and a flow path 34 that communicates with the recess 32. That is, the opening 32o of the recess 32 is formed in the upper surface 30a.

[0020] The recess 32 has a bottom surface 32b that is substantially parallel to the upper surface 51 (upper surface 30a of the well member 30) or the lower surface 52 (lower surface 11b of the substrate 11) of the cell culture vessel 50A, and a side surface 32l that includes an inclined portion 32s that is inclined relative to the bottom surface 32b. The bottom surface 32b is, for example, a flat surface that is substantially parallel to the xy plane. The bottom surface 32b may be, for example, a surface that is substantially parallel to the lower surface 30b of the well member 30 or the upper surface 11a of the substrate 11. In the thickness direction of the well member 30 (the z direction in the figure), the bottom surface 32b is located between the lower surface 30b and the upper surface 30a. That is, in the thickness direction of the well member 30, the distance between the bottom surface 32b and the upper surface 30a is smaller than the distance between the lower surface 30b and the upper surface 30a. In this example, the shape of the bottom surface 32b is substantially circular. However, the shape of the bottom surface 32b is not limited to this and may be, for example, polygonal. The bottom surface 32b is provided with an opening 32p that communicates with the flow path 34. The opening 32p may be provided at an end of the bottom surface 32b, as in the illustrated example, or may be provided in the center of the bottom surface 32b.

[0021] The inclined portion 32s of the side surface 32l is configured so that the cross-sectional area of ​​the recess 32 in a plane perpendicular to the normal direction of the upper surface 30a (cross-sectional area in the xy plane in the figure) increases with increasing distance from the bottom surface 32b. In this example, the shape of the inclined portion 32s in a cross-sectional view is linear. However, the shape of the inclined portion 32s in a cross-sectional view is not limited to a linear shape, and may be, for example, a shape including steps (e.g., a staircase shape) or a shape including a curve.

[0022] In this example, the side surface 32l has, in addition to the inclined portion 32s, a vertical portion 32v perpendicular to the upper surface 30a. In the example shown, the recess 32 has a first portion 32A having the inclined portion 32s on the side surface and a second portion 32B having the vertical portion 32v on the side surface. The first portion 32A includes the bottom surface 32b of the recess 32, and the second portion 32B includes the opening 32o of the recess 32. In this example, the first portion 32A has a substantially truncated cone shape, and the second portion 32B has a substantially cylindrical shape. The shapes of the first portion 32A and the second portion 32B of the recess 32 are not limited to the example shown. The second portion 32B may be omitted.

[0023] The flow path 34 has a first portion 34a and a second portion 34b. The first portion 34a extends in a direction parallel to the bottom surface 32b (in this example, a direction parallel to the xy plane in the figure) and is located on the substrate 11 side of the bottom surface 32b. The first portion 34a is defined by the upper surface 11a of the substrate 11 and an inner wall surface 34h facing the upper surface 11a. In other words, the upper surface 11a of the substrate 11 forms the bottom of the first portion 34a of the flow path 34. The second portion 34b connects the first portion 34a to an opening 32p provided in the bottom surface 32b. The second portion 34b extends in a direction intersecting (orthogonal to) the bottom surface 32b. The second portion 34b is disposed so as to at least partially overlap the bottom surface 32b in a top view. The second portion 34b has an inner wall surface 34l perpendicular to the bottom surface 32b. The inner wall surface 34l may be inclined with respect to the bottom surface 32b. The bottom surface 32b of the recess 32 is connected to the inner wall surface 34l of the second portion 34b of the flow path 34, and a step is formed between the bottom surface 32b and the inner wall surface 34l. The "step" here includes a bend or discontinuity between the bottom surface 32b and the inner wall surface 34l.

[0024] In this example, the well member 30 further has a through-hole 38 that communicates with the flow path 34. The through-hole 38 opens to both the upper surface 30a and the lower surface 30b of the well member 30. An opening 38o in the upper surface 30a of the through-hole 38 does not overlap with the opening 32o of the recess 32. In this example, the through-hole 38 has a substantially cylindrical shape. However, the shape of the through-hole 38 is not limited to this and may be, for example, a polygonal shape.

[0025] 2A, 2B, 2C, and 2D are schematic cross-sectional views illustrating a method for culturing nerve cells using cell culture vessel 50 A. The reason why nerve cells can be cultured efficiently by using cell culture vessel 50 A will be explained with reference to FIGS. 2A to 2D.

[0026] As described below, neuronal aggregates (i.e., aggregated masses of neuronal cell bodies) are seeded in the cell culture vessel 50A and cultured, allowing axons extending from the cell bodies to self-organize into bundles. Culture using the cell culture vessel 50A can yield neuronal aggregates with bundled axons. When culturing neurons using the cell culture vessel 50A, surfaces of the cell culture vessel 50A to which cells can adhere (e.g., surfaces within the recesses 32, the channels 34, and the through-holes 38) may be surface-treated with a coating solution containing a cell-adherent component, such as an extracellular matrix (ECM), to which the cells have preference.

[0027] As shown in FIG. 2A , neural cell aggregates 74 are transferred (seeded) into a cell culture vessel 50A, for example, from another vessel, using a pipette 62. At this time, the wells 32 of the cell culture vessel 50A are filled with a liquid medium (or culture solution) 72. The liquid medium containing the aggregates 74 is dripped from the pipette 62 into the liquid medium 72 in the wells 32. The dripping of the liquid medium containing the aggregates 74 from the pipette 62 creates a flow in the liquid medium 72 in the wells 32, as indicated by the bold arrow in FIG. 2A . The aggregates 74 float along this flow in the liquid medium 72 in the wells 32. Because neural cells are adhesive cells, they require adhesion to a scaffold for proliferation and growth. That is, when neural cells are suspended in the liquid medium 72, they become inactive and their axons do not extend.

[0028] As shown in FIG. 2B , as the flow in the liquid medium 72 in the recess 32 subsides, the aggregates 74 sink within the liquid medium 72 in the recess 32, land on the bottom surface 32b of the recess 32, and adhere thereto. At this time, since the side surface 321 of the recess 32 includes the inclined portion 32s, the aggregates 74 can be guided to the bottom surface 32b by moving along the inclined portion 32s. In other words, the likelihood that the aggregates 74 will adhere to the bottom surface 32b can be increased. Note that the bottom surface 32b of the recess 32 is preferably surface-treated with a coating solution containing a palatable component to which the cells have a preference, as described above.

[0029] As shown in FIG. 2C, the culture is performed in a state where aggregates 74 are adhered to bottom surface 32b. As described above, a step is formed between bottom surface 32b and inner wall surface 34l of second portion 34b of flow channel 34. Adherent cells are more likely to proliferate and grow on a surface having an uneven structure (e.g., including convex portions, concave portions, protrusions, steps, etc.) than on a flat surface. Therefore, by culturing aggregates 74 in a state where they are adhered to bottom surface 32b, the culture of aggregates 74 can be performed efficiently.

[0030] Furthermore, by using the cell culture vessel 50A, it is easy to control the direction in which the axons 76 extend during culture. At least some of the axons 76 extending from the aggregates 74 extend into the flow channel 34 through the openings 32p provided in the bottom surface 32b. The axons 76 extend away from the aggregates 74 along the direction in which the first portion 34a of the flow channel 34 extends, and then bundle within the first portion 34a of the flow channel 34. In this way, the aggregates 74 and the axons 76 can be cultured at a distance from each other, thereby improving biological compatibility with neural tissue in vivo. Note that some of the axons 76 extending from the aggregates 74 may be formed within the recess 32, as shown in the figure.

[0031] Furthermore, by using cell culture vessel 50A, liquid medium 72 can easily reach underneath aggregates 74, compared to when aggregates 74 are cultured on upper surface 11a of substrate 11, as in the cell culture method according to the comparative example described below, and therefore nutrients in liquid medium 72 can more easily reach aggregates 74. Therefore, the cell culture efficiency can be improved.

[0032] As shown in FIG. 2D , a step of exchanging the liquid medium 72 in the wells 32 (sometimes referred to as a "medium exchange step") may be further performed during the culture. Specifically, a step of providing (adding) the liquid medium to the wells 32, and a step of absorbing a portion of the liquid medium 72 from the wells 32 before providing (adding) the liquid medium, may be further performed as needed during the culture. The cells consume necessary nutrients from the liquid medium 72 and excrete waste products into the liquid medium 72. Therefore, it is preferable to perform the medium exchange step when the proportion of nutrients in the liquid medium 72 decreases and / or when the amount of waste products in the liquid medium 72 increases during the culture.

[0033] 3A and 3B are schematic cross-sectional views illustrating a cell culture vessel 950A according to a comparative example and a method for culturing nerve cells using the cell culture vessel 950A. Differences from the cell culture vessel 50A of the present embodiment and the cell culture method using the cell culture vessel 50A will be mainly described.

[0034] 3A and 3B, the cell culture vessel 950A according to the comparative example differs from the cell culture vessel 50A having the well member 30 in the structure and shape of the well member 930. The well member 930 has a through-hole 932 that opens to an upper surface 930a and a lower surface 930b, and a flow path 934 that communicates with the through-hole 932. A side surface 932l of the through-hole 932 is perpendicular to the upper surface 930a and does not include a portion that is inclined relative to the upper surface 930a. In other words, the through-hole 932 has a substantially cylindrical or prismatic shape. The through-hole 932 exposes a first region 11r that is a portion of the upper surface 11a of the substrate 11.

[0035] A method for culturing nerve cells using the cell culture vessel 950A according to a comparative example will be described.

[0036] As shown in Fig. 3A, nerve cell aggregates 74 are transferred (seeded) from, for example, another container into cell culture container 950A according to the comparative example using pipette 62. Liquid medium containing aggregates 74 is dripped from pipette 62 into liquid medium 72 in through-hole 932. The dripping of liquid medium containing aggregates 74 from pipette 62 causes a flow in liquid medium 72 in through-hole 932 as indicated by the thick arrow in Fig. 3A. Aggregates 74 float in liquid medium 72 in through-hole 932 along this flow.

[0037] As shown in FIG. 3B , as the flow in the liquid medium 72 subsides, the aggregates 74 sink in the liquid medium 72 in the through-holes 932 and land on and adhere to the first regions 11r exposed by the through-holes 932 on the upper surface 11a of the substrate 11. At this time, the positions in the first regions 11r to which the aggregates 74 adhere are determined randomly. From the viewpoint of extending axons extending from the aggregates 74 along the direction of extension of the flow channels 934, it is preferable that the aggregates 74 adhere to the first regions 11r near the flow channels 934. However, as shown in FIG. 3B , if the aggregates 74 adhere to a position far from the flow channels 934, it may be difficult to form axons extending from the aggregates 74 in the flow channels 934. In such cases, it may be difficult to control the direction in which the axons extend, and the axons may not be successfully bundled.

[0038] In contrast, according to the cell culture vessel 50A and the cell culture method using the cell culture vessel 50A of this embodiment, as described above, the possibility of the aggregate 74 adhering to the bottom surface 32b can be increased, thereby solving the problems that arise in the cell culture vessel 950A and the cell culture method using the cell culture vessel 950A of the comparative example.

[0039] 4 is a schematic cross-sectional view illustrating a cell culture vessel 950B according to another comparative example and a method for culturing nerve cells using the cell culture vessel 950B. Differences from the cell culture vessel 950A and the cell culture method using the cell culture vessel 950A will be mainly described.

[0040] As shown in FIG. 4 , in a cell culture vessel 950B according to another comparative example, the area of ​​the first region 11r of the upper surface 11a of the substrate 11, which is exposed by the through-hole 932, in a top view is smaller than that of the cell culture vessel 950A. By using the cell culture vessel 950B instead of the cell culture vessel 950A, it becomes easier to control the position where the aggregate 74 adheres to the first region 11r, near the flow path 934. However, as shown in FIG. 4 , when the cell culture vessel 950B is used, the distance between the aggregate 74 and the pipette 64 used for medium replacement becomes shorter during the medium replacement process during culture. This increases the possibility that the aggregate 74 will be accidentally aspirated by the pipette 64 when sucking up the liquid medium 72 or that the aggregate 74 will be affected by the flow generated when the liquid medium is added from the pipette 64 to the through-hole 932. If this occurs, the cell culture efficiency will decrease. Alternatively, attempts to reduce the possibility of this may reduce the efficiency of the medium replacement process.

[0041] In contrast, the cell culture vessel 50A and the cell culture method using the cell culture vessel 50A can solve the problems that arise in the cell culture vessel 950B and the cell culture method using the cell culture vessel 950B according to the comparative example. In the cell culture vessel 50A, the side surface 32l of the recess 32 has the inclined portion 32s, so that the area of ​​the opening 32o of the recess 32 is larger than the area of ​​the bottom surface 32b of the recess 32. Therefore, in the medium replacement step of the cell culture method using the cell culture vessel 50A shown in FIG. 2D, the medium replacement can be performed while maintaining a distance between the pipette 64 that performs the medium replacement and the aggregate 74. By using the cell culture vessel 50A, the influence (damage) on the aggregate 74 during the medium replacement step can be reduced without reducing the efficiency of the medium replacement process.

[0042] The shape and preferred size of the cell culture vessel 50A will be described with reference to Fig. 5A, which is a schematic cross-sectional view of the cell culture vessel 50A.

[0043] The diameter A1 of the bottom surface 32b of the recess 32 is, for example, about 500 μm. The diameter A1 of the bottom surface 32b of the recess 32 can be, for example, 300 μm or more and 500 μm or less. The diameter A1 of the bottom surface 32b of the recess 32 can be, for example, 1.5 to 2.5 times the diameter of the neuronal cell body (or neuronal aggregate) seeded in the cell culture vessel 50A. Here, the diameter of the neuronal aggregate in the cell culture vessel 50A is, for example, about 200 μm, but the diameter of the neuronal aggregate is, for example, 5 μm or more and 1 mm or less. If the shape of the bottom surface 32b in top view is a shape other than a circle, the equivalent diameter of the area of ​​the bottom surface 32b in top view can be set within the above-mentioned range of the diameter A1.

[0044] Diameter A2 of opening 32p provided in bottom surface 32b is, for example, 200 μm or less. For example, it may be 0.1 to 0.5 times diameter A1 of bottom surface 32b of recess 32. If the shape of opening 32p in top view is a shape other than a circle, the diameter of the circle equivalent in area of ​​opening 32p in top view may be set within the above-mentioned range of diameter A2.

[0045] Distance B1 between bottom surface 32b of recess 32 and upper surface 11a of substrate 11 in the thickness direction of well member 30 is, for example, 10 μm or more and 100 μm or less. Distance B1 can also be said to be the distance between bottom surface 32b of recess 32 and lower surface 30b of well member 30 in the thickness direction of well member 30. When distance B1 is 10 μm or more, a step is formed between bottom surface 32b and inner wall surface 341 of second portion 34b of flow channel 34, allowing cells to be cultured efficiently.

[0046] The distance B2 between the bottom surface 32b of the recess 32 and the inner wall surface 34h of the first portion 34a of the flow channel 34 in the thickness direction of the well member 30 is, for example, 0 μm or more and 100 μm or less. The height B3 of the first portion 34a of the flow channel 34 (the distance in the thickness direction of the well member 30) is, for example, the value obtained by subtracting the distance B2 from the distance B1 (B3 = B1 - B2). The height B3 of the first portion 34a is, for example, 10 μm or more and 100 μm or less. The distance B2 and the height B3 can be set appropriately depending on the diameter of the axon bundle formed in the first portion 34a of the flow channel 34. The diameter of the axon bundle is, for example, approximately 50 μm.

[0047] The inclination angle θ of the inclined portion 32s of the side surface 32l of the recess 32 is, for example, 20° or more and 80° or less. The inclination angle of the inclined portion 32s is determined as the angle between a line segment connecting the lower end and upper end of the inclined portion 32s in a cross-sectional view (on the xz plane in the figure) and a straight line parallel to the upper surface 30a of the well member 30. Even if the shape of the inclined portion 32s in a cross-sectional view is other than linear, the inclination angle of the inclined portion 32s can be determined in the same way.

[0048] [Variations] A cell culture vessel 50A1 according to a modified example of this embodiment will be described with reference to Fig. 5B, which is a schematic cross-sectional view of the cell culture vessel 50A1.

[0049] Cell culture vessel 50A1 differs from cell culture vessel 50A, which has substrate 11 and well member 30, in that it has well member 53. Well member 53 has a shape and structure in which substrate 11 and well member 30 of cell culture vessel 50A are integrally formed.

[0050] The cell culture vessel 50A1 also provides the same effects as the cell culture vessel 50A.

[0051] [Embodiment 2] A cell culture vessel 50B according to this embodiment will be described with reference to Figures 6A and 6B. Figure 6A is a schematic cross-sectional view of the cell culture vessel 50B, and Figure 6B is a schematic top view of a recess 32 provided in the cell culture vessel 50B. Below, differences from the previous embodiment will be mainly described.

[0052] The cell culture vessel 50B differs from the cell culture vessel 50A in that the side surface 32l of the recess 32 has a protrusion 44 that protrudes into the recess 32. In the illustrated example, the side surface 32l of the recess 32 has multiple protrusions 44, but at least one protrusion 44 may be provided. Each protrusion 44 extends from the side surface 32l of the recess 32 in a direction parallel to the bottom surface 32b (in this example, a direction parallel to the xy plane in the figure). In the illustrated example, the multiple protrusions 44 are arranged at equal intervals at the intersection of the side surface 32l and a predetermined plane parallel to the bottom surface 32b (in this example, a plane parallel to the xy plane). In FIG. 6B, the intersection of the plane parallel to the bottom surface 32b and the inclined portion 32s of the side surface 32l is indicated by a dashed circle. 6B, the plurality of protrusions 44 (four protrusions 44 in this example) are arranged on the side surface 32l of the recess 32 at equal intervals (at 90° central angles in this example) along the circumference of a circle that is the intersection of the side surface 32l and a plane parallel to the bottom surface 32b. The plurality of protrusions 44 include a pair of protrusions 44a that are symmetrical with respect to the center CP of the recess 32 in a top view. In the example shown, the plurality of protrusions 44 further include a pair of protrusions 44b that are symmetrical with respect to the center CP of the recess 32 in a top view. The one or more protrusions 44 are formed integrally with the side surface 32l of the recess 32, for example, using a known injection molding technique.

[0053] In the cell culture vessel 50B, similar to the cell culture vessel 50A according to the first embodiment, nerve cells can be cultured efficiently.

[0054] Furthermore, the cell culture vessel 50B, having the protrusions 44, offers a wider range of diameters for the neuronal aggregates 74 that can be cultured therein than the cell culture vessel 50A without the protrusions. As shown in FIG. 7 , in the cell culture vessel 50A, if the diameter of the aggregates 74 is too large, the openings 32p in the bottom surfaces 32b of the recesses 32 may be at least partially filled (blocked) by the aggregates 74, which may impede smooth movement of the liquid medium 72 between the recesses 32 and the flow channels 34 via the openings 32p. In such cases, for example, even if a medium exchange step is performed, nutrients in the liquid medium 72 may not be sufficiently replenished and waste products may not be sufficiently removed, resulting in reduced culture efficiency. Alternatively, attempting to sufficiently replenish nutrients and remove waste products from the liquid medium 72 may reduce the efficiency of medium exchange. In contrast, cell culture vessel 50B can hold aggregate 74 at a position closer to top surface 30a than bottom surface 32b by protrusion 44, and can therefore maintain a gap between aggregate 74 and bottom surface 32b. Therefore, even if aggregate 74 has a large diameter, opening 32p of bottom surface 32b is prevented from being partially filled with aggregate 74.

[0055] 6C, 6D, and 6E are schematic diagrams illustrating other examples of protrusions 44 of a cell culture vessel 50B. FIGS. 6C and 6D are schematic top views of a recess 32 of the cell culture vessel 50B, and FIG. 6E is a schematic cross-sectional view of the cell culture vessel 50B. As shown in FIGS. 6C, 6D, and 6E, the shape, number, and position of the protrusions 44 on the side surface 321 can be adjusted appropriately depending on, for example, the diameter of the neuronal aggregate 74 to be cultured in the cell culture vessel 50B. For example, as shown in FIG. 6C, the number of protrusions 44 may be an odd number, such as three or five. As shown in FIG. 6D, the protrusions 44 protruding from the side surface 321 of the recess 32 may be connected (continuous) to one another. In the example of FIG. 6D, the protrusions 44 have a generally cross shape when viewed from above. In the example of Fig. 6D, the protrusions 44 protrude from a plurality of points equally spaced along the circumference of a circle that is the intersection of a plane parallel to the bottom surface 32b and the side surface 32l, and are connected to each other at the center CP of the recess 32 in a top view. Also, as shown in the example of Fig. 6E, the plurality of protrusions 44 may extend from the side surface 32l toward the upper surface 51 of the cell culture vessel 50B (in this example, the upper surface 30a of the well member 30).

[0056] [Variations] A cell culture vessel 50B1 according to a modified example of this embodiment will be described with reference to Figures 8A and 8B. Figure 8A is a schematic cross-sectional view of the cell culture vessel 50B1, and Figure 8B is a schematic top view of a recess 32 included in the cell culture vessel 50B1.

[0057] The cell culture vessel 50B1 differs from the cell culture vessel 50B in that the protrusion 46 is supported by a support 47 that is disposed in contact with the side surface 32l of the recess 32 and matches the side surface 32l.

[0058] The support portion 47 has a shape that matches the side surface 32l of the recess 32 between the opening 32o and the bottom surface 32b in a top view and has a perforated portion that allows liquid to flow through. In the example shown, the support portion 47 has a ring shape. Each of the multiple protrusions 46 supported by the ring-shaped support portion 47 extends toward the inside of the ring. The support portion 47 may be made of a metal such as titanium.

[0059] The protrusions 46 may have a structure similar to that of the protrusions 44 of the cell culture vessel 50B. Each protrusion 46 extends from the side surface 321 of the recess 32 into the recess 32 in a direction parallel to the upper surface 30a (a direction parallel to the xy plane in the figure). The multiple protrusions 46 include a pair of protrusions 46a that are symmetrical with respect to the center CP of the recess 32 in a top view. In the example shown, the multiple protrusions 46 further include a pair of protrusions 46b that are symmetrical with respect to the center CP of the recess 32 in a top view.

[0060] The cell culture vessel 50B1 also provides the same effect as the cell culture vessel 50B. In the cell culture vessel 50B1, the protrusions 46 can hold the aggregates 74 at a position closer to the top surface 30a than the bottom surface 32b, so that a gap can be maintained between the aggregates 74 and the bottom surface 32b. Therefore, even if the diameter of the aggregates 74 is large, the openings 32p of the bottom surface 32b are prevented from being partially filled with the aggregates 74.

[0061] A member 48 having one or more protrusions 46 and a support member 47 that supports one or more protrusions 46 can be formed as a member separate from the well member 30. Therefore, when changing the shape, number, and position on the side surface 32l of the protrusions 46, for example, depending on the diameter of the neuronal aggregate 74 to be cultured in the cell culture vessel 50B1, it is sufficient to change the shape of only the member 48. Compared to the cell culture vessel 50B in which the protrusions 44 are formed integrally with the side surface 32l of the recess 32, the cell culture vessel 50B1 can be manufactured at a lower cost.

[0062] [Embodiment 3] A cell culture vessel 50C according to this embodiment will be described with reference to Fig. 9. Fig. 9 is a schematic cross-sectional view of the cell culture vessel 50C. Below, differences from the previous embodiment will be mainly described.

[0063] In the cell culture vessel 50C, the side surface 32l of the recess 32 differs from the cell culture vessel 50A in that it has a first portion 32lc and a second portion 32ld located on the upper surface 30a side of the first portion 32C and having lower cell adhesiveness than the first portion 32lc.

[0064] In the cell culture vessel 50C, the portion of the side surface 32l of the recess 32 having the first portion 32lc is referred to as the first portion 32C, and the portion of the side surface 32l of the recess 32 having the second portion 32ld is referred to as the second portion 32D. In this example, the well member 30 has the first portion 32C and the second portion 32D. The first portion 32C and the second portion 32D may be formed from different materials, with the second portion 32D being formed from a material with lower cell adhesiveness than the first portion 32C. Alternatively, the recess 32 may be formed from a single material, with the first portion 32lc of the side surface 32l having a cell adhesive layer on its surface, and the second portion 32ld of the side surface 32l not having the cell adhesive layer. The cell adhesion layer may be formed by surface treatment with a coating solution containing a cell-adhesive component, such as an extracellular matrix, to which the cells have preference, and the cell adhesion layer also contains the cell-adhesive component. Examples of the palatable components contained in the coating solution and the cell adhesion layer include proteins such as collagen, laminin, fibronectin, vitronectin, and gelatin, synthetic amino acids such as poly-D-lysine and poly-L-lysine, and artificially synthesized peptides. Any one of these may be contained, or two or more may be contained in combination.

[0065] The first portion 32C and the second portion 32D of the cell culture vessel 50C may or may not coincide with the first portion 32A of the recess 32 having an inclined portion 32s on the side and the second portion 32B of the recess 32 having a vertical portion 32v on the side shown with reference to Figure 1.

[0066] In the cell culture vessel 50C, similar to the cell culture vessel 50A according to the first embodiment, nerve cells can be cultured efficiently.

[0067] Furthermore, in the cell culture vessel 50C, the extension direction of the axons 76 can be controlled more effectively than in the cell culture vessel 50A. Since the side surface 321 of the recess 32 has the first portion 32C and the second portion 32D, the axons 76 can be prevented from extending into the second portion 32D, which has relatively low cell adhesiveness. Because the axons 76 can be prevented from extending toward the upper surface 30a (i.e., away from the flow channel 34), more axons 76 can be extended into the flow channel 34, and thicker bundles of axons 76 can be obtained.

[0068] [Variations] A cell culture vessel 50C1 according to a modified example of this embodiment will be described with reference to Fig. 10. Fig. 10 is a schematic cross-sectional view of the cell culture vessel 50C1.

[0069] In the cell culture vessel 50C1, the second portion 32ld of the side surface 32l has a microrelief structure 32d on its surface, while the first portion 32lc of the side surface 32l does not have a microrelief structure on its surface. In Fig. 10, the second portion 32ld of the side surface 32l is shown with a thick line to indicate that it has a microrelief structure 32d on its surface, and a portion of the second portion 32ld of the side surface 32l is shown enlarged in a speech bubble in Fig. 10. The microrelief structure is, for example, a moth-eye structure.

[0070] In the cell culture vessel 50C1, the second portion 32ld of the side surface 32l has the microrelief structure 32d on its surface, and therefore has better water repellency than the first portion 32lc, which does not have the microrelief structure. When a coating solution for forming a cell adhesion layer is applied to the entire surface of the side surface 32l of the recess 32, application of the coating solution to the second portion 32ld is suppressed, and therefore the second portion 32ld has lower cell adhesiveness than the first portion 32lc. The cell culture vessel 50C1 also achieves the same effects as the cell culture vessel 50C.

[0071] [Embodiment 4] A cell culture vessel 50D according to this embodiment will be described with reference to Fig. 11. Fig. 11 is a schematic cross-sectional view of the cell culture vessel 50D. Below, differences from the previous embodiment will be mainly described.

[0072] The cell culture vessel 50D differs from the cell culture vessel 50A in that it further includes one or more electrodes 42 disposed within the first portion 34a of the flow channel 34. In this example, the electrodes 42 are supported on the upper surface 11a of the substrate 11.

[0073] In the cell culture vessel 50D, similar to the cell culture vessel 50A according to the first embodiment, nerve cells can be cultured efficiently.

[0074] Furthermore, in the cell culture vessel 50D, the direction in which the axons 76 grow can be more effectively controlled. During culture, the axons 76 can be attracted into the first portion 34a of the flow channel 34 by supplying a voltage to the electrode 42. If the axons 76 are negatively charged, the axons 76 will be attracted to the electrode 42 by supplying a positive voltage to the electrode 42.

[0075] A plurality of electrodes 42 may be provided. The plurality of electrodes 42 may be connected to a plurality of electrically independent wirings. In such a case, different voltages can be supplied to the plurality of electrodes 42, and thus different electrodes 42 can be supplied with voltages depending on the state of the axons 76 during culture. The axons 76 can be more effectively attracted into the first portion 34a of the flow channel 34. By including an ionic attractant in the liquid medium 72, the attractant can aggregate on the electrodes 42 to which voltage is supplied, thereby enhancing the effect of attracting the axons 76. The electrodes 42 may also be used to detect and externally extract electrical signals (action potentials) emitted by the cells during culture. By controlling the direction of axon 76 extension, thicker axon bundles 76 can be obtained, allowing for more accurate detection of cell action potentials.

[0076] It is also possible to combine two or more of the above-described embodiments 1 to 4. For example, by combining embodiment 3 and embodiment 4, the direction in which the axon 76 extends can be controlled more effectively.

[0077] A cell culture vessel according to an embodiment of the present invention may have a plurality of sets of the recesses 32 and channels 34 described above. For example, a cell culture vessel according to an embodiment of the present invention may have a substrate 11 and a single well member 30 supported by the substrate 11, and a plurality of sets of recesses 32 and channels 34 may be arranged and formed in the single well member 30. Alternatively, a plurality of well members 30, each having one set of recesses 32 and channels 34, may be prepared and connected to each other. A cell culture vessel according to an embodiment of the present invention may be formed from only a single well member having a plurality of sets of recesses 32 and channels 34. [Industrial Applicability]

[0078] The cell culture vessel according to the embodiment of the present invention is used for culturing, for example, nerve cells, and can improve the efficiency of culturing nerve cells. [Explanation of symbols]

[0079] 11: Substrate 30: Well member 32: Recess 32l: Side surface 32s: Inclined portion 32b: Bottom surface 32p: Opening 34: Flow path 34a: First portion (of flow path) 34b: Second portion (of flow path) 38: Through-hole 42: Electrode 44: Protrusion 46: Protrusion 50A, 50A1, 50B, 50B1, 50C, 50C1, 50D: Cell culture vessel 72: Liquid medium 74: Aggregate of nerve cells 76: Axon

Claims

1. an upper surface and a lower surface opposite the upper surface; a recessed portion that opens to the upper surface and has a bottom surface that is substantially parallel to the upper surface or the lower surface and a side surface that includes an inclined portion that is inclined with respect to the bottom surface; a flow path communicating with the recess; and The flow path is a first portion extending in a direction parallel to the bottom surface and located on the lower surface side of the bottom surface; a second portion connecting the first portion and an opening provided in the bottom surface; A cell culture vessel comprising:

2. The cell culture vessel according to claim 1 , wherein the second portion extends in a direction intersecting the bottom surface.

3. The cell culture vessel according to claim 1 , wherein the second portion is disposed so as to overlap the bottom surface in a top view.

4. The cell culture vessel according to claim 1 , wherein the side surface has one or more protrusions that protrude into the recess.

5. The cell culture vessel according to claim 4 , wherein the protrusion extends from the side surface in a direction parallel to the bottom surface or toward the top surface.

6. the one or more protrusions are a plurality of protrusions, The cell culture vessel according to claim 4 , wherein the plurality of protrusions are arranged at equal intervals at an intersection between the side surface and a predetermined plane parallel to the bottom surface.

7. The cell culture vessel according to claim 6 , wherein the plurality of protrusions include a pair of protrusions symmetrical with respect to the center of the recess in a top view.

8. The cell culture vessel according to claim 4 , wherein the protrusion is formed integrally with the side surface.

9. The recess further includes a support portion that is disposed in contact with the side surface of the recess and matches the side surface, The cell culture vessel according to claim 4 , wherein the protrusion is supported by the support.

10. the side surface has a first portion and a second portion located on the upper surface side of the first portion of the side surface, The cell culture vessel according to claim 1 , wherein the second portion of the side surface has a lower cell adhesiveness than the first portion of the side surface.

11. the second portion of the side surface has a fine uneven structure on the surface, The cell culture vessel according to claim 10 , wherein the first portion of the side surface does not have a fine uneven structure on the surface.

12. the first portion of the side surface has a cell adhesion layer having cell adhesive properties on a surface thereof, The cell culture vessel according to claim 10 , wherein the second portion of the side surface does not have the cell adhesion layer on its surface.

13. The cell culture vessel according to claim 10 , wherein the portion of the side surface having the second portion is formed from a material having lower cell adhesiveness than the portion of the side surface having the first portion.

14. The cell culture vessel according to claim 1 , further comprising one or more electrodes disposed within the first portion of the flow path.

15. a substrate having a top surface; a well member supported on the upper surface of the substrate, the well member having the recess and the channel; and The cell culture vessel according to claim 1 , wherein the upper surface of the substrate forms a bottom of the first portion of the channel.

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