Cell culturing device and cell culturing method

Abstract

A generation unit (5) generates a dispersion (DI) in a state where droplets (DR) in which a culture solution that contains cells is encapsulated are dispersed in a particular solution (S) having a specific gravity greater than that of the culture solution. A substrate (11) has a main surface (111), and the dispersion (DI) is held on the main surface (111). A mesh member (2) is disposed apart from the main surface (111) of the substrate (11). In the mesh member (2), a plurality of through holes (23) are formed, in a state where the mesh member (2) is positioned above the substrate (11) in the vertical direction, to trap droplets (DR) which have been introduced to the substrate (11) and floated.

Description

Cell culture device and cell culture method

[0001] The present disclosure relates to a cell culture device and a cell culture method, and more particularly to a technique for culturing cells in droplets.

[0002] Recently, a cell culture technique has been gaining attention, in which cells are cultured in droplets of water containing cells and a culture medium dispersed in an oil phase. In this technique, droplets are used as independent culture vessels separated by an oil phase.

[0003] Non-Patent Documents 1 and 2 disclose techniques for separating multiple droplets in mineral oil, which has a lower specific gravity than a culture medium. Non-Patent Document 1 discloses a technique for introducing mineral oil containing droplets onto a microwell chip and submerging the droplets one by one into the holes of the microwell chip. Non-Patent Document 2 also discloses a technique for submerging droplets in mineral oil one by one into the spaces between pillars of a microcage array chip. By individually capturing (trapping) droplets in this way, desired droplets can be collected and further cultured or analyzed.

[0004] Eujin Um et al., “Mesh-integrated microdroplet array for simultaneous merging and storage of single-cell droplets”, Lab on a Chip, 2012, 12, pp. 1594-1597. Jin-Gang Xu et al., “Forming a Large-Scale Droplet Array in a Microcage Array Chip for High-Throughput Screening”, Analytical Chemistry, 2019, 91, 16, pp. 10757-10763.

[0005] In cell culture using droplets, it is preferable to use fluorinated oil rather than mineral oil because it is easier to supply oxygen into the droplets.

[0006] However, since the specific gravity of fluorinated oil is greater than that of the culture solution, the droplets cannot be separated by sinking them as in Non-Patent Documents 1 and 2.

[0007] Given the above circumstances, there has been a strong demand for technology that can individually trap droplets floating in liquids with a higher specific gravity than the culture medium, such as fluorinated oil.

[0008] A cell culture device according to one aspect of the present disclosure includes a generation unit, a substrate, and a mesh member. The generation unit generates a dispersion in which droplets containing cells and a culture medium are dispersed in a specific solution having a specific gravity greater than that of the culture medium. The substrate has a main surface and holds the dispersion on the main surface. The mesh member is disposed at a distance from the main surface of the substrate. The mesh member has a plurality of through-holes formed therein, which trap droplets that are introduced to the substrate and float up, with the mesh member positioned vertically above the substrate.

[0009] A cell culture method according to another aspect of the present disclosure includes the steps of: generating a dispersion in which droplets containing cells and a culture medium are dispersed in a specific solution having a specific gravity greater than that of the culture medium; retaining the dispersion on a main surface of a substrate having a main surface; and disposing a mesh member spaced apart from the main surface of the substrate, the mesh member having a plurality of through-holes formed therein, the mesh member being positioned vertically above the substrate, for trapping droplets that are introduced to the substrate and float up.

[0010] The culture device according to the present disclosure can individually trap droplets floating in a specific solution having a higher specific gravity than the culture medium.

[0011] FIG. 1 is a schematic configuration diagram of a culture unit according to an embodiment. FIG. 2 is a cross-sectional view of a culture section according to an embodiment. FIG. 3 is a diagram showing an example of an outline of an observation image of a droplet in a culture section. FIG. 4 is a flowchart showing an example of a culture method according to an embodiment. FIG. 5 is a diagram for explaining droplet trapping. FIG. 6 is a perspective view showing a culture system according to another embodiment. FIG. 7 is a view of the structure around the culture section and a linear guide as seen obliquely from above. FIG. 8 is a cross-sectional view showing the structure of the culture section. FIG. 9 is a cross-sectional view showing the structure around the culture section.

[0012] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.

[0013] [Configuration of Culture Unit and Culture Apparatus] FIG. 1 is a schematic diagram of a culture unit 100 according to the present embodiment. The culture unit 100 includes a microscope 7, a capillary 8, a control device 9, and a culture apparatus 15. The culture apparatus 15 corresponds to an example of a "cell culture apparatus." The culture apparatus 15 includes a generation unit 5 and a culture unit 10. FIG. 2 is a cross-sectional view of the culture unit 10 according to the embodiment. The culture unit 10 includes a substrate 11 and a mesh member 2. FIGS. 1 and 2 are diagrams of the culture unit 100 and the culture unit 10 with droplets DR trapped on the mesh member 2. In FIGS. 1 and 2, the direction perpendicular to the substrate 11 is the Z axis, and the plane parallel to the substrate 11 is the XY plane. In one example, the culture unit 10 is used in a state where it is installed so that the Z axis is approximately parallel to the direction of gravity (vertical direction).

[0014] The generating unit 5 generates a dispersion liquid DI in which droplets DR containing cell-containing culture fluid are dispersed in a specific solution S having a specific gravity greater than that of the culture fluid. In one embodiment, the generating unit 5 includes a microchannel device 50 having microchannels 51A and 51B formed therein. The microchannels 51A and 51B intersect at an intersection 52. A predetermined amount of cell-containing culture fluid intermittently flows into the microchannel 51A from an end 511A. The specific solution S flows into the microchannel 51B. More specifically, the specific solution S is supplied from the microchannel 51B to sandwich the microchannel 51A from both sides. This generates droplets DR in which a predetermined amount of culture fluid is encapsulated in the specific solution S. In other words, a dispersion liquid DI in which the droplets DR are dispersed in the specific solution S is generated. The dispersion liquid DI then flows out from an end 512A. End 512A is located on the opposite side of end 511A of microchannel 51A across intersection 52. The dispersion liquid DI is introduced into culture section 10, for example, manually by a user. However, culture unit 100 may also include a device (not shown) that transports the dispersion liquid DI from production section 5 to culture section 10, and this device may be controlled by control device 9.

[0015] In the culture unit 10, the dispersion liquid DI is introduced and then held on the main surface 111 of the substrate 11. The main surface 111 is the surface of the substrate 11 facing the positive Z-axis direction (vertically upward). The specific solution S has a higher specific gravity than the culture solution. The mesh member 2 is disposed at a distance from the main surface 111 of the substrate 11. The mesh member 2 has a plurality of through-holes 23 formed therein, which trap droplets DR introduced to and floating up on the substrate 11 while the mesh member 2 is positioned vertically above the substrate 11. More specifically, the mesh member 2 has a plurality of through-holes 23 formed therein, each of which is sized to individually trap the droplets DR. With this configuration, each droplet DR floating in the specific solution S floats up into the through-hole 23. In other words, the droplets DR can be individually trapped in the through-hole 23. This allows cells to be cultured and observed in an isolated state.

[0016] In one embodiment, the cells are microorganisms. Some microorganisms in the environment are closely related to humans, and some are used not only in daily life but also in industry and manufacturing. However, most microorganisms in the environment have not been successfully cultured, and they have not been effectively utilized as biological resources. Furthermore, the physiological characteristics of many microorganisms under specific environments have not been elucidated. Therefore, culturing microorganisms and elucidating their properties may be applied to the development of medicine, industry, etc. In another embodiment, the cells are animal cells. In this case, culturing animal cells and elucidating their properties may also be applied to the development of medicine, industry, etc. In yet another embodiment, the cells are plant cells.

[0017] In one embodiment, the culture medium includes cells and a liquid medium. The culture medium may also include a marker for visually indicating the characteristics or amount of the cells or a metabolite produced by the cells. The marker may be, for example, a substance that indicates the presence of a predetermined metabolite by color or fluorescence.

[0018] The droplets DR are, for example, water droplets with a diameter of about 30 to 130 μm, but are not limited thereto. The droplets DR in the specific solution S can be produced in the hundreds of thousands at a time using a dedicated device.

[0019] In one embodiment, the specific solution S is a fluorine oil. Examples of fluorine oils include FC40, Novec (trademark, the same applies hereinafter) 7500, and combinations thereof. Fluorine oils have a higher oxygen permeability than mineral oils. Furthermore, surfactants suitable for creating droplets DR in fluorine oil exist. Users add these surfactants to at least one of the aqueous phase (in the culture medium) and the oil phase (in the fluorine oil). For example, users use Span (trademark) 80, Tween (trademark) 20, etc. as surfactants for the aqueous phase. Furthermore, users use 008-FluoroSurfactant, Pico-surf (trademark) 1, Kryto x (trademark), etc. as surfactants for the oil phase. As described above, using fluorine oil as the specific solution S facilitates the supply of oxygen necessary for cell culture to the droplets DR. Furthermore, the surfactant can be used to prevent droplets DR from coalescing and maintain them as independent compartments stably over the long term. This facilitates long-term cell culture and observation in an isolated state for each droplet DR. However, the specific solution S is not limited to the above example and can be any liquid with a higher specific gravity than the culture medium. Examples of such liquids include ionic liquids and liquid mercury. Furthermore, the specific solution S is a liquid that is immiscible with the culture medium. In one embodiment, the amount of dispersion liquid DI introduced onto the substrate 11 is sufficient to trap the droplets DR in the through-holes 23. Specifically, the amount is such that the height of the dispersion liquid DI from the substrate 11 is approximately the same as the height of the mesh member 2. For example, a micropillar from Yodaka Giken Co., Ltd. can be used as the micropillar device 1. The micropillar device 1 may be fabricated by a casting method using polydimethylsiloxane, for example.

[0020] In one embodiment, the culture unit 10 is disposed on a substrate 11 and further includes a protrusion 12 protruding upward from the substrate 11. The mesh member 2 is disposed above the protrusion 12. This configuration facilitates the mesh member 2 to be disposed above the substrate 11 at a distance. More specifically, it is easy to maintain a certain distance between the mesh member 2 and the substrate 11. In the example of FIG. 2, the upper end of the protrusion 12 contacts the mesh member 2, and the protrusion 12 directly supports the mesh member 2. However, if a dispersion liquid DI is present between the substrate 11 and the mesh member 2, the dispersion liquid DI may enter between the mesh member 2 and the protrusion 12, causing the mesh member 2 to be spaced apart from the protrusion 12 (floating on the dispersion liquid DI). Furthermore, the substrate 11 and the protrusion 12 are preferably formed integrally. This configuration facilitates the mesh member 2 to be stably held above the substrate 11 at a distance, making the entire culture unit 10 easier to handle. 2, a micropillar device 1 including a substrate 11 and protrusions 12 is used. However, the configuration for disposing the mesh member 2 above the substrate 11 in the culture unit 10 at a distance is not limited to the above example, and the mesh member 2 and protrusions 12 may be formed integrally, or the substrate 11, protrusions 12, and mesh member 2 may all be formed integrally. Furthermore, instead of the protrusions 12, the culture unit 10 may have a configuration in which the mesh member 2 is clamped from the side and held at a predetermined height from the substrate 11.

[0021] In one embodiment, the substrate 11 is a container having a wall 112 that rises vertically upward at its peripheral edge. By providing the wall 112 at the peripheral edge of the substrate 11, the possibility of the dispersion liquid DI spilling from the peripheral edge is reduced compared to when the wall 112 is not provided.

[0022] The mesh member 2 is typically a plate-like member having a plurality of through-holes 23 formed therein, each of which is large enough to trap a droplet DR individually. One example of the mesh member 2 is a mesh member having a lattice-like mesh, in which case the mesh corresponds to the through-holes 23. In one embodiment, the mesh member 2 and droplets DR are used such that the ratio of the diameter of the droplets DR to the mesh is 80% to 120%, preferably 90% to 110%. Another example of the mesh member 2 is a member having circular through-holes 23 formed therein. Preferably, the through-holes 23 of the mesh member 2 are regularly arranged on the substrate 11. This facilitates identifying and continuously observing each of the droplets DR trapped in the mesh member 2.

[0023] The microscope 7 is a device for observing the droplets DR in the culture unit 10. The microscope 7 is, for example, an optical microscope, and the culture unit 10 is placed on a stage (not shown), and the droplets DR in the culture unit 10 are observed through a lens (not shown). FIG. 3 is a diagram showing an example of an outline of an observation image of the droplets DR in the culture unit 10. In the observation image of FIG. 3, a white circle 600 is indicated at the portion corresponding to the droplet DR. As shown in FIG. 3, the user can observe the droplets DR trapped in the through-holes 23 of the mesh member 2 from above in a magnified view.

[0024] The culture unit 10 may be stored in an incubator (not shown) when observation with the microscope 7 is not being performed. In one embodiment, the culture unit 10 may be stored in an incubator set at a predetermined temperature of 25° C. to 40° C. for a predetermined period of time of several hours to several tens of hours, before observation with the microscope 7 is performed. In this embodiment, it is preferable that the culture unit 10 be stored in a state where it has been treated to prevent it from drying out. Possible treatments for this prevention include, for example, covering the top surface of the culture unit 10 with a cover.

[0025] In one embodiment, the capillary 8 is moved under the control of the control device 9 to individually collect (pick) desired droplets DR. This configuration allows for easier and more accurate control of the position of the capillary 8 compared to when the user manually moves the capillary 8. The control device 9 may be configured to move the tip of the capillary 8 to the position of a droplet DR at a predetermined position in the field of view of the microscope 7 in conjunction with the microscope 7. The capillary 8 corresponds to one embodiment of a "collection device." The capillary 8 may be, but is not limited to, a capillary 8 having an inner diameter at the tip that is approximately the same as the diameter of the droplet DR. The capillary 8 can also suck in droplets DR having a diameter that is approximately a predetermined percentage larger than the through-holes 23 of the mesh member 2 (e.g., a diameter that is 110% to 120% of the size of the through-holes 23). The capillary 8 may be configured to suck in droplets DR using capillary action, or may suck in droplets DR at a predetermined suction amount using a suction unit (not shown) that supplies suction force.

[0026] The user can transfer the cells contained in the droplet DR collected by the capillary 8 to another container for further culturing, observation and / or analysis.

[0027] [Comparison with conventional droplet-based cell culture methods] A technique for culturing cells using minute droplets dispersed in an oil phase (water-in-oil droplets) has been attracting attention in recent years. This technique makes it possible to culture a large number of different types of microorganisms in a small space. In this regard, Non-Patent Documents 1 and 2 disclose a technique for separating droplets in mineral oil, which has a lower specific gravity than the culture medium.

[0028] Non-Patent Document 1 discloses a technique that uses a microwell chip and a mesh to isolate and align microdroplets generated in mineral oil. Specifically, a mesh is placed on a microwell plate with a spacer on the top surface. The mesh helps to fill the microwells with microdroplets.

[0029] Non-Patent Document 2 discloses a technique using a microcage array chip to isolate and align droplets generated in mineral oil. Specifically, mineral oil containing droplets is spread on a microcage array chip with a pillar structure, causing the droplets to sink between the pillars.

[0030] On the other hand, when culturing cells in droplets, fluorinated oil is preferable to mineral oil in that it easily supplies oxygen to the droplets and contains surfactants that stabilize the droplets for a long period of time. However, unlike mineral oil, fluorinated oil has a higher specific gravity than the culture medium. Therefore, the techniques described in Non-Patent Documents 1 and 2 are techniques for trapping droplets that sink in mineral oil, which has a lower specific gravity than the culture medium, but have the problem that they cannot be applied to droplets floating in fluorinated oil. For these reasons, a technology for isolating droplets floating in a liquid, such as fluorinated oil, which has a higher specific gravity than the culture medium has been desired.

[0031] Isolating droplets floating in fluorine oil posed the following challenges. First, the position of droplets floating in fluorine oil was unstable, making it difficult to isolate specific droplets. Also, droplets floating on the fluorine oil tended to aggregate, making it difficult to manipulate individual droplets. Furthermore, the droplets had to be in a state where they could be sucked in by the capillary.

[0032] Therefore, in the culture unit 10 according to this embodiment, a mesh member 2 is placed on a substrate 11, and a dispersion liquid DI, in which droplets DR are dispersed in a specific solution S such as fluorinated oil, is introduced onto the substrate 11. This causes the droplets DR to float up into the through-holes 23 of the mesh member 2 and can be individually trapped. This allows the droplets DR in the specific solution S to be dispersed and fixed. Furthermore, a capillary 8 is brought into contact with the droplets DR from above the through-holes 23 and can be sucked in.

[0033] [Flowchart of Cell Culture Method] FIG. 4 is a flowchart showing an example of a cell culture method according to an embodiment.

[0034] In step (hereinafter referred to as "ST") 1, the user generates a dispersion liquid DI in which droplets DR, each containing a culture solution containing cells, are dispersed in a specific solution S. The specific solution S has a higher specific gravity than the culture solution.

[0035] In ST2, the user applies the dispersion liquid DI onto the main surface 111 of the substrate 11 having the main surface 111, and then holds the dispersion liquid DI thereon.

[0036] In ST3, the user places the mesh member 2 at a distance from the main surface 111 of the substrate 11. The mesh member 2 is formed with a plurality of through-holes 23 that trap droplets DR introduced to the substrate 11 and floating up, with the mesh member 2 positioned vertically above the substrate 11. In one embodiment, the user places the mesh member 2 above a protrusion 12 that is disposed on the substrate 11 and protrudes upward from the substrate ( FIG. 5 ). After placing the mesh member 2, the user may add the specific solution S or dispersion liquid DI to the culture unit 10 or apply appropriate vibration to the culture unit 10 to promote dispersion and / or floating of the droplets DR in the specific solution S and facilitate individual trapping in the through-holes 23. Preferably, the liquid level of the dispersion liquid DI introduced into the culture unit 10 is adjusted to be located between the lower surface of the mesh member 2 and a position that is higher than the upper surface of the mesh member 2 by the radius of the droplets DR. In other words, it is preferable that an amount of the dispersion liquid DI between the lower limit value and the upper limit value described below be introduced into the culture unit 10. The lower limit value is the minimum amount at which the dispersion liquid DI comes into contact with the mesh member 2, and the upper limit value is the amount at which the liquid surface of the dispersion liquid DI is positioned higher than the upper surface of the mesh member 2 by the radius of the droplet DR.

[0037] In ST4, the user cultures the cells in the droplet DR under predetermined conditions. In one embodiment, the user places the culture unit 10 in an incubator set to a predetermined temperature for a predetermined period of time.

[0038] In ST5, the user observes the droplets DR in the culture unit 10. In one embodiment, the user observes the droplets DR trapped in the through-holes 23 in ST3 using the microscope 7. In one embodiment, the user selects the droplets DR to be collected in ST6 based on the observation results.

[0039] In ST6, the user individually collects droplets DR. In one embodiment, the user contacts the capillary 8 with any droplet DR selected through the observation in ST5 and aspirates it. The aspirated droplet DR is then transferred to a predetermined container for further culturing and / or analysis.

[0040] In the process of Figure 4, the order of ST2 and ST3 may be reversed. Specifically, after the mesh member 2 is placed above the substrate 11 at a distance, the dispersion liquid DI may be introduced and maintained between the mesh member 2 and the substrate 11. Furthermore, when using a culture unit 10 having a structure in which the micropillar device 1 and the mesh member 2 are integrated as described above, the mesh member 2 is placed above the substrate 11 at the time of ST2, and therefore the process of ST3 is omitted. Furthermore, after collecting some of the droplets DR from the culture unit 10, the droplets DR remaining in the culture unit 10 may be subsequently cultured and / or observed. At least a portion of the process of Figure 4 may be automated by an experimental device, and the device may be controlled by a control device 9.

[0041] According to the process shown in FIG. 4 , droplets DR can be fixed in position while dispersed in a specific solution S. Furthermore, while observing the fixed droplets DR under a microscope, any droplets DR can be sucked into the capillary 8. Specifically, differences in the state of the droplets DR due to the type of encapsulated cells can be observed, and droplets DR containing desired types of cells can be collected. For example, a user can observe the turbidity, color, etc. of the droplets DR to isolate cells with a high proliferation rate, cells that produce a large amount of a predetermined metabolite, etc. Thus, the cell culture method according to this embodiment can be considered an efficient screening method for easily selecting and culturing desired types of cells from a large number of types of cells cultured in a small space.

[0042] 6 to 9, a culture system 200 having a configuration for suppressing reduction of the specific solution S due to drying during cell culture and observation will be described. The culture system 200 is used as a culture unit together with a microscope 7, a capillary 8, a control device 9, and a generation unit 5.

[0043] Fig. 6 is a perspective view showing a culture system 200 according to another embodiment. Fig. 7 is a view of the culture system 200 of Fig. 6 excluding the lid 6, the first connecting member 41, and the second connecting member 42, showing the structure of the culture unit 10A and the linear guide 34 from an obliquely upper view. Fig. 8 is a cross-sectional view showing the structure of the culture unit 10A. Fig. 9 is a cross-sectional view showing the structure of the culture unit 10A and the surrounding structure.

[0044] The culture system 200 includes a culture unit 10A and a main body 210 that houses the culture unit 10A. The main body 210 includes a lid 6 and a base unit 3.

[0045] The base unit 3 includes a base 32, a linear guide 34, a handle 36, a carriage 38, a first connecting member 41, a second connecting member 42, a third connecting member 43, a spacer 45, an inlet pipe 46, and a pump 47.

[0046] 6 and subsequent figures, the extension direction of the linear guide 34 is the X-axis, the direction perpendicular to the base 32 is the Z-axis, and the plane parallel to the base 32 is the XY plane. In one embodiment, the culture system 200 is used in a state where it is installed so that the Z-axis is approximately parallel to the vertical direction. For example, the culture system 200 is placed in an incubator or the like for culture, or placed on the observation stage of a microscope 7 for observation, with the Z-axis approximately parallel to the vertical direction.

[0047] The culture unit 10A includes a mesh device 20A and a substrate device 1A. The substrate device 1A includes a substrate 11A and a substrate holding unit 12A.

[0048] The substrate 11A has a main surface 111A and holds the dispersion liquid DI on the main surface 111A. In one embodiment, the substrate 11A is a plate-like member made of transparent glass.

[0049] The substrate holder 12A holds the substrate 11A. In one embodiment, the substrate holder 12A is a plate-like member having a through-hole 121A formed therein, and the substrate 11A is fitted into the through-hole 121A.

[0050] In one embodiment, the thickness (length in the Z-axis direction) of the substrate holding part 12A is greater than the thickness (length in the Z-axis direction) of the substrate 11A. With this configuration, when the lower surface 122A of the substrate holding part 12A and the lower surface 112A of the substrate 11A are fitted together with their Z-axis positions aligned (so that they are flush), a recess 15A is formed in the substrate device 1A, with the main surface 111A of the substrate 11A as its bottom surface and surrounded by the substrate holding part 12A. The dispersion liquid DI is introduced into this recess 15A.

[0051] The mesh device 20A includes a mesh member 2A and a mesh holding portion 21A. The mesh device 20A is placed on the substrate device 1A so that the mesh holding portion 21A is located above the substrate 11A. This positions the mesh member 2A at a distance from the main surface 111A of the substrate 11A. The substrate holding portion 12A of the substrate device 1A also corresponds to an example of a "protrusion."

[0052] The mesh member 2A has a plurality of through holes formed therein for trapping droplets DR that are introduced onto the substrate 11A and float up, with the mesh member 2A positioned vertically above the substrate 11A.

[0053] In one embodiment, the mesh device 20A is a plate-like member in which a mesh member 2A is formed at a position corresponding to the substrate 11A when the mesh device 20A is placed above the substrate device 1A.

[0054] With this configuration, each droplet DR floating in the specific solution S floats up into the through-holes of the mesh member 2A. In other words, the droplets DR can be individually trapped in the through-holes. This allows cells to be cultured and observed in an isolated state for each droplet DR.

[0055] 9, a spacer 45 is provided on the upper surface of the mesh holding portion 21A so as to surround the upper surface of the mesh member 2A. In one embodiment, the spacer 45 is a plate-like member having a through-hole that is slightly larger than the mesh member 2A, and is disposed on the mesh device 20A so that the through-hole is positioned above the mesh member 2A. By providing the spacer 45, even if the dispersion liquid DI flows out onto the upper surface of the mesh holding portion 21A when the mesh device 20A is placed on the substrate device 1A, the spacer 45 can be blocked by the spacer 45. The space below the upper end 451 of the spacer 45 into which the dispersion liquid DI is introduced is also referred to as a "pool."

[0056] The inlet pipe 46 is made of, for example, silicon and is a flow path for introducing the specific solution S, fluidly connected to the main body 210. As an example, the tip 461 of the inlet pipe 46 is positioned in contact with the vicinity of the upper surface 201A inside the spacer 45 of the mesh device 20A. In this case, the tip 461 is provided with multiple notches in the circumferential direction. Alternatively, the tip 461 is cut to have a tapered shape. By configuring the tip 461 in this manner, even when the specific solution S is introduced at a low flow rate, droplets of the specific solution S can be prevented from remaining on the tip 461, allowing the specific solution S to be quickly dispersed within the pool. As another example, the tip 461 is positioned at a predetermined distance from the upper surface 201A so that droplets formed on the tip 461 do not separate from the upper surface 201A. Note that in this case, multiple notches in the circumferential direction or a tapered cut on the tip 461 are not necessarily required. Even with this configuration, even if the specific solution S is introduced at a low flow rate, droplets of the specific solution S can be prevented from remaining at the tip 461, and the specific solution S can be quickly diffused within the pool.

[0057] The pump 47 is connected to the end of the inlet pipe 46 opposite to the tip 461, and delivers the specific solution S to the inlet pipe 46. The pump 47 is, for example, a syringe pump.

[0058] By providing a mechanism for supplying a constant amount of specific solution S to the main body 210, such as the inlet pipe 46 and pump 47, it is possible to supply new dispersion liquid DI to make up for any shortage that occurs due to drying, thereby enabling long-term cell cultivation and observation.

[0059] In one embodiment, the position of the tip 461 of the inlet tube 46 is fixed with respect to the upper surface 201A of the mesh device 20A. As a result, even when the culture section 10A including the mesh device 20A is translated as described below, the position of the tip 461 with respect to the position of the upper surface 201A is maintained.

[0060] The second connecting member 42 is joined to the upper part of the spacer 45. In one embodiment, the second connecting member 42 is a plate-like member having a through hole that is slightly larger than the mesh member 2A, and is disposed on the spacer 45 so that the through hole is positioned above the mesh member 2A. The upper surface of the second connecting member 42 is disposed so as to contact the lower surface of the lid 6.

[0061] The spacer 45 and the second connecting member 42 seal the gap between the culture section 10A and the lid 6, thereby preventing the specific solution from evaporating from the gap.

[0062] An opening 62 is formed in the lid 6. In one embodiment, the lid 6 is a plate-like member having the opening 62 and a size that allows the lid 6 to always cover the top of the culture unit 10A even when the culture unit 10A is moved horizontally.

[0063] The opening 62 is a slit having a width smaller than that of the mesh member 2A. In this specification, "width" refers to the length in the X direction. The width of the opening 62 is configured to be a width that allows a capillary 8 to be inserted through the opening 62 to collect samples or for observation using a microscope 7. In this way, by providing the lid 6 in which the opening 62 having a width smaller than that of the mesh member 2A is formed, it is possible to both prevent the dispersion liquid DI from drying and enable suitable observation and collection of cells.

[0064] Preferably, the opening 62 is configured to have the smallest width (for example, a few mm) that allows the capillary 8 to be inserted through it to collect samples or for observation using a microscope 7, thereby minimizing drying of the dispersion liquid DI while still allowing suitable observation and collection of cells.

[0065] The relative positional relationship between the mesh member 2A and the opening 62 of the lid 6 is changed in the horizontal direction perpendicular to the vertical direction by the linear guide 34, the handle 36, and the carriage 38. The linear guide 34, the handle 36, and the carriage 38 correspond to an example of a "translation mechanism." In this specification, "translation" refers to parallel movement in the X-axis direction along the linear guide.

[0066] The linear guide 34 is fixed on the base 32. The carriage 38 moves translationally on the linear guide 34. The handle 36 controls the position of the carriage 38 on the linear guide 34.

[0067] The carriage 38 and the culture unit 10A are connected by connecting members 41 to 43. In one embodiment, the connecting members 41 to 43 are configured as follows. The first connecting member 41 is a plate-like member having a through-hole formed therein into which the third connecting member 43 is embedded. The first connecting member 41 is fixed to the upper surface of the carriage 38. After the dispersion liquid DI is introduced into the culture unit 10A, the second connecting member 42 is fixed to a portion of the upper surface of the first connecting member 41. The third connecting member 43 is a plate-like member fitted into the first connecting member 41. The substrate device 1A of the culture unit 10A is fixed to the upper surface of the third connecting member 43. With this configuration, when the carriage 38 on the linear guide 34 is translated, the culture unit 10A also moves translationally.

[0068] As described above, the position of the mesh member 2A relative to the opening 62 of the lid 6 can be changed in the horizontal direction using the linear guide 34, the handle 36, and the carriage 38. Therefore, it becomes possible to observe and collect cells at a desired position on the mesh member 2A.

[0069] Preferably, in the culture system 200, the substrate 11A is formed of a material with a light transmittance equal to or greater than a predetermined value, and components vertically below the substrate 11A are also configured to transmit light. For example, the substrate 11A is formed of a transparent glass material, and a through-hole 430 is formed in the third connection member 43 at a position corresponding to the substrate 11A (a position having the same XY coordinates), and a through-hole 320 is formed in the base 32 at a position corresponding to the substrate 11A. A through-hole is also formed in the first connection member 41 at a position corresponding to the substrate 11A, or a recess for transmitting light is formed at a position corresponding to the substrate 11A (not shown). Furthermore, the mesh member 2A of the mesh device 20A is formed with multiple through-holes and configured to transmit light. This configuration allows light incident from the bottom surface of the main body 210 to be irradiated onto the culture unit 10A. This facilitates microscopic observation of the culture unit 10A from above the opening 62.

[0070] In the culture system 200, the user places the dispersion liquid DI on the substrate device 1A, then places the mesh member 2A thereon, closes the lid 6, and performs culture and observation.

[0071] As a more specific example, the user places the dispersion liquid DI on the substrate device 1A connected to the carriage 38, and then fixes the mesh member 2A, spacer 45, and second connecting member 42 to the substrate device 1A. Furthermore, the user fixes the lid 6 to the base 32. With this configuration, it becomes possible to culture cells, observe cells at each position on the mesh member 2A, and collect cells while minimizing drying of the dispersion liquid DI.

[0072] The configuration of each part of the culture system 200 may be changed as appropriate within the scope of achieving the above-mentioned effects.

[0073] For example, the outer shape of at least one of the lid 6 and the connecting members 41 to 43 may be rectangular. At least two of the substrate device 1A, the third connecting member 43, the first connecting member 41, and the carriage 38 may be integrally formed. At least two of the mesh member 2A, the spacer 45, and the second connecting member 42 may be integrally formed.

[0074] The carriage 38 may be moved relative to the linear guide 34 by manually turning the handle 36 or by driving an actuator. Alternatively, the carriage 38 may be moved in translation by pushing the carriage 38 on the linear guide 34 in the X-axis direction without providing the handle 36.

[0075] An additional drain pipe (not shown) may also be provided to drain oil from the pool.

[0076] Aspects It will be understood by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0077] (Item 1) A cell culture device according to one aspect includes a generation unit, a substrate, and a mesh member. The generation unit generates a dispersion in which droplets containing cells and a culture medium are dispersed in a specific solution having a specific gravity greater than that of the culture medium. The substrate has a main surface and holds the dispersion on the main surface. The mesh member is disposed at a distance from the main surface of the substrate. The mesh member has a plurality of through-holes formed therein, which trap droplets introduced to the substrate and float up, with the mesh member positioned vertically above the substrate.

[0078] According to the cell culture device described in paragraph 1, droplets suspended in the specific solution having a specific gravity greater than that of the culture solution rise up into the through-holes, thereby allowing the droplets to be individually trapped in the through-holes of the mesh member.

[0079] (Item 2) The cell culture device according to item 1 further includes a protrusion disposed on the substrate and protruding upward from the substrate. The mesh member is disposed above the protrusion.

[0080] According to the cell culture device described in paragraph 2, it is easy to dispose the mesh member above the substrate at a distance. More specifically, it is easy to stably maintain the distance between the mesh member and the substrate.

[0081] (Item 3) In the cell culture device according to item 1 or 2, the specific solution contains fluorine oil.

[0082] According to the cell culture device described in item 3, it is easy to culture and observe cells for a long period of time in an isolated droplet state.

[0083] (Item 4) In the cell culture device according to item 2, the substrate and the protrusion are integrally formed.

[0084] According to the cell culture device described in item 4, it becomes easy to stably hold the mesh member at a distance above the substrate, and handling of the entire cell culture device becomes easy.

[0085] (Item 5) In the cell culture device according to any one of items 1 to 4, the cells include microorganisms or animal cells.

[0086] According to the cell culture device described in item 5, culturing microorganisms and clarifying their properties may be applied to the development of medicine, industry, etc. Alternatively, culturing animal cells and clarifying their properties may be applied to the development of medicine, industry, etc. (Item 6) The cell culture device according to any one of items 1 to 5 further comprises a main body that holds the substrate and the mesh member and contains a dispersion liquid, and an introduction flow path for a specific solution that is fluidly connected to the main body.

[0087] According to the cell culture device described in paragraph 6, by providing a mechanism for supplying a constant amount of a specific solution to the main body, it is possible to culture and observe cells for a long period of time by supplying new solution to make up for any shortage of the dispersion liquid that has dried out. (paragraph 7) The cell culture device according to any one of paragraphs 1 to 6 further includes a lid disposed vertically above the mesh member, and the lid has an opening with a width smaller than that of the mesh member.

[0088] According to the cell culture device described in paragraph 7, by providing a lid having an opening formed therein whose width is smaller than the width of the mesh member, it is possible to both prevent the dispersion liquid from drying out and enable suitable observation and collection of the cells. (Item 8) The cell culture device according to paragraph 7 further includes a translation mechanism that horizontally changes the position of the mesh member relative to the opening of the lid.

[0089] According to the cell culture device described in item 8, by changing the relative positional relationship between the mesh member and the opening of the lid, it becomes possible to observe and collect cells at a desired position.

[0090] (Item 9) A culture unit comprising a cell culture device according to any one of items 1 to 8, a microscope for observing droplets in the cell culture device, and a collection device for individually collecting droplets.

[0091] According to the culture unit described in paragraph 9, a user can observe the droplets trapped in the through-holes of the mesh member from above in a magnified manner. The user can then use the capillary to individually collect desired droplets. Furthermore, the user can transfer the cells contained in the droplets collected with the capillary to another container for further culture, observation, and / or analysis.

[0092] (Item 10) A cell culture method according to another aspect includes the steps of: generating a dispersion in which droplets containing cells and a culture medium are dispersed in a specific solution having a specific gravity greater than that of the culture medium; retaining the dispersion on a main surface of a substrate having a main surface; and disposing a mesh member spaced apart from the main surface of the substrate, the mesh member having a plurality of through-holes formed therein, the mesh member being positioned vertically above the substrate, for trapping droplets that are introduced to the substrate and float up.

[0093] According to the cell culture method described in paragraph 10, droplets suspended in the specific solution having a specific gravity greater than that of the culture solution rise up into the through-holes, thereby allowing the droplets to be individually trapped in the through-holes of the mesh member.

[0094] (Item 11) In the cell culture method according to item 10, the placing step includes a step of placing a mesh member above a protrusion that is placed on the substrate and protrudes upward from the substrate.

[0095] According to the cell culture method described in item 11, it is easy to dispose the mesh member above the substrate at a distance, and more specifically, it is easy to stably maintain the distance between the mesh member and the substrate.

[0096] (Item 12) The cell culture method according to item 10 or 11, further comprising the steps of observing the droplets trapped in the mesh member and individually collecting the droplets.

[0097] According to the cell culture method described in paragraph 12, a user can observe droplets trapped in the through-holes of the mesh member from above using a microscope or the like. The user can then individually collect desired droplets using a capillary or the like. Furthermore, the user can transfer the cells contained in the droplets collected using the capillary or the like to another container for further culture, observation, and / or analysis.

[0098] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present disclosure is defined by the claims, not by the description of the above embodiments, and is intended to include all modifications within the meaning and scope of the claims.

[0099] DESCRIPTION OF SYMBOLS 1 Micropillar device, 1A Substrate device, 2, 2A Mesh member, 3 Base unit, 5 Generation unit, 6 Lid, 7 Microscope, 8 Capillary, 9 Control device, 10, 10A Culture unit, 11, 11A Substrate, 12 Protrusion, 12A Substrate holder, 15 Culture device, 15A Recess, 20A Mesh device, 21A Mesh holder, 23, 121A, 320, 430 Through hole, 32 Base, 34 Linear guide, 36 Handle, 38 Carriage, 41 First connecting member, 42 Second connecting member, 43 Third connecting member, 45 Spacer, 46 Inlet tube, 47 Pump, 50 Microchannel device, 51A, 51B Microchannel, 52 Intersection position, 62 Opening, 100, 100A Culture unit, 111, 111A Main surface, 112 Wall, 112A, 122A lower surface, 200 culture system, 201A upper surface, 210 main body, 451 upper end, 461 tip portion, 511A, 512A end portion, DI dispersion liquid, DR droplet, S specific solution.

Claims

1. A cell culture device comprising: a generation unit that generates a dispersion liquid in which droplets containing cells and culture liquid are dispersed in a specific solution having a specific gravity greater than that of the culture liquid; a substrate having a main surface and holding the dispersion liquid on the main surface; and a mesh member arranged at a distance from the main surface of the substrate, the mesh member having a plurality of through holes formed therein for trapping the droplets that are introduced to the substrate and float up, with the mesh member positioned vertically above the substrate.

2. The cell culture device according to claim 1, further comprising a protrusion disposed on said substrate and protruding upward from said substrate, said mesh member being disposed above said protrusion.

3. The cell culture device according to claim 1, wherein the specific solution includes fluorine oil.

4. The cell culture device of claim 2, wherein the substrate and the protrusion are integrally formed.

5. The cell culture device of claim 1, wherein the cells include microbial or animal cells.

6. The cell culture device according to claim 1, further comprising: a main body that holds the substrate and the mesh member and contains the dispersion liquid; and an introduction flow path for the specific solution that is fluidly connected to the main body.

7. The cell culture device according to claim 1, further comprising a lid arranged vertically above the mesh member, the lid having an opening having a width smaller than that of the mesh member.

8. The cell culture device of claim 7, further comprising a translation mechanism for horizontally changing the position of the mesh member relative to the opening in the lid.

9. A culture unit comprising: a cell culture device according to claim 1; a microscope for observing the droplets in the cell culture device; and a collection device for individually collecting the droplets.

10. A cell culture method comprising the steps of: generating a dispersion liquid in which droplets containing cells and culture liquid are dispersed in a specific solution having a specific gravity greater than that of the culture liquid; retaining the dispersion liquid on a main surface of a substrate having a main surface; and arranging a mesh member at a distance from the main surface of the substrate, wherein the mesh member has a plurality of through holes formed therein for trapping the droplets that are introduced to the substrate and float up, with the mesh member positioned vertically above the substrate.

11. The cell culture method according to claim 10, wherein the placing step includes a step of placing the mesh member above a protrusion that is placed on the substrate and protrudes upward from the substrate.

12. The cell culture method according to claim 10, further comprising the steps of: observing the droplets trapped in the mesh member; and individually collecting the droplets.