Culture instrument, culture kit, and tissue culture method

Abstract

To provide a culture instrument that enables tissue culture to be continued for a long period of time; to provide a culture kit that includes such a culture instrument and makes it easy to continue tissue culture for a long period of time; and to provide a tissue culture method that makes it easy to continue tissue culture for a long period of time.SOLUTION: A culture device comprises a cylindrical cell culture insert having a bottom, and a supply member that is inserted into the cell culture insert and supplies culture medium to tissue being cultured on the top surface of the bottom. The supply member has a main body having a flow path through which the culture medium flows, a holding part that holds the height position of the supply member within the cell culture insert, and a microneedle part that is provided at the other end side of the main body to cover the opening of the flow path. The microneedle part has a microneedle that communicates with the flow path. The microneedle is separated from the bottom when the supply member is inserted into the cell culture insert, and punctured into the tissue.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The present invention relates to a culture device, a culture kit, and a method for culturing tissues.

Background Art

[0002] Conventionally, in the field of life sciences, various studies have been conducted using tissues cultured. These tissues are usually cultured in a state of being immersed in a culture medium stored in a culture container such as a well plate. At that time, a method of culturing the tissue to be cultured on a membrane of a cell culture insert is widely adopted (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the conventional culture method as described above, nutrients are supplied from the culture medium disposed around the tissue to be cultured to the tissue. Therefore, in the grown tissue, compared with the tissue before growth, it becomes difficult for the culture medium (nutrients contained therein) to reach the inside of the tissue from the culture medium. As a result, in the grown tissue, necrosis and inflammation are likely to occur from the inside of the tissue, and it has been difficult to continue the experiment for a long time.

[0005] The present invention has been made in view of such circumstances, and an object thereof is to provide a culture device that enables long-term continuation of tissue culture. Another object is to provide a culture kit equipped with such a culture device and that facilitates long-term continuation of tissue culture. Furthermore, an object is to provide a method for culturing tissues that facilitates long-term continuation of tissue culture.

Means for Solving the Problems

[0006] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0007] [1] A cylindrical cell culture insert having a bottom, and a supply member inserted into the cell culture insert for supplying a culture medium to a tissue cultured on the upper surface of the bottom, the supply member having a main body portion having a flow path through which the culture medium flows, a holding portion for holding the height position of the supply member in the cell culture insert, and a micro needle portion provided to cover an opening of the flow path on the other end side of the main body portion, the micro needle portion having a micro needle communicating with the flow path, the micro needle being spaced apart from the bottom in a posture in which the supply member is inserted into the cell culture insert and being punctured into the tissue.

[0008] [2] The culture device according to [1], further comprising a separation portion provided on an outer surface of the main body portion for separating the main body portion from an inner surface of the cell culture insert.

[0009] [3] The culture device according to [1] or [2], further comprising a supply pipe connected to one end side of the main body portion and communicating with the flow path.

[0010] [4] The culture device according to [3], further comprising a connection member for connecting the main body portion and the supply pipe.

[0011] [5] The connection member has a first connection portion provided on the main body portion and a second connection portion provided at an end of the supply pipe, and the first connection portion and the second connection portion are combined to connect the main body portion and the supply pipe. The culture device according to [4].

[0012] [6] The culture device according to any one of [3] to [5], further comprising a support member for supporting the supply pipe.

[0013] [7] The micro-needle part is provided so as to be replaceable with respect to the main body part, and the culture device according to any one of [1] to [6].

[0014] [8] It has a second micro-needle part that is replaceable with the micro-needle part, and the micro-needles of the second micro-needle part have a configuration different from that of the micro-needles of the micro-needle part, and the culture device according to [7].

[0015] [9] A culture kit having the culture device according to any one of [1] to [8], and a well plate having a plurality of wells for storing a culture medium in the wells.

[0016]

[10] A method for culturing a tissue, which comprises immersing the tissue in a culture medium and supplying the culture medium into the tissue from a supply member disposed inside the tissue.

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a culture device that enables long-term continuous tissue culture. Further, it is possible to provide a culture kit that includes such a culture device and is easy to continuously perform tissue culture over a long period of time. Furthermore, it is possible to provide a method for culturing a tissue that is easy to continuously perform tissue culture over a long period of time.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

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Figure 4

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Figure 8

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Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, with reference to FIGS. 1 to 11, a culture device, a culture kit, and a tissue culture method according to the present embodiment will be described. In all the following drawings, for ease of viewing, the dimensions and ratios of each component are appropriately different.

[0020] In the following description, an xyz orthogonal coordinate system is set, and the positional relationship of each member will be described with reference to this xyz orthogonal coordinate system. Here, a predetermined direction in the horizontal plane is defined as the x-axis direction, a direction orthogonal to the x-axis direction in the horizontal plane is defined as the y-axis direction, and a direction orthogonal to each of the x-axis direction and the y-axis direction (i.e., the vertical direction) is defined as the z-axis direction. "Plan view" means a view for observing an object in a field of view from the +z direction.

[0021] Also, the +z direction may be referred to as "up" and the -z direction may be referred to as "down". Also, when explaining each component, the upper end of a member may be referred to as "one end" and the lower end of a member may be referred to as "the other end".

[0022] 《Culture Kit, Culture Apparatus》 FIG. 1 is a schematic perspective view of a culture kit 1000. The culture kit 1000 includes a culture apparatus 100 of the present embodiment and a well plate 50.

[0023] [Well Plate] The well plate 50 includes a top plate 51, a frame 52 that supports the top plate 51, and a plurality (12 in the figure) of wells 53 provided on the top plate 51. When the culture kit 1000 is used, the culture apparatus 100 is inserted into the well 53.

[0024] The top plate 51 is rectangular in plan view and is connected to and supported by the inner peripheral surface of a cylindrical frame 52 that also presents a rectangular shape in plan view.

[0025] The well 53 stores a culture medium used for cell culture. The wells 53 are arranged in a 3-row and 4-column matrix on the top plate 51. The bottom of the well 53 can adopt various known shapes. The bottom of the well 53 may be flat, round, or V-shaped.

[0026] The well plate 50 is formed of a material known as a well plate material. For example, the material of the well plate 50 can adopt polystyrene.

[0027] FIG. 2 is an exploded perspective view of the culture apparatus 100. FIG. 3 is an exploded cross-sectional view of the culture apparatus 100. As shown in FIGS. 2 and 3, the culture apparatus 100 includes a cell culture insert 10 and a supply member 20A. In the following description, the cell culture insert may sometimes be abbreviated as "insert".

[0028] [Cell Culture Insert] The insert 10 is a cylindrical member having one end open and the other end having a bottom 120. Specifically, the insert 10 has a cylindrical side wall 110 with one end 110x open, and a bottom 120 provided at the other end 110y of the side wall 110.

[0029] The side wall 110 has a cylindrical first side wall portion 111 provided at one end side, and a cylindrical second side wall portion 112 connected to the first side wall portion 111 at the other end side of the first side wall portion 111. The first side wall portion 111 has a larger opening diameter than the second side wall portion 112.

[0030] The side wall 110 is formed of a material known as a material for cell culture inserts. For example, the material of the side wall 110 can be polystyrene.

[0031] A resin membrane is used for the bottom 120. Cells are seeded and cultured on the upper surface 120a of the bottom 120 to form tissue X. The medium in the well 53 is supplied to the tissue X being cultured through the bottom 120.

[0032] In the operations using the culture kit 1000 and the culture instrument 100, various tissues can be cultured as tissue X. Examples of tissue X include human tissues such as cultured skin, cultured cartilage, and cultured myocardial sheets, tissues of animals other than humans such as mice, and artificial tissues such as artificial organs mimicking animal tissues.

[0033] [Supply member] The supply member 20A has a supply part 21 and a micro needle part 25, and has a flow path 20x through which the medium flows. A supply pipe 30 for supplying the medium, which communicates with the flow path 20x, is connected to the supply member 20A. The supply member 20A is inserted into the insert 10 and has a function of supplying the medium to the tissue X cultured on the upper surface 120a of the bottom 120.

[0034] (Supply part) The supply unit 21 includes a main body part 211, a holding part 212, a coupling part 213, a spacing part 214, a first connection part 215, and a second connection part 216. The main body part 211, the holding part 212, the coupling part 213, the spacing part 214, and the first connection part 215 are integrally formed, and the second connection part 216 is configured as a separate member.

[0035] The main body part 211 is a cylindrical member with both ends open, and has a flow path 211x through which the culture medium flows. One end side of the flow path 211x communicates with a hole 211y having a diameter larger than that of the flow path 211x. The other end side of the flow path 211x communicates with a tapered hole 211z whose diameter gradually increases downward. The flow path 211x and the tapered hole 211z constitute a part of the flow path 20x of the supply member 20A.

[0036] Although the main body part 211 of the present embodiment is a cylindrical member, various shapes can be adopted without limitation as long as the effects of the invention are not impaired.

[0037] The holding part 212 is a disk-shaped (flange-shaped) part in a plan view that extends radially outward from the outer surface 211a of the main body part 211 at one end side of the main body part 211. In the posture where the supply member 20A is inserted into the insert 10, the lower surface 212a (the surface on the insert 10 side) of the holding part 212 contacts the end surface 10a at one end side of the insert 10 and holds the height position of the supply member 20A within the insert 10.

[0038] A notch 212x is formed at the peripheral edge of the holding part 212. In FIG. 2, one notch 212x is shown, but it is not limited thereto, and two or more notches may be provided. The notch 212x functions as an air hole connecting the inside and the outside of the insert 10 in the posture where the supply member 20A is inserted into the insert 10.

[0039] The joint portion 213 is a disk-shaped (flange-shaped) portion in a plan view that extends radially outward from the outer surface 211a of the main body portion 211 on the other end side of the main body portion 211. The joint portion 213 has a flange-shaped base portion 213a and a convex portion 213b provided in an annular shape in a plan view at the peripheral edge of the lower surface of the base portion 213a. Further, an annular groove portion 213c is provided on the lower surface of the base portion 213a inside the convex portion 213b. The micro needle portion 25 is joined to the joint portion 213.

[0040] The separation portion 214 is provided on the outer surface 211a of the main body portion 211. The separation portion 214 has a flat plate-like structure extending in the same direction as the axial direction of the main body portion 211, and is arranged at equal intervals from each other at a plurality of locations in the circumferential direction of the main body portion 211 on the outer surface 211a of the main body portion 211. The plurality of separation portions 214 have the same shape and the same size. In the present embodiment, the separation portions 214 are arranged at four locations at equal intervals in the circumferential direction of the main body portion 211.

[0041] The separation portion 214 separates the main body portion 211 from the inner surface 110a of the insert 10 by interfering with the inner surface 110a of the insert 10 in the posture in which the supply member 20A is inserted into the insert 10. Therefore, the supply member 20A can easily arrange the main body portion 211 at the center of the insert 10 in the posture of being inserted into the insert 10. Since the tissue X cultured in the insert 10 is usually arranged at the center of the bottom portion 120 of the insert 10, the supply member 20A can easily face the tissue X because the supply portion 21 has the separation portion 214.

[0042] The first connection portion 215 and the second connection portion 216 correspond to the "connection member" of the present invention that connects the supply portion 21 (main body portion 211) and the supply pipe 30.

[0043] The first connection portion 215 is provided in the supply portion 21 (main body portion 211) and connects the supply portion 21 and the supply pipe 30. The first connection portion 215 is provided at the center in a plan view on the upper surface 212b of the holding portion 212. The first connection portion 215 is provided in a cylindrical shape and has a through hole communicating with the hole 211y of the main body portion 211.

[0044] The second connection part 216 is provided at the end of the supply pipe 30. The second connection part 216 has a cylindrical first member 216a with one end closed and the other end open, a cylindrical second member 216b inserted inside the first member 216a, and a cylindrical third member 216c provided at the center of the plane on the upper surface of the first member 216a. The outer peripheral surface of the upper end of the third member 216c is formed in a frustum of a cone shape, and it has a reverse 216d below the end of the frustum of a cone shape.

[0045] The second connection part 216 has a through hole 216x that communicates from the upper end of the third member 216c to the lower end of the second member 216b. The through hole 216x constitutes a part of the flow path 20x of the supply member 20A.

[0046] The second connection part 216 can be connected to the first connection part 215 in a liquid-tight manner by inserting the second member 216b with a sealing member (O-ring) OR inserted into the hole 211y of the first connection part 215. The first connection part 215 and the second connection part 216 adopt a known luer lock connection method. The connection method between the first connection part 215 and the second connection part 216 is not limited to this, and other known connection methods such as a screwing method or a luer slip method may also be used.

[0047] (Micro needle part) FIG. 4 is a cross-sectional view of the micro needle part 25. FIG. 5 is a bottom view of the micro needle part 25.

[0048] As shown in FIGS. 4 and 5, the micro needle part 25 is a member circular in plan view. The micro needle part 25 has a disk-shaped base part 251 in plan view, a convex part 252 provided in an annular shape in plan view at the peripheral part of the upper surface of the base part 251, and a plurality of micro needles provided on the lower surface of the base part 251. The micro needles are needles with a diameter and length of less than 1 mm (in the order of μm).

[0049] At the center of the base part 251 in plan view, a convex part 251a that bulges upward is provided.

[0050] The micro needle portion 25 inserts the convex portion 252 into the groove portion 213c of the connecting portion 213 and is liquid-tightly connected to the supply portion 21.

[0051] In the posture where the micro needle portion 25 is connected to the supply portion 21, a space 25x is formed between the micro needle portion 25 and the supply portion 21. Further, the convex portion 251a is disposed at a position facing the flow path 211x (the enlarged diameter hole 211z). The space 25x constitutes a part of the flow path 20x of the supply member 20A.

[0052] The micro needle portion 25 has, as a micro needle, a first micro needle 255 and a second micro needle 256. Four first micro needles 255 and four second micro needles 256 are provided respectively, and are alternately and equiangularly arranged in the circumferential direction in a plan view on the lower surface of the base portion 251. In FIGS. 4 and 5, the micro needle portion 25 is assumed to have four first micro needles 255 and four second micro needles 256 respectively, but it is not limited thereto.

[0053] The tips of the first micro needle 255 and the second micro needle 256 are inclined surfaces facing the inside of the micro needle portion 25 in a plan view.

[0054] The first micro needle 255 has a through hole 255x reaching the upper surface of the base portion 251. The base portion 251 side of the through hole 255x gradually expands toward the base portion 251 side.

[0055] On the other hand, the second micro needle 256 does not have a through hole.

[0056] The height of the first micro needle 255 (the distance in the Z direction from the bottom surface of the base portion 251 to the needle tip) and the height of the second micro needle 256 may be different, but are preferably the same. When the height of the first micro needle and the height of the second micro needle are “the same”, a forming error is allowed.

[0057] The heights of the first micro-needle 255 and the second micro-needle 256 are, for example, 10 μm or more and 100 μm or less.

[0058] In such a micro-needle part 25, the culture medium L flowing through the flow path 211x collides with the convex part 251a of the micro-needle part 25 and diffuses into the space 25x, and is supplied from the tip of the first micro-needle 255 through the through hole 255x.

[0059] The micro-needle part 25 may be provided so as to be replaceable with respect to the main body part 211, or may be fixed to the coupling part 213 so as not to be removable.

[0060] When the micro-needle part 25 is replaceable, the culture device 100 may be a kit having a second micro-needle part that is replaceable with or a spare for the micro-needle part 25. In that case, the culture device 100 may have a plurality of second micro-needle parts and may be replaceable for each experiment.

[0061] The micro-needles included in the second micro-needle part may have the same configuration as the micro-needles included in the micro-needle part 25, or may have a different configuration. The "configuration" of the micro-needles includes both the configuration of individual micro-needles and the configuration of the entire micro-needle.

[0062] Examples of the configuration of individual micro-needles include the length, diameter, inclination angle of the inclined surface at the tip, and diameter of the through hole 255x of the first micro-needle 255.

[0063] Examples of the configuration of the entire micro-needle include the arrangement of a plurality of micro-needles, the total number of micro-needles, the total number of the first micro-needles 255, and the total number of the second micro-needles 256.

[0064] Since the "configuration" of the microneedle is different between the microneedle part and the second microneedle part, culturing can be performed using an appropriate microneedle part according to the experimental content.

[0065] For example, in FIGS. 4 and 5, although the microneedle part 25 is shown as having four first microneedles 255, when the number of the first microneedles 255 increases, the discharge resistance during the supply of the culture medium L decreases. Therefore, in order to realize the stable injection of the culture medium L according to the type and supply rate of the culture medium L, a second microneedle part with different numbers of the first microneedles 255 may be prepared and appropriately exchanged.

[0066] Regarding the diameter of the through hole 255x of the first microneedle part 255, it can be understood in the same way as the number of the first microneedles 255 described above.

[0067] [Tissue culture method] FIG. 6 is a schematic diagram showing the usage method of the culture kit 1000 and the culture instrument 100, and is an explanatory diagram of the tissue culture method of the present embodiment.

[0068] In the tissue culture method of the present embodiment, the tissue X is immersed in the culture medium L, and the culture medium L is supplied into the tissue X from the supply member 20A disposed inside the tissue X. In order to implement such a method, the above-described culture kit 1000 and culture instrument 100 are used as follows.

[0069] It will be described that skin cells are cultured as the tissue X at the bottom 120 of the insert 10. The tissue X is, for example, a tissue grown to a thickness of 1 mm.

[0070] First, the supply member 20A is inserted into the insert 10 to assemble the culture device 100. The supply member 20A has its height position within the insert 10 held by the holding portion 212 contacting the edge at the upper end of the insert 10. The micro needles (first micro needle 255, second micro needle 256) of the supply member 20A are spaced apart from the bottom 120 and puncture the tissue X in the posture in which the supply member 20A is inserted into the insert 10 (the height position held by the holding portion 212).

[0071] When the micro needle portion 25 punctures the tissue X, the first micro needle 255 and the second micro needle 256 below the micro needle portion 25 are pressed against the tissue X. At this time, the second micro needle 255 bites into the tissue X and functions as an "anti-slip" to suppress the displacement of the micro needle portion 25 in the surface direction of the tissue X.

[0072] In addition, since the micro needle portion 25 has the second micro needle 256, the tissue X is stretched and spread between the adjacent second micro needles 256 in the circumferential direction during puncture, giving an appropriate tension to the tissue X. Therefore, compared with the case where the micro needle portion 25 has four first micro needles 255 and no second micro needle 256, it is easier to puncture the soft tissue X or the highly elastic tissue X with the first micro needle 255.

[0073] The culture device 100 is inserted into the well 53 of the well plate 50 in which the culture medium L is stored. The culture device 100 contacts the edge of the well 53 at the upper end portion of the outer surface of the insert 10, and its height position is held within the well 53. The culture medium L in the well 53 is supplied to the outer surface of the tissue X through the membrane bottom 120. The tissue X absorbs the culture medium L from the outer surface.

[0074] On the other hand, the culture medium L is supplied to the supply member 20A from a storage portion (not shown) through the supply pipe 30. A micro pump such as a syringe pump may be appropriately connected to the storage portion (not shown). The culture medium L may be supplied intermittently or continuously in small amounts.

[0075] At this time, when attaching a plurality of culture devices 100 to the well plate 50, due to the elasticity and weight of the supply pipes 30 of each culture device 100, the culture kit 1000 may move easily and it may be difficult to keep it stationary. Therefore, the culture device 100 may have a support member that supports the plurality of supply pipes 30.

[0076] FIG. 7 is an explanatory diagram of the support member 60. The support member 60 has a pair of column portions 61 and a support portion 62 spanned between the column portions 61. The support portion 62 has a plurality of concave portions 62a. The plurality of supply pipes 30 are caught in the concave portions 62a of the support member 60, are supported, and their movement is restricted.

[0077] Note that the support member 60 shown here is an example, and various configurations can be adopted as long as it is possible to support the supply pipe 30. The support member 60 is configured to support the plurality of supply pipes 30 simultaneously, but is not limited to this, and a configuration in which one supply pipe 30 is supported by one support member may also be possible.

[0078] The culture medium L is supplied into the tissue X from the tip of the first micro needle 255 punctured into the tissue X through the flow path 20x (the flow path 20x in which the through hole 216x, the flow path 211x, the enlarged diameter hole 211z, the space 25x, and the through hole 255x communicate) of the supply member 20A. The tissue X absorbs the culture medium L inside.

[0079] At this time, since the first micro needles 255 are arranged at equal angular intervals in a plan view, the culture medium L can be supplied without bias at the contact portions of the supply member 20A in contact with the tissue X.

[0080] That is, the culture kit 1000 (culture device 100) can supply the culture medium L to the outer surface and the inside of the tissue X to be cultured. Thereby, in the method for culturing a tissue using the culture kit 1000 (culture device 100), even for the tissue X after growth, the culture medium can be effectively supplied to the inside of the tissue, and the experiment can be continued for a long time.

[0081] In addition, in the present embodiment, as a connecting member for connecting the main body portion 211 and the supply pipe 30, a configuration in which the first connecting portion 215 and the second connecting portion 216 are connected by a luer lock method is shown, but the present invention is not limited to this.

[0082] FIGS. 8 and 9 are exploded perspective views showing modified examples of the supply member. The supply member 20B shown in FIG. 8 is different from the above-described supply member 20A in that no first connecting portion is provided at the center of the holding portion 212 in plan view, and instead, a hole 212y is formed. The hole 212y communicates with a flow path 211x (not shown).

[0083] In addition, a connecting member 217 is provided at the end of the supply pipe 30. The connecting member 217 is a frustum-shaped member whose outer peripheral surface is a tapered surface, and has a through hole (not shown) that communicates with the internal space of the supply pipe 30.

[0084] In the supply member 20B, the main body portion 211 and the supply pipe 30 are connected by inserting such a connecting member 217 into the hole 212y.

[0085] The supply member 20C shown in FIG. 9 is different from the above-described supply member 20A in that no second connecting portion is provided at the end of the supply pipe 30. In addition, no first connecting portion is provided at the center of the holding portion 212 in plan view, and instead, a connecting member 218 is formed. The connecting member 218 is a cylindrical member having a through hole 218x, and the outer peripheral surface of the upper end is formed in a frustum shape. The through hole 218x communicates with a flow path 211x (not shown).

[0086] In the supply member 20C, the main body portion 211 and the supply pipe 30 are connected by inserting the supply pipe 30 into such a connecting member 218.

[0087] In addition, in the present embodiment, it is assumed that the main body portion 211 has four flat plate-shaped separation portions 214, but the present invention is not limited to this. FIG. 10 is a cross-sectional view showing a modified example of the supply member.

[0088] The supply member 20D shown in FIG. 10 does not have the flat separation part 214 unlike the above-described supply member 20A. Further, the supply member 20D has a bowl-shaped holding part 219 instead of the holding part 212 of the supply member 20A.

[0089] The holding part 219 has a flange part 219a, a tapered part 219b, and an annular part 219c. The flange part 219a has a disk-shaped structure in plan view that extends radially outward from the outer surface 211a of the main body part 211.

[0090] The tapered part 219b is a frustum-shaped tubular part that is connected to the outer edge of the flange part 219a and has an increasing diameter upward.

[0091] The annular part 219c is an annular structure that is connected to the outer edge of the tapered part 219b.

[0092] In such a supply member 20D, in the posture where the supply member 20D is inserted into the insert 10, the annular part 219c contacts the end face 10a on one end side of the insert, and holds the height position of the supply member 20D within the insert 10. Further, since the outer peripheral surface 219x of the holding part 219 interferes with the inner surface 110a of the insert 10, the main body part 211 and the inner surface 110a of the insert 10 are separated. Therefore, the supply member 20D can easily arrange the main body part 211 at the center of the insert 10 in the posture where it is inserted into the insert 10. That is, the holding part 219 of the supply member 20D also functions as a separation part.

[0093] Also, in the present embodiment, although it has been described that the above-described culture kit 1000 and culture instrument 100 are used when implementing the tissue culture method, the culture kit 1000 and culture instrument 100 are not essential for implementing the tissue culture method.

[0094] FIG. 11 is an explanatory diagram of a tissue culture method according to a modification. In the tissue culture method shown in FIG. 11, a culture kit having a storage part (not shown) and a pipe 200 is used.

[0095] In the configuration shown in FIG. 11, cells are seeded on the outer surface 200a of the pipe 200 through which the culture medium L flows, and the tissue X is cultured. A plurality of fine through-holes 200x are provided in the pipe wall of the pipe 200, and the tissue X covers the through-holes 200x. Further, the pipe 200 is immersed in the culture medium L stored in a storage portion (not shown).

[0096] Even in such a method for culturing a tissue, the tissue X absorbs the culture medium L from the outer surface and also absorbs the culture medium L from the inside of the tissue X. Therefore, even for the grown tissue X, the culture medium can be effectively supplied to the inside of the tissue, and the experiment can be continued for a long time.

[0097] As described above, the preferred embodiments according to the present invention have been described with reference to the accompanying drawings, but the present invention is not limited to such examples. The various shapes, combinations, etc. of the constituent members shown in the above examples are merely examples, and various changes can be made based on the design, specifications, etc. without departing from the gist of the present invention.

Example

[0098] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0099] In this example, a skin tissue was cultured using the culture device 100 (culture kit 1000) having the supply member 20A described above. That is, in the example, the culture medium was supplied to the surface of the skin tissue, and the culture medium was also supplied to the inside of the skin tissue by the supply member 20A.

[0100] As a comparative example, a skin cell was cultured using a known culture kit using a cell culture insert and a well plate without using the above-described supply member. That is, in the comparative example, the culture medium was supplied only to the surface of the skin tissue.

[0101] Commercially available human abdominal skin pieces (Biopredic International) with a diameter of approximately 12 mm and a thickness of approximately 900 μm were used in the experiment. Each skin piece was placed in a cell culture insert (manufactured by Thermo Fisher Scientific), and the culture medium (Dulbecco’s Modified Eagle’s Medium, manufactured by Thermo Fisher Scientific) was allowed to penetrate through the membrane at the bottom of the insert, and cultured at 37 °C under 5% CO2 for up to 11 days. During the culture period, the medium was changed daily.

[0102] In the example, the microneedles of the supply member 20A were inserted into the upper part of the skin (epidermal side), and the medium was supplied into the dermis of the skin at a rate of 2 ml / day using a syringe pump (manufactured by AS ONE).

[0103] (Appearance of cultured tissue) The skin cells of the example and the comparative example were stained by a known method and observed.

[0104] At the start of culture, and 5 days and 11 days after the start of culture, the skin tissues were fixed with 4% paraformaldehyde, sliced thinly to a thickness of approximately 1 mm, and blocked with a blocking buffer (Starting Block, manufactured by ThermoFisher Scientific).

[0105] As the primary antibody, an anti-elastin antibody (Santa Cruz Biotechnology) was used for visualization of dermal elastic fibers, and an anti-COL VII antibody (Abcam) was used for visualization of the basement membrane.

[0106] As the secondary antibody, an anti-mouse IgG H&L antibody (Alexa Fluor 488, manufactured by ThermoFisher Scientific) and an anti-rabbit IgG antibody (Alexa Fluor 647, manufactured by ThermoFisher Scientific) were used.

[0107] Furthermore, DAPI (Dojindo Laboratories) was used for cell nucleus staining.

[0108] Before imaging, tissue clearing was performed using Rapiclear 1.49 (manufactured by Sanei Labo). Imaging was performed three-dimensionally using a dry lens with a magnification of 40 by a scanning confocal laser microscope AX R (manufactured by Nikon Corporation).

[0109] Figure 12 is a magnified photograph showing the skin tissue of the example, and Figure 13 is a magnified photograph showing the skin tissue of the comparative example. In Figures 12 and 13, reference sign A indicates the epidermis, reference sign B indicates the basement membrane, and reference sign C indicates the dermis. Also, in Figures 12 and 13, (a) shows the skin tissue at the start of culture (day 0), (b) shows the skin tissue at 5 days from the start of culture, and (c) shows the skin tissue at 11 days from the start of culture. Figure 12(a) and Figure 13(a) are the same photograph showing the state at the start of culture.

[0110] As the applicant's finding, it has been found that when culturing skin tissue by a conventional method, the skin tissue usually partially dies within 3 to 4 days, or at most about one week, and the dermis and the epidermis are separated, making it impossible to continue the culture.

[0111] Based on the above finding and by checking Figure 12, it was confirmed that the skin tissue of the example was appropriately cultured without separation of the dermis and the epidermis even on the 11th day from the start of culture.

[0112] On the other hand, by checking Figure 13, it was confirmed that in the skin tissue of the comparative example, a portion where the dermis and the epidermis were separated (indicated by reference sign α) was found on the 11th day from the start of culture, and as per the conventional finding, it was confirmed that the subsequent culture could not be continued. Also, by observing Figure 13(b) (5 days from the start of culture) in detail, it was confirmed that the shape of the cell nucleus had changed from an elliptical shape to a shape close to a perfect circle, indicating that the skin cells were weakened.

[0113] (Confirmation of cell products) For the skin cells of the example and the comparative example, the cell products were confirmed by a known method. The confirmation method was as follows. The culture media on days 5 and 11 after the start of culturing were collected, and the amount of serum lactate dehydrogenase (LDH) in the culture media, which is an index of cytotoxicity, was measured using a commercially available measurement kit (FUJIFILM Wako Pure Chemical Corporation) based on the attached instruction manual.

[0114] Also, the amount of inflammatory cytokines in the culture media 11 days after culturing was measured using a commercially available measurement kit (BD Cytometric Bead Array (CBA) Human Inflammatory Cytokine Kit (Becton, Dickinson and Company)) based on the attached instruction manual.

[0115] Figures 14 to 16 are graphs showing the amounts of substances produced by skin cells. Figure 14 is a graph showing the amount of interleukin 6 (IL-6), which is an inflammatory cytokine. Figure 15 is a graph showing the amount of interleukin 8 (IL-8), which is an inflammatory cytokine. IL-6 and IL-8 are used as markers indicating the degree of inflammation occurring in tissues.

[0116] In the skin tissue of the examples, the value of IL-6 decreased from day 1 to day 11 of culturing (Figure 14), and the increase in the value of IL-8 was suppressed (Figure 15). Therefore, it can be seen that in the examples, the inflammation of the skin tissue is suppressed.

[0117] On the other hand, in the skin tissue of the comparative examples, the values of both IL-6 and IL-8 increased from day 1 to day 11 of culturing, indicating that inflammation has occurred in the skin tissue.

[0118] Figure 16 is a graph showing the amount of serum lactate dehydrogenase (LDH), which is a cytotoxicity marker. Since the detected amount of LDH increases as cell death increases, it is used as a marker of cytotoxicity.

[0119] In the skin tissue of the examples, the increase in the amount of LDH was suppressed until day 11 of culturing, indicating that cell death of skin cells was suppressed.

[0120] On the other hand, in the skin tissue of the comparative example, a large amount of LDH that is already comparable to that on the 11th day of culture is detected on the 5th day of culture. As described above, in the skin tissue of the comparative example, detachment between the epidermis and the dermis could not be confirmed on the 5th day of culture. However, considering the change in the shape of the cell nucleus in FIG. 13(b) described above and the sharp increase in the amount of LDH shown in FIG. 16, it can be seen that many cells have already died on the 5th day of culture.

[0121] From the above results, it was confirmed that cell culture can be continuously carried out for a long time according to the present invention, and it was found that the present invention is useful.

Explanation of Reference Numerals

[0122] 10…Cell culture insert, 10a…End face, 20x, 211x…Flow path, 20A, 20B, 20C, 20D…Supply member, 21…Supply part, 25…Microneedle part, 25x…Space, 30…Supply pipe, 50…Well plate, 51…Top plate, 52…Frame body, 53…Well, 60…Support member, 61…Column part, 62…Support part, 62a…Recess, 100…Culture instrument, 110…Side wall, 110a…Inner surface, 110x…One end, 110y…The other end, 111…First side wall part, 112…Second side wall part, 120…Bottom, 120a, 212b…Upper surface, 200…Pipe, 200a, 211a…Outer surface, 200x, 216x, 218x, 255x…Through hole, 211…Main body part, 211y, 212y…Hole, 211z…Diameter-expanded hole, 212, 219…Holding part, 212a…Lower surface, 212x…Notch, 213…Coupling part, 213a, 251…Base part, 213b, 252…Convex part, 213c…Groove part, 214…Separation part, 215…First connection part, 216…Second connection part, 216a…First member, 216b…Second member, 216c…Third member, 216d…Opposite, 217, 218…Connection member, 219a…Flange part, 219b…Taper part, 219c…Annular part, 219x…Outer peripheral surface, 251a…Convex part, 255…First microneedle, 256…Second microneedle, 1000…Culture kit, L…Culture medium, OR…Sealing member (O-ring), X…Tissue

Claims

1. A cylindrical cell culture insert having a bottom, and a supply member inserted into the cell culture insert and supplying a culture medium to a tissue cultured on the upper surface of the bottom, The supply member has a main body portion having a flow path through which the culture medium flows, a holding portion that holds the height position of the supply member within the cell culture insert, and a micro needle portion provided to cover an opening of the flow path on the other end side of the main body portion. The micro needle portion has a micro needle communicating with the flow path, The micro needle is spaced apart from the bottom in a posture in which the supply member is inserted into the cell culture insert and is a culture instrument punctured into the tissue.

2. The culture instrument according to claim 1, further comprising a separation portion provided on an outer surface of the main body portion to separate the main body portion from an inner surface of the cell culture insert.

3. The culture instrument according to claim 1 or 2, further comprising a supply pipe connected to one end side of the main body portion and communicating with the flow path.

4. The culture instrument according to claim 3, further comprising a connection member that connects the main body portion and the supply pipe.

5. The connection member has a first connection portion provided on the main body portion and a second connection portion provided at an end of the supply pipe, and The culture instrument according to claim 4, wherein the first connection portion and the second connection portion are combined to connect the main body portion and the supply pipe.

6. The culture instrument according to claim 3, further comprising a support member that supports the supply pipe.

7. The culture instrument according to claim 1 or 2, wherein the micro needle portion is provided to be replaceable with respect to the main body portion.

8. It has a second micro-needle part that is replaceable with the micro-needle part, The culture device according to claim 7, wherein the micro-needles of the second micro-needle part have a different configuration from the micro-needles of the micro-needle part.

9. The culture device according to claim 1 or 2, A culture kit having a well plate provided with a plurality of wells for storing a culture medium in the wells.

10. A method for culturing a tissue, which comprises immersing the tissue in a culture medium and supplying the culture medium into the tissue from a supply member disposed inside the tissue.

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