Cell culture inserts and culture kits

Abstract

We provide a cell culture insert that allows for accurate evaluation of the penetration of a test substance into tissue. [Solution] The cell culture insert (2) comprises a substantially cylindrical side wall portion (2a) and a bottom portion (2b). The bottom portion (2b) comprises a porous membrane (2b1) to which cells can adhere and a cell non-adhesion layer (2b3) located above the porous membrane (2b1). The cell non-adhesion layer (2b3) has a plurality of through holes (2b4) formed therein.

Description

Technical Field

[0001] The present invention relates to a cell culture insert and a culture kit.

Background Art

[0002] As disclosed in, for example, Patent Document 1, cell culture inserts that are inserted into containers such as dishes, flasks, and cell culture plates to enable cell culture for various purposes such as co-culture and air-liquid interface culture have been conventionally known.

[0003] A cell culture insert generally has a bottomed cylindrical shape, and is provided with a flange portion for suspending on the upper edge of a container at the upper edge of a substantially cylindrical side wall portion. A porous membrane is disposed on the inner bottom of the insert, and a culture solution can pass through the membrane, while cells cannot pass therethrough, so that cells can be adhesively cultured on the membrane.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problem to be Solved by the Invention

[0005] On the other hand, there is a demand for evaluating the permeability of test substances such as anticancer drug candidate substances and the permeability of metabolites of test substances into various tissues such as cancer tissue.

[0006] The present invention has been made in view of such circumstances, and an object of the present invention is to provide a cell culture insert capable of accurately evaluating the permeability of a test substance and a metabolite thereof into tissue, and a culture kit including the cell culture insert.

Means for Solving the Problem

[0007] To solve the above problems, one aspect of the present invention includes the following aspects. [1] A cell culture insert comprising a substantially cylindrical side wall portion and a bottom portion, wherein the bottom portion comprises a porous membrane to which cells can adhere and a cell non-adhesion layer located above the porous membrane, and the cell non-adhesion layer has a plurality of through holes formed therein.

[0008] [2] The cell culture insert according to [1], wherein the diameter of the through hole is 50 to 500 μm.

[0009] [3] The cell culture insert according to [1] or [2], wherein the diameter of the through hole is 100 to 200 μm.

[0010] [4] The cell culture insert according to any one of [1] to [3], wherein two or more through holes are formed in an array in the cell non-adherent layer.

[0011] [5] A cell culture insert according to any one of [1] to [4], comprising a cell adhesion layer to which cells can adhere between the cell non-adhesion layer and the porous membrane, wherein the through-hole penetrates the cell non-adhesion layer and the cell adhesion layer substantially in the vertical direction, and the cell adhesion layer is exposed within the through-hole.

[0012] [6] The cell culture insert according to [5], wherein the thickness of the cell adhesion layer in the vertical direction is 20 μm or more.

[0013] A culture kit comprising a cell culture insert described in any one of [7][1] to [6], and a container for receiving material that has passed through the porous membrane of the cell culture insert. [Effects of the Invention]

[0014] According to the cell culture insert and culture kit of the present invention, substances can penetrate downward through through-holes and a porous membrane in a limited area at the bottom of the cell culture insert, while other areas are covered with a non-adherent cell layer, preventing substance passage. Therefore, by culturing the tissue to be evaluated in the area where the through-holes are formed, the permeability of the test substance and its metabolites into the tissue can be accurately evaluated. [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic perspective view of a culture kit according to a preferred embodiment of the present invention. [Figure 2] Figure 1 is a schematic plan view showing the bottom of the cell culture insert. [Figure 3] Figure 2 is a schematic cross-sectional view of the bottom section, taken longitudinally at the position of line XX. [Figure 4] This is a schematic side view showing how tissue is cultured in a through-hole formed at the bottom of a cell culture insert. [Figure 5] This is a schematic side view showing an example of the procedure for manufacturing the bottom. [Figure 6] Figure 1 shows a schematic cross-sectional end view of the culture kit, cross-sectioned longitudinally at the YY line indicated. [Modes for carrying out the invention]

[0016] In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0017] The following describes in detail cell culture inserts and culture kits according to preferred embodiments of the present invention, with reference to drawings as appropriate. However, the present invention is not limited to the following embodiments. In all the following drawings, the dimensions and proportions of each component have been appropriately changed for clarity.

[0018] Fig. 1 is a schematic perspective view of a culture kit 1 according to a preferred embodiment of the present invention. As shown in Fig. 1, the culture kit 1 includes a cell culture insert 2 and a container 3 that receives a permeate that has passed downward from the cell culture insert 2. The container 3 has a bottomed cylindrical shape. The cell culture insert 2 is suspended on the upper edge of the container 3, and at least the lower part thereof can be inserted into and removed from the container 3 in a freely detachable manner. Fig. 1 shows a state where the cell culture insert 2 is inserted into the container 3 and a state where the cell culture insert 2 is taken out from the container 3.

[0019] <セルカルチャーインサート2> The cell culture insert 2 includes a substantially cylindrical side wall portion 2a and a bottom portion 2b connected to the side wall portion 2a. The bottom portion 2b and the side wall portion 2a may be integrally molded, but it is preferable that they are formed as separate members and connected to each other. Only the bottom portion 2b of the cell culture insert 2 is configured to allow a culture solution, a test substance and the like to pass therethrough, but does not allow cells to pass through.

[0020] An operator of the culture kit 1 adds a test substance, for which permeability into the inside of an arbitrary tissue is desired to be evaluated, into the cell culture insert 2 while culturing the tissue on the bottom portion 2b of the cell culture insert 2. Thereafter, by measuring whether the test substance or a metabolite thereof is contained in the culture solution stored in the container 3 and measuring the concentration thereof, the permeability of the test substance or the metabolite thereof into the inside of the tissue can be evaluated.

[0021] As used herein, the term "metabolite" refers to a product produced by metabolizing a test substance in cells. The type of test substance for which permeability into tissue is evaluated using the cell culture insert 2 is not particularly limited, and examples thereof include anticancer agents and candidate substances thereof.

[0022] (Side wall portion 2a) The side wall portion 2a is formed of a material that does not allow cells, liquids such as culture medium, or the test substance to pass through (in other words, does not allow permeation). The side wall portion 2a may be cylindrical, rectangular, or tapered, with the diameter becoming smaller towards the bottom or top. Preferably, the side wall portion 2a is made of resin and is transparent or translucent. However, as long as the side wall portion 2a does not allow liquids or the test substance to pass through, the material, shape, color, transparency, etc., are not particularly limited.

[0023] The side wall portion 2a has a flange portion 2a1 at its upper end that protrudes radially outward, allowing the operator to suspend the flange portion 2a1 on the upper edge 3c of the container 3 and support the cell culture insert 2 in the container 3. When the flange portion 2a1 is suspended on the upper edge 3c of the container 3, the bottom portion 2b of the cell culture insert 2 is located inside the container 3. At this time, the lower surface of the bottom portion 2b is spaced apart from the upper surface of the bottom of the container 3 (see reference numeral 3b1 in Figure 6, described later).

[0024] It is not necessarily required that the side wall portion 2a has a flange portion 2a1; for example, at least a part of the outer surface of the side wall portion may contact the inner surface of the container 3, thereby enabling the bottom of the cell culture insert to be held inside the container 3.

[0025] (bottom 2b) Figure 2 is a schematic plan view showing the bottom 2b of the cell culture insert 2 shown in Figure 1. Figure 3 is a schematic cross-sectional end view of the bottom 2b, taken longitudinally at the position of line XX shown in Figure 2.

[0026] As shown in Figure 3, the bottom portion 2b comprises a porous membrane 2b1 to which cells can adhere, a cell adhesion layer 2b2 located above the porous membrane 2b1, and a non-cell adhesion layer 2b3 located above the cell adhesion layer 2b2. The non-cell adhesion layer 2b3 is located above the porous membrane 2b1.

[0027] The porous membrane 2b1 and the cell adhesion layer 2b2 exhibit cell adhesion, while the non-cell adhesion layer 2b3 does not. "Cell adhesion" specifically refers to the property of a membrane or layer such that, when cells are deposited against it, they adhere to the membrane or layer at certain adhesion points. Without cell adhesion, most cells will not spread or proliferate.

[0028] The cell adhesion layer 2b2 and the non-cell adhesion layer 2b3 do not allow liquids such as culture medium or test substances to pass through in the thickness direction of the bottom 2b.

[0029] Here, multiple through-holes 2b4 that penetrate approximately vertically are formed in the cell adhesion layer 2b2 and the non-cell adhesion layer 2b3. In the cell adhesion layer 2b2 and the non-cell adhesion layer 2b3, areas other than those where the through-holes 2b4 are formed do not allow liquids such as culture medium or the test substance to pass through, whereas the through-holes 2b4 allow liquids such as culture medium or the test substance to pass through.

[0030] Therefore, at least when no cells or tissues are seeded on the bottom 2b, the internal space of the cell culture insert 2 (in other words, the space immediately above the bottom 2b) and the space below the bottom 2b are in communication through the through-holes 2b4 of the bottom 2b and the pores of the porous membrane 2b1 located below it.

[0031] Therefore, in this situation, the test substance and its metabolites added to the cell culture insert 2 penetrate into the space below the bottom 2b, i.e., into the container 3, through the through-holes 2b4 and the pores of the porous membrane 2b1. From the above, as shown in Figures 2 and 3, the region in the bottom 2b where the through-holes 2b4 are formed can be called the penetration region 2ba, and the region in the bottom 2b other than the penetration region 2ba can be called the non-penetration region 2bb.

[0032] As shown in Figure 3, the side surface of the cell adhesion layer 2b2 is exposed within the through-hole 2b4 and faces the space within the through-hole 2b4.

[0033] Figure 4 is a schematic side view showing how tissue 4 is cultured in the through-hole 2b4 formed in the bottom 2b of the cell culture insert 2. The non-cell-adherent layer 2b3, located at the top of the bottom 2b, does not exhibit cell adhesion, while the porous membrane 2b1 does. Therefore, at the bottom 2b, the tissue 4 is located within the through-holes 2b4. For this reason, the through-holes 2b4 serve as wells for containing the tissue 4 in which the permeability of the test substance or its metabolites is evaluated.

[0034] The cells constituting tissue 4 do not adhere to the side surface 2b3a of the non-adherent cell layer 2b3 within the through-hole 2b4, but adhere to the side surface 2b2a of the cell adhesion layer 2b2 and the upper surface 2b1a of the porous membrane 2b1. As a result, the pathways connecting the inside of the cell culture insert 2 and the space located below the porous membrane 2b1 are blocked by the cells.

[0035] Therefore, if, while culturing tissue 4 in the through-hole 2b4, the test substance or its metabolites added to the inside of the cell culture insert 2 are detected in the culture medium stored in the container 3, it can be determined that the test substance or its metabolites were transported downwards to the cells by transcytosis, endocytosis, and exocytosis, and then moved downwards to the bottom 2b through the pores of the porous membrane 2b1. In this case, it can be said that the test substance or its metabolites have permeability into the tissue 4.

[0036] Therefore, by culturing the tissue 4 in the through-hole 2b4, adding the test substance to the inside of the cell culture insert 2, and measuring whether the liquid stored in the container 3 (the liquid outside the cell culture insert 2) contains the test substance or its metabolites, and measuring their concentrations, the permeability of the test substance into the tissue 4 can be accurately evaluated.

[0037] In evaluating permeability, tissue 4 may be placed in the through-holes 2b4 after formation, but it is preferable to seed cells in the through-holes 2b4 and form tissue 4 within the through-holes 2b4. The type of tissue 4 cultured in the through-holes 2b4 is not particularly limited, but may be, for example, spheroids or organoids, or spheroids of cancer cells. The types of cells constituting tissue 4 may be one type, two types, or three or more types.

[0038] It is not necessarily required that the bottom portion 2b has a cell adhesion layer 2b2 between the cell non-adhesion layer 2b3 and the porous membrane 2b1; for example, the cell non-adhesion layer 2b3 may be positioned in contact with the porous membrane 2b1.

[0039] However, because the bottom portion 2b is equipped with a cell adhesion layer 2b2, the cell adhesion layer 2b2 exposed within the through-hole 2b4 adheres to the tissue 4. As a result, leakage of the test substance or its metabolites to the bottom of the porous membrane 2b1 without passing through the cells is effectively suppressed.

[0040] Furthermore, the bottom portion 2b may have a cell-non-adherent laminate layer between the cell adhesion layer 2b2 and the porous membrane 2b1. The presence of the laminate layer facilitates the bonding process between the cell adhesion layer 2b2 and the porous membrane 2b1 during the manufacturing of the bottom portion 2b. Alternatively, the laminate layer may be omitted, allowing the cell adhesion layer 2b2 and the porous membrane 2b1 to be in direct contact.

[0041] The diameter of the through-holes 2b4 is not particularly limited as long as it allows the test substance and its metabolites to pass through, but is for example 50 to 500 μm, preferably 100 to 200 μm. By configuring the diameter of the through-holes 2b4 within the above range, the tissue 4 can be accommodated in the through-holes 2b4, and the tissue 4 and the cell adhesion layer 2b2 can be properly bonded.

[0042] The number of through-holes 2b4 is not particularly limited as long as it is 2 or more, but is preferably 10 or more. The more through-holes 2b4 there are, the more tissue can be evaluated. As a result, the amount of the test substance or its metabolites that can pass below the bottom 2b increases, allowing for a more accurate evaluation of the presence and degree of permeability. The through-holes 2b4 may be arranged in an array when viewed from above, as shown in Figure 2.

[0043] The spacing between multiple through holes 2b4 (in other words, the length of the gap between the holes) is not particularly limited, but may be in the range of 50 to 1000 μm or in the range of 50 to 500 μm.

[0044] The material of the porous membrane 2b1 is not particularly limited, as long as it exhibits cell adhesion and allows the test substance or its metabolites to pass through its pores, but is preferably a resin such as polyester, polycarbonate, or polyimide. The porous membrane is preferably a track-etched membrane.

[0045] The size (diameter) of the pores formed in the porous membrane 2b1 is not particularly limited, but may be, for example, 0.01 to 8 μm, 0.01 to 5 μm, 0.1 to 3 μm, or 0.1 to 1 μm. The size of the pores can be confirmed, for example, by microscopy.

[0046] The thickness (length in the vertical direction) of the porous membrane 2b1 is not particularly limited, but may be, for example, 1 to 500 μm, 1 to 150 μm, 2 to 50 μm, 3 to 25 μm, or 5 to 20 μm.

[0047] The material of the cell adhesion layer 2b2 is not particularly limited as long as cells can adhere to it, but it is preferably a resin, and preferably polyethylene terephthalate, polycarbonate, polyimide, polystyrene, or polycycloolefin.

[0048] The vertical thickness of the cell adhesion layer 2b2 is not particularly limited, but is preferably 20 μm or more, more preferably 23 μm or more, and may be, for example, 20 to 30 μm or 23 to 27 μm. By making the thickness of the cell adhesion layer 2b2 20 μm or more, cells adhere firmly to the sides of the cell adhesion layer 2b2, increasing the sealing force of the through-holes 2b4 by the tissue, and enabling more accurate evaluation.

[0049] The cell non-adhesion layer 2b3 is preferably formed by coating the upper surface of the cell adhesion layer 2b2 with a cell non-adhesion substance. If the cell adhesion layer 2b2 is not provided, the cell non-adhesion layer 2b3 may be formed by coating the upper surface of the porous membrane 2b1 with a cell non-adhesion substance.

[0050] Examples of cell-non-adherent material layers include non-adherent hydrogel membranes and monolayers. Examples of non-adherent hydrogel materials include, but are not limited to, cell-non-adherent polysaccharides such as cellulose and agarose, and cell-non-adherent proteins such as 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, polyethylene glycol, hydroxyethyl methacrylate (HEMA), segmented polyurethane (SPC), and albumin.

[0051] The thickness of the cell non-adherent layer 2b3 in the vertical direction is not particularly limited, but is preferably 1 μm or less.

[0052] An example of the procedure for preparing cell culture insert 2, configured as described above, is explained in detail below.

[0053] Figure 5 is a schematic side view showing an example of the procedure for fabricating the bottom portion 2b. As shown in Figure 5, first, a sheet to be used as the cell adhesion layer 2b2, such as a PET sheet, is prepared (step (a)). A laminating material 2b5 may be attached to the lower surface of this sheet to facilitate adhesion with the porous membrane 2b1 later. A laminating adhesive may be used for attachment.

[0054] Next, a non-adhesive surface treatment is applied to the upper surface of the cell adhesion layer 2b2, which is the sheet described above, to form a cell non-adhesive layer 2b3 (step (b)). After that, a plurality of through holes 2b4 that penetrate in a roughly vertical direction are formed (step (c)). Methods for forming the through holes 2b4 include, for example, formation by laser irradiation or formation by punching.

[0055] After the formation of the through-holes 2b4, the cell adhesion layer 2b2 and the cell non-adhesion layer 2b3 (and the laminate material) are attached to the porous membrane 2b1, such as a track-etched membrane (step (d)). Laminating adhesive may be used during attachment. If the cell adhesion of the porous membrane 2b1 is low, the cell adhesion may be improved beforehand by hydrophilization treatment or the like. Examples of hydrophilization treatments include, but are not limited to, polyvinylpyrrolidone (PVP) treatment.

[0056] The resulting bottom portion 2b can be cut out to match the shape of the side wall portion 2a and joined to the lower end of the side wall portion 2a to obtain the cell culture insert 2.

[0057] <Container 3> Figure 6 is a schematic cross-sectional end view of culture kit 1, cross-sectioned longitudinally at the position of the YY line shown in Figure 1. As shown in Figure 6, the container 3 comprises a substantially cylindrical container side wall portion 3a and a disc-shaped container bottom portion 3b that closes the lower end of the container side wall portion 3a, and receives the material that passes through the bottom portion 2b of the cell culture insert 2. The container side wall portion 3a and the container bottom portion 3b may be integrally molded or they may be molded separately and then joined to each other, but integral molding is preferred. The shape of the container 3 is not particularly limited as long as it can support the cell culture insert 2 and store the culture medium and test substance that has passed down the bottom portion 2b.

[0058] The type of container 3 is not particularly limited, but may be a dish, a flask, or a cell culture well plate such as a 24-well, 48-well, or 96-well plate. If container 3 is a cell culture well plate with multiple wells, the culture kit 1 may have as many cell culture inserts 2 as there are wells, and these multiple cell culture inserts 2 may be connected to each other horizontally.

[0059] When evaluating permeability, the evaluation may be performed while adhering and culturing cells inside the container 3 and outside the cell culture insert 2. The upper surface 3b1 of the container bottom 3b may or may not exhibit cell adhesion.

[0060] The present invention is not limited to the embodiments described above, and it goes without saying that various modifications are possible within the scope of the invention as described in the claims, and these modifications are also included within the scope of the present invention. [Industrial applicability]

[0061] According to the present invention, in a limited area at the bottom of the cell culture insert, substances can pass (permeate) downward through through-holes and a porous membrane, while other areas are covered with a non-cell-adherent layer, preventing substance passage. Therefore, by blocking the through-holes with the tissue to be evaluated during culture, the permeability of the test substance or its metabolites into the tissue can be accurately assessed. Accordingly, the present invention is industrially applicable. [Explanation of symbols]

[0062] 1…Culture kit, 2…Cell culture insert, 2a…Side wall, 2a1…Flange, 2b…Bottom, 2b1…Porous membrane, 2b1a…Top surface of porous membrane, 2b2…Cell adhesion layer, 2b2a…Side surface of cell adhesion layer, 2b3…Non-adherent cell layer, 2b3a…Side surface of non-adherent cell layer, 2b4…Through-hole, 2b5…Laminate material, 2ba…Penetration area, 2bb…Non-penetration area, 3…Container, 3a…Container side wall, 3b…Container bottom, 3b1…Top surface of container bottom, 3c…Container upper edge, 4…Tissue

Claims

1. It comprises a roughly cylindrical side wall and a bottom, The bottom is, A porous membrane to which cells can adhere, The system comprises a cell non-adhesion layer located above the porous membrane, The cell culture insert has multiple through-holes formed in the cell non-adherent layer.

2. The cell culture insert according to claim 1, wherein the diameter of the through hole is 50 to 500 μm.

3. The cell culture insert according to claim 2, wherein the diameter of the through hole is 100 to 200 μm.

4. The cell culture insert according to claim 1, wherein two or more through holes are formed in an array-like manner in the cell non-adherent layer.

5. A cell adhesion layer to which cells can adhere is provided between the cell non-adhesion layer and the porous membrane. The through-hole penetrates the cell non-adherent layer and the cell adherent layer in a substantially vertical direction. The cell culture insert according to claim 1, wherein the cell adhesion layer is exposed within the through-hole.

6. The cell culture insert according to claim 5, wherein the thickness of the cell adhesion layer in the vertical direction is 20 μm or more.

7. A cell culture insert according to any one of claims 1 to 6, A culture kit comprising a container for receiving material that has passed through the porous membrane of the cell culture insert.

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