Medical device, kit, and culture-embedding method for culturing biological samples and embedding them in embedding material.

Abstract

It is difficult to produce thin biological samples that are embedded in an embedding material without using an expensive microtome. The present invention relates to a medical device that comprises: a base part that has an opening part; and a holder that holds the base part on a culture plate. The base part also has a channel that is in fluid communication with the opening part. Biological samples are cultured and embedded in an embedding material at the opening part.

Description

Technical Field

[0001] The present invention relates to a medical device, a kit, and a culturing and embedding method for culturing a biological sample and embedding the biological sample in an embedding material.

Background Art

[0002] Age-related macular degeneration is a disease that causes vision loss due to the macula that has changed due to damage accumulated with aging. Age-related macular degeneration is generally classified into two types: atrophic age-related macular degeneration caused by atrophy of macular tissue, and exudative age-related macular degeneration caused by damage to the macula due to neovascularization under the retina. [[ID=​​​​​​​​​​​​​​​​[Non-Patent Document 1] Ben M'Barek et al., Sci. Transl. Med. 9, eaai7471 (2017) [Overview of the project] [Problems that the invention aims to solve]

[0006] In the conventional technology described above, it is necessary to culture the cell sheet for transplantation, excavate it, and then embed it. However, these processes require separate equipment, which is expensive and requires skilled work, and there is a risk of contamination of the cell sheet. [Means for solving the problem]

[0007] The inventors have developed an implantation device and a culture / embedding method that enable the continuous cultivation and embedding of biological samples. The present invention A foundation with an opening, The above base is equipped with a holder for fixing it to a culture dish, The above-mentioned base portion is equipped with a flow path that communicates with the above-mentioned opening. A medical device that performs the cultivation of a biological sample and the embedding of the biological sample in an embedding material at the above-mentioned opening. Regarding.

[0008] Such medical devices allow for the continuous and easy cultivation and embedding of biological samples, preventing contamination of the biological samples and eliminating the need for expensive equipment and skilled labor, thus enabling the easy creation of hydrogel-embedded cell sheets for transplantation. Furthermore, cultured biological samples are not wasted.

[0009] The holder may have a straight body portion with a through hole and a recess at one of the ends of the straight body portion capable of accommodating at least a portion of the base portion. The through hole and the opening may be in fluid communication. The base portion may include a spacer and a spacer support portion that abuts the spacer. The spacer support portion may have at least two grooves on the surface that abuts the spacer. The flow path may be formed by the grooves being covered by the spacer. The spacer may be made of an elastic material. The medical device may further include a pressing jig having a size that can be inserted into the through hole. The pressing jig includes at least a head portion and a body portion.

[0010] Furthermore, the present invention relates to a kit comprising the above-mentioned medical device.

[0011] Furthermore, the present invention is This invention relates to a culture and embedding method for culturing a biological sample using the above-mentioned medical device and embedding the biological sample with an embedding material, wherein the above method is A setting step of setting the base part and the holder in the culture dish, A culture step in which a biological sample is cultured in the aforementioned opening, An embedding step in which the cultured biological sample is embedded in the opening with the embedding material, The process includes a removal step for removing the embedded biological sample.

[0012] The culture embedding method described above may further include a discharge step of discharging excess embedding material by inserting a pressing jig into the holder. The culture dish may be a temperature-responsive culture dish. The wells of the temperature-responsive culture dish may be coated with laminin. The embedding material may be a hydrogel. In the embedding step, a hydrogel at a temperature at which it is fluid may be filled into the opening. In the removal step, the embedded biological sample may be removed at a temperature below the temperature at which the hydrogel solidifies. The temperature of the fluid hydrogel may be a temperature suitable for culturing the biological sample.

Brief Description of the Drawings

[0013] [Figure 1] Figure 1 shows a development view (plane-side perspective view) of the medical device 1 according to the present embodiment. [Figure 2] Figure 2 shows the spacer support portion 100 on the side that abuts the spacer 200. [Figure 3] Figure 3 shows a development view (bottom-side perspective view) of the medical device 1 according to the present embodiment. [Figure 4] Figure 4 shows a side view of the assembled medical device 1 according to the present embodiment. [Figure 5] Figure 5 shows a cross-sectional view of the assembled medical device 1 according to the present embodiment. [Figure 6] Figure 6 shows a state where the medical device 1 according to the present embodiment is set in the well 401 of the culture dish 400. [Figure 7] Figure 7 is a partial end view of the medical device 1 set in the well 401 of the culture dish 400. [Figure 8] Figure 8 is a partial end view of the medical device 1 and the culture dish 400, and the biological sample 500 and the culture solution 600 are contained in the culture section 800. [Figure 9] Figure 9 is a partial end view of the medical device 1 and the culture dish 400, and the biological sample 500 wrapped with the embedding material 700 is contained in the culture section 800. [Figure 10] Figure 10 shows the holder 30A according to another embodiment. [Figure 11] Figure 11 shows the holder 30B according to yet another embodiment. [Figure 12] Figure 12 shows the holder 30C according to yet another embodiment. [Figure 13] Figure 13 shows the holder 30D according to yet another embodiment. [Figure 14] Figure 14 shows the spacer support portion 100a according to another embodiment. [Figure 15] FIG. 15 shows a spacer support portion 100b according to yet another embodiment. [Figure 16] FIG. 16 shows a spacer support portion 100c according to yet another embodiment. [Figure 17] FIG. 17 shows a developed view of a medical device 1001 according to another embodiment. [Figure 18] FIG. 18 shows a developed view of the medical device 1001 based on a perspective different from that of FIG. 17. [Figure 19] FIG. 19 shows a holder 1030 of the medical device 1001. [Figure 20] FIG. 20 shows the holder 1030 of the medical device 1001 based on a perspective different from that of FIG. 19. [Figure 21A] FIG. 21A shows a pressing jig 1900 of the medical device 1001. [Figure 21B] FIG. 21B shows a head portion 1920 of the pressing jig 1900. [Figure 22] FIG. 22 shows the holder 1030 with the pressing jig 1900 attached thereto. [Figure 23] FIG. 23 shows a cross-sectional view of the holder 1030 with the pressing jig 1900 attached thereto, cut between the first arm 1305 and the second arm 1306 so as to pass through the central axis of the head portion 1920. [Figure 24] FIG. 24 shows the holder 1030 with the pressing jig 1900 and the base portion 1010 attached thereto. [Figure 25] FIG. 25 shows the pressing jig 1900 in contact with the base portion 1010. [Figure 26] FIG. 26 shows the pressing jig 1900 in contact with the base portion 1010 based on a perspective different from that of FIG. 25. [Figure 27] FIG. 27 shows a cross-sectional view of the holder 1030 with the pressing jig 1900 and the base portion 1010 attached thereto. [Figure 28]Figure 28 shows a retaining jig 1900A according to another embodiment. [Figure 29] Figure 29 shows a cross-sectional view of the clamping jig 1900A. [Modes for carrying out the invention]

[0014] definition For convenience, the specific terms used in this application are gathered here. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Unless otherwise specified in the context, the singular forms "a," "an," and "the" include plural references.

[0015] The numerical ranges and parameters shown in this invention are approximations, although the numerical values ​​shown in specific examples are described as accurately as possible. However, all numerical values ​​inherently contain certain errors that inevitably arise from the standard deviation observed in each test measurement. Furthermore, the term "about" as used herein generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means that it is within an acceptable standard error, as considered by those skilled in the art.

[0016] The embodiments of the present invention will now be described. The following embodiments are illustrative, and the scope of the present invention is not limited to those shown in the following embodiments. In order to avoid repetition and complexity, explanations of similar content will be omitted as appropriate.

[0017] Medical device 1 Figure 1 shows an exploded view (plan view) of the medical device 1 according to this embodiment. The medical device 1 according to this embodiment comprises a base part 10 having an opening 20 and a holder 30. The holder 30 is used to fix the base part 10 to a culture dish 400. The base part 10 is provided with a flow path 150 that communicates with the opening 20.

[0018] The opening 20 is a through-opening. The base portion 10 may consist of a spacer 200 and a spacer support portion 100 that contacts the spacer 200. The medical device 1 may be disposable.

[0019] Spacer support part 100 The spacer support portion 100 includes a first opening 101. The spacer support portion 100 includes four protrusions 120. The spacer support portion 100 has four grooves 110 on the surface that contacts the spacer 200 (the surface with the protrusions 120). The four grooves 110 are arranged at equal intervals. The grooves 110 are gaps between two protrusions 120. The grooves 110 include an inlet portion 130 and an outlet portion 140. The grooves 110 communicate fluidly with the first opening 101 via the inlet portion 130 and with the outside of the first opening 101 via the outlet portion 140.

[0020] The spacer support portion 100 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic) or a combination thereof, but it is preferable that it be made of a material with high thermal conductivity (e.g., metal (e.g., silicon, aluminum, or stainless steel)) in order to improve the heating and cooling efficiency of the medical device 1 (especially the biological sample 500 in the medical device 1).

[0021] In this embodiment, the shape of the spacer support portion 100 is circular, but it may also be elliptical or a polygon including a quadrilateral. In this embodiment, the shape of the first opening 101 of the spacer support portion 100 is circular, but it may also be elliptical or a polygon including a quadrilateral. The thickness of the spacer support portion 100 and the area of ​​the first opening 101 can be changed according to the biological sample 500 to be cultured.

[0022] Figure 2 shows the spacer support portion 100 on the side that contacts the spacer 200. For ease of explanation, the center point C is clearly indicated at the center of the opening 101, and auxiliary lines X passing through the center point C and auxiliary line Y perpendicular to auxiliary line X and also passing through the center point C are clearly indicated.

[0023] In this embodiment, the four inlets 130 are spaced apart at equal angles with respect to the center point C. The spacer support portion 100 has at least one groove 110, preferably an even number of grooves (e.g., 4, 6, 8, 10, and 12). Because each inlet 130 is spaced apart at equal angles with respect to the center point C, excess embedding material 700 is discharged evenly, and as a result, displacement of the biological sample 500 within the embedding material 700 can be prevented.

[0024] The width and depth of the grooves 110 can be changed according to the type and size of the biological sample 500 and the number of grooves 110. For example, the width of the grooves 110 may be in the range between two values ​​selected from the group consisting of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mm. The depth of the grooves 110 may be in the range between two values ​​selected from the group consisting of 0.3, 0.5, 0.7, and 1.0 mm.

[0025] Spacer 200 The spacer 200 is provided with a second opening 201. The second opening 201 is in fluid communication with the first opening 101.

[0026] The spacer 200 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic), or a combination thereof, but it is preferably made of an elastic material.

[0027] In this embodiment, the shape of the spacer 200 is circular, but it may also be elliptical or a polygon including a quadrilateral. In this embodiment, the shape of the second opening 201 of the spacer 200 is circular, but it may also be elliptical or a polygon including a quadrilateral. The area of ​​the second opening 201 can be changed according to the biological sample 500 to be cultured.

[0028] Holder 30 Figure 3 shows an exploded view (bottom perspective view) of the medical device 1 according to this embodiment. The holder 30 includes a straight body portion 300 having a through hole 301 and a recess 302 at one end of the straight body portion 300 capable of accommodating at least a part of the base portion 10. The through hole 301 is in fluid communication with the opening 20 (first opening 101 and second opening 201). The depth of the recess 302 of the holder 30 may be the same as the thickness of the spacer 200, or it may be shallower than the thickness of the spacer 200. The inner diameter of the recess 302 of the holder 30 may be the same as or approximately the same as the outer diameter of the spacer 200, or it may be larger than the outer diameter of the spacer 200. The spacer 200 may be detachable from the recess 302 of the holder 30 by fitting or screwing, or it may be fixed to the recess 302 of the holder 30 by fixing means (e.g., adhesive).

[0029] The outer diameter of the holder 30 is larger than the outer diameter of the base 10. Furthermore, the outer diameter of the holder 30 is preferably such that the medical device 1 does not detach from the well 401 of the culture dish 400 even when the culture dish 400 is inverted while the medical device 1 is set in the well 401 of the culture dish 400. However, it is not limited to a size that prevents detachment, as long as it is secured to the culture dish by another means. The holder 30 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic), or a combination thereof, but it is preferably made of a material with high thermal conductivity (e.g., metal (e.g., silicon, aluminum, or stainless steel)) in order to improve the heating and cooling efficiency of the medical device 1 (especially the biological sample in the medical device 1).

[0030] Assembled medical device 1 Figure 4 shows a side view of the assembled medical device 1 according to this embodiment, and Figure 5 shows a cross-sectional view of the assembled medical device 1 according to this embodiment. In the assembled medical device 1, the spacer 200 is located between the recess 302 of the holder 30 and the surface of the spacer support portion 100 that has a protrusion 120. Preferably, the flow path 150 is not covered by the holder 30.

[0031] Culture and embedding method The culture embedding method according to this embodiment comprises a setting step of setting the base part 10 and the holder 30 in a culture dish 400, a culture step of culturing the biological sample 500 in the opening 20, an embedding step of embedding the biological sample 500 cultured in the opening 20 with an embedding material 700, and an extraction step of removing the embedded biological sample 500. The culture embedding method according to this embodiment allows for the cultivation of a biological sample 500 using a medical device 1 comprising a base part 10 with an opening 20 and a holder 30 for fixing the base part 10 to a culture dish 400, and the embedding of the biological sample 500 with an embedding material 700.

[0032] 1. Setting process: Set the base unit 10 and holder 30 into the culture dish 400. Figure 6 shows the medical device 1 according to this embodiment set in the well 401 of the culture dish 400. Figure 7 is a partial end view of the medical device 1 set in the well 401 of the culture dish 400. When the medical device 1 is set in the well 401 of the culture dish 400 so that the spacer support portion 100 contacts the bottom of the well 401, a culture compartment 800 is formed, defined by the first opening 101 of the spacer support portion 100 and the well 401 of the culture dish 400.

[0033] The culture dish 400 is preferably a functional culture dish that facilitates the removal of the biological sample 600 from the bottom of the well 401. The functional culture dish is preferably a temperature-responsive culture dish (e.g., Upcell®, Cellseed Co., Ltd.) in which the well 401 is coated with a temperature-responsive polymer (e.g., poly-N-isopropylacrylamide (PIPAAm)), and more preferably a temperature-responsive culture dish in which laminin is further coated on the temperature-responsive polymer coated on the well. In a culture dish 400 in which a laminin-coated temperature-responsive polymer is coated on the bottom of the well 401 of the culture dish 400, the biological sample adheres well to the culture dish 400, allowing it to be cultured in a sheet-like manner, and it becomes easier to remove the cultured sample from the bottom of the well 401. Note that the chemical substance coated on the temperature-responsive polymer is not limited to laminin.

[0034] 2. Culture process for culturing biological samples in the opening. Figure 8 is a partial end view of the medical device 1 and culture dish 400, in which the biological sample 500 and culture medium 600 are contained in the culture compartment 800. The culture medium 600 and biological sample 600 are added to the culture compartment 800 through the through-hole 301 of the holder 30. The biological sample 500 is not particularly limited, but may be a cell sheet created by culture or a piece of biological sample. The biological sample 500 may contain a scaffold (receiving material) such as collagen. The culture medium 600 may contain a drug with pharmacological effects (e.g., an anti-inflammatory agent or other cell-based drug). The conditions in the culture process (type of culture medium, temperature, etc.) can be appropriately changed according to the biological sample 500. After the culture is complete, the culture medium 600 can be removed using a dispensing device such as a pipette. The dispensing device can access the culture medium 600 through the through-hole 301 of the holder 30. After removing the culture medium 600, the biological sample 500 may be washed again with fresh culture medium 600.

[0035] 3. Embedding process: Embedding the cultured biological sample in the opening with embedding material. Figure 9 is a partial end view of the medical device 1 and culture dish 400, in which a biological sample 500 wrapped in embedding material 700 is contained in the culture compartment 800. The embedding material 700 is preferably biocompatible and can be a hydrogel (e.g., gelatin, hyaluronic acid, collagen, polyacrylamide gel, collagen gel, hyaluronic acid crosslinked gel, alginate gel, thermoreversible hydrogel (such as PNIPAM polymer), and PVA gel). The embedding material 700 is filled at a temperature at which it is fluid, but a temperature suitable for culturing the biological sample 500 (e.g., 37°C) is preferred. The embedding material 700 can be filled using a dispensing device such as a pipette. The dispensing device can access the biological sample 500 through the through-hole 301 of the holder 30.

[0036] Excess embedding agent 700 is discharged through the channel 150, resulting in an embedded biological sample 500 with a certain thickness. The thickness of the embedded biological sample 500 can be adjusted by the depth of the groove 100.

[0037] 4. Extraction process for removing embedded biological samples. The embedded biological sample 500 is removed at a temperature below the temperature at which the embedding material 700 hardens (loses its fluidity) (e.g., 4°C). The embedded biological sample 500 can be removed through the through-hole 301 of the holder 30. Alternatively, the embedded biological sample 500 can be removed after the holder 30, the holder 30 and the spacer 200 or medical device 1 have been removed from the culture dish 400. If a temperature-responsive culture dish is used, the embedded biological sample 500 can be peeled from the bottom of the well 401 of the culture dish 400 by heating or cooling the culture dish 400 to an appropriate temperature. If only one side of the biological sample 500 is covered with the embedding material 700, both sides of the biological sample 500 can be covered with the embedding material 700 by performing the above embedding process with the biological sample 500 repositioned in the culture compartment 800 so that the side of the biological sample 500 covered with the embedding material 700 is in contact with the bottom of the well 401 of the culture dish 400.

[0038] When the embedded biological sample 500 is implanted into a living site using a syringe, for example by forming it into a tube, the elasticity of the embedding material 700 causes the biological sample 500 to return to its original shape at the living site. The embedding material 700 becomes fluid due to body temperature and is absorbed by the body.

[0039] Variations of Holder 30 Figure 10 shows a holder 30A according to another embodiment. The holder 30A comprises a straight body portion 300A, a recess 302 provided at one end of the straight body portion 300A, and a circular plate 303A provided at the other end of the straight body portion 300A. The straight body portion 300A has a through hole 301A that passes from the recess 302 through to the circular plate 303A. The outer diameter of the circular plate 303A is larger than that of the straight body portion 300A. The circular plate 303A functions as a support member to assist in removing the holder 30A from the well 401 of the culture dish 400 with fingers or a tool (e.g., tweezers).

[0040] Figure 11 shows a holder 30B according to yet another embodiment. The holder 30B comprises a straight body portion 300B, a recess 302 provided at one end of the straight body portion 300B, and a projection 304B extending from the other end of the straight body portion 300B. The straight body portion 300B has a through hole 301B through the recess 302 to the projection 304B. The cross-sectional area of ​​the projection 304B perpendicular to the axis of the through hole 301B is smaller than that of the straight body portion 300B. Two of the side walls of the projection 304B in Figure 11 are configured to be in contact with the inner wall of the well 401 of the culture dish 400. In one embodiment, only one of the side walls of the projection 304B is configured to be in contact with the inner wall of the well 401 of the culture dish 400. In another embodiment, none of the side walls of the projection 304B are in contact with the inner wall of the well 401 of the culture dish 400. The ratio of the length of the projection 304B to the thickness of the straight body 300B is variable. The projection 304B functions as a support member to assist in removing the holder 30B from the well 401 of the culture dish 400 with fingers or a tool (e.g., tweezers).

[0041] Figure 12 shows a holder 30C according to yet another embodiment. The holder 30C comprises a straight body portion 300B, a recess 302 provided at one end of the straight body portion 300C, and two arms (first arm 305C and second arm 306C) extending from the other end of the straight body portion 300C. The straight body portion 300C has a through hole 301C through the recess 302. Both one side wall of the first arm 305C and one side wall of the second arm 306C in Figure 12 are configured to contact the inner wall of the well 401 of the culture dish 400. In one embodiment, neither arm may be configured to contact the inner wall of the well 401 of the culture dish 400. The ratio of the length of the arms to the thickness of the straight body portion 300C is variable. The length of the first arm 305C may be different from the length of the second arm 306C, but it is preferable that they be the same. The first arm 305C and the second arm 306C function as support members to assist in removing the holder 30C from the well 401 of the culture dish 400 with fingers or a tool (e.g., tweezers). The holder 30C allows for a thinner straight section 300C, making it easier to access the biological sample 500 through the through-hole 301C.

[0042] Figure 13 shows a holder 30D according to yet another embodiment. The holder 30D comprises a straight body 300D having two flat side walls 330D, a recess 302 at one end of the straight body 300D, two arms (first arm 305D and second arm 306D) extending from the other end of the straight body 300D, and a protruding portion 340D extending from one end of each arm. The straight body 300D has a through hole 301D passing through the recess 302. The two flat side walls 330D do not contact the inner wall of the well 401 of the culture dish 400. Each arm 305D, 306D has a first arc side wall 310D and a second arc side wall 320D, the second arc side wall 320D protruding more than the first arc side wall 310D in a direction perpendicular to the central axis of the through hole 301D. The first arc-shaped side wall 310D does not contact the inner wall of the well 401 of the culture dish 400. The second arc-shaped side wall 320D contacts the inner wall of the well 401 of the culture dish 400. The longest distance from the central axis of the through hole 301D to the first arc-shaped side wall 310D is shorter than the shortest distance from the central axis to the second arc-shaped side wall 320D. The protruding portion 340D extends from one end of the second arc-shaped side wall 320D (the end opposite to the end where the first arc-shaped side wall 310D connects to the second arc-shaped side wall 320D) in a direction perpendicular to the central axis. The ratio of the area of ​​the second arc-shaped side wall 320D to the area of ​​the first arc-shaped side wall 310D can be changed as appropriate. By changing the area of ​​the second arc-shaped side wall 320D, the force required to remove the holder 30D from the well 401 of the culture dish 400 can be changed. The length of the first arm 305D may differ from the length of the second arm 306D, but it is preferable that they be the same. Each arm 305D, 306D and the overhang 340D function as a support member to assist in removing the holder 30D from the well 401 of the culture dish 400 with fingers or a tool (e.g., tweezers). The holder 30D can have a thinner straight section 300D, making it easier to access the biological sample 500 through the through-hole 301D. The first arm 305D and the second arm 306D may not have a first arc side wall 310D, and may consist only of a second arc side wall 320D.

[0043] Variations of the spacer support part 100 Figure 14 shows a spacer support 100a according to another embodiment. The width of the inlet 130a of the spacer support 100a is smaller than the width of the corresponding outlet 140a (in other words, the width of the groove 110a widens from the inlet 130a to each outlet 140a). The spacer support 100a can facilitate the discharge of the embedding material 700 and suppress the outflow of the biological sample 500.

[0044] Figure 15 shows a spacer support portion 100b according to yet another embodiment. The spacer support portion 100b includes a set of notches 160b provided on the exit portion 140b side of the groove 110b. The spacer support portion 100b allows the length of the groove 110b to be shortened, making it easier to discharge the embedding material 700.

[0045] Figure 16 shows a spacer support 100c according to yet another embodiment. The spacer support 100c comprises a circular spacer support 102c having a first opening 101c, and two rectangular spacer support 103c extending in a direction perpendicular to the central axis of the first opening 101c. The spacer support 100c has four grooves 110c on the surface that contacts the spacer 200 (the surface having a protrusion 120c composed of two first protrusions 121c and two second protrusions 122c). Each groove 110c is a gap between the first protrusion 121c and the second protrusion 122c. The grooves 110c have an inlet 130c and an outlet 140c. The groove 110c comprises a first groove 111c that communicates fluidly with the inlet 130c, a second groove 112c that communicates fluidly with the outlet 140c, and a bend 114c between the first groove 111c and the second groove 112c where the direction of fluid flow changes. The outlet 140c is connected to a partial groove 113c. The partial groove 113c has walls and a bottom surface derived from the rectangular spacer support 102c, but does not have walls derived from the circular spacer support 101c. The partial groove 113c can distribute the positions where the embedding material 700 flows out onto the bottom surface of the well 401 of the culture dish 400, thereby suppressing clogging of the embedding material 700 at the outlet 140c, even when the viscosity of the embedding material 700 is high.

[0046] Medical device 1001 Figures 17 and 18 show exploded views of a medical device 1001 according to another embodiment. The medical device 1001 according to this embodiment comprises a base portion 1010 having an opening 1020, a holder 1030, and a pressing jig 1900. The base portion 1010 according to this embodiment does not have a spacer, but it may have a spacer. The base portion 1010 has substantially the same structure as the spacer support portion 100c, but it may be a different spacer support portion. In the following, only the differences between medical devices 1 and 1001 will be shown.

[0047] Holder 1030 Figures 19 and 20 show a holder 1030 according to this embodiment. The holder 1030 comprises (1) a straight body portion 1300 having two flat side walls 1330, a surface 1300A, a back surface 1300B located opposite to the surface 1300A, and a through hole 1301 extending from the surface 1300A to the back surface 1300B; (2) two arms (first arm 1305 and second arm 1306) extending from the surface 1300A so as to sandwich the through hole 1301; (3) protruding portions 1340 extending from the tips of each arm in a direction perpendicular to and away from the central axis of the through hole 1301; and (4) two legs (first leg 1307 and second leg 1308) extending from the back surface 1300B so as to sandwich the through hole 1301.

[0048] The first arm 1305 and the first leg 1307 are provided with a common first arc side wall 1310. The second arm 1306 and the second leg 1308 are provided with a common first arc side wall 1310. Each arm 1305, 1306 is further provided with a second arc side wall 1320, the second arc side wall 1320 protruding more than the first arc side wall 1310 in a direction perpendicular to and away from the central axis of the through hole 1301. The holder 1030 is provided with a recess 1302 defined by the back surface 1300B, the first leg 1307 and the second leg 1308, the recess 1302 being able to accommodate at least a portion of the base 1010.

[0049] Pressing jig 1900 Figure 21A shows a pressing jig 1900 according to this embodiment. The pressing jig 1900 comprises a main body 1910, a head portion 1920 protruding from one end of the main body 1910, and two protruding plates 1911 extending from the other end of the main body 1910 in a direction perpendicular to and away from the central axis of the head portion 1920.

[0050] Figure 21B shows the head portion 1920. The head portion 1920 comprises a frustum 1921 having a base and a top surface, a cylindrical base 1922 extending from the base and having the same diameter as the base, a cylindrical tip 1923 protruding from the top surface and having a smaller diameter than the diameter of the top surface, and an annular projection 1924 protruding from the top surface and having an outer shape smaller than the diameter of the top surface and an inner diameter larger than the diameter of the cylindrical tip. Auxiliary line X is parallel to the central axis Z of the head portion 1920 and extends along the side wall of the cylindrical base 1922, and auxiliary line Y extends along the side wall of the frustum 1921. The frustum 1921 has a frustum shape in which its diameter decreases from the base to the top surface, such that there is an angle θ between auxiliary line X and auxiliary line Y. That is, the top surface is approximately parallel to the base surface and has a smaller area than the base surface. The angle θ is, for example, 5 ± 1 degrees, but is not limited to these values. The center of the top surface lies on a line extending perpendicularly to the center of the bottom surface. The diameter of the cylindrical tip 1923 is smaller than the diameter of the opening 1020 of the base 1010. The diameter of the head 1920 is approximately the same as the diameter of the through hole 1301 of the straight body 1300. It is preferable that the central axis of the frustocone 1921, the central axis of the cylindrical base 1922, the central axis of the cylindrical tip 1923, and the central axis of the annular projection 1924 are located approximately on the same line. The cylindrical tip 1923 protrudes more than the annular projection 1924. The projection plates 1911 are not essential, but it is preferable that the retaining jig 1900 has two projection plates 1911 so that the retaining jig 1900 can be easily removed from the holder 1030.

[0051] The pressing jig 1900 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic) or a combination thereof, but it is preferable that it be made of a material with high thermal conductivity (e.g., metal (e.g., silicon, aluminum, or stainless steel)) in order to improve the heating and cooling efficiency of the medical device 1001 (especially the biological sample 500 in the medical device 1001).

[0052] Figure 22 shows the holder 1030 with the clamping jig 1900 attached. Figure 23 shows a cross-sectional view of the holder 1030 with the clamping jig 1900 attached, cut between the first arm 1305 and the second arm 1306 so as to pass through the central axis of the head portion 1920. In the cross-sectional view shown in Figure 23, the first arm 1305 and the first leg 1307 are shown behind the clamping jig 1900. The clamping jig 1900 is inserted into the straight body portion 1300 from between the first arm 1305 and the second arm 1306 so as to insert the head portion 1920 of the clamping jig 1900 into the through hole 1301 of the straight body portion 1300.

[0053] The body 1910 of the pressing jig 1900 in this embodiment is a rectangular parallelepiped, but it may have other shapes. For example, the shape of the body of a pressing jig suitable for holders 30, 30A, and 30B is a cylinder suitable for the shape of the respective through holes 301, 301A, and 301B. In other words, the pressing jig must be sized to be insertable into the through holes of the holder and must have at least a head portion and a body. The head portion and body may be integrated.

[0054] The thickness of the clamping jig 1900 is preferably the same as or slightly less than the distance between the first arm 1305 and the second arm 1306. The height of the clamping jig 1900 may be the same as, greater than, or less than the height of the first arm 1305 and the second arm 1306. The width of the clamping jig 1900 can be any size as long as it can fit into the well 401 of the culture dish 400. Parts of the head portion 1920, in particular the cylindrical tip portion 1923 and the annular projection portion 1924, pass through the through hole 1301 of the straight body portion 1300 and protrude from the back surface 1300B of the straight body portion 1300.

[0055] Figure 24 shows the holder 1030 with the pressing jig 1900 and base 1010 attached. Figures 25 and 26 show the pressing jig 1900 in contact with the base 1010. To clearly show the relationship between the pressing jig 1900 and the base 1010, the holder 1030 is omitted in Figures 25 and 26. Figure 27 shows a cross-sectional view of the holder 1030 with the pressing jig 1900 and base 1010 attached. The base 1010 is provided between the first leg 1307 and the second leg 1308 such that the protrusion 120c faces the back surface 1300B of the base 1010. The annular projection 1924 of the head 1920 is in contact with the protrusion 120c. The cylindrical tip 1923 of the head 1920 is partially inserted into the opening 1020 of the base 1010.

[0056] The embedding material 700 present in the culture compartment defined by the culture dish 400 and the opening 1020 is discharged through the groove in the base 1010 when the cylindrical tip 1923 of the head 1920 is inserted into the opening 1020, thereby removing any excess embedding material 700. Embedding material 700 not located near the groove in the base 1010 moves into the groove in the base 1010 through the annular groove defined between the cylindrical tip 1923 and the annular projection 1924. The annular projection 1924 is not essential, but it is preferable that the head 1920 be equipped with the annular projection 1924 in order to discharge the embedding material 700 quickly and efficiently. By using the holding jig 1900, the embedding material 700 can be discharged efficiently and quickly, and the embedded biological sample 500 can be made thinner. The amount of embedding material 700 discharged can be varied by the diameter of the cylindrical tip 1923 and the length of the cylindrical tip 1923 protruding from the annular projection 1924.

[0057] Pressing jig 1900A Figure 28 shows a pressing jig 1900A according to another embodiment. The portion showing multiple parallel thin lines indicates that it is made of transparent material. Figure 29 shows a cross-sectional view of the pressing jig 1900A. Below, only the differences between the pressing jig 1900 and 1900A are shown.

[0058] The pressing jig 1900A comprises a main body 1910A, a head portion 1920A protruding from one end of the main body 1910A, two protruding plates 1911A extending from the other end of the main body 1910A in a direction perpendicular to and away from the central axis of the head portion 1920A, and a transparent body 1912A that penetrates the main body 1910A along the central axis of the head portion 1920A. In this embodiment, the transparent body 1912A also serves as the cylindrical tip portion 1923A of the head portion 1920A, but may also serve as a part of the cylindrical tip portion 1923A. Through the transparent body 1912A, the embedding material 700 and the biological sample 500 present in the culture compartment defined by the culture dish 400 and the opening 1020 can be directly observed. In this embodiment, the transparent body 1912A has a cylindrical shape, but may have other shapes (for example, a rectangular prism). Its interior may be hollow. The transparent material 1912A is not particularly limited as long as it is a transparent material, but examples include glass, plastic, acrylic glass, and polycarbonate.

[0059] Culture and embedding method The culture embedding method according to this embodiment comprises a setting step of setting the base part 1010 and the holder 1030 in the culture dish 400; a culture step of culturing the biological sample 500 in the opening 1020; an embedding step of embedding the biological sample 500 cultured in the opening 1020 with embedding material 700; a discharge step of discharging excess embedding material 700 by inserting a pressing jig 1900 into the holder 1030; and a removal step of removing the embedded biological sample 500. The culture embedding method according to this embodiment allows for the discharge of excess embedding material 700, making the embedded biological sample 500 thinner.

[0060] kit The kit according to this embodiment comprises the medical device 1 or 1001 described above. The kit may comprise one medical device 1 or 1001, or multiple medical devices 1 or 1001. The kit may also comprise a culture dish 400. [Explanation of Symbols]

[0061] 1. 10001 Medical Devices 10, 1010 Foundation part 20, 1020 opening 30, 30A, 30B, 30C, 1030 holder 100, 100a, 100b, 100c Spacer support section 101, 101a, 101b, 101c First opening 102c Circular spacer support 103c Rectangular spacer support 110, 110a, 110b, 110c groove 111c First groove 112c Second groove 113c Partial groove 114c Bend section 120, 120a, 120b, 120c protrusions 121c First protrusion 122c Second convex part 130, 130a, 130b, 130c entrance section 140, 140a, 140b, 140c outlet section 150 channels 160b Notch 200 Spacer 201 Second opening 300, 300A, 300B, 300C, 300D, 1300 Straight body 301, 301A, 301B, 301C, 301D, 1301 through hole 302, 1302 recess 303A Circular Plate 304B Protrusion 305C, 305D, 1305 First Arm 306C, 306D, 1306 Second Arm 310D, 1310 First arc side wall 320D, 1320 Second arc side wall 330D, 1330 flat sidewall 340D, 1340 overhang 400 culture dishes 401 Wells in a culture dish 500 biological samples 600 Culture solution 700 Embedding material 800 culture compartments 1300A surface 1300B back side 1307 First Leg 1308 Second Leg 1900, 1900A Pressing jig 1910, 1910A Main Unit 1911, 1911A protruding plate 1912A transparent body 1920, 1920A Head section 1921, 1921A frustum of a cone 1922, 1922A Cylindrical base 1923, 1923A Cylindrical tip 1924, 1924A Annular projection

Claims

1. A foundation with an opening, The base portion is equipped with a holder for fixing it to a culture dish, The aforementioned foundation is provided with a groove, The groove comprises an inlet portion and an outlet portion, The holder comprises a straight body portion having a through hole and a recess at one of the ends of the straight body portion capable of accommodating at least a part of the base portion. The through hole and the opening are in fluid communication. The groove communicates fluidly with the opening via the inlet portion and with the outside of the opening via the outlet portion. A medical device for culturing a biological sample and embedding the biological sample in an embedding material at the aforementioned opening.

2. The medical device according to claim 1, further comprising a spacer that covers the groove.

3. The medical device according to claim 2, wherein the groove has at least two.

4. The medical device according to claim 2, wherein the spacer is made of an elastic material.

5. The system further comprises a pressing jig having a size that can be inserted into the aforementioned through hole, The aforementioned pressing jig comprises a head portion and a body portion, the medical device according to claim 1.

6. A kit comprising the medical device described in any one of claims 1 to 5.

7. A culture and embedding method for culturing a biological sample and embedding the biological sample with an embedding material, using a medical device comprising a base having an opening and a holder for fixing the base to a culture dish, A setting step of setting the base part and the holder in the culture dish, A culture step in which a biological sample is cultured in the aforementioned opening, An embedding step in which the cultured biological sample is embedded in the opening with the embedding material, A discharge step is performed by inserting a pressing jig into the holder to discharge excess embedding material, The process includes an extraction step for removing the embedded biological sample, The holder comprises a straight body portion having a through hole and a recess at one of the ends of the straight body portion capable of accommodating at least a part of the base portion. The through hole and the opening are in fluid communication. Culture embedding method.

8. The culture dish is a temperature-responsive culture dish. The method for embedding culture according to claim 7.

9. The method for embedding culture according to claim 8, wherein the temperature-responsive culture dish has its wells coated with laminin.

10. The aforementioned embedding material is hydrogel. A method for embedding culture according to any one of claims 7 to 9.

11. The aforementioned embedding step involves filling the opening with a hydrogel at a temperature that allows it to flow, The aforementioned removal step involves removing the embedded biological sample at a temperature below the temperature at which the hydrogel solidifies. The method for embedding culture according to claim 10.

12. The temperature of the fluid hydrogel is suitable for culturing the biological sample. The culture treatment method according to claim 11.

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