A biological sample manufacturing device, a peeling tool used therein, and a biological sample manufacturing method.

Abstract

The objective is to provide a device necessary for culturing and extracting biological samples on a culture vessel, as well as a method for producing biological samples. [Solution] A medical device comprising a base with an opening for culturing cells, a holder for fixing the base to a culture vessel, a biosample manufacturing device comprising a peeling tool for separating the peripheral portion of the biological sample from the culture vessel or medical device before embedding with embedding material, and a holding jig for removing excess embedding material, makes it possible to manufacture biological samples without the need for advanced techniques such as slicing with lasers or microtomes.

Description

Technical Field

[0001] The present invention relates to a device necessary for culturing and collecting a biological sample, and a method for producing a biological sample. Background Art

[0002] Regenerative medicine using cell sheets has entered clinical research and clinical application for disease treatment in various organs including the heart, esophagus, articular cartilage, periodontal ligament, cornea, and retina. Various cell sheets have been approved as products for regenerative medicine and the like, and such cell sheets, including epidermal cell-derived cell sheets, limbal epithelium-derived cell sheets, oral mucosa-derived epithelial cell sheets, and skeletal muscle-derived cell sheets, have come to be used in treatment. Particularly in the field of ophthalmology, research is being conducted on the regeneration of the cornea, retina, and optic nerve, and this is expected to provide new options for the treatment of age-related macular degeneration and retinitis pigmentosa.

[0003] Retinal diseases such as age-related macular degeneration and retinitis pigmentosa are diseases that cause decreased visual acuity and may lead to blindness. Although treatment methods such as drug administration have been established, in Japan, age-related macular degeneration accounts for the fourth leading cause of visual impairment and is the top cause of blindness among elderly people aged 60 years or older. However, even in advanced retinal diseases, if the function of photoreceptor cells is maintained, the transplantation of retinal pigment epithelium (RPE) cells can be expected to maintain and recover visual function, and thus extensive research has been conducted in this area.

[0004] In recent years, therapeutic methods involving transplanting a retinal pigment epithelium (RPE) cell sheet derived from human embryonic stem (ES) cells or human induced pluripotent stem (iPS) cells into patients with age-related macular degeneration have been studied. However, RPE cell sheets are extremely fragile, often bend during transplantation, and are difficult to handle, so methods for embedding them in hydrogel have been developed.

[0005] Non-patent document 1 discloses that when RPE cells damaged by neovascularization are removed along with the neovascularization, and the RPE cell sheet embedded in a hydrogel such as gelatin or amniotic membrane is transplanted under the retina in a folded state, it spontaneously unfolds and engrafts. Although this method reduces the time required to prepare the PRE cell sheet and makes transplantation easier, it has not yet been put into practical use.

[0006] Conventional techniques require culturing, cutting, and embedding cell sheets for transplantation, but these processes involve the use of separate devices and other equipment, necessitating expensive equipment and skilled labor. The present inventors have developed a transplantation device and culture / embedding method that allows for the continuous cultivation and embedding of biological samples, and have already published these findings (Patent Documents 1 and 2).

[0007] Patent documents 1 and 2 disclose medical devices that have a space for assisting in the embedding of biological samples in embedding material. Conventionally, it was necessary for a skilled specialist to thinly slice a gelatin block in which a PRE cell sheet was embedded using a microtome to create an RPE cell sheet, but by using this medical device, the process of slicing with a microtome is no longer necessary.

[0008] Furthermore, in cases of advanced age-related macular degeneration, both photoreceptor cells and retinal pigment epithelium are damaged. Therefore, visual recovery cannot be expected unless neuroretina (NR) and retinal pigment epithelial cells are transplanted simultaneously. The present inventors have disclosed a method for producing a composite graft including an NR sheet and a PRE sheet (Patent Document 3). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2020 / 189528 [Patent Document 2] International Publication No. 2022 / 065204 [Patent Document 3] International Publication No. 2020 / 218480 [Non-patent literature]

[0010] [Non-Patent Document 1] Ben M'Barek K. etal., Sci. Transl. Med., 2017, 9(421), eaai7471 [Overview of the project] [Problems that the invention aims to solve]

[0011] The inventions disclosed in Patent Documents 1-3 have made it possible to culture cells within a medical device, embed them in hydrogel, and extract the biological sample as a cell sheet. It was thought that cell sheets combining NR and PRE would also eliminate the need for microtome slicing, and that hydrogel-attached cell sheets could be easily produced. However, in reality, it became clear that most of the cells in the produced cell sheets had undergone cell death and were unusable.

[0012] The inventors investigated the causes of cell death and developed a method for manufacturing cell sheets in a way that prevents cell death. The object of this invention is to provide a method for manufacturing biological samples, a peeling device used therefor, and a biological sample manufacturing device equipped with the peeling device. [Means for solving the problem]

[0013] The present invention relates to a biological sample manufacturing device including a peeling instrument and a medical device, and to a biological sample manufacturing method. (1) A peeling instrument for peeling a biological sample from a medical device comprising a base having an opening and a holder for fixing the base to a culture vessel, wherein the medical device performs culture of a biological sample and embedding of the biological sample in an embedding material at the opening, wherein the peeling instrument comprises a holder-fitting portion that fits into the holder of the medical device, and is configured to rotate integrally with the medical device by rotating the holder-fitting portion. By equipping a medical device with a peeling mechanism, it is possible to obtain a sheet-like biological sample without causing cell death.

[0014] (2) A biological sample manufacturing device comprising a medical device and a peeling instrument, wherein the medical device comprises a base portion having an opening and a holder for fixing the base portion to a culture vessel, the base portion having a groove, the groove having an inlet portion and an outlet portion, the groove having fluid communication with the opening via the inlet portion and fluid communication with the outside of the opening via the outlet portion, and the device performing the cultivation of a biological sample and the embedding of the biological sample with an embedding material in the opening, and the peeling instrument comprising a holder fitting portion that fits into the holder, and the peeling instrument configured to rotate integrally with the medical device by rotating the holder fitting portion. A biological sample manufacturing device equipped with a peeling tool for culturing biological samples and embedding them in an embedding material allows the entire process, from culturing the biological sample to peeling off the sheet-like biological sample, to be carried out consistently on a single culture vessel.

[0015] (3) A method for manufacturing a biological sample, comprising a biological sample manufacturing device equipped with a medical device, a peeling instrument, and a pressing jig, wherein the medical device comprises a base portion having an opening and a holder for fixing the base portion to a culture container, the peeling instrument comprises a holder fitting portion that fits into the holder of the medical device, the method comprising a setting step of setting the base portion and the holder of the medical device in the culture container, a culture step of culturing the biological sample in the opening, a peeling step of fitting the holder fitting portion of the peeling instrument into the holder and rotating the peeling instrument and the medical device together to peel off the peripheral portion of the biological sample from the culture container, an embedding step of embedding the cultured biological sample in the opening with an embedding material, a discharge step of discharging excess embedding material by inserting a pressing jig into the holder, and a discharge step of removing the peeled biological sample. After setting a medical device in a culture vessel and culturing a biological sample, before embedding with an embedding material, the biological sample adhering to the sidewall or the like of the culture vessel is mechanically peeled off with a peeling instrument, whereby a biological sample can be obtained without causing cell death even when embedded with the embedding material.

[0016] (4) A biological sample production kit comprising a medical device consisting of a base portion and a holder, a peeling instrument, a pressing jig, and a culture vessel. By using a biological sample production kit comprising a medical device, a peeling instrument, a pressing jig, and a culture vessel, the processes from culturing to recovering a biological sample can be consistently performed in a single culture vessel. [BRIEF DESCRIPTION OF DRAWINGS]

[0017] [Figure 1A] A perspective view of the medical device according to Embodiment 1. [Figure 1B] A perspective view of a spacer support portion of the medical device according to Embodiment 1. [Figure 2] A cross-sectional view of the medical device according to Embodiment 1 set in a culture vessel. [Figure 3A] A perspective view of the peeling instrument according to Embodiment 1. [Figure 3B] A diagram schematically illustrating a method of using the peeling instrument according to Embodiment 1. [Figure 4A] A perspective view of the pressing jig for use in the medical device according to Embodiment 1. [Figure 4B] A front view of a head portion of the pressing jig for use in the medical device according to Embodiment 1. [Figure 5A] An assembly drawing of the medical device and the pressing jig according to Embodiment 1. [Figure 5B] A cross-sectional view showing a state where the pressing jig is inserted into the medical device according to Embodiment 1 set in a culture vessel. [Figure 6] A diagram schematically showing an example of a biological sample production method. [MODE FOR CARRYING OUT THE INVENTION]

[0018] In this invention, a biological sample refers to cells or cells cultured in a sheet form (cell sheet). The following examples show an example using PRE cells to treat retinal diseases, but it goes without saying that various cells can be used depending on the disease. Furthermore, the target disease is not limited to retinal diseases; any disease can be treated using a cell sheet. Depending on the target disease, the cells to be used and the size of the cell sheet to be manufactured should be selected as appropriate.

[0019] The numerical ranges and parameters shown in this invention are approximations, but the numerical values ​​shown in the 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.

[0020] [Investigating the causes of cell death] The inventors investigated the cause of cell death and hypothesized that when gelatin is applied to cells that have spread flat to cover gaps in the culture plane during culture, even slight mechanical strain applied when the gelatin-covered cells are subsequently isolated exceeds the tolerance range of the flat-extended cell membrane, causing it to tear and leading to cell death. This hypothesis is consistent with previous verification results, as (1) when a cultured cell sheet is peeled off, the cell sheet shrinks, (2) if the gelatin is dissolved without peeling after application, cell death does not occur, and (3) cell death occurs immediately after mechanical peeling with gelatin applied. Furthermore, based on this hypothesis, they formulated a new hypothesis and conducted experiments to see if peeling the periphery of the cell sheet from the side wall of the medical device used for culture or the culture vessel before embedding the cell sheet would absorb some strain and prevent cell death. They found that no cell death occurred. Based on the results of their investigation, the inventors revised the method for manufacturing biological samples and developed a peeling device for peeling cell sheets. As a result, it is now possible to perform everything from cell culture to cell sheet preparation within a single culture vessel.

[0021] The culture equipment and instruments shown below are examples, and it goes without saying that equipment and instruments with similar functions can be used. The size, angle, etc. of the peeling instrument disclosed below can also be applied to the medical devices disclosed by the present inventors in Patent Documents 1 to 3, by appropriately changing them.

[0022] The biological sample manufacturing device according to this embodiment consists of a medical device comprising a holder used when culturing a biological sample, a base part (spacer, spacer support part), a pressing jig for removing excess embedding material, and a peeling device for separating the peripheral portion of the biological sample from the culture vessel. The biological sample manufacturing kit may also include a culture vessel, a biological sample manufacturing device sized to match the culture vessel, and other necessary components for cultivation.

[0023] The culture method according to this embodiment includes a setting step of setting the base and holder in a culture vessel, a culture step of culturing a biological sample in an opening provided in the base, a peeling step of peeling the peripheral portion of the biological sample from the culture vessel by fitting the holder fitting portion of the peeling device into the holder and rotating the peeling device and medical device together, an embedding step of embedding the biological sample with embedding material, a discharge step of discharging excess embedding material by inserting a pressing jig into the holder, and a removal step of removing the embedded biological sample. The instruments used for manufacturing cell sheets and the culture method will be described in order below.

[0024] First, the biological sample manufacturing device used in the present invention will be described. The biological sample manufacturing device consists of a medical device comprising a holder and a base, and a peeling instrument and a pressing jig.

[0025] [Medical devices] Figure 1A shows a medical device 1 according to Embodiment 1. The medical device 1 consists of a holder 2 and a base 3. The base 3 further consists of a spacer 31 and a spacer support 32.

[0026] The holder 2 comprises a straight body 202 having two side walls 201, a recess 203 at one end of the straight body 202, two arms (first arm 204 and second arm 205) extending from the other end of the straight body 202, and a protruding portion 206 extending from one end of each arm. The straight body 202 has a through hole 207 passing through the recess 203. The two flat side walls 201 do not contact the inner wall of the well of the culture vessel (not shown). Each arm 204, 205 is shaped to contact the inner wall of the well of the culture vessel. The protruding portion 206 extends from one end of each arm 204, 205 (the end opposite to the straight body 202) in a direction perpendicular to the central axis. Each arm 204, 205 and the protruding portion 206 function as support members to assist in removing the holder 2 from the well of the culture vessel with fingers or a tool (e.g., tweezers). The holder 2 allows for easier access to biological samples through the through-hole 207 by adjusting the thickness of the straight body portion 202.

[0027] The base section 3 consists of a spacer 31 and a spacer support section 32. When the base section 3 is installed on the holder 2, it has an opening 33 that communicates with the through hole 207. The base section 3 is used by being installed on the culture vessel together with the holder 2, and is shaped to be housed in the recess 203 of the holder 2.

[0028] Here, a silicone rubber ring is used as the spacer 31, but any elastic material is preferable as long as it is in close contact with the holder 2 and the spacer support part 32. Furthermore, the spacer 31 has a second opening 302, and the shape of the second opening 302 is acceptable as long as it does not block the through hole 207 of the holder 2 and the first opening 301 of the spacer support part 32.

[0029] Figure 1B shows the spacer support portion 32 of Embodiment 1. The spacer support portion 32 includes a first opening 301. The spacer support portion 32 includes four protrusions 303. The spacer support portion 32 has four grooves 304 on the surface that contacts the spacer 31 (the surface with the protrusions 303). The four grooves 304 are arranged at equal intervals. The grooves 304 are gaps between two protrusions 303. The grooves 304 include an inlet portion 305 and an outlet portion 306. The grooves 304 communicate fluidly with the first opening 301 via the inlet portion 305 and with the outside of the first opening 301 via the outlet portion 306.

[0030] In this embodiment, the shape of the spacer support portion 32 is square, but it may be a polygon including a quadrilateral, or it may be circular or elliptical. In this embodiment, the shape of the first opening 301 of the spacer support portion 32 is circular, but it may be elliptical or a polygon including a quadrilateral. The spacer indicator portion can take on various variations. For example, by providing a notch on the outlet side and shortening the length of the groove, it is possible to create a form that facilitates the discharge of the embedding material. Alternatively, the width of the entrance of the spacer support portion may be made smaller than the width of the corresponding outlet portion; in other words, the width of the groove may be configured to widen from the entrance portion to each outlet portion. By widening the outlet portion, the spacer support portion can facilitate the discharge of the embedding material and suppress the outflow of the biological sample. In addition, the area of ​​the first opening can be changed according to the size of the biological sample to be cultured or the biological sample required for treatment.

[0031] The holder 2, spacer 31, and spacer support 32, which are components of the medical device 1, may be made of materials commonly used in this art, such as glass, metal, elastic materials (e.g., silicone rubber and plastic), or a combination thereof. When using a culture vessel coated with a temperature-responsive polymer, it is preferable to use a material with high thermal conductivity (e.g., metal (e.g., silicon, aluminum, or stainless steel)) to improve the heating and cooling efficiency of the biological sample.

[0032] Figure 2 shows a cross-sectional view of the medical device 1 according to Embodiment 1, set in the well 1000 of the culture vessel. Although only one well is shown here, a culture vessel of an appropriate size can be selected according to the area of ​​the biological sample to be prepared, such as a 12-well plate, a 24-well plate, or a petri dish, and a medical device that matches the size of the well can be used. The spacer support portion 32 has a convex portion 303 on its upper surface, and the medical device 1 is set in the well 1000 of the culture vessel so that the opposite surface is in contact with the bottom of the well 1000. A culture compartment 1010 is formed by the first opening 301 of the spacer support portion 32 and the well 1000 of the culture vessel.

[0033] A functional culture vessel is preferred for which the biological sample can be easily detached from the bottom of the wells. The functional culture vessel is preferably a temperature-responsive culture vessel in which the wells are coated with a temperature-responsive polymer (e.g., poly-N-isopropylacrylamide (PIPAAm)) (e.g., Upcell®, Cellseed Co., Ltd.), and more preferably a temperature-responsive culture vessel in which an extracellular matrix (ECM) protein such as laminin, a polycationic polymer such as poly-L-lysine, peptides, synthetic matrix, FBS, etc., is further coated on the temperature-responsive polymer coating the wells can be used. By coating the culture vessel in which the wells are coated with a temperature-responsive polymer with a substance that enhances the adhesion of these cells, it is possible to culture cells without them detaching during the culture period.

[0034] Figure 2 is a cross-sectional view of the holder 2 of the medical device 1 set in well 1000 of the culture vessel at the center line (A in Figure 1). A biological sample, such as a cell sample, is seeded and cultured in the culture compartment 1010 along with the culture medium through the through-hole 207 and opening 33 of the holder 2. The culture compartment 1010 is connected to the area outside the culture compartment 1010 of the well 1000 by a groove 304 provided in the spacer support part 32.

[0035] The culture medium used to cultivate the biological sample may contain a scaffold (supporting material) such as collagen. The culture medium may also contain pharmacologically active drugs (e.g., anti-inflammatory agents or other cell-based therapies). The conditions in the culture process (type of culture medium, temperature, etc.) can be appropriately changed depending on the biological sample. After the culture is complete, the culture medium can be removed using a dispensing device such as a pipette. The dispensing device can access the culture medium through the through-hole 207 of the holder 2. After removing the culture medium, the biological sample may be further washed with fresh culture medium or physiological saline.

[0036] [Peeling tools] After culturing for a certain period, the cells are cultured in a sheet-like form. A peeling process is then performed to mechanically detach the cells that have adhered to and proliferated on the edges of the culture vessel and spacer support. The peeling tool 4 used in the peeling process is equipped with a gripping part 401 and a holder fitting part 402 (Figure 3A). The holder fitting part 402 is sized and shaped to fit into the upper space of the first arm 204 and the second arm 205 of the holder 2. Here, the holder fitting part 402 is shaped to fit into the upper space formed by the first arm 204 and the second arm 205 of the holder 2, but as long as the force generated by rotating the peeling tool 4 is transmitted to the holder 2, it can be shorter in length, or longer to reach the top surface of the straight body part 202, or any other size and shape is acceptable.

[0037] Figure 3B schematically illustrates the method of using the delamination device 4. For clarity, the thickness of the culture vessel well 1000 is ignored, and only the inner wall is shown. The delamination device 4 is used by fitting the holder fitting part 402 into the upper space between the first arm 204 and the second arm 205 of the holder 2 of the medical device 1. When the operator grasps the gripping part 401 and slowly rotates it along the well, the medical device 1 rotates within the well 1000 together with the delamination device 4. As it rotates, the cells that were adhering to and proliferating in the well and spacer support are mechanically detached and cut from the cell sheet. As a result, the sheet-like cells that were proliferating in the culture section 1010 are separated from the cells that were proliferating in other areas. Performing the delamination process makes it possible to suppress distortion caused by the solidification of the hydrogel, which causes cell death during embedding.

[0038] The peeling instrument 4 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic), or a combination thereof. Since the peeling instrument does not come into direct contact with the culture compartment, it is intended for repeated use. Therefore, it is preferable that the material be one that can be repeatedly sterilized.

[0039] [Pressing jig] After the peeling process, the embedding process is performed. Figure 4A shows a pressing jig 5 according to this embodiment used in the embedding process. The pressing jig 5 comprises a main body 501, a head portion 502 protruding from one end of the main body, and two protruding plates 503 extending from the other end of the main body 501 in a direction perpendicular to the central axis of the head portion and away from the central axis.

[0040] Figure 4B shows the head portion 502. The head portion 502 has a frustum of a cone 504 with a base and a top surface, a cylindrical base 505 extending from the frustum of a cone and having the same diameter as the top and base surfaces, and an annular projection 507 and a cylindrical tip 506 on the opposite side of the cylindrical base with the frustum of a cone 204 in between. The cylindrical tip 506 protrudes from the annular projection 507 and has a smaller diameter than the annular projection. Auxiliary line X is parallel to the central axis Z of the head portion 502 and extends along the side wall of the cylindrical base 505, and auxiliary line Y extends along the side wall of the frustum of a cone 504. The frustum of a cone 504 has a frustum shape in which its diameter decreases toward the cylindrical tip side, 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 toward the cylindrical tip side. The angle θ is, for example, 5 ± 1 degrees, but is not limited to these values. The center of the top surface of the frustum of the cone lies on a line extending perpendicularly to the center of the base. The diameter of the cylindrical tip 506 is smaller than the diameter of the opening 301 of the spacer support portion 32 of the base. The diameter of the head portion 502 is approximately the same as the diameter of the through hole 207 of the straight body portion 202. It is preferable that the central axis of the frustum of the cone 504, the central axis of the cylindrical base 505, the central axis of the cylindrical tip 506, and the central axis of the annular projection 507 are located on approximately the same line. The cylindrical tip 506 protrudes more than the annular projection 507. The projection plates 503 are not essential, but it is preferable to have two projection plates 503 so that the retaining jig 5 can be easily removed from the holder 2.

[0041] The pressing jig 5 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 and the biological sample cultured in the medical device.

[0042] Figure 5A is a schematic diagram showing how the pressing jig 5, holder 2, and base part 3 (spacer 31, spacer support part 32) are assembled, and Figure 5B is a central cross-sectional view showing the pressing jig attached to the holder 2 which is mounted on the well 1000 of the culture vessel. A biological sample is cultured in the culture compartment 1010 formed by the medical device 1 consisting of the holder 2, spacer 31, and spacer support part 32 and the well 1000 of the culture vessel. After cutting and peeling with a peeling instrument, an embedding material is added, and the pressing jig 5 is inserted between the first and second arms 204 and 205 of the holder 2.

[0043] The cross-sectional view shown in Figure 5B shows the head portion 502 of the pressing jig 5 inserted from between the first arm 204 and the second arm 205 toward the straight body portion 202 so as to be inserted into the through hole 207 of the straight body portion 202. The thickness of the body 51 of the pressing jig 5 is preferably the same as or slightly less than the distance between the first arm 204 and the second arm 205. The height of the pressing jig 5 may be the same as the height of the first arm 204 and the second arm 205, or it may be higher or lower, as long as it can push out excess embedding material. Part of the head portion 502, in particular the cylindrical tip portion 506 and the annular projection portion 507, pass through the through hole 207 of the straight body portion 202 and protrude from the back surface of the straight body portion 202.

[0044] The embedding material present in the culture compartment 1010, defined by the wells 1000 and opening 33 of the culture vessel, is discharged through the groove in the base 3 when the cylindrical tip 506 of the head 502 is inserted into the opening 33. Embedding material not located near the groove in the base 3 moves into the groove in the base 3 through the annular groove defined between the cylindrical tip 506 and the annular projection 507. The annular projection 507 is not essential, but it is preferable that the head 502 be equipped with the annular projection 507 in order to discharge the embedding material quickly and efficiently. By using the holding jig 5, the embedding material can be discharged efficiently and quickly, and the embedded biological sample can be made thinner.

[0045] [Example 1: Method for producing a cell sheet] A method for producing a cell sheet using the biological sample production device of Embodiment 1 will be described. While the preparation of a PRE sheet will be described in detail here, other sheets can be produced in the same manner. The following numbers correspond to each step in Figure 6. 1. Prepare a 24-well cell culture plate (Upcell 24-well plate, CellSeed) with a temperature-responsive polymer immobilized on its surface. 2. Place 500uL / well of coating solution (ECM) into a 2.24-well plate and leave it standing in a 37°C incubator for at least 12 hours to coat the surface of the equipment. <day1> 3-1. Detach RPE cells using 0.25% Trypsin EDTA. 3-2. Count the cells and collect them by centrifugation. 3-3. The cells are 5 × 10 4 Adjust the volume to 10 μL per cell. 3-4. Completely remove the coating liquid from the 24-well plate. 3-5. Set the holder (fixture) and base (sillicone rubber ring and microculture chip) in a 24-well plate. 4-1. 5 x 10 in the central circle 4 Seed cells at a rate of 10 μL and placed in an incubator. 4-2. After 30 minutes to 1 hour, add 450 μL of culture medium. <day2> 4-3. The day after sowing, replace with maintenance medium. <day3> 5. Insert the peeling tool into the holder and rotate it 180 degrees. Leave at 6-1.4°C for 30-60 minutes. 6-2. Remove the sheet by pipetting. 6-3. Stop when 80-90% of the sheet has peeled off, without completely removing it. Spread the sheet while removing the culture medium, and remove all of the culture medium. 7-1. Prepare 10% gelatin (medizelatin, nippi) in advance and warm it to 37°C. 7-2. Place the plate on a preheated plate heater and slowly pour 100 μL of gelatin onto the cell sheet. 8-1. Using a preheated pressing jig, extrude the gelatin and firmly press it into the holder. Leave at 8-2.4°C for 20 minutes. 9-1. Remove the holding fixture from the holder. Remove the holder and detach the base from the well. Carefully remove the gelatin-embedded cell sheet from the base and trim off any excess gelatin.

[0046] Although 7% FBS is used as the coating solution here, various components and concentrations of coating agents can be used depending on the adhesion between the cells and the culture equipment. Other coating agents commonly used in cell culture can be selected and used, such as laminin, fibronectin, collagen, gelatin, ε-poly-L-lysine, and VitroCol(trademark) collagen (Advanced BioMatrix).

[0047] As the embedding material, gelatin is used here as the hydrogel, but biocompatible materials can preferably be used. Examples of such hydrogels include, in addition to gelatin, hyaluronic acid, collagen, polyacrylamide gel, collagen gel, hyaluronic acid crosslinked gel, alginate gel, thermoreversible hydrogel (such as PNIPAM polymer, and PVA gel), etc.

[0048] The culture medium composition used here is as follows; when simply referred to as "culture medium," it means a mixture of adhesion medium and maintenance medium in a 1:1 ratio, to which Y27632 is added at a final concentration of 10 μM. Adhesion medium: DMEM / F12 450mL, FBS 50mL, PC / SM 5mL Maintenance medium: DMEM 350mL, F12 Ham 150mL, L-glutamin 5mL, PC / SM 5mL, B27 10mL

[0049] As described above, by using the peeling device shown in this embodiment, it is possible to peel off the cells as a cell sheet without causing cell death. Furthermore, using this method, it is possible to manufacture cells from culture to cell sheet collection in a single container without requiring advanced technology. [Explanation of Symbols]

[0050] 1…Medical devices 2…Holder 3…Foundation part 4…Peeling tools 5…Pressing jig 31...Spacer 32...Spacer support part 33…Opening 201…Side wall 202...straight body part 203…recess 204, 205...arms (204...first arm, 205...second arm) 206...Protruding section 207…Through hole 301...First opening 302...Second opening 303... protruding part 304… Groove 305... Entrance 306...Exit part 401...Gripping part 402...Holder fitting part 501...Main unit 502... Head section 503...Protruding plate 504...Truncated cone 505... Base of the cylinder 506...Cylinder tip 507... Annular projection 1000...wells of the culture vessel 1010...Culture compartment

Claims

1. A peeling device for peeling a biological sample from a medical device comprising a base having an opening and a holder for fixing the base to a culture vessel, wherein the biological sample is cultured and embedded in an embedding material at the opening, The aforementioned peeling device The medical device is equipped with a holder fitting portion that fits into the holder, A peeling instrument configured to rotate integrally with the medical device by rotating the holder mating portion.

2. The peeling device according to claim 1, wherein the peeling device comprises a gripping portion that is integrally formed with the holder fitting portion.

3. A biological sample preparation device comprising a medical device and a peeling instrument, The aforementioned medical device, A foundation with an opening, The base portion is equipped with a holder for fixing it to the culture vessel, The aforementioned base portion is provided with a groove, The groove comprises an inlet portion and an outlet portion, The groove communicates fluidly with the opening via the inlet portion and with the outside of the opening via the outlet portion. This device performs the cultivation of a biological sample at the aforementioned opening and the embedding of the biological sample with an embedding material. The aforementioned peeling device It is equipped with a holder fitting portion that fits into the aforementioned holder, A biological sample preparation device, which is a peeling instrument configured to rotate integrally with the medical device by rotating the holder mating portion.

4. The biological sample manufacturing device according to claim 3, wherein the peeling device comprises a gripping portion integrally formed with the holder fitting portion.

5. The holder of the medical device 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 portion of the base portion. The biological sample manufacturing device according to claim 3, wherein the through hole and the opening are in fluid communication.

6. The biological sample manufacturing device according to claim 3, further comprising a spacer that covers the groove of the medical device.

7. The medical device further comprises a pressing jig having a size that can be inserted into the through-hole of the aforementioned medical device. The aforementioned pressing jig comprises a head portion and a body portion, the biological sample manufacturing device according to claim 5.

8. A method for producing a biological sample, comprising a biological sample production device equipped with a medical device, a peeling instrument, and a pressing jig, wherein the biological sample is cultured and removed from the biological sample. The medical device comprises a base having an opening and a holder for fixing the base to a culture vessel. The peeling device includes a holder fitting portion that fits into the holder of the medical device, The medical device is set up by setting the base and holder in the culture vessel, A culture step in which a biological sample is cultured in the aforementioned opening, A peeling step is performed by fitting the holder-fitting portion of the peeling instrument into the holder and rotating the peeling instrument and the medical device together to peel the peripheral portion of the biological sample from the culture vessel. An embedding step in which the cultured biological sample is embedded in the opening with an embedding material, A discharge step is performed by inserting a pressing jig into the holder to discharge excess embedding material, A method for producing a biological sample, comprising a step of removing a detached biological sample.

9. The culture vessel is a temperature-responsive culture vessel. The method for producing a biological sample according to claim 8.

10. The method for producing a biological sample according to claim 9, wherein the temperature-responsive culture vessel has its wells coated with a coating agent.

11. The method for producing a biological sample according to claim 10, wherein the coating agent is at least one of an extracellular matrix (ECM) protein, a polycationic polymer, a peptide, a synthetic matrix, or FBS (fetal bovine serum).

12. The aforementioned embedding material is hydrogel. The method for producing a biological sample according to claim 8.

13. The embedding step involves filling the opening with the hydrogel at a temperature that allows it to flow. The method for producing a biological sample according to claim 12, wherein the removal step involves removing the embedded biological sample at a temperature below the temperature at which the hydrogel solidifies.

14. The temperature of the fluid hydrogel is suitable for culturing the biological sample. The method for producing a biological sample according to claim 13.

15. The aforementioned biological sample is a cell, A cell sheet manufactured by the manufacturing method described in any one of claims 8 to 14.

16. A biological sample preparation kit comprising a medical device consisting of a base and a holder, a peeling tool, a holding jig, and a culture vessel.

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