Biological sample production device, detachment tool used therefor, and biological sample production method

Abstract

Provided are: a device necessary for culturing a biological sample in a culture vessel and removing said sample from the culture vessel; and a biological sample production method. With respect to medical devices provided with a base portion having an opening for culturing cells and a holder for fixing the base portion to a culture vessel, there has been a problem in that cell death occurs when the biological sample is embedded in an embedding material and then removed. By detaching the biological sample, before embedding same in the embedding material, using a detachment tool for detaching the peripheral edge of the biological sample from the culture vessel or a medical device, it is possible to produce a biological sample without the need for advanced techniques such as slicing with a laser or a microtome.

Description

Biological specimen production device, peeling instrument used therein, and biological specimen production method

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

[0002] Regenerative medicine using cell sheets has started clinical research and clinical application for the treatment of diseases in various organs including the heart, esophagus, articular cartilage, periodontal ligament, cornea, and retina. Various cell sheets have come to be used in therapy as regenerative medical products approved as such, including epidermal cell-derived cell sheets, limbal-derived epithelial cell sheets, oral mucosa-derived epithelial cell sheets, and skeletal muscle-derived cell sheets. In particular, 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 therapeutic options for 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 including medication have been established, in Japan, age-related macular degeneration accounts for the fourth leading cause of visual impairment, and is the first leading cause of blindness among elderly people aged 60 years or older. However, even for advanced retinal diseases, if the function of photoreceptors is maintained, maintenance and recovery of visual function can be expected through transplantation of retinal pigment epithelium (RPE) cells, and thus many studies have been conducted on this approach.

[0004] In recent years, therapeutic methods for 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 after removing RPE cells damaged by new blood vessels together with the new blood vessels, when an RPE cell sheet embedded in a hydrogel such as gelatin or amnion is transplanted into the subretinal space in a folded state, the sheet spontaneously unfolds and engrafts. Although this method shortens the working time required to produce an RPE cell sheet and reduces the difficulty of transplantation, 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 using 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 an RPE 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 an RPE sheet (Patent Document 3).

[0009] International Publication No. 2020 / 189528, International Publication No. 2022 / 065204, International Publication No. 2020 / 218480

[0010] Ben M'Barek K. etal., Sci. Transl. Med., 2017, 9(421), eaai7471

[0011] The inventions disclosed in Patent Documents 1 to 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 RPE would also eliminate the need for microtome slicing, and that cell sheets with added hydrogel 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 producing cell sheets in a way that prevents cell death. The object of this invention is to provide a method for producing biological samples, a peeling device used therefor, and a biological sample production device equipped with the peeling device.

[0013] The present invention relates to a biological sample manufacturing device including a peeling device and a medical device, and a method for manufacturing a biological sample. (1) A peeling device for peeling a biological sample from a medical device comprising a base part having an opening and a holder for fixing the base part to a culture vessel, wherein the medical device performs culture of the biological sample and embedding of the biological sample in an embedding material at the opening, wherein the peeling device comprises a holder-fitting part that fits into the holder of the medical device, and the medical device and the peeling device are configured to rotate together by fitting the holder-fitting part into the holder and rotating the peeling device. By equipping the medical device with a peeling device, a sheet-like biological sample can be obtained 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 holder having a straight body portion having a through hole and a recess at one end 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 is in fluid communication with the opening via the inlet portion and is in fluid communication with the outside of the opening via the outlet portion, and the device is used for culturing a biological sample and embedding the biological sample in an embedding material at the opening, and the peeling instrument comprises a holder-fitting portion that fits into the holder, and the device is configured such that the medical device and the peeling instrument rotate together by fitting the holder-fitting portion into the holder and rotating the peeling instrument. 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 producing a biological sample, comprising a biological sample production 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, a setting step of setting the medical device, the base portion and the holder in the culture container, a culture step of culturing the biological sample in the opening, and fitting the holder fitting portion of the peeling instrument into the holder, and the peeling instrument A method for producing a biological sample comprising: a peeling step of peeling the peripheral portion of a biological sample from the culture vessel by rotating the peeling device and the medical device together; an embedding step of embedding the cultured biological sample with an embedding material in the opening; a discharge step of discharging excess embedding material by inserting a pressing jig into the holder; and a removal step of removing the peeled biological sample, wherein the holder has 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 part of the base portion, and the through hole and the opening are in fluid communication. A biological sample can be obtained without cell death occurring when embedded with an embedding material by mechanically peeling off the biological sample adhering to the side wall of the culture vessel etc. with a peeling device before embedding with an embedding material by setting the medical device in the culture vessel and culturing the biological sample.

[0016] (4) A biological sample manufacturing kit comprising a medical device consisting of a base and a holder, a peeling tool, a pressing jig, and a culture vessel. By using a biological sample manufacturing kit comprising a medical device, a peeling tool, a pressing jig, and a culture vessel, the entire process from culturing to harvesting of a biological sample can be carried out in a single culture vessel.

[0017] Perspective view of the medical device according to Embodiment 1. Perspective view of the spacer support portion of the medical device according to Embodiment 1. Cross-sectional view of the medical device according to Embodiment 1 set in a culture vessel. Perspective view of the peeling tool according to Embodiment 1. Diagram schematically showing how to use the peeling tool according to Embodiment 1. Perspective view of the pressing jig used with the medical device according to Embodiment 1. Front view of the head portion of the pressing jig used with the medical device according to Embodiment 1. Assembly diagram of the medical device and pressing jig according to Embodiment 1. Cross-sectional view of the medical device according to Embodiment 1 with the pressing jig inserted into the device set in a culture vessel. Diagram schematically showing an example of a biological sample manufacturing method.

[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 RPE 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] [Investigation of the cause 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 the 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 the 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, a new hypothesis was formulated: if the peripheral edge of the cell sheet is peeled off from the side wall of the medical device used for culture or the culture vessel before embedding the cell sheet, some strain may be absorbed and cell death may not occur. Experiments were conducted with this hypothesis and it was 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 in 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 biological samples, 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 instrument into the holder, gripping and rotating the peeling instrument to rotate the peeling instrument 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 Device] 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 portion 202 having two side walls 201, a recess 203 at one end of the straight body portion 202, two arms (first arm 204 and second arm 205) extending from the other end of the straight body portion 202, and a protruding portion 206 extending from one end of each arm. The straight body portion 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 portion 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). By adjusting the thickness of the straight body portion 202 of the holder 2, access to the biological sample through the through-hole 207 becomes easier.

[0027] The base section 3 consists of a spacer 31 and a spacer support section 32. The base section 3 has an opening 33 that communicates with the through hole 207 when it is installed on the holder 2. The base section 3 is used by being installed on a 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 any shape is acceptable as long as the second opening 302 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 has a first opening 301. The spacer support portion 32 has 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 have 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 produced, 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 the 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 in communication with 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 drugs 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] [Removal device] After culturing for a certain period, when the cells have been cultured in a sheet-like form, a removal process is performed to remove the cells that have adhered to and proliferated on the edges of the culture vessel and spacer support by mechanical force. The removal device 4 used in the removal 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 of rotating the removal device 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 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 device 4 may be made of glass, metal, elastic material (e.g., silicone rubber and plastic), or a combination thereof. Since the peeling device 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 the 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 504 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 is located on a line extending perpendicularly to the center of the base. The diameter of the tip of the cylinder 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 cylinder base portion 505, the central axis of the tip of the cylinder 506, and the central axis of the annular projection 507 are located approximately on the same line. The tip of the cylinder 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, an elastic material (for example, silicone rubber and plastic), or a combination thereof. In order to improve the heating and cooling efficiency of the medical device 1 and the biological sample cultured in the medical device, it is preferably made of a material with high thermal conductivity (for example, a metal (e.g., silicon, aluminum or stainless steel)).

[0042] FIG. 5A is a diagram schematically showing how the pressing jig 5, the holder 2, and the base portion 3 (the spacer 31 and the spacer support portion 32) are assembled, and FIG. 5B is a central cross-sectional view showing the pressing jig attached to the holder 2 mounted in the well 1000 of a culture container. A biological sample is cultured in the culture compartment 1010 formed by the medical device 1 consisting of the holder 2, the spacer 31, and the spacer support 32 and the well 1000 of the culture container. 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, 205 of the holder 2.

[0043] The cross-sectional view shown in FIG. 5B shows the pressing jig 5 inserted from between the first arm 204 and the second arm 205 toward the straight body portion 202 such that the head portion 502 of the pressing jig 5 is inserted into the through hole 207 of the straight body portion 202. The thickness of the main body 51 of the pressing jig 5 is preferably equal to or slightly smaller than the distance between the first arm 204 and the second arm 205. The height of the pressing jig 5 may be equal to, higher than, or lower than the height of the first arm 204 and the second arm 205, as long as excess embedding material can be extruded. A part of the head portion 502, particularly the cylindrical tip portion 506 and the annular protruding portion 507, passes through the through hole 207 of the straight body portion 202 and protrudes from the back surface of the straight body portion 202.

[0044] The embedding material present in the culture compartment 1010, defined by the well 1000 and opening 33 of the culture vessel, is discharged through the groove of 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 of the base 3 moves into the groove of 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 Manufacturing Cell Sheets] A method for manufacturing cell sheets using the biological sample manufacturing device of Embodiment 1 will be described. Here, the preparation of RPE sheets will be described in detail, but sheets other than RPE sheets can be manufactured in the same manner. The following numbers correspond to each step in Figure 6. 1. Prepare a 24-well culture plate (Upcell 24-well plate, CellSeed) on which a temperature-responsive polymer is immobilized on the surface of the equipment. 2. Place 500 μL / well of coating solution (ECM) into the 24-well plate and leave it in a 37°C incubator for 12 hours or more to coat the surface of the equipment. <Day 1> 3-1. Detach RPE cells with 0.25% Trypsin EDTA. 3-2. Count the cells and collect them by centrifugation. 3-3. The cells are 5 × 10⁻⁶. ４ Adjust to cells / 10 μL. 3-4. Completely aspirate the coating solution from the 24-well plate. 3-5. Set the holder (fixture) and base (silicone rubber ring and microculture chip) (medical device) in the 24-well plate. 4-1. 5 × 10 in the central circle (opening). ４Seed cells at cells / 10 μL and place in an incubator. 4-2. After 30 minutes to 1 hour, add 450 μL of medium. <Day2> 4-3. On the day after seeding, replace with maintenance medium. <Day3> 5. Fit the detachment tool to the holder and rotate it 180 degrees. 6-1. Leave at 4°C for 30 to 60 minutes. 6-2. Detach the sheet by pipetting. 6-3. Stop when 80 to 90% of the sheet is detached so as not to completely detach the sheet, spread the sheet while removing the medium, and completely remove the medium. 7-1. Prepare 10% gelatin (Medigelatin, Nippi) in advance and warm it to 37°C. 7-2. Place the plate on a pre-warmed plate heater, and slowly add 100 μL of gelatin onto the cell sheet. 8-1. Extrude the gelatin with a pre-warmed pressing jig, and firmly fit it to the holder. 8-2. Leave at 4°C for 20 minutes. 9-1. Remove the pressing jig from the holder. Remove the holder, and remove the base from the well. Carefully remove the gelatin-embedded cell sheet from the base, and trim off excess gelatin.

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

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

[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 450 mL, FBS 50 mL, PC / SM 5 mL Maintenance medium: DMEM 350 mL, F12 Ham 150 mL, L-glutamine 5 mL, PC / SM 5 mL, B27 10 mL

[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.

[0050] 1...Medical device 2...Holder 3...Base 4...Peeling tool 5...Pressing jig 31...Spacer 32...Spacer support 33...Opening 201...Side wall 202...Straight body 203...Recess 204, 205...Arms (204...First arm, 205...Second arm) 206...Protruding part 207...Through hole 301...First opening 302...Second opening 303...Convex part 304...Groove part 305...Inlet part 306...Outlet part 401...Gripping part 402...Holder fitting part 501...Main body 502...Head part 503...Protruding plate 504...Truss of cone 505...Cylinder base 506...Cylinder tip 507...Annular projection 1000...Well of 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 medical device performs culture of a biological sample and embedding of the biological sample in an embedding material at the opening, wherein the peeling device comprises a holder-fitting portion that fits into the holder of the medical device, and the device and the peeling device are configured to rotate together by fitting the holder-fitting portion into the holder and rotating the peeling device.

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 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 holder having a straight body portion having a through hole and a recess at one end 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 is in fluid communication with the opening via the inlet portion and is in fluid communication with the outside of the opening via the outlet portion, and the device is for culturing a biological sample and embedding the biological sample in an embedding material at the opening, and the peeling instrument comprises a holder-fitting portion that fits into the holder, and the peeling instrument is configured such that the medical device and the peeling instrument rotate together by fitting the holder-fitting portion into the holder and rotating the peeling instrument.

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

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

6. The biological sample manufacturing device according to claim 3, further comprising a pressing jig having a size that can be inserted into the through hole of the medical device, wherein the pressing jig comprises a head portion and a body portion.

7. A method for producing a biological sample, comprising a biological sample production device equipped with a medical device, a peeling tool, 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 tool 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 tool into the holder and rotating the peeling tool to rotate the peeling tool and the medical device together, thereby peeling 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 removal step of removing the peeled biological sample. A method for producing a biological sample, wherein 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, and the through hole and the opening are in fluid communication.

8. The method for producing a biological sample according to claim 7, wherein the culture vessel is a temperature-responsive culture vessel.

9. The method for producing a biological sample according to claim 8, wherein the temperature-responsive culture vessel has its wells coated with a coating agent.

10. The method for producing a biological sample according to claim 9, 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).

11. The method for producing a biological sample according to claim 7, wherein the embedding material is a hydrogel.

12. The method for producing a biological sample according to claim 11, wherein the embedding step involves filling the opening with the hydrogel at a temperature at which it is fluid, and the removal step involves removing the embedded biological sample at a temperature below the temperature at which the hydrogel solidifies.

13. The method for producing a biological sample according to claim 12, wherein the temperature of the fluid hydrogel is a temperature suitable for culturing the biological sample.

14. The biological sample is a cell, and the cell sheet is manufactured by the manufacturing method described in any one of claims 7 to 13.

15. A biological sample preparation kit comprising a medical device consisting of a base and a holder, a peeling instrument, a pressing jig, and a culture vessel.

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