JP7068382B2 - Biological tissue cutting method, biological tissue cutting device and biological tissue cutting substrate - Google Patents

Description

The present invention relates to a method for cutting a biological tissue, a biological tissue cutting device, and a substrate for cutting a biological tissue, and more particularly to a technique for finely cutting a soft tissue collected from a living body.

Culture, treatment, testing, etc. are carried out on biological tissues extracted from organisms such as the human body, specifically soft tissues (hereinafter, simply referred to as "tissues"). As a method for culturing a tissue, for example, a CTOS (Cancer tissue-originated Spheroid) method can be mentioned. Tissue treatment includes, for example, enzymatic treatment. Tissue testing includes, for example, drug testing. Prior to culturing, treating, testing, etc., the tissue is cut, thereby processing the tissue into multiple pieces or chunks.

Tissues are generally very soft, and when external forces are applied to them, the tissues are easily deformed. Therefore, it is not easy to cut the tissue finely, especially evenly. Manual chopping of tissue with a scalpel and tweezers limits the degree of fineness and at the same time causes non-negligible crushing in the tissue.

Conventionally, an apparatus for automatically cutting a tissue has been put into practical use, but it is very difficult to cut the tissue finely (for example, with a thickness of 1 mm or less) in such a conventional apparatus. In addition, the conventional device cannot cut the tissue step by step. For example, the sheet obtained by cutting the block-shaped tissue cannot be cut into smaller pieces.

In addition, Patent Document 1 discloses a biological tissue cutting device for slicing an object to be cut. The object to be cut is a columnar complex consisting of tissue and the agarose that surrounds it. The end face of the object to be cut constitutes a vertical plane, and the end face is exposed in the water tank. It is not possible to cut the tissue into small pieces in stages using this device, and in particular, it is not possible to cut the tissue in two dimensions.

Special Table 2010-502983 Gazette

An object of the present disclosure is to realize a technique capable of finely cutting a biological tissue. Alternatively, an object of the present disclosure is to realize a technique capable of cutting a biological tissue while preventing or reducing the crushing of the biological tissue.

The method for cutting a biological tissue according to the present disclosure includes a step of providing a biological tissue on a plate, a step of providing a gel layer covering the biological tissue and its surroundings on the plate, and the gel layer on the plate by a cutting blade. It is characterized by including a step of cutting the biological tissue together with the above.

The biological tissue cutting apparatus according to the present disclosure includes a stage for holding a plate provided with a biological tissue and a cover layer surrounding the biological tissue, and a cutter provided with a blade for cutting the biological tissue together with the cover layer. It is characterized by including.

The biological tissue cutting substrate according to the present disclosure is a plate and a base sheet on which the biological tissue to be cut is placed, which is deformed or cut when the edge of the cutting blade hits the plate. It is characterized by including a base sheet composed of.

According to the present disclosure, biological tissues can be finely cut. Alternatively, according to the present disclosure, it is possible to cut a biological tissue while preventing or reducing the crushing of the biological tissue.

It is a perspective view which shows the biological tissue cutting apparatus which concerns on 1st Embodiment. It is a figure which shows the plate which put the tissue embedding body. It is a figure which shows the state which the sample set is held on the stage. It is a side view which shows the biological tissue cutting apparatus which concerns on 1st Embodiment. It is a figure which shows an example of the cube array manufacturing method. It is a perspective view which shows a part of a cube array. It is a flowchart which shows the biological tissue cutting method which concerns on embodiment. It is a figure which shows the arrangement of a plate in a petri dish. It is a figure which shows the arrangement of a base sheet and a frame on a plate. It is a flowchart which shows the detail of a cutting process. It is a figure which shows the modification of the cutter. It is a figure which shows another example of the cube array manufacturing method. It is a schematic diagram which shows the biological tissue cutting apparatus which concerns on 2nd Embodiment. It is a figure which shows the 1st example of a substrate. It is a figure which shows the 2nd example of a substrate. It is a figure which shows the 3rd example of a substrate. It is a figure which shows the 1st modification of the biological tissue cutting method. It is a figure which shows the 2nd modification of the biological tissue cutting method. It is a perspective view which shows the modification of the holder. It is a perspective view which shows the 1st example of the biological tissue cutting method using a holding member. It is sectional drawing which shows the 1st example of the biological tissue cutting method using a holding member. It is sectional drawing which shows the 2nd example of the biological tissue cutting method using a holding member.

Hereinafter, embodiments will be described with reference to the drawings.

(1) Outline of Embodiment The biological tissue cutting method according to the embodiment includes a preparation step and a cutting step. The preparatory step includes a step of providing the biological tissue on the plate and a step of providing the biological tissue and a gel layer surrounding the biological tissue on the plate. In the cutting step, the biological tissue is cut together with the gel layer by the cutting blade on the plate.

According to the above method, on the plate, the entire biological tissue to be cut is wrapped by the gel layer. The gel layer functions as a cover layer. The gel layer suppresses the deformation and migration of biological tissue. By pressing the blade against the tissue-embedded body containing the biological tissue and the gel layer, the biological tissue is cut together with the gel layer. Tissue implants are usually less likely to collapse than biological tissue or have better morphological retention than biological tissue. By targeting such a tissue-embedded body to be cut, it becomes possible to cut the biological tissue more finely than in the conventional case. At that time, the crushing of biological tissues can be avoided or alleviated. Further, according to the above configuration, since the biological tissue is placed on the plate, it is easy to cut the biological tissue step by step and evenly.

In an embodiment, the plate is a member having a plane (mounting surface) extending in the first horizontal direction and the second horizontal direction. The plate is basically composed of a hard material that does not break or deform when hit by a blade. In embodiments, the gel layer exhibits better morphological retention than biological tissue, which is easier to cut than biological tissue. If a transparent gel layer is used, the biological tissue can be visually recognized from the outside through the gel layer. The gel may be removed from the individual pieces or lumps produced after cutting, but the removal of the gel may be omitted.

In the embodiment, the steps of providing the gel layer include a step of providing a frame surrounding the biological tissue on the plate, a step of pouring a material having fluidity into the frame, and a step of gelling the material poured into the frame. The steps of forming a gel layer and the like are included. According to these steps, the pouring of the material causes the gel layer to naturally adhere to the biological tissue. In other words, the biological tissue and the gel layer are naturally integrated. In addition, the gel layer is in direct or indirect contact with the plate by pouring. Thereby, the deformation suppressing action and the movement suppressing action of the gel layer can be further enhanced. The frame may be made of a material that can be cut (for example, a gel material). In that case, it is not necessary to remove the frame after pouring the fluid material into the frame.

In the embodiment, when the blade is pulled up from the biological tissue after cutting the biological tissue, the lifting of the biological tissue is suppressed by the holding member. This configuration prevents the biological tissue from being pulled up with the blade. The pressing action may be exerted by the weight of the pressing member. The holding member may be mechanically held or driven. Holding members may be provided on one side of the blade, or holding members may be provided on both sides of the blade.

In embodiments, the biological tissue has a flat morphology that extends along the plate. The gel layer has a central portion covering the biological tissue and a peripheral portion surrounding the biological tissue. The perimeter is in direct or indirect contact with the plate. In the embodiment, the cutting step is repeatedly executed. This processes the biological tissue into a two-dimensional cube array. Since the biological tissue on the plate has a flat morphology and its deformation and movement are suppressed by the gel layer, it becomes easy to cut the biological tissue into small pieces. In particular, it becomes easy to cut the biological tissue into small pieces in stages.

In embodiments, a flexible basesheet is provided on the plate and biological tissue is provided on the basesheet. The base sheet is composed of a material that is softer than the plate, for example, a material that is deformed by the contact of the edge of the blade or a material that is cut by the contact. According to the above configuration, even if the plate is made of a hard material, the blade can be easily passed through the biological tissue, so that incomplete cutting of the biological tissue can be avoided. A modification in which the plate itself is made of a material having a certain degree of flexibility is also conceivable. In that case, the plate functions like a cutting board used in cooking.

In embodiments, the plate has a horizontal orientation. Biological tissue is cut by lowering the blade vertically and abutting the edge of the blade against the top surface of the plate while maintaining the vertical posture of the blade. In the embodiment, a very thin blade with a sharp edge is utilized as the blade.

The biological tissue cutting device according to the embodiment includes a stage and a cutter. A stage is a member that holds a plate provided with a cover layer that covers the biological tissue and its surroundings. The cutter comprises a blade that cuts biological tissue along with a cover layer.

According to the above configuration, the cover layer suppresses the deformation and movement of biological tissue. In such a state, the biological tissue is cut by the blade. Therefore, it becomes easy to cut the biological tissue thinly or finely. In addition, it is possible to effectively prevent or reduce the crushing of biological tissue at the time of cutting. Since the biological tissue is placed on the plate, it is possible to cut the biological tissue into small pieces in stages. Further, the cutting direction can be easily changed by changing the direction of the blade with respect to the plate. The cutter with the blade may be moved manually or automatically. The cutter may be provided with a plurality of blades.

A cover layer may be formed by providing a frame surrounding the biological tissue on the plate, pouring the material into the frame, and hardening or gelling the material. Examples of the material constituting the cover layer include gelling materials, resins, and the like. A cover layer with recesses for accommodating biological tissue may be used.

In embodiments, a base sheet is provided between the plate and the biological tissue. The base sheet is composed of a material that deforms or is cut when the edge of the blade hits it. This configuration allows the edges of the blade to pass completely through the biological tissue, thus preventing incomplete cutting of the biological tissue.

In the embodiment, a holding member for suppressing the floating of the biological tissue is provided when the blade is pulled up from the biological tissue after cutting the biological tissue. According to this configuration, it is possible to prevent a part of the biological tissue (and a part of the cover layer) from being pulled up together with the blade. It makes it easier to maintain the alignment of multiple pieces in biological tissue.

In an embodiment, the plate has a horizontal position on the stage. A guide member is provided to allow the cutter to perform vertical movement while maintaining the vertical posture of the blade. In embodiments, the blade has a first horizontal extending edge. A slide mechanism for changing the blade position in the second horizontal direction orthogonal to the first horizontal direction is provided. This configuration facilitates the production of a plurality of strips or cubes having a uniform size.

The biological tissue cutting substrate according to the embodiment includes a plate and a base sheet provided on the plate. The base sheet is a member on which the biological tissue to be cut is placed. The base sheet is composed of a material that is deformed or cut when the edge of the cutting blade is hit. The base sheet is a member for ensuring complete cutting of biological tissue. If the base sheet is pre-placed on the plate, the work of placing the base sheet on the plate prior to cutting the biological tissue becomes unnecessary. If the base sheet is adhered to the plate, the work of removing the sheet pieces adhering to the tissue pieces after cutting the biological tissue becomes unnecessary.

The biological tissue cutting substrate according to the embodiment is provided on a plate and includes a frame surrounding the biological tissue. The frame is composed of a material that can be cut by a cutting blade. This configuration eliminates the need to remove the frame after pouring a fluid material into the frame and curing it.

(2) Details of the Embodiment FIG. 1 schematically shows a biological tissue cutting apparatus according to the first embodiment. The biological tissue cutting device is a device for cutting a tissue (soft tissue) collected from an organism, and in particular, a device for processing the tissue into a cube array described later. The tissue to be processed is, for example, a tumor containing cancer cells, a normal tissue, or the like. Of course, that is just an example. Tissues of animals other than humans may be the target of amputation.

The biological tissue cutting device according to the first embodiment is a device that supports manual cutting of biological tissue. This will be described in detail below. In FIG. 1, the X direction is the first horizontal direction, the Y direction is the second horizontal direction, and the Z direction is the vertical direction (vertical direction).

In FIG. 1, the biological tissue cutting device 10 is roughly classified into a cutting mechanism 11 and a sample set 12. The sample set 12 is composed of a plate 14, a tissue embedding body 15, and the like. The plate 14 is a transparent hard glass plate, specifically a glass slide. The upper surface and the lower surface of the plate 14 are planes extending in the X direction and the Y direction, respectively. The plate 14 is made of a material that is chemically stable and does not break or deform when the edge of the blade 43 abuts during tissue cutting. Seen from above, the shape of the plate 14 is rectangular, specifically rectangular. A circular plate may be utilized. A plate made of resin, metal, or the like may be used. It is also conceivable that the plate 14 is made of a material that is deformed in the surface layer when the edge of the blade 43 abuts.

The tissue-embedded body 15 is composed of a tissue 16 placed on the upper surface of the plate 14 and a gel layer 18 covering the tissue 16 and its surroundings. The structure 16 extends along the upper surface of the plate 14, specifically has a generally uniform thickness and has a flat or sheet-like morphology. Seen from above, the tissue 16 has a rectangular or circular morphology. A slicer or the like may be used in producing the sheet-shaped structure 16. The tissue 16 is very soft, which is easily crushed or deformed by an external force.

The gel layer 18 functions as a cover layer that covers the entire tissue 16 and thus encloses the entire tissue 16. As will be described later, the gel layer 18 is composed of a central portion covering the upper surface of the tissue 16 and a peripheral portion covering the periphery of the tissue 16 when viewed from the Z direction. The central portion of the gel layer 18 is in close contact with the upper surface of the tissue 16, and the peripheral portion of the gel layer 18 is in close contact with the upper surface of the plate 14. In the central portion of the gel layer 18, there is a downward recess formed as a result of pouring, which will be described later, and the tissue 16 is embedded in the recess.

The gel layer 18 and the tissue 16 are integrated to form a tissue-embedded body 15 as a complex. The tissue-embedded body 15 is less deformable than the tissue 16, in other words, has better morphological retention than the tissue 16. In the process of cutting the tissue 16 using the blade 43, the gel layer 18 exerts an action of suppressing the collapse and movement of the tissue 16.

The gel layer 18 is composed of a specific material that gels at a temperature below the melting point. Specifically, the specific material is a material that becomes fluid or liquid by heating and gels by cooling. For example, the temperature at the time of heating is 37 ° C. or higher. For example, the cooling temperature is 4 degrees or more and 10 degrees or less. Each numerical value given in the present specification is an example.

Specifically, the specific material is a material containing gelatin derived from the subcutaneous tissue of pig as a main component. It is also conceivable to use other gelling materials such as gelatin and agarose. Instead of the gel layer 18, a cover layer made of a material other than the gelling material may be provided. For example, such a material may be a resin that can be cut by a blade. The gel layer 18 has transparency, and the tissue 16 existing inside can be visually recognized through the gel layer.

The cutting mechanism 11 includes a base 20, a slide mechanism 22, an L-shaped bracket 28, a holder 34, a cutter 38, and the like. The L-shaped bracket 28 is composed of a horizontal plate 28A and a vertical plate 28B. In FIG. 1, the positioning mechanism is not shown.

The base 20 is a rectangular pedestal that extends in the X and Y directions. One end of the base 20 constitutes the stage 20A. A sheet-shaped holder 34 is arranged on the stage 20A. A slide mechanism 22 is mounted on the other end of the base 20. The slide mechanism 22 is composed of a lower portion 24 and an upper portion 26 in the illustrated configuration example. The lower portion 24 is fixed to the base 20, and the upper portion 26 is fixed to the horizontal plate 28A. The upper part 26 slides relative to the lower part 24. The direction of motion is the X direction. A mechanism for relatively sliding both of the lower portion 24 and the upper portion 26 is provided, but the mechanism is not shown in FIG. The mechanism includes, for example, a plurality of rails, a plurality of sliders, a rack, a pinion, and the like.

The base 20 and the lower portion 24 constitute the fixing mechanism 30, and the upper portion 26 and the L-shaped bracket 28 constitute the movable mechanism 32. The sliding motion of the movable mechanism 32 is shown by X1 in FIG. By adjusting the slide position of the movable mechanism 32, the position of the vertical plate 28B in the X direction is determined. The vertical plate 28B has a vertical surface to which the low friction film 46 is attached. The surface of the low friction film 46 functions as a guide surface. The low friction film 46 is made of, for example, polytetrafluoroethylene which is a fluororesin. The coefficient of friction of the material is very small.

The cutter 38 is composed of a blade 43 and two plates 40 and 42 in the illustrated configuration example. The lower end of the blade 43 is an edge, which cuts the tissue implant 15. The edge corresponds to a straight line parallel to the Y direction. As the blade 43, high stainless steel manufactured by Feather (product number: FH-10) may be used. The thickness of the product is 0.1 mm, which is composed of stainless steel. The surface of the blade 43 is coated, if necessary. The surface of the above product is coated with platinum alloy and resin.

The blade 43 is sandwiched between the two plates 40 and 42. The two plates 40, 42 are fastened to each other, for example, by four screws. The two plates 40, 42 may be made of resin, metal or other material.

In the illustrated example, the back surface 42A of the plate 42 is in close contact with the front surface of the low friction film 46. In that state, the cutter 38 is in the vertical position, that is, the blade 43 is in the vertical position. When the cutter 38 is moving, the front surface of the low friction film 46 functions as a guide surface. The downward and upward movements of the cutter 38 are manually performed so that the back surface 42A of the plate 42 is in perfect contact with the front surface and the edges are kept horizontal. It is shown by Z1 in FIG.

The stage 20A is a horizontal table in the illustrated example, and the holder 34 is arranged therein. The holder 34 is a sheet-like member having an opening 36. For example, the holder 34 is made of resin. The opening 36 has a cross shape in which the vertical groove 36X and the horizontal groove 36Y are combined. The vertical groove 36X and the horizontal groove 36Y each have a form for accommodating the plate 14. When the sample set 12 is arranged in the flute 36X, the longitudinal direction of the plate 14 is parallel to the X direction, and the lateral direction of the plate 14 is parallel to the Y direction. When the sample set 12 is arranged in the lateral groove 36Y, the longitudinal direction of the plate 14 is parallel to the Y direction, and the lateral direction of the plate 14 is parallel to the X direction. The thickness of the holder 34 is thinner than the thickness of the sample set 15, and more specifically, it is equal to or less than the thickness of the plate 14.

The sample set 12 is held by the holder 34, and the sample set 12 is in a predetermined posture. Although a member is provided to ensure the holding of the sample set 12, the illustration thereof is omitted in FIG. Another holding mechanism or holding means may be provided in place of the holder 34.

FIG. 2 shows the tissue implant 15 mounted on the plate 14. (A) shows the upper surface of the plate 14, and (B) shows the cross section of the plate 14. The x direction is the longitudinal direction of the plate 14, and the y direction is the lateral direction of the plate 14. The z direction is the thickness direction of the plate 14.

A rectangular base sheet 44 is arranged on the upper surface of the plate 14. It is composed of a flexible resin sheet, for example it is a silicone sheet. The base sheet 44 has the same or larger size as the tissue 16. The tissue 16 has a size of, for example, 10 mm × 10 mm when viewed from above (see reference numeral 46), and its thickness is, for example, in the range of 1 to 3 mm (see reference numeral 54). The base sheet 44 has, for example, a size of 10 m × 10 mm or more when viewed from above (see reference numeral 48), and its thickness is, for example, 0.5 mm (see reference numeral 52). The diameter of the gel layer 18 is, for example, 25 mm (see reference numeral 50), and the thickness thereof is, for example, 4 mm (see reference numeral 56).

The gel layer 18 includes a central portion 18A that covers the tissue 16 and a peripheral portion 18B that covers the periphery of the tissue 16. As described above, below the central portion 18A, there is a recess that is open to the bottom as a result of pouring, and the tissue 16 is embedded in the recess. By pouring a fluid material, specifically a gelling material, the inner surface of the recess is in close contact with the outer surface of the structure 16, whereby the gel layer 18 and the structure 16 are integrated.

The peripheral portion 18B is in close contact with the portion of the base sheet 44 protruding outward from the structure 16 and is in close contact with the portion existing on the outside of the base sheet 44 on the upper surface of the plate 14. The entire base sheet 44 is covered with the gel layer 18. The form of the gel layer 18 may be a flat plate instead of a disk.

FIG. 3 shows a sample set 12 arranged in the lateral groove 36Y. The opening 36 has a shape in which the vertical groove 36X and the horizontal groove 36Y are combined. One end of the sample set 12 is held down by the belt 60. Both ends of the belt 60 are fixed to the holder 34 by a pair of fixing tools 62 and 64. Even when the sample set 12a is arranged in the vertical groove 36X, one end thereof is suppressed by the belt 60a. Reference numeral 62 indicates a cutting line formed by the blade.

FIG. 4 shows a positioning mechanism 66 included in the cutting mechanism 11. The positioning mechanism 66 is provided straddling between the lower portion 24 and the upper portion 26, and is a mechanism for changing the position of the upper portion 26 in the X direction with respect to the lower portion 24. In the illustrated example, the positioning mechanism 66 has a tubular member 68, a pedestal 70, a knob 72, a shaft 74, a pedestal 76, and the like. The pedestal 70 is fixed to the lower 24. The tubular member 68 is fixed to the pedestal 70. The shaft 74 is rotatably inserted through the tubular member 68. A knob 72 is connected to the end of the shaft 74. The tip of the shaft 74 is rotatably connected to the pedestal 76. A screw structure is provided on the outer surface of the shaft 74, and a screw structure is also provided on the inner surface of the tubular member 68. The two threaded structures are in mesh.

When the knob 72 is rotated, the shaft 74 moves relative to the tubular member 68. As a result, the upper part 26 slides with respect to the lower part 24. For example, at 0.1 mm, 0.25 mm, 0.5 mm pitch, 0.75 mm pitch, or 1.0 mm pitch, the upper part 26 is gradually slid with respect to the lower part 24, and the cutter 38 is moved at each movement position. By pulling down vertically, the tissue implant 15 is cut by the blade 43.

At that time, the cutter 38 is pulled down while maintaining a state in which the back surface of the cutter 38 is in close contact with the front surface of the vertical plate 28B. That is, the blade 43 is pulled down vertically while maintaining the vertical posture of the blade 43. The pair of plates 40, 42 are connected by a plurality of fastening members 78. A positioning mechanism other than the illustrated positioning mechanism 66 may be provided. A mechanism for generating a sliding motion in both the X direction and the Y direction may be provided.

FIG. 5 shows a method for manufacturing a two-dimensional cube array. As described above, the tissue implant 15 is composed of the tissue 16 and the gel layer 18. The gel layer 18 has a peripheral portion 80 that covers the outside of the tissue 16. In the illustrated example, the peripheral portion 80 is roughly classified into an inner peripheral portion 80A that covers the protruding portion of the base sheet 44 and an outer peripheral portion 80B that covers the periphery of the protruding portion of the base sheet 44. The inner peripheral portion 80A is indirectly in close contact with the upper surface of the plate 14 via the base sheet 44. The outer peripheral portion 80B is in direct contact with the upper surface of the plate 14.

When viewed from above, if the size of the base sheet 44 is matched to the size of the structure 16, it is possible to increase the area in which the gel layer 18 is in direct contact with the upper surface of the plate 14. When the size of the base sheet 44 is made smaller than the size of the structure 16, cutting defects are likely to occur in a part of the structure 16. Therefore, it is desirable that the size of the base sheet 44 be the same as the size of the tissue 16 or slightly larger than the size of the tissue 16.

The cutting line row 82 is composed of a plurality of cutting lines 82a arranged in the y direction. The cutting line row 82 is produced by arranging the sample set so that the longitudinal direction of the plate 14 is parallel to the blade and then performing repeated cutting while sequentially changing the blade positions. The pitch at that time is Δy.

The cutting line row 84 is composed of a plurality of cutting lines 84a arranged in the x direction. The cutting line row 84 is produced by arranging the sample set so that the lateral direction of the plate 14 is parallel to the blade and then performing repeated cutting while sequentially changing the blade positions. The pitch at that time is Δx. Δy and Δx are, for example, in the range of 200 to 1000 μm, and in the embodiment, 300 μm.

Seen from above, each of the cutting lines 82a, 84a has a length that completely exceeds the tissue inclusion body 15 (ie, the gel layer 18). The cutting lines 82a and 84a may be formed so that the tissue 16 is completely cut and a part of the peripheral portion is not cut in the gel layer 18.

As a result of forming the cutting line row 82 and the cutting line row 84, the tissue 16 is processed into a two-dimensional cube array (which may be called a two-dimensional microcube array). For individual cutting, the edge of the blade reaches and abuts on the base sheet 44. The base sheet 44 is deformed at that time, or is cut at that time. Lowering the blade until the edge of the blade completely hits the top surface of the plate 14 yields a plurality of small pieces from the base sheet 44. Usually, those pieces are removed ex post facto from multiple cubes.

In the embodiment, a flat tissue-embedded body 15 extending along the upper surface of the plate 14 is provided on the plate 14, and such a tissue-embedded body 15 is targeted for cutting. The tissue-embedded body 15 has better morphological stability than the exposed tissue. Therefore, it is possible to cut the tissue-embedded body 15 into small pieces in stages. That is, it is possible to form one of the cutting line trains 82 and 84 and then form the other. At that time, since the gel layer 18 exerts an action of limiting the movement and deformation of the plurality of small pieces, it is possible to prevent the arrangement and morphology thereof from being significantly disrupted.

FIG. 6 shows a fragment array 86 including a cube array 89. The fragment array 86 is made from a tissue implant and is composed of a plurality of fragments 88 arranged two-dimensionally. Each fragment 88 is composed of a cube 16a, which is a part of biological tissue, and a gel part 18a, which is a part of a gel layer. The cube array 89 is composed of a plurality of cubes 16a arranged two-dimensionally. In each fragment 88, its widths 94,96 are, for example, 500 μm. Its height 90 is, for example, 4 mm. The height of the cube 16a is, for example, 2.0 mm. On the other hand, the base sheet is two-dimensionally cut to generate a plurality of small pieces 44a.

FIG. 7 shows a flowchart of the biological tissue cutting method according to the embodiment. In S10, gelatin is produced. Under heating at 56 ° C., gelatin powder (15 w%) is added to the sterile phosphate buffer solution to dissolve the gelatin powder. The gelatin solution thus produced is contained in a plurality of sterilizing tubes of 1 ml each. Those sterile tubes are stored in the refrigerator. During the cooling process, the gelatin solution gels in each sterile tube. As the gelatin powder, for example, pig subcutaneous gelatin powder (Gelatin Porcine, Type A, G1890) manufactured by Sigma Co., Ltd. is used.

On the other hand, in S12, a tissue block having a size of 10 × 10 × 10 mm is repeatedly sliced by a slicer, whereby a plurality of tissue sheets are produced. The thickness of each tissue sheet is, for example, in the range of 1.0 to 3.0 mm. A thinner tissue sheet may be made. The plurality of tissue sheets are stored in the culture medium in a cooled state. A plurality of tissue sheets may be produced using the configuration according to the embodiment.

In S14, for example, as shown in FIG. 8, the slide glass (plate) 106 is arranged in the transparent petri dish 102. In that case, the petri dish 102 is placed on the slide glass 100 having the mark 104, and then the slide glass 106 is arranged so that the slide glass 106 completely overlaps the slide glass 100.

In S16 shown in FIG. 7, the frame 108 is arranged on the slide glass 106 as shown in FIG. The frame 108 is a circular formwork and is a ring. At that time, the frame 108 is arranged so that the mark 104 coincides with the center of the frame 108. The thickness of the frame 108 is, for example, 2 mm. The thickness defines the thickness of the gel layer. If it is desired to change the thickness of the gel layer, a frame having a scale may be used. The inner diameter of the frame 108 is, for example, 25 mm.

In S18 shown in FIG. 7, the base sheet 44 is arranged on the slide glass 106 as shown in FIG. At that time, the base sheet 44 is arranged so that the mark 104 coincides with the center of the base sheet 44. The base sheet 44 may be arranged before the arrangement of the frame 108.

In S20 shown in FIG. 7, the tissue sheet produced in S12 is arranged in the frame and on the base sheet. In S21, the gelatin solution is poured into the frame. Specifically, the required number of tubes (eg, two tubes) are removed from the refrigerator and placed under heating at 56 ° C. This returns the gelatin gel to the gelatin solution. After that, the temperature of the gelatin solution is changed to 42 ° C. The temperature is a temperature that does not adversely affect the tissue. Gelatin solution is poured into the frame. The pouring is performed with the plate cooled to, for example, 4 ° C. The cooling may be omitted.

Then, in S22, the petri dish is placed in the refrigerator. The temperature inside the refrigerator is, for example, 4 ° C. As it cools, the gelatin solution gels, forming a gel layer, that is, a tissue embedding body. After gelation, the frame on the glass slide is removed.

In S24, cutting of the tissue implant is repeated to produce a cube array. The details will be described later. The cutting process is performed with the tissue implants cooled. The cooling temperature is, for example, 4 ° C. Cooling of the tissue inclusions may be omitted.

In S26, post-processing is applied to the produced cube array. Post-treatment includes removal of the plurality of basesheet pieces resulting from the basesheet, removal of the plurality of gels resulting from the gel layer, and the like. Those processes may be omitted. For example, gel removal may be omitted if enzymatic treatment is applied to individual cubes.

FIG. 10 shows the detailed contents of S24 shown in FIG. 7. In S30, a sample set having the first posture is arranged on the stage. That is, the sample set is arranged so that the y direction (the short side direction of the slide glass) is parallel to the blade. In S32, the blade position (cutting position) in the x direction is positioned at the initial position x1. In S34, the tissue implant is cut by the vertical descent of the cutter. The cutter is then pulled up vertically. In S36, it is determined whether or not the blade position exceeds the final position x2, and if not, the blade position is shifted in the x direction by Δx in S36. After that, S34 and subsequent steps are executed again. By repeatedly executing S34, a plurality of cutting lines parallel to the y direction are formed.

In S37, the sample set is arranged on the stage in the second posture. That is, the sample set is arranged so that the x direction (longitudinal direction of the slide glass) is parallel to the blade. In S38, the blade position (cutting position) in the y direction is positioned at the initial position y1. In S40, the tissue implant is cut by the vertical descent of the cutter. The cutter is then pulled up vertically. In S42, it is determined whether or not the blade position exceeds the final position y2, and if not, the blade position is shifted in the y direction by Δy in S44. After that, S40 and subsequent steps are executed again. By repeatedly executing S40, a plurality of cutting lines parallel to the x direction are formed. As a result of the above, a two-dimensional cube array is produced from one tissue sheet. Desirable values can be set as Δx and Δy.

FIG. 11 shows a modified example of the cutter. The cutter 110 has a blade row 112 consisting of four blades arranged in parallel. The plurality of blades are held by a block 114 composed of a plurality of plates. The cutter 110 may be provided with more blades. A blade body having a matric form may be provided on the cutter.

FIG. 12 shows a modified example of the cube array manufacturing method. The tissue implant 15 is placed on the plate 14. The cutting line matrix 116 is composed of a cutting line sequence consisting of a plurality of cutting lines parallel to the x direction and a cutting line sequence consisting of a plurality of cutting lines parallel to the y direction. The formation of the cutting line matrix 116 forms the cube array 117. The cut line matrix 116 occurs in a local area within the tissue implant 15 and does not extend to the entire area. Similarly, the cutting line matrix 116 occurs in the portion of the base sheet 44 excluding the peripheral portion, and does not extend to the peripheral portion. One end of the plurality of cutting lines parallel to the x direction may be extended in the x direction, or one end of the plurality of cutting lines parallel to the y direction may be extended in the y direction. That is, the cut line matrix 116 may be configured so that no cut is made across the entire tissue implant 15.

In the illustrated example, the entire tissue is cut, but the entire base sheet 44 is not cut, resulting in a frame-shaped uncut portion. The gel layer also has a relatively large frame-shaped uncut portion. As a result, the decrease in the tissue-suppressing action of the gel layer with the progress of the cutting process is reduced. Further, after cutting, the base sheet 44 tends to remain on the plate 14. A cube array manufacturing method other than that described above may be adopted.

FIG. 13 schematically shows the biological tissue cutting apparatus according to the second embodiment. The illustrated biological tissue cutting device is a device that automatically creates a cube array. The biological tissue cutting device has a cutting mechanism 120. The operation of the cutting mechanism 120 is controlled by the control unit 134. The control unit 134 has a processor that executes a program.

A swivel stage 124 is mounted on the XY stage 122. The horizontal position of the swivel stage 124 is determined by the XY stage 122. The sample set 12 is arranged on the swivel stage 124. The posture (rotation angle) of the sample set 12 is changed by the swivel stage 124. The elevating mechanism 126 holds the cutter 128 and elevates it. The ascending / descending motion of the cutter 128 is indicated by Z1. The blade 129 of the cutter 128 cuts the tissue inclusions on the substrate. A camera 136 is provided that captures the tissue implant as a still image or a moving image.

The pretreatment unit 130 automatically produces a plurality of tissue sheets from the tissue blocks. In addition, the pretreatment unit 130 automatically prepares a tissue-embedded body after placing the tissue sheet on the plate. The sample set 12 thus produced is transferred onto the swivel stage 124 by the transfer mechanism 132. After repeating the cutting step, the sample set 12 including the cube array is transferred to the post-processing unit 131 by the transfer mechanism 132. The post-treatment unit 131 has a function of removing gel, removing small pieces of the base sheet, and the like. In the post-treatment section, individual cubes may be immersed in the culture medium, or enzymatic treatment may be applied to the individual cubes.

A tissue cutting substrate can be manufactured by pre-bonding a base sheet onto the plate. FIG. 14 shows a first example of such a substrate. (A) shows the upper surface of the substrate, and (B) shows the cross section of the substrate. The substrate is composed of a plate 140 and a base sheet 142 bonded to the plate 140. The base sheet 142 has a circular shape when viewed from above. The plate 140 is made of a hard material that does not cut or deform when hit by the edge of the blade, while the base sheet 142 is made of a soft material that is crushed, deformed or cut when the edge of the blade hits. Will be done.

FIG. 15 shows a second example of the substrate. (A) shows the upper surface of the substrate, and (B) shows the cross section of the substrate. The substrate is composed of a plate 140 and a base sheet 144 adhered to the plate 140. The base sheet 144 has a square shape when viewed from above, and the width thereof matches the width of the plate 140. The base sheet 144 is made of the same material as the base sheet 142 shown in FIG. The plate and the base sheet may be separated (that is, a substrate kit is configured), and the substrate may be configured by adhering or placing the base sheet on the plate at the time of use.

FIG. 16 shows a third example of the substrate. The substrate 146 is made of a material having a certain elasticity. Specifically, it is composed of a material that is slightly dented and deformed in the surface layer when the edge of the blade hits. For example, it is composed of a relatively hard resin.

In the cutting process, the substrate 146 is placed on a rigid stage. The biological tissue 150 is directly placed on the upper surface of the substrate 146 and is covered by the cover layer 152. Such tissue inclusions are cut by the blade.

FIG. 17 shows a first modification of the biological tissue cutting method. In the modified example, the cover layer 160 in which the recess 162 is formed in advance is used. After arranging the base sheet 156 on the plate 154, the tissue 158 is arranged on the base sheet 156. After that, the cover layer 160 is arranged on the plate 154 so that the tissue 158 is housed in the recess 162.

At least one of the shape of the recess 162 and the shape of the structure 158 is operated so that the shape of the recess 162 and the shape of the structure 158 match, that is, the inner surface of the recess 162 and the outer surface of the structure 158 are in close contact with each other. .. The base sheet may be attached to the plate 154 in advance. A frame may be provided around the cover layer 160 to limit its horizontal movement. In that case, the frame may be pre-fixed to the plate 154.

In the past, it has been extremely difficult to automatically or manually produce a plurality of tissue pieces having an even thickness of, for example, 1 mm or less. Even if such multiple thin pieces of tissue could be produced, there was considerable crushing. On the other hand, according to the above-mentioned apparatus and the above-mentioned method, a plurality of thin tissue pieces having an even thickness can be easily produced, and in that case, the crushing of the tissue can be significantly reduced. Further, according to the above-mentioned apparatus and the above-mentioned method, it is possible to cut the tissue on the plate step by step, that is, it is possible to prepare a cube array from the tissue sheet. Very small and healthy cubes can be used in tissue culture, tissue treatment, tissue testing and the like.

FIG. 18 shows a second modification of the tissue cutting method. A gelled frame 172 is provided on the plate 170, and a base sheet 174 is provided. The frame 172 has a circular or rectangular shape when viewed from above. A tissue 176 is provided on the base sheet 174. A gelling material having fluidity is poured into the inside of the frame 172 by heating. By cooling the gelling material, a gel layer 178 is formed. The gel layer 178 covers and encloses the tissue 176. The tissue 176 and the gel layer 178 make up the tissue implant. The tissue implant is surrounded by a frame 172 made of gelled material. As indicated by reference numeral 182, when the blade 180 is pulled down, the frame 172 is cut together with the tissue implant.

According to the second modification shown in FIG. 18, there is an advantage that the frame does not need to be removed from the plate 170. Further, at the time of cutting, the frame 172 exerts an action of indirectly suppressing the movement and deformation of the tissue 176. That is, the frame 172 functions like a part of the gel layer 178. The inner circumference of the frame 172 and the outer circumference of the structure 176 may be brought close to each other. A substrate consisting of a plate 170 and a frame 172 preformed on the plate 170 may be utilized.

FIG. 19 shows a modified example of the holder. Four stoppers 186 to 192 are fixed in a predetermined arrangement on a relatively large sheet 184. Each stopper 186 to 192 is a rectangular sheet piece. A cross groove is formed by four stoppers 186 to 192. The cross groove is composed of a groove 193A and a groove 193B. In the illustrated example, the plate 194 is arranged in the groove 193A. The end side 194A is aligned with the end side 184A of the sheet 184. A flexible L-shaped sheet 196 is arranged across the three stoppers 188, 190, 192. The L-shaped sheet 196 exerts an effect of suppressing the floating of the plate 194.

When the plate 194 is arranged in the groove 193B, the edge 194A of the plate 194 is aligned with the edge 184B of the sheet 184. A tissue implant 202 is placed on the plate 194. The tissue-embedded body 202 is composed of the tissue 198 and the gel layer 200 that encloses the tissue 198. At the time of cutting, the configuration shown in FIG. 19 is arranged on the base 20 shown in FIG. At that time, the sheet 184 and the plate 194 may be fixed on the base 20 by an instrument such as a clip. The thickness of the sheet 184 is, for example, 1 mm, and the thickness of each stopper 186 to 192 is, for example, 1 mm. The thickness of the plate 194 is, for example, 0.9 to 1.2 mm. The thickness of the L-shaped sheet 196 is, for example, 400 μm.

20 and 21 show a first example of a method for cutting a biological tissue using a holding member. In FIG. 20, a pressing piece 204 as a pressing member is placed on the gel layer 200. The holding piece 204 is a small piece having a trapezoidal shape when viewed from above in the illustrated example. However, the shape is an example.

The tip surface 204A of the holding piece 204 is in contact with or close to one side surface of the blade 206 immediately before being pulled up. In the illustrated state, the rear end portion 204B of the holding piece 204 protrudes horizontally from the gel layer 200. The rear end portion 204B can be grasped with a tool such as tweezers, and the position and posture of the holding piece 204 can be adjusted by the tool. However, as the cut surface progresses, the rear end portion 204B of the holding piece 204 does not protrude in the horizontal direction from the gel layer 200.

More specifically, in the cross-sectional view shown in FIG. 21, stoppers 186 and 188 are provided on the sheet 184, and the positions and postures of the plates 194 are held by them. The base sheet 208 and the structure 198 are laminated on the plate 194, and the laminate is wrapped with the gel layer 200. This constitutes the tissue embedding body 202. The end of the plate 194 is held down by the L-shaped sheet 196.

The blade 206 is pulled down as shown by S50 in a state where the holding piece 204 is positioned slightly away from the cutting position. This cuts the tissue implant 202. After that, as shown in S52, the tip surface of the holding piece 204 is abutted against one side surface of the blade 206. Then, as shown in S54, the blade 206 is pulled upward. Even if a part of the structure 198 or a part of the gel layer 200 is attached to one side surface of the blade 206, those deposits are peeled off from one side surface of the blade 206 by the pressing action of the pressing piece 204. This makes it possible to prevent or reduce the disorder of the arrangement of one or fragment caused by cutting. In addition, the influence of the deposits on the next cutting action is prevented or reduced. After the blade 206 is pulled up, the blade is moved to the next cutting position, as shown in S56. After that, the same series of steps as described above is repeated. According to the experiment, it has been confirmed that the residue of the deposit on the blade can be significantly reduced by the weight of the holding piece only by placing the holding piece of 0.15 to 0.20 g. Depending on the situation, the weight of the holding piece may be a weight other than the above range. Further, depending on the situation, the form of the holding piece may be a form other than the above. In general, it is desirable that the width of the tip surface of the holding piece be the same as or wider than the width of the tissue-embedded body. A pair of holding members may be placed on both sides of the blade. The thickness of the holding piece 204 is, for example, 1 mm.

FIG. 22 shows a second example of a method for cutting a biological tissue using a holding member. The cutter 210 has a blade row 212 composed of a plurality of blades arranged at equal intervals. The blade row is fixed to the block 214, and as shown by reference numeral 222, the blade row 212 is moved up and down by moving the block 214 up and down. Illustration of the mechanism for holding the block 214 is omitted.

The cutter 210 has a holding member 216. The pressing member 216 is composed of a plurality of pressing pieces 218-1 to 218-5. They are fixed to the member 220. As shown by reference numeral 224, the pressing member 216 is moved up and down by raising and lowering the member 220. Illustration of the mechanism for holding the member 220 is omitted. The plurality of holding pieces 218-1 to 218-5 are composed of holding pieces 218-1,218-5 at both ends and holding pieces 218-2 to 218-4 between the blades.

After the blade row 212 is lowered to cut the tissue-embedded body, the holding member 216 is lightly abutted against the upper surface of the tissue to maintain that state. In that state, the blade row 212 is pulled up. A part of the tissue or gel layer attached to the side surface of each blade is scraped off from the side surface of each blade by the action of the pressing member 216.

The presser member 216 may be applied to the surface of the tissue when or before lowering the blade row 212. The contact force at that time may be detected by the sensor, and the descending position of the holding piece may be feedback-controlled by the detected value. It is desirable to adopt a configuration in which the pressing member 216 independently moves up and down with respect to the blade row 212.

10 Biological tissue cutting device, 12 sample set, 14 plate, 15 tissue embedding, 16 tissue, 18 gel layer, 20 base, 20A stage, 22 slide mechanism, 24 lower part, 26 upper part, 28 L-shaped bracket, 30 fixing mechanism , 32 Movable mechanism, 34 holder, 36 openings, 38 cutters, 44 base sheet.

Claims (14)

The process of providing biological tissue on the plate and
A step of providing the biological tissue and a gel layer surrounding the biological tissue on the plate, and
A step of cutting the biological tissue together with the gel layer with a cutting blade on the plate.
A method for cutting a biological tissue, which comprises. In the biological tissue cutting method according to claim 1,
The step of providing the gel layer is
The step of providing a frame surrounding the biological tissue on the plate, and
The process of pouring a fluid material into the frame and
The step of gelling the material poured into the frame to form the gel layer, and
including,
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
The biological tissue has a flat morphology that extends along the plate.
The gel layer has a central portion that covers the biological tissue and a peripheral portion that covers the periphery of the biological tissue.
The peripheral portion is in direct or indirect contact with the plate.
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
The cutting step is repeatedly performed, whereby the biological tissue is processed into a two-dimensional cube array.
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
A flexible base sheet is provided on the plate, and the biological tissue is provided on the base sheet.
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
When the blade is pulled up from the biological tissue after cutting the biological tissue, the lifting of the biological tissue is suppressed by the pressing member.
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
The plate has a horizontal posture
The biological tissue is cut by lowering the blade vertically and abutting the edge of the blade against the plate while maintaining the vertical posture of the blade.
A method for cutting biological tissue. A biological tissue cutting device for carrying out the biological tissue cutting method according to claim 1.
A stage for holding the plate provided with the biological tissue and the gel layer surrounding the biological tissue, and
A cutter with the blade that cuts the biological tissue together with the gel layer,
A biological tissue cutting device characterized by containing. In the biological tissue cutting apparatus according to claim 8,
A base sheet made of a material provided between the plate and the biological tissue and which is deformed or cut when the edge of the blade is hit.
A biological tissue cutting device characterized by that. In the biological tissue cutting apparatus according to claim 8,
A holding member that suppresses the floating of the biological tissue when the blade is pulled up from the biological tissue after cutting the biological tissue.
A biological tissue cutting device characterized by that. In the biological tissue cutting apparatus according to claim 8,
The plate has a horizontal posture on the stage and
A guide member is provided to allow the cutter to perform a vertical motion while maintaining the vertical posture of the blade.
A biological tissue cutting device characterized by that. In the biological tissue cutting apparatus according to claim 8,
The blade has a first horizontal extending edge
A slide mechanism for changing the blade position in the second horizontal direction orthogonal to the first horizontal direction is provided.
A biological tissue cutting device characterized by that. In the biological tissue cutting method according to claim 1,
The plate has a plane extending in the first horizontal direction and the second horizontal direction.
The biological tissue and the gel layer are provided on the plane.
A method for cutting biological tissue. In the biological tissue cutting method according to claim 1,
The cutting step is repeatedly executed while sequentially changing the cutting position, and the pitch of the cutting line train generated thereby is in the range of 200 to 1000 μm.
A method for cutting biological tissue.

Images