WO2003044489A1 - Cone plate type cell separation evaluating device - Google Patents

Cone plate type cell separation evaluating device Download PDF

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Publication number
WO2003044489A1
WO2003044489A1 PCT/JP2002/012049 JP0212049W WO03044489A1 WO 2003044489 A1 WO2003044489 A1 WO 2003044489A1 JP 0212049 W JP0212049 W JP 0212049W WO 03044489 A1 WO03044489 A1 WO 03044489A1
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WO
WIPO (PCT)
Prior art keywords
cone
test material
cell detachment
microscope
evaluation device
Prior art date
Application number
PCT/JP2002/012049
Other languages
French (fr)
Japanese (ja)
Inventor
Katsuko Sakai
Tetsuya Tateishi
Takashi Ushida
Original Assignee
National Institute Of Advanced Industrial Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute Of Advanced Industrial Science And Technology filed Critical National Institute Of Advanced Industrial Science And Technology
Priority to AU2002349385A priority Critical patent/AU2002349385A1/en
Publication of WO2003044489A1 publication Critical patent/WO2003044489A1/en

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/6456Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458Fluorescence microscopy
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N11/10Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by moving a body within the material
    • G01N11/14Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by moving a body within the material by using rotary bodies, e.g. vane
    • G01N11/142Sample held between two members substantially perpendicular to axis of rotation, e.g. parallel plate viscometer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N19/00Investigating materials by mechanical methods
    • G01N19/04Measuring adhesive force between materials, e.g. of sealing tape, of coating
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0092Monitoring flocculation or agglomeration

Definitions

  • the present invention provides a cone plate capable of peeling the adhered material and the cell, obtaining data at that time, and observing the state of cell detachment.
  • the present invention relates to a type cell detachment evaluation device. Background art
  • Vascular diseases are known to cause serious disabilities, such as life-threatening dysfunction and limb amputation, which can severely reduce the quality of life of people.
  • many elderly people suffer from vascular diseases.
  • the probability of how to treat vascular diseases has become an important issue.
  • As a treatment method for these diseases of the vascular system it is often effective to replace the diseased site with a small-diameter artificial blood vessel with a vessel inner diameter of 4 mm or less. None that can withstand this has been developed.
  • blood vessels in a living body are composed of vascular endothelial cells, vascular smooth muscle cells, and fibroblasts.
  • an antithrombotic substance that constantly prevents blood from adhering to vascular endothelial cells and an anticoagulant that dissolves thrombus formed after blood adheres to vascular endothelial cells
  • an anticoagulant that dissolves thrombus formed after blood adheres to vascular endothelial cells
  • vascular endothelial cells and a material exhibits excellent antithrombotic properties under in vitro conditions with no flow.
  • the vascular endothelial cells on the surface of the material that have been tested are easily detached by blood flow in the materials tested so far, and as a result, sufficient antithrombotic activity can be achieved even with the hybrid material. I can't get sex.
  • vascular endothelial cells It is considered important to search for a material that enhances the adhesion to the bottom surface of, or a culture environment that enhances the adhesion of vascular endothelial cells to the bottom surface. Improving the adhesion of cells to carriers, etc., is not a problem limited to hybrid type artificial blood vessels made of vascular endothelial cells and materials. This is an important issue. Recently, the goal of artificial organ production by the braid engineering technique has been spreading to all organs, including artificial cartilage, bone, nerve, esophagus, liver, kidney, heart, and bladder. It is necessary to develop a means for quantitatively evaluating the adhesive strength.
  • the present invention has been made in view of the above circumstances, and its main purpose is to closely resemble the environment in a living body, and to easily quantify and visualize the detached state of cells adhered to a carrier due to shear stress in real time. It is an object of the present invention to provide an apparatus for evaluating cell detachment that can be performed easily.
  • Another object of the present invention is to provide a cell detachment evaluation apparatus capable of accurately and easily evaluating various materials such as a plate, a film, and a three-dimensional carrier under the same conditions.
  • Another object of the present invention is to provide a cell exfoliation evaluation apparatus capable of evaluating the tendency of cells to adhere to a material under an environment at a temperature close to that of a living body. Disclosure of the invention
  • the cone-plate type cell detachment evaluation device comprises a bottom plate for fixing a test material in which cells are seeded on a carrier, a cone made of a transparent material and arranged in close proximity to the test material, and the cone inside.
  • a cone holder having a rotation drive unit on the outer periphery thereof, a driving device for rotating the cone holder via the rotation drive unit, and an incident light for observing seeded cells and the like on the test material from above the cone. And a fluorescence microscope.
  • the test material is placed in a depression of a transparent sample mounting plate, and is fixed to the bottom plate by the elastic holding member in a pressed state via the loading plate.
  • the test material to be mounted on the sample mounting plate is a cell seeded on a material such as a plate, a film, or a three-dimensional carrier, and the diameter is set to a small diameter of about 15 mm.
  • a radial groove is formed in the holding member so that a liquid can be introduced from the outside, and a hole communicating with an outer peripheral portion of the groove of the holding member is formed in the bottom plate.
  • the cone holder is provided at the center of the cone holder. including.
  • the rotation driving unit of the cone holder is a ring gear, and includes rotating the ring gear via an intermediate gear by a gear of the driving device.
  • the inner periphery of the bottom plate includes a screw that is screwed to the bottom plate support member, and includes a height adjusting device that adjusts the height of the cone with respect to the bottom plate.
  • the diameter of the cone is set to a small diameter of about 10 mm, and a laser displacement sensor for measuring a gap between the cone and the test material is provided.
  • the cone can be swung between a position directly below the microscope and other parts by a movable arm that fixes the bottom plate and rotatably supports the cone holder.
  • the driving device includes a moving device for focusing a microscope on the surface of the test material and an xy- Z- axis movable device for changing an observation site on the surface of the test material.
  • the microscope includes an epifluorescence microscope equipped with an ultra-long focal length objective lens for observing the surface of the test material from above, and further, the microscope emits light from the surface side of the test material. It includes a lighting device and a lighting device that projects light from the back side.
  • at least the test material, the test material support, and the microscope are housed in a box capable of controlling the inside to a predetermined temperature.
  • the microscope includes being mounted on an integrated vibration isolator.
  • the microscope includes an ultra-sensitive camera and is connected to an image analyzer, a video, and a monitor.
  • the cell detachment evaluation apparatus includes a combustor for controlling the rotation of the cone.
  • the cone-plate type cell detachment evaluation apparatus utilizes the shearing force generated by the rotation of the cone as described above. Peeling test can be performed, and quantitative tests can be easily performed under the same conditions as in the actual cell environment, and the condition can be observed in real time with an epifluorescence microscope. Quantitative evaluation of cell adhesion tendency Can be.
  • test material is placed on the bottom plate and fixed to the rib plate in a pressed state by an elastic pressing member, any material such as a plate, a film, and a three-dimensional carrier can be mounted. As a result, it is possible to quantitatively analyze the detachment phenomenon due to the shear stress of cells on all materials including small materials.
  • the cell detachment evaluation device of the present invention includes that at least the test material, its supporting portion, and the microscope are housed in a box whose inside can be controlled to a predetermined temperature.
  • the temperature can be easily and accurately set to 37 ° C as the environment, and accurate measurement and observation can be performed.
  • FIG. 1 is a schematic diagram showing the overall configuration of one embodiment of the cell detachment evaluation device according to the present invention.
  • FIG. 2 is a cross-sectional view showing an assembled state of a cone support portion and a test material support portion of the cell detachment evaluation device of FIG.
  • FIG. 3 (a) is a partial cross-sectional side view showing a disassembled state of a cone holding portion including a cone holding member, a cone, and a cone holder of the cell detachment evaluation device of FIG.
  • FIG. 3 (b) is a partial cross-sectional side view showing a disassembled state of a sample supporting portion composed of a holding plate, a sample mounting plate, and a bottom plate of the cell detachment evaluation device of FIG.
  • FIG. 4 (a) is a plan view of the bottom plate of FIG. 3 (b).
  • FIG. 4 (b) is a cross-sectional view taken along line 4a-4a of the bottom plate of FIG. 4 (a).
  • FIG. 5 (a) is a plan view of the sample mounting plate of FIG. 3 (b).
  • FIG. 5 (b) is a cross-sectional view of the sample mounting plate of FIG. 5 (a) taken along line 5a-5a.
  • FIG. 6 (a) is a plan view of the holding plate of FIG. 3 (b).
  • FIG. 6 (b) is a cross-sectional view of the holding plate of FIG. 6 (a) taken along line 6a-6a.
  • FIG. 7 (a) is a plan view of the O-ring fitted to the holding plate of FIG. 3 (b).
  • FIG. 7 (b) is a cross-sectional view of the O-ring of FIG. 7 (a) taken along line 7a-7a.
  • FIG. 8 (a) is a plan view of the cone holder of FIG. 3 (a).
  • FIG. 8 (b) is a cross-sectional view along the line 8a-8a in FIG. 8 (a).
  • FIG. 9 (a) is a plan view of the cone holding member of FIG. 3 (a).
  • FIG. 9 (b) is a cross-sectional view of the cone holding member of FIG. 9 (a) taken along line 9a-9a.
  • FIG. 10 (a) is a plan view of the cone of FIG. 3 (a).
  • FIG. 10 (b) is a side view of the cone of FIG. 10 (a).
  • FIG. 1 is a simplified view of an embodiment of the cell detachment evaluation device of the present invention
  • FIG. 2 is a test material support portion for supporting a test material and a tip rotating close to the material.
  • the cone support part which supports the cone to be enlarged is shown.
  • FIG. 3 is an exploded view of the cone support portion and the test material support portion of FIG. 2, and
  • FIGS. 4 to 10 are plan views of the members constituting the support portions. It is a side view including a partially broken state.
  • the bottom plate 1 has an opening 2 in the center, a screw hole 4 in a land 3 around the bottom plate, and a fixed wall on the outer periphery. 5 is provided, and can be screwed and fixed to the tip of the support member 7 by screws 6 formed on the inner periphery of the fixing wall 5.
  • screws 6 formed on the inner periphery of the fixing wall 5.
  • a transparent glass or quartz sample mounting plate 8 mounted on the bottom plate 1 is provided with a depression 9 at the center of the upper surface, and the depression 9 has an outer periphery.
  • the test material 10 molded roughly in conformity with the shape of the depression 9 is dropped and placed. In addition, this test material can be set to any size.
  • the size of the depression 9 is set to accommodate the test material.
  • the holding plate 11 has a hole 12 in the center, an O-ring groove 13 is formed around the hole 12, and the outer peripheral portion corresponds to the screw hole 4 of the bottom plate M. A screw hole 19 is formed in the hole.
  • An O-ring 14 as shown in FIG. 7 is fitted into the O-ring groove.
  • the holding plate 11 is made of an elastic polyimide material so that test materials of various shapes can be securely fixed.
  • two grooves 15 in the figure extending from the outer periphery to the center through hole 12 are formed on the surface of the holding plate 11, and these grooves 15 are O-rings.
  • the culture solution and the like are supplied to the surface of the test material 10 from the outer peripheral side of the groove 15 in a state where all are assembled. And discharge it as necessary.
  • the land portion 3 of the bottom plate 1 is formed with a through hole 16 as shown in FIG. 4, and the through hole 16 is aligned with the outer peripheral end of the groove 15 so that the through hole 16 is formed.
  • the culture solution and the like can be introduced and discharged from the connecting pipe force connected to the lower surface of the cell.
  • the test material support is composed of the holding plate 11, the O-ring 14, the sample mounting plate 8, and the bottom plate 1 as described above. Place the test material 10 on the bottom plate 1, place it on the bottom plate 1, place the 0 ring 14 on top of the holding plate 11 fitted in the O-ring groove 13, and screw the bottom plate 1 with the screw 17. 1 and the holding plate 1 1 are fixed together. At this time, the test material 10 is pressed onto the sample mounting plate 8 and fixed by the O-ring 14 protruding from the lower surface of the holding play M1.
  • any material such as a plate, a film, and a three-dimensional carrier can be easily mounted.
  • the cone support portion has a cone inlet 22 formed in the center of the bottom plate 21 of the cone holder 20.
  • a small-diameter portion 24 of a cone 23 made of a transparent material made of glass or quartz is fitted into the inlet 22.
  • the small-diameter portion 24 can be set to an appropriate size.
  • the small-diameter portion 24 is set to a size of about 1 Omm.
  • a locking flange 25 is provided on the upper portion of the cone 23, and the cone 25 is provided by the locking flange 25. It is placed on the bottom plate 21 of the holder 20.
  • a screw 26 is provided on the inner periphery of the cone holder 20, and a cone holding member 27 screwed into this screw is screwed into the cone holder 20, so that the cone holder 20 is screwed.
  • ⁇ ⁇ ⁇ The cone 23 is firmly fixed to the cone holder 20 by pressing the locking flange 25 of the cone 23 from above from the holding groove 29 on the bottom surface of the through hole 28 provided in the center of the bottom wall of the member 27. I have. Also, by using the cone holder 20, the horizontality of the cone and the plate can be accurately adjusted.
  • the bottom plate M in the test material support section assembled as described above is fixed to a support member 7, and in the illustrated embodiment, the support member 7 is rotatably attached to the frame of the motor 30.
  • the support plate 31 is provided at the tip of the support plate 31.
  • Various other means can be adopted as the means for fixing the support plate 31.
  • An intermediate gear 33 is engaged with the gear 32 of the motor 30, and a ring gear 34 fixed to the flange of the cylindrical cone holder 20 by a port is engaged with the intermediate gear 33.
  • the ring gear 34 to which the cone 33 and the cone holder 20 are fixed is supported on the support plate 31 via a bearing. It is preferable that these bearings have a driving resistance as low as possible and are supported by a preferable material such as a belt bearing or a floating air bearing.
  • the tip of the cone 23 fixed to the cone holder 20 rotated and driven by the motor 30 is close to the surface of the test material 10 on the bottom plate 1 screwed and fixed to the support member 7 of the support plate 31, They are arranged as shown in Figure 2.
  • the cone 23 is formed small and the angle between the cone 23 and the surface of the test material "! 0" is set small, so that the lower surface of the cone 23, the upper surface of the test material 10 and the surrounding area are reduced.
  • the space 35 formed by the wall surface is made small, so that the environment in which the culture solution flows can be formed and the test can be performed using small test materials and a small amount of culture solution.
  • the computer 40 shown in FIG. 1 measures the torque of the motor 8 so that the shear force can be calculated and displayed on the monitor 41 in order to detect the shear force applied to the culture solution as a change in the driving force. I have to.
  • the cone holder 20 and the bottom plate 1, which are swingably supported by the motor 30 as described above, are arranged in the observation section of the upright epi-illumination fluorescence microscope 42 in the state shown in FIG.
  • the entire microscope can be housed in a box 43 made of acrylic or the like in a substantially sealed state, and the inside of the box 43 is controlled by a temperature control device 50, for example, under physiological conditions. A predetermined temperature such as 7 ° C can be maintained.
  • the erecting epi-illumination fluorescence microscope 42 is fixed to a vibration isolating plate 44 in a box 43 so as not to generate image noise due to external vibration. At this time, it is preferable to provide an anti-vibration plate also in the support portion of the motor 30.
  • a fine adjustment device 39 may be provided between the vibration isolating plate 44 and the lower surface of the upright epi-illumination fluorescence microscope 42 as needed.
  • the support plate 31 is fixed to the frame of the motor 30 as described above so that it can swing about 90 degrees about the drive shaft of the motor 30, so as to mount the test material and adjust the cone.
  • the cover 45 of the box 43 is opened, the support plate 31 is rotated to the front side and moved, and then returned to a predetermined position of the upright epi-illumination fluorescence microscope 20 as shown in FIG. So that they can be blocked.
  • the motor 30 is supported by a height adjusting device 46 using a piezoelectric element or the like so that the microscope can be focused, and these are fixed on an XY stage 47, and as a whole, X—Y— The Z stage is configured.
  • the XY stage 47 is fixed so as to be movable in the X-axis direction and the Y-axis direction, so that the position viewed by the microscope can be freely adjusted.
  • a laser displacement sensor-148 is used as a device for measuring the distance. As a result, the gap between the test material and the cone can be separately adjusted, and the gap can be adjusted in sub-micron units.
  • a computer 40 is connected to the motor 30 so that its rotation is controlled to a predetermined state.
  • the computer 40 detects the torque of the motor 30 and changes the state of the load received by stirring the liquid when the cone 23 rotates. Is detected.
  • the computer 40 calculates the shearing force between the culture solution and the test material or the shearing force in another test as necessary, displays the calculated result on the display device, and prints out.
  • These devices constitute a conical-flat plate type shear stress loading device 49.
  • an ultra-long focal length objective lens 51 of an erectile epi-illumination fluorescence microscope 42 is located above the transparent cone 23, and the light from the mercury lamp 52 is filtered by a filter 53, a half mirror 54, an objective lens 51, and a cone 51. It is possible to project the test material 10 through each of the 23.
  • the state of the cell force adhered to the surface of the test material 10 illuminated in this manner in the flowing culture solution is measured by the high-precision SIT camera 56 via the objective lens 51 and the half mirror 54.
  • Shooting, image analysis and image processing by image processor 57 The image is output to an image recording device equipped with a timer 58, a VTR 59, and the like, and is appropriately recorded. Further, the monitor 60 can monitor the state in real time and also observe a slight change in cell morphology.
  • the upright epi-illumination fluorescence microscope 42 allows the light of a halogen lamp 61 to be irradiated from the back side of the test material 10 through a condenser 62 and a transparent sample mounting plate 8, and if necessary, an inverted transmission microscope. So that it can be used as Industrial applicability
  • a cone plate type cell detachment evaluation device comprises a bottom plate for fixing a test material in which cells are seeded on a carrier, a cone made of a transparent material and arranged in close proximity to the test material, A cone holder fixed inside and provided with a rotation driving unit on the outer periphery, a driving device for rotating the cone holder via the rotation driving unit, and observing seeded cells and the like on the test material from above the cone. Equipped with an epi-fluorescence microscope, it is possible to perform a detachment test of seeded cells on the material surface under the shear stress environment of the cells obtained by the specified flow, which is equivalent to the actual cell environment. Quantitative tests under conditions can be easily performed. In addition, the state can be observed in real time by an epifluorescence microscope.
  • test material is placed in the recess of the transparent sample mounting plate, the mounting of the test material is facilitated, and the force of the test material is also reduced.
  • the light from the test material can be transmitted, and the condition of the test material can be observed with a microscope.
  • test material is placed on the bottom plate and fixed in a pressed state on the bottom plate by an elastic pressing member, so that any material such as a plate, a film, and a three-dimensional carrier can be mounted.
  • any material such as a plate, a film, and a three-dimensional carrier can be mounted.
  • a radial groove is formed in the holding member so that a liquid can be introduced from outside.
  • a liquid such as a culture solution can be introduced into the space between the test material and the cone after assembling the test device, and an easy-to-use cell detachment evaluation device can be obtained.
  • the cone holder is provided with a cone ⁇ entrance at the center thereof, and after inserting a small diameter portion of the cone into the cone ⁇ entrance, the upper portion of the cone is pressed and fixed by the cone holding member, so the cone is fixed to the cone holder. It can be fixed securely.
  • the rotation drive unit of the cone holder is a ring gear, and the ring gear is rotationally driven by the gear of the drive device via an intermediate gear, a cone without a drive device above the cone is provided. Can easily be formed.
  • test material is cells seeded on a material such as a plate, a film, or a three-dimensional carrier
  • a material such as a plate, a film, or a three-dimensional carrier
  • the bottom plate is provided with a screw screwed to the bottom plate support member on the outer periphery, the bottom plate on which the test material is fixed can be easily fixed to the bottom plate support member.
  • the diameter of the cone is set to a small diameter of about 1 Omm, it is possible to test the detachment of cells from a small test material in a small amount of liquid, and it is possible to perform the test at low cost and easily.
  • the diameter of the test material is set to a small diameter of about 15 mm, the small test material can be tested for cell detachment in a small amount of liquid, and the test can be performed at low cost and easily.
  • the present invention is provided with a height adjusting device for adjusting the height of the cone with respect to the bottom plate. Therefore, by adjusting the gap between the cone and the test material, the flow of the liquid to the cells is set to a predetermined state. can do.
  • the gap between the cone and the test material can be accurately adjusted in submicron units. This makes it possible to precisely set the flow of the liquid to the cells to a predetermined state.
  • the cone is swingably supported between immediately below the microscope and other portions by a movable arm that fixes the bottom plate and rotatably supports the cone holder. Therefore, the cone can be rotated from the position directly below the microscope and moved to a position where it can be easily handled. After various operations, the cone can be returned to the position directly below the microscope and the microscope can be easily observed.
  • the moving device that focuses the microscope on the surface of the test material is provided in the driving device, the microscope can be easily focused without performing the operation on the microscope.
  • the present invention includes an X-y-z-axis movable device for changing the observation site on the surface of the test material, and therefore, the site to be observed with a microscope while the test is being performed by rotating the cone. Can be moved freely.
  • the microscope is an epifluorescence microscope equipped with an ultra-long focal length objective lens for observing the surface of the test material from above, the surface of the test material can be easily observed from above with a microscope.
  • the microscope is provided with an illuminating device for projecting light from the front side of the test material and an illuminating device for projecting light from the back surface side, an upright-type epi-illumination fluorescence microscope according to the characteristics of the test material is provided. Also, it can be used as an inverted transmission microscope.
  • test material test material support, and microscope are housed in a box that can be controlled to a predetermined temperature. It can be set easily and accurately, and accurate measurement and observation can be performed.
  • the influence on the vibration of the microscope can be reduced, and the image noise generated by receiving external vibration is reduced. Can be.
  • the microscope is equipped with an ultra-high sensitivity camera, it is possible to accurately photograph the state of blood and the like stirred by the cone.
  • an image analyzer is connected to the microscope, an image obtained by the microscope can be easily analyzed, and the image data can be appropriately processed. Further, since a video is connected to the microscope, an observation image obtained by the microscope can be recorded for a long period of time, and can be output and observed when necessary. In addition, since a monitor is connected to the microscope, an image obtained by the microscope can be viewed on the monitor, and observation can be performed easily.
  • the present invention since the present invention includes the computer that controls the rotation of the cone, the rotation of the cone can be reliably controlled to a predetermined value.

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Abstract

A cone plate type cell separation evaluating device, wherein a test material (10) having cells disseminated therein is held between a specimen loading plate (8) and a retaining plate (11) with O-ring (14) and fixed to a bottom plate (1), a cone (23) formed of transparent material is disposed close to the test material and fixed to the inside of a cone holder (20) having a ring gear (34) on the outer periphery thereof, the cone holder is rotated by a motor (30) through the ring gear in the state of the cone positioned close to the test material, and the state of separation of the cells disseminated in the test material under the flow of fluid caused by the cone is observed, in real time, on a monitor (60) through an erect type epi-fluorescence microscope (42) disposed on the upper side of the cone.

Description

明 細 書 コーンプレー卜型細胞剥離評価装置 技術分野  Description Corn plate type cell detachment evaluation system Technical field
本発明は細胞と各種材料との接着力を定量的に評価するために、接着されている材 料と細胞を剥離しそのときのデータを得ると共に細胞の剥離状態を観察することのでき るコンーンプレート型細胞剥離評価装置に関する。 背景技術  In order to quantitatively evaluate the adhesive force between a cell and various materials, the present invention provides a cone plate capable of peeling the adhered material and the cell, obtaining data at that time, and observing the state of cell detachment. The present invention relates to a type cell detachment evaluation device. Background art
血管系の疾患は命に関わるだけでなぐ生体の機能不全や四肢の切断など、人々の 生活の質を著しく低下させる重大な障害を引き起こすことが知られている。近年、血管 系の疾患を患う高齢者は多ぐこれから本格的な高齢者社会を迎えるにあたり、血管 系の疾患の治療方法の確率は重要な課題となっている。これらの血管系の疾患の治 療方法として、血管内径力4mm以下の小口径の人工血管による疾病部位の置換手 術が有効である場合が多いが、今までに小口径領域の人工血管で臨床に耐えうるも のは開発されていない。  Vascular diseases are known to cause serious disabilities, such as life-threatening dysfunction and limb amputation, which can severely reduce the quality of life of people. In recent years, many elderly people suffer from vascular diseases. With the coming of a full-fledged elderly society, the probability of how to treat vascular diseases has become an important issue. As a treatment method for these diseases of the vascular system, it is often effective to replace the diseased site with a small-diameter artificial blood vessel with a vessel inner diameter of 4 mm or less. Nothing that can withstand this has been developed.
一方、生体内の血管は、血管内皮細胞、血管平滑筋細胞、繊維芽細胞から構成され ている。その最内腔面に存在する血管内皮細胞からは、常に血液の血管内皮細胞へ の粘着を抑制する抗血栓性の物質や血液が血管内皮細胞に粘着した後に形成される 血栓を溶かす抗凝固性の物質が放出されることによって、内腔面に血栓が付着しない 平滑な状態が一生涯に亘つて維持されている。したがって、近年では、血管内皮細胞 と材料とのハイブリッド型の人工血管の開発が注目されている。血管内皮細胞と材料 とのハイブリッド型の材料は流れのない in vitroの条件下では確かに優れた抗血栓性 を発揮することが多くの研究者によって確かめられているが、その血管内皮細胞と材 料とのハイブリッド型の材料を生体内に移植すると、今までに試験されてきた材料では 材料表面の血管内皮細胞が血流によって容易に剥離し、その結果ハイブリッド型の材 料でも十分な抗血栓性を獲得できない。  On the other hand, blood vessels in a living body are composed of vascular endothelial cells, vascular smooth muscle cells, and fibroblasts. From the vascular endothelial cells present on the innermost luminal surface, an antithrombotic substance that constantly prevents blood from adhering to vascular endothelial cells and an anticoagulant that dissolves thrombus formed after blood adheres to vascular endothelial cells By releasing this substance, a smooth state in which thrombus does not adhere to the lumen surface is maintained for a lifetime. Therefore, in recent years, attention has been focused on the development of hybrid artificial blood vessels of vascular endothelial cells and materials. Many researchers have confirmed that a hybrid material composed of vascular endothelial cells and a material exhibits excellent antithrombotic properties under in vitro conditions with no flow. When a hybrid material is implanted into a living body, the vascular endothelial cells on the surface of the material that have been tested are easily detached by blood flow in the materials tested so far, and as a result, sufficient antithrombotic activity can be achieved even with the hybrid material. I can't get sex.
したがって、抗血栓性に優れた血液適合性材料を開発するためには、血管内皮細胞 の底面への接着力が増強する材料、または血管内皮細胞の底面への接着力が増強 する培養環境を探索することが重要であると考えられる。また、細胞の担体などへの接 着力の向上は血管内皮細胞と材料とのハイブリッド型の人工血管に限った問題ではな ぐ組織再構築型の人工臓器の設計を目指すいわゆる組繊工学の分野でも重要な課 題となっている。最近では組繊工学的手法による人工臓器作製の目標は、人工軟骨、 骨、神経、食道、肝臓、腎臓、心臓、膀胱をはじめとするあらゆる臓器に広がりつつあり、 上述のあらゆる細胞と材料との接着力を定量的に評価する手段の開発が必要となつ ている。 Therefore, in order to develop a blood-compatible material with excellent antithrombotic properties, vascular endothelial cells It is considered important to search for a material that enhances the adhesion to the bottom surface of, or a culture environment that enhances the adhesion of vascular endothelial cells to the bottom surface. Improving the adhesion of cells to carriers, etc., is not a problem limited to hybrid type artificial blood vessels made of vascular endothelial cells and materials. This is an important issue. Recently, the goal of artificial organ production by the braid engineering technique has been spreading to all organs, including artificial cartilage, bone, nerve, esophagus, liver, kidney, heart, and bladder. It is necessary to develop a means for quantitatively evaluating the adhesive strength.
血管内皮細胞の底面への接着力が増強する材料、または血管内皮細胞の底面への 接着力が増強する培養環境を探索するために、従来は血管内皮細胞を播種した人工 血管を実験動物に移植するなどの方法が用いられてきたが、実験材料を実験ごとに血 管状に加工しなければならない煩雑さ、実験動物の犠牲、実験スケール、実験期間な ど多くの問題があった。  Conventionally, artificial blood vessels seeded with vascular endothelial cells are transplanted into experimental animals to search for a material that enhances the adhesive strength of the vascular endothelial cells to the bottom surface or a culture environment that enhances the adhesive strength to the bottom surface of the vascular endothelial cells. Although various methods have been used, there are many problems such as the necessity of processing experimental materials into tubes for each experiment, the sacrifice of experimental animals, the scale of experiments, and the duration of experiments.
最終的には動物実験、臨床治検によって作製した人工血管の性能を評価しなければ ならないが、 in vitroによる評価系が確立されれば詳細な人工血管の設計が可能にな る。 in vitroの評価方法として、従来は平行平板型チェンバーといわれている 2枚の板 の間にポンプによって流れを起こし、細胞の材料表面からの剥離の程度を評価する装 置も報告されてし、るが、このシステムではポンプで培養液を送り出さなければならなしゝ ので、大量の培養液が必要であり、さらに回路内から空気を抜く作業が必要である問 題がある。  Ultimately, the performance of artificial blood vessels created by animal experiments and clinical trials must be evaluated, but if an in vitro evaluation system is established, detailed artificial blood vessel design will be possible. As an in vitro evaluation method, a device has been reported that uses a pump to generate a flow between two plates, conventionally called a parallel plate type chamber, and evaluates the degree of detachment of cells from the material surface. However, in this system, since the culture medium must be sent out by a pump, a large amount of the culture medium is required, and there is a problem that it is necessary to evacuate the air from the circuit.
また、マイクロピペットで細胞を一つずつ弓 Iつ張って、剥離させる方法も既に報告され てし、る力 一度に大量の材料を簡便に評価する点において問題がある。  In addition, a method of stretching cells one by one with a micropipette and peeling them has already been reported, and there is a problem in that a large amount of material can be easily evaluated at a time.
磁気ビーズで細胞にツイストと呼ばれる力を負荷させることによって細胞を剥離させ る方法も存在するが、生理的な細胞の剥離現象とはかけ離れている問題点があり、今 までに報告された装置では簡便に材料への細胞の接着強度を計測できない現状があ る。  There is also a method of detaching cells by applying a force called twist to the cells with magnetic beads, but there is a problem that is far from the physiological cell detachment phenomenon, and the devices reported so far have At present, it is not possible to easily measure the adhesive strength of cells to materials.
血管内皮細胞を有するタイプの人工血管を開発するために、生理的な条件にできる だけ近い環境下で細胞の接着傾向を定量的に評価する必要力《ある。  In order to develop a type of artificial blood vessel having endothelial cells, it is necessary to quantitatively evaluate the tendency of cells to adhere under physiological conditions as close as possible.
更に、培養皮膚、培養軟骨、培養血管をはじめとする、あらゆる人工臓器では材料へ の細胞の接着力を定量的に解析することによって、各種臓器、各種細胞に最適な材料 設計、細胞の培養方法の指針を得ることができる。また、血管内皮細胞の剥離と動脈 硬化との間に相関があることから、血管内皮の細胞の剥離状態を定量的に評価するこ とにより、動脈硬化を含めた血管系の疾患の新薬の創出に寄与することができること になる。 Furthermore, it is a material for all artificial organs, including cultured skin, cultured cartilage, and cultured blood vessels. By quantitatively analyzing the adhesive force of the cells, it is possible to obtain guidelines for designing materials optimal for various organs and cells and culturing cells. In addition, since there is a correlation between the detachment of vascular endothelial cells and arteriosclerosis, a new drug for vascular diseases including arteriosclerosis can be created by quantitatively evaluating the detachment of vascular endothelial cells. Can be contributed to.
この発明は、上記に鑑みなされたもので、その主目的は、生体内の環境に酷似して し、る剪断応力による担体に接着している細胞の剥離状態をリアルタイムで定量化及び 可視化を簡便にできる細胞剥離評価装置を提供することにある。  The present invention has been made in view of the above circumstances, and its main purpose is to closely resemble the environment in a living body, and to easily quantify and visualize the detached state of cells adhered to a carrier due to shear stress in real time. It is an object of the present invention to provide an apparatus for evaluating cell detachment that can be performed easily.
この発明の他の目的は、板状、膜状、 3次元担体などの多様な材料の評価を同一条 件下で正確且つ簡便に行うことができる細胞剥離評価装置を提供することにある。  Another object of the present invention is to provide a cell detachment evaluation apparatus capable of accurately and easily evaluating various materials such as a plate, a film, and a three-dimensional carrier under the same conditions.
更に、この発明の他の目的は、小さな材料でも確実に評価することができる経済的な 細胞剥離評価装置を提供することにある。  It is a further object of the present invention to provide an economical cell detachment evaluation apparatus that can reliably evaluate even a small material.
この発明の他の目的は、生体内に近い温度の環境下で細胞の材料への接着傾向 を評価することのできる細胞剥離評価装置を提供することにある。 発明の開示  Another object of the present invention is to provide a cell exfoliation evaluation apparatus capable of evaluating the tendency of cells to adhere to a material under an environment at a temperature close to that of a living body. Disclosure of the invention
本発明に依る、コーンプレート型細胞剥離評価装置は、担体に細胞を播種した試験 材料を固定するボトムプレートと、透明材料からなり前記試験材料に近接して配置され るコーンと、前記コーンを内部に固定し、外周に回転駆動部を備えたコーンホルダーと、 前記回転駆動部を介してコーンホルダーを回転する駆動装置と、前記コーンの上部か ら前記試験材料上の播種細胞等を観察する落射蛍光顕微鏡とを備えたことから成る。 前記試験材料は透明な試料載置プレートの窪みに載置され、上記積載プレートを介 して、弾性を有する押さえ部材によりボトムプレートに押圧状態に固定されることを含む。 また、前記試料載置プレートに載置する試験材料は板状、膜状、 3次元担体などの材 料に播種した細胞であり、直径は 1 5mm程度の小径に設定したことを含む。  The cone-plate type cell detachment evaluation device according to the present invention comprises a bottom plate for fixing a test material in which cells are seeded on a carrier, a cone made of a transparent material and arranged in close proximity to the test material, and the cone inside. , A cone holder having a rotation drive unit on the outer periphery thereof, a driving device for rotating the cone holder via the rotation drive unit, and an incident light for observing seeded cells and the like on the test material from above the cone. And a fluorescence microscope. The test material is placed in a depression of a transparent sample mounting plate, and is fixed to the bottom plate by the elastic holding member in a pressed state via the loading plate. Further, the test material to be mounted on the sample mounting plate is a cell seeded on a material such as a plate, a film, or a three-dimensional carrier, and the diameter is set to a small diameter of about 15 mm.
また、前記押さえ部材と試験材料との間には Oリングを備えたことを含む  In addition, including that an O-ring is provided between the holding member and the test material.
前記押さえ部材には放射状の溝を形成し、外部から液体を導入することができるよう にすると共に前記ボトムプレー卜には、前記押さえ部材の溝の外周部に連通する孔を 形成したことを含む。 また本発明の細胞剥離評価装置は、前記コーンホルダーの中心にコーン揷入口を設 【ナ、前記コーン揷入口にコーンの小径部を挿入した後、コーン押さえ部材によりコーン の上部を押圧固定したことを含む。 A radial groove is formed in the holding member so that a liquid can be introduced from the outside, and a hole communicating with an outer peripheral portion of the groove of the holding member is formed in the bottom plate. . Further, in the cell detachment evaluation device of the present invention, the cone holder is provided at the center of the cone holder. including.
前記コーンホルダーの回転駆動部はリングギアであり、前記リングギアを前記駆動装 置のギアにより中間ギアを介して回転駆動することを含む。  The rotation driving unit of the cone holder is a ring gear, and includes rotating the ring gear via an intermediate gear by a gear of the driving device.
前記ボトムプレー卜の内周にはボトムプレー卜支持部材に螺合するねじを備え、前記 ボトムプレートに対するコーンの高さ調節を行う高さ調節装置を備えたことを含む。 前記コーンの直径は 1 0mm程度の小径に設定したことを含み、前記コーンと試験材 料との間隙を測定するレーザー変位センサーを設けたことを含む。  The inner periphery of the bottom plate includes a screw that is screwed to the bottom plate support member, and includes a height adjusting device that adjusts the height of the cone with respect to the bottom plate. The diameter of the cone is set to a small diameter of about 10 mm, and a laser displacement sensor for measuring a gap between the cone and the test material is provided.
更に、本発明の細胞剥離評価装置は、前記ボトムプレートを固定すると共に前記コー ンホルダーを回転可能に支持する可動型アームにより、コーンを顕微鏡直下とそれ以 外の部分との間に揺動自在に支持したことを含む。  Further, in the cell detachment evaluation apparatus of the present invention, the cone can be swung between a position directly below the microscope and other parts by a movable arm that fixes the bottom plate and rotatably supports the cone holder. Includes support for
前記駆動装置は、試験材料表面に顕微鏡の焦点を合わせる移動装置及び前記試験 材料表面上の観察部位を変更する x—y—Z軸方向可動装置を備えたことを含む。 また、前記顕微鏡は、前記試験材料の表面を上方から観察する超長焦点対物レンズ を備えた落射蛍光顕微鏡であることを含み、更に、前記顕微鏡は、前記試験材料の表 面側から投光する照明装置と、裏面側から投光する照明装置とを備えたことを含む。 また、少なくとも前記試験材料、試験材料支持部、及び前記顕微鏡は、内部を所定の 温度に制御することができるボックス内に収容されていることを含む。 The driving device includes a moving device for focusing a microscope on the surface of the test material and an xy- Z- axis movable device for changing an observation site on the surface of the test material. Further, the microscope includes an epifluorescence microscope equipped with an ultra-long focal length objective lens for observing the surface of the test material from above, and further, the microscope emits light from the surface side of the test material. It includes a lighting device and a lighting device that projects light from the back side. In addition, at least the test material, the test material support, and the microscope are housed in a box capable of controlling the inside to a predetermined temperature.
また、前記顕微鏡は、一体型防振板上に載置されていることを含む。  In addition, the microscope includes being mounted on an integrated vibration isolator.
また、前記顕微鏡には超高感度カメラを備え、画像解析装置、ビデオ、及びモニタを 接続したことを含む。  In addition, the microscope includes an ultra-sensitive camera and is connected to an image analyzer, a video, and a monitor.
更に、本発明に依る細胞剥離評価装置は、前記コーンの回転を制御するコンビユー タを備えたことを含む。  Further, the cell detachment evaluation apparatus according to the present invention includes a combustor for controlling the rotation of the cone.
本発明に依るコーンプレー卜型細胞剥離評価装置は、上述の如 コーンの回転によ リ生ずる剪断力を利用したので、簡便な構成で、細胞の剪断応力環境下で材料表面 上の播種細胞等の剥離試験を行うことができ、実際の細胞の環境下と同等の条件で の定量的な試験を容易に行うこと力できると共に、その状態を落射蛍光顕微鏡によつ てリアルタイムに観察することが可能となり、細胞の接着傾向も定量的に評価すること ができる。 The cone-plate type cell detachment evaluation apparatus according to the present invention utilizes the shearing force generated by the rotation of the cone as described above. Peeling test can be performed, and quantitative tests can be easily performed under the same conditions as in the actual cell environment, and the condition can be observed in real time with an epifluorescence microscope. Quantitative evaluation of cell adhesion tendency Can be.
また、前記試験材料はボトムプレートに載置され、弾性を有する押さえ部材によリボト 厶プレートに押圧状態に固定されるので、板状、膜状、 3次元担体などのあらゆる材料 を装着できる。その結果、小さな材料を含み、あらゆる材料上の細胞の剪断応力によ る剥離現象を定量的に解析することが可能である。  In addition, since the test material is placed on the bottom plate and fixed to the rib plate in a pressed state by an elastic pressing member, any material such as a plate, a film, and a three-dimensional carrier can be mounted. As a result, it is possible to quantitatively analyze the detachment phenomenon due to the shear stress of cells on all materials including small materials.
更に、本発明の細胞剥離評価装置は、少なくとも試験材料及びその支持部並びに前 記顕微鏡は、内部を所定温度に制御できるボックス内に収容されてし、ることを含むの で、例えば生体の標準環境としての 37°C等に容易に且つ正確に設定することができ、 正確な測定及び観察を行うことができる。  Further, the cell detachment evaluation device of the present invention includes that at least the test material, its supporting portion, and the microscope are housed in a box whose inside can be controlled to a predetermined temperature. The temperature can be easily and accurately set to 37 ° C as the environment, and accurate measurement and observation can be performed.
この発明の他の目的、その他の特徴は、図面に基づき詳細に説明する。 図面の簡単な説明  Other objects and other features of the present invention will be described in detail with reference to the drawings. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、本発明による細胞剥離評価装置の一実施例の全体構成を示す概要図で あ 。  FIG. 1 is a schematic diagram showing the overall configuration of one embodiment of the cell detachment evaluation device according to the present invention.
第 2図は、第 1図の細胞剥離評価装置のコーン支持部及び試験材料支持部の組立 状態を示す断面図である。  FIG. 2 is a cross-sectional view showing an assembled state of a cone support portion and a test material support portion of the cell detachment evaluation device of FIG.
第 3図 (a)は、第 1図の細胞剥離評価装置のコーン押さえ部材、コーン、及びコーンホ ルダ一から成るコーン支持部の分解状態を示す一部断面とした側面図である。  FIG. 3 (a) is a partial cross-sectional side view showing a disassembled state of a cone holding portion including a cone holding member, a cone, and a cone holder of the cell detachment evaluation device of FIG.
第 3図 (b)は、第 1図の細胞剥離評価装置の押さえプレー卜、試料載置プレート及び ボトムプレートから成る試料支持部の分解状態を示す一部断面とした側面図である。 第 4図(a)は、第 3図 (b)のボトムプレー卜の平面図である。  FIG. 3 (b) is a partial cross-sectional side view showing a disassembled state of a sample supporting portion composed of a holding plate, a sample mounting plate, and a bottom plate of the cell detachment evaluation device of FIG. FIG. 4 (a) is a plan view of the bottom plate of FIG. 3 (b).
第 4図 (b)は、第 4図 (a)のボトムプレー卜の 4a— 4a線に沿った断面図である。  FIG. 4 (b) is a cross-sectional view taken along line 4a-4a of the bottom plate of FIG. 4 (a).
第 5図 (a)は、第 3図 (b)の試料載置プレートの平面図である。  FIG. 5 (a) is a plan view of the sample mounting plate of FIG. 3 (b).
第 5図(b)は、第 5図(a)の試料載置プレートの 5a— 5a線に沿った断面図である。 第 6図(a)は、第 3図 (b)の押さえプレー卜の平面図である。  FIG. 5 (b) is a cross-sectional view of the sample mounting plate of FIG. 5 (a) taken along line 5a-5a. FIG. 6 (a) is a plan view of the holding plate of FIG. 3 (b).
第 6図(b)は、第 6図(a)の押さえプレートの 6a— 6a線に沿った断面図である。  FIG. 6 (b) is a cross-sectional view of the holding plate of FIG. 6 (a) taken along line 6a-6a.
第 7図(a)は、第 3図 (b)の、押さえプレートに嵌合してし、る Oリングの平面図である。 第 7図(b)は、第 7図(a)の Oリングの 7a— 7a線に沿った断面図である。  FIG. 7 (a) is a plan view of the O-ring fitted to the holding plate of FIG. 3 (b). FIG. 7 (b) is a cross-sectional view of the O-ring of FIG. 7 (a) taken along line 7a-7a.
第 8図(a)は、第 3図 (a)のコーンホルダーの平面図である。 第 8図(b)は、第 8図(a)の 8a— 8a線に沿った断面図である。 FIG. 8 (a) is a plan view of the cone holder of FIG. 3 (a). FIG. 8 (b) is a cross-sectional view along the line 8a-8a in FIG. 8 (a).
第 9図(a)は、第 3図(a)のコーン押さえ部材の平面図である。  FIG. 9 (a) is a plan view of the cone holding member of FIG. 3 (a).
第 9図(b)は、第 9図(a)のコーン押さえ部材の 9a— 9a線に沿った断面図である。 第 1 0図 (a)は、第 3図(a)のコーンの平面図である。  FIG. 9 (b) is a cross-sectional view of the cone holding member of FIG. 9 (a) taken along line 9a-9a. FIG. 10 (a) is a plan view of the cone of FIG. 3 (a).
第 1 0図(b)は、第 1 0図(a)のコーンの側面図である。 発明を実施するための最良の形態  FIG. 10 (b) is a side view of the cone of FIG. 10 (a). BEST MODE FOR CARRYING OUT THE INVENTION
器型をしたプレートと逆円錐形のコーンから構成されるコーンプレート型の粘度計が 既に存在し、この粘度計はシンプルな構造を有し、従来は液体の粘度を計測するため に開発されたものである。このコーンプレート型の粘度計では、角速度は中心からの距 離が遠くなるに従い速くなるが、コーンは特定の角度を有するために、底面に一様なず リ速度、剪断応力を負荷できる優れた特徴がある。そこで、本発明においてはこの点に 着目し、コーンプレー卜型の粘度計を基に、更に検討の結果、板状、膜状、不織布、ポ 一ラス担体などのあらゆる 3次元担体への細胞の接着強度を簡便に定量的に計測で きるコーンプレート型細胞剥離評価装置を開発した。  There is already a cone-plate type viscometer composed of a container-shaped plate and an inverted conical cone.This viscometer has a simple structure and was conventionally developed to measure the viscosity of liquids. Things. In this cone plate type viscometer, the angular velocity increases as the distance from the center increases, but since the cone has a specific angle, it has an excellent ability to apply uniform velocity and shear stress to the bottom surface. There are features. Therefore, the present invention pays attention to this point, and based on a cone plate type viscometer, as a result of further studies, found that cells were transferred to all three-dimensional carriers such as plate-like, membrane-like, non-woven fabric, and porous carriers. We have developed a cone-plate type cell detachment evaluation system that can easily and quantitatively measure the adhesive strength.
第 1図は本発明の細胞剥離評価装置の一実施例を簡略化して示したものであり、第 2図は試験材料を支持する試験材料支持部とこの材料に対して先端が近接して回転 するコーンを支持するコーン支持部を拡大して示している。また、第 3図には第 2図のコ -ン支持部及び試験材料支持部の分解図をそれぞれ示し、第 4図〜第 1 0図は、上記 各支持部を構成する各部材の平面図と一部破断状態を含む側面図である。  FIG. 1 is a simplified view of an embodiment of the cell detachment evaluation device of the present invention, and FIG. 2 is a test material support portion for supporting a test material and a tip rotating close to the material. The cone support part which supports the cone to be enlarged is shown. FIG. 3 is an exploded view of the cone support portion and the test material support portion of FIG. 2, and FIGS. 4 to 10 are plan views of the members constituting the support portions. It is a side view including a partially broken state.
第 2図、第 3図及び第 4図に示されるように、ボトムプレー卜 1は中央に開口 2を形成し、 その周囲のランド部 3にねじ通孔 4を備え、また外周には固定壁 5を備えており、固定 壁 5の内周に形成したねじ 6により支持部材 7の先端にねじ込み固定することができる ようにしている。なお、このねじ 6のねじ込みにより、後述するようなコーンの先端と材料 表面との間隔調節を行うことができ、 l OOnmの精度で位置の調節が可能となる。  As shown in FIGS. 2, 3, and 4, the bottom plate 1 has an opening 2 in the center, a screw hole 4 in a land 3 around the bottom plate, and a fixed wall on the outer periphery. 5 is provided, and can be screwed and fixed to the tip of the support member 7 by screws 6 formed on the inner periphery of the fixing wall 5. By screwing in the screw 6, the distance between the tip of the cone and the surface of the material as described later can be adjusted, and the position can be adjusted with an accuracy of 100 nm.
このボトムプレート 1に載置する透明なガラスあるいは石英製の試料載置プレート 8 には第 5図に示すように、上面の中央部分に窪み 9を備えており、この窪み 9にはその 外周を窪み 9の形状に概略合わせて成形した試験材料 1 0を落とし込んで載置する。 なお、この試験材料は任意の大きさに設定することができるが 1 5mm程度の大きさと し、窪み 9はこの試験材料が入る大きさに設定する。押さえプレー卜 1 1は中心部に通 孔 1 2を備え、その通孔 1 2の周囲に Oリング溝 1 3を形成し、外周部分には前記ボトム プレー Mのねじ通孔 4に対応する位置にねじ孔 1 9を形成している。前記 Oリング溝に は第 7図に示されるような Oリング 14を嵌入する。この押さえプレート 1 1は弾性のある ポリイミド製材料からなり、各種形状の試験材料を確実に固定することができるように なっている。 As shown in FIG. 5, a transparent glass or quartz sample mounting plate 8 mounted on the bottom plate 1 is provided with a depression 9 at the center of the upper surface, and the depression 9 has an outer periphery. The test material 10 molded roughly in conformity with the shape of the depression 9 is dropped and placed. In addition, this test material can be set to any size. The size of the depression 9 is set to accommodate the test material. The holding plate 11 has a hole 12 in the center, an O-ring groove 13 is formed around the hole 12, and the outer peripheral portion corresponds to the screw hole 4 of the bottom plate M. A screw hole 19 is formed in the hole. An O-ring 14 as shown in FIG. 7 is fitted into the O-ring groove. The holding plate 11 is made of an elastic polyimide material so that test materials of various shapes can be securely fixed.
また、第 6図に示すように押さえプレー卜 1 1の表面には外周から中心の通孔 1 2に延 びる図中 2本の溝 1 5を形成しており、この溝 1 5は Oリング溝 1 3より幾分深く形成する ことによって、 Oリング 1 4を Oリング溝 1 3に嵌入し全てが組み立てられた状態で培養 液等を溝 1 5の外周側から試験材料 10の表面に供給し、また必要に応じてこれを排出 することができるようにしている。なお、前記ボトムプレート 1のランド部 3には、第 4図に 示すように貫通孔 1 6形成しており、この貫通孔 1 6が前記溝 1 5の外周端に一致させる ことにより、貫通孔 1 6の下面に結合した連結パイプ力、ら前記培養液等を導入し、また 排出することができる。  Also, as shown in FIG. 6, two grooves 15 in the figure extending from the outer periphery to the center through hole 12 are formed on the surface of the holding plate 11, and these grooves 15 are O-rings. By forming the O-ring 14 into the O-ring groove 13 by forming it somewhat deeper than the groove 13, the culture solution and the like are supplied to the surface of the test material 10 from the outer peripheral side of the groove 15 in a state where all are assembled. And discharge it as necessary. The land portion 3 of the bottom plate 1 is formed with a through hole 16 as shown in FIG. 4, and the through hole 16 is aligned with the outer peripheral end of the groove 15 so that the through hole 16 is formed. The culture solution and the like can be introduced and discharged from the connecting pipe force connected to the lower surface of the cell.
第 3図 (b)に示すように試験材料支持部は上記のように押さえプレート 1 1、 Oリング 1 4、試料載置プレート 8、ボトムプレート 1で構成され、試料載置プレート 8の窪み 9に試 験材料 1 0を載置し、これをボトムプレート 1に載置すると共にその上から 0リング 1 4を Oリング溝 1 3に嵌入した押さえプレート 1 1を被せ、ねじ 1 7によりボトムプレート 1と押さ えプレート 1 1とを一体的に固定する。その際、押さえプレー M 1の下面から突出する O リング 1 4により試験材料 1 0が試料載置プレート 8上に押さえ込まれ、固定される。こ のような構成の試験材料支持部を用いることにより、板状、膜状、 3次元担体などのあ らゆる材料を容易に装着することができる。  As shown in Fig. 3 (b), the test material support is composed of the holding plate 11, the O-ring 14, the sample mounting plate 8, and the bottom plate 1 as described above. Place the test material 10 on the bottom plate 1, place it on the bottom plate 1, place the 0 ring 14 on top of the holding plate 11 fitted in the O-ring groove 13, and screw the bottom plate 1 with the screw 17. 1 and the holding plate 1 1 are fixed together. At this time, the test material 10 is pressed onto the sample mounting plate 8 and fixed by the O-ring 14 protruding from the lower surface of the holding play M1. By using a test material support having such a configuration, any material such as a plate, a film, and a three-dimensional carrier can be easily mounted.
一方、コーン支持部は第 3図 (a)、第 8図〜第 1 0図から明らかなように、コーンホルダ 一 20の底板 21には中心にコーン揷入口 22を形成しており、コーン揷入口 22にガラス あるいは石英製の透明材料からなるコーン 23の小径部 24を嵌揷する。この小径部 2 4は適宜の大きさに設定することができるが、例えば" 1 Omm程度の大きさに設定する。 コーン 23の上部には係止フランジ 25を備え、この係止フランジ 25によりコーンホルダ 一 20の底板 21に載置される。コーンホルダ一 20の内周にはねじ 26を備え、このねじ に螺合するコーン押さえ部材 27をコーンホルダー 20にねじ込むことにより、コーン押さ ぇ部材 27の底壁の中心部に設けた通孔 28下面の押さえ溝 29に、コーン 23の係止フ ランジ 25を上側から押さえることによって、コーン 23をコーンホルダー 20に強固に固 定している。また、コーンホルダー 20を用いることによって、コーンとプレートの水平さ を正確に合わせることができる。 On the other hand, as is clear from FIG. 3 (a) and FIGS. 8 to 10, the cone support portion has a cone inlet 22 formed in the center of the bottom plate 21 of the cone holder 20. A small-diameter portion 24 of a cone 23 made of a transparent material made of glass or quartz is fitted into the inlet 22. The small-diameter portion 24 can be set to an appropriate size. For example, the small-diameter portion 24 is set to a size of about 1 Omm. A locking flange 25 is provided on the upper portion of the cone 23, and the cone 25 is provided by the locking flange 25. It is placed on the bottom plate 21 of the holder 20. A screw 26 is provided on the inner periphery of the cone holder 20, and a cone holding member 27 screwed into this screw is screwed into the cone holder 20, so that the cone holder 20 is screwed. コ ー ン The cone 23 is firmly fixed to the cone holder 20 by pressing the locking flange 25 of the cone 23 from above from the holding groove 29 on the bottom surface of the through hole 28 provided in the center of the bottom wall of the member 27. I have. Also, by using the cone holder 20, the horizontality of the cone and the plate can be accurately adjusted.
第 1図において、上記のようにして組み立てられた試験材料支持部におけるボトムプ レー Mは支持部材 7に固定され、この支持部材 7は図示実施例においてはモータ 30 のフレームに回動自在に取り付けられた支持プレート 31の先端に設けられている。な お、この支持プレー卜 31の固定手段としてはその他種々の手段を採用することができ る。  In FIG. 1, the bottom plate M in the test material support section assembled as described above is fixed to a support member 7, and in the illustrated embodiment, the support member 7 is rotatably attached to the frame of the motor 30. The support plate 31 is provided at the tip of the support plate 31. Various other means can be adopted as the means for fixing the support plate 31.
モータ 30のギア 32には中間ギア 33が嚙み合し、、この中間ギア 33には円筒状のコ ーンホルダー 20のフランジにポル卜で固定したリングギア 34が嚙み合し、、特に中間ギ ァ 33及びコーンホルダー 20が固定されるリングギア 34は支持プレー卜 31上にベアリ ングを介して支持されている。なお、これらのベアリングはできる限り駆動抵抗の少な し、ものが好ましぐ例えばベルトべアリング、浮動式のエアべアリング等によって支持す ることが好ましい。  An intermediate gear 33 is engaged with the gear 32 of the motor 30, and a ring gear 34 fixed to the flange of the cylindrical cone holder 20 by a port is engaged with the intermediate gear 33. The ring gear 34 to which the cone 33 and the cone holder 20 are fixed is supported on the support plate 31 via a bearing. It is preferable that these bearings have a driving resistance as low as possible and are supported by a preferable material such as a belt bearing or a floating air bearing.
モータ 30によって回転駆動されるコーンホルダー 20に固定したコーン 23の先端は、 支持プレート 31の支持部材 7に対してねじ込み固定しているボトムプレート 1上の試験 材料 10の表面に近接して、第 2図に示すように配置される。このコーン 23は小さく形 成されており、且つそのコーン 23と試験材料"! 0の表面のなす角度を小さく設定するこ とにより、コーン 23の下面と試験材料 1 0の上面、及ぴ周囲の壁面とで形成される空間 35を小さなものとし、小さな試験材料と少ない培養液により、培養液が流れている環 境を形成し試験を行うことができるようにしている。なお、このときのコーンが培養液に 与える剪断力をその駆動力の変化として検出するため、第1図のコンピュータ 40により モータ 8のトルクを測定することにより剪断力を演算してモニタ41に表示することがで きるようにしている。 The tip of the cone 23 fixed to the cone holder 20 rotated and driven by the motor 30 is close to the surface of the test material 10 on the bottom plate 1 screwed and fixed to the support member 7 of the support plate 31, They are arranged as shown in Figure 2. The cone 23 is formed small and the angle between the cone 23 and the surface of the test material "! 0" is set small, so that the lower surface of the cone 23, the upper surface of the test material 10 and the surrounding area are reduced. The space 35 formed by the wall surface is made small, so that the environment in which the culture solution flows can be formed and the test can be performed using small test materials and a small amount of culture solution. The computer 40 shown in FIG. 1 measures the torque of the motor 8 so that the shear force can be calculated and displayed on the monitor 41 in order to detect the shear force applied to the culture solution as a change in the driving force. I have to.
前記のようにモータ 30に対して揺動自在に支持されているコーンホルダー 20とボト ムプレート 1は、第 1図に示す状態では正立型落射蛍光顕微鏡 42の観測部に配置さ れ、これらの顕微鏡全体を略密封状態でアクリル等の材料からなるボックス 43に収納 できるようにし、ボックス 43内を温度制御装置 50により、例えば生理的な条件である 3 7°C等の所定の温度に維持することができるようにしている。また、正立型落射蛍光顕 微鏡 42はボックス 43内において防振板 44に固定し、外部からの振動を受けることに よる画像ノイズの発生を生じることがないようにしている。このとき、モータ 30の支持部 分にも防振板を設けることが好ましい。なお、必要に応じて防振板 44と正立型落射蛍 光顕微鏡 42の下面との間に微動調整用装置 39を設けても良い。 The cone holder 20 and the bottom plate 1, which are swingably supported by the motor 30 as described above, are arranged in the observation section of the upright epi-illumination fluorescence microscope 42 in the state shown in FIG. The entire microscope can be housed in a box 43 made of acrylic or the like in a substantially sealed state, and the inside of the box 43 is controlled by a temperature control device 50, for example, under physiological conditions. A predetermined temperature such as 7 ° C can be maintained. Further, the erecting epi-illumination fluorescence microscope 42 is fixed to a vibration isolating plate 44 in a box 43 so as not to generate image noise due to external vibration. At this time, it is preferable to provide an anti-vibration plate also in the support portion of the motor 30. Note that a fine adjustment device 39 may be provided between the vibration isolating plate 44 and the lower surface of the upright epi-illumination fluorescence microscope 42 as needed.
図示実施例においては支持プレート 31は前記のようにモータ 30のフレームに固定さ れ、モータ 30の駆動軸を中心に 90度程度揺動できるようにしており、試験材料の取り 付け、コーンの調整等の作業に際してはボックス 43の蓋 45を開放し、支持プレート 31 を手前側に回転して移動させ、その後、図示するような正立型落射蛍光顕微鏡 20の 所定の位置に戻し、蓋 45で封鎖することができるようにしている。  In the illustrated embodiment, the support plate 31 is fixed to the frame of the motor 30 as described above so that it can swing about 90 degrees about the drive shaft of the motor 30, so as to mount the test material and adjust the cone. When performing such operations, the cover 45 of the box 43 is opened, the support plate 31 is rotated to the front side and moved, and then returned to a predetermined position of the upright epi-illumination fluorescence microscope 20 as shown in FIG. So that they can be blocked.
モータ 30は圧電素子等を用いた高さ調節装置 46に支持されて顕微鏡の焦点を合わ せることができるようにし、これらは X—Yステージ 47上に固定されており、全体として X —Y— Zステージが構成される。 X— Yステージ 47は X軸方向及び Y軸方向に移動自在 に固定され、それにより顕微鏡で見る位置を自由に調節することができるようにしてい る。またあらゆる材料とコーンとの間の距離を一定に設定するためにこれを計測する装 置としてレーザー変位センサ一 48を用いる。それにより試験材料とコーンとの間隙を 別途調節し、サブミクロン単位に間隙調節を行うことができる。  The motor 30 is supported by a height adjusting device 46 using a piezoelectric element or the like so that the microscope can be focused, and these are fixed on an XY stage 47, and as a whole, X—Y— The Z stage is configured. The XY stage 47 is fixed so as to be movable in the X-axis direction and the Y-axis direction, so that the position viewed by the microscope can be freely adjusted. In order to set the distance between any material and the cone constant, a laser displacement sensor-148 is used as a device for measuring the distance. As a result, the gap between the test material and the cone can be separately adjusted, and the gap can be adjusted in sub-micron units.
モータ 30にはコンピュータ 40が接続され、これによりその回転が所定の状態に制御 されると共に、コンピュータ 40はモータ 30のトルクを検出し、コーン 23が回転するとき に液体の撹拌によって受ける負荷の状態を検出する。それによりコンピュータ 40は必 要に応じて培養液と試験材料との剪断力、あるいは他の試験における剪断力を演算し て表示装置に表示し、また印刷出力等を行う。これらの装置により円錐一平板型剪断 応力負荷装置 49が構成される。  A computer 40 is connected to the motor 30 so that its rotation is controlled to a predetermined state. In addition, the computer 40 detects the torque of the motor 30 and changes the state of the load received by stirring the liquid when the cone 23 rotates. Is detected. As a result, the computer 40 calculates the shearing force between the culture solution and the test material or the shearing force in another test as necessary, displays the calculated result on the display device, and prints out. These devices constitute a conical-flat plate type shear stress loading device 49.
一方、透明なコーン 23の上部には正立型落射蛍光顕微鏡 42における超長焦点の 対物レンズ 51が位置しており、水銀ランプ 52の光をフィルタ一 53とハーフミラー 54、 対物レンズ 51、コーン 23を各々介して試験材料 1 0に投射することカ《できるようになつ ている。また、このようにして照明された試験材料 1 0としての材料の表面に接着した細 胞力 流動する培養液中における状態を、対物レンズ 51、ハーフミラ一 54を介して高 精度の SITカメラ 56で撮影し、イメージプロセッサ 57によって画像解析及び画像処理し、 タイマ 58、 VTR59等を備えた画像記録装置に出力して適宜録画し、更にモニタ 60に その状態がリアルタイムに、しかも微弱な細胞の形態の変化も観察することができるよ うにしている。 On the other hand, an ultra-long focal length objective lens 51 of an erectile epi-illumination fluorescence microscope 42 is located above the transparent cone 23, and the light from the mercury lamp 52 is filtered by a filter 53, a half mirror 54, an objective lens 51, and a cone 51. It is possible to project the test material 10 through each of the 23. In addition, the state of the cell force adhered to the surface of the test material 10 illuminated in this manner in the flowing culture solution is measured by the high-precision SIT camera 56 via the objective lens 51 and the half mirror 54. Shooting, image analysis and image processing by image processor 57, The image is output to an image recording device equipped with a timer 58, a VTR 59, and the like, and is appropriately recorded. Further, the monitor 60 can monitor the state in real time and also observe a slight change in cell morphology.
この正立型落射蛍光顕微鏡 42はハロゲンランプ 61の光をコンデンサー 62、透明な 試料載置プレート 8を通して試験材料 1 0の裏面から照射することができるようにし、必 要に応じて倒立型透過顕微鏡として使用できるようにしている。 産業上の利用可能性  The upright epi-illumination fluorescence microscope 42 allows the light of a halogen lamp 61 to be irradiated from the back side of the test material 10 through a condenser 62 and a transparent sample mounting plate 8, and if necessary, an inverted transmission microscope. So that it can be used as Industrial applicability
本発明に依るコーンプレー卜型細胞剥離評価装置は、担体に細胞を播種した試験材 料を固定するボトムプレートと、透明材料からなり前記試験材料に近接して配置される コーンと、前記コーンを内部に固定し、外周に回転駆動部を備えたコーンホルダーと、 前記回転駆動部を介してコーンホルダーを回転する駆動装置と、前記コーンの上部か ら前記試験材料上の播種細胞等を観察する落射蛍光顕微鏡とを備えたことにより、所 定の流れによって得られる細胞の剪断応力環境下で材料表面上の播種細胞等の剥 離試験を行うことができ、実際の細胞の環境下と同等の条件での定量的な試験を容易 に行うことができる。しかもその状態を落射蛍光顕微鏡によってリアルタイムに観察す ることが可能となる。  A cone plate type cell detachment evaluation device according to the present invention comprises a bottom plate for fixing a test material in which cells are seeded on a carrier, a cone made of a transparent material and arranged in close proximity to the test material, A cone holder fixed inside and provided with a rotation driving unit on the outer periphery, a driving device for rotating the cone holder via the rotation driving unit, and observing seeded cells and the like on the test material from above the cone. Equipped with an epi-fluorescence microscope, it is possible to perform a detachment test of seeded cells on the material surface under the shear stress environment of the cells obtained by the specified flow, which is equivalent to the actual cell environment. Quantitative tests under conditions can be easily performed. In addition, the state can be observed in real time by an epifluorescence microscope.
また、前記試験材料は透明な試料載置プレートの窪みに載置するようにしたので、試 験材料の装着が容易となり、し力、も透明な試料載置プレートを用いてし、るので下方か らの光を透過させることができ、顕微鏡によって試験材料の状態を観察することもでき る。  In addition, since the test material is placed in the recess of the transparent sample mounting plate, the mounting of the test material is facilitated, and the force of the test material is also reduced. The light from the test material can be transmitted, and the condition of the test material can be observed with a microscope.
前記試験材料はボトムプレートに載置され、弾性を有する押さえ部材によりボトムプ レートに押圧状態に固定されるので、板状、膜状、 3次元担体などのあらゆる材料を装 着できる。その結果、あらゆる材料上の細胞の剪断応力による剥離現象を定量的に解 析することが可能である。  The test material is placed on the bottom plate and fixed in a pressed state on the bottom plate by an elastic pressing member, so that any material such as a plate, a film, and a three-dimensional carrier can be mounted. As a result, it is possible to quantitatively analyze the detachment phenomenon due to shear stress of cells on any material.
また、前記押さえ部材と試験材料との間には Oリングを介在させたので、試験材料を 押さえ部材により押さる際、材料の表面形状に関わらず確実なシール状態で強固に固 定すること力できる。  In addition, since an O-ring is interposed between the holding member and the test material, when the test material is pressed by the holding member, it must be firmly fixed in a reliable sealing state regardless of the surface shape of the material. it can.
また、前記押さえ部材には放射状の溝を形成し、外部から液体を導入することができ るようにしたので、試験装置を組み立てた後で試験材料とコーンの間の空間に培養液 等の液体を導入することが可能となり、使用し易い細胞剥離評価装置とすることができ る。 Further, a radial groove is formed in the holding member so that a liquid can be introduced from outside. As a result, a liquid such as a culture solution can be introduced into the space between the test material and the cone after assembling the test device, and an easy-to-use cell detachment evaluation device can be obtained.
また、前記ボトムプレー卜には、前記押さえ部材の溝の外周部に連通する孔を形成し たので、試験装置を組み立てた後で試験材料とコーンの間の空間に培養液等の液体 を導入することが容易となる。  In addition, since a hole communicating with the outer periphery of the groove of the holding member is formed in the bottom plate, a liquid such as a culture solution is introduced into a space between the test material and the cone after the test apparatus is assembled. It becomes easy to do.
また、前記コーンホルダーはその中心にコーン揷入口を設け、前記コーン揷入口にコ ーンの小径部を挿入した後、コーン押さえ部材によリコーンの上部を押圧固定したので、 コーンをコーンホルダーに確実に固定することができる。  In addition, the cone holder is provided with a cone 揷 entrance at the center thereof, and after inserting a small diameter portion of the cone into the cone 揷 entrance, the upper portion of the cone is pressed and fixed by the cone holding member, so the cone is fixed to the cone holder. It can be fixed securely.
また、前記コーンホルダーの回転駆動部はリングギアであり、前記リングギアを前記 駆動装置のギアにより中間ギアを介して回転駆動するようにしたので、コーンの上方に 駆動装置を設けることのないコーンの駆動機構を容易に形成することができる。  In addition, since the rotation drive unit of the cone holder is a ring gear, and the ring gear is rotationally driven by the gear of the drive device via an intermediate gear, a cone without a drive device above the cone is provided. Can easily be formed.
また、前記試験材料は板状、膜状、 3次元担体などの材料に播種した細胞であるの で、各種形状の試験材料の表面に播種した血管内皮細胞等が血流によって剥離する 状態を定量的に観察することができる。  In addition, since the test material is cells seeded on a material such as a plate, a film, or a three-dimensional carrier, the state in which vascular endothelial cells or the like seeded on the surface of the test material having various shapes are separated by blood flow is determined. Can be visually observed.
また、前記ボトムプレー卜の外周にはボトムプレート支持部材に螺合するねじを備え たので、試験材料を固定したボトムプレートを容易にボトムプレート支持部材に固定す ることができる。  Further, since the bottom plate is provided with a screw screwed to the bottom plate support member on the outer periphery, the bottom plate on which the test material is fixed can be easily fixed to the bottom plate support member.
また、前記コーンの直径は 1 Omm程度の小径に設定したので、小型試験材料を少 量の液体中で細胞の剥離を試験することができ、安価に且つ容易に試験を行うことが できる。  Further, since the diameter of the cone is set to a small diameter of about 1 Omm, it is possible to test the detachment of cells from a small test material in a small amount of liquid, and it is possible to perform the test at low cost and easily.
また、前記試験材料の直径は 1 5mm程度の小径に設定したので、小型試験材料を 少量の液体中で細胞の剥離を試験することができ、安価に且つ容易に試験を行うこと 力できる。  In addition, since the diameter of the test material is set to a small diameter of about 15 mm, the small test material can be tested for cell detachment in a small amount of liquid, and the test can be performed at low cost and easily.
また、本発明は、前記ボトムプレートに対するコーンの高さ調節を行う高さ調節装置を 備えたので、コーンと試験材料との間隙の調節を行うことにより、細胞に対する液体の 流動を所定状態に設定することができる。  Further, the present invention is provided with a height adjusting device for adjusting the height of the cone with respect to the bottom plate. Therefore, by adjusting the gap between the cone and the test material, the flow of the liquid to the cells is set to a predetermined state. can do.
また、本発明は、前記コーンと試験材料との間隙を測定するレーザー変位センサーを 設けたので、コーンと試験材料との間隙の調節をサブミクロン単位で正確に行うことが 可能となり、細胞に対する液体の流動を所定状態に厳密に設定することができる。 また、本発明は、前記ボトムプレートを固定すると共に前記コーンホルダーを回転可 能に支持する可動型アームにより、コーンを顕微鏡直下とそれ以外の部分との間に揺 動自在に支持するようにしたので、コーンを顕微鏡直下部分から回転して取り扱いの 容易な位置に移動することができ、各種の操作終了後には元の顕微鏡直下部分に戻 して顕微鏡による観察を容易に行うこと力《できる。 Further, in the present invention, since the laser displacement sensor for measuring the gap between the cone and the test material is provided, the gap between the cone and the test material can be accurately adjusted in submicron units. This makes it possible to precisely set the flow of the liquid to the cells to a predetermined state. Further, according to the present invention, the cone is swingably supported between immediately below the microscope and other portions by a movable arm that fixes the bottom plate and rotatably supports the cone holder. Therefore, the cone can be rotated from the position directly below the microscope and moved to a position where it can be easily handled. After various operations, the cone can be returned to the position directly below the microscope and the microscope can be easily observed.
また、本発明は、試験材料表面に顕微鏡の焦点を合わせる移動装置を前記駆動装 置部分に設けたので、顕微鏡における操作を行うことなく容易に顕微鏡の焦点を合わ せることができる。  In addition, according to the present invention, since the moving device that focuses the microscope on the surface of the test material is provided in the driving device, the microscope can be easily focused without performing the operation on the microscope.
また、本発明は、前記試験材料表面上の観察部位を変更する X— y— z軸方向可動装 置を備えたので、コーンを回転して試験を行っている状態で、顕微鏡で観察する部位を 自由に移動することが可能となる。  In addition, the present invention includes an X-y-z-axis movable device for changing the observation site on the surface of the test material, and therefore, the site to be observed with a microscope while the test is being performed by rotating the cone. Can be moved freely.
また、前記顕微鏡は、前記試験材料の表面を上方から観察する超長焦点対物レンズ を備えた落射蛍光顕微鏡であるので、試験材料の表面を顕微鏡により上方から容易 に観察することができる。  Further, since the microscope is an epifluorescence microscope equipped with an ultra-long focal length objective lens for observing the surface of the test material from above, the surface of the test material can be easily observed from above with a microscope.
また、前記顕微鏡は、前記試験材料の表面側から投光する照明装置と、裏面側から 投光する照明装置とを備えたので、試験材料の特性に合わせて正立型落射蛍光顕微 鏡として、また倒立型透過顕微鏡として使用することができる。  Further, since the microscope is provided with an illuminating device for projecting light from the front side of the test material and an illuminating device for projecting light from the back surface side, an upright-type epi-illumination fluorescence microscope according to the characteristics of the test material is provided. Also, it can be used as an inverted transmission microscope.
また、上記試験材料、試験材料支持部及び上記顕微鏡は、所定の温度に制御するこ とができるボックス内に収容するようにしたので、ボックス内を例えば生体の標準環境 としての 37°C等に容易に且つ正確に設定することができ、正確な測定及び観察を行う ことができる。  In addition, the test material, test material support, and microscope are housed in a box that can be controlled to a predetermined temperature. It can be set easily and accurately, and accurate measurement and observation can be performed.
また、少なくとも前記顕微鏡は、一体型防振板上の載置するようにしたので、顕微鏡 の振動に対する影響を少なくすることができ、外部から振動を受けることにより発生す る画像ノイズを少なくすることができる。  In addition, since at least the microscope is mounted on the integrated vibration isolating plate, the influence on the vibration of the microscope can be reduced, and the image noise generated by receiving external vibration is reduced. Can be.
また、前記顕微鏡には超高感度カメラを備えたので、コーンによって撹拌される血液 等の状態を正確に撮影することができる。  Further, since the microscope is equipped with an ultra-high sensitivity camera, it is possible to accurately photograph the state of blood and the like stirred by the cone.
また、前記顕微鏡には画像解析装置を接続したので、顕微鏡で得られた画像を容易 に解析することができ、その画像データを適宜加工することもできる。 また、前記顕微鏡にはビデオを接続したので、顕微鏡により得られた観察画像を長 期間記録しておくことができ、必要なときにこれを出力して観察することができる。 また、前記顕微鏡にはモニタを接続したので、顕微鏡により得られた画像をモニタで 見ることができ、容易に観察を行うことができる。 Further, since an image analyzer is connected to the microscope, an image obtained by the microscope can be easily analyzed, and the image data can be appropriately processed. Further, since a video is connected to the microscope, an observation image obtained by the microscope can be recorded for a long period of time, and can be output and observed when necessary. In addition, since a monitor is connected to the microscope, an image obtained by the microscope can be viewed on the monitor, and observation can be performed easily.
また、本発明は、前記コーンの回転を制御するコンピュータを備えたので、コーンの回 転を所定の値に確実に制御することができる。  In addition, since the present invention includes the computer that controls the rotation of the cone, the rotation of the cone can be reliably controlled to a predetermined value.

Claims

請 求 の 範 囲 The scope of the claims
1 . 担体に細胞を播種した試験材料 ( 1 0)を固定するボトムプレート( 1 )と、 1. A bottom plate (1) for fixing a test material (10) in which cells are seeded on a carrier,
透明材料からなり前記試験材料に近接して配置されるコーン (23)と、  A cone (23) made of a transparent material and placed in close proximity to the test material;
前記コーンを内部に固定し、外周に回転駆動部(34)を備えたコーンホルダー (20)と、 前記回転駆動部を介してコーンホルダーを回転する駆動装置 (30)と、  A cone holder (20) having the cone fixed therein and having a rotation drive unit (34) on the outer periphery, and a drive unit (30) for rotating the cone holder via the rotation drive unit;
前記コーンの上部から前記試験材料上の播種細胞を観察する落射蛍光顕微鏡 (4 2)とを備えたことを特徴とするコーンプレート型細胞剥離評価装置。  An epi-fluorescence microscope (42) for observing seeded cells on the test material from above the cone (42).
2. 前記試験材料 (1 0)は、透明な試料載置プレー卜 (8)の窪み (9)に載置することを 特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  2. The cell detachment evaluation apparatus according to claim 1, wherein the test material (10) is placed in a depression (9) of a transparent sample mounting plate (8).
3. 前記試験材料 (10)は、ボトムプレート (1 )に載置され、弾性を有する押さえプレー ト (1 1 )によりボトムプレート【こ押圧状態に固定されることを特徴とする請求の範囲第 1 項記載の細胞剥離評価装置。  3. The test material (10) is placed on a bottom plate (1) and is fixed to the bottom plate (pressed state) by an elastic holding plate (1 1). Item 2. The cell detachment evaluation device according to Item 1.
4. 前記押さえプレート (1 1 )と試験材料(1 0)との間には◦リング (1 4)を介在させた ことを特徴とする請求の範囲第 3項記載の細胞剥離評価装置。  4. The cell detachment evaluation device according to claim 3, wherein a ◦ ring (14) is interposed between the holding plate (11) and the test material (10).
5. 前記押さえプレー卜 (1 1 )には放射状の溝 (1 5)を形成し、外部から液体を導入す ることができるようにしたことを特徴とする請求の範囲第 3項記載の細胞剥離評価装置。  5. The cell according to claim 3, wherein a radial groove (15) is formed in the holding plate (11) so that a liquid can be introduced from the outside. Peeling evaluation device.
6. 前記ボトムプレー卜 ( 1 )には、前記押さえ部材 (27)の溝 (1 5)の外周部に連通す る孔 (1 6)を形成したことを特徴とする請求の範囲第 5項記載の細胞剥離評価装置。  6. The bottom plate (1) is provided with a hole (16) communicating with an outer peripheral portion of the groove (15) of the holding member (27). The cell detachment evaluation device according to the above.
7. 前記コーンホルダー (20)は、その中心にコーン揷入口(22)を有し、前記コーン 挿入口にコーン (23)の小径部 (24)を挿入した後、コーン押さえ部材 (27)によリコー ンの上部を押圧固定したことを特徴とする請求の範囲第 1項記載の細胞剥離評価装  7. The cone holder (20) has a cone inlet (22) at its center, and after inserting the small diameter portion (24) of the cone (23) into the cone insertion opening, the cone holder (27) The cell detachment evaluation device according to claim 1, wherein the upper part of the ribbon is pressed and fixed.
8. 前記コーンホルダー (20)の回転駆動部はリングギア (34)であり、前記リングギ ァを前記駆動装置 (30)のギア (32)により中間ギア (33)を介して回転駆動することを 特徴とする請求の範囲第 1項記載の細胞剥離評価装置。 8. The rotary drive unit of the cone holder (20) is a ring gear (34), and the rotary drive unit drives the ring gear via the intermediate gear (33) by the gear (32) of the drive unit (30). The cell detachment evaluation device according to claim 1, wherein the cell detachment evaluation device is characterized in that:
9. 前記試験材料 (10)は、板状、膜状、 3次元担体の材料に播種した細胞であること を特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  9. The cell detachment evaluation apparatus according to claim 1, wherein the test material (10) is a cell seeded on a plate-like, film-like, or three-dimensional carrier material.
1 0.前記ボトムプレート(1 )の内周にはボトムプレー卜支持部材 (7)に螺合するねじ (6)を備えたことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。 10. On the inner periphery of the bottom plate (1), screws screwed to the bottom plate support member (7) 2. The cell detachment evaluation device according to claim 1, comprising (6).
1 1 .前記コーン (23)の直径は、 1 0mm程度の小径に設定したことを特徴とする請求 の範囲第 1項記載の細胞剥離評価装置。  11. The cell detachment evaluation device according to claim 1, wherein the diameter of the cone (23) is set to a small diameter of about 10 mm.
1 2.前記試験材料 ( 1 0)の直径は、 1 5mm程度の小径に設定したことを特徴とする請 求の範囲第 1項記載の細胞剥離評価装置。  12. The cell detachment evaluation device according to claim 1, wherein the diameter of the test material (10) is set to a small diameter of about 15 mm.
1 3.前記ボトムプレー卜 ( 1 )に対するコーン (23)の高さ調節を行う高さ調節装置 (46) を更に備えたことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  1 3. The apparatus for evaluating cell detachment according to claim 1, further comprising a height adjusting device (46) for adjusting the height of the cone (23) with respect to the bottom plate (1).
1 4.前記コーン (23)と試験材料 ( 1 0)との間隙を測定するレーザ一変位センサー (4 8)を更に設けたことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  1 4. The cell detachment evaluation device according to claim 1, further comprising a laser displacement sensor (48) for measuring a gap between the cone (23) and the test material (10). .
1 5.前記ボトムプレート ( 1 )を固定すると共に前記コーンホルダー (20)を回転可能に 支持する可動型アーム (31 )により、コーン (23)を顕微鏡 (42)直下とそれ以外の部 分との間に揺動自在に支持したことを特徴とする請求の範囲第 1項記載の細胞剥離評 価装置。  1 5. The cone (23) is moved directly under the microscope (42) and other parts by a movable arm (31) that fixes the bottom plate (1) and rotatably supports the cone holder (20). 2. The cell exfoliation evaluation device according to claim 1, wherein the cell exfoliation evaluation device is supported so as to be swingable.
1 6.試験材料表面に顕微鏡の焦点を合わせる移動装置 (46)を前記駆動装置 (30) に設けたことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  1 6. The cell detachment evaluation device according to claim 1, wherein a moving device (46) for focusing a microscope on the surface of the test material is provided in the driving device (30).
1 7.前記試験材料表面上の観察部位を変更する X— y— z軸方向可動装置 (47)を備 えたことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  1 7. The cell detachment evaluation apparatus according to claim 1, further comprising an X-y-z-axis movable device (47) for changing an observation site on the surface of the test material.
1 8.前記顕微鏡 (42)は、前記試験材料の表面を上方から観察する超長焦点対物レ ンズ (51 )を備えた落射蛍光顕微鏡であることを特徴とする請求の範囲第 1項記載の 細胞剥離評価装置。  18. The microscope according to claim 1, wherein the microscope (42) is an epi-fluorescence microscope equipped with an ultra-long focal length objective lens (51) for observing the surface of the test material from above. Cell detachment evaluation device.
1 9.前記顕微鏡 (42)は、前記試験材料の表面側から投光する照明装置 (52)と、裏 側から投光する照明装置 (61 )とを備えたことを特徴とする請求項 1記載の細胞剥離  1 9. The microscope (42) includes a lighting device (52) for projecting light from the front side of the test material and a lighting device (61) for projecting light from the back side. Cell detachment as described
20.前記試験材料、試験材料支持部、コーンプレート支持部及び前記顕微鏡を収容 する内部を所定温度に制御することができるボックス (43)を更に備えたことを特徴と する請求の範囲第 1項記載の細胞剥離評価装置。 20. The apparatus according to claim 1, further comprising a box (43) capable of controlling the inside of the test material, the test material support, the cone plate support, and the inside of the microscope to a predetermined temperature. The cell detachment evaluation device according to the above.
21 .前記顕微鏡 (42)を載置する一体型防振板 (44)を備えたことを特徴とする請求 の範囲第 1項記載の細胞剥離評価装置。  21. The apparatus for evaluating cell detachment according to claim 1, further comprising an integrated vibration isolator (44) on which the microscope (42) is mounted.
22.前記コーン (23)の回転を制御するコンピュータ (40)を備えたことを特徴とする請 求の範囲第 1項記載の細胞剥離評価装置。 22. A contractor comprising a computer (40) for controlling the rotation of the cone (23). 2. The device for evaluating cell detachment according to claim 1.
23.前記落射顕微鏡 (42)には超高感度カメラ (56)を備えたことを特徴とする請求の 範囲第 1項記載の細胞剥離評価装置。  23. The apparatus for evaluating cell detachment according to claim 1, wherein the epi-illumination microscope (42) is provided with an ultra-sensitive camera (56).
24.前記落射顕微鏡 (42)には画像解析装置 (57)を接続したことを特徴とする請求 の範囲第 1項記載の細胞剥離評価装置。  24. The apparatus for evaluating cell detachment according to claim 1, wherein an image analyzer (57) is connected to the epi-illumination microscope (42).
25.前記落射顕微鏡 (42)にはビデオ (59)を接続したことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  25. The cell detachment evaluation device according to claim 1, wherein a video (59) is connected to the epi-illumination microscope (42).
26.前記落射顕微鏡 (42)にはモニタ (60)を接続したことを特徴とする請求の範囲第 1項記載の細胞剥離評価装置。  26. The apparatus for evaluating cell detachment according to claim 1, wherein a monitor (60) is connected to the epi-illumination microscope (42).
PCT/JP2002/012049 2001-11-19 2002-11-19 Cone plate type cell separation evaluating device WO2003044489A1 (en)

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