WO2003044489A1

WO2003044489A1 - Cone plate type cell separation evaluating device

Info

Publication number: WO2003044489A1
Application number: PCT/JP2002/012049
Authority: WO
Inventors: Katsuko Sakai; Tetsuya Tateishi; Takashi Ushida
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2001-11-19
Filing date: 2002-11-19
Publication date: 2003-05-30
Also published as: AU2002349385A1; JP2003156484A; JP3829179B2

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

Description Corn plate type cell detachment evaluation system Technical field

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

In addition, including that an O-ring is provided between the holding member and the test material.

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.

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.

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

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.

In addition, the microscope includes being mounted on an integrated vibration isolator.

In addition, the microscope includes an ultra-sensitive camera and is connected to an image analyzer, a video, and a monitor.

Further, the cell detachment evaluation apparatus according to the present invention includes a combustor for controlling the rotation of the cone.

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.

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.

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.

Other objects and other features of the present invention will be described in detail with reference to the drawings. BRIEF DESCRIPTION OF THE FIGURES

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). BEST MODE FOR CARRYING OUT THE INVENTION

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.

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.

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.

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.

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.

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.

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.

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.

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. 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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

The scope of the claims

1. A bottom plate (1) for fixing a test material (10) in which cells are seeded on a carrier,

A cone (23) made of a transparent material and placed in close proximity to the test material;

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;

An epi-fluorescence microscope (42) for observing seeded cells on the test material from above the cone (42).

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. 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. 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. 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. 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. 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. 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. 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.

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).

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.

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. 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. 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. 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. 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. 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.

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. 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. 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. 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. 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. 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. The apparatus for evaluating cell detachment according to claim 1, wherein an image analyzer (57) is connected to the epi-illumination microscope (42).

25. The cell detachment evaluation device according to claim 1, wherein a video (59) is connected to the epi-illumination microscope (42).

26. The apparatus for evaluating cell detachment according to claim 1, wherein a monitor (60) is connected to the epi-illumination microscope (42).