CN114018667A - Cell-retaining substrate holder for producing observation specimen, kit including same, and method for producing observation specimen - Google Patents
Cell-retaining substrate holder for producing observation specimen, kit including same, and method for producing observation specimen Download PDFInfo
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- CN114018667A CN114018667A CN202111301485.4A CN202111301485A CN114018667A CN 114018667 A CN114018667 A CN 114018667A CN 202111301485 A CN202111301485 A CN 202111301485A CN 114018667 A CN114018667 A CN 114018667A
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- cell
- filter
- retaining
- support plate
- cells
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Optics & Photonics (AREA)
- Hydrology & Water Resources (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The present invention provides a cell-retaining substrate holder for producing an observation specimen and a cell-retaining substrate kit, which can easily attach and detach a cell-retaining substrate, and provides a method for producing an observation specimen, which can simultaneously solve three problems of cell detachment, drying, and three-dimensional disappearance, and can sufficiently observe a cell organelle using an optical microscope. The cell-retaining substrate holder includes: a support plate (1) having a cell-retaining substrate arrangement part (12), the cell-retaining substrate arrangement part (12) having a window part through which water can pass; and (two) clamping plates (2), (3) which have a window through which water can pass, and which are detachably fixed to the cell holding substrate arrangement part (12) so as to be capable of clamping the cell holding substrate (9) in cooperation with the support plate (1). The cell-retaining substrate kit (10) comprises the cell-retaining substrate holder, and a cell-retaining substrate (9). In the method, cells are captured by an inorganic fiber aggregate, and wet fixation, dyeing, and sealing with a sealing agent having a refractive index equal to that of the inorganic fiber are directly performed.
Description
The present application is filed on 2016 (9/14/9/2016) under the application No. 201680050931.X, entitled cell-retaining substrate holder for observation sample preparation, a kit including the same, and a divisional application of a method for preparing an observation sample. Priority of Japanese prior applications JP2015-180832, JP2015-180833 and JP2016-114067 is required, with priority dates of 9/14/2015, 9/14/2015 and 6/8/2016.
Technical Field
The present invention relates to: a cell-retaining substrate holder and a kit including the same, which can be used for producing an observation specimen produced for the purpose of observing cells by various stains and using a microscope (optical or fluorescent light), and a method for producing an observation specimen, more specifically, the present invention relates to: a method for preparing an observation specimen was performed, in which wet fixation and subsequent staining using a staining bath (papanicolaou staining, PAS staining, alcian blue staining, and the like) were performed in pathological staining performed in cell diagnosis (cytodiagnosis).
Background
The field is mainly used for diagnosing cancer and is implemented according to the following steps: an observation specimen is prepared from cells collected from a patient, and abnormal cells (abnormal cells) are detected by microscopic examination of a person qualified by a cytodiagnosis doctor, a cytodiagnosis specialist, a pathology specialist, or the like. In addition, this field is mainly performed in medical institutions such as hospitals, and a large number of specimens (observation specimens and microscopic specimens) need to be prepared and examined every day.
The cell diagnosis is classified according to the collection method of the cells, and comprises the following steps: a diagnosis of exfoliated cells (sputum, urine, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid, bile, etc.) in which body fluid mixed with exfoliated cells is collected; exfoliative cytodiagnosis (cervical or uterine body, bronchus, bile duct, pancreatic duct, etc.) in which a brush, a cotton swab, etc. are inserted into the body and rubbed across the affected part to collect cells; a puncture aspiration cytodiagnosis (breast, thyroid, lymph node, liver, etc.) in which a fine needle is inserted into a lesion and aspirated to collect cells, and the like, and an observation specimen is prepared by applying the collected cells to an observation substrate (slide glass). The slide glass is often applied in a state in which cells are dispersed in a liquid, and when the collected state is not a liquid such as a body fluid, the slide glass may be applied after being dispersed in a fixed liquid (liquid specimen cytology).
"cell staining" has a variety of fields in addition to this field. The staining method, reagent, instrument, and equipment are completely different depending on the purpose of use (degree of completion) of the prepared observation specimen. Examples of the purpose of dyeing include: staining the tissue with different colors according to the cell types, thereby defining the respective positions (for example: immunohistostaining); based on some markers and morphological characteristics of cells, the number and staining intensity of the cells of interest can be detected by using the device (e.g., high content screening, flow cytometry); among the above objects, observation is carried out by the human eye (e.g., cell counting by a hemocytometer); staining the target substance inside and outside the cell to observe the site (for example, fluorescence microscope observation); observing the shape and color of the organelles (for example: cell diagnosis); further, although the details of the internal structure of the organelle and the structure thereof are observed (for example, electron microscope observation), in this field, observation is performed by observation under an optical microscope, and it is required to obtain an observation image capable of discriminating the shape of the organelle, the color of the dyeing, and the shade at an observation magnification (about 200 to 400 times) capable of sufficiently observing the organelle.
In addition, in the conventional observation specimen preparation step in this field, a unique operation step as described below, which has not been performed in other staining fields, is required due to the state of the supplied cell sample, the nature of the reagent used, the degree of completion of the specimen required, and the like.
Application of the composition
The provided cell sample is applied to an observation substrate (slide glass). Including a method of directly applying to a slide glass (a pull tab method, a push tab method, a centrifugal direct application method, etc.), and a method of transferring to a slide glass after capturing cells with a filter (a filter method). The former pull tab method and push tab method are simple and convenient, but are difficult to apply to a narrow range, and also have a problem that the cell density becomes low because cells adhere to the glass side used for the push tab, and a large range must be observed during the observation operation. In addition, the latter filter method has a problem that all captured cells cannot be transferred to a slide glass, and thus there is a problem that the cells are lost, and there is a problem that the shape of the cells is denatured by the pressure at the time of transfer operation.
Wet fixation
The procedure is to immerse the cells in a reagent (fixing solution) and fix the cells so as to prevent denaturation of the applied cells. In this field, wet fixation is important, and when cells are denatured by drying, the staining property changes, which affects diagnosis. Typically, cells applied to a slide must be wet fixed within a few seconds. Therefore, it is difficult to apply a large number of samples simultaneously. Further, the centrifugal direct application method and the filter method using the apparatus for application have the following problems: during removal from the apparatus, the slide rack and also during the transfer operation of the filter method. In addition, the cells immediately after application are easily peeled off from the slide glass, and when immersed in a fixing solution, a large number of cells are peeled off from the slide glass. In the case of cells with clumpy stackability, it is more difficult to hold them on a slide. In order to solve the problem of the peeling, there is also a proposal of a slide glass to which a coating for preventing cell peeling (silane coating or the like) is applied, but in practice, sufficient effects are not obtained. In order to solve the problems of peeling and drying at the same time, a method of spraying or dropping a fixative solution having moisture retention properties to coat the cells and the slide glass has been proposed. However, there is a new problem that the staining property of cells is changed and the diagnosis is affected because the cells are peeled off at the moment of spraying the fixing solution and a moisturizing component (such as PEG) coats the cells.
Dyeing (including solvent change, separation, color development)
Several slide glasses on which smearing and wet fixing have been completed are vertically set in a staining basket, and are put into a staining bath containing a reagent such as a staining solution or a pad covered with tap water, and allowed to stand or sink. The dyeing process is long and complicated, and for example, triple dyeing is performed in a method of pasteurizing dyeing, and although there is a little difference depending on the apparatus used, the total process requires about 20 to 25 steps. This is due to: in addition to dyeing, a step of replacing a solvent with a dyeing reagent, a washing step (separation) of washing off an excess dyeing solution, a step of immersing in a solution for color development, and the like are also included. In addition, the time of each staining step and the number of times of appearance and disappearance in the reagent are preferably performed in a strictly set state, and in the staining in this field in which the target such as a cell organelle is stained separately, it is very important to obtain the stability and reproducibility of the staining. In this way, in the staining in this field, it is necessary to treat a large number of patient specimens uniformly and rapidly, and to perform staining with stability and reproducibility, and in this case, it is useful to use a staining basket and a staining bath. Since the staining basket can hold a plurality of slide glasses without damaging the application surface, the operation of moving and sinking between staining tanks is facilitated. Further, in order to prevent uneven dyeing and uneven decoloring, it is necessary to quickly immerse the application surface in a large volume of a dyeing liquid or a separation liquid (cleaning liquid), but such unevenness can be avoided by performing an operation of loading and unloading using a dyeing basket and a dyeing tank. In this way, in the staining step, since the contact with the reagent is performed through several steps, there is a problem that the cells applied to the slide glass are peeled off similarly to the wet fixing step.
Dewatering and color clarification
The dehydration was performed by gradually transferring from a low-concentration ethanol tank to a pure ethanol tank, and finally immersing in a xylene tank. This operation makes the tissue transparent, and the specimen is suitable for microscopic examination. Since a solvent having a strong dissolving power is used for the color clarification, when cells on a polycarbonate film mainly used in the filter method are directly clarified, the film is dissolved and clouded, and therefore, the film cannot be used as a substrate for observation, and it is necessary to transfer the captured cells to a slide glass.
Sealing and fixing
The application surface is injected with a sealing agent (resin dissolved in a solvent), and cells are sandwiched between a cover glass and a slide glass. By sealing the stained cells with a sealing agent, physical impact on the painted surface during microscopic examination, deterioration due to microscopic illumination, and discoloration with time can be prevented, and the cells can be stored for a long period of time.
Observation of
Using an optical microscope. For example, in Papanicolaou staining, the chromatin structure in nuclei needs to be observed, while in PAS staining, the color of particles needs to be observed, and thus it is necessary to obtain a clear observation image 200 to 400 times as much as possible.
In this field, the preparation of a normal observation specimen has been carried out in the above-described steps. Such a dyeing process using a dyeing basket or a dyeing vessel is carried out manually or by an automatic dyeing apparatus.
In this field, it is conventionally known that an observation specimen is prepared by being applied to a slide glass. The slide glass has good observation performance, and cells are attached to the glass surface in a slightly diffused state and distributed, so that the inside of the cells is easily observed, and the slide glass is flat and has an advantage that the sealing operation is easily performed. On the other hand, due to the planar structure, cells cannot be held in the process of preparing the observation specimen, and peeling occurs. Moreover, the composition lacks moisture retention and is liable to cause cell degeneration due to desiccation. In addition, since the cells have a stretched shape, there is a problem that it is difficult to detect the shape of the cells and the dimensional irregularity of the cell nucleus, which are characteristics of cancer cells. Thus, there has been no method for producing an observation specimen that can simultaneously solve the three problems of cell detachment, drying, and three-dimensional disappearance, and can sufficiently observe a cell organelle with an optical microscope.
Further, a method of capturing cells using a filter and preparing a sample for analysis and diagnosis has been proposed as described below, but the above-mentioned technical problem cannot be solved.
On the other hand, when the filter in which the cells are trapped is observed on the microscope stage, it is preferable that only the filter is taken out from the filter device or the like and placed on a thin substrate such as a slide glass, thereby completing the observation of the specimen. The reason is that: the sealing operation is required, and the distance between the objective lens and the observation target becomes short when observation is performed at a high magnification, so that the entire observation specimen needs to be thinned. In order to prevent physical damage to the cell-solidified surface during microscopic operation, damage due to microscopic illumination, and discoloration with time, the sealing operation can be performed by sealing with an oily substance mainly composed of glycerin or liquid paraffin, or a reagent called a sealing agent composed of a resin dissolved in a solvent. Since the visibility is deteriorated if air bubbles are mixed during sealing, cells are sandwiched between two flat glass sheets (glass slides, coverslips) so that air bubbles are less likely to adhere to and remain on the sealing surface, and the gap is sealed with a sealing agent.
Although the specimen holder disclosed in patent document 4 describes taking out a window capable of capturing cells, the method of fixing the window at the time of cell capture, the method of taking out the window, and the mechanism therefor are not specifically described. In this way, in the case of a filter holder in which only the filter cannot be easily attached and detached, it is not possible to obtain an observation specimen having a desired finish in this field.
In the filter module disclosed in patent document 5, the filter is adhered to the frame (resin frame) with an adhesive so as to maintain the shape of the filter at the time of cell trapping, and cannot be easily attached and detached, and when the filter in which cells are trapped is sealed with the frame sandwiched between glass, bubbles tend to remain in the stepped portion between the filter and the frame, and the volume of the filter is reduced while the sealing agent is dried, so that a gap is formed between the upper and lower glass, and bubbles are generated, thereby deteriorating the visibility. Moreover, the observation specimen itself becomes thick, and microscopic examination at high magnification is not possible. Therefore, although it is necessary to remove only the filter from the frame, in this case, the filter cannot be removed without performing a complicated operation such as cutting the filter, and there is a possibility that the filter is distorted and cells adhering to the filter are peeled off when the cutting operation is performed.
Documents of the prior art
Patent document
Patent document 1: japanese laid-open patent publication No. 4-165321
Patent document 2: japanese patent laid-open No. 2012-75383
Patent document 3: japanese patent laid-open publication No. 2004-298158
Patent document 4: japanese Kohyo publication Hei 11-507724
Patent document 5: japanese Kokai publication No. 2008-537485
Disclosure of Invention
Technical problem to be solved
Accordingly, an object of the present invention is to provide a cell-holding substrate holder and a kit for producing a cell observation specimen, which enable easy attachment and detachment of a cell-holding substrate such as a filter, and to provide a method for producing an observation specimen, which can solve three problems of cell detachment, drying, and three-dimensional disappearance at the same time and can sufficiently observe a cell organelle using an optical microscope.
(II) technical scheme
The technical problem is solved by the following solution of the present invention.
[1] A cell-retaining substrate holder for observing specimen preparation, comprising:
(1) a support plate having a cell-retaining substrate arrangement section having a window section through which water can pass; and
(2) and a detachable clamping plate which has a window through which water can pass, and which clamps and fixes the cell-retaining substrate to the cell-retaining substrate arrangement part so as to be engaged with the support plate.
[2] The cell-retaining substrate holder for observation specimen preparation according to [1], wherein the support plate has a cell-retaining substrate arrangement part and a frame part.
[3] The cell-retaining substrate holder according to [1] or [2], wherein the support plate has recesses (preferably through holes) on both sides with the window interposed therebetween, and the clamping plate is a cover plate having claws that can be fitted into the recesses (preferably through holes).
[4] The cell-retaining substrate holder according to [2], wherein the clamping plate is a flange plate having a flange portion capable of abutting against the frame portion of the support plate, and the cell-retaining substrate holder further comprises a clip capable of clamping and fixing the frame portion of the support plate and the flange portion of the flange plate.
[5] The cell-retaining substrate holder according to [4], wherein the flange plate has a cup portion into which the sample can be introduced and which can communicate with the window portion of the flange portion.
[6] The cell-retaining substrate holder according to [5], wherein the cup portion is detachable.
[7] The cell-retaining substrate holder according to any one of [3] to [6], which comprises a cover plate, a flange plate, and a clip.
[8] The cell-retaining substrate holder according to any one of [1] to [7], wherein the window portion of the support plate has a support member capable of supporting the cell-retaining substrate.
[9] The cell-retaining substrate holder according to any one of [1] to [8], wherein the support plate has a rectangular shape and a size capable of being accommodated in the staining basket.
[10] The cell-retaining substrate holder according to any one of [1] to [9], wherein the support plate and/or the holding plate has a recess (preferably a through-hole) into which the tip of the forceps can be inserted.
[11] The cell-retaining substrate holder according to any one of [1] to [10], wherein any of the materials is composed of an organic resin.
[12] The cell-retaining substrate holder according to [1] or [2], wherein the holding plate is a cover plate that can be fitted, clamped, or pivoted to the support plate.
[13] The cell-retaining substrate holder according to any one of [3] and [7] to [12], wherein the cover plate has a force point portion capable of applying a force.
[14] The cell-retaining substrate holder according to any one of [3] and [7] to [13], wherein the cell-retaining substrate arrangement part and/or the cover plate of the support plate has a liquid passing structure.
[15] A kit for preparing an observation specimen, comprising the cell-retaining base material holder according to any one of [1] to [14], and a cell-retaining base material.
[16] The kit according to [15], wherein the cell-retaining substrate is an inorganic fiber sheet having a porosity of 90% or more.
[17] The kit for producing an observation specimen according to [15] or [16], wherein the shape and size of the cell-retaining base material when the cell-retaining base material is attached to the cell-retaining base material holder are rectangular shapes that can be stored in the staining basket.
[18] A method for producing an observation specimen, which comprises capturing cells with an inorganic fiber aggregate, directly performing wet fixation, staining, and sealing with a sealing agent having a refractive index equivalent to that of the inorganic fiber.
(III) advantageous effects
According to the cell-retaining substrate holder of [1] of the present invention, since the cell-retaining substrate can be held and fixed by the support plate and the holding plate, and the cell-retaining substrate can be easily removed from the support plate, it is possible to move only the cell-retaining substrate onto the slide glass, realize a stable sealing operation, and make the entire observation specimen thin.
According to the cell-retaining substrate holder of [2] of the present invention, the support plate has a frame portion, and the frame portion can be provided with: various functions are added to the cell holding substrate, such as an insertion recess for detachably fixing the holding plate, a recess into which the tip of the forceps can be inserted when the cell holding substrate is collected, and a storage recess capable of storing the projection of the holding plate.
According to the cell-retaining substrate holder of [3] of the present invention, since the cell-retaining substrate can be fixed to the support plate by the claws of the cover plate, no shear force acts on the cell-retaining substrate. Therefore, even if an inorganic cell-retaining substrate (for example, an inorganic fiber sheet) such as glass is used, there is no fear of breakage, and the cell-retaining substrate can be easily attached and detached. Further, the present invention has excellent workability because it has a cover plate and can be used as it is in a staining process using a staining basket in a state where the cell-retaining base material is sandwiched and fixed between the cover plate and the cell-retaining base material arrangement part in cooperation with the support plate.
According to the cell-retaining substrate holder of [4] of the present invention, since the cell-retaining substrate can be fixed to the support plate by the clip, no shear force acts on the cell-retaining substrate. Therefore, even if an inorganic cell-retaining substrate (for example, an inorganic fiber sheet) such as glass is used, there is no fear of breakage, and the cell-retaining substrate can be easily attached and detached.
According to the cell-retaining substrate holder of [5] of the present invention, in the case where the cell-retaining substrate is used as a filter, cells can be captured by filtration even in the case of a large amount of liquid specimen.
According to the cell-retaining substrate holder of [6] of the present invention, since the cup portion is separable, the volume of the kit can be reduced.
According to the cell-retaining substrate holder of [7] of the present invention, since the cell-retaining substrate can be used as it is in the staining process using the staining basket in a state where the cell-retaining substrate is clamped and fixed to the cell-retaining substrate arrangement portion in cooperation with the support plate, the workability is excellent, and since no shearing force acts on the cell-retaining substrate, the cell-retaining substrate can be attached and detached so as to avoid damage to the cell-retaining substrate.
According to the cell-retaining substrate holder of [8] of the present invention, since the window portion of the support plate is provided with the support member capable of supporting the cell-retaining substrate, the cell-retaining substrate is less likely to be damaged by water pressure generated by operations such as cell filtration and staining.
According to the cell-retaining substrate holder of item [9] of the present invention, since the support plate can be accommodated in the staining basket, it can be applied to a staining basket and an automatic staining apparatus which have been conventionally used for staining cells applied to slides.
According to the cell-retaining substrate holder of [10] of the present invention, since the cell-retaining substrate can be attached and detached using forceps, the workability is good and the possibility that the cell-retaining substrate is damaged by the tip of the forceps is reduced.
The cell-retaining substrate holder according to [11] of the present invention is made of an organic resin, and therefore, the cell-retaining substrate holder is easy to discard after use.
According to the cell-retaining substrate holder of [12] of the present invention, since the cell-retaining substrate can be fixed to the support plate by fitting, sandwiching, or pivoting the cover plate, no shearing force acts on the cell-retaining substrate. Therefore, even if an inorganic cell-retaining substrate (for example, an inorganic fiber sheet) such as glass is used, there is no fear of breakage, and the cell-retaining substrate can be easily attached and detached. In addition, the cover plate can be used directly in the staining process using the staining basket in a state where the cell-retaining base material is sandwiched and fixed between the cover plate and the support plate, and thus the workability is excellent.
According to the cell-retaining substrate holder of [13] of the present invention, since the cover plate has a force point portion capable of applying a force and the clamping and fixing action between the cover plate and the support plate can be released by applying a force to the force point portion, the cell-retaining substrate can be easily attached and detached without being damaged. For example, if the force point portion is a protruding portion that can catch a finger or a notched portion into which a finger can be inserted, the force can be applied to the protruding portion or the notched portion, thereby releasing the clamping and fixing action between the cover plate and the support plate, and the cell-retaining substrate can be easily attached and detached without being damaged.
According to the cell-retaining base material holder of [14] of the present invention, since the cell-retaining base material arrangement part and/or the cover plate has a liquid passing structure, it is possible to facilitate the washing and removal of the residual staining solution which is easily generated at the time of staining and which is present in the portion where the cover plate of the cell-retaining base material and the cell-retaining base material arrangement part are sandwiched and fixed. Therefore, the residual staining solution is less likely to diffuse and stain, and thus the microscopic observation performance is excellent.
According to the kit of [15] of the present invention, since the cell-retaining base material can be held and fixed by the support plate and the holding plate and the cell-retaining base material can be easily removed from the support plate, it is possible to move only the cell-retaining base material onto the slide glass, realize a stable sealing operation, and make the entire observation specimen thin. In addition, when the cell holding base material is a porous sheet, concentration of floating cells and quasi-solidification on the cell holding base material can be performed in one step of cell filtration by gravity without requiring expensive equipment or special operation skills, thereby producing a cell observation specimen.
According to the kit of [16] of the present invention, since the cell-retaining base material is composed of the inorganic fiber sheet and the cells can be fixed in the internal space of the cell-retaining base material, the operation of immersing the cells in the reagent can be realized in the same manner as the cells in the adhered state. Further, since the porosity is 90% or more, not only is it easy to fix cells to the internal voids of the cell-retaining base material, but also the water permeability is excellent.
According to the kit of [17] of the present invention, since the cell-retaining base material can be stored in the staining basket in a state of being attached to the cell-retaining base material holder, the kit can be applied to a staining basket and an automatic staining apparatus which have been conventionally used for staining cells applied to a slide glass, and can stain the cells with good operability.
According to the method for producing an observation specimen of [18] of the present invention, since cells are stably held in the internal voids of the inorganic fiber aggregate having excellent rigidity, the cells can be prevented from being peeled off and from being stereoscopically lost. In addition, it is also useful for retaining cell masses having overlapping properties.
Further, since the inorganic fibers are hydrophilic and the inorganic fiber aggregate has a certain degree of water retention, cells are not dried even when left for several minutes, and stable wet fixation can be achieved. Therefore, the present invention is useful for simultaneously preparing a large number of samples and applying the samples to a device.
Further, since the stereoregularity of cells can be maintained, it is also useful for observing cell masses having a stereoregularity and overlapping property of cells and nuclei.
Further, since the inorganic fiber aggregate exhibits resistance to a solvent (ethanol, methanol, xylene) included in a reagent for cell diagnosis, and can be used as an observation substrate and sealed directly after cell capture, a transfer operation by a conventional filter method using a membrane filter is not required. Therefore, there is no fear of denaturation of the cell shape by the pressure of transfer, and the loss of cells generated at this time is avoided. Further, since the sealing is performed by the sealing agent having a refractive index matching the fiber, the diffuse reflection caused by the curved fiber surface can be suppressed, and a clear observation image can be obtained to the extent that the organelles can be discriminated even under an optical microscope.
Drawings
Fig. 1 is a perspective view schematically showing one embodiment of a filter kit according to the present invention.
Fig. 2 is a photograph showing a state before use (a state before holding a filter) of one embodiment of the filter holder (support plate and cover plate) of the present invention included in the filter kit of the present invention shown in fig. 1, in place of the drawings.
Fig. 3 is a photograph showing an alternative drawing showing a state in which the filter is held by being sandwiched between the support plate and the cover plate shown in fig. 2.
Fig. 4 is a photograph showing an alternative drawing showing a state in which a flange plate (flange portion and cup portion are integrated) included in another embodiment of the filter kit of the present invention is assembled with a gasket.
Fig. 5 is a photograph showing an alternative drawing in a state where the filter kit of the present invention shown in fig. 1 (in which the flange plate shown in fig. 4 is used as the flange plate) is assembled.
Fig. 6 is a photograph showing an alternative drawing of a filter kit (support plate, cover plate, and filter) according to another embodiment of the present invention in a state before use (state before holding the filter).
FIG. 7 is a photomicrograph showing a cell image obtained by observing the observation specimen of example 1 with an optical microscope (magnification: 100 times).
FIG. 8 is a photomicrograph showing a cell image obtained by observing the observation specimen of example 1 with an optical microscope (magnification: 400 times).
FIG. 9 is a photomicrograph showing a cell image obtained by observing the observation specimen of example 2 with an optical microscope (magnification: 100 times).
FIG. 10 is a photomicrograph showing a cell image obtained by observing the observation specimen of example 2 with an optical microscope (magnification: 400 times).
FIG. 11 is a photomicrograph showing a cell image obtained by observing the observation specimen of comparative example 1 with an optical microscope (magnification: 100 times).
FIG. 12 is a photomicrograph showing a cell image obtained by observing the observation specimen of comparative example 1 with an optical microscope (magnification: 400 times).
FIG. 13 is a photomicrograph showing a cell image obtained by observing the observation specimen of comparative example 2 with an optical microscope (magnification: 100 times).
FIG. 14 is a photomicrograph showing a cell image obtained by observing the observation specimen of comparative example 2 with an optical microscope (magnification: 400 times).
Detailed Description
Next, a filter holder for an observation specimen preparation for floating cells (hereinafter, may be referred to as a filter holder of the present invention) which is one of the cell holder for an observation specimen preparation of the present invention and in which a cell holder exhibits a filter function, and a filter kit (hereinafter, may be referred to as a kit of the present invention) which is one of the kits for an observation specimen preparation of the present invention including the cell holder for an observation specimen preparation will be described with reference to the drawings, and a method for preparing an observation specimen of the present invention will be described.
The filter kit of the present invention comprises a filter for capturing cells, and the filter holder of the present invention. The present invention will be described below with reference to a kit, but the description is directly applicable to the filter holder of the present invention, except that the kit of the present invention includes a filter.
One aspect of the kit 10 of the present invention shown in fig. 1 is comprised of a support plate 1; a cover plate 2; a flange portion 31 and a cup portion 32 which can constitute the flange plate 3; two clips 4a, 4 b; a gasket 5; and a filter 9.
The support plate 1 has a filter arrangement 12 and can further have a frame part 13, the filter arrangement 12 having a window part 11 through which water can pass. The window 11 may be provided with a support member 14 capable of supporting the filter 9. Although the window portion 11 shown in fig. 1 is provided with the X-shaped support member 14, the kit of the present invention may be provided with support members other than the X-shape, for example, in a X-shape, a triangular shape, an I-shape, a Y-shape, a two-shape, a lattice shape, a mesh shape, and the like.
Further, the recess 17 into which the tip of the forceps can be inserted is preferably provided adjacent to the filter arrangement portion 12 so that the filter 9 can be taken out. In this way, if the recess 17 into which the tip of the forceps can be inserted is provided in either the support plate 1 or the holding plate, the filter 9 can be removed without damage even if the filter 9 is stuck to either the support plate 1 or the holding plate when the holding plate is removed. Further, since the protruding portion 24 of the lid plate 2 can be housed in the housing recess 18 and no projection is formed on the filter holder, it can be easily housed in a dyeing basket for use as in the conventional art.
In fig. 1, the filter arrangement portion 12 is a rectangular shape similar to the filter 9, and is formed of a recess having an outer shape with substantially the same area. Therefore, the filter 9 can be closely accommodated in the filter arrangement portion 12, and the filter 9 can be prevented from being displaced. In fig. 6, the filter arrangement portion is a right circular shape similar to the filter, and is formed of a recess having an outer shape with substantially the same area. Therefore, the filter can be closely accommodated in the filter arrangement portion, and the filter can be prevented from being displaced. In this way, the filter arrangement portion is preferably formed of a recess having the same shape as the filter and having an outer shape with substantially the same area.
However, the filter arrangement portion may be a recess portion that is different from the filter shape in an extent to avoid the filter from being displaced or that has a larger area than the filter. In this case, since a space is left between the filter arrangement portion and the filter and the tweezers can be inserted between the filter arrangement portion and the filter, the filter can be easily set in the filter arrangement portion without being damaged, and the filter can be easily removed from the filter setting portion. For example, in the case of a perfect circle-shaped filter, the filter arrangement portion can be constituted by a rectangular recess having the same diameter as the filter or a longer longitudinal length and a longer lateral length than the diameter of the filter, and tweezers can be inserted into a space formed between the filter arrangement portion and the filter, so that the filter can be attached and detached without damage.
In addition, when there is a possibility that the filter may be displaced due to the filter arrangement portion being a concave portion having a shape different from that of the filter or a larger area than that of the filter, the filter arrangement portion preferably has a convex portion at a position contactable with an outer edge of the filter so as to be locally contactable with the outer edge of the filter to prevent displacement of the filter.
Further, as shown in fig. 6, the filter arrangement portion 12 preferably has a through hole 19 as a liquid passing structure. This is due to: by providing the through-holes in this manner, liquid permeability is improved through the through-holes, and in the cleaning step after the dyeing step, the cleaning liquid easily reaches the filter 9 and is easily drained after cleaning, so that the residual dyeing liquid is easily cleaned and removed. That is, if the staining solution remains in the portion sandwiched between the cover plate 2 of the filter and the filter arrangement portion 12, the staining solution spreads to generate staining spots and the observation performance under the microscope is degraded, but the structure (through-hole 19) of the through-flow allows the remaining staining solution to be easily cleaned and removed, and thus the effect of excellent observation performance under the microscope is obtained.
Although the liquid passing structure in fig. 6 is the through hole 19, it is not necessarily the through hole, and may be a groove provided on the surface contacting the filter and communicating with the outer edge of the filter arrangement portion 12 (including the fitting recesses 15a, 15b, and the recess 17). When the liquid passage structure is a groove, the liquid passage property is improved through the groove, and therefore, the same effect as in the case of a through hole is exhibited, and the effect of excellent observation property under a microscope is obtained.
As will be described later, when the gasket 5 capable of preventing leakage is provided between the window portion 35 of the flange plate 3 and the window portion 11 of the support plate 1 (the window portion 21 of the cover plate 2 when the cover plate 2 is used), the liquid passage structure (the through hole 19, the groove, or the like) is preferably located outside the position where the gasket 5 contacts so as to prevent leakage of cells in a floating state during cell filtration.
The cover plate 2 has a window 21 through which water can pass, and a support member 22 capable of supporting the filter 9 can be provided in the window 21. Examples of the shape of the support member 22 include an X shape, a star shape, a triangular shape, an I shape, a Y shape, a two-letter shape, a lattice shape, and a mesh shape. The support member 22 of the cover plate 2 does not necessarily have to be the same shape as the support member 14 of the support plate 1.
The cover plate 2 may be provided with fitting claws 23a and 23b that can be fitted into the two fitting recesses 15a and 15b of the support plate 1. As shown in fig. 3, the cover plate 2 can clamp and fix the filter 9 to the filter arrangement portion 12 of the support plate 1 in cooperation with the support plate 1. That is, after the filter 9 is disposed on the filter disposition portion 12 of the support plate 1, the fitting claws 23a and 23b of the cover plate 2 are fitted into the fitting recesses 15a and 15b, and the support plate 1 and the cover plate 2 are sandwiched therebetween, whereby the filter 9 can be fixed. Therefore, no shearing force acts on the filter, and there is no fear of breakage even if an inorganic filter (for example, an inorganic fiber sheet) such as glass is used.
Further, since the cover plate 2 in fig. 1 has the protruding portion 24 capable of hooking a finger, the cover plate 2 can be easily removed from the support plate 1 by pulling the finger upward while hooking the finger on the protruding portion 24, and the filter 9 can be taken out without damage. In particular, in fig. 1, the cover plate 2 has a protruding portion 24 capable of hooking a finger, and one of the fitting claws (23 a in fig. 1) of the cover plate 2 is shorter in length than the other fitting claw (23 b in fig. 1), and the fitted state of the one fitting claw 23a and the fitting recess 15a is shallow, so that the cover plate 2 can be easily removed and the filter 9 can be taken out without damage by hooking the finger to the protruding portion 24 and pulling it upward.
Further, since the protruding portion 24 of the cover plate 2 is smaller than the receiving recess 18 of the support plate 1 and a gap is formed between the protruding portion 24 and the receiving recess 18 in a state where the cover plate 2 is attached to the support plate 1, a finger can be inserted into the gap and pulled upward by hooking the finger on the protruding portion 24, the cover plate 2 can be easily removed from the support plate 1, and the filter 9 can be removed without damage.
The protruding portion 24 of the cover 2 in fig. 1 is a force point portion protruding in the surface direction of the cover 2, but the protruding portion may protrude not in the surface direction of the cover 2 but in the thickness direction. In the case of such a force point portion protruding in the thickness direction, the force point portion is grasped with fingers and pulled, whereby the filter 9 can be released from the clamping by the cover plate 2 and the support plate 1, and the filter can be taken out without damage.
In fig. 1, the force point portion of the cover plate 2 is a form of a protruding portion 24 capable of hooking a finger, and the cover plate 2 may have a force point portion formed of a notched portion capable of inserting a finger instead of the protruding portion 24. When the filter has a notch, a finger is inserted into the notch, and the finger is hooked on the cover plate 2 and pulled upward, whereby the filter 9 can be released from being sandwiched between the cover plate 2 and the support plate 1, and the filter can be removed without damage. The notch preferably extends from the outer edge of the cover plate 2 toward the window 21. The cutout may be a complete cutout (i.e., a through hole) or a partial cutout (i.e., a depression) in the thickness direction of the cover plate.
The support member 22 of the cover plate 2 is preferably arranged so as not to overlap the support member 14 of the support plate 1 so as to support the filter 9 against water pressure from both sides of the filter surface generated in the subsequent dyeing step and to disperse the load applied to the filter 9.
Further, as shown in fig. 1 and 2, since the cover plate 2 has a recess 27 into which the tip of the forceps can be inserted, and the recess 27 is close to the window 21 between the fitting claws 23b, the filter 9 can be removed without damage even if the filter 9 is stuck to the cover plate 2 when the cover plate 2 is removed from the support plate 1.
In fig. 1 and 6, the outer shape of the cover plate 2 corresponds to the outer shape of the filter arrangement portion 12 of the support plate 1, and the cover plate 2 is in a state of being able to be accommodated in the filter arrangement portion 12. Therefore, the filter 9 can be sandwiched and fixed by the filter arrangement portion 12 and the cover plate 2, and the filter 9 can be prevented from being displaced. In this way, the outer shape of the cover plate 2 is preferably equivalent to the outer shape of the filter arrangement portion, and the cover plate 2 can be accommodated in the filter arrangement portion 12.
In addition, as described above, when the filter arrangement portion 12 has a convex portion, the cover plate 2 is preferably formed as a concave portion at a corresponding position, and the convex portion can be accommodated therein. This is because: since the filter arrangement portion 12 can be stored in this manner, the thickness of the filter arrangement portion 12 when the cover 2 is attached can be made substantially the same as that of the frame portion 13, and therefore, the filter arrangement portion can be easily stored in a dyeing basket for use as in the related art.
Further, as shown in fig. 6, the cover plate 2 preferably also has a through hole 29 as a liquid passing structure. This is due to: the same effect as when the filter arrangement portion 12 of the support plate 1 has a through hole is exhibited, and the observation under a microscope is excellent.
Further, although the liquid passing structure of the cover plate 2 in fig. 6 is the through hole 29, it is not necessarily a through hole, and may be a groove communicating with the outer edge portion of the cover plate provided on the surface contacting the filter. In the case of such a groove, since the liquid permeability is improved through the groove, the same effect as in the case of the through-hole is exhibited, and the observation performance under a microscope is excellent.
Further, similarly to the case where the filter arrangement portion 12 of the support plate 1 has a liquid passing structure, in the case where the gasket 5 capable of preventing liquid leakage is provided between the window portion 35 of the flange plate 3 and the window portion 21 of the cover plate 2 as described later, the liquid passing structure (the through hole 29, the groove, or the like) is preferably located outside the position where the gasket 5 contacts so as to prevent leakage of cells in a floating state during cell filtration.
The liquid passing structure in the filter arrangement portion of the support plate 1 may be the same as or different from the liquid passing structure in the cover plate 2. For example, the liquid passing structures may be identical or different in shape, size, position, through hole, groove, or the like. However, as shown in fig. 6, it is preferable that the liquid passing structure in the filter arrangement portion of the support plate 1 and the liquid passing structure in the cover plate 2 are in opposite positions. This is due to: by being located at the opposite position in this way, the cleaning liquid easily reaches the filter and is easily drained after cleaning, so that the staining solution is easily prevented from remaining and the observation under a microscope is excellent. Further, although the liquid passing structure may be provided only in the filter arrangement portion of the support plate 1 or only in the cover plate 2, the above-described effects are more excellent when both have the liquid passing structure.
In the filter holder of fig. 1, the support plate 1 has two fitting recesses 15a and 15b facing each other in the longitudinal direction through the window 11, and the fitting claws 23a and 23b of the lid plate 2 are fitted to hold and fix the filter 9 therebetween, but the fitting positions do not necessarily have to face each other in the longitudinal direction through the window 11 of the support plate 1. For example, the window portions 11 may face each other in the short side direction through the support plate 1, and the window portions may be arranged at a predetermined angle such as 60 °, 72 °, 90 °, 120 ° with respect to the center of the window portion 11 and the adjacent fitting positions.
In addition, the mode in fig. 1 is: the support plate 1 has four fitting recesses at four positions, and the cover plate has four fitting claws corresponding to the recesses, but the four positions are not necessarily required, and two or more positions may be fitted. Preferably, the filter 9 is fitted at 3 to 6 positions so as to be stably held and fixed.
Further, although the filter holder in fig. 1 can clamp and fix the filter 9 by fitting the fitting claws 23a and 23b of the cover plate 2 into the two fitting recesses 15a and 15b of the support plate 1, it is not always necessary to fix the filter 9 by fitting the cover plate 2 to the support plate 1. For example, the following method is also possible: the filter device is constituted by a cover plate having a window portion through which water can pass and a support plate having a filter arrangement portion having a window portion through which water can pass, and the cover plate is arranged at the filter arrangement portion and can be interposed by a thin-walled clip capable of sandwiching the support plate and the cover plate as described later. Alternatively, the following may be used: the filter device is composed of a cover plate having a window portion through which water can pass, a pivot shaft at one end portion, and a convex portion (projection or the like) or a concave portion (through hole, recess or the like) at the other end portion, and a support plate having a filter arrangement portion having a window portion through which water can pass, a bearing at one end portion of the filter arrangement portion, and a concave portion (through hole, recess or the like) or a convex portion (projection or the like) at the other end portion, wherein the pivot shaft of the cover plate can be inserted into and pivoted to the bearing of the support plate at one end portion, and the convex portion and the concave portion can be fitted in at the other end portion. In these systems, since no shearing force acts on the filter 9, the filter can be attached and detached without damage.
In addition, when the cover plate 1 has a cover plate that can be interposed or a cover plate that can be pivoted, it is also preferable that the cover plate has a force point portion such as a protruding portion that can catch a finger, a protruding portion that can be grasped with a finger, or a notch portion into which a finger can be inserted, so that the filter can be easily removed without damage.
The shape and size of the support plate 1 are preferably rectangular in shape and size so that a conventionally known dyeing basket and dyeing vessel can be used and can be accommodated in the dyeing basket. That is, the shape and size of the slide glass for specimen preparation are preferably matched to those of conventionally known observation specimens, and more specifically, the slide glass is preferably about 76mm in length, about 26mm in width, and about 1mm in thickness. The cover plate is preferably of a shape and size that does not protrude from the support plate so that conventionally known dyeing baskets and dyeing baths can be used.
As shown in fig. 1, the flange plate 3 is composed of a flange portion 31 and a cup portion 32 into which a sample can be introduced and which can communicate with a window portion 35 of the flange portion 31, and may be designed so that the flange portion 31 and the cup portion 32 are separable or integrally formed as shown in fig. 4.
The flange portion 31 shown in fig. 1 is composed of a flange 33 having a window portion 35 through which water can pass at a central portion thereof and being capable of coming into contact with the frame portion 13 of the support plate 1, and a connecting portion 34 capable of connecting the inside of the cup portion 32 and the window portion 35 of the flange portion 31 so as to communicate with each other. Since the flange portion 31 is disposed at a predetermined position with respect to the support plate 1 in the flange 33, the connection projection 36 can be provided at a position corresponding to the connection hole 16 of the support plate 1.
In the kit 10 of the present invention, the frame portion 13 of the support plate 1 and the flange 33 of the flange portion 31 of the flange plate 3 can be clamped and fixed by sliding the clips 4a and 4b having コ -shaped cross sections with respect to the support plate 1 (preferably, the support plate 1 in a state in which the filter 9 is fixed by the cover plate 2) and the flange plate 3 in a state in which the filter 9 is held by the filter arrangement portion 12, as shown in fig. 5, and therefore, the support plate 1 and the flange plate 3 can be fitted to clamp and fix the filter 9 to the filter arrangement portion 12 without damage. Further, since the flange plate 3 can be removed by releasing the clamped state of the filter 9 by sliding the clips 4a and 4b, the filter 9 can be removed without damage. In this case, if the spacer 5 capable of preventing leakage is provided between the window portion 35 of the flange plate 3 and the window portion 11 of the support plate 1 (the window portion 21 of the cover plate 2 when the cover plate 2 is used), leakage of cells in a floating state can be easily prevented during cell filtration. Further, even in the case of clips that can be biased by a spring, which are different from those shown in fig. 5, the clips 4a and 4b can hold and fix the filter 9 without damage, and can take out the filter 9 without damage. The flange plate 3 may be inserted or pivoted to the support plate by a clip as shown in fig. 5.
Further, since the flange plate 3 of the kit 10 of fig. 1 and 5 has the cup portion 32, a liquid sample including cells can be introduced into the cup portion 32 in a large amount and the curing step can be performed, but the flange plate 3 without the cup portion 32 can be used even when the curing step is performed using a small amount of liquid sample as in the case of introducing a liquid sample by a pipette. In this case, the coupling portion 34 of the flange plate 3 can perform the same function as the cup portion 32. That is, the connection portion 34 can temporarily store the liquid specimen without causing lateral leakage of the liquid specimen, and supply the liquid specimen to the window portion 35 of the flange plate 3 to solidify the cell.
In addition, the flange plate 3 shown in fig. 1 includes: the flange plate 3 may be constituted by only the flange 33, although the flange 31 having the flange 33 and the coupling portion 34, and the cup portion 32 are provided. That is, the coupling portion 34 and the cup portion 32 may be absent. In this case, as long as the flange plate 3 has a sufficient thickness to temporarily store the liquid specimen, the window portion 35 can filter the liquid specimen while temporarily storing the liquid specimen and solidify the cells without causing lateral leakage of the liquid specimen.
Further, as shown in fig. 1, the kit of the present invention may include both the cover plate 2 and the flange plate 3 as the clamping plates, or may include only one of them. When both the cover plate 2 and the flange plate 3 are used, the cover plate 2 functions mainly as a clamping plate and the flange plate 3 functions secondarily as a clamping plate. When only one of the cover plate 2 and the flange plate 3 is used, the one functions as a clamping plate. In the case where only the flange plate 3 is used as the clamping plate, it is preferable that the same support member as the cover plate 2 that can support the filter be provided in the window portion 35 of the flange portion 31 of the flange plate 3.
Further, in the case of using only the flange plate 3, if the recess into which the tip of tweezers can be inserted is provided adjacent to the position corresponding to the filter arrangement portion 12 of the support plate 1, as in the case of the cover plate 2, the filter 9 can be removed without damage even if the filter 9 is attached to the flange plate 3 when the flange plate 3 is removed from the support plate 1. Further, as described above, when the filter arrangement portion 12 has the convex portion, the flange plate 3 is preferably in a state in which the convex portion can be accommodated in the concave portion at the corresponding position, as in the case of the cover plate 2.
The support plate 1 of the filter holder of fig. 1 has a filter arrangement 12 and a frame part 13, which has: fitting recesses 15a and 15b for detachably fixing the clamping plates (cover plate 2 and flange plate 3); recesses 17, 27 capable of being inserted into the forceps tips when the filter 9 is recovered; the holding fixture, the attaching/detaching property, and/or the dyeing property of the filter 9 are excellent because the holding recess 18 capable of holding the protruding portion 24 of the holding plate (the cover plate 2, the flange plate 3) can be used as the holding fixture portion of the clips 4a, 4b when the flange plate 3 is held and fixed. For example, in the case of using clamping plates (cover plate 2, flange plate 3) that can be fitted, sandwiched, or pivoted to the filter arrangement portion 12, in the case of protruding the filter 9 from the filter arrangement portion 12, in the case of having a notch portion as a force point portion in the clamping plates (cover plate 2, flange plate 3), and/or in the case of not using the flange plate 3, the support plate 1 does not have to have a frame portion.
The filter 9 included in the kit of the present invention is not particularly limited as long as it can capture cells by a filtration operation and can perform a subsequent staining process, sealing process, and the like, and for example, an inorganic fiber sheet is preferable because it can fix cells in the internal space of the filter and can perform an operation of immersing the filter in a reagent. In particular, an inorganic fiber sheet having a porosity of 90% or more is preferable because it is easy to fix cells in the internal space of the filter and has excellent water permeability. As such an inorganic fiber sheet having a void ratio of 90% or more, for example, an inorganic fiber nonwoven fabric described in japanese patent laid-open publication No. 2010-185164 can be used.
Examples of the material constituting the fibers of the inorganic fiber nonwoven fabric include SiO2、Al2O3、B2O3、TiO2、ZrO2、CeO2、FeO、Fe3O4、Fe2O3、VO2、V2O5、SnO2、CdO、LiO2、WO3、Nb2O5、Ta2O5、In2O3、GeO2、PbTi4O9、LiNbO3、BaTiO3、PbZrO3、KTaO3、Li2B4O7、NiFe2O4、SrTiO3Etc. canThe oxide-based resin composition may be composed of an oxide of one of the components, or may be composed of two or more oxides. For example, it may be made of SiO2-Al2O3Consists of two components.
The inorganic fiber nonwoven fabric preferably has a void ratio of 91% or more, more preferably 92% or more, still more preferably 93% or more, and still more preferably 94% or more. On the other hand, although the upper limit of the porosity is not particularly limited, it is preferably 99.9% or less in order to obtain excellent morphological stability.
In order to make the inorganic fiber nonwoven fabric less likely to be broken by water pressure at the stage of cell filtration, dyeing, or the like and to have excellent handleability, the tensile breaking strength is preferably 0.2MPa or more, more preferably 0.3MPa or more, further preferably 0.4MPa or more, further preferably 0.5MPa or more, and further preferably 0.55MPa or more. The tensile breaking strength is a product of a shear load divided by a cross-sectional area of the inorganic fiber nonwoven fabric. The shear load is a value measured under the following conditions, and the cross-sectional area is a value obtained from the product of the width and the thickness of the test piece at the time of measurement.
Product name: small-sized tensile testing machine
The model is as follows: TSM-01-cre SEARCH co., LTD. (サーチ co.) manufacture test dimensions: 5mm wide by 40mm long
The distance between the chucks: 20mm
Stretching speed: 20mm/min.
Initial load: 50mg/1d
The average fiber diameter of the fibers constituting the inorganic fiber nonwoven fabric is not particularly limited, but is preferably 3 μm or less, more preferably 2 μm or less, further preferably 1 μm or less, and further preferably 0.8 μm or less, in order to facilitate formation of pores of a size that can easily hold cells. The lower limit of the average fiber diameter is not particularly limited, but is preferably 0.01 μm or more. In the present invention, "average fiber diameter" means an arithmetic average of the fiber diameters at 50 points, and "fiber diameter" means the thickness of a fiber measured based on an electron micrograph of an inorganic fiber nonwoven fabric taken in a field of view in which 10 or more fibers are projected.
Although the average weight per unit area of the inorganic fiber nonwoven fabric is not particularly limited, if the weight per unit area is excessively high, the water-draining property in the cell trapping step and the dyeing step is deteriorated, and the time for trapping and the dyeing unevenness are easily caused, so that it is preferable to be 20g/m2Hereinafter, more preferably 15g/m2Hereinafter, it is more preferably 10g/m2The following. The lower limit of the average weight per unit area is not particularly limited, but is preferably 1g/m2The above. In the present invention, "average weight per unit area" means an arithmetic average of weights per unit area of 18 samples (inorganic fiber nonwoven fabric), and "weight per unit area" means an area and a mass of a surface having the largest area measured and converted into an average weight per 1m based on the area and the mass2The value of the mass of the area.
The average thickness of the inorganic fiber nonwoven fabric is not particularly limited, but if it is too thick, the volume reduction accompanying drying of the sealing agent increases the possibility of air bubbles being generated in the specimen to be observed, and therefore, it is preferably 400 μm or less, more preferably 300 μm or less, and still more preferably 200 μm or less. The lower limit of the average thickness is not particularly limited, but is preferably 20 μm or more. In the present invention, "average thickness" refers to an arithmetic average of 54 positions of the thickness of a sample (inorganic fiber nonwoven fabric), and "thickness" refers to a thickness measured by a micrometer method [ load: 0.5N (measurement area: diameter: 14.3mm) was measured for the face having the largest area and the length of the face.
The average pore diameter of the inorganic fiber nonwoven fabric is not particularly limited, but is preferably 2 to 40 μm, more preferably 4 to 20 μm, and still more preferably 6 to 10 μm in order to easily hold a normal cell having a diameter of about 20 μm. The average pore diameter is a value of the average flow pore diameter obtained by the method specified in AS TM — F316, and can be measured by the average flow point method using, for example, a pore diameter meter (Polometer, Coulter, inc.).
The constituent fibers of the inorganic fiber nonwoven fabric are preferably continuous fibers. This is due to: if the constituent fibers are short fibers, the cells may be damaged at the ends of the inorganic fiber when the inorganic fiber nonwoven fabric is distorted in a dyeing step or the like or when the cells held in the pores of the inorganic fiber nonwoven fabric move, but this problem is not caused in the case of continuous fibers. The "continuous fibers" are fibers whose ends cannot be confirmed when an electron micrograph of the nonwoven fabric made of inorganic fibers is taken at 5,000 times.
Preferably, the inorganic fiber nonwoven fabric is bonded with an inorganic adhesive. This is due to: the membrane has excellent morphological stability, is easy to maintain pores for holding cells, and has the effect of preventing the filter from being damaged in each process. In particular, in the entire inorganic fiber nonwoven fabric including the inside, if the fibers are bonded together with an adhesive without forming a coating film therebetween, the water-repellent property is good in the cell trapping step and the dyeing step, and the reduction of the filtration time and the uneven dyeing can be suppressed, which is preferable.
The inorganic fiber nonwoven fabric that can be used in the method of the present invention can be produced by a known electrospinning method, preferably an electrospinning method in which a sol-gel method and a neutralization spinning method are combined, for example, the production method described in japanese patent application laid-open No. 2010-185164. The production method described in japanese patent laid-open publication No. 2010-185164 includes the following steps:
(1) a step of spinning an inorganic gel-like fiber by an electrospinning method from an inorganic sol solution for spinning containing a compound mainly containing an inorganic component;
(2) irradiating and collecting ions having a polarity opposite to that of the inorganic gel-like fibers to form a gel-like fiber web;
(3) firing the gel-like fiber web to form an inorganic fiber web;
(4) a step of supplying an inorganic sol solution for bonding containing a compound mainly containing an inorganic component to the entire inorganic fiber web including the inside thereof, and removing the remaining inorganic sol solution for bonding by ventilation to form an inorganic fiber web containing the inorganic sol solution for bonding;
(5) and a step of forming an inorganic fiber nonwoven fabric bonded with an inorganic binder in the entire body including the inside by heat-treating the inorganic fiber web containing the bonding inorganic sol solution.
The shape of the filter 9 of the present invention is not particularly limited, but may be, for example, a square shape such as a square shape as shown in fig. 1 or a circular shape such as a perfect circle as shown in fig. 6.
The materials of the components other than the filter, that is, the support plate 1, the cover plate 2, the flange plate 3, the clips 4a, 4b, and the gasket 5, which are included in the kit of the present invention, are not particularly limited AS long AS floating cells can be captured, and examples thereof include organic resins (e.g., polyamide, polybutylene terephthalate, polycarbonate, polyethylene terephthalate, acrylic, polyacetal, polypropylene, polyphenylene ether, polyphenylene sulfide, polystyrene, polyvinyl chloride, ABS resin, AS resin, chlorotrifluoroethylene, 1-difluoroethylene, perfluoroalkoxy fluororesin, and the like), and from the viewpoint of disposability after use of the kit, it is preferable that the materials of the components other than the filter are all made of organic resins.
After the kit of the present invention is assembled as shown in fig. 5, the suspension of the cells to be used for preparing the specimen for observation is introduced into the cup 32, and filtration is performed by gravity or suction as desired, whereby the cells can be trapped in the filter 9. The filter 9 with the captured cells can be directly subjected to a cell fixing step or a staining step using a staining basket or a staining bath in a state of being fixed between the support plate 1 and the cover plate 2, and after staining, the filter 9 is removed from the support plate 1, moved onto a slide glass, and subjected to a sealing step. In this way, if the shape and size of the cell-holding base material when it is attached to the cell-holding base material holder are rectangular shapes that can be stored in the staining basket, the cell-holding base material can be stored in the staining basket in a state in which it is attached to the cell-holding base material holder.
The above description of the filter holder for observing a suspended cell preparation as one of the cell holding base holders for observing preparation of the present invention and the filter kit as one of the kits for observing preparation of the present invention including the cell holding base holder for observing preparation of the present invention is that when the cell holding base functions as a filter, the cell holding base holder for observing preparation of the present invention and the kit for observing preparation of the present invention can be used even when the cell holding base does not function as a filter, for example, when the cell holding base is a cell culture base or a cell adsorption base. For example, after the cell-retaining substrate solidification step of cells on the cell-retaining substrate is performed using a cell-retaining substrate such as a cell culture substrate or a cell-adsorbing substrate without using the filter holder of the present invention as described above, the cell-retaining substrate holding cells can be transferred to a cell fixing step and a staining step using a staining basket or a staining bath in a state where the cell-retaining substrate holding cells is held between a support plate and a holding plate (particularly, a cover plate). In this case, the cell-retaining substrate may have a thickness suitable for observing the specimen with a microscope, and for example, a glass substrate, a thin film, an inorganic fiber sheet, or the like may be used. The cell-retaining base material can be used for the cell-retaining base material in the following steps: culturing and adhering adherent cells on a cell culture substrate; a step of placing a porous cell-adsorbing base material such as an inorganic fiber sheet used as the filter on the bottom surface of the culture container, injecting the cell dispersion, and allowing the cell dispersion to stand, thereby allowing the cells to naturally settle and adsorb; or a step of performing electrostatic adsorption by utilizing the electric charges between the cell surface and the cell adsorption substrate.
The cell-retaining base material holder for producing an observation specimen and the kit for producing an observation specimen of the present invention can be used in the method for producing an observation specimen of the present invention.
The method for producing an observation specimen of the present invention (hereinafter, may be referred to as the method of the present invention) may comprise a cell trapping step using an inorganic fiber aggregate, a wet fixing step, a staining step, a color clarification step, and a sealing step.
The cell trapping step in the method of the present invention uses an inorganic fiber aggregate to trap cells. The method of trapping is not particularly limited as long as the cells in the sample can be trapped in the inorganic fiber aggregate in a sufficient amount for cell diagnosis.
For example, a cell trapping plate having a filter unit formed in the center of a substrate can be produced by applying an inorganic fiber aggregate having an appropriate size to the surface of at least one (preferably one) of the substrates having a window in the center thereof so as to completely cover the window, and a sample can be passed (filtered) through the filter unit. Alternatively, two substrates provided with a window at the center are prepared, and the substrates are attached to each other with an inorganic fiber aggregate of an appropriate size sandwiched therebetween, whereby a cell-trapping plate having a filter unit formed at the center of the substrate can be produced, and the sample can be passed (filtered) through the filter unit. Although the size and thickness of the substrate are not particularly limited, it is preferable to use a slide glass for specimen preparation observation as a reference when considering the use of a conventional staining basket.
More specifically, in the case of a liquid specimen, cells can be captured by directly or after diluting the specimen with an appropriate liquid (e.g., physiological saline, a cell fixing solution, etc.) and dropping the diluted specimen onto the upper surface of a filter unit composed of an inorganic fiber aggregate, and then filtering the diluted specimen by gravity or suction as desired. In particular, the gravity-based filtration method is preferable because it is less likely to damage cells and denature cells. When the amount of the liquid sample is large, a sufficient amount of cells can be captured by performing a filtration operation of the liquid sample in a state where a liquid storage unit (for example, a tube having a hollow portion penetrating therethrough) is disposed on the filter unit. In addition, the filtration operation may be performed after removing excess liquid by centrifuging the liquid sample in advance.
When the sample is not in a liquid state, the capturing operation may be performed after dispersing the sample in an appropriate liquid (e.g., physiological saline, a cell fixing solution, etc.).
As the inorganic fiber aggregate used in the method of the present invention, a flat and thin inorganic fiber sheet capable of trapping cells by a filtration operation is preferable. The inorganic fiber sheet is preferably an inorganic fiber nonwoven fabric, for example, because cells can be fixed in the internal space of the filter and the operation of immersing the filter in a reagent can be performed. In particular, an inorganic fiber nonwoven fabric having a porosity of 90% or more is preferable because it is easy to fix cells in the internal voids of the filter and has excellent water permeability. The above description relating to the inorganic fiber nonwoven fabric that can be used in the kit of the present invention can be directly applied to the inorganic fiber nonwoven fabric that can be used in the method of the present invention.
The wet fixation step in the method of the present invention is a step of immersing the inorganic fiber aggregate in which cells are trapped in a reagent (a fixing solution, for example, 95% ethanol) to fix the inorganic fiber aggregate in order to avoid denaturation of the cells trapped in the inorganic fiber aggregate, and can be performed according to a wet fixation operation in a conventionally known ordinary observation specimen preparation method using a slide glass. In the wet fixing operation in the conventionally known observation specimen preparation method, wet fixing is required to be performed within several seconds because cells applied to a slide glass are easily dried, but in the method of the present invention, since the inorganic fiber aggregate has water retentivity, cells are not dried even if left to stand for several minutes (for example, 3 minutes to 10 minutes), and stable wet fixing can be performed.
The staining step in the method of the present invention is a step of staining cells trapped in an inorganic fiber aggregate after completion of wet fixation by an appropriately selectable staining method according to the purpose of use (examination purpose), and can be performed according to a staining procedure in a conventionally known general observation specimen preparation method using a slide glass. Although the dyeing basket and the dyeing bath are not necessarily used in the dyeing step of the method of the present invention, the dyeing basket and the dyeing bath are preferably used in order to be able to collectively treat a large amount of inorganic fiber aggregates.
Examples of the dyeing method that can be used in the dyeing step in the method of the present invention include papanicolaou dyeing, PAS dyeing, and alcian blue dyeing.
The color clarification step in the method of the present invention is a step of dehydrating the cells trapped in the inorganic fiber aggregate, and then immersing the cells in xylene or the like to thereby make the cells transparent. The method can be performed according to the color clarification operation in a conventional and well-known method for preparing an observation specimen using a slide glass.
The sealing step in the method of the present invention is a step of sealing the inorganic fiber aggregate carrying the stained cells on a cover glass with a sealing agent. In the method of the present invention, a sealing agent having a refractive index equivalent to that of the constituent fibers of the inorganic fiber aggregate is used. Here, the equivalent means within a range of ± 0.05 of the refractive index thereof. As the blocking agent which can be used in the method of the present invention, for example, Neo-Mount (registered trademark) (merck #109016, refractive index: 1.46), Softmount (registered trademark) (Wako Pure Chemical Industries, Ltd. (and optical Pure drugs) #192-16301, refractive index: 1.50), blocking agent New M.X (Matsunami Glass Ind., Ltd. (Sonlange electronics industry) # FX00100, refractive index: 1.545), blocking agent MGK-S (Matsunami Glass Ind., Ltds. (Sonlange electronics industry) # FK00100, refractive index: 1.545), Multi-Mount (Sumatami Glass Ind. # 480, Mitdna Glass Ind., Ltd. # FM48001, refractive index: 1.49), Miunt-Mount (Miss Mr. # FM 4801, Miss # EMiMire) # FM 22091, Mi # FM 33, Mi # 140, Mi # FM 22084, Mi # 01, Mi # 49, Mi # Mi et Mi # I Mi # 01, Mi # 01, Mi # Mi.
The method of producing an observation specimen of the present invention can be carried out using the cell-retaining base material holder for producing an observation specimen and the kit for producing an observation specimen of the present invention.
[ examples ]
The present invention will be specifically described below with reference to examples, but these do not limit the scope of the present invention.
Cells were captured or smeared and fixed under the conditions described in examples 1 and 2 and comparative examples 1 and 2 below. Subsequently, the reagents were used for Papanicolaou staining with a staining basket and a staining bath according to the procedure described in the attached specification, using Papanicolaou/hematoxylin staining solution (Wako Pure Chemical Industries, Ltd. (and Wako Pure Chemical Industries) # 168-. The color clarification after the coloring was carried out by using a xylene tank in accordance with the procedure described in the specification. Then, the specimen was sealed with a commercially available sealing agent (Softmount (registered trademark), Wako Pure Chemical Industries, Ltd. (Wako Pure Chemical Industries, Ltd.), # 192-.
EXAMPLE 1
In the whole including the inside of the inorganic fiber web obtained by combining the sol-gel method and the neutralization spinning method, which is manufactured by supplying a silica sol solution to the whole including the inside and performing a heat treatment, a silica continuous fiber assembly (average weight per unit area: 7.42 g/m) bonded by a silica binder without forming a coating film is formed2Average thickness: 142 μm, average pore diameter: 7 μm, average fiber diameter: 0.73 μm, porosity: 95% shear load per unit area weight: 0.57MPa, refractive index of fiber material: 1.46) was cut into a rectangular shape having a width of 30mm and a length of 26mm, holes of an aluminum plate having a width of 76mm and a length of 26mm, each of which was provided with a hole having a diameter of 20mm, were covered with a silica continuous fiber aggregate, and the covered holes were bonded with an epoxy resin adhesive to prepare a filter (filter surface: diameter of 20mm and area of about 3.1cm2). Then, a cartridge having a diameter of 20mm was fixed by a clip so that the hole of the filter and the hollow portion of the cartridge were communicated with each other through an O-ring (gasket).
Then, 10mL of a physiological saline dispersion (5X 10) of HepG2 cells (human liver cancer-derived cell line) as a liquid specimen was put into the hollow portion of the cylinder4cells/mL), filtered by gravity. Immediately after the cells were trapped by the silica continuous fiber aggregate of the filter, the cells were immersed in a 95% ethanol tank and fixed.
EXAMPLE 2
Cells were trapped in the silica continuous fiber aggregate in the same manner as in example 1, left at room temperature for 3 minutes, and then immersed in a 95% ethanol bath to fix the cells.
Comparative example 1
Will comprise 5 x 105cell pellets were prepared by centrifugation of HepG2 cells in physiological saline to remove the supernatant. It was applied to about 9.3cm of a slide glass (martial arts, #511617) on which a treatment of cell-peeling-preventing coating was applied by a pull tab method2The range of (1). After the application, the coated fabric was immediately immersed in a 95% ethanol bath and fixed.
Comparative example 2
Cells were smeared onto the slide as in comparative example 1. After standing at room temperature for 3 minutes, the plate was immersed in a 95% ethanol bath and fixed.
Results of comparison
Fig. 7 (magnification: 100 times) and fig. 8 (magnification: 400 times) show cell images obtained by observing the observation specimen of example 1 (observation specimen obtained by immediately fixing cells after cell capture) with an optical microscope.
Fig. 9 (magnification: 100 times) and 10 (magnification: 400 times) show cell images obtained by observing the observation specimen of example 2 (observation specimen obtained by capturing cells and then standing at room temperature for 3 minutes and fixing) with an optical microscope.
Fig. 11 (magnification: 100 times) and fig. 12 (magnification: 400 times) show cell images obtained by observing the observation specimen of comparative example 1 (observation specimen in which cells are fixed immediately after being smeared) with an optical microscope.
The cell images obtained by observing the observation specimen of comparative example 2 (observation specimen obtained by applying cells and then leaving the cells at room temperature for 3 minutes and fixing the cells) with an optical microscope are shown in fig. 13 (magnification: 100 times) and fig. 14 (magnification: 400 times).
In fig. 7 to 14, the gray cells appearing dark and light are actually stained blue. In addition, in fig. 14, cells that appear dark gray to black due to their swelling were actually stained scarlet to brown.
1. Cell retention
The 5 positions of the capturing surface of the observation specimen of example 1 and the 5 positions of the applying surface of the observation specimen of comparative example 1 were randomly photographed under 100-fold observation using an optical microscope (olympus inverted microscope IX73PI-22 FL/PH). In example 1, at least 1000 or more cells were observed uniformly in all the fields of view (fig. 7), whereas in comparative example 1, 2 fields in which about 500 cells were observed, and almost 50 or less cells were observed, and it was found that 3 fields in which cell exfoliation occurred were observed. Fig. 11 is a photomicrograph in which about 500 cells were observed.
2. Resistance to drying
In example 2, the staining property did not change even when left at room temperature for 3 minutes (fig. 10), whereas in comparative example 2, the swelling of the cells occurred and the staining property changed (fig. 14).
3. Observability (intracellular)
In example 1, even in the observation at a high magnification (400 times), an observation image capable of sufficiently discriminating the organelles was obtained (fig. 8). On the other hand, in comparative example 1, the cells slightly spread, and organelles were easily observed.
4. Observability (three-dimensional)
In example 1, the three-dimensional properties of each cell and the three-dimensional positional relationship between cells were maintained (fig. 8). On the other hand, in comparative example 1, all the cells slightly spread so as to stick to the slide glass, and therefore the original three-dimensional structures of the cells were lost (fig. 12).
These results are shown in table 1.
[ Table 1]
| Examples 1 and 2 | Comparative examples 1 and 2 | |
| Cell retention | ◎ | × |
| Resistance to drying | ◎ | × |
| Observability (intracellular) | ○ | ◎ |
| Observability (three-dimensional) | ◎ | × |
Industrial applicability
The cell-retaining substrate holder and cell-retaining substrate kit for producing an observation specimen and the method for producing an observation specimen according to the present invention can be used in the field of pathological diagnosis such as cell diagnosis and cell-using research such as medical science, pharmacy, and department of life science.
The present invention has been described above in terms of specific embodiments, but modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
Description of the reference numerals
10-a filter kit for preparing an observation specimen for floating cells;
1-a support plate; 2-cover plate;
3-a flange plate; 4a, 4 b-clips;
5-lining; 9-a filter; 11-a window portion;
12-a filter arrangement portion; 13-a frame part;
14-a support member; 15a, b-embedding depressions;
16-a hole for connection; 17-dishing; 18-a receiving recess;
19-through holes (liquid passing structure); 21-a window section; 22-a support member;
23a, b-embedding claws; 24-a protrusion; 27-dishing;
29-through holes (liquid passing structure); 31-a flange portion;
32-a cup portion; 33-a flange; 34-a joint;
35-a window portion; 36-connecting protrusions.
Claims (15)
1. A cell-retaining base material holder for observing specimen preparation, the cell-retaining base material being composed of an inorganic fiber sheet, the cell-retaining base material holder for observing specimen preparation comprising:
(1) a support plate having a cell-holding base material arrangement portion having a window portion through which a staining solution can be passed; and
(2) a clamping plate which has a window through which a staining solution can be passed, and which is detachably fixed to the cell holding substrate arrangement part so as to be capable of clamping the cell holding substrate in cooperation with the support plate,
the support plate has recesses on both sides with a window interposed therebetween, and the clamping plate is a cover plate having claws fittable into the recesses
Both the cell holding base material arrangement part and the cover plate of the support plate have a liquid passing structure in a portion where the cell holding base material can be held and fixed by the cell holding base material arrangement part and the cover plate, the liquid passing structure being different from the window part through which the staining solution can pass, and the staining solution remaining in the portion where the cell holding base material is held and fixed by the cell holding base material arrangement part and the cover plate of the support plate can pass through the liquid.
2. The cell-retaining substrate holder according to claim 1, wherein the support plate has a cell-retaining substrate arrangement portion and a frame portion.
3. The cell-holding substrate holder according to claim 2, further comprising: the flange plate has a flange portion capable of abutting against the frame portion of the support plate, and a clip capable of clamping and fixing the frame portion of the support plate and the flange portion of the flange plate.
4. The cell-holding substrate holder according to claim 3, wherein the flange plate has a cup portion into which the sample can be introduced and which can communicate with the window portion of the flange portion.
5. The cell-holding substrate holder according to claim 4, wherein the cup portion is detachable.
6. The cell-retaining substrate holder according to claim 1, wherein a support member capable of supporting the cell-retaining substrate is provided in the window portion of the support plate.
7. The cell-holding substrate holder according to claim 1, wherein the support plate has a rectangular shape and a size capable of being accommodated in the staining basket.
8. The cell-holding substrate holder according to claim 1, wherein the support plate and/or the holding plate has a recess into which the tip of the forceps can be inserted.
9. The cell-holding substrate holder according to claim 1, wherein any material is composed of an organic resin.
10. The cell-retaining substrate holder according to claim 1, wherein the cover plate has a force point portion capable of applying a force.
11. A kit for preparing an observation specimen, comprising the cell-retaining substrate holder according to any one of claims 1 to 10 and a cell-retaining substrate.
12. The kit according to claim 11, wherein the cell-retaining substrate is an inorganic fiber sheet having a porosity of 90% or more.
13. The kit according to claim 11, wherein the shape and size of the cell-retaining substrate when mounted on the cell-retaining substrate holder are rectangular so as to be receivable in the staining basket.
14. The kit according to claim 11, wherein the cell-retaining substrate is any one selected from the group consisting of a filter, a cell culture substrate, and a cell adsorption substrate.
15. A method for producing an observation specimen, which comprises using the kit according to claim 11, using an inorganic fiber nonwoven fabric having a porosity of 90% or more as a cell-retaining base material, capturing cells with the inorganic fiber nonwoven fabric, and directly performing wet fixation, dyeing, and sealing with a sealing agent having a refractive index equivalent to that of the inorganic fiber.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015180833 | 2015-09-14 | ||
| JP2015180832A JP6671681B2 (en) | 2015-09-14 | 2015-09-14 | How to make a microscope specimen |
| JP2015-180832 | 2015-09-14 | ||
| JP2015-180833 | 2015-09-14 | ||
| JP2016-114067 | 2016-06-08 | ||
| JP2016114067A JP6738012B2 (en) | 2015-09-14 | 2016-06-08 | Cell-holding substrate holder for preparing cell observation specimen and kit containing the same |
| CN201680050931.XA CN107923821A (en) | 2015-09-14 | 2016-09-14 | Observe preparation of specimen cell holding substrate holder and the external member including it and the production method for observing sample |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201680050931.XA Division CN107923821A (en) | 2015-09-14 | 2016-09-14 | Observe preparation of specimen cell holding substrate holder and the external member including it and the production method for observing sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114018667A true CN114018667A (en) | 2022-02-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111301485.4A Pending CN114018667A (en) | 2015-09-14 | 2016-09-14 | Cell-retaining substrate holder for producing observation specimen, kit including same, and method for producing observation specimen |
Country Status (2)
| Country | Link |
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| CN (1) | CN114018667A (en) |
| WO (1) | WO2017047617A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7072798B2 (en) * | 2018-03-05 | 2022-05-23 | 国立大学法人滋賀医科大学 | Holder for immunostaining |
| WO2020008563A1 (en) * | 2018-07-04 | 2020-01-09 | オリンパス株式会社 | Cell inspection device and cell inspection method |
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| JPH041433U (en) * | 1990-04-19 | 1992-01-08 | ||
| US5578459A (en) * | 1993-11-24 | 1996-11-26 | Abbott Laboratories | Method and apparatus for collecting a cell sample from a liquid specimen |
| US6436662B1 (en) * | 2000-04-04 | 2002-08-20 | Digene Corporation | Device and method for cytology slide preparation |
| JP2006010437A (en) * | 2004-06-24 | 2006-01-12 | Hiroyuki Kishi | Liquid leakage preventive tool for microchip, and microchip holder |
| JP5410307B2 (en) * | 2009-01-14 | 2014-02-05 | 日本バイリーン株式会社 | Inorganic fiber nonwoven fabric and method for producing the same |
| EP2902476B1 (en) * | 2012-09-28 | 2021-01-06 | Sysmex Corporation | Sample preparation device and cell analysis device |
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| US3615257A (en) * | 1968-10-14 | 1971-10-26 | Becton Dickinson Co | Filter cassette and holder therefor |
| JPS62121331A (en) * | 1985-11-21 | 1987-06-02 | Johoku Ika Kiki Seisakusho:Kk | Sample for microscope observation and its preparing tool |
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| WO2017047617A1 (en) | 2017-03-23 |
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