JP7153365B2 - Isolated cell specimen, method for producing isolated cell specimen, and method for detecting target cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to an isolated cell specimen, a method for producing an isolated cell specimen, and a method for detecting target cells.

In a wide variety of fields, there is a need for techniques for detecting only specific cells from a sample containing multiple cells of various types. In particular, in research and development and clinical sites in fields related to medicine and pharmacology, target cells to be detected (e.g., cells containing specific substances, cells producing specific substances, cells infected with pathogenic bacteria, etc.) Such detection techniques are very important for experiments and diagnostics in order to confirm the presence or absence of .

However, it is difficult to detect cells contained only in minute amounts in a sample. Such cells include Circulating Tumor Cells (also called “CTCs”), and the like. Normally, in order to detect circulating cancer cells, etc., it is necessary to analyze white blood cells in the blood. A few to a few thousand compared to about 50 million white blood cells contained.

Non-Patent Literature 1 proposes a technique in which a cell sample is placed in a microarray chip and circulating cancer cells are detected by immunostaining. The immunostaining method is a technique for detecting cells by binding antigens contained in cells to be detected using antibodies corresponding to the antigens through antigen-antibody reaction.

However, the method described in Non-Patent Document 1 requires a special microarray chip and lacks convenience, so there is a need for a technique that can more easily detect target cells.

As a simpler method, there is a method in which a cell sample is immobilized on a slide glass or the like and subjected to immunostaining (Patent Document 1, etc.).

WO2015/093116

Yamamura, et al., Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip, PLoS ONE, March 2012, Volume 7, e32370

However, for immunostaining, it has been said that the isolated cell preparation with the attached cell sample should not be dried. Therefore, it has been difficult to store or transport an isolated cell specimen to be immunostained before immunostaining.

An object of the present invention is to provide an isolated cell specimen that is suitable for immunostaining and the like and that can be easily stored and transported before being subjected to immunostaining and the like.

As a result of intensive studies by the present inventors, the inventors have found that the above problems can be solved by an isolated cell specimen adjusted so that the average thickness of observation target cells satisfies a predetermined condition, and have completed the present invention. rice field. That is, the present invention includes the following aspects.

<1> An isolated cell specimen in which observation target cells are attached to the surface of a flat substrate,
substantially no liquid is present on the surface of the flat substrate on which the cells are arranged;
The average thickness of the cells to be observed satisfies the following formula (1),
An isolated cell preparation used for immunostaining and/or nuclear staining.
(t/tw)≧0.95 Formula (1)
(In formula (1),
t represents the average thickness of the observed cells attached to the surface of the flat substrate,
tw represents the average thickness of cells of the same type as the observed cells that have not undergone the drying process and adhered to the surface of the flat substrate. )

<2> The isolated cell specimen according to <1>, wherein the cells to be observed include peripheral blood mononuclear cells.

<3> The isolated cell specimen according to <1> or <2>, wherein the surface of the flat substrate on which the cells to be observed are arranged has a water contact angle of 10° or less at 25°C.

<4> A developing step of developing the observation target cell-containing liquid on the flat substrate;
After the developing step, a cell removing step of removing cells not attached to the surface of the flat substrate;
After the cell removal step, a drying step of naturally drying the flat substrate until there is substantially no liquid left on the surface of the flat substrate on which the cells are arranged;
A method for producing an isolated cell specimen used for immunostaining and/or nuclear staining, comprising:

<5> The method for producing an isolated cell specimen according to <4>, wherein the average thickness of the cells to be observed satisfies the following formula (2) before and after the drying step.
(t'/tw')≧0.95 Formula (2)
(In formula (2),
t 'represents the average thickness of the cells to be observed after the drying process attached to the surface of the flat substrate,
tw' represents the average thickness of the observed cells attached to the surface of the flat substrate before the drying step. )

<6> The manufacturing method according to <4> or <5>, wherein in the cell removal step, the cells are removed using water or an aqueous solution, and the water or the aqueous solution has a specific resistance value of 10 Ω·cm or more. .

<7> A method for detecting a target cell, comprising the step of subjecting the isolated cell sample according to any one of <1> to <3> to immunostaining and/or nuclear staining.

<8> The detection method according to <7>, wherein the cells to be observed include peripheral blood mononuclear cells, and the target cells are circulating cancer cells.

According to the present invention, it is possible to provide an isolated cell specimen suitable for immunostaining and the like, and easy to store and transport before being subjected to immunostaining and the like.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline of the measuring method of the average thickness of the cell in this invention. FIG. 10 is an example plot of fluorescence signals for determining average cell thickness. FIG. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline of the manufacturing method of the isolated cell sample of this invention. FIG. 4 is a diagram schematically showing the arrangement of cells in the manufacturing process of an isolated cell sample. 1 is a diagram showing the results of measurement of water content on the surface of an isolated cell specimen in Example 1. FIG. FIG. 2 shows the results of immunostaining using the isolated cell specimen of Example 1. FIG. FIG. 2 shows the results of immunostaining using the isolated cell specimen of Comparative Example 1. FIG. FIG. 10 shows the results of immunostaining using the isolated cell specimen of Comparative Example 2. FIG. FIG. 10 shows the results of immunostaining using the isolated cell specimen of Comparative Example 3. FIG. FIG. 10 shows the results of immunostaining using the isolated cell specimen of Comparative Example 4. FIG. FIG. 10 shows the results of immunostaining using the isolated cell specimen of Comparative Example 5. FIG.

The present invention will be described in detail below.

<Isolated cell sample>
The isolated cell specimen of the present invention is an isolated cell specimen in which cells to be observed adhere to the surface of a flat substrate, and substantially no liquid is present on the surface of the flat substrate on which the cells are arranged, and the cells are observed. It is an isolated cell specimen whose average thickness of target cells satisfies the following formula (1) and is used for immunostaining and/or nuclear staining.
(t/tw)≧0.95 Formula (1)
(In formula (1),
t represents the average thickness of the observed cells attached to the surface of the flat substrate,
tw represents the average thickness of cells of the same type as the observed cells that have not undergone the drying process and adhered to the surface of the flat substrate. )

In the isolated cell specimen of the present invention, cells adhere to the surface of the flat substrate. This cell is an object to be prepared and observed as a specimen, and is referred to as an "observation object cell" in the present invention. In addition, the “observation target cell” means a cell group containing a plurality of cells of the same or different type.

The isolated cell specimen of the present invention is characterized in that the average thickness of observation target cells satisfies formula (1). Formula (1) is (t / tw), that is, the average thickness (t) of the observation target cells attached to the surface of the flat substrate is the observation target that has not undergone the drying process attached to the surface of the flat substrate. It means that the value obtained by dividing the cell by the average thickness (tw) of cells of the same type is 0.95 or more. This formula states that the average thickness of the observed cells is slightly less than the average thickness of homogeneous cells kept in wet conditions, i.e., the original average thickness of the observed cells, or the original Means equal to the average thickness.

Excessive drying of the cells reduces (t/tw). When (t/tw) is small, the cells do not have their original thickness, size, shape, etc. (e.g., cells are flattened, etc.), and cannot be used for various staining and various detections. means However, as a result of examination by the present inventors, when the surface of the flat substrate is dried so that the average thickness of the cells to be observed satisfies the formula (1), the substrate is no longer a wet system. An unexpected finding was found that the cells retain their original thickness and the like, and that immunostainability equal to or higher than that of the wet system can be achieved. Furthermore, it was found that such an isolated cell specimen can have good staining efficiency even without the membrane permeabilization treatment (treatment with saponin or the like) normally required for the detection of intracellular antigens. rice field. Such an isolated cell sample has a characteristic not found in conventional samples for immunostaining in that it is extremely easy to store and transport before immunostaining.

In the present invention, "substantially no liquid exists on the surface of the flat substrate on which the cells are arranged" means that there is no visually observable amount of liquid on the surface of the flat substrate on which the cells are arranged. do. However, in the isolated cell specimen of the present invention, it is not excluded that a small amount of liquid exists on the surface of the flat substrate on which the cells are arranged. Examples of liquids include any liquids that can retain the structure of cells, such as buffers, water, and media. Note that the observation target cells adhering to the surface of the flat substrate contain enough water to satisfy the formula (1).

Whether or not there is substantially no liquid on the surface of the flat substrate on which the cells are arranged can be determined by the following method.
(1) The amount of moisture on the surface of the flat substrate at any time is measured with an infrared moisture meter (for example, "fiber type infrared moisture meter IM-3SCV MODEL-2000" manufactured by Fujiwork Co., Ltd.) at 25 ° C. and a relative humidity of 50%. Measured under the conditions of The moisture content is output as an IM-D value (unit: mV), which is a moisture meter output value corresponding to the moisture content.
(2) After 5 minutes from the time of (1) above under the conditions of 25° C. and 50% relative humidity, the water content on the surface of the flat substrate is measured with an infrared moisture meter.
(3) Compare the measured values of (1) and (2) above, and if the difference between the two is within 3% (preferably within 1%), the liquid is substantially present on the surface of the flat substrate on which the cells are arranged. It is determined that it does not exist in

The configuration of the isolated cell specimen of the present invention will be described in detail below.

(flat board)
Examples of flat substrates include substrates having any material and shape that are commonly used in cell observation. In the present invention, the term "flat plate" means that the surface of the substrate is so smooth as to allow cells to adhere to it, and the substrate surface may not be partially uneven.

The material of the flat substrate is not particularly limited. Considering the easiness of cell observation using microscopy, etc., the flat substrate is preferably molded from a transparent material. Examples of transparent materials include various glasses and various resins (thermoplastic resins, thermosetting resins, etc.).

Thermoplastic resins include polyethylene, polypropylene, polystyrene, polyvinyl chloride, acrylate polymers, methacrylate polymers, cyclic olefin copolymers (norbornene, etc.), aromatic polyesters (polyethylene terephthalate, polybutylene terephthalate, etc.). , polycarbonate, polyamide and the like.

Examples of thermosetting resins include silicone resins (such as those having a polyorganosiloxane skeleton), phenol resins, melamine resins, and epoxy resins.

Among the above, polystyrene, methacrylic acid ester polymers, cyclic olefin copolymers, and silicone resins (having a polyorganosiloxane skeleton) are preferred.

The size of the flat substrate is not particularly limited, and can be appropriately selected according to the use of the isolated cell sample, the device for observing the observation target cell, and the like.

In the flat substrate, the surface on which the cells to be observed are arranged is preferably hydrophilic from the viewpoint that the liquid containing the cells to be observed can be easily spread on the flat substrate. Specifically, the contact angle of water on the surface of the flat substrate on which cells to be observed are arranged is preferably 10° or less, more preferably 5° or less at 25°C.

When the surface of the flat substrate is not hydrophilic, the surface can be subjected to a surface treatment (hydrophilization treatment), if necessary. Any surface treatment method can be employed, including plasma treatment, ultraviolet treatment, electron beam treatment, corona discharge treatment, ozone treatment, and the like. Among these, plasma treatment is preferred, and oxygen plasma treatment is particularly preferred.

The contact angle of water on the surface of the flat substrate is measured using the θ/2 method. In the θ/2 method, the angle of the straight line connecting the left and right end points and the vertex of the droplet dropped on the surface of the flat substrate is obtained with respect to the surface of the flat substrate, and the value obtained by doubling this is specified as the contact angle. It is a way to For example, when determining the contact angle of water on the surface of a flat substrate, distilled water (eg, 1 to 2 μl) is dropped at a plurality of different points (eg, 5 or more points) on the surface, and each of the dropped droplets , and the average value of the obtained values may be treated as the contact angle of water. The contact angle may be specified using a contact angle meter (manufactured by Kyowa Interface Science Co., Ltd., etc.).

(Observation target cell)
Observation target cells are cells that adhere to the surface of the flat substrate and are the target of cell observation. The type of cells to be observed is not particularly limited. For example, preferred cells include peripheral blood mononuclear cells (also called PBMCs).

For t and tw defined in formula (1), the cells for which the average thickness is specified may be of the same species as long as they are derived from the same individual. No need. However, it is preferably derived from the same species of organism. For example, if t is the average thickness of peripheral blood mononuclear cells, tw also means the average thickness of peripheral blood mononuclear cells.

tw may be a predetermined numerical value depending on the cell type. For example, the tw of peripheral blood mononuclear cells can be about 15 μm. In such cases, the lower limit of t for peripheral blood mononuclear cells is 14.2 μm or greater (ie (t/tw)≧0.95), preferably 14.4 μm or greater (ie (t/tw)≧0.96 ), more preferably 14.5 μm or more (that is, (t/tw)≧0.97). In this case, the upper limit of t for peripheral blood mononuclear cells is about 15.7 μm, taking into consideration variations in measurement results and individual differences.

Observation target cells may be in any form that is commonly used for cell observation. For example, cells to be observed may be those isolated from tissues (eg, epithelium) or body fluids (eg, blood) by a known method. The method for isolating the cells to be observed is not particularly limited as long as it does not impair the original size and shape of the cells, and examples thereof include centrifugation (density gradient centrifugation, etc.) and methods using reagents. In addition, since the cells to be observed in the present invention are isolated cells, they do not include aspects such as tissue sections.

A separation solution can be used in density gradient centrifugation. Examples of the separation solution include a lymphocyte separation solution and the like. The separation solution is usually used by overlaying it on the blood sample. In such a case, a reagent or the like adjusted to a density of 1.077 g/ml can be used as the separation solution.

For example, the separation solution can be used to remove most of red blood cells, granulocytes (neutrophils, eosinophils, basophils) that are part of white blood cells, and platelets contained in whole blood. As a result, it is possible to obtain a fraction of mononuclear cells mainly composed of lymphocytes (T cells, B cells, NK cells) and monocytes which are part of leukocytes.

A commercially available separation solution can be used for the density gradient centrifugation method. Commercially available separation solutions include, for example, the trade name “LymphoPrep” (manufactured by Alere Technologies), the trade name “NycoPrep 1.077” (manufactured by Alere Technologies), the trade name “OptiPrep” (manufactured by Alere Technologies), the trade name "Histopaque 1077" (Sigma-Aldrich), trade name "Ficoll-Paque" (GE Healthcare), and the like. In "OptiPrep", it is necessary to adjust the solution density to 1.077 g/ml by mixing appropriate amounts of reagents, medium, etc. in advance.

An example of a technique of density gradient centrifugation for obtaining a fraction containing peripheral blood mononuclear cells is given. First, whole blood is collected in a blood collection tube containing an anticoagulant, diluted with physiological saline (0.9% NaCl solution), and then the separation solution is dispensed. This is centrifuged to recover the mononuclear cell fraction. This mononuclear cell can be treated as a fraction containing peripheral blood mononuclear cells.

When the target cells to be detected are circulating cancer cells, the cells to be observed preferably include peripheral blood mononuclear cells.

(average cell thickness)
In the present invention, the "average cell thickness" refers to an isolated cell specimen in which cells are attached to a smooth surface of a flat substrate (usually arranged in a single layer), immunostained using a fluorescently labeled antibody (fluorescent staining ), and then the fluorescence image under fluorescence microscope observation is analyzed using image analysis software.

FIG. 1 shows an outline of the method for measuring the average cell thickness. First, an isolated cell sample attached with cells whose average thickness is to be determined is set horizontally on the observation table of a fluorescence microscope. Next, the field of view is fixed while the focus is on the substrate surface to which the cells are attached, and the objective lens is moved upward (in the Z-axis direction) every 1.05 μm to acquire a fluorescence image. The acquired images are analyzed using image analysis software (Image J, Ver1.52a, NIH, https://imagej.nih.gov/ij/) to calculate the average fluorescence signal intensity of each image. By plotting the fluorescence signal intensity against the distance in the Z-axis direction, the distance from the lower end of the cell (substrate surface) to the upper end of the cell is obtained (the method for identifying the upper end of the cell from the plot will be described later using FIG. 2). do). This distance corresponds to the average cell thickness. The average cell thickness measured by this method is calculated based on the sum of the average fluorescence signal intensities of the cells contained in the isolated cell sample. Therefore, this method has the advantage that the results obtained are less susceptible to variations in the size of individual cells, individual differences in cell sample providers, and the like.

FIG. 2 is an example plot of fluorescence signal intensity against distance in the Z-axis direction used to determine the average cell thickness. The bottom end of the cell (substrate surface) is identified as a point with a Z-axis value of "0 μm". The top end of the cell is specified as the point with the minimum value that satisfies all of the following relationships among the points counted from the bottom end of the cell (substrate surface) in the plot.
|Y _N-2 -Y _N-1 |>|Y _N-1 -Y _N |
Y _N-2 −Y _N-1 >0
|Y _N−1 −Y _N |/Y _MAX ≦0.04
(Wherein, “Y _x ” (x is any of the three consecutive points N−2, N−1, and N in the plot.) is the point x on the Y axis (average signal intensity) "Y _MAX " means the maximum value on the Y-axis in the plot.)
For example, in FIG. 2, the point with the smallest value that satisfies all of the above relationships is "n".

The average thickness of cells arranged in a single layer on the flat substrate of the present invention satisfies the following formula (1), as described above.
(t/tw)≧0.95 Formula (1)
(In formula (1),
t represents the average thickness of the observed cells attached to the surface of the flat substrate,
tw represents the average thickness of cells of the same type as the observed cells that have not undergone the drying process and adhered to the surface of the flat substrate. )

t is specified by determining the average thickness of the cells adhered to the surface of the flat substrate of the isolated cell sample by the method described above.

tw is specified by determining the average cell thickness of cells of the same type as the cells to be observed, which have not undergone a drying process, and which have adhered to the surface of the flat substrate, by the above method. The flat substrate for specifying tw is not particularly limited as long as cells can adhere to its smooth surface, but it is preferably made of the same material as the flat substrate for specifying the corresponding t. In the present invention, "the cells have not undergone a drying process" means that the cells are retained only in water or an aqueous buffer solution (phosphate buffered saline (PBS), etc.), and the original size and structure of the cells are maintained. means that it holds When determining tw, measurements are taken while the cells are maintained in a wet system. Methods for maintaining cells in a wet system include, for example, a method in which a frame is provided on the substrate and a liquid (water or aqueous buffer solution (PBS, etc.)) that does not affect the structure of the cells is placed in the frame.

When trying to produce the isolated cell specimen of the present invention, tw can be specified by maintaining a part of the cells to be observed in a wet system and using this to determine the average thickness of the cells.

tw in any isolated cell preparation can be determined as follows.
(1) Identify the type and origin of the cells attached to the isolated cell sample by microscopic observation or the like.
(2) Obtain cells of the same kind as the cells specified in (1) above.
(3) The cells obtained in (2) above are adhered to the surface of a flat substrate, and tw is identified while the cells are maintained in a wet system.

The lower limit of (t/tw) is preferably (t/tw)≧0.96, more preferably (t/tw)≧0.97. When (t/tw) is less than 0.95, it generally indicates that the cells to be observed have atrophied due to loss of intracellular water due to drying or the like during preparation of the specimen.

The upper limit of (t/tw) is usually 1, as understood from the definition of (t/tw). However, it may be a value of about 1.05.

Whether or not (t/tw) satisfies the formula (1) is an index of whether or not the observed cell maintains its original structure. For example, when cells are dried when immunostaining is performed using an antibody, the drying destroys the membrane structure of the cells, which may prevent the cells from maintaining their original structures. As a result, the localization of the protein to be detected changes, and there is a possibility that accurate results cannot be obtained. Since dry cells usually decrease in thickness and size due to water loss, equation (1), which is based on the original thickness of the cell (tw), is used to identify changes in size of the observed cells. effective for

From the viewpoint of specimen examination efficiency, etc., it is preferable that cells to be observed are arranged as densely as possible on a flat substrate. The density of the cells arranged in a monolayer on the plate substrate is preferably 4000-12000 cells/mm ² , more preferably 6000-10000 cells/mm ² , still more preferably 7000-9000 cells/mm ² .

<Uses of isolated cell samples>
The isolated cell specimen of the present invention is used for immunostaining and/or nuclear staining. However, the isolated cell specimen of the present invention can also be used for any other purpose for cell observation.

The following methods are known as conventional immunostaining methods (see, for example, Non-Patent Document 1). First, cells diluted with a liquid medium are dropped onto the surface of a substrate, and then the substrate is washed with the liquid medium to arrange the cells in a monolayer on the surface of the substrate. The cells are subjected to immunostaining using an antibody in the presence of a liquid medium, and microscopic observation is performed. And all these steps should be done in a wet system to avoid drying the cells. This is because if the cells dry out, the structure of the cells and the like may change, making it impossible to obtain accurate results. Therefore, in the conventional immunostaining method, it was difficult to stably store and transfer the cell sample for observation before immunostaining.

In contrast, in the isolated cell specimen of the present invention, the cells adhered to the substrate retain moisture and maintain the original structure of the cells, while the liquid (medium or buffers, etc.) are substantially absent. This means that, according to the present invention, it is not necessary to maintain the cells arranged on the surface of the substrate in a wet system as in conventional immunostaining methods. As a result, according to the present invention, the isolated cell specimen can be stably stored for a desired period of time (e.g., one day or more) or the isolated cell specimen can be transported to a distant place until immunostaining is performed. be able to.

When the isolated cell specimen of the present invention is subjected to immunostaining, a known method can be appropriately selected as the method, and antibodies and conditions according to the marker or the like to be detected can be used.

Conventionally, when immunostaining is performed using an antibody against a protein inside a cell, it has been essential to permeabilize the cell with saponin or the like. In contrast, in the present invention, sufficient dyeing can be performed without such treatment. However, in the present invention, the aspect of performing membrane permeabilization in immunostaining is not excluded.

As a nuclear staining method, any fluorescent dye can be used, such as DAPI (4',6-diamidino-2-phenylindole).

<Method for producing an isolated cell sample>
The method for producing an isolated cell specimen of the present invention includes a spreading step of spreading a liquid containing cells to be observed on a flat substrate, and cell removal of removing cells that are not attached to the surface of the flat substrate after the spreading step. and a drying step of air-drying the flat substrate after the cell removal step, and the obtained isolated cell specimen is used for immunostaining and/or nuclear staining.

FIG. 3 shows an example of the method for preparing the isolated cell preparation of the present invention. A detailed description will be given below with reference to FIG.

(1) Preparation of Flat Substrate A flat substrate is prepared and subjected to hydrophilic treatment such as oxygen plasma treatment as necessary.

(2) Developing Step A liquid containing cells to be observed is developed on a flat substrate. Any method that can arrange the cells on the flat substrate without destroying the cells can be adopted as the method for developing the observation target cell-containing liquid. For example, a method of dropping onto the surface of a flat substrate using a micropipette or the like can be mentioned. Examples of the cell-containing solution to be observed include cell-containing fractions such as mononuclear cell fractions, and diluted fractions thereof. The developed observation target cell-containing liquid spreads on the surface of the flat substrate, enabling observation over a wide range.

Various aqueous buffer solutions (PBS, etc.), pure water, etc. can be used as liquid media for diluting the cells in the observation target cell-containing solution.

In the cell-containing solution to be observed, the cell concentration is not particularly limited, but the concentration is preferably such that the cells on the substrate do not overlap excessively. For example, the cell concentration is preferably 1×10 ⁶ to 1×10 ⁷ cells/ml, more preferably 2×10 ⁶ to 5×10 ⁶ cells/ml.

Cells arranged on a flat substrate are usually not arranged in a single layer, but are arranged in a partially overlapping state. In this state, cells that are in direct contact with the surface of the planar substrate bind to the surface of the planar substrate by physical adsorption. That is, in the expansion step, at least part of the observation target cells arranged on the substrate surface adheres to the surface of the flat substrate.

(3) Cell Removal Step After the expansion step, cells that are not adhered to the surface of the flat substrate are removed. Cells may be layered on the plate-shaped substrate after the development step, but cells in such a state interfere with the detection of the target cells by microscopic observation, so they are layered in this step. Excess cells are removed by washing or the like. As a result, only the cells adhering to the surface of the flat substrate are arranged on the flat substrate in a substantially monolayer without agglomeration. FIG. 4 shows a schematic diagram of the state of the cells layered after the expansion step and the state of the cells arranged in a single layer after the cell removal step.

The method for removing cells not adhering to the surface of the flat substrate is not particularly limited, but from the viewpoint of preventing cell damage, a method of contacting the substrate with a liquid is preferred. Examples thereof include a method of immersing a flat substrate in a container containing a cell-removing liquid, a method of pouring a cell-removing liquid over the surface of the flat substrate, and the like.

The cell removal liquid is preferably water or an aqueous solution. The lower limit of the specific resistance value of water or an aqueous solution is preferably 10 Ω·cm or more, more preferably 10 ⁴ Ω·cm or more, and still more preferably 10 ⁶ Ω·cm or more. If the specific resistance value of water or aqueous solution is less than 10 Ω·cm, the content of ionic substances in the liquid for cell removal increases, and solutes precipitate in the drying process described later, or the solutes are difficult to observe with a microscope. may hinder. The upper limit of the specific resistance value of water or aqueous solution is preferably 100 Ω·cm or less.

Aqueous solutions having a specific resistance within the above range include liquid media, tap water (15 to 16 kΩ·m), and various buffer solutions (phosphate buffered saline (62 to 63 Ω·m), etc.).

Water containing almost no ionic substances with a specific resistance value of 10 ⁴ Ω·cm or more is preferable as the cell removal liquid because it is less likely to affect precipitation in the drying process described later and during microscopic observation. It is more preferable to use pure water with a resistance value of 10 ⁶ Ω·cm or more.

(4) Drying Step After the cell removal step, the flat substrate is dried until substantially no liquid remains on the surface of the flat substrate on which the cells are arranged. The drying process can reduce the amount of liquid on the surface of the flat substrate. Accordingly, the cells to be observed can be arranged on the substrate in a state in which there is substantially no liquid on the surface of the flat substrate on which the cells are arranged.

Drying in the drying process is natural drying. Natural drying means drying at a temperature near room temperature (for example, 1 to 40°C). The method of drying is not particularly limited as long as the specimen can be exposed to a temperature near room temperature. Examples include a method of placing a specimen.

The temperature conditions for natural drying can be adjusted according to the length of drying time and the like. The temperature conditions for natural drying may be within the range of room temperature, for example, preferably 1 to 40°C, more preferably 10 to 30°C, still more preferably 15 to 29°C, still more preferably 20 to 28° C., most preferably 22-24° C. (around 23° C.). If the temperature is too high, the drying of the cells progresses, the membrane structure of the cells is destroyed, and the original structure cannot be maintained, which may affect subsequent observations. If the temperature is too low, the drying time may become too long.

The humidity conditions for natural drying are preferably 10 to 70% relative humidity, more preferably 20 to 60%, still more preferably 20 to 50%.

The pressure conditions for natural drying are preferably 0.5 to 2.0 atmospheres, more preferably 0.8 to 1.2 atmospheres, and still more preferably 1 atmosphere.

The natural drying time is preferably 30 minutes or longer, more preferably 35 minutes or longer, still more preferably 50 minutes or longer, and even more preferably 1 hour or longer. Although the natural drying time may be longer, it is usually preferably 100 hours or less, more preferably 90 hours or less, and still more preferably 80 hours or less from the viewpoint of work efficiency.

Air speed conditions for natural drying may be 0.0 to 1.0 m/sec. Higher wind speeds may allow shorter drying times.

Before and after the drying process, the average thickness of the cells to be observed satisfies the following formula (2).
(t'/tw')≧0.95 Formula (2)
(In formula (2),
t ' represents the average thickness of the cells to be observed after the drying process attached to the surface of the flat substrate,
tw' represents the average thickness of observed cells attached to the surface of the flat substrate before the drying process. )

The details of formula (2) conform to the description in <Isolated Cell Specimen> above. However, the "average thickness of the cells to be observed after the drying process" corresponds to the "average thickness of the cells to be observed attached to the surface of the flat substrate" in the <isolated cell specimen> above. "Average thickness of cells to be observed before the drying process" is equivalent to "Average thickness of cells of the same type as the cells to be observed that have not undergone the drying process and attached to the surface of the flat substrate" in the above <isolated cell specimen>. corresponds to Note that in formula (2), t' and tw' are values in the same cell group.

Completion of the drying step provides the isolated cell preparation of the present invention. This isolated cell specimen is used for immunostaining and/or nuclear staining.

(5) Staining step The isolated cell specimen obtained as described above is subjected to immunostaining and/or nuclear staining. If necessary, the isolated cell specimen of the present invention can be subjected to various analyzes by methods other than immunostaining and/or nuclear staining.

The isolated cell specimen obtained as described above has good immunostaining and/or nuclear staining. In the present invention, the phrase "good immunostaining and/or nuclear staining" means that the stained cells maintain their original structure and provide sufficient signal intensity for detection. .

<Method for detecting target cells>
The isolated cell specimen of the present invention is obtained through each of the above steps. The obtained isolated cell specimen is subjected to immunostaining and/or nuclear staining. As the method of immunostaining, arbitrary conditions can be adopted according to the type of target cells, and examples thereof include methods using fluorescence-labeled antibodies and the like. As a nuclear staining method, arbitrary conditions can be adopted according to the type of target cells, and examples thereof include a method using DAPI or the like. Target cells include, for example, circulating cancer cells. When the cells of interest are circulating cancer cells, the cells to be observed preferably include peripheral blood mononuclear cells.

For example, in the detection of circulating cancer cells, EpCAM (surface marker of epithelial cells) and Cytokeratin (intracellular protein marker of epithelial cells) are stained with respective fluorescence-labeled antibodies. In addition, in order to avoid false positives due to non-specific adsorption of antibody molecules, EpCAM-positive, Cytokeratin-positive, and CD45-negative cells can be narrowed down by simultaneously performing staining using an antibody against CD45 (a surface marker of leukocytes). . In addition, the cells narrowed down as circulating cancer cell candidates by these antibody staining are subjected to nuclear staining with DAPI to evaluate the normality of the nuclear structure, thereby increasing the risk of cancer metastasis and recurrence. can identify the presence of cancer cells with high The above stained isolated cell specimen is subjected to the detection and analysis of the target cells using a fluorescence microscope, etc., and the results of these staining and observations are comprehensively evaluated to determine the cells of the isolated cell specimen. contains circulating cancer cells.

When immunostaining the isolated cell specimen obtained by the production method of the present invention, membrane permeabilization may not be performed. However, in the present invention, a mode in which membrane permeabilization is performed upon immunostaining is not excluded. In normal immunostaining, when the staining target is not the cell surface (for example, when staining with Cytokeratin antibody for intracellular proteins or DAPI for cell nuclei), membranes using saponin etc. It is necessary to perform transparency processing. That is, staining for obtaining intracellular information usually requires membrane permeabilization as a pretreatment. In contrast, the isolated cell specimen obtained by the production method of the present invention can be stained without such membrane permeabilization treatment, and can greatly contribute to the simplification and efficiency of the examination process.

EXAMPLES The present invention will be described below with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

<Test 1: Examination of various isolated cell specimens>
Based on the following method, isolated cell specimens were prepared under various conditions, and the test efficiency was examined.

(Example 1)
An isolated cell sample was prepared by the following procedure and stained.

[Preparation of isolated cell specimen]
(1) Preparation of Flat Substrate A plastic plate made of polystyrene and having dimensions of 76 mm×25 mm×1 mm (thickness) was used. Using a soft plasma etching device (trade name: SEDE-P, manufactured by Meiwaforsys), oxygen gas was applied to the plastic plate at a flow rate of 0.25 L/min (pressure: equivalent to 0.2 kg/m ³ ). Plasma irradiation was performed for 15 minutes under conditions of an internal pressure of 6 to 10 Pa and an output of 5 mA to 25 mA. The average contact angle of water on the surface of the plate-like substrate after plasma irradiation was 5° or less at five points.
(2) Preparation of Observation Target Cells In this example, lung cancer cells were used as a circulating cancer cell model, and a cell group containing a small number of lung cancer cells was prepared as observation target cells. Specifically, human lung cancer cells (H1650) and human leukemia cells (CEM) were mixed so that the number of H1650 cells: the number of CEM cells = 1:100, and the cell concentration was 3.33 × 10. A liquid containing ⁶ cells/ml of cells to be observed was prepared.
(3) Development step 3 ml of the cell-containing liquid to be observed was dropped onto the flat substrate using a micropipette. After being dropped, the observed cell-containing liquid spread over almost the entire surface of the substrate.
(4) Cell removal step The plate-like substrate after the development step is immersed in ultrapure water with a specific resistance of 18.2 MΩ cm to wash excess cells, so that they are not attached to the surface of the plate-like substrate. Cells were removed.
(5) Drying Step After the cell removal step, the flat substrate was naturally dried for 50 minutes in a room at 25°C and a relative humidity of 50% to obtain an isolated cell sample of Example 1. When observed visually and with a stereoscopic microscope, no liquid was observed on the surface of the flat substrate on which the cells were arranged. In addition, using an infrared moisture meter (trade name "fiber type infrared moisture meter IM-3SCV MODEL-2000", manufactured by Fujiwork Co., Ltd.), the water content on the surface of the flat substrate was measured at 25 ° C. and a relative humidity of 50%. was measured over time, and it was confirmed that the surface of the flat substrate on which the cells were arranged had no liquid after 50 minutes of natural drying (Fig. 5).
(6) Measurement of Average Thickness of Cells Before and after the drying process, the average thickness of cells to be observed attached to the surface of the flat substrate was measured. Specifically, Z-stack observation (using a 20x objective lens, Interval: 1.05 μm) is performed using a confocal laser scanning microscope (trade name “FV-3000”, manufactured by Olympus Corporation), and the signal Based on the intensity, the average thickness of the observed cells on the flat substrate was measured.

[Dyeing process]
The obtained isolated cell specimen was subjected to immunostaining and nuclear staining by the following method.
For immunostaining, EpCAM (surface marker for epithelial cells), Cytokeratin (intracellular protein marker for epithelial cells), CD45 (surface marker for leukocytes) were used. By using CD45 as a marker, false positives due to nonspecific adsorption of antibody molecules can be avoided, so EpCAM-positive, Cytokeratin-positive, and CD45-negative cells can be identified.
In addition, nuclear staining was performed with DAPI.
(1) Preparation of staining solution A PBS solution containing all of the following was prepared as a staining solution.
Anti-EpCAM antibody: trade name "Alexa488-labeled anti-EpCAM antibody", Thermo Fisher Scientific Co., Ltd., 50-fold dilution Anti-Cytokeratin antibody: PE-labeled anti-Cytokeratin antibody, 50-fold dilution Anti-CD45 antibody: Trade name "Alexa647-labeled anti-CD45 antibody ”, 30-fold dilution DAPI: 2 μg/ml
(2) Staining method 3 ml of the staining solution was spread so as to come into contact with the cells on the surface of each specimen, and the staining was performed for 1 hour at room temperature under light shielding. Then, after removing excess staining solution by decantation, each specimen was immersed in PBS and washed. Before the PBS on the specimen dried, it was quickly covered with a cover glass and sealed with a cover glass sealant (trade name “CoverGrip”, manufactured by Biotium).
(3) Evaluation of stainability The stainability of each marker was evaluated according to the following criteria. Whether or not the stained cells maintained their original structure was judged from the outline of the stained cells, the stained sites, and the like.
A: Stained cells maintained their original structure and the signal intensity of the staining was sufficient for detection.
B: Although the stained cells maintained their original structure, the intensity of the staining signal was weak.
C: Stained cells did not maintain their original structure and/or no staining signal was observed.

(Comparative example 1)
In the drying step, instead of natural drying, the same operation as in Example 1 was performed except that hot air was applied from a distance of 1.5 cm for 10 seconds using a 1200 W dryer. was made. When observed visually and with a stereoscopic microscope, no liquid was observed on the surface of the flat substrate on which the cells were arranged.

(Comparative example 2)
In the drying step, instead of natural drying, a 1200 W dryer was used to apply hot air for 30 seconds from a distance of 10 cm.The same operation as in Example 1 was performed to prepare an isolated cell specimen of Comparative Example 2. did. When observed visually and with a stereoscopic microscope, no liquid was observed on the surface of the flat substrate on which the cells were arranged.

(Comparative Example 3)
In the drying step, the same operation as in Example 1 was performed except that cold air was applied from a distance of 1.5 cm for 50 seconds using a 1200 W dryer instead of natural drying. was made. When observed visually and with a stereoscopic microscope, no liquid was observed on the surface of the flat substrate on which the cells were arranged.

(Comparative Example 4)
A flat substrate similar to that of Example 1 was used, and an acrylic frame (width 2 mm, height 2 mm) for holding the liquid was fixed to the periphery of the flat substrate with an adhesive. Observation target cells were prepared in the same manner as in Example 1. After arranging the cells to be observed on the substrate, the cells to be observed were kept in a buffer solution at all times, and then subjected to staining and observation, which will be described later.

(Comparative Example 5)
The same operation as in Comparative Example 4 was performed except that the membrane permeabilization treatment using saponin, which is a conventional method, was performed before the staining step described later.

(result)
Table 1 shows the evaluation results. Also, the results of immunostaining and nuclear staining are shown in FIGS. For Example 1 and Comparative Examples 1 to 3, (t/tw) was specified as follows. That is, the cell thickness of Comparative Example 4 was treated as (tw).
(t / tw) = "Average thickness (t) of observation target cells attached to the surface of the flat substrate" / "Average thickness (tw) of observation target cells attached to the surface of the flat substrate of Comparative Example 4 ”

Note that (t'/tw') specified below for Example 1 and Comparative Examples 1 to 3 was also the same value as (t/tw).
(t′/tw′)=“Average thickness (t′) of observation target cells attached to the surface of the flat substrate after the drying process”/“Observation before the drying process attached to the surface of the flat substrate Average thickness of target cells (tw′)”

Example 1 (FIG. 6) is a specimen in which the average thickness of the cells to be observed is within an appropriate range and substantially no liquid exists on the surface of the flat substrate on which the cells are arranged. In this specimen, four types of markers for identifying circulating cancer cells could be clearly detected, and the cell density on the specimen was dense.

Comparative Example 1 (Fig. 7) and Comparative Example 2 (Fig. 8) are specimens prepared in the same manner as in Example 1, except that hot air drying was performed. In this specimen, the average thickness of the cells to be observed adhered to the surface of the flat substrate was flat within the specified range, the cells did not maintain their original structure, and the stainability with the CD45 antibody was remarkable. Decreased. In addition, in Comparative Example 1, the cell density on the specimen was low (bad). On the other hand, when cold air drying was performed as in Comparative Example 3 (Fig. 9), the average thickness of the cells to be observed adhered to the surface of the flat substrate was less than the specified range, as in the case of hot air drying. It became flattened and the stainability with CD45 antibody was remarkably decreased.

Comparative Example 4 (FIG. 10) and Comparative Example 5 (FIG. 11) are the results of staining specimens in the same manner as immunostaining that is commonly performed. In preparing these specimens, all operations were performed in a buffer solution from beginning to end, so the average thickness of the cells was maintained at the original thickness and was within the appropriate range. This is a specimen in which a liquid with a liquid level that greatly exceeds the average thickness of the adhered cells to be observed coexists. Comparative Example 4 is a sample that was not subjected to membrane permeabilization with saponin, and could not be stained with Cytokeratin antibody and DAPI. On the other hand, in Comparative Example 5, membrane permeabilization with saponin was performed by a conventional method. there were.

Example 1 is a comparative example that is a general test method in terms of excellent stainability with a CD45 antibody, and in that nuclear staining such as DAPI is possible without membrane permeabilization and inspection efficiency is good. Better than 4.

Furthermore, even when samples were repeatedly prepared in the same manner as in Example 1, the cells could be stably arranged in a dense monolayer on the flat substrate. On the other hand, in the methods of Comparative Examples 4 and 5, although the cells could be densely arranged on the flat substrate in some cases, the cells were unevenly distributed to form sparse portions in some cases. Therefore, the method of the present invention is excellent in test efficiency in that cells can be stably arranged in a monolayer on a flat substrate.

<Test 2: Consideration of natural drying conditions>
Four isolated cell specimens were prepared in the same manner as in Experimental Example 1 of Test 1 above, except that the natural drying time was varied within the range of 1 to 72 hours.

Antibody staining was performed in the same manner as in Test 1 above for the isolated cell specimen (wet system) that was not naturally dried and the isolated cell specimen after natural drying.

After antibody staining, Z-stack observation (using a 20x objective lens, Interval: 1.05 μm) is performed using a confocal laser scanning microscope (trade name “FV-3000”, manufactured by Olympus Corporation), and the signal intensity is Based on, the average thickness of cells on the flat substrate was measured. Table 2 shows the results.

All air-dried isolated cell preparations were approximately 15 μm thick, within ±3% compared to the wet system. Surprisingly, the results showed that even when dried for an extended period of time, the cell thickness of the specimens was comparable to that of the wet system.

Claims (8)

The cells to be observed are attached to the surface of a flat substrate, the cells not attached to the surface of the flat substrate are removed with water or an aqueous solution having a specific resistance of 10 Ω·cm or more, and the isolated cell specimen is air-dried. and
substantially no liquid is present on the surface of the flat substrate on which the cells are arranged;
The average thickness of the cells to be observed satisfies the following formula (1),
An isolated cell preparation used for immunostaining and/or nuclear staining.
(t/tw)≧0.95 Formula (1)
(In formula (1),
t represents the average thickness of the observed cells attached to the surface of the flat substrate,
tw represents the average thickness of cells of the same type as the observation target cells that are not air-dried and attached to the surface of the flat substrate. ) 2. The isolated cell preparation according to claim 1, wherein said cells to be observed include peripheral blood mononuclear cells. The isolated cell specimen according to claim 1 or 2, wherein the contact angle of water on the surface of the flat substrate on which the cells to be observed are arranged is 10° or less at 25°C. a spreading step of spreading a liquid containing cells to be observed on a flat substrate;
After the developing step, a cell removing step of removing cells not adhering to the surface of the flat substrate using a cell removing liquid, wherein the cell removing liquid has a specific resistance of 10 Ω·cm or more. a step of being water or an aqueous solution;
After the cell removal step, a drying step of naturally drying the flat substrate until there is substantially no liquid left on the surface of the flat substrate on which the cells are arranged;
A method for producing an isolated cell specimen used for immunostaining and/or nuclear staining, comprising: 5. The method for producing an isolated cell specimen according to claim 4, wherein the average thickness of the observation target fine cells contained in the observation target cell-containing liquid satisfies the following formula (2) before and after the drying step.
(t'/tw')≧0.95 Formula (2)
(In formula (2),
t 'represents the average thickness of the cells to be observed after the drying process attached to the surface of the flat substrate,
tw' represents the average thickness of the observed cells attached to the surface of the flat substrate before the drying step. ) 6. The production method according to claim 4, wherein in said cell removal step, cells are removed using water or an aqueous solution, and said water or said aqueous solution has a specific resistance value of 10 Ω·cm or more. A method for detecting a target cell, comprising the step of subjecting the isolated cell sample according to any one of claims 1 to 3 to immunostaining and/or nuclear staining. 8. The detection method according to claim 7, wherein the cells to be observed include peripheral blood mononuclear cells, and the target cells are circulating cancer cells.

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