JP2010107373A - Method for detecting cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily detecting a small amount of cells contained in biological samples, such as cancer cells, from a sufficient amount of the biological samples without extracting/collecting cells. <P>SOLUTION: The method for detecting cells in biological samples includes the steps of: (a) adding buffers in biological samples to prepare suspended particles; (b) labeling cells in the suspended particles prepared with a labeled substance in the step (a); (c) solidifying the suspended particles after the step (b); and (d) detecting the cells labeled by detecting labeled particles in the solidified, suspended particles obtained in the step (c) with an image analysis method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for easily detecting cells contained in a biological sample without extracting and collecting the cells from a sufficient amount of the biological sample.

  As in Europe and the United States, the number of patients with colorectal cancer has increased rapidly year by year, and colorectal cancer has become one of the top cancer mortality rates. This is thought to be caused by the fact that the Japanese diet has become a Western-style meat center. Specifically, about 60,000 people suffer from colorectal cancer every year, and the number of deaths by organ is the third largest after stomach cancer and lung cancer, and further increases are expected in the future. . On the other hand, unlike other cancers, colorectal cancer is a cancer that can be cured by nearly 100% by treating it at the beginning of onset. Therefore, it is extremely meaningful to make colorectal cancer a target for early cancer screening, and research and development of test methods for early detection of colorectal cancer are actively conducted.

As an inspection method for early detection of colorectal cancer, for example, enema inspection, colonoscopy and the like are performed. The enema examination is an examination in which barium is injected into the large intestine, adhered to the mucosal surface of the large intestine, irradiated with X-rays, and the surface irregularities are photographed to observe the surface of the large intestine. On the other hand, the colonoscopy is an examination in which the inside of the large intestine is directly observed with an endoscope. In particular, colonoscopy has high sensitivity and specificity, and has the advantage that polyps and early cancer can be removed.
However, these inspection methods have a problem that the cost is high, the burden on the subject is large, and there is a risk of complications. For example, enema examination involves the risk of X-ray exposure and bowel obstruction. In addition, the colonoscopy is invasive because the endoscope is placed directly into the large intestine, and the endoscope operation requires skill and the facilities that can be examined are limited. For this reason, it is difficult to say that these examination methods are suitable for colorectal cancer examinations for asymptomatic general people such as regular medical examinations.

  In recent years, fecal occult blood tests have been widely performed as a primary screening method for colorectal cancer. The fecal occult blood test is a test for examining the presence of hemoglobin derived from red blood cells contained in feces and is a method for indirectly predicting the presence of colorectal cancer. The fecal occult blood test has the advantage of being non-invasive and low-cost, but has a problem that the sensitivity is as low as about 25% and the probability of overlooking colorectal cancer is high. Furthermore, the positive predictive value is low, and the proportion of patients who are actually colorectal cancer patients among fecal occult blood test positive subjects is 10% or less, and includes many false positives. Therefore, development of a new inspection method with higher reliability is strongly desired.

  As a new non-invasive, simple and reliable test method suitable for regular medical examinations, tests that check for the presence of cancer cells in the stool and the presence of cancer cell-derived genes have attracted attention. ing. For example, there is a report that only cancer cells were separated from spontaneous excretion of colorectal cancer patients by Parcoll centrifugation, and the presence of proteins abnormally expressed in cancer cells was confirmed (for example, Non-Patent Document 1). reference.). However, the Parcoll centrifugation method has a problem that cells are easily damaged and the cell recovery rate is low. Therefore, as a method with less damage to cells, (1) magnetic beads carrying a monoclonal antibody that recognizes cancer cells are dispersed in a stool sample and bound to cancer cells, and then the magnetic beads are used. A method for recovering cancer cells by recovering a cell using a magnet has been established (for example, see Patent Document 1). There is also a report that sensitivity was 71% and specificity was 88% as a result of recovering cells from a stool sample using this method and carrying out a genetic test (see, for example, Non-Patent Document 2).

  In addition, as a method of collecting cells for directly examining cancer cells, for example, (2) a method of separating cells from stool, a) a step of cooling the stool to a temperature below its gel freezing point And b) collecting cells from the stool while maintaining the stool at a temperature below its gel freezing point so that the stool remains substantially intact. (For example, refer to Patent Document 2). (3) A stool filtration device for filtering a sample in which a buffer solution is added to the collected spontaneously discharged stool and collecting cancer cells from the filtrate, the mixture of the spontaneously discharged stool and the buffer solution The filter for filtering the filter is a conical or cylindrical filter, has a porous or mesh structure supporting mechanism for mounting the filter, has a mechanism for rotating the filter and the supporting mechanism, and has an outer periphery of the supporting mechanism. A stool filtration apparatus characterized by having a container for collecting the filtrate filtered by centrifugal force in the section is disclosed (for example, see Patent Document 3).

In addition, there is a method using a flow cytometer as a method for directly examining cancer cells. The flow cytometer measures individual fluorescence intensity of a cell while flowing the cell, and determines whether each cell is a cancer cell or a normal cell based on the measurement result. The flow cytometer is generally used for the separation of living cells, fixed culture cells, whole blood cells, microorganisms, and the like. In addition, kits such as BD Quantitrite (registered trademark) PE beads, QuantBrite (manufactured by Becton Dickinson), etc., which recognize and label the surface antigens of white blood cells and sort them with a flow cytometer are commercially available.
Yamao, T., 7 others, GASTROENTEROLOGY, 1998, 114, 1196-1205. Matsushita, H., 14 others, GASTROENTEROLOGY, 2005, 129, 1918-1927. JP 2005-46065 A Japanese National Patent Publication No. 11-511982 JP 2005-241520 A

The amount of cells contained in a biological sample such as feces is very small compared to the whole biological sample. For this reason, it is necessary to detect cells from a sufficient amount of a biological sample, particularly in the case of a test requiring high accuracy such as a cancer test.
Actually, in Non-Patent Document 2, 5 to 10 g of stool is collected, suspended in 200 mL of buffer and homogenized, and then divided into five through a filter, and cells are collected from each. Thus, in order to obtain sufficient sensitivity, a large amount of stool is required, and as a result, there is a disadvantage that the amount of solution to be processed increases. Furthermore, although the method described in Non-Patent Document 2 performs gene analysis and has higher test accuracy than the conventional method, it requires a process of extracting genes from the collected cells and a subsequent gene analysis process. There is a problem that a complicated operation is required and time and cost are required.

  In the method (2), various devices such as freezing stool and centrifugation are required, and it is time consuming and complicated, and it is difficult to process a large number of stool samples. It is. Furthermore, in the method (3), since cancer cells are filtered and collected from stool containing a large amount of contaminants, in addition to being very cumbersome, the device must be washed for each stool sample. However, there is also a problem that there is a risk of contamination and infection during the cleaning operation.

  In addition, in the method using a flow cytometer, since the measurement sample is passed through a very narrow flow path, a sample with a large amount of contaminants such as stool can be obtained only by performing simple pretreatment such as dilution. In addition to the difficulty of separating the gas and the foreign matter from each other, there is a high possibility that the flow path will be blocked. For this reason, when detecting cells labeled from stool using a flow cytometer, a step of once collecting cells from stool is required by some method, and it is difficult to say that cells can be detected quickly and easily. .

  It is an object of the present invention to provide a method for easily detecting cells contained in a trace amount in a biological sample such as cancer cells from a sufficient amount of the biological sample without extracting and collecting the cells. To do.

  As a result of diligent research to solve the above problems, the present inventor prepared a suspension by adding a buffer to a sufficient amount of a biological sample, and labeled cells to be detected in the suspension. By detecting the labeled cells in the solidified suspension obtained by solidification using a three-dimensional analysis method, the cells can be detected easily and accurately without extracting and collecting the cells. The present invention was completed.

That is, the present invention
(1) A method for detecting cells in a biological sample, comprising: (a) a step of adding a buffer to the biological sample to prepare a suspension; and (b) the suspension prepared in step (a). A step of labeling the cells therein with a labeling substance, (c) a step of solidifying the suspension after the step (b), and (d) a solidified suspension obtained in the step (c). Detecting a labeled cell by detecting a labeling substance in an object using an image analysis method, and a method for detecting a cell,
(2) The method for detecting a cell according to (1), wherein the step (c) is a step of solidifying the suspension by gelation,
(3) The step (a) is a step of preparing a suspension by adding a buffer to the biological sample so that the final concentration of the biological sample component is 2 to 10 (w / v)%. The method for detecting a cell according to (1) or (2) above,
(4) The method for detecting a cell according to any one of (1) to (3), wherein the suspension prepared in the step (a) is 5 to 25 mL.
(5) The method for detecting a cell according to any one of (2) to (4), wherein the gelation of the suspension is performed using a polymer gelling agent or a polymerizable monomer,
(6) The gelation of the suspension in the step (c) is performed by lowering the suspension temperature using agarose or gelatin as a polymer gelling agent. ) To (4), the method for detecting a cell according to any one of
(7) The gelation of the suspension in the step (c) is performed by polymerizing a polymerizable monomer in the presence of a crosslinking agent, any one of (2) to (4) above Cell detection method,
(8) The method for detecting cells according to (7), wherein the polymerizable monomer is acrylamide and the crosslinking agent is N, N′-methylenebisacrylamide,
(9) The method for detecting cells according to any one of (1) to (8), wherein the biological sample is feces or body fluids,
(10) The detection of a cell according to any one of (1) to (9), wherein the labeling of the cell in the step (b) is performed using a labeled antibody that specifically recognizes the cell. Method,
(11) The detection of a cell according to (10), wherein the labeled antibody is an antibody labeled with one selected from the group consisting of a fluorescent substance, a radioisotope, and a paramagnetic substance. Method,
(12) The cell detection method according to any one of (1) to (11), wherein the image analysis method is a three-dimensional image analysis method,
(13) After the step (d), (e) a step of again detecting the labeling substance in the solidified suspension analyzed in the step (d) using an image analysis method. The method for detecting a cell according to any one of (1) to (12),
(14) Any of the above (1) to (13), wherein the step (d) is a step of detecting a labeling substance in a part of the solidified suspension obtained in the step (c). Or a method for detecting a cell according to claim 1,
Is to provide.

  The cell detection method of the present invention is a method for detecting cells from a suspension obtained by diluting a biological sample in a buffer, and even when the volume of the suspension is as large as several mL, the cells The cells in the suspension can be detected without extracting and collecting the. For this reason, only a very small amount of cells to be detected are contained in the biological sample, and it is necessary to detect cells from a large amount of biological sample in order to obtain a reliable detection result. In addition, the cells can be easily detected using the cell detection method of the present invention.

The cell detection method of the present invention is a method for detecting cells in a biological sample, comprising: (a) adding a buffer to the biological sample to prepare a suspension; and (b) said step (a). A step of labeling the cells in the suspension prepared in step with a labeling substance, (c) a step of solidifying the suspension after step (b), and (d) in step (c). And a step of detecting a labeled cell by detecting a labeled substance in the obtained solidified suspension using an image analysis method. By using an image analysis method, labeled cells can be detected from the entire suspension of the biological sample without extracting and collecting the cells, so that the amount of the biological sample used for detection is increased. Can be easily detected. In particular, when detecting a cell from a relatively non-uniform sample such as a biological sample, as the amount of the sample used for detection increases, a more reliable and stable detection result can be obtained. That is, the cell detection method of the present invention can detect cells easily from a sufficient amount of a biological sample, and thus can obtain a detection result excellent in reliability and stability as never before.
Hereinafter, it demonstrates for every process.

First, as step (a), a buffer is added to a biological sample to prepare a suspension.
The biological sample used for the cell detection method of the present invention is not particularly limited as long as it is a sample collected from a living organism and can be expected to contain cells. Examples of the biological sample include stool, urine, body fluid, and organ washing solution. Examples of the body fluid include blood, bone marrow fluid, lymph fluid, saliva, ascites, exudate, and amniotic fluid. The biological sample used in the cell detection method of the present invention is preferably a biological sample generally used for clinical examinations such as feces or body fluids.

  The biological sample used in the cell detection method of the present invention is particularly preferably feces. Mammalian cells detached from the gastrointestinal tract and the like contained in the stool are extremely small and may not be detected if the amount of sample used for detection is too small. Moreover, since feces are heterogeneous, that is, various components are present non-uniformly, mammalian cells may not be included depending on the site from which the sample is collected. In order to avoid such an influence of cell localization, it is preferable to mix samples collected from various parts of stool and use them for detection. In other words, in order to detect cells with high accuracy from heterogeneous stool with a low content of cells to be detected, it is necessary to use a large amount of stool collected from a plurality of sites as samples. The cell detection method of the present invention that can easily detect cells from a large amount of biological sample is very suitable.

  The biological sample may be a sample collected from a living organism or may be a prepared sample. Note that “samples collected from living organisms” include samples that have been refrigerated or stored at room temperature after collection in addition to samples immediately after collection. Further, the preparation method is not particularly limited as long as it does not damage the cells contained in the biological sample, and the preparation method usually used for the biological sample can be performed. it can. For example, it may be plasma or serum obtained by centrifugation from blood, or may be a homogenized tissue or the like.

Furthermore, a biological sample that has been previously immobilized may be used. Cells in biological samples are subject to deterioration and damage over time, but immobilization treatment after collection makes it possible to influence the passage of time even if there is time between collection and implementation of the detection method. It is possible to obtain a detection result that excludes. The immobilization treatment can be appropriately selected from immobilization treatments generally performed on cells before staining in a cell staining method or the like. For example, the cells in the biological sample are immobilized and stabilized by immersing the biological sample in an immobilization treatment solution such as an ethanol solution, an alcohol solution such as a methanol solution, an aldehyde solution such as a formaldehyde solution, or a paraformaldehyde solution. Can be saved.
In the case where the biological sample is immobilized in advance, the biological sample after the immobilization treatment is washed with an appropriate buffer before the step (a) in order to suppress the influence of the immobilization treatment liquid on the subsequent steps. It is preferable to keep it.

  The buffer added to the biological sample is not particularly limited as long as it does not inhibit subsequent labeling or detection of cells, and is appropriately selected from buffers generally used for dilution of biological samples. Can be used. Examples of the buffer include a phosphate buffer, a tris buffer, a citrate buffer, a HEPES buffer, and a HUNKS buffer. The pH of the buffer added to the biological sample is not particularly limited as long as it does not impair the labeling of cells, and in particular, when a fluorescent substance is used for labeling cells, the fluorescence of the fluorescent substance used is not limited. It is preferable to use a buffer adjusted to a pH range suitable for the characteristics. In general, the pH is 6 to 8, preferably about 7.

  An arbitrary component may be added to the buffer as long as subsequent labeling and detection of cells are not inhibited. For example, when using a biological sample that has been subjected to immobilization in advance, the biological sample can be suspended more easily by adding a buffer containing a chaotropic salt or a surfactant. The surfactant that can be added to the buffer is preferably a nonionic surfactant. Examples of the nonionic surfactant include Tween 80, CHAPS (3- [3-colamidopropyldimethylammonio] -1-propanesulfonate), Triton X-100, Tween 20, and the like. Examples of chaotropic salts that can be added to the buffer include guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, sodium perchlorate, and sodium trichloroacetate. The type and concentration of the chaotropic salt and surfactant added to the buffer are not particularly limited as long as the concentration does not inhibit the labeling or detection of the target cells. It can be determined appropriately in consideration of the type and the like.

  The amount of buffer added to the biological sample is not particularly limited as long as the biological sample can be sufficiently dispersed so that cells in the suspension can be labeled in a later step. Instead, it can be appropriately determined in consideration of the type and amount of the biological sample, the type of buffer, and the like. For example, it is preferable to prepare a suspension by adding a buffer to the biological sample such that the final concentration of the biological sample component is 2 to 10 (w / v)%. More specifically, for example, a suspension in which a final concentration of biological sample components is 2 to 10 (w / v)% by adding 5 to 25 mL of buffer to 0.5 g of biological sample. Can be prepared.

  The method for preparing the suspension is not particularly limited as long as it can disperse the biological sample in a buffer without damaging the cells contained in the biological sample. It can be carried out by appropriately selecting from physical methods used in preparing the product. For example, the biological sample may be placed in a sealable container in which a buffer has been placed in advance and sealed, and the container may be mixed and suspended by turning the container upside down. It may be mixed and suspended by applying a shaker. Further, the biological sample and the buffer may be mixed and suspended in the presence of the mixing particles.

  The amount of the biological sample used for the preparation of the suspension is not particularly limited as long as it can be expected to obtain a reliable detection result, and the type and state of the biological sample, the collection method, etc. It can be appropriately determined in consideration. For example, when detecting mammalian cells detached from a digestive tract or the like present in a relatively small amount in stool, the stool used is preferably 0.1 to 5 g, and preferably 0.1 to 1 g. More preferably, it is more preferably 0.1 to 0.5 g.

  The amount of the suspension obtained in the step (a) is not particularly limited, but is preferably 5 mL or more because detection by a three-dimensional image analysis method described later is easy. The upper limit of the amount of suspended matter can be determined as appropriate according to the standard of the three-dimensional image analysis apparatus used, but is preferably about 25 mL or less from the viewpoint of shortening the detection time.

  Next, as step (b), the cells in the suspension prepared in step (a) are labeled with a labeling substance. In the present invention, “labeling a cell (using a labeling substance)” means binding a labeling substance directly or indirectly to the cell. The labeling substance is not particularly limited as long as it is a substance that can detect cells by detecting and analyzing signals emitted from the substance, and labels biological components such as cells, proteins, and nucleic acids. In this case, it can be appropriately selected from labeling substances generally used.

  In the present invention, since the detection by the image analysis method is easy, the labeling substance for labeling the cells is preferably a fluorescent substance, a radioisotope, a paramagnetic substance, or the like. The fluorescent material is not particularly limited as long as it is generally used for labeling biomolecules such as proteins, and can be appropriately selected from known fluorescent materials. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine) and the like. In addition, fluorescent semiconductor quantum dots such as CdSe may be used. The paramagnetic substance is not particularly limited as long as it can be used as an MRI (Magnetic Resonance Imaging) contrast agent. Examples thereof include paramagnetic metals such as gadolinium (Gd) and iron. . In addition, chemiluminescent substances, enzymes used in chemiluminescent reactions, and the like may be used as labeling substances. The chemiluminescent substance used for labeling may be a substance that emits light by an inorganic reaction such as luminol, or may be a luminescent substance that is catalyzed by an enzyme such as luciferin or coelenterazine. Furthermore, the enzyme used for labeling can be appropriately selected from enzymes usually used in detection of a target substance utilizing an enzyme reaction. Examples of such enzymes include alkaline phosphatase, peroxidase, luciferase and the like. In the present invention, since the detection sensitivity is excellent, the labeling substance for labeling cells is more preferably a fluorescent substance.

  The cell labeling method is not particularly limited as long as it is a method capable of labeling the detection target cell, and is appropriately selected from methods generally used in the fields of cell biology and clinical examinations. Can be used. In particular, since a cell to be detected can be specifically labeled separately from other cells, it is preferable to label by a immunological technique using a labeled antibody that specifically recognizes the cell. The “labeled antibody” means an antibody to which a labeling substance is bound. The labeled antibody and cells in suspension can be bound by a conventional method.

  Specifically, a labeled antibody labeled with a fluorescent substance or the like that specifically recognizes cells to be detected is added to the suspension prepared in step (a), incubated, and suspended in the suspension. The cells can be labeled by binding the labeled cells to the labeled antibody. Thereafter, the labeled antibody bound to the cells and the unbound labeled antibody can be separated by separating into a supernatant and a precipitate by centrifugation or the like. Furthermore, the obtained precipitate is washed with an appropriate buffer or the like, and the labeled antibody that is non-specifically bound to the components in the suspension is removed to reduce the detection background. Can do.

In the present invention, “specifically recognize” means that the substance can be specifically bound to such an extent that it can be normally used for detection or purification of the substance to be recognized, and is used for cell detection. It may intersect with other substances that do not have an influence.
The antibody used in the present invention may be a monoclonal antibody or a polyclonal antibody. In addition, a commercially available antibody may be used as such an antibody, and an antibody prepared by a conventional method after immunizing an experimental animal such as a mouse with an antigen may be used.

  After step (b), the suspension is solidified as step (c). “Solidify the suspension” means that in the subsequent cell detection step, the fluidity is lowered to such an extent that the cells in the suspension do not migrate. In the present invention, it is particularly preferable to obtain a solidified suspension by gelling the suspension. For example, by gelling the suspension, the fluidity of the suspension can be sufficiently reduced while keeping the influence on the labeled cells in the suspension low.

  The suspension can be gelled using a polymer gelling agent such as gelatin or agarose (agar). In addition, the suspension can be gelled by polymerization using a polymerizable monomer.

  For example, when agarose or gelatin is used as the polymer gelling agent, the suspension can be gelled by lowering the temperature of the suspension containing the polymer gelling agent. More specifically, for example, the melted polymer gelling agent solution cooled to about 37 ° C. is added to the suspension containing the labeled cells obtained in the step (b) and rapidly mixed. By cooling, the suspension can be gelled to obtain a solidified suspension. In addition, the suspension containing the labeled cells obtained in the step (b) collects only the solid component (precipitate) by centrifugation or the like, and is cooled to about 37 ° C. to obtain a molten polymer gel. The suspension can also be gelled by adding the agent solution, mixing rapidly and cooling. The concentration of agarose or gelatin added to the suspension is not particularly limited as long as the concentration is sufficient for the entire suspension to gel, and may be, for example, about 1%.

  Moreover, when using a polymerizable monomer, for example, after adding and mixing a polymerizable monomer solution to the suspension containing labeled cells obtained in step (b), a crosslinking agent or a polymerization initiator is added. By further adding, the polymerization reaction can be started and a solidified suspension can be obtained. In addition, only the solid component (precipitate) of the suspension is recovered by centrifugation or the like, and after adding and mixing the polymerizable monomer solution, the suspension is further added by adding a crosslinking agent and mixing. Can be gelled. In addition, a polymerizable monomer is added in advance to the buffer added to the biological sample in the step (a), and the suspension containing the labeled cells obtained in the step (b) is added with a crosslinking agent or a polymerization initiator. Etc. can be added and mixed to gel the suspension.

  The polymerizable monomer used in the present invention is a monomer having at least one polymerizable group such as a vinyl group or an allyl group in one molecule and capable of performing a polymerization reaction without damaging the labeled cells. If it is, it will not specifically limit, It can select from the polymerizable monomer generally used in field | areas, such as a clinical test and a formulation, and can use it suitably. In addition, one type of polymerizable monomer may be used, or two or more types may be used. For example, the suspension can be gelled by using acrylamide as the polymerizable monomer and N, N′-methylenebisacrylamide as the crosslinking agent. In this case, it is also preferable to use ammonium persulfate or the like as a polymerization initiator. The concentration of the polymerizable monomer, crosslinking agent, polymerization initiator and the like added to the suspension is not particularly limited as long as the concentration is sufficient for the entire suspension to gel.

  Thereafter, as a step (d), the labeled cells are detected by detecting the labeling substance in the obtained solidified suspension using an image analysis method. That is, by detecting the labeling substance in the solidified suspension by the image analysis method, the cells labeled with the labeling substance are detected. Unlike the detection of cells fixed to almost one layer such as a conventional slide glass, the cells are detected from a solid suspension that is a thicker solid, so that cells can be easily detected from a larger amount of sample. .

  The image analysis method used for detecting the labeling substance may be a two-dimensional image analysis method or a three-dimensional image analysis method. Specifically, the detection using the three-dimensional image analysis method means that the cross section of the solidified suspension is shifted in a direction perpendicular to the cross section and two-dimensional images (tomographic images) of the respective cross sections are continuously obtained. Capture and acquire, and combine them to reconstruct a 3D image. Thereby, the labeled cell in the solidified suspension which has thickness and the cell exists in the multilayer can be detected easily. As a method for acquiring a tomographic image, an imaging means suitable for the type of cell labeling can be appropriately selected from known imaging means. For example, when cells are labeled using a gadolinium-labeled antibody, a three-dimensional image of the solidified suspension is obtained by a conventional method using a general-purpose MRI apparatus or the like, and the labeled cells are detected. (See, for example, Journal of surgical oncology, 2006, 94 (2), p144-8). In addition, when a cell is labeled using a fluorescent label or a luminescent labeled antibody, it can be detected using a light detection device described in Japanese Patent No. 3786903. When a radioisotope is used as the labeling substance, it can be detected using a positron emission tomography (PET) apparatus or the like.

  In the present invention, since the detected cells are fixed in the solidified suspension, the position of the cells in the sample does not vary and is stable for a relatively long time. For this reason, after acquiring an image from the solidified suspension, it is possible to easily acquire an image of the same part again, so re-examination is easy, and cells in the solidified suspension are detected with higher accuracy. can do. More specifically, based on a tomographic image acquired from the entire solidified suspension, or a three-dimensional image constructed therefrom, a tomographic image of only the suspicious part of the part where the labeling substance is detected is acquired again, By checking, the accuracy of cell detection can be increased. In addition, it is difficult to determine whether the “suspected part” is a labeling substance that is labeling a cell or merely a labeling substance that causes non-specific aggregation. It means that part.

  That is, in the method for detecting a cell of the present invention, after step (d), as step (e), the labeled substance in the solidified suspension analyzed in step (d) is again used by image analysis. A detecting step. Thus, when detecting the labeling substance again (acquiring an image of a site where the labeling substance exists), it is preferable to pick up an image with a higher resolution.

  Moreover, you may detect a cell not only from the whole solidified suspension but only from the part. Specifically, acquisition of tomographic images may be performed on only a part of the solidified suspension. For example, when a sufficient amount of cells are present in the solidified suspension, the time required for the detection method can be shortened by detecting from only a part of the solidified suspension.

  Thus, by using the cell detection method of the present invention, cells can be easily detected from a relatively large amount of biological sample. For this reason, it is particularly suitably used when it is required to detect a cell having a very small amount in a biological sample, such as a cancer test, with high specificity. This is because detection from a sufficient amount of biological sample enables stable and reliable detection. In addition, it is also suitably used for tests for examining the presence or absence of infections.

  In the cell detection method of the present invention, as described above, a two-dimensional or three-dimensional image showing the position of the labeled cell in the prepared solidified suspension is obtained. Based on the above, the labeled cells can be recovered by cutting out the region containing the labeled cells from the solidified suspension. The recovered cells can be subjected to various analysis methods as with other biological samples. For example, in the case of a solidified suspension gelled with agarose, gelatin, etc., the labeled cell-derived nucleic acid is recovered from the gel obtained by heating and melting the excised solidified suspension. In contrast, known nucleic acid analysis such as PCR can be performed.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
2 g of stool collected from a colon cancer patient was suspended in 5 mL of Tris buffer (pH 7.5) to prepare a suspension. Thereafter, an appropriate amount of FITC-labeled anti-CD34 antibody (manufactured by Beckman Coulter, Inc.), which is an antibody against CD34 expressed specifically in cancer cells and labeled with FITC, is added to this suspension and mixed. The cancer cells were labeled by reacting with the cancer cells contained in the stool. Thereafter, a 2% agarose solution once dissolved and cooled to about 40 ° C. was added to the suspension, mixed, and then cooled to obtain a solidified suspension (sample). As a control, an appropriate amount of FITC-labeled anti-CD34 antibody was added to and mixed with a suspension prepared by suspending 2 g of stool collected from a healthy person in 5 mL of Tris buffer (pH 7.5). % Agarose solution was added to obtain a solidified suspension (control).

A tomographic fluorescence image of the obtained solidified suspension was obtained using IVIS200 (manufactured by Xenogen), and fluorescently labeled cells were detected. FIG. 1 is a tomographic fluorescence image obtained from a solidified suspension (sample), and FIG. 2 is a tomographic fluorescence image obtained from a solidified suspension (control). As a result, although fluorescence was detected as a whole, in the tomographic fluorescence image acquired from the solidified suspension (sample), a portion where the fluorescence intensity is locally high (the portion circled in FIG. 1) ) Was observed. The moiety is a cell labeled with a FITC-labeled anti-CD34 antibody. On the other hand, in the tomographic fluorescence image acquired from the solidified suspension (control), a portion of local increase in fluorescence intensity detected in the solidified suspension (sample) was not observed.
From these results, it was confirmed that cells can be easily detected from a sufficient amount of biological sample by the method of the present invention.

  By using the cell detection method of the present invention, cells can be easily detected from a relatively large amount of biological sample, so that there are only a very small number of cells to be detected, particularly in cancer tests. It can be used in the fields of clinical examination, cell biology, pharmacy, etc., in which cells are detected and analyzed using a biological sample that is not present.

In Example 1, it is a tomographic fluorescence image acquired from the solidification suspension (sample). In Example 1, it is a tomographic fluorescence image acquired from the solidification suspension (control).

Claims (14)

A method for detecting cells in a biological sample, comprising:
(A) adding a buffer to the biological sample to prepare a suspension;
(B) labeling the cells in the suspension prepared in step (a) with a labeling substance;
(C) after the step (b), solidifying the suspension;
(D) detecting a labeled cell by detecting the labeling substance in the solidified suspension obtained in the step (c) using an image analysis method;
A method for detecting a cell, comprising:   The method for detecting cells according to claim 1, wherein the step (c) is a step of solidifying the suspension by gelation.   The step (a) is a step of adding a buffer to the biological sample and preparing a suspension so that the final concentration of the biological sample component is 2 to 10 (w / v)%. The method for detecting a cell according to claim 1 or 2.   The method for detecting cells according to any one of claims 1 to 3, wherein the suspension prepared in the step (a) is 5 to 25 mL.   The method for detecting cells according to any one of claims 2 to 4, wherein the gelation of the suspension is performed using a polymer gelling agent or a polymerizable monomer.   Gelation of the suspension in the step (c) is performed by lowering the suspension temperature using agarose or gelatin as a polymer gelling agent. The method for detecting a cell according to any one of the above.   The method for detecting a cell according to any one of claims 2 to 4, wherein the gelation of the suspension in the step (c) is performed by polymerizing a polymerizable monomer in the presence of a crosslinking agent.   The method for detecting cells according to claim 7, wherein the polymerizable monomer is acrylamide, and the crosslinking agent is N, N'-methylenebisacrylamide.   The method for detecting cells according to any one of claims 1 to 8, wherein the biological sample is feces or body fluid.   The cell detection method according to claim 1, wherein the labeling of the cell in the step (b) is performed using a labeled antibody that specifically recognizes the cell.   The method for detecting a cell according to claim 10, wherein the labeled antibody is an antibody labeled with one selected from the group consisting of a fluorescent substance, a radioisotope, and a paramagnetic substance.   The cell detection method according to claim 1, wherein the image analysis method is a three-dimensional image analysis method. After the step (d),
(E) It has the process of detecting again the labeling substance in the solidified suspension analyzed in the said process (d) using an image-analysis method, The one of Claims 1-12 characterized by the above-mentioned. Cell detection method.   The cell detection according to any one of claims 1 to 13, wherein the step (d) is a step of detecting a labeling substance in a part of the solidified suspension obtained in the step (c). Method.

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