JP6485783B2 - Medical inspection apparatus and cell inspection method - Google Patents

Description

The present invention relates to a medical test apparatus and a cell test method capable of capturing specific cells in blood and body fluids (such as cancer cells present in blood and body fluids).

It is known that cancer cells will eventually come out in the blood and body fluids, and the cancer cells that have come out in the blood are called circulating tumor cells (CTC). Confirmation of cancer treatment effects by examining circulating tumor cells in the blood, prognostic life span, prediction of anticancer drug effects before administration, examination of treatment methods using genetic analysis of cancer cells, etc. Expected.

However, there are problems that circulating tumor cells in blood are very few (several to several hundreds / 1 mL of blood) and it is difficult to capture cancer cells.

For example, a technique called the Cell Search system is known as a technique for capturing circulating tumor cells in the blood. This technique uses an antigen-antibody reaction (captured with an EpCAM antibody), and thus expresses EpCAM. Only cancer cells can be captured, and the types of cancer cells that can be captured are limited.

Japanese translation of PCT publication No. 2005-523981

An object of the present invention is to solve the above-mentioned problems and to provide a medical test apparatus and a cell test method capable of capturing many cancer cells including cancer cells that do not express EpCAM.

The present invention is a medical examination apparatus having a well portion, wherein a hydrophilic polymer layer is formed on at least a part of the inner surface of the well portion, and fibronectin is adsorbed on the hydrophilic polymer layer. Relates to the device.

（式中、Ｒ^１は水素原子又はメチル基、Ｒ^２はアルキル基を表す。ｍは１〜５、ｎは繰り返し数を表す。） The hydrophilic polymer layer is preferably formed of at least one hydrophilic polymer selected from the group consisting of a polymer represented by the following formula (I) and poly (meth) acryloylmorpholine.

(In the formula, R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkyl group, m represents 1 to 5, and n represents a repeating number.)

（式中、Ｒ^１、Ｒ^２、ｍは前記と同様。） The hydrophilic polymer layer is a copolymer of at least one hydrophilic monomer selected from the group consisting of a compound represented by the following formula (I-1) and (meth) acryloylmorpholine and another monomer. Preferably it is formed.

(Wherein R ¹ , R ² and m are the same as above)

It is preferable that the medical examination apparatus further includes a part on which fibrinogen is adsorbed.

The present invention also relates to a cytological examination method for examining cells in blood or body fluid using the medical examination apparatus described above.
In the cell inspection method, it is preferable that blood or body fluid is brought into contact with a part to which fibrinogen is adsorbed before being introduced into a medical inspection apparatus.
The part on which the fibrinogen is adsorbed is preferably a blood collection syringe, a blood collection tube or a centrifuge tube.
The cell inspection method is preferably a method in which the blood and body fluid are subjected to centrifugation to remove the supernatant and / or sediment before being introduced into the medical inspection apparatus.

According to the present invention, there is provided a medical examination apparatus having a well part, wherein a hydrophilic polymer layer is formed on at least a part of the inner surface of the well part, and fibronectin is adsorbed on the hydrophilic polymer layer. Since it is an inspection apparatus, many cancer cells can be captured, including cancer cells that do not express EpCAM. Therefore, for example, specific cells such as cancer cells can be sufficiently captured from blood and body fluids, and adhesion and adhesion of other proteins and cells can be suppressed at the same time. Therefore, it becomes possible to selectively capture cancer cells and the like.

It is an example of the schematic diagram of the multiwell plate (medical test | inspection apparatus) which has a well part in which the fibronectin adsorption hydrophilic polymer layer was formed in the inner surface.

The present invention is a medical examination apparatus having a well portion, wherein a hydrophilic polymer layer is formed on at least a part of the inner surface of the well portion, and fibronectin is adsorbed on the hydrophilic polymer layer. Device.

In addition to forming a hydrophilic polymer layer on at least a part of the inner surface of the well part, fibronectin is also adsorbed to the hydrophilic polymer layer, so adhesion of specific cells such as cancer cells of the hydrophilic polymer (adsorption) ) Capability can be improved significantly. Therefore, the capturing ability of specific cells such as cancer cells is greatly improved, the capturing ability of platelets and the like is lowered, and there is a selective capturing effect of specific cells that can hardly be exhibited in a state where there are many proteins.

More specifically, tumor cells (cancer cells, etc.) that have appeared in body fluids such as circulating tumor cells in the blood (several to several hundreds / ml of blood 1 mL) are very few and are used for examination. It is important to capture as many tumor cells as possible in the collected body fluid. In the present invention, in addition to the hydrophilic polymer layer, fibronectin that promotes adhesion (adsorption) of tumor cells is further adsorbed on the surface of the hydrophilic polymer layer, so that a body fluid such as blood is brought into contact therewith, More tumor cells and the like in the body fluid are adsorbed and adhered to the hydrophilic polymer layer. Then, by measuring the number of specific cells such as adsorbed tumor cells, the number of specific cells in blood or body fluid can be determined, and confirmation of cancer treatment effects can be expected. In addition, by culturing the captured specific cells and confirming the efficacy of the anticancer agent etc. in the cultured cells, before the administration of the anticancer agent etc., the efficacy of the anticancer agent etc. outside the body. Can also be used to select anticancer drugs.

Hereinafter, an example of a preferred embodiment of the present invention will be described with reference to the drawings.
A medical examination apparatus 1 (multiwell plate 1) shown in FIGS. 1A and 1B is an apparatus used for capturing specific cells such as cancer cells. Wells 11 are arranged in a so-called matrix. Multiwell plate 1. The multiwell plate 1 has a plurality of wells 11 opened in a circular shape. The well 11 is a recess for injecting blood, body fluid, and the like, and can confirm the presence or absence of specific cells in the injected blood or body fluid, measure the number of specific cells, culture specific cells, and confirm and select the effectiveness of drugs.

FIGS. 1A and 1B show a 24-well plate having 24 wells 11 in 4 rows and 6 columns as an example, but the multiwell plate 1 has at least two wells 11 or more. The number of wells 11 is arbitrary. In addition to the 24-well plate, a general-purpose multi-well plate having six, 96, 384 wells 11 may be used.
The well 11 may be a single well 2 (medical examination apparatus 2) (FIG. 1C).

As a constituent material of the multi-well plate 1 and the single well 2, those having high transparency in observation at the time of cell examination are preferable, and acrylic resins such as polymethyl acrylate, polymethyl methacrylate, polyacrylic acid, and polymethacrylic acid ( Polyacrylic resin), cycloolefin resin (polycycloolefin), carbonate resin (polycarbonate), polyester resin such as styrene resin (polystyrene), polyethylene terephthalate (PET), polydimethylsiloxane and glass (borosilicate glass, soda lime glass) Etc.). For coating with a hydrophilic polymer, the constituent material should be highly hydrophilic, and polyacrylic resin or soda-lime glass is preferred.

The well 11 is a non-through hole, and is opened on the surfaces of the multiwell plate 1 and the single well 2. Blood and body fluid are injected into the well 11 from the opening. Further, when the presence of specific cells such as cancer cells is confirmed, it is possible to inject a culture solution for culturing the specific cells.

The diameter R and depth D of the opening of the well 11 are not particularly limited, and R and D of the normal multiwell plate 1 can be adopted. In FIG. 1, the inner surface of the well 11 is substantially perpendicular to the front and back surfaces of the multiwell plate 1 and the single well 2, but the well 11 may have a shape in which the inner surface is inclined and narrows from the opening to the bottom surface. good. Moreover, the shape which an inner side surface inclines and expands from an opening to a bottom face may be sufficient.

In FIG. 1, the well 11 is opened in a circular shape, but the opening shape of the well 11 is arbitrary, and may be opened in an arbitrary shape such as a quadrangle.

As the multi-well plate 1, a plate in which a plurality of wells 11 can be separated can be suitably used. When there are multiple wells, it can be used separately for specific cell count measurement wells and specific cell culture wells. For example, the presence of cancer cells was confirmed after confirming the presence or absence of cancer cells in the measurement wells. In some cases, cancer cells are cultured in a culture well, and the effectiveness of the drug can be confirmed with the cells.

In the multiwell plate 1 (medical examination apparatus 1) and single well 2 (medical examination apparatus 2), a hydrophilic polymer layer is formed on at least a part of the inner surface of the well 11, and the hydrophilic polymer layer Has adsorbed fibronectin. FIG. 1 shows an example in which a hydrophilic polymer layer 21 is formed on a part of the bottom and side surfaces of a well, and fibronectin 31 is further adsorbed on the hydrophilic polymer layer 21.

When blood or body fluid is introduced into the well 11, specific cells such as cancer cells contained therein are adsorbed to the hydrophilic polymer layer 21 to which fibronectin 31 is adsorbed, and adsorption of platelets, erythrocytes and the like is suppressed. Is done. Therefore, specific cells can be adsorbed to the hydrophilic polymer layer 21 by holding for a predetermined time after introduction of blood or body fluid and then washing. Then, by measuring the number of adsorbed specific cells, the number of cancer cells in blood or body fluid can be determined, and confirmation of cancer treatment effects and the like are expected.

The film thickness of the hydrophilic polymer layer 21 (layer formed of a hydrophilic polymer) is preferably 2 to 200 nm, more preferably 20 to 180 nm. By adjusting within the above-mentioned range, it is possible to obtain good protein and low adsorptivity to cells and selective adsorptivity and adhesion to cancer cells.

As the hydrophilic polymer, a hydrophilic polymer can be appropriately selected. Examples thereof include homopolymers and copolymers of one or more hydrophilic monomers, copolymers of one or more hydrophilic monomers and other monomers, and the like. Examples of the homopolymer and copolymer include polyacrylic acid, polyacrylic acid ester, polymethacrylic acid, polymethacrylic acid ester, polyacryloylmorpholine, polymethacryloylmorpholine, polyacrylamide, and polymethacrylamide.

As the hydrophilic monomer, various monomers having a hydrophilic group can be used. Examples of the hydrophilic group include known hydrophilic groups such as an amide group, a sulfuric acid group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group, an amino group, an amide group, and an oxyethylene group.

Specific examples of the hydrophilic monomer include (meth) acrylic acid, (meth) acrylic acid ester, alkoxyalkyl (meth) acrylate such as (methoxyethyl (meth) acrylate), and hydroxyalkyl (meth) acrylate such as hydroxyethyl (meth) acrylate. ) Acrylate), (meth) acrylamide, (meth) acrylamide derivatives having a cyclic group (such as (meth) acryloylmorpholine), and the like.

Other monomers may be appropriately selected within a range that does not inhibit the action and effect of the hydrophilic polymer. For example, aromatic monomers such as styrene, vinyl acetate, N-isopropylacrylamide capable of imparting temperature responsiveness, and the like can be given.

（式中、Ｒ^１は水素原子又はメチル基、Ｒ^２はアルキル基を表す。ｍは１〜５、ｎは繰り返し数を表す。） Among them, the hydrophilic polymer is preferably at least one selected from the group consisting of a polymer represented by the following formula (I) and poly (meth) acryloylmorpholine.

(In the formula, R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkyl group, m represents 1 to 5, and n represents a repeating number.)

The carbon number of the alkyl group of R ² is 1 to 10 preferably 1 to 5 is more preferable. Among these, R ² is particularly preferably a methyl group or an ethyl group. m is preferably 1 to 3. n (the number of repeating units) is preferably 15 to 1,000, and more preferably 30 to 500.

（式中、Ｒ^１、Ｒ^２、ｍは前記と同様。） Further, as a hydrophilic polymer, a copolymer of at least one hydrophilic monomer selected from the group consisting of a compound represented by the following formula (I-1) and (meth) acryloylmorpholine and another monomer is also used. It can be used suitably.

(Wherein R ¹ , R ² and m are the same as above)

The weight average molecular weight (Mw) of the hydrophilic polymer is preferably 4,000 to 150,000, more preferably 5,000 to 100,000, and still more preferably 8,000 to 50,000 from the viewpoint of selective adsorptivity and adhesion to cancer cells. In this specification, Mw is gel permeation chromatography (GPC) (GPC-8000 series, manufactured by Tosoh Corp., detector: differential refractometer, column: TSKGEL SUPERMALTPORE HZ-M manufactured by Tosoh Corp.) Can be determined by standard polystyrene conversion based on the measured value of

From the viewpoint of adsorbing fibronectin 31 on the hydrophilic polymer layer 21, it is preferable to use a solution, dispersion, or the like in which the fibronectin concentration is preferably adjusted to 0.5 to 500 μg / ml, more preferably 1 to 250 μg / ml. Is preferred. By adjusting to a concentration within the above-mentioned range, good adsorption to proteins and cells and selective adsorption / adhesion to cancer cells can be obtained.

The medical examination apparatus of the present invention includes, for example, a multiwell plate 1 or a single well 2 provided with a well 11 having a hydrophilic polymer layer 21 having fibronectin 31 adsorbed on the inner surface thereof in FIG. It can be manufactured by adding other members (parts).

Specifically, in the case of the multiwell plate 1 and the single well 2 in which the hydrophilic polymer layer 21 is formed, (1) a hydrophilic polymer solution / dispersion solution obtained by dissolving and dispersing a hydrophilic polymer in various solvents is added to the well 11 The inner surface of the well 11 by a known method, such as a method of injecting into the inner surface of the well 11 and holding it for a predetermined time, or (2) a method of coating (spraying) the hydrophilic polymer solution / dispersion on the inner surface of the well 11 Alternatively, a multiwell plate or a single well in which a polymer layer formed of a hydrophilic polymer is formed can be produced by coating a hydrophilic polymer solution / dispersion on a part thereof.

Conventionally known materials and methods can be applied to the solvent, the injection method, the coating (spraying) method, and the like.
The retention time of (1) and (2) may be appropriately set depending on the size of the well 11, the type of liquid to be introduced, etc., but is preferably 5 minutes to 10 hours, more preferably 10 minutes to 5 hours, Minutes to 2 hours are more preferable. After the holding, the excess hydrophilic polymer solution / dispersion may be appropriately discharged and dried.

Next, the method for adsorbing fibronectin 31 to the formed hydrophilic polymer layer 21 is not particularly limited, and a known method can be applied. For example, the hydrophilic polymer layer 21 is contacted with a buffer solution of fibronectin 31 (phosphate buffered saline PBS or the like) by a known method, left at a predetermined temperature for a predetermined time, and adsorbed by a method of washing as necessary. Can be made. What is necessary is just to set temperature and time suitably, for example, it can implement at 10-60 degreeC and about 0.1 to 10 hours.

Then, by adding other parts as necessary to the multiwell plate 1 and the single well 2 in which the hydrophilic polymer layer 21 to which the produced fibronectin 31 is adsorbed is formed on a part of the inner surface of the well 11, Medical inspection devices can be manufactured.

In the present invention, it is preferable to use a component on which fibrinogen is adsorbed as another component. By bringing test blood or body fluid into contact with a part to which fibrinogen related to the adhesion of blood cells is adsorbed, the number of blood cells decreases in advance, and the adhesion / adsorption of specific cells such as cancer cells can be improved.

The cell inspection method of the present invention is a cell inspection method for examining cells in blood or body fluid using the above-described medical inspection apparatus. As described above, the medical examination apparatus according to the present invention improves the capturing ability of cancer cells and the like, decreases the capturing ability of platelets and the like, and obtains a selective capturing effect of cancer cells and the like. By examining cells in blood or body fluids using, you can expect the confirmation of the above-mentioned cancer treatment effect, confirmation of the efficacy of anticancer agents outside the body, selection of anticancer agents, etc. .

From the viewpoint of reducing the number of blood cells and improving the adhesion / adsorption properties of cancer cells and the like, the cell inspection method may be performed by introducing blood or body fluid before introduction (injection, dropping, etc.) into a medical examination apparatus. A method of contacting a part on which fibrinogen is adsorbed is preferable. For example, by using a blood collection syringe, a blood collection tube, or a centrifuge tube as a part to which fibrinogen is adsorbed, the above-described effect can be obtained remarkably.

In addition, from the viewpoint of reducing the number of blood cells and improving the adhesion / adsorption properties of cancer cells and the like, the cell inspection method may be performed before introducing (injection, dripping, etc.) into a medical inspection apparatus. A method of centrifuging body fluid to remove supernatant and / or sediment (sediment) is preferred.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited only to these.

Example 1
Using AIBN (azobisisobutyronitrile), 2-methoxyethyl acrylate was thermally polymerized at 80 ° C. for 6 hours to prepare poly-2-methoxyethyl acrylate (molecular weight Mn: about 15000, Mw: about 50000). And the 2.5 W / V% methanol solution of the obtained poly 2-methoxyethyl acrylate was produced.
Inject the prepared poly-2-methoxyethyl acrylate solution (2.5 W / V%) into a commercially available PMMA plate, leave it at room temperature for 30 minutes, and then suck out the solution with a pipette and dry it. Was made.
Furthermore, fibronectin was adsorbed on the portion (hydrophilic polymer layer) coated with poly-2-methoxyethyl acrylate. Specifically, 1 μl / ml of fibronectin in PBS (phosphate buffered saline) is prepared, left at 40 ° C. for 1 hour, and washed with PBS solution to form the fibronectin-adsorbing hydrophilic polymer layer in FIG. An inspection apparatus having a prepared multi-well plate was produced.

(Example 2)
An inspection apparatus was prepared in the same manner as in Example 1 except that the PBS solution was changed to 10 μl / ml.

(Example 3)
An inspection device was prepared in the same manner as in Example 1 except that the PBS solution was changed to 100 μl / ml.

Example 4
Separately, the inner surface of the pipette was coated with a 10 μl / ml solution of fibrinogen, washed with a PBS solution, and then a pipette (a blood collection tube to which fibrinogen was adsorbed) that had been sufficiently drained was prepared. An inspection device was prepared.

(Example 5)
An inspection device was prepared in the same manner as in Example 1 except that the PBS solution was changed to 200 μl / ml.

(Comparative Example 1)
An inspection apparatus was produced in the same manner as in Example 1 except that only the poly-2-methoxyethyl acrylate layer was formed.

The medical examination apparatus produced by the Example and the comparative example was evaluated with the following method. In Example 4, the evaluation was made in the same manner except that blood was sucked with a prepared pipette in advance and held for 10 minutes and then injected into the well.

[Film thickness of hydrophilic polymer layer (coating layer)]
The film thickness of the hydrophilic polymer layer on the inner surface of the well was measured (photographed) using TEM at an acceleration voltage of 15 kV and 1000 times.

[Platelet adsorption]
A well was mixed with plasma and mixed with platelets (seed density) adjusted to 4 × 10 ⁷ cells / cm ² and allowed to stand at 37 ° C. for 1 hour. The interior was washed with phosphate buffered saline and fixed with 1% glutaraldehyde (left at 37 ° C. for 2 hours). Then, it was washed again with phosphate buffered saline and distilled water.
This sample was observed by SEM, and the number of adsorbed platelets was counted. In addition, the number of the comparative example 1 was set to 1.0 and it compared by the relative value.

[Counting the number of cancer cells]
Fibrosarcoma (HT-1080) is suspended using a stripping solution, a part is made turbid in PBS solution, the number of cells is counted with a hemocytometer, and this value is used to add fibrosarcoma (HT The stripping solution containing -1080) was made turbid. At this time, the blood was turbid so that the seeding density (concentration) was calculated to 2850 cells / cm ² .
Using this blood, 1 ml was injected into each well and allowed to adhere at 37 ° C. for 1 hour. Thereafter, unadhered cells were washed with a PBS solution. Subsequently, immunostaining was performed, and the number of cancer cells adhered with a fluorescence microscope was counted. In addition, the number of adhesion of the comparative example 1 was set to 1.0, and it compared by the relative value.

[Spike blood test with cancer cells added to whole blood]
Stained human colon cancer-derived epithelial cells (HT-29) were suspended in whole blood at 100 cells / ml of blood (spike blood). This was diluted with an equal volume of liquid medium (diluted spike blood). Next, a mononuclear cell solution (LymphoPrep: density 1.077 ± 0.001 g / mL) is put into a 15 mL centrifuge tube, and the above-described diluted spiked blood is added thereto, followed by centrifugation at 800 g for 20 minutes. Separation was performed. And the mononuclear cell layer was separated. A phosphate buffer (PBS) solution was added to the separated mononuclear cell layer, and centrifugation was performed again for concentration. The clot formed in the lowermost layer after centrifugation was suspended in a liquid medium supplemented with 10% FBS (fetal bovine serum) (the same volume as the initial whole blood volume). Using this suspension, 1 ml was injected into each well and allowed to adhere at 37 ° C. for 1 hour. Thereafter, unadhered cells were washed with a PBS solution. Subsequently, the number of cancer cells adhered with a fluorescence microscope was counted. In addition, the adhesion number of the comparative example 1 was set to 1.00, and it compared by the relative value.

In Examples 1 to 3 in which the hydrophilic polymer layer to which fibronectin was adsorbed was formed, the adhesion amount of cancer cells was significantly improved as compared with Comparative Example 1 in which only the hydrophilic polymer layer was formed. This was presumed that cancer cells preferentially adhered because the protein fibronectin related to cancer cell adhesion was adsorbed onto the poly-2-methoxyethyl acrylate layer.

In Example 4 in which blood was previously brought into contact with a container (inner pipette surface) on which fibrinogen was adsorbed on the surface, the amount of adhesion was further improved. This is because blood was injected into a pipette that had previously adsorbed fibrinogen related to the adhesion of blood cells to the inner surface of the container before dropping into the well, so the number of blood cells was reduced and the adhesion of cancer cells was further improved It was guessed.

Further, in the Examples, while the amount of adsorbed platelets was small, the amount of cancer cells adhered was large, and the selectivity (cancer cell adhesion amount / platelet adsorbed amount) was also good.

According to Table 2, Example 4 in which the adhesion of cancer cells in Table 1 is good, and Example 5 in which the fibronectin concentration is increased are the same in the spike blood test in which cancer cells are added to whole blood. Excellent adhesion.

1 Medical testing equipment (multiwell plate)
2 Medical testing equipment (single well)
11 Well 21 Hydrophilic polymer layer 31 Fibronectin

Claims (7)

A medical examination apparatus having a well part,
A hydrophilic polymer layer is formed on at least a part of the inner surface of the well portion, and fibronectin is adsorbed on the hydrophilic polymer layer;
The hydrophilic polymer layer, the following formula (I) represented by polymers and poly (meth) acrylate of at least one hydrophilic polymer formed which do that physicians Ryoyo testing device is selected from the group consisting of acryloyl morpholine.

(In the formula, R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkyl group, m represents 1 to 5, and n represents a repeating number.) A medical examination apparatus having a well part,
A hydrophilic polymer layer is formed on at least a part of the inner surface of the well portion, and fibronectin is adsorbed on the hydrophilic polymer layer;
The hydrophilic polymer layer is a copolymer of at least one hydrophilic monomer selected from the group consisting of a compound represented by the following formula (I-1) and (meth) acryloylmorpholine and another monomer. formed have that doctor Ryoyo inspection apparatus.

(Wherein R ¹ , R ² and m are the same as above) A medical examination apparatus having a well part,
A hydrophilic polymer layer is formed on at least a part of the inner surface of the well portion, and fibronectin is adsorbed on the hydrophilic polymer layer;
Further, that having a fibrinogen is adsorbed components Medical Ryoyo inspection apparatus. Using medical examination apparatus according to any one of claims 1 to 3 cell inspection method of examining cells of the blood or body fluid. The cell inspection method according to claim 4 , wherein blood or body fluid is brought into contact with a part on which fibrinogen is adsorbed before being introduced into the medical inspection apparatus. 6. The cytological test method according to claim 5 , wherein the part to which fibrinogen is adsorbed is a blood collection syringe, a blood collection tube or a centrifuge tube. The cell inspection method according to any one of claims 4 to 6 , wherein the supernatant and / or sediment is removed by centrifuging blood or body fluid before introduction into the medical inspection apparatus.

Images