JP2020115787A - Cell culture apparatus and cell culture method - Google Patents

Abstract

To provide a cell culture apparatus and a cell culture method in which many cancer cells including a cancer cell that does not express EpCAM can be captured and cultured.SOLUTION: A cell culture apparatus in which a hydrophilic polymer layer is formed at least on a part of a surface of the apparatus, and fibronectin is adsorbed on the hydrophilic polymer layer. The hydrophilic polymer layer is formed of at least one type of hydrophilic polymer selected from a group consisting of [a], [b] and [c] below. [a] polymer having a specific structure, [b] poly(meth)acryloyl morpholine, and [c] a copolymer of at least one type of hydrophilic monomer selected from a group consisting of a compound having another specific structure (meth)acryloyl morpholine and another monomer.SELECTED DRAWING: None

Description

The present invention relates to a cell culture device and a cell culture method capable of capturing and culturing specific cells in blood and body fluid (such as cancer cells present in blood and body fluid).

If specific cells such as cancer cells can be captured and cultured, it is expected to be useful for elucidating the mechanism of diseases, developing new drugs based on them, and examining treatment methods. In particular, it is known that when cancer cells occur, they will soon come out in the blood/body fluid, and the cancer cells that come out in the blood are called circulating tumor cells in the blood (CTC). .. And, confirming the therapeutic effect of cancer by examining this circulating tumor cell in the blood, prognosis lifespan, predicting the effect of anticancer drug before administration, examining the therapeutic method using gene analysis of cancer cells, etc. Is expected.

However, circulating tumor cells in the blood are very small in number (several to several hundreds per 1 mL of blood), and there is a problem that it is difficult to capture cancer cells. There is also a problem that it is difficult to culture and increase the captured cells.

For example, as a technique for capturing circulating tumor cells in the blood, a technique called Cell Search system is known. Since this is a technique that uses an antigen-antibody reaction (captured by EpCAM antibody), EpCAM is expressed. Only cancer cells can be captured, the types of cancer cells that can be captured are limited, and the culturing of the captured cancer cells is also limited (see Patent Document 1, etc.).

Japanese Patent Publication No. 2005-523981

It is an object of the present invention to solve the above problems and provide a cell culture device and a cell culture method capable of capturing and culturing many cancer cells, including cancer cells that do not express EpCAM.

（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。）
〔ｂ〕ポリ（メタ）アクリロイルモルホリン
〔ｃ〕下記式（Ｉ−１）で表される化合物及び（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性モノマーと、他のモノマーとの共重合体

（式中、Ｒ^１、Ｒ^２、ｍは前記と同様。） The present invention is a cell culture device in which a hydrophilic polymer layer is formed on at least a part of the device surface, and fibronectin is adsorbed to the hydrophilic polymer layer, wherein the hydrophilic polymer layer has the following [a]: , [B] and [c], a cell culture device formed of at least one hydrophilic polymer selected from the group consisting of:
[A] Polymer represented by the following formula (I)

(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 the number of repetitions.)
[B] poly(meth)acryloylmorpholine [c] 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: Copolymer of

(In the formula, R ¹ , R ² and m are the same as above.)

In the cell culture device, the hydrophilic polymer layer preferably has a thickness of 10 to 800 nm.
The cell culture device is preferably a device for culturing cells contained in blood or body fluid.

（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。）
〔ｂ〕ポリ（メタ）アクリロイルモルホリン
〔ｃ〕下記式（Ｉ−１）で表される化合物及び（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性モノマーと、他のモノマーとの共重合体

（式中、Ｒ^１、Ｒ^２、ｍは前記と同様。） The present invention is also a cell culturing method in which a sample containing cells is brought into contact with a hydrophilic polymer layer on which fibronectin is adsorbed to capture and culture the cells, wherein the hydrophilic polymer layer comprises the following [a]: , [B] and [c], a cell culture method formed of at least one hydrophilic polymer selected from the group consisting of:
[A] Polymer represented by the following formula (I)

(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 the number of repetitions.)
[B] poly(meth)acryloylmorpholine [c] 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: Copolymer of

(In the formula, R ¹ , R ² and m are the same as above.)

In the cell culture method, it is preferable that the sample is brought into contact with the hydrophilic polymer layer to adhere and/or adsorb the specific cells, and then the specific cells are cultured.

In the cell culture method, the sample is preferably at least one selected from the group consisting of the following (1), (2) and (3).
(1) Blood or body fluid (2) Sample obtained by separating and/or concentrating specific cells in blood or body fluid from blood or body fluid (3) Separation and/or concentration of specific cells in blood or body fluid from blood or body fluid And suspended sample

The separation and/or concentration is preferably performed by cell size separation and/or centrifugation.
It is preferable to separate and/or concentrate the specific cells by the centrifugation using a separation liquid.

It is preferable to mix a hemolytic agent with the sample during the separation and/or concentration.
It is preferable that the blood cells in the sample are aggregated during the separation and/or the concentration.

In the cell culture method, it is preferable that a liquid medium is used for the culture and the medium is exchanged at least once every four days.

According to the invention, a hydrophilic polymer layer is formed on at least a part of the device surface, and a cell culture device in which fibronectin is adsorbed to the hydrophilic polymer layer, wherein the hydrophilic polymer layer is Since it is a cell culture device formed of at least one hydrophilic polymer selected from the group consisting of a], [b] and [c], many cells including cancer cells not expressing EpCAM can be obtained. Can capture and culture cancer cells.

FIG. 1 is an example of a schematic view of a cell culture device having a fibronectin-adsorbing hydrophilic polymer layer formed on the inner surface.

The present invention is a cell culture device in which a hydrophilic polymer layer is formed on at least a part of the surface of the device, and fibronectin is adsorbed to the hydrophilic polymer layer, wherein the hydrophilic polymer layer comprises the above [a]. , [B] and [c], a cell culture device formed of at least one hydrophilic polymer selected from the group consisting of:

Not only is a hydrophilic polymer layer formed on at least a part of the surface of the cell culture device, but fibronectin is also adsorbed on the hydrophilic polymer layer, so that hydrophilic polymer cells (specific cells such as cancer cells, etc. It is possible to remarkably improve the adhesion (adsorption) ability of (1). Therefore, the ability to capture specific cells such as cancer cells is greatly improved, the ability to capture platelets and the like is lowered, and a selective capturing effect of specific cells that cannot be exerted at all in a protein-rich state is exerted. Further, not only the hydrophilic polymer layer is formed on at least a part of the surface of the cell culture device, but fibronectin is also adsorbed on the hydrophilic polymer layer, so that the trapped cells (specific cells etc.) are preferably cultured. it can.

Specifically, the number of tumor cells (cancer cells, etc.) that have appeared in body fluids such as circulating tumor cells in the blood (several to several hundreds/1 mL of blood) is very small, and are suitable for testing. It is important to capture as much as possible the tumor cells present in the collected body fluid. In the present invention, in addition to the hydrophilic polymer layer, since fibronectin that promotes adhesion (adsorption) of tumor cells is further adsorbed on the surface of the hydrophilic polymer layer, by contacting it with a body fluid such as blood, Tumor cells and the like in body fluid are more adsorbed and adhered to the hydrophilic polymer layer. The presence of fibronectin promotes the differentiation of the adsorbed/adhered tumor cells and promotes the culture. Then, by confirming the effect of the anticancer drug or the like on the cultured cells, it is possible to confirm the effect of the anticancer drug or the like outside the body before administration of the anticancer drug or the like, and at the same time, It is also useful for selecting drugs. Furthermore, the cultured cells can be used to elucidate the mechanism of cancer cell growth and to develop new drugs (anticancer drugs, molecular target drugs, etc.).

Hereinafter, an example of a preferred embodiment of the present invention will be described with reference to the drawings.
The cell culture device 1 of FIG. 1 is a device used for the purpose of capturing and culturing cells. Examples of the cells include specific cells such as cancer cells (arbitrary cancer cells including cancer cells that do not express EpCAM). Examples of the cancer cells include circulating tumor cells in blood (CTC).

It has a shape for injecting a solution containing cells into well-shaped recesses to capture the cells and then culturing the cells. The well 11 may be a single well or a multiwell (plate) in which wells are arranged in a matrix. The shape of the well portion is not particularly limited, and examples thereof include depressions (recesses) having a shape such as a circle, an ellipse, and a polygon such as a quadrangle. Further, the lateral wall of the well portion may be structured so as to be separated from the bottom portion. The well can be used for confirmation (observation) of the presence or absence of cells (specific cells, etc.), measurement of the number of cells (specific cell numbers, etc.), culturing cells (specific cells, etc.), confirmation/selection of drug efficacy, etc. In addition, it may be easier to observe, measure, confirm and select when the lateral wall of the well portion is removed, and it is easier to take out the cultured cells and the like.

As a constituent material of the well 11, a material having high transparency in observing cells is preferable, and acrylic resin (polyacrylic resin) such as polymethyl acrylate, polymethyl methacrylate, polyacrylic acid, polymethacrylic acid, cycloolefin resin. (Polycycloolefin), carbonate resin (polycarbonate), styrene resin (polystyrene), polyester resin such as polyethylene terephthalate (PET), polydimethylsiloxane and glass (borosilicate glass, soda lime glass, etc.) and the like. For the coating of the hydrophilic polymer, the constituent material preferably has high hydrophilicity, and polyacrylic resin or soda lime glass is preferable.

The well 11 is a non-through hole, and the upper portion is opened. A solution containing cells (sample (blood containing specific cells, body fluid, etc.)) is injected into the well 11 through 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 length L (each side when the opening is rectangular), the diameter R (diameter when the opening is circular), and the depth D of the opening of the well 11 are not particularly limited. From the viewpoint of containing a solution (sample), the depth D (height) is preferably 3 to 25 mm. In FIG. 1, the inner side surface of the well 11 is substantially perpendicular to the front and back surfaces of the well 11, but the well 11 may have a shape in which the inner side surface is inclined and narrows from the opening to the bottom surface. Further, the inner side surface may be inclined so that it extends from the opening to the bottom surface. Further, the bottom may be flat or uneven. Concave dents tend to collect cells, which is advantageous for culture. This is because a plurality of clumps give some signals to promote the culture, rather than a single cell.

In the case of a multi-well, a plurality of wells may be separable. If you have multiple wells, you can separate and use them for cell number (specific cell number etc.) measurement wells and cell (specific cell etc.) culture wells. For example, check the presence or absence of cancer cells in the measurement wells. Then, if the presence is confirmed, the cancer cells are cultured in a culture well, and the efficacy of the drug can be confirmed in the cells.

In the cell culture device 1, the hydrophilic polymer layer 21 is formed on at least a part of the surface of the device (the surface of the well 11), and the hydrophilic polymer layer 21 is adsorbed with the fibronectin 31. In the case of the well 11, the surface of the device is preferably the inner surface thereof. FIG. 1 shows an example in which a hydrophilic polymer layer 21 is formed on a part of the bottom surface and the side surface of the well 11, and fibronectin 31 is 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 and adhered to the hydrophilic polymer layer 21 having the fibronectin 31 adsorbed thereto, and blood cells such as platelets Adsorption is suppressed. Therefore, the specific cells can be adsorbed and adhered to the hydrophilic polymer layer 21 by holding for a predetermined time after the introduction of blood or body fluid and then washing. Then, by measuring the number of adsorbed/adhered specific cells, the number of cancer cells in blood or body fluid can be known. Furthermore, since the specific cells are adsorbed and adhered to the hydrophilic polymer layer on which fibronectin is also adsorbed, the specific cells can be efficiently cultured. Therefore, the use of the cultured specific cells is expected to confirm the cancer treatment effect.

The film thickness of the hydrophilic polymer layer 21 (layer formed of a hydrophilic polymer) is preferably 10 to 800 nm, more preferably 30 to 550 nm. By adjusting the amount within the above range, good low adsorptivity for proteins and cells, and selective adsorptivity/adhesiveness for cancer cells can be obtained. Moreover, cancer cells can be efficiently cultured.

（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。）
〔ｂ〕ポリ（メタ）アクリロイルモルホリン
〔ｃ〕下記式（Ｉ−１）で表される化合物及び（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性モノマーと、他のモノマーとの共重合体

（式中、Ｒ^１、Ｒ^２、ｍは前記と同様。） The hydrophilic polymer forming the hydrophilic polymer layer 21 is at least one selected from the group consisting of the following [a], [b] and [c].
[A] Polymer represented by the following formula (I)

(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 the number of repetitions.)
[B] poly(meth)acryloylmorpholine [c] 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: Copolymer of

(In the formula, R ¹ , R ² and m are the same as above.)

In formulas (I) and (I-1), the carbon number of the alkyl group of R ² is preferably 1 to 10, and more preferably 1 to 5. Of these, R ² is particularly preferably a methyl group or an ethyl group. 1-3 are preferable as m. 15-1000 are preferable and, as for n (number of repeating units), 30-500 are more preferable.

In the copolymer [c], the other monomer may be appropriately selected within a range that does not impair the action and effect of the hydrophilic polymer. Examples thereof include aromatic monomers such as styrene, vinyl acetate, and N-isopropylacrylamide capable of imparting temperature response.

The number average molecular weight (Mn) of the hydrophilic polymer is preferably 8000 to 150,000, more preferably 10,000 to 60,000, and further preferably from the viewpoint of selective adsorption/adhesiveness to cancer cells and culturing properties of cancer cells. It is 12,000 to 50,000. In the present specification, Mn means gel permeation chromatography (GPC) (GPC-8000 series manufactured by Tosoh Corporation, detector: differential refractometer, column: TSKGEL SUPERMALPTORE HZ-M manufactured by Tosoh Corporation). It can be determined by standard polystyrene conversion based on the measured value by.

From the viewpoint of adsorbing the fibronectin 31 on the hydrophilic polymer layer 21, it is preferable to use a solution or dispersion in which the fibronectin concentration is adjusted to 0.5 to 500 μg/ml, more preferably 1 to 250 μg/ml. It is suitable. By adjusting the concentration within the above range, good low adsorptivity for proteins and cells, selective adsorptivity/adhesiveness for cancer cells, and cultivability of cancer cells can be obtained.

The cell culture device 1 includes, for example, a well 11 in which a hydrophilic polymer layer 21 having a fibronectin 31 adsorbed on the inner surface of FIG. 1 is formed, a multi-well plate including a plurality of wells 11, and, if necessary, other wells. It can be manufactured by adding members (parts).

Specifically, in the case of a device having a well 11 in which a hydrophilic polymer layer 21 is formed, (1) a hydrophilic polymer solution/dispersion liquid in which a hydrophilic polymer is dissolved/dispersed in various solvents is placed inside the well 11. The whole or a part of the inner surface of the well 11 is prepared by a known method such as a method of injecting and maintaining for a predetermined time, (2) a method of coating (spraying) the hydrophilic polymer solution/dispersion solution on the inner surface of the well 11. By coating the hydrophilic polymer solution/dispersion liquid on the well, the well 11 having the polymer layer formed of the hydrophilic polymer can be manufactured.

Conventionally known materials and methods can be applied to the solvent, the injection method, the coating (spraying) method, and the like.
The holding 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 5 minutes to 10 hours is preferable, 10 minutes to 5 hours is more preferable, and 15 Minutes to 2 hours are more preferable. After the holding, the excess hydrophilic polymer solution/dispersion may be appropriately discharged or may be dried without being discharged.

Next, the method of adsorbing the 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 physiological saline PBS, etc.) by a known method, left at a predetermined temperature for a predetermined time, and washed as necessary. Can be made. The temperature and time may be set as appropriate, and for example, it may be carried out at 10 to 50° C. for about 0.1 to 24 hours.

Then, 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, and if necessary, other components are added to manufacture the cell culture device.

The cell culture method of the present invention is a cell culture method in which a cell-containing sample (such as blood or body fluid containing specific cells) is brought into contact with the hydrophilic polymer layer to capture the cells, and the hydrophilic polymer layer is cultivated. It is a method of forming with at least one hydrophilic polymer selected from the group consisting of the above [a], [b] and [c].

As described above, the cell culture device has an improved ability to capture cancer cells and the like, has a reduced ability to capture blood cells such as platelets, and has a selective capture effect on cancer cells and the like. Cancer cells can be efficiently cultured. Therefore, for example, using the device, blood or body fluid (sample containing cells) is brought into contact with a hydrophilic polymer layer having adsorbed fibronectin to adhere and/or adsorb specific cells such as cancer cells, By culturing the specific cells and examining the cultivated specific cells, confirmation of the above-mentioned cancer treatment effect, confirmation of the efficacy of anticancer agents etc. outside the body, selection of anticancer agents, etc. Expected to elucidate the mechanism of development and disease.

Examples of the sample containing cells include (1) blood or body fluid (blood itself or body fluid itself), (2) sample obtained by separating and/or concentrating blood or body fluid from blood or specific cells in body fluid, (3) blood or A sample obtained by separating and/or concentrating specific cells in the blood or body fluid from the body fluid and further suspending it can be preferably used.

In the above (2) and (3), separation and/or concentration of specific cells such as cancer cells can be carried out by separation/concentration according to cell size, separation/concentration by centrifugation and the like. Examples of separation and concentration according to cell size include a method using a filter and the like.

A known method can be adopted for the centrifugation, and for example, it can be carried out by a known centrifugation device. The centrifugal force in centrifugation, the time and temperature of centrifugation may be appropriately set from the viewpoint of separability of blood cells, cancer cells and the like. By centrifuging the collected blood or body fluid and separating the intermediate mononuclear cell layer, platelets, erythrocytes and polynuclear leukocytes are separated and removed, and a sample with an increased concentration of specific cells such as cancer cells is obtained. Can be made.

It should be noted that before the centrifugation, other treatments for reducing the protein concentration in blood or body fluid may be performed. Other treatment methods for decreasing the protein concentration in blood or body fluid include, for example, a method of diluting the collected blood or body fluid. Here, as a method of dilution, there is a method of using a buffer solution such as human blood pH (about 7.4) phosphate buffered saline (PBS) or a liquid medium such as DMEM (Dulbecco's modified Eagle medium). Specifically, by adding a buffer solution to the collected blood or body fluid to dilute it, or by adding the collected blood or body fluid to a liquid medium and diluting it, the protein concentration can be increased compared to the collected blood or body fluid. It can be reduced.

In centrifugation, it is preferable to separate and/or concentrate specific cells by centrifugation using a separation liquid. Thereby, it is possible to preferably separate into a mononuclear cell layer containing specific cells (cancer cells and the like), a layer containing red blood cells and the like. Then, the mononuclear cell layer is separated, the specific cells in the layer are captured by the hydrophilic polymer layer having adsorbed fibronectin, and then the cells are cultured, whereby the specific cells can be suitably cultured.

The separation liquid used in the density gradient centrifugation method may be one prepared so as to have a specific gravity suitable for fractionating cells in blood, and an osmotic pressure and a pH that do not destroy cells. As the medium, a medium that can be used in the density gradient centrifugation method can be used. The separated liquid preferably has a specific gravity at 20° C. of 1.076 to 1.078 g/ml. The pH of the separated liquid is preferably 4.5 to 7.5.

Representative media (separation liquids) include sucrose, ficoll (copolymer of sucrose and epichlorohydrin), and percoll (colloidal silica product having a polyvinylpyrrolidone coating). Ficoll is Ficoll-Paque PLUS (Pharmacia Biotech Co., Ltd.), Histopaque-1077 (Sigma Aldrich Japan Co., Ltd.), Lymphoprep (Lymphoprep) (Nikomed, Oslo (Norway)), etc.; Percoll is Percoll (Sigma Aldrich). Japan Co., Ltd.; products are commercially available.

In the above (3), for the suspension treatment, a method using a liquid medium or the like can be adopted.

In the above (2) and (3), it is preferable to mix (add) a hemolytic agent to the sample at the time of separating and/or concentrating the specific cells, since at least a part of the red blood cells in the sample can be removed. A hemolytic agent is a reagent that physically or chemically acts on red blood cells to lyse them. As the hemolytic agent, those conventionally used can be used, and examples thereof include ammonium chloride, synthetic surfactants and alcohols.

In the above (2) and (3), it is preferable that the blood cells are aggregated in the sample when the specific cells are separated and/or concentrated. The method for aggregating blood cells is not particularly limited as long as it is a method capable of such aggregation, and among them, a method utilizing an antigen-antibody reaction can be preferably used. Specifically, an agglutination reaction method such as a hemagglutination reaction can be preferably used.

In such an aggregating step, when cells are aggregated in blood or body fluid by utilizing a hemagglutination reaction, when the sample containing the produced aggregate is then subjected to a centrifugal separation treatment, the agglutinates containing the blood cells are aggregated. Things are removed. Therefore, specific cells (such as cancer cells) remaining at a high concentration in the sample can be effectively captured by the hydrophilic polymer layer.

For aggregation of blood cells, for example, a method of mixing (adding) a compound that aggregates blood cells can be adopted. Specifically, a red blood cell and white blood cell agglutinating antibody reagent (a red blood cell and a white blood cell agglutinating antibody composition) can be used. During centrifugation, the separation of some leukocytes whose specific gravity is close to that of specific cells such as cancer cells becomes poor. However, when the antigen-antibody reaction is used using the above antibody composition, erythrocytes and leukocytes are bound and aggregated. Also, not only erythrocytes and platelets having a specific gravity different from that of the specific cells, but also the separation of the white blood cells and the specific cells is improved, and the adhesion/capturing property of the specific cells is improved.

In the cell culture method, a method of contacting a sample containing cells with a hydrophilic polymer layer on which fibronectin is adsorbed to capture the cells is any method capable of contacting the sample and the hydrophilic polymer layer. it can. For example, when an apparatus having a well in which a hydrophilic polymer layer is formed is used, a method of injecting a sample into the well may be mentioned. After contact (after injection), cells can be trapped (adsorbed or adhered) to the hydrophilic polymer layer by holding the cells under predetermined conditions. The predetermined condition may be set appropriately, and for example, it may be held at 34 to 40° C. for 10 minutes to 15 hours.

In the cell culture method, a method of culturing the cells may be appropriately adopted as a method of culturing the captured cells. For example, cells can be suitably cultured by adding a liquid medium to the fibronectin-adsorbing hydrophilic polymer layer in which cells such as specific cells are trapped and maintaining it under predetermined conditions. The predetermined condition may be set appropriately, and for example, it may be held at 34 to 40° C. for 1 to 10 weeks.

In the cell culturing method, it is preferable to use a liquid medium for culturing and exchange the medium once or more every four days. Thereby, it is possible to preferably carry out the culturing of cells such as specific cells.

The present invention will be specifically described based on Examples, but the present invention is not limited thereto.

(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 15,000, Mw: about 50,000). Then, a 2.5 W/V% methanol solution of the obtained poly-2-methoxyethyl acrylate was prepared.
The prepared poly-2-methoxyethyl acrylate solution (2.5 W/V%) was injected into a commercially available PMMA plate (depth D (height) 17.5 mm), left at room temperature for 30 minutes, and then the solution was pipetted. Then, the hydrophilic polymer layer was prepared by sucking it out and drying it.
Furthermore, fibronectin was adsorbed to the portion (hydrophilic polymer layer) coated with poly 2-methoxyethyl acrylate. Specifically, 100 μg/ml of fibronectin PBS solution (phosphate buffered saline) was prepared, left at 40° C. for 1 hour, and then washed with PBS solution to obtain a fibronectin-adsorbed hydrophilic polymer layer as shown in FIG. A cell culture device having a multi-well plate in which cells were formed was produced.

(Example 2)
A cell culture device was produced in the same manner as in Example 1 except that poly-2-methoxyethyl acrylate (molecular weight Mn: about 30,000, Mw: about 95,000) was used.

(Example 3)
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 15,000, Mw: about 50,000). Then, a 0.25 W/V% methanol solution of the obtained poly-2-methoxyethyl acrylate was prepared.
A commercially available chamber slide (2 chamber type, a type in which the chamber on the side of the well part can be removed and the bottom surface is a glass substrate (soda lime glass), depth D (height) 12 mm), the prepared poly 2-methoxyethyl acrylate solution ( 0.25 W/V%) was injected, and the mixture was allowed to stand at room temperature for 5 minutes and then vacuum dried at 40° C. to prepare a hydrophilic polymer layer.
Furthermore, fibronectin was adsorbed to the portion (hydrophilic polymer layer) coated with poly 2-methoxyethyl acrylate. Specifically, 200 μg/ml of a PBS solution (phosphate buffered saline) of fibronectin was prepared, left at 40° C. for 1 hour, and then washed with the PBS solution to obtain a fibronectin-adsorbed hydrophilic polymer layer as shown in FIG. A cell culture device in which the cells were formed was produced.

(Example 4)
A cell culture device was produced in the same manner as in Example 3 except that poly-2-methoxyethyl acrylate (molecular weight Mn: about 30,000, Mw: about 95,000) was used.

(Comparative Example 1)
A commercially available chamber slide (2 chamber type, a type in which the chamber on the side of the well can be removed and the bottom surface is a glass substrate (soda lime glass)) was used.

(Comparative example 2)
Fibronectin was adsorbed on a commercially available chamber slide (two-chamber type, a type in which the chamber on the side of the well can be removed and the bottom surface was a glass substrate (soda lime glass)). Specifically, a cell culture device in which a fibronectin adsorption layer was formed by preparing 200 μg/ml of a fibronectin PBS solution (phosphate buffered saline), leaving it at 40° C. for 1 hour, and then washing with a PBS solution It was made.

The cell culture devices produced in Examples and Comparative Examples were evaluated by the following methods.

[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 a TEM at an acceleration voltage of 15 kV and 1000 times.

[Spike blood test in which cancer cells are added to whole blood: measurement of the number of adherent cells]
The stained human colon cancer-derived epithelial cells (HT-29) were suspended in whole blood at 100 cells/blood (1 mL) (spike blood). An equal amount of liquid medium was added to this and diluted (diluted spiked blood). Next, the separated liquid (LymphoPrep: density 1.077±0.001 g/mL) was put into a 15 mL centrifuge tube, the diluted spiked blood was put thereon, and centrifugation was carried out under the condition of 800 g for 20 minutes. .. Then, the mononuclear cell layer was separated. A phosphate buffer (PBS) solution was added to the separated mononuclear cell layer, centrifugation was performed again, and concentration was performed. The clot formed in the lowermost layer after centrifugation was suspended in a liquid medium containing 10% FBS (fetal bovine serum) (the same amount as the initial whole blood amount). Using this suspension, 1 ml was injected into each well, and adhered at 37° C. for 1 hour. Then, the PBS solution was used to wash the non-adherent cells. Then, the number of adhered cancer cells was counted with a fluorescence microscope.
In addition, the cancer cell adhesion number of Comparative Example 1 was set to 1.00 and compared by the relative value.

[Spike blood test in which cancer cells are added to whole blood: measurement of number of cultured cells]
Human colon cancer-derived epithelial cells (HT-29) (unstained) were suspended in whole blood at 100 cells/1 mL of blood (spike blood). An equal amount of liquid medium was added to this and diluted (diluted spiked blood). Next, the separated liquid (LymphoPrep: density 1.077±0.001 g/mL) was put into a 15 mL centrifuge tube, the diluted spiked blood was put thereon, and centrifugation was carried out under the condition of 800 g for 20 minutes. .. Then, the mononuclear cell layer was separated. A phosphate buffer (PBS) solution was added to the separated mononuclear cell layer, centrifugation was performed again, and concentration was performed. The clot formed in the lowermost layer after centrifugation was suspended in a liquid medium containing 10% FBS (fetal bovine serum) (the same amount as the initial whole blood amount). Using this suspension, 1 ml was injected into each well, and adhered at 37° C. for 1 hour. Then, the PBS solution was used to wash the non-adherent cells. Then, a liquid medium was added and the cells were cultured at 37°C for 3 weeks. Also, the liquid medium was changed once every two days during the culture. After the culture was completed, the cells were detached with trypsin, and the cells were counted with a hemocytometer.
In addition, the number of cultured cancer cells increased in Comparative Example 1 was set to 1.00, and comparison was made by relative values.

In Examples 1 to 4 in which the hydrophilic polymer layer having fibronectin adsorbed was formed, the adhesion amount of cancer cells was significantly improved as compared with Comparative Example 1. It was speculated that the cancer cells were preferentially adhered because the fibronectin, which is a protein associated with adhesion of cancer cells, was adsorbed on the poly-2-methoxyethyl acrylate layer.

In Examples 1 to 4 in which the hydrophilic polymer layer adsorbing fibronectin was formed, the number of cells increased in the culture of cancer cells was significantly improved as compared with Comparative Example 1. This was presumed to be because the culture of cancer cells was promoted because fibronectin, a protein associated with the adhesion and culture promotion of cancer cells, was adsorbed on the poly-2-methoxyethyl acrylate layer.

1 Cell Culture Device 11 Well 21 Hydrophilic Polymer Layer 31 Fibronectin

Claims (11)

A cell culture device in which a hydrophilic polymer layer is formed on at least a part of the device surface, and fibronectin is adsorbed to the hydrophilic polymer layer,
The cell culture device in which the hydrophilic polymer layer is formed of at least one hydrophilic polymer selected from the group consisting of the following [a], [b] and [c].
[A] Polymer represented by the following formula (I)

(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 the number of repetitions.)
[B] poly(meth)acryloylmorpholine [c] 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: Copolymer of

(In the formula, R ¹ , R ² and m are the same as above.) The cell culture device according to claim 1, wherein the hydrophilic polymer layer has a thickness of 10 to 800 nm. The cell culture device according to claim 1 or 2, which is a device for culturing cells contained in blood or body fluid. A method for culturing cells, comprising contacting a sample containing cells with a hydrophilic polymer layer having fibronectin adsorbed thereon to capture the cells, and culturing the cells.
The cell culture method, wherein the hydrophilic polymer layer is formed of at least one hydrophilic polymer selected from the group consisting of the following [a], [b] and [c].
[A] Polymer represented by the following formula (I)

(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 the number of repetitions.)
[B] poly(meth)acryloylmorpholine [c] 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: Copolymer of

(In the formula, R ¹ , R ² and m are the same as above.) The cell culture method according to claim 4, wherein the specific cells are adhered and/or adsorbed by bringing the sample into contact with the hydrophilic polymer layer, and then the specific cells are cultured. The cell culture method according to claim 4 or 5, wherein the sample is at least one selected from the group consisting of the following (1), (2), and (3).
(1) Blood or body fluid (2) Sample obtained by separating and/or concentrating specific cells in blood or body fluid from blood or body fluid (3) Separation and/or concentration of specific cells in blood or body fluid from blood or body fluid And suspended sample The cell culture method according to claim 6, wherein the separation and/or concentration is performed by separation according to cell size and/or centrifugation. The cell culture method according to claim 7, wherein the specific cells are separated and/or concentrated by the centrifugation using a separation liquid. The cell culture method according to claim 6, wherein a hemolytic agent is mixed with the sample during the separation and/or the concentration. The cell culture method according to claim 6, wherein the blood cells in the sample are aggregated during the separation and/or the concentration. The cell culture method according to any one of claims 6 to 10, wherein a liquid medium is used for culturing, and the medium is replaced at least once every four days.

Images