JP2016131561A - Method of recovering cells, and polymer used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that is capable of efficiently bonding and concentrating cancer cells, stem cells or the like circulating in blood or lymph in a body in a floating manner and easily recovers the bonded cells or the like, and polymer preferably used for the method.SOLUTION: A recovery method of target cells includes: a process of bringing an anti-thrombogenic material, which has lower limit critical solution temperature within a range of 0 to 37°C and has cell adhesiveness, into contact with cell suspension in which a target cell is suspended at the temperature of lower limit critical solution temperature of the material or higher, and bonding the target cell on a surface of the material; and a process in which the target cell is suspended in aqueous medium and recovered by keeping the material at the temperature of the lower limit critical solution temperature or lower in the aqueous medium and dissolving the material into the aqueous medium.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for selectively adsorbing and recovering cancer cells, stem cells, and the like, and a polymer used therefor. More specifically, a cell suspension such as blood using a polymer that has an adsorptivity for selectively adsorbing cancer cells and stem cells suspended in an aqueous medium and whose solubility in an aqueous medium changes with temperature. The present invention relates to a method for concentrating and recovering cancer cells or stem cells suspended in a liquid, and a polymer having such properties.

  In advanced cancer that causes metastasis, circulating circulating tumor cells (Circulating Tumor Cells: CTCs) infiltrated into the blood from the primary lesion are present. The clinical significance of detecting and recovering CTCs is great and can be applied to cancer diagnosis and development of new anticancer agents. For example, the therapeutic effect can be determined from early detection of metastatic cancer, prediction of response before treatment, and measurement of the amount of residual tumor after treatment, which is a useful means for determining the progression of cancer pathology. Currently, the usefulness of determination of therapeutic effect and prognosis prediction using CTCs is recognized in metastatic cancer cases such as breast cancer, prostate cancer, and colon cancer.

  However, in the case of a general cancer patient who has started metastasis, there is only about 1 CTCs per 10 billion blood cells in the blood. On the other hand, blood contains an overwhelmingly large amount of blood components such as red blood cells and white blood cells. Therefore, in order to detect extremely low concentrations of CTCs, high sensitivity, high efficiency, and high specific analysis means are required. Therefore, methods of concentrating CTCs by a method using magnetic beads, density gradient centrifugation, micro flow path, and flow cytometer have been tried. However, these methods require complicated processing and a recovery rate. There is a problem that is bad.

  One technique for isolating and identifying CTCs is a method using an antibody against a cell surface marker, and a method using an antibody against an epithelial cell adhesion factor (EpCAM) expressed on the cell surface is approved by the US Food and Drug Administration (FDA). Has been. However, when the detectability of various breast cancer cell subtypes was investigated using this antibody, there was a report that normal cell-like breast cancer cells could not be recognized, and a novel method comprising an antibody capable of specifically recognizing this subtype The necessity of this is pointed out (for example, refer nonpatent literature 1). In addition, since cancer cells such as CTCs are one size larger than blood cells in blood such as red blood cells, white blood cells, or platelets, these blood cell components are removed by mechanical filtration. Cells can be concentrated. In such a method, a technique for separating and concentrating CTCs using a filter made of a novel nanomaterial such as carbon nanotube or nanofiber has also been proposed (see, for example, Non-Patent Document 2). However, even when these nanoscale materials are used, it is difficult to capture CTCs having a small cell size and separation from blood cells is still difficult.

  The present inventor has focused on a polymer capable of forming a special hydration structure from the past, and that the polymer can be hydrated in a form called intermediate water that behaves differently from normal water molecules, and It has been clarified that when the surface of the water-containing polymer is brought into contact with a biological substance, thrombus formation and hemolysis due to activation of the complement system and platelet activation can be prevented and the properties of the antithrombotic polymer are exhibited. (For example, see Patent Document 1). In addition, by optimizing the hydration structure of the antithrombotic polymer and reducing the amount of intermediate water that can be contained, it is possible to compare cancer cells, vascular endothelial cells, stem cells, etc. while preventing thrombus formation and hemolysis It has been clarified that cells having high adhesiveness can be adhered (see, for example, Patent Document 2).

  In addition, by applying a polymer with such a specific hydration structure to the surface of a cancer cell concentration filter, the adhesion of cancer cells to the filter surface is improved and the concentration rate of cancer cells is improved. (For example, refer to Patent Document 3).

WO2004 / 087228 pamphlet JP 2012-105579 A WO2012 / 173097 pamphlet

A M. Sieuwerts et al., "Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells" J Natl Cancer Inst 2009; 101: 61-66 H J. Yoon et al., "Emerging Role of Nanomaterials in Circulating Tumor Cell Isolation and Analysis" ACS Nano, 2014, 8 (3), pp.1995-2017

In the cancer cell concentration filter coated with a predetermined polymer described in Patent Document 3, in order to selectively adhere to high cancer cells and the like while suppressing the adhesion of blood cell components Although it is useful in concentrating and capturing CTCs circulating in the blood, when the captured CTCs are used after culturing, cancer cells adhered to the filter by a method such as exfoliation with trypsin are used. It was necessary to peel and collect. On the other hand, when a method such as exfoliation using trypsin is used, the exfoliated cancer cells are damaged, and thus there is a problem that a proper result cannot always be obtained in various evaluations using CTCs.
Therefore, the present invention is capable of efficiently attaching and concentrating target cells such as cancer cells and stem cells that suspend and circulate in blood or lymph in the body, and easily attach the attached cells and the like. It is an object to provide a method of collecting. It is another object of the present invention to provide a polymer suitably used in the method.

  In order to solve the above-mentioned problems, various polymers exhibiting temperature responsiveness can be used alone or in combination of two or more to select a polymer that is optimal for the separation and recovery of target cells such as cancer cells and stem cells, and the recovery conditions As a result of using the antithrombotic material having a lower critical solution temperature in the range of 0 to 37 ° C. and having cell adhesiveness, the target cells can be captured and the material can be cooled extremely. The present invention was completed by finding that target cells can be easily and efficiently recovered.

  That is, the method for recovering target cells of the present invention has an objective antithrombotic material having a lower critical solution temperature in the range of 0 to 37 ° C. and having cell adhesion at a temperature equal to or higher than the lower critical solution temperature of the material. Contacting the cell suspension in which the cells are suspended to adhere the target cells to the surface of the material; and holding the material in a solution having a temperature equal to or lower than the lower critical solution temperature in the aqueous medium. And the step of suspending and recovering the target cells in the aqueous medium by dissolving the material therein.

  In another aspect of the present invention, the antithrombotic material having cell adhesion that can be used in the above method includes a copolymer containing a repeating unit selected from the structure represented by the following general formula (I). A temperature responsive polymer is provided.

In the general formula, R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is 2 or 3, and n represents the number of repetitions.

In a preferred embodiment of the present invention, in the above formula (I), R ¹ is a hydrogen atom, R ² is an ethyl group, and m is a repeating unit of 2, R ¹ is a methyl group, R ² is a methyl group, and and a temperature-responsive polymer including a repeating unit in which m is 2.

  According to the present invention, target cells such as cancer cells and stem cells can be selectively captured without being disturbed by blood cells in the blood. Since the cell-adhesive and antithrombotic material used in the method of the present invention has a lower critical solution temperature in the range of 0 to 37 ° C., the target cell captured at a temperature equal to or higher than the lower critical solution temperature is used as the material. It is possible to efficiently recover by simply lowering the temperature.

It is the schematic which shows one Embodiment for collect | recovering CTCs in the blood using the cell collection material of this invention. The adhesion / adhesion number of (a) platelets or (b) cancer cells suspended in plasma on a substrate coated with various polymers is shown. The temperature change in the cell culture medium when left in an incubator at 10 ° C. or 15 ° C. is shown. The change of the static contact angle with the water of a board | substrate after leaving the board | substrate which apply | coated various polymers still in the phosphate buffered saline cooled to 10 degreeC or 15 degreeC is shown. The recovery rate of the cancer cell from the board | substrate apply | coated using various polymer solutions is shown. The recovery rate of the cancer cell from the board | substrate which apply | coated various polymers in different temperature and time is shown. The cytotoxicity with respect to the cancer cell at the time of culture | cultivating in the culture medium for cell culture containing a temperature-responsive polymer is shown. The adhesion form of the cell after making blood contact the board | substrate which apply | coated various polymers is shown.

Hereinafter, preferred embodiments will be described with reference to the drawings as the case may be.
When the polymer described in Patent Document 1 has a predetermined hydration structure that can contain water molecules defined as “intermediate water” as shown in Table 1, when it comes into contact with a biological substance, For example, it has been confirmed that biocompatibility is exhibited such that adhesion of blood cell components such as platelets is suppressed. At the same time, there exists a phase transition temperature (lower critical solution temperature (LCST)) determined according to each polymer structure, and in the temperature range below the temperature, the polymer molecule exhibits water solubility, while the phase transition temperature is higher than the phase transition temperature. It has been confirmed that the polymer molecules are insoluble in the temperature range. In addition, the polymers A to F in Table 1 are described in Patent Document 1, and the general formula (I) in the present invention:

In which R ¹ , R ² and m each have a structure shown in the table. Moreover, LCST shown in Table 1 etc. is LCST which each polymer shows in a pure water. Although the reason why the lower critical solution temperature (LCST) exists in the polymer described in Patent Document 1 is not clear, the hydrophobic group contained in the main chain and the side chain, the ethylene glycol unit contained in the side chain, etc. Hydrophobic at a predetermined temperature or higher as a result of a kind of phase transformation in which the hydrophobic group part or the hydrophilic group part is exposed to the outside depending on the temperature due to the balance with the hydrophilic group due to It is considered that a polymer exhibiting specific characteristics becomes hydrophilic and water-soluble at or below the temperature. Since LCST varies with the polymer structure and the polarity of the surrounding environment, in an aqueous solution containing various solutes such as isotonic solution, LCST is about several degrees Celsius depending on its components and concentration. There is a tendency to decrease.

  Table 2 shows LCSTs of copolymers (copolymers) each containing 50% of the repeating units constituting the polymers A to F. As shown in Table 2, it was confirmed that the copolymer containing a repeating unit included in the general formula (I) shows an LCST intermediate between LCSTs of a homopolymer composed only of each repeating unit constituting the copolymer. By using this, it is possible to obtain a polymer having an appropriate LCST by making a copolymer containing a repeating unit contained in the general formula (I). At this time, even if it is a repeating unit that does not have an obvious LCST when it is made into a homopolymer, such as the above-mentioned homopolymer F, there is a repeating unit that shows LCST when made into a copolymer. Such repeating units are believed to have a potential LCST at temperatures below 0 ° C.

  In addition, as described in Patent Document 2, a substance that hardly shows hydration and does not form a surface water layer, or even a hydrating composition, the surface water layer is composed of antifreeze water or free water. On the surface of a substance that is substantially free of intermediate water, it is known that all cells and plasma proteins present in body fluids such as blood are adsorbed at a high adsorption frequency. On the other hand, on the polymer surface containing intermediate water, even if the amount of intermediate water is relatively small, adsorption of relatively small floating cells such as platelets and plasma proteins is suppressed, and compensation is performed. It has become clear that thrombus formation and hemolysis due to system activation and platelet activation can be prevented, and so-called biocompatibility is exhibited. On the other hand, even on the surface where the adsorption of floating cells and plasma proteins is suppressed due to the presence of intermediate water, relatively large and highly adsorbable cells such as cancer cells and stem cells still cause adsorption. It has been found that it is necessary to contain a greater proportion of intermediate water to prevent adsorption. Thus, by using the fact that the amount of intermediate water whose adsorption frequency decreases greatly depending on the type of cells, etc., the use of a hydrating composition having an appropriate amount of intermediate water as an adsorbent for cells, etc. It is possible to selectively adsorb the corresponding cells.

  Based on the knowledge as described above, the present inventor has intermediate water as described in Patent Document 1 and is also present in body fluids such as blood by selecting an appropriate polymer from polymers having LCST. As a result of performing various studies on the assumption that the adsorbed cells can be easily recovered in an aqueous medium using LCST while selectively adsorbing cancer cells and stem cells to be absorbed. The invention has been completed.

  That is, as a result of various studies by the present inventor, cell adsorption using a homopolymer comprising the same repeating unit among polymers containing intermediate water and having LCST and being dissolved at a temperature below body temperature. When the base material for use is used, selective adhesion of cancer cells and the like is confirmed. On the other hand, when the polymer is cooled to a temperature of LCST or lower and dissolved in an aqueous medium, It was observed that the rate released into the aqueous medium was low and most remained on the substrate. On the other hand, when a cell-adsorbing substrate using a copolymer containing different repeating units is used, selective adhesion of cancer cells and the like is similarly confirmed, and the polymer is used in a low-temperature aqueous medium. It was clarified that cancer cells and the like were released into an aqueous medium at a high rate when dissolved in and recovered.

  As mentioned above, despite having intermediate water and showing LCST, depending on the structure of the polymer, cells adhered to the polymer remain on the substrate or released into the aqueous medium upon dissolution of the polymer The reason for this difference is not clear, but it is thought to be caused by the difference in behavior when the polymer film dissolves depending on the polymer structure. That is, when the polymer is a copolymer, it is considered that in the cooling process in the aqueous medium, the portion having a higher LCST is first hydrophilized and dissolved, so that the dissolution of the polymer film proceeds non-uniformly. As a result, the polymer coating partially dissolved and swollen and having a reduced adhesion is released from the substrate and cells, and then the dissolution of the coating is completed, so that cell release is promoted in the process. Conceivable. On the other hand, when the polymer is a homopolymer, the polymer coating dissolves sequentially from the surface in the cooling process in an aqueous medium, so that the dissolution progresses with cells adhering to the surface of the polymer coating due to hydrophobic interaction or the like. As a result, it is presumed that the cells are reattached to the surface of the base material after the polymer coating is dissolved and are not released into the aqueous medium.

  Utilizing the adhesion and peeling behavior of cancer cells and the like when using a polymer having a predetermined structure as described above, the method for recovering cancer cells according to the present invention can contain a predetermined amount of intermediate water. And using a substrate having at least a part of the surface coated with a copolymer having an LCST of 37 ° C. or lower, which is a body temperature, in a cell suspension in which cells to be collected are suspended, It includes a step of contacting (adsorbing) cancer cells, stem cells and the like to the surface of the polymer by bringing the polymer surface into contact with a cell suspension such as blood or lymph at a temperature of LCST or higher. In this process, when the polymer used contains a predetermined amount of intermediate water, adsorption of blood cells such as platelets and proteins is suppressed, while relatively large cells such as cancer cells and stem cells are removed. It can be selectively adhered to the polymer surface. In the present specification, the terms “adhesion” and “adsorption” are sometimes used complementarily with respect to the behavior of cells with respect to the polymer surface.

  Next, the substrate having cancer cells and stem cells adhered to the polymer surface as described above is transferred into an aqueous medium for collecting the cancer cells and the like, and the polymer is dissolved at a temperature equal to or lower than the LCST, A step of detaching cancer cells and the like adhered to the polymer surface from the substrate and collecting them in an aqueous medium. When dissolving the polymer, by using a copolymer containing two or more different repeating units, the recovery rate of cells in an aqueous medium is increased as compared with the case of using a homopolymer consisting of a single repeating unit. It becomes possible. In addition, according to the present invention, when recovering the adhered cancer cells or the like in an aqueous medium, a treatment that actively impairs the cell adhesion mechanism such as trypsin treatment is not required, and the cells adhere. Since the existing scaffold is eluted from the substrate, the cancer cells can be recovered in an aqueous medium in a more healthy state. Further, as the present inventor separately clarifies, since the polymer surface having a hydration structure that suppresses adhesion of platelets and the like is good as a substrate for cell culture, cancer cells and the like are used as substrates in the present invention. It is considered that the soundness can be maintained even when adhered to the film.

  As the polymer used in the cell recovery method according to the present invention, a polymer having a LCST of 0 ° C. to 37 ° C. in a cell suspension such as blood to be collected such as cancer cells is preferably used. Further, from the viewpoint of increasing the dissolution rate of the polymer when cooled in an aqueous medium, and preventing the decrease in cell activity due to excessive cooling, LCST is preferably 5 ° C or higher, more preferably 10 ° C or higher. desirable. On the other hand, when used in contact with blood in a living body, LCST is preferably 35 ° C. or lower, more preferably from the viewpoint of reliably preventing dissolution of the polymer during contact with blood and stabilizing the polymer surface. It is desirable to use a polymer of 30 ° C or lower.

As a target for recovering cancer cells and the like by applying the method for recovering cells according to the present invention, a cell suspension in which cancer cells or the like to be recovered are present (or suspected of being present) in an aqueous medium. It is not particularly limited as long as it is a turbid liquid. For example, a part of blood circulating in the body may be extracorporeally circulated, or a substrate coated with a predetermined polymer may be placed in the body to substantially It can be applied to blood in a state existing in a living body. In addition, for blood and lymph extracted outside the body, other body fluids, cell suspensions obtained by processing the body fluids, etc., cell suspensions containing cancer cells separated by processing biological tissues, etc. In addition, cancer cells and the like may be adhered to the polymer surface by contacting the substrate coated with the polymer according to the present invention at a temperature equal to or higher than the LCST of the polymer.
Further, in order to efficiently adhere cancer cells and the like to the polymer coated with the substrate, it is preferable to perform an operation for increasing the contact frequency of the cells to be adhered to the polymer surface. That is, especially when trying to adhere and recover cancer cells from whole blood, etc., because the frequency of contact between the cancer cells and the polymer surface may decrease due to the high density of blood cells, If the density of cancer cells is low, dilute the whole blood with appropriate plasma or isotonic solution as necessary to ensure that the target cancer cells are in contact with the polymer surface. It is preferable. In addition, by bringing a medium containing cancer cells etc. into contact with the polymer surface at a predetermined flow rate, the frequency of cancer cells etc. coming into contact with the polymer surface is increased, and blood cells etc. cause physical adsorption on the polymer surface. It is also preferable to prevent this.

  As an aqueous medium for recovering cancer cells and the like adhered to the polymer surface according to the present invention, an aqueous medium appropriately selected according to the purpose of recovery can be used. For example, when the collected cells are subsequently cultured or used for gene mutation analysis, expression gene analysis, or production protein analysis, generally cells containing physiological saline, phosphate buffered saline, or serum are used. A culture medium used for culture, a culture medium not containing serum, and the like can be used. The recovery of cancer cells and the like from a substrate having cancer cells and the like adhered to the polymer surface is performed by bringing the substrate to a temperature below the LCST of the polymer in the aqueous medium, so that the polymer is dissolved in the aqueous medium, Along with this, it can be performed by utilizing the fact that cancer cells and the like are released into the aqueous medium. Cancer cells and the like released in an aqueous medium can be handled according to their purpose, as in conventional cell samples. For example, cancer cell growth culture, anticancer drug resistance test using cancer cells after growth culture, gene mutation analysis using collected cancer cells, transporter involved in the expression of anticancer drug resistance, etc. Quantitative analysis, semi-quantitative analysis, qualitative analysis, protein production analysis, and tissue staining such as hematoxylin-eosin staining generally used for pathological examinations, and antibodies It can be used for characterization of cancer cells by immunostaining.

  As described above, the polymer used when recovering cancer cells or the like by the method according to the present invention has a predetermined antithrombotic property and does not adhere blood cells existing at high density in blood. On the other hand, any copolymer can be used without particular limitation as long as it exhibits a certain adhesion to cancer cells and the like and LCST is present in the range of 0 to 37 ° C. In one embodiment, when a cancer cell or the like is collected by the method according to the present invention, a polymer containing a repeating unit represented by the following general formula (I) can be used.

In the formula, R ¹ is hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is 2 or 3, n represents the number of repetitions.

  By containing the repeating unit represented by the general formula (I) at a predetermined ratio or more, as shown in Tables 1 and 2, the anti-thrombotic property can be obtained by being able to contain intermediate water, while cancer cells and A relatively large and highly adherent cell such as a stem cell can be adhered, and at the same time, a copolymer exhibiting a lower critical solution temperature depending on its structure can be obtained. At this time, it is more preferable that all of the repeating units constituting the copolymer are represented by the general formula (I) in terms of easy adjustment of the content of intermediate water and LCST. Accordingly, the copolymer can include repeating units other than those represented by general formula (I).

  The polymer containing the repeating unit represented by the general formula (I) is typically suitable for a solution containing at least one monomer selected from the structure represented by the following general formula (II). It can be produced by adding an initiator and polymerizing by a general method such as random polymerization, ionic polymerization, photopolymerization, or polymerization using a macromer. The temperature at which the polymerization reaction is performed is preferably 40 ° C to 100 ° C, more preferably 60 ° C to 90 ° C, and still more preferably 70 ° C to 80 ° C. The pressure for conducting the polymerization reaction is preferably normal pressure. The polymerization reaction may be random copolymerization by mixing the respective monomers, or block copolymerization in which the respective monomers are polymerized to some extent and then both are mixed.

In the general formula (II), the definitions of R ¹ , R ² and m are the same as those in the general formula (I).

  In the polymerization reaction, as the solvent, a solvent capable of dissolving at least two types of monomers selected from the structure represented by the general formula (II) can be used. For example, an aliphatic or aromatic organic solvent, more specifically, ether solvents such as dioxane, tetrahydrofuran, diethyl ether, halogenated aromatic hydrocarbons such as ortho-dichlorobenzene, N, N-dimethylformamide, etc. Amides, sulfoxides such as dimethyl sulfoxide, aromatic hydrocarbons such as benzene and toluene, and aliphatic hydrocarbons such as hexane and pentane, with ether solvents such as dioxane being preferred.

In the repeating unit described in the general formula (I), as shown in the LCST and the intermediate water amount described in Table 1, depending on R ¹ , R ² and m in the general formula (I), the LCST or intermediate It is possible to change the amount of water. In general, the LCST shifts to a higher temperature side and the amount of intermediate water that can be contained tends to increase as the hydrophilicity of each part increases, for example, R ¹ is changed from a methyl group to hydrogen and R ² is changed from an ethyl group to a methyl group. It is done. Further, the amount of intermediate water that can be contained by changing LCST to a high temperature side by increasing the number of repetitions (m) of ethylene glycol part (C ₂ H ₄ —O), which is considered to be hydrophilic, from 2 to 3. There is a tendency to increase. In the method for recovering cells according to the present invention, the LCST in an aqueous solution in which cancer cells to be recovered are present is particularly in the range of 0 to 37 ° C. by utilizing the characteristics inherent to the structure of each repeating unit. It is preferable to synthesize and use a copolymer containing a plurality of types of repeating units.

  In the LCST described in Tables 1 and 2, each polymer was dissolved in Milli-Q water (pure water) at a concentration of 1.0 wt% as a polymer solution, and 500 μL of this polymer solution was placed in a microcell. Using a V-630 spectrophotometer (Nippon Bunko Co., Ltd., Tokyo) while measuring the transmittance of visible light with a wavelength of 500 nm, the temperature was increased at a rate of 0.5 ° C./min to precipitate each polymer. Thus, the temperature when the light transmittance of the polymer solution becomes 50% of the transmittance of Milli-Q water in which the polymer is not dissolved is measured. The LCST varies depending on the polarity of the surrounding environment, and in an aqueous solution containing various solutes such as an isotonic solution, the LCST tends to decrease by about 2 to 5 ° C. depending on its component and concentration. Moreover, the amount of intermediate water described in Tables 1 and 2 is measured by the measuring method described in Patent Document 2 and the like for each polymer. That is, from the amount of heat generated near −40 ° C. observed in the process of reheating to room temperature after cooling each saturated water-containing polymer to about −100 ° C. and the amount of endotherm observed between −20 ° C. and 0 ° C. The weight of the intermediate water contained in the polymer is obtained and divided by the dry weight of the polymer.

  Water molecules in a state called “intermediate water” are also contained in biological materials such as phospholipids, and non-specific adsorption of proteins in biological tissues is caused by the presence of intermediate water on the surface of the material. It is clear that it is prevented. Intermediate water is characterized by, for example, an exothermic peak derived from the transfer of latent heat based on low-temperature crystallization of water, typically in the vicinity of −40 ° C., during a temperature rising process from a low temperature of about −100 ° C., for example. . This low temperature crystallization is considered to be a transformation from amorphous ice to crystalline ice. Intermediate water is thought to be a water molecule that is weakly constrained by a specific interaction with the polymer chain, and this is thought to result in a unique behavior not observed with water molecules alone, such as low-temperature crystallization. . Depending on the content of such intermediate water, it can prevent the adsorption of proteins in the living body, blood cells such as platelets, and various somatic cells, while substances that are present in the living body by appropriately adjusting the amount of intermediate water It is becoming clear that can be selectively adsorbed.

  That is, the polymers used in the present invention are known to have existed so far (1) “free water” which has weak interaction with the polymer and solidifies and melts at 0 ° C., (2) In addition to “antifreeze water” that has strong interaction and does not freeze even at −100 ° C., (3) “intermediate water that freezes at a temperature lower than 0 ° C. due to an intermediate interaction between free water and antifreeze water. It is thought that there exists a hydrated structure including “ This hydration structure suppresses normal blood cells from adhering to the substrate surface, whereas cancer cells and stem cells hydrate on the polymer surface because the expression of sugar chains on the cell surface is different from normal cells. It is thought that the structure is disturbed and the adhesion to the molecules forming the polymer and the substrate surface is expressed. Also in the polymer used in the present invention, by containing this intermediate water in a predetermined amount ratio, protein adsorption denaturation and activation / adhesion are suppressed when it comes into contact with a tissue in a living body or blood. On the other hand, cancer cells and stem cells can be selectively adhered. In particular, CTCs that circulate in blood and adhere to tissues in the living body to cause cancer metastasis have high adhesiveness, and the method for recovering cancer cells according to the present invention is suitably applied. It is considered a thing.

  Due to the mechanism described above, the polymer used when recovering cancer cells and the like by the method according to the present invention adheres to blood cells and the like that exist in high density in blood by having a predetermined antithrombotic property. On the other hand, any copolymer can be used without particular limitation as long as it has a certain adhesion to cancer cells and the like and LCST is present in the range of 0 to 37 ° C. By using the copolymer containing the repeating unit shown in I), a method for recovering cancer cells and the like according to the present invention can be carried out more easily.

  In addition, when a copolymer containing a repeating unit represented by the general formula (I) is used, a plurality of repeating units represented by the general formula (I) may be combined to form a copolymer. The copolymer may be combined with the repeating unit shown in (1). In that case, as the repeating unit of the other structure, it is not always necessary to use a repeating unit containing a single intermediate water and giving a polymer showing LCST, or a repeating unit giving a water-soluble polymer. Thus, it is possible to obtain a copolymer using an appropriate repeating unit without departing from the gist of the present invention. That is, when a repeating unit containing a polar group having a strong polarity at a high density is combined with a repeating unit represented by the general formula (I), the repeating unit represented by the general formula (I) is affected by the polarity. There is a case where the originally exhibited behavior is inhibited and the inclusion of intermediate water is inhibited. On the other hand, when a copolymer is formed by combining a repeating unit having a polarity or the like below a predetermined unit with the repeating unit represented by the general formula (I), the amount of intermediate water contained is reduced according to the ratio, and the LCST is reduced. Since there is a tendency to lower the temperature, it is particularly useful in adjusting the properties of the polymer used in the present invention. Arrangement between each repeating unit in the copolymer used in the present invention is not particularly limited, and can be used in the form of a random copolymer, a block copolymer, a graft copolymer or the like. In particular, by making it a form of block copolymer or graft copolymer, the polymer chain is partially insolubilized, and when it is cooled to a temperature of LCST or less, a part of the polymer chain is adhered to the substrate surface. Thus, it is also preferable to prevent dissolution of the polymer in the aqueous medium and prevent contamination of the aqueous medium while peeling cancer cells and the like adhered to the polymer surface to the aqueous medium.

In order to combine the repeating unit represented by the general formula (I) with the repeating unit having another structure, a monomer that can be polymerized with a monomer selected from the structure represented by the general formula (II) is a copolymer obtained by polymerization. Is not particularly limited as long as it can contain a predetermined amount of intermediate water and can produce a copolymer having LCST in the temperature range of 0 to 37 ° C., acrylamide, t-butylacrylamide, n-butylacrylamide, i -Alkyl acrylamide such as butyl acrylamide, hexyl acrylamide, heptyl acrylamide, N, N-dialkyl acrylamide such as N, N-dimethylacrylamide, N, N-diethylacrylamide, aminomethyl acrylate, aminoethyl acrylate, aminoisopropyl acrylate Aminoalkyl acrylate, diaminomethyl acrylate, diaminoethyl acrylate, diaminoalkyl acrylate such as diaminobutyl acrylate, N, N-dialkyl methacrylamide such as methacrylamide, N, N-dimethylmethacrylamide, N, N-diethylmethacrylamide, Aminoalkyl methacrylate such as aminomethyl methacrylate, aminoethyl methacrylate, diaminoalkyl methacrylate such as diaminomethyl methacrylate, diaminoethyl methacrylate, alkyl acrylate such as methyl acrylate, ethyl acrylate, isopropyl acrylate, butyl acrylate, hexyl acrylate, 2-ethylhexyl acrylate, Methyl methacrylate, ethyl methacrylate, buty Methacrylate, alkyl methacrylates such as hexyl methacrylate, methoxy (meth) alkoxy (meth) acrylates such as acrylates, alkoxyalkyl (meth) acrylates such as methoxyethyl (meth) acrylate, glycidyl methacrylate and propylene. Further, from the viewpoint of independently adjusting the intermediate water content and LCST of the polymer used in the present invention, the intermediate water is contained in the monomer selected from the structure represented by the general formula (II). (Meth) acrylamide compounds showing LCST, N-acryl substituted (meth) acrylamide derivatives such as N-isopropyl (meth) acrylamide, N, N-dialkyl substituted (meth) such as N, N-dimethyl (meth) acrylamide It is possible to use acrylamide derivatives, N-heterocyclic group-substituted (meth) acrylamide derivatives such as 1- (1-oxa-2-propenyl) -pyrrolidine, and the like.
The ratio of each repeating unit in the copolymer used in the present invention is such that the LCST of the synthesized copolymer is 0 to 37 ° C., depending on the LCST inherent in the repeating unit used, the amount of intermediate water that can be contained, and the like. The amount of intermediate water is not particularly limited as long as it can be contained, but it is mainly at least from the viewpoint of ensuring cell detachability when lysing at a temperature below LCST in an aqueous medium. The ratio of the repeating unit is preferably 90% or less. By doing so, the film made of the copolymer causes non-uniform dissolution, and the cells adhered to the polymer can be peeled well. Further, by making the ratio of the main repeating unit 70%, more preferably 60% or less, it is possible to improve the cell detachability. Furthermore, it is also preferable to make the ratio of the main repeating unit 40%, further 30% or less by making it a copolymer containing 3 or more types of repeating units.

  Moreover, as a polymer used for collection | recovery of a cancer cell etc. by this invention, you may use the polymer formed by mixing multiple types of polymer in the range which does not deviate from the meaning of this invention. In particular, it is preferable to use a mixture of polymers having different LCSTs in that the dissolution process of the polymer in the aqueous medium becomes non-uniform and the peeling of cancer cells and the like adhered to the surface is promoted. At that time, by adjusting the mixed form of polymers having different LCSTs, it is expected that exfoliation of cancer cells and the like adhered to the surface is promoted by using a homopolymer as each polymer.

  The method for recovering cancer cells and the like according to the present invention can be applied to cancer cells showing adhesion to a polymer surface containing intermediate water, but it is suitable for cancer derived from epithelial cells that are liable to cause invasion into blood vessels. It is particularly preferable to apply to digestive organ cancers such as lung cancer, colon cancer, stomach cancer or esophageal cancer, breast cancer, and prostate cancer, which can be suitably applied. In addition to cancer cells, the method for recovering cancer cells and the like according to the present invention can also be used for stem cells, vascular endothelial cells, etc. existing in blood and the like.

  In one embodiment, the present invention provides a cell recovery material in which at least a part of a substrate material surface having an arbitrary shape is coated with the polymer described above. The shape and form of the cell recovery material can be appropriately determined according to the application. For example, a predetermined polymer can be applied to the surface of a dish for cell culture, and a cell suspension can be used. It is also possible to use a tube or mesh, or a material in which a predetermined polymer is applied to the inner surface of the substrate material on the stent, and circulate a cell suspension such as blood inside. Alternatively, a predetermined polymer may be applied to at least a part of the surface of a substrate material such as a filter having a plurality of through holes or a micro device, and a cell suspension such as blood may be circulated for use. Furthermore, a predetermined polymer may be applied to at least a part of the filter surface for collecting cancer cells and the like by utilizing the difference in size depending on the cells, and used as an auxiliary means for cell recovery.

  As a method for applying the polymer of the present invention to the surface of the cell recovery material, any suitable form can be used without limitation, but coating is the most common. The coating is performed by attaching a solution in which a polymer to be used is dissolved at a predetermined concentration to the substrate surface by a dipping method, a spray method, a spin coating method, or the like, and then removing (drying) the solvent. The film thickness after drying is not particularly limited, but typically 0.01 to 100 μm is preferable, 0.05 to 50 μm is more preferable, and 0.2 to 10 μm is still more preferable. If the film thickness is less than 0.01 μm, non-adhesiveness with blood cell components and selectivity with cancer cells may not be sufficiently developed. Also, there may be cases where the adhered cancer cells or the like do not peel well. When the film thickness exceeds 100 μm, it may take a long time to dissolve the copolymer by cooling.

  In one embodiment, the material and shape of the substrate used by coating the polymer of the present invention are not particularly limited, and for example, a porous body, a fiber, a nonwoven fabric, a film, a sheet, and a tube can be used. Substrate materials include natural polymers such as cotton and linen, nylon, polyester, polyacrylonitrile, polyolefin, halogenated polyolefin, polyurethane, polyamide, polysulfone, polyethersulfone, poly (meth) acrylate, and halogenated polyolefin ethylene-polyvinyl. Examples thereof include synthetic polymers such as alcohol copolymers and butadiene-acrylonitrile copolymers, or mixtures thereof. Moreover, a metal, ceramics, these composite materials, etc. can be illustrated and may be comprised from the some board | substrate.

Examples of the metal include, but are not limited to, noble metals such as gold and silver, base metals such as copper, aluminum, tungsten, nickel and chromium, and alloys of these metals. The metal may be used alone, or may be used as an alloy with another metal or an oxide of a metal in order to impart functionality. From the viewpoint of price and availability, it is preferable to use nickel, copper, and metals containing these as main components.
Further, the method of applying the polymer of the present invention to the surface of the cell recovery material is not limited to coating. For example, cancer cells and the like are adhered in a state in which the polymer of the present invention formed into a film is fixed by an appropriate means. Thereafter, the present invention can be carried out in such a form that the polymer is dissolved in an aqueous medium.

  FIG. 1 is a schematic view showing an embodiment for recovering CTCs and the like in blood using the cell recovery material of the present invention. The surface of the substrate material coated with the polymer of the present invention is brought into contact with a cell suspension such as blood. As used herein, the term “cell suspension” includes any type of tissue or fluid removed from an organism, including a human or other mammal. Specifically, the cell suspension may be whole blood, serum, plasma, urine, cerebrospinal fluid or saliva, or derived therefrom, and includes cells obtained by processing living tissue. Refers to an appropriate suspension. The temperature at the time of contact is not particularly limited as long as the polymer on the surface of the substrate is not dissolved, but the sample temperature at the time of collection is preferable. In the case of humans, treatment at a temperature around 37 ° C. Preferred for preventing denaturation.

  The polymer surface provided in the cell recovery material of the present invention suppresses adhesion of blood cell components such as red blood cells, white blood cells, and platelets, and therefore hardly adsorbs these even when brought into contact with a sample containing a large amount of blood components. On the other hand, since it has adhesion to cancer cells, stem cells, etc., cancer cells such as CTCs, stem cells, etc. can be selectively adhered. Therefore, in the present specification, “target cells” include, but are not limited to, cancer cells and stem cells having such adhesion. The cell collection material after contacting with a predetermined amount of the sample is subsequently cooled in an appropriate aqueous medium, whereby the polymer provided on the substrate surface is dissolved. The cooling temperature and cooling time can be appropriately adjusted depending on the type of polymer used.

  For example, by setting the LCST of the polymer to be used in the range of 15 ° C. to 20 ° C., cells adhered to the substrate surface can be released by treatment in a lower temperature aqueous medium. The lower limit of the treatment temperature is not particularly limited, but is preferably 0 ° C. or higher, more preferably 5 ° C. or higher, and more preferably 10 ° C. or higher in order to minimize the influence on the cells to be collected. The time for performing the low temperature treatment is not particularly limited as long as it is sufficient for cells to be released, but is preferably within 90 minutes, more preferably within 60 minutes, and even more preferably within 30 minutes. Since the optimum low-temperature treatment temperature and time are related to each other, those skilled in the art can appropriately set the temperature based on the LCST of the polymer used for the surface treatment.

  Cancer cells and the like released in an aqueous medium can be handled according to their purpose, as in conventional cell samples. For example, cancer cell growth culture, anticancer drug resistance test using cancer cells after growth culture, gene mutation analysis using collected cancer cells, transporter involved in the expression of anticancer drug resistance, etc. Quantitative analysis, semi-quantitative analysis, qualitative analysis, protein production analysis, and tissue staining such as hematoxylin-eosin staining generally used for pathological examinations, and antibodies It can be used for characterization of cancer cells by immunostaining.

  The details of the method for recovering the target cells of the present invention and the temperature-responsive copolymer used therein will be described below with reference to examples. The present invention is not limited to these examples.

[Example 1] Production of cell recovery material (1.1) Substrate As a cell recovery material according to the present invention, a PET (polyethylene terephthalate) film (Mitsubishi Resin, Diafoil catalog number T100E125) is used as a base material. What coated each of various polymers was used. The PET film was punched into a circle having a diameter of 14 mm using a hand press (Fukui Machine Industry Co., Ltd., CAN SYSTEM HANDPRESS FK-HP200). This PET substrate was immersed in methanol (Kanto Chemical Co., Inc.) and washed.

(1.2) Synthesis of Polymer Poly [2- (2-ethoxyethoxy) ethyl acrylate] (hereinafter referred to as “homopolymer A”) was prepared by using monomer 2- (2-ethoxyethoxy) ethyl acrylate (Tokyo). Kasei Kogyo Co., Ltd.) 15 g (molecular weight 188.22, 0.080 mol) was dissolved in 1,4-dioxane 60 g, and after nitrogen bubbling for 30 minutes, AIBN (15 mg, 0.091 mmol) as an initiator was added. Polymerization was carried out at 75 ° C. for 6 hours while adding nitrogen dissolved in a small amount of 1,4-dioxane. The polymer produced | generated by superposition | polymerization was collect | recovered as precipitation by dripping the polymerization solvent to 1000 mL of n-hexane. The obtained product was purified by repeating the precipitation operation three times in a THF / n-hexane system. After purification, the solvent was removed by an evaporator, and finally, vacuum drying was performed at 60 ° C. for 18 hours. By performing free radical polymerization and reprecipitation, the desired poly [2- (2-ethoxyethoxy) ethyl acrylate] (Pet2A) (yield 11.99 g, yield 80.0%, colorless and transparent viscous product) Got. Note that 2- (2-ethoxyethoxy) ethyl acrylate has a structure in which R ¹ ; H, R ² ; ethyl group, m = 2 in the general formula (I).

Preparation of poly (2- (2-ethoxyethoxy) ethyl acrylate-co-2- (methoxyethoxy) ethyl methacrylate (hereinafter referred to as “copolymer AB”) is a monomer, 2- (2-ethoxyethoxy) ethyl acrylate. (Tokyo Chemical Industry Co., Ltd.) 7.5 g (molecular weight 188.22, 0.04 mol), 2- (2-methoxyethoxy) ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.) 7.5 g (molecular weight 188.22, 0.04 mol) ) (Molar ratio 1: 1) was dissolved in 60 g of 1,4-dioxane and subjected to nitrogen bubbling for 30 minutes, and then α, α′-azobisisobutyronitrile (AIBN) as an initiator. ) (15 mg, 0.091 mmol) dissolved in a small amount of 1,4-dioxane and added, and polymerized at 75 ° C. for 6 hours while bubbling nitrogen. The polymer produced by the polymerization was recovered as a precipitate by dropping the polymerization solvent into 1000 mL of n-hexane, and the resulting product was purified by repeating the precipitation operation three times in a THF / n-hexane system. The solvent was removed by an evaporator, and finally a copolymer was obtained by vacuum drying at 60 ° C. for 18 hours, wherein 2- (2-ethoxyethoxy) ethyl acrylate is represented by R ¹ in the general formula (I); H, R ² ; ethyl group, m = 2, and 2- (methoxyethoxy) ethyl methacrylate has R ¹ ; methyl group, R ² ; methyl group, m = 2.

  The preparation of poly (2- (2-ethoxyethoxy) ethyl acrylate-co-2- [2- (2-methoxyethoxy) ethoxy] ethyl acrylate (hereinafter referred to as “copolymer AC”) is a monomer 2 -(2-Ethoxyethoxy) ethyl acrylate (Tokyo Chemical Industry Co., Ltd.) 7 g (molecular weight 188.22, 0.037 mmol) and 2- [2- (2-methoxyethoxy) ethoxy] ethyl acrylate (Kyoeisha Chemical Co., Ltd.) 8 g A total of 15 g (molecular weight 218.244, 0.037 mmol) (molar ratio 1: 1) was dissolved in 60 g of 1,4-dioxane, and nitrogen bubbling was performed for 30 minutes, after which initiator AIBN (15 mg, 0.091 mmol) Was dissolved in a small amount of 1,4-dioxane, and polymerization was carried out at 75 ° C. for 6 hours with nitrogen bubbling.

The polymer produced | generated by superposition | polymerization was collect | recovered as precipitation by dripping the polymerization solvent to 1000 mL of n-hexane. The obtained product was purified by repeating the precipitation operation three times in a THF / n-hexane system. After the purification, the solvent was removed by an evaporator, and finally, vacuum drying was performed at 60 ° C. for 18 hours to obtain a copolymer. Note that 2- (2-ethoxyethoxy) ethyl acrylate has a structure in which R ¹ ; H, R ² ; ethyl group, m = 2 in the general formula (I), and 2- [2- (2-methoxy Ethoxy) ethoxy] ethyl acrylate has a structure in which R ¹ ; H, R ² ; methyl group, m = 3.

  PMEA (poly (2-methoxyethyl acrylate)) used as a comparative example was synthesized by the method disclosed in M. Tanaka and A. Mochizuki (J Biomed Mater Res, 68A, pp 684-695, 2004). In addition, poly N-isopropylacrylamide (PNIPAAm) used was sold by SCIENCE POLYMER ((Cat. 963)). Further, UpCell obtained by modifying PNIPAAm into a cell culture plate by electron beam cross-linking was used from Cell Seed. Poly (2-methacryloyloxyethyl phosphorylcoline-co-N-butyl methacrylate) used was provided by NOF Corporation.

(1.3) Measurement of polymer molecular weight (unit: g / mol) Gel permeation chromatography calibrated with standard polystyrene having a known peak molecular weight for copolymer AB synthesized in section (1.2). (GPC) (“HLC-8220” manufactured by Tosoh Corporation, column configuration: Tosoh TSK-gels super AW5000, super AW4000, super AW3000), the number average molecular weight (Mn) and the weight average molecular weight (Mw) of the polymer Was measured. (Solvent: THF, temperature: 40 ° C., flow rate: 1.0 mL / min).
The weight average molecular weight of copolymer AB (Mw (g / mol) was Mw (g / mol) = 145,000, and the molecular weight distribution was Mw / Mn = 5.5.

(1.5) Structural Analysis of Polymer For structural analysis of homopolymer A and copolymer AB, ¹ H-NMR measurement was performed using an NMR measuring apparatus (JEOL 500 MHz JNM-ECX, manufactured by JEOL Ltd.). The chemical shift was based on CDCl 3 ( ¹ H: 7.26 ppm).
The ¹ H-NMR data of homopolymer A, copolymer AB and copolymer AC synthesized in this example are as follows.

Homopolymer A (PEt2A)
¹ H-NMR (500MHz, CDCl ₃ , ppm): δ 1.2 (s, 3H, -O-CH ₂ -CH ₃ of PEt2A), 1.4-2.6 (br, polymer backbone of PEt2A), 3.5-3.8 (m, 8H, -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₃ of PEt2A), 4.2 (br, 2H, -COOCH ₂ -of PEt2A)

Copolymer AB
¹ H-NMR (500 MHz, CDCl ₃ , ppm): δ 0.8-1.1 (m, 3H, α-CH ₃ of PMe2MA), 1.2 (s, 3H, -O-CH ₂ -CH ₃ of PEt2A), 1.4- 2.3 (br, polymer backbone of PEt2A and PMe2MA), 3.38 (s, 3H, -O-CH ₃ of PMe2MA), 3.5-3.8 (m, 14H, -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₃ of PEt2A and -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₃ of PMe2MA), 4.0-4.2 (br, 4H, -COOCH ₂ -of PEt2A and PMe2MA)

Copolymer AC
¹ H-NMR (500 MHz, CDCl ₃ , ppm): δ 1.2 (s, 3H, -O-CH ₂ -CH ₃ of PEt2A), 1.4-1.9 (br, 2H, -CH ₂ -CH- of polymer backbone , 2.2-2.5 (br, 1H, -CH ₂ -CH- of polymer backbone), 3.4 (s, 3H, -O-CH ₃ of PMe3A) 3.5-3.8 (m, 18H, -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CH ₃ of PEt2A and -CH ₂ -O-CH ₂ -CH ₂ -O-CH _2- CH ₂ -O-CH ₃ of PMe3A), 4.0-4.3 (m, 4H, -COOCH ₂ -of PEt2A and PMe3A)

(1.6) Measurement of LCST of Polymer 0.10 g of each of the copolymer synthesized above and analogs thereof and a homopolymer composed of a single repeating unit constituting them were added, and 10 mL of solvent was added to add 1.0 wt. A polymer solution of / vol% was prepared. 500 μL of the prepared polymer solution is put in a microcell, and the transmittance for visible light having a wavelength of 500 nm is measured at a temperature of 0.5 ° C./min using a V-630 spectrophotometer (JASCO Corporation, Tokyo). The measurement was performed while raising the value. The transmittance of MilliQ water in which no polymer was dissolved was defined as 100%, and LCST was defined as the temperature at which the transmittance was 50%. The measurement results of LCST when using MilliQ water as the solvent are as shown in Tables 1 and 2 above. In addition, when the polymer was dissolved using LCST using various media as an aqueous medium, the same measurement was performed using each medium as a solvent.

(1.7) Polymer coating Polymer coating on a PET substrate was performed as follows. A washed PET substrate is placed in a spin coater (Mikasa Corporation, catalog number MS-A100), and 40 μL of a polymer solution (concentration is 5 wt% or 1 wt%) prepared using methanol as a solvent is added dropwise to T. Hoshiba et al. Advanced Healthcare Material, 3, 775, 2014). Briefly, the spin coating conditions were 500 rpm for 5 seconds, 2,000 rpm for 10 seconds, 2000 to 4000 rpm rotational speed slope up for 5 seconds, 4000 rm for 10 seconds, and 4000 to 0 rpm rotational speed slope down for 5 seconds. It was. The substrate coated once was allowed to stand and dried. After the second coating was performed in the same procedure, the substrate was dried overnight. Transfer the polymer substrate to a 24-well cell culture plate (Asahi Glass Co., Ltd., IWAKI, Catalog No. 3820-024), and disinfect the lamp in the clean bench (Sanyo Electric Co., Ltd., Catalog No. MCV-B131F). No. GL15) was sterilized by irradiation with ultraviolet light for 2 hours.

  When coating a 96-well cell culture plate, add 11.2 μL of the prepared polymer solution (concentration: 5 wt%) per well, cover the plate, and then use parafilm to separate the gap between the lid and the plate. Then, each polymer was cast coated by allowing it to stand in a constant temperature bath at 37 ° C. for 3 days or longer.

[Example 2] Examination of adhesion between blood cells and cancer cells in plasma Examination of adhesion between blood cells and CTCs in plasma was performed using each polymer solution having a concentration of 5 wt% in section (1.7). The coated PET substrate, UpCell, cell culture plate, and untreated PET substrate surface were examined as follows.
The adhesion of blood cells was determined by pipetting 0.2 mL of human fresh platelet-rich plasma containing platelets anticoagulated with sodium citrate onto each substrate with a seeding density of 4 × 10 ⁷ cells / cm ^2. And left at 37 ° C. for 60 minutes. Subsequently, after rinsing with phosphate buffered saline and fixing with 1% glutaraldehyde, each substrate was observed with a scanning electron microscope, and the number of platelets adhered to an area of 1 cm ² was counted.

The adhesion of cancer cells was evaluated using a human fibrosarcoma cell line HT-1080 purchased from the Human Science Research Resource Bank as a model for CTCs. After suspending HT-1080 in human fresh platelet poor plasma, seeded on the PET substrate coated with each polymer solution with a concentration of 5 wt% in Section (1.7), UpCell, cell culture plate, and PET substrate surface 0.2 mL of the suspension was dropped with a pipette so that the density was 1 × 10 ⁴ pieces / cm ^2, and the mixture was allowed to stand at 37 ° C. for 60 minutes. Subsequently, after rinsing with phosphate buffered saline and fixing with 1% glutaraldehyde, the substrate was observed with a scanning electron microscope, and the number of HT-1080 adhered to an area of 1 cm ² was counted.

FIG. 2a shows the number of platelet adhesion to each substrate 60 minutes after seeding. The horizontal axis represents the polymer or the like coated on each substrate, and the vertical axis represents the number of platelet adhesion (unit: 10 ⁵ / cm ² ). As shown in FIG. 2a, platelets were seeded at a density of 4 × 10 ⁷ cells / cm ² , whereas an untreated PET substrate had a density of about 6.5 × 10 ⁵ cells / cm ² , a cell culture plate ( Polystyrene adhered at a density of 2 × 10 ⁵ cells / cm ² for polystyrene and 1 × 10 ⁵ cells / cm ² for UpCell. On the other hand, in the PET substrate coated with PMEA, copolymer, PMPC and PNIPAAm, the adhesion density of platelets is about 0.5 × 10 ⁵ pieces / cm ² or less, indicating that platelet adhesion does not substantially occur. It was.

FIG. 2b shows the number of adhesions of HT-1080 to each substrate 60 minutes after sowing. The horizontal axis represents the polymer or the like coated on each substrate, and the vertical axis represents the number of adhered platelets (unit: 10 ³ / cm ² ). HT-1080 cells were seeded at a density of 1 × 10 ⁴ cells / cm ² , but as shown in FIG. 2b, the untreated PET substrate had a density of about 6 × 10 ³ cells / cm ² , PMEA or copolymer AB. HT-1080 cells adhere at a density of about 8 × 10 ³ cells / cm ² and about 7.5 × 10 ³ cells / cm ² , respectively, and most of the seeded HT-1080 cells adhere. Was shown to do. On the other hand, the adhesion density of HT-1080 to a PET substrate coated with PNIPAAm and PMPC, UpCell, and cell culture plate is 0.5 × 10 ³ cells / cm ² or less, and the seeded HT-1080 cells are large. It was shown that the part was removed by rinsing with phosphate buffered saline without sticking.

  As is apparent from FIGS. 2a and 2b, in the PET substrate coated with a thrombotic material containing intermediate water of copolymer AB or PMEA, HT-1080 cells were adhered at a high ratio while platelets were not adhered, and cancer cells It was shown to produce a selective adhesion. On the other hand, the other polymers tested were classified as those that adhere to each other or those that do not adhere to both, indicating that selective adhesion of cancer cells does not occur.

[Example 3] Examination of adhesion between blood cells and cancer cells in human whole blood Cell adhesion in human whole blood in which cancer cells (HT1080 cells) were suspended was coated with copolymers AB and PMEA, respectively. Using a PET substrate and an untreated PET substrate, the following examination was made. The HT-1080 cell suspension obtained for the density of HT-1080 cells was diluted with human whole blood (collected from males or women in their 20s) to prepare HT1080 cell suspension whole blood, which was then added to a temperature of 37 ° C. using a water bath. Warm up. HT-1080 cell suspension whole blood was dropped in 200 μL each on the surface of PET substrate coated with copolymer AB and PMEA using a polymer solution having a concentration of 5 wt% in section (1.7) and untreated PET substrate. It left still for 1 hour in the heat-retaining incubator. The seeding density of HT-1080 cells at this time was 1 × 10 ⁴ cells / cm ² . After 1 hour, the substrate was tilted to remove HT-1080 cell suspension whole blood and washed twice with PBS solution. The washed substrate was immersed in a dish containing a 1% glutaraldehyde PBS solution and allowed to stand in a 37 ° C. incubator for 2 hours, thereby fixing the cells adhered on the substrate. Thereafter, the substrate was immersed in a PBS solution for 10 minutes and a solution prepared in PBS solution: MilliQ water = 1: 1 for 8 minutes, and further, glutaraldehyde was washed and removed by repeating the immersion in MilliQ water twice for 8 minutes. . The washed substrate was air-dried overnight, and platinum-palladium was vacuum-deposited with an ion coater (JFC-1200 FINE COATER, JEOL), and observed as a scanning electron microscope sample at 200 times by SEM.

  FIG. 8 shows a scanning electron microscope image of each sample. As shown in FIG. 8, it was observed that many cells adhered to the PET surface by contact with HT-1080 cell suspension whole blood, whereas the cell adhesion density was low on the surface of PMEA and copolymer AB. It was. From the contrast with the seeding density of HT-1080 cells, most of the cells adhering to the PET surface are considered to be blood cells, and even when whole blood is used, the adhesion of blood cells to the surface of PMEA and copolymer AB is It was shown to be suppressed.

[Example 4] Confirmation of solubility of polymer showing LCST in aqueous medium As a premise for examining the detachability of cells adhered to various polymer surfaces, polymer showing LCST by the following method using copolymer AB Was confirmed to be soluble in an aqueous medium.
Using a 24-well cell culture plate used for examining the solubility of a polymer exhibiting LCST in an aqueous medium in association with cooling, the state of temperature change during the examination of solubility was measured in advance. In FIG. 3, 1 mL of serum-free DMEM / F-12 medium is placed in each hole of the used 24-well cell culture plate and heated to 37 ° C., and then immediately 10 ° C. or 15 ° C. Fig. 6 shows the results of measuring the temperature change of the culture medium when it is left in a pre-maintained container with a probe thermometer (As One CT1200D) over time. FIG. 3 also shows the LCST (18 ° C.) of copolymer AB when DMEM / F-12 medium is used as a solvent, as an example of LCST.

  As shown in FIG. 3, after standing in a 10 ° C. container, the temperature of the medium decreased to 10 ° C. and stabilized in about 30 minutes. Similarly, when left in a 15 ° C. container, the temperature of the medium decreased to 15 ° C. and stabilized in about 30 minutes. In addition, as shown in FIG. 3, for example, if the copolymer AB has an LCST of 18 ° C., it is about 10 minutes when left in a 10 ° C. container, and is left in a 15 ° C. container. It was thought that the dissolution started at a temperature below LCST in about 15 minutes.

  Next, in order to examine the dissolution behavior of the polymer having LCST when the temperature change occurs, the time-dependent change in the static contact angle between the PET substrate coated with the copolymer AB and the untreated PET substrate with water is shown. It was measured. Specifically, the PET substrate coated with the copolymer AB using each polymer solution having a concentration of 1 wt% in section (1.7) and the untreated PET substrate are placed in the 24-well cell culture plate, and 1 mL of phosphate buffered saline heated to 37 ° C. was added to each hole, and immediately left in a container maintained at 10 ° C. or 15 ° C. for a predetermined time (maximum 90 minutes). Phosphate buffered saline is removed from the cell culture plates that have been allowed to stand for each time, each substrate is washed with 1 mL of ultrapure water at 70 ° C., and then left overnight at room temperature to dry each substrate. The static contact angle with water was measured. The static contact angle is measured by using a contact angle measuring device (Eruma Sales Co., Ltd., ERMA, catalog number G-1-1000), dropping 2 μL of pure water and measuring the contact angle after 30 seconds as θ / 2. Measured by the method.

  FIG. 4 shows the static contact angle between the untreated PET substrate subjected to the above treatment and the water of the PET substrate coated with the copolymer AB, with respect to the stationary state. The static contact angle of the PET substrate with water was not observed after any treatment. On the other hand, the PET substrate coated with copolymer AB had a static contact angle with water of about 34 ° before treatment, whereas when cooled at 10 ° C, it started to rise 10 minutes after the start of cooling. After 30 minutes, the value was almost the same as that of the PET substrate. In the case of cooling at 15 ° C., the static contact angle started to increase 15 minutes after the start of cooling, and showed almost the same value as that of the PET substrate 55 minutes later.

  Since the concentration of the salt contained in the DMEM / F-12 medium and the phosphate buffered saline is equivalent, and it is considered that there is no significant change in the LCST, the comparison with FIGS. It is considered that the coated copolymer AB starts to dissolve quickly when it reaches a temperature of LCST or lower in an aqueous medium, and the PET substrate is exposed after a certain time. Moreover, since the degree of supercooling increases at lower temperatures, the dissolution rate is considered to increase.

[Example 5] Evaluation of recoverability of cancer cells adhering to the surface of the polymer For each of the polymers AB, Copolymer AC, Homopolymer A, and PMEA in which selective adhesion of cancer cells and the like occurs, adhesion to the polymer surface The recoverability of the treated cancer cells in an aqueous medium was examined by the following method.
A PET substrate coated with each polymer using each polymer solution having a concentration of 1 wt% or 5 wt% was washed with phosphate buffered saline, and then placed in a DMEM / F-12 medium containing 10% fetal bovine serum. Acclimatized by immersion for 1 hour. Thereafter, the human fibrosarcoma cell line HT-1080 suspended in DMEM / F-12 medium containing 10% fetal bovine serum was seeded at 1 × 10 ⁴ cells / cm ² and incubated at 37 ° C. for 24 hours. Cultured to allow HT-1080 cells to adhere to the polymer surface. Subsequently, after each substrate was allowed to stand at 10 ° C. or 15 ° C. for 45 minutes or 90 minutes in the above medium, the cells that had not adhered were shaken at 500 rpm for 30 seconds using a shaker (IKA). A sample was separated from the substrate. Similarly, a sample that was only subjected to a shaker treatment at 37 ° C. without standing at 10 ° C. or 15 ° C. was produced.

Each sample was rinsed twice with PBS, and the cells adhered to the substrate with 0.1% glutaraldehyde were fixed. Then, the cells were stained and visualized with a 0.2% crystal violet solution. Each sample was observed under an optical microscope, and the number of HT-1080 adhered to an area of 1 × 10 ⁴ μm ² was counted. The recovery rate of HT-1080 on various substrates is the number of cells adhering to the substrate subjected to only the shaker treatment (A), adhering to the substrate subjected to the stationary treatment and the shaker treatment at 10 ° C. or 15 ° C. The number of cells (B)
It was calculated as (1-([number of cells (B)] / [number of cells (A)])) × 100 (%).

  FIG. 5 shows the recovery rate of HT-1080 cells adhered on various substrates showing selective adhesion to cancer cells and the like by performing a static treatment at 10 ° C. for 90 minutes in the above medium. (Peeling rate). In addition, from the examination result in Example 4, it is considered that the dissolution of the copolymers AB, AC, and homopolymer A having LCST is completed by the standing treatment in the medium at 10 ° C. for 90 minutes.

As shown in FIG. 5, HT-1080 cells were recovered in the medium at a high rate by allowing the copolymers AB and AC having LCST to stand for 10 minutes at 10 ° C. in the medium for lysis. On the other hand, the homopolymer A has an LCST in the medium of about 13 ° C., and although it is considered that the HT-1080 cells are dissolved in the same manner as the copolymers AB and AC, a large proportion of HT-1080 cells are on the substrate. It was difficult to recover in the medium. In addition, since PMEA does not show LCST, it is considered that PMEA film is present on the substrate even after the treatment, and HT-1080 cells are adhered.
Further, as a result of coating using polymer solutions having different concentrations for copolymers AB and AC, when the concentration of the polymer solution is low and the polymer coating is thin, the recovery rate of HT-1080 cells tends to decrease. As can be seen, in order to obtain a sufficient recovery rate, it was inferred that a predetermined film thickness was required.

  FIG. 6 shows the cell recovery rate of HT-1080 cells adhered on a PET substrate coated with copolymer AB when the temperature at which the polymer is dissolved is changed. Cells can be efficiently collected in a short time by dissolving the polymer at 10 ° C., which has a large degree of supercooling against LCST. However, by dissolving the polymer over a sufficient period of time, A recovery was shown to be obtained. Moreover, in PMEA which does not show LCST, the significant change was not produced in the collection | recovery rate of the cell by hold | maintaining at 10 to 15 degreeC.

[Example 6] Evaluation of cytotoxicity of polymer The copolymer AB showing good selective adhesion and recoverability to cancer cells and the like was evaluated for cytotoxicity to cells as follows. (1) A PET substrate coated with copolymer AB by the method described in section 1.7 is immersed in a 24-well cell culture plate containing 1 mL of DMEM / F-12 medium containing 10% fetal calf serum. The copolymer AB was sufficiently dissolved in the medium by allowing it to stand at 10 ° C. overnight. The human fibrosarcoma cell line HT-1080 is suspended in a medium in which the copolymer AB is dissolved, and the cells are seeded in a 96-well cell culture plate (Asahi Glass, IWAKI) so that the number of cells is 1 × 10 ⁴ per 1 cm ^2. Culture was performed for 1 or 2 days. Use Cell Counting Kit (WST-8 method: Dojindo), which reacts with formazan salt according to the mitochondrial cell metabolic activity and colors before culturing, after culturing for 1 day, after culturing for 2 days. The absorbance was measured by measuring the absorbance of the colored medium at a wavelength of 450 nm.

  FIG. 7 shows the evaluation results of cytotoxicity. Regardless of the dissolution of the copolymer AB, almost equivalent cell growth was observed in each cell culture medium. This result indicates that copolymer AB is not cytotoxic.

Claims (8)

A cell suspension in which a target cell is suspended is brought into contact with an antithrombotic material having a lower critical solution temperature in the range of 0 to 37 ° C. and having cell adhesion properties at a temperature equal to or higher than the lower critical solution temperature of the material. Attaching the target cell to the surface of the material;
Maintaining the material in an aqueous medium at a temperature below the lower critical solution temperature and dissolving the material in the aqueous medium to suspend and recover the target cells in the aqueous medium. A method for recovering a target cell.   The method for recovering target cells according to claim 1, wherein the antithrombotic material is a polymer containing a copolymer. The antithrombotic material is represented by the general formula (I):

[Wherein, R ¹ represents a hydrogen atom or a methyl group, R ² represents a methyl group or an ethyl group, m represents 2 or 3, and n represents the number of repetitions] The method for recovering a target cell according to claim 1 or 2, wherein the polymer comprises a copolymer containing a repeating unit selected from the group consisting of:   The method for recovering a target cell according to any one of claims 1 to 3, wherein the target cell is a cancer cell or a stem cell. Formula (I):

[Wherein, R ¹ represents a hydrogen atom or a methyl group, R ² represents a methyl group or an ethyl group, m represents 2 or 3, and n represents the number of repetitions] A temperature-responsive polymer comprising a copolymer comprising a repeating unit selected from:   6. The temperature-responsive polymer according to claim 5, wherein the temperature-responsive polymer is used for recovering target cells into an aqueous medium. In the formula (I), a repeating unit in which R ¹ is a hydrogen atom, R ² is an ethyl group, and m is 2, a repeating unit in which R ¹ is a methyl group, R ² is a methyl group, and m is 2, The temperature-responsive polymer according to claim 5 or 6. In the formula (1), a repeating unit in which R ¹ is a hydrogen atom, R ² is an ethyl group, and m is 2, a repeating unit in which R ¹ is a hydrogen atom, R ² is a methyl group, and m is 3, The temperature-responsive polymer according to claim 5 or 6.