JP2012105579A - Method for separating cell, for separating cell from solution, and hydratable composition for cell sorting - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple method for separating cells, having high selectivity, and a hydratable composition usable as a tool for cell sorting.SOLUTION: This method for separating cells is characterized by bringing the hydratable composition having ≤30 wt.% of intermediate water in contact with a solution, and adsorbing the cells in the solution on the surface of the hydratable composition to separate the cells.

Description

  The present invention relates to a cell separation method for selectively adsorbing and separating predetermined cells existing in a living body, and a hydratable composition used in the method.

Separation / recovery methods such as selective separation / recovery of target substances such as cells, bioactive substances and proteins from biological tissue fluids such as blood and body fluids, and separation / removal of bacteria and viruses from biological tissue fluids The technology is used for autoimmune diseases, AIDS, and prevention of acute rejection after transplantation. Also, isolation and recovery of hematopoietic stem cells expressing CD34 ⁺ , collection of lymphocytes with autoreactive antigen receptors for the treatment of autoimmune diseases, and lymphocytes from bone marrow transplantation cells Removal and the like are effectively used for hematopoietic malignant tumor, cancer treatment and the like.
Furthermore, separation of cells is an important technique for cell medicine such as detection and treatment of leukemia cells. In recent years, it has been shown that stem cells differentiate and proliferate into various organs, and it is possible to isolate and collect stem cells, progenitor cells, various differentiation-inducing factors / growth factors, and other physiologically active substances without damaging them. This technology is indispensable for the field of treatment and diagnosis of diseases.
Currently used cell and bioactive substance separation methods include membrane separation, centrifugation, electrical separation, killing cells other than target cells, and immobilizing ligands that have affinity for target cells There are a method for binding target cells to the carrier, a separation method using magnetic beads, etc., each of which is used for a specific application.

As a method for separating cells using a material that selectively adsorbs cells, Patent Document 1 discloses a cell separation material having a polymer substrate, a stimulus-responsive polymer, and a site that selectively adsorbs target cells. The adsorbent is shown.
Among the conventional techniques for selectively separating and recovering a target substance from the biological tissue fluid described above, the techniques for sorting specific cells in particular have problems due to the principle used for cell separation, and are not always common. It cannot be said that it can be used.
For example, the membrane separation method and the centrifugal separation method are methods that separate according to the difference in cell size and specific gravity. Therefore, separation is possible when there are large differences in physical properties such as white blood cells, red blood cells, and platelets. However, it cannot be used when the physical difference between the target substances is small, such as separation of a subpopulation of leukocytes (for example, separation of T and B cells), and there is a disadvantage that the specificity is low.
In addition, the electrical separation method uses a difference in charge characteristics and dielectric characteristics of cells in an electric field, but also has limited cell selectivity. Various methods for killing cells other than the target cells have been studied and developed, but the target cells may be damaged and cause side effects.
In the magnetic bead method, a target cell is labeled with magnetic beads by incubating a cell mixture with magnetic beads to which antibodies are bound, and the labeled cells are separated from unlabeled cells using a magnetic device. It is. In this method, it is necessary to incubate for a long time in order to efficiently bind the beads to the target cells. As a result, nonspecific adsorption of cells other than the target may occur, leading to a decrease in the purity of the target cells. In addition, it is difficult to recover target cells adsorbed on small magnetic beads.
In the immunosorbent column method, a ligand such as an antibody against a membrane antigen of a target cell is immobilized on the surface of a substrate such as a bead, and this is packed into a column to perform cell separation. As a method using the interaction between ligands such as immunoadsorption, a method using a bond between biotin and avidin has been disclosed, but since the binding force between biotin and avidin is very strong, separation is performed. It is difficult to collect the collected cells, and there are disadvantages such as a reduction in the collection efficiency. Since these methods use antibodies, they have the advantage of high selectivity to target cells. However, since recovery of adsorbed cells uses enzymes such as protease and papain, the recovery rate is low and the cells themselves are damaged. Has the disadvantage of being large.

On the other hand, when it is considered that the biological tissue fluid such as blood and body fluid after selectively separating and collecting the desired target substance is returned to the human body, the blood that has contacted the medical device during the separation and collection There is a need to avoid bodily fluids being adversely affected. On the known surface, which is said to have little adhesion of blood components when coming into contact with medical devices, medical materials based on 2-hydroxyethyl methacrylate (HEMA) and polyvinyl alcohol are known (Nobuo Nakabayashi, Kazuhiko Ishihara, Yasuhiko Iwasaki: Biomaterials, Corona (1999) or Tsuruta, T. Adv. Polym. Sci. Contemporary topics in polymeric materials for biomedical applications 1996, 126, 1-51. It is used as a carrier for artificial vitreous and drug delivery systems. However, since it has a hydroxyl group, activation of the complement system occurs and is a problem. In addition, on the surface where polyacrylamide, a hydrophilic gel used as an electrophoresis gel, is introduced, platelets are activated upon contact with blood, and when the blood is returned to the body, thrombus formation is induced. It was a problem to do.
Other medical materials include polyethylene glycol (poly (ethylene glycol), PEG) (Mori, Y .; Nagaoka, S .; Takiguchi T, Kikuchi, T, Noguchi N, Tanzawa, H .; Noishiki, Y. A new antithrombogenic material with long polyethylene oxide chains, Trans. ASAIO. 1982, 28, 459- 463. Or Harris JM, ed, Poly (ethylene glycol) chemistry, Biotechnical and Biomedical Applications Plenum Press, New York (1992). PEG has very good biocompatibility, and many applied studies in the medical field have been conducted. However, the target biological substance or the like could not be selectively adsorbed.

  Patent Documents 2 and 3 and Non-Patent Document 1 describe the use of materials that are highly adaptable to the human body, mainly focusing on the surface structure of polymer materials.

JP 2006-158305 A WO2004 / 087228 JP 2004-161954 A

Biomaterial 28-1, 2010

The present invention aims to provide a simple and highly selective cell separation method and a hydratable composition that can be used in a cell sorting instrument, compared to the conventional techniques described above.
In addition, a cell separation method capable of suppressing adverse effects on blood or body fluids to be separated, and a hydratable composition that can be used for a cell sorting device, particularly when the cells are separated. With the goal.

In order to solve the above problems, the present invention has the following features.
<1> A hydratable composition having an intermediate water amount of 30 wt% or less is brought into contact with the solution, and cells in the solution are adsorbed on the surface of the hydratable composition and separated from the solution. Cell separation method.
<2> The cell separation method as described above, further characterized in that the amount of intermediate water in the hydratable composition is 1 wt% or more.
<3> The cell separation method as described above, wherein the hydratable composition is a polymer containing 60 mol% or more of poly (2-methoxyethyl acrylate).
<4> Hydrating composition for cancer cell sorting characterized in that the amount of intermediate water is 30 wt% or less <5> Stem cell sorting water characterized in that the amount of intermediate water is 30 wt% or less <6> A tube for circulating blood, characterized in that at least part of the surface in contact with blood is made of a hydratable composition in which the amount of intermediate water is 1 to 30 wt%. Tube to do.

  According to the adhesive device or the material of the medical device, the adhesive device, and the bonding method of the present invention, it is possible to bond the target biological substance or the like to the bonding surface selectively and without alteration. Furthermore, it is possible to provide a blood safety and a highly safe adhesive device such as a material or a biological substance, a medical device, or the like.

It is a graph which shows the result of the DSC measurement in the water-containing PMEA. It is a graph which shows the result of DSC measurement in PMEA which changed moisture content. It is a schematic diagram which shows the structure of the water layer formed in the hydratable composition surface. It is a figure explaining the characteristic of each layer which comprises the water layer formed in the hydratable composition surface. It is a graph which shows the result of the DSC measurement in a MEA-HEMA copolymer. It is a graph which shows the structure of the surface water layer of a MEA-HEMA copolymer. It is a graph which shows the relationship between the amount of intermediate water on the surface of a hydration composition, and the frequency which various cells adsorb | suck.

As a result of examining the adsorptivity to various substances for various cells present in blood and body fluid in the body, the present inventor determined that the surface according to the state of the surface water layer formed on the surface of the substance It was found that the cell adsorption frequency when adsorbing on the cell changes, and that the correlation between the surface water layer state and the cell adsorption frequency varies greatly depending on the cell type. By utilizing this phenomenon, it becomes possible to selectively adsorb and separate various cells present in the body fluid in the body, and the present invention has been completed. This will be described in detail below.
By bringing various substances into contact with water, a certain amount of water is constrained in a predetermined form on the surface of the substance, so that the surface of the substance is hydrated and hydrated, and the surface has a predetermined structure. It is known that an aqueous layer is formed. Further, it is known that the structure and thickness of the surface water layer change depending on the structure near the surface of the substance. In this way, as a hydratable composition capable of hydrating by constraining a certain amount of water on its surface, typically, in addition to substances that can be hydrated with a large amount of water, such as gelatin and collagen, In addition, it is known that various organic and inorganic substances can form a surface water layer by constraining a certain amount of water on the surface by hydration.

About the poly (2-methoxyethyl acrylate (PMEA)) as an example of the hydration composition in which these surface water layers were formed, the characteristic measurement result at the time of measuring by a differential scanning calorimeter (DSC) Is shown in FIG. FIG. 1 shows that poly (2-methoxyethyl acrylate (PMEA)) containing 9 wt% water was cooled to −100 ° C. and then heated from −100 ° C. to 50 ° C. at a rate of 2.5 ° C. per minute. The state of heat absorption and heat generation observed at the time is shown.
As shown in FIG. 1, when a hydratable composition containing a predetermined amount of water is once sufficiently cooled and then heated at a relatively slow rate, it is determined at a specific temperature range of 0 ° C. or lower. It is clarified that endotherm is observed in a wide temperature range from around −10 ° C. to 0 ° C. (see, for example, Non-Patent Document 1). In FIG. 1, a remarkable heat generation occurs in the vicinity of −40 ° C. When normal frozen water (ice) is heated, an endotherm is generated as heat of fusion at its melting point of 0 ° C., and transformation occurs in liquid water. It is understood that the phenomenon described above is caused by the unique behavior of water restrained by the surface water layer on the surface of the hydrated composition containing water. In addition, when the surface water layer of the hydratable composition is heated from a sufficiently low temperature to 0 ° C. or higher, the total endotherm (= endotherm−exotherm) measured by DSC measurement is Since it does not match the water content, it is also known that there is water that is not related to the measurement of DSC measurement by not causing transformation by heating or cooling.

In FIG. 2, the result of having performed DSC measurement about what made water of water each different in PMEA is shown. When the water content is 1.8 wt% or less, the exotherm near −40 ° C. is not observed, but the exotherm increases until the water content reaches about 7 wt%, but the water content further increases. However, it has been observed that the calorific value is saturated and does not change. On the other hand, it is known that the moisture content of PMEA varies depending on the moisture content, such that the endotherm in the vicinity of 0 ° C. becomes noticeable when the moisture content becomes 7 wt% or more.
Regarding the heat generation near −40 ° C., since it is known that the glass transition point of PMEA exists in the vicinity of −40 ° C., the heat generated from the water solidified in a metastable state by supercooling is It is presumed to be caused by the occurrence of ordering (cold crystallization) due to heating above the glass transition point of PMEA. In addition, the calorific value associated with the cold crystallization is presumed to be proportional to the amount of water causing the ordering, and the surface state of the hydratable composition can be evaluated by measuring the calorific value. It is considered a thing.
Table 1 shows the calorific value (ΔH _CC ) accompanying cold crystallization and the endothermic amount (ΔHm) observed between −20 ° C. and 0 ° C. obtained from the results of each DSC measurement shown in FIG.

As is apparent from Table 1, when the water content is 1.8 wt% or less, no latent heat due to the water contained in the water is observed, whereas when the water content is higher, the cold crystal An endotherm (ΔH _m ) of almost the same amount as the calorific value (ΔH _CC ) associated with the rise is observed. Further, it can be seen that when the water content is 7.0 wt% or more, the heat generation amount associated with cold crystallization is kept constant, and the heat absorption amount (ΔH _m ) near 0 ° C. increases.

  Based on the above findings, the present inventors have clarified the structure of the surface water layer present on the surface of the hydratable composition. 3 and 4 show an outline of the structure of the surface water layer on the surface of the hydratable composition clarified by the present inventors. As shown in FIG. 3, the surface of the water-containing hydratable composition is constrained by strong interaction with the composition surface, so that phase transformation such as freezing / thawing is performed at least in the range of −100 ° C. to room temperature. A layer of water that does not generate water is formed, and the inventors define water belonging to this layer as “non-freezing water”. As shown in FIG. 4, since this antifreeze water is strongly restrained by forming some bond with the surface of the hydratable composition, it cannot perform free behavior as water. It is considered that no phase transformation such as freezing / thawing occurs in the range of. On the other hand, it is inferred that a part of the saturated water content is formed by desorption by strong heating in a dry atmosphere.

Further, it is presumed that a layer of “intermediate water” constrained by the interaction with the composition surface and antifreeze water exists outside the antifreeze water (FIG. 4). The details of the interaction of intermediate water with the composition surface and antifreeze water are not clear, but based on various findings, water molecules contained as intermediate water have a certain degree of freedom above 0 ° C. On the other hand, it is presumed that at least at a temperature of about −40 ° C. or less, there is a strong tendency to order by interaction with the composition surface and antifreeze water. Then, due to the presence of a strong tendency to order, in the heating from the state solidified in an irregular state, heat is generated due to the ordering near −40 ° C., and again in the range from around −10 ° C. to 0 ° C. It is presumed that it becomes irregular and generates heat.
Furthermore, it is speculated that there is a weakly constrained “free water” layer outside the intermediate water, which causes a sharp endothermic peak at 0 ° C. during the DSC measurement (FIG. 4). When the hydrating composition is placed in water, this free water can be exchanged relatively freely with the water in the surrounding aqueous phase (bulk water), while it interacts with intermediate water. It is considered that it is difficult to remove from the surface and forms a surface water layer.

In addition, the structure of the surface water layer on the surface of PMEA characterized by the results of DSC measurement of the PMEA has been observed also in hydrating compositions other than PMEA. In addition, it is known that the amounts of antifreeze water, intermediate water, and free water constituting the surface water layer vary depending on the type of hydratable composition. FIG. 5 shows a random copolymer obtained by copolymerizing by a conventional method by changing the ratio of 2-methoxyethyl acrylate (MEA) and 2-hydroxyethyl methacrylate (HEMA) used above. The results of DSC measurement as described above are shown. By increasing the ratio of HEMA with respect to MEA, the peak of exotherm accompanying cold crystallization, which is observed at around -40 ° C., is flattened, and in the case of containing 50 mol% or more of HEMA, the peak is observed. Will not be.
FIG. 6 shows the contents of antifreeze water, intermediate water and free water in the copolymer obtained by mixing MEA and HEMA shown in FIG. The contents of the antifreeze water, intermediate water, and free water can be obtained from the behavior of the exothermic peak accompanying cold crystallization and the total water content as shown in FIG. As shown in FIG. 6, in the copolymer of MEA and HEMA, the amount and ratio of antifreeze water, intermediate water, free water contained in each copolymer are changed by appropriately changing the ratio. can do.

  And this invention is based on having discovered that adsorption | suction frequency of various cells etc. changed according to the quantity of the intermediate water which exists in the surface of a hydration composition as shown in a following example. . In other words, substances that show little hydration and do not form a surface water layer, or even a hydrating composition, the surface water layer is composed of antifreeze water or free water, and there is virtually no intermediate water. On the surface of the substance, adsorption occurs at a high adsorption frequency for all cells and plasma proteins tested, whereas on the surface of a hydrating composition with a large amount of intermediate water, the adsorption frequency of those cells and plasma proteins is high. A downward trend was observed. In addition, since the amount of intermediate water whose adsorption frequency decreases greatly varies depending on the type of cells, etc., by selecting a hydratable composition having an appropriate amount of intermediate water according to the purpose and using it as an adsorbent for cells, etc., It becomes possible to selectively adsorb and collect cells.

  Although the reason why the adsorption frequency of cells and the like changes depending on the amount of intermediate water present on the surface of the hydratable composition is not clear, the intermediate water does not move due to its weak interaction with the surface of the hydratable composition. It is presumed to exist in a state where the degree of freedom is limited, and it is presumed that it acts as a kind of cushioning material that prevents cells and the like from directly contacting the surface of the hydratable composition. In other words, biological components such as cells and proteins are stabilized by forming hydration shells in the blood and body fluids, and these hydration shells are in direct contact with the surface of foreign substances and their antifreeze water to disrupt or destroy them. If this is done, it is thought that this will trigger the adsorption and activation of biological components on the surface of the material, but it is presumed that the adsorption is suppressed by the presence of intermediate water, and that the strength of the adsorption is affected ( Figure 2).

The amount of intermediate water on the surface of a hydratable composition suitable for adsorbing various cells well varies depending on the type of cell and the purpose of adsorption, but relatively large cells such as so-called cancer cells and stem cells. Can be adsorbed by using a hydratable composition in which the amount of intermediate water is 30 wt% or less of the hydratable composition when the hydratable composition is sufficiently hydrated in water. In particular, by using a hydratable composition in which the amount of intermediate water is 10 wt% or less, the adsorption frequency is saturated and a high adsorption ability can be obtained. In addition, when trying to adsorb relatively small floating cells or plasma proteins such as platelets, a hydrating composition in which the amount of intermediate water is 3 wt% or less can be used with a sufficient adsorption frequency. Can be adsorbed.
On the other hand, if a hydrating composition with an excessively small amount of intermediate water is used, the adsorbed cells can be damaged by the surface of the hydrating composition and the antifreeze water present there. Therefore, it is not preferable when the adsorbed and separated cells are used in culture. On the other hand, by using a hydrating composition in which the amount of intermediate water is 1 wt% or more, adsorption can be performed without damaging cancer cells or stem cells while avoiding adsorption of platelets and plasma proteins. It becomes possible. In particular, by using a hydrating composition in which the amount of intermediate water is 3 wt% or more, adsorption is performed without altering cancer cells and stem cells while effectively avoiding adsorption of platelets and plasma proteins. Is possible.

  The substance that can be separated and recovered according to the present invention is not particularly limited as long as it is a substance that forms a hydration shell in blood or body fluid. For example, metastatic cancer cells contained in blood or the like Tumor cells such as leukemia cells. Stem cells, platelets, vascular endothelial cells, neurons, macrophages, dendritic cells, mononuclear cells, neutrophils, smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle cells, liver parenchymal cells, liver non-parenchymal cells, pancreas In addition to functional cells such as ra islet cells, application to plasma proteins and the like is also possible.

In addition, the hydrating composition used in the present invention may have any amount of intermediate water depending on the purpose and may have no adverse effect on the adsorbed cells. For example, it has predetermined intermediate water. Examples thereof include inorganic substances such as organic polymers and silica gel, proteins such as gelatin, collagen and albumin, polysaccharides such as hyaluronic acid and chondroitin sulfate. In addition, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polyvinyl methyl ether (PVME), poly (2-methacryloyloxyethyl phosphorylcholine), polytetrahydrofurfuryl acrylate, etc., which are known as biocompatible polymers, It can be used in a state where the influence on is suppressed. Further, examples of preferable polymers include poly (2-ethoxyethyl acrylate), poly (2-methoxyethyl acrylate), and poly [2- (2-methoxyethoxy) ethyl represented by the following formula (1). Methacrylate], poly [2- (2-ethoxyethoxy) ethyl acrylate], poly [2- (2-methoxyethoxy) ethoxy] ethyl methacrylate], poly [2- (2- (2-methoxyethoxy) ethoxy) ethyl acrylate ], Poly [2- (2-ethoxyethoxy) ethyl methacrylate], polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl methyl ether (PVME), methoxyethyl (meth) acrylamide, methoxyethyl vinyl ether, polytetrahydrofurfuryl (Meth) acrylate and the like are included.
[Wherein, R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, m is 1 to 3, and n is a repeating unit]
Among these polymers, poly (2-methoxyethyl acrylate (PMEA)) represented by the following formula (2) is particularly preferable because of excellent biocompatibility.
Moreover, you may use as a material which has a desired intermediate | middle water amount by mixing the monomer which comprises these polymers with another monomer, and making it a copolymer.

  The above polymers only have ether bonds and ester bonds as polar groups. These polar groups are different from nitrogen atoms (amino groups, imino groups), carboxyl groups, etc. Has no interaction. For this reason, the above-mentioned polymer has low activity against blood and does not have a large hydrophobic group, and therefore has a small hydrophobic interaction. From the above, it is estimated that the above-described polymer exhibits excellent biocompatibility. For example, considering the contact between the surface of a synthetic polymer material and a protein in living tissue or blood, a surface that does not undergo protein adsorption / denaturation or activation is preferable, and hydrophobic interaction, which is a large interaction between substances, is preferable. It is considered useful to reduce the action and electrostatic interaction. Therefore, also from this, the adhesive device and medical device of the present invention using the above-described polymer as a material can have a suitable surface structure.

  Moreover, the surface of a medical device or the like using the above-described polymer as a material has moderate hydrophilicity without the presence of a hydroxyl group. For this reason, when it is used as a material for a medical device used for the purpose of blood purification or the like, adhesion of platelets becomes slight when it comes into contact with blood, and excellent blood compatibility is exhibited. Furthermore, it is possible to prevent interaction with biological components due to hydrogen bonding caused by hydroxyl groups, denaturation of adsorbed proteins, and the like.

  In the cell separation method according to the present invention, cells and the like separated from an aqueous solution such as a body fluid by being adsorbed to the hydratable composition are detached from the hydratable composition by an appropriate method, for example, cancer If it is a cell, it can be used for screening of an anticancer agent by culturing it, or if it is a stem cell, it can be used for differentiation and proliferation to a desired organ. Thus, when the adsorbed cells are used for various purposes, it is preferable to suppress damage when the cells are detached from the hydratable composition. It is also preferable to remove the adsorbed cells from the hydratable composition by increasing the amount of intermediate water of the hydratable composition by changing the temperature or the like after adsorbing.

  For example, poly [2- (2-ethoxyethoxy) ethyl acrylate] (PEEA) has a low critical solution temperature (LCST) of 14 ° C. with excellent biocompatibility, and it can be dissolved in an aqueous solution at a temperature below this temperature. It has the characteristic of dissolving. For this reason, for example, when cells are adsorbed from blood collected from a human body to a hydratable composition mainly composed of PEEA at a temperature substantially equal to the body temperature, the adhesive device is heated to a temperature lower than 14 ° C. Etc. can be easily detached from the adhesive device or the like. Thus, without causing a special reaction, cells or the like are contained in the blood by quickly detaching the target biological substance such as cells or the target physiologically active substance from the hydratable composition. It can be used for subsequent tests and evaluations as it is. In addition, a substance that changes from hydrophobic to hydrophilic according to temperature by having a lower critical solution temperature (LCST) as described in Patent Document 2 is used as a hydrating composition. It is possible to use.

  On the other hand, when a hydrating composition that selectively adsorbs vascular endothelial cells and separates them from blood while preventing the adsorption of blood cells such as platelets, an artificial blood vessel with a small inner diameter is formed. Which is advantageous. In other words, vascular endothelial cells contained in blood adhere to the inner wall of the blood vessel, so that platelet function and coagulation and fibrinolysis are controlled to prevent thrombus formation. Therefore, there is a problem in that blood vessel endothelial cells do not necessarily adhere well, so that thrombus formation is likely to occur, and in particular, an artificial blood vessel having a small inner diameter tends to be clogged by thrombus. On the other hand, for example, by providing at least a part of the hydratable composition in which the amount of intermediate water is 3 to 10 wt% on the surface in contact with blood, vascular endothelial cells are selectively placed on the inner wall of the blood vessel According to the present invention, it is possible to make it adhere, and it is possible to provide an artificial blood vessel or the like that is less likely to be clogged by a thrombus. This is effective when forming a fine artificial blood vessel having an inner diameter of 10 mm or less, and is particularly effective when forming an artificial blood vessel having an inner diameter of about 4 mm.

  The cell separation method according to the present invention is a kit for detecting metastatic cancer cells in blood by using a hydrating composition having a predetermined intermediate water amount as it is or by coating on a predetermined substrate, It can be applied to a detection instrument for detecting various biological substances and / or physiologically active substances such as immunochromatography, a cell separation system, and the like. Moreover, a desired added value can be given by applying to an artificial blood vessel, a vascular system stent, an artificial heart lung, etc.

  The cell separation method according to the present invention can be used for adsorbing and separating predetermined cells from blood and body fluids taken out of the human body outside the body, as well as used for adsorbing and separating predetermined cells in the human body. It is. Furthermore, it is also possible to use it to adsorb and separate desired cells in a solution in which multiple types of cells contained in the tissue are separated and dispersed in an appropriate support solution for cancer tissues taken out from the human body. It is.

  The material and shape of the substrate to which the hydratable composition used in the present invention is applied are not particularly limited, and examples thereof include porous bodies, fibers, nonwoven fabrics, particles, films, sheets, tubes, hollow fibers and powders. Either is fine. The materials include natural polymers such as cotton and hemp, nylon, polyester, polyacrylonitrile, polyolefin, halogenated polyolefin, polyurethane, polyamide, polysulfone, polyethersulfone, poly (meth) acrylate, ethylene-polyvinyl alcohol copolymer, Examples thereof include synthetic polymers such as butadiene-acrylonitrile copolymer or a mixture thereof. Moreover, a metal, ceramics, those composite materials, etc. can be illustrated and you may comprise from several base material.

Examples of a method for retaining the hydratable composition on the surface of a medical device include a coating method, a graft polymerization using radiation, electron beams and ultraviolet rays, and a method of introducing the chemical compound with a functional group of a substrate. There are known methods. Among these, the coating method is particularly preferable because it is easy to manufacture. Furthermore, there are coating methods, spraying methods, dipping methods and the like as coating methods, and any of them can be applied without any particular limitation.
For example, the coating treatment by the application method of the hydratable composition can be performed by simply immersing the filter medium in a coating solution in which the copolymer is dissolved in an appropriate solvent, removing the excess solution, and then air-drying. Can be implemented by operation. In addition, in order to more firmly fix the hydratable composition on the filter medium, heat can be applied to the filter medium after coating to further improve the adhesion between the filter medium and the hydratable composition. Moreover, you may fix | immobilize by bridge | crosslinking the surface. As a method for crosslinking, a crosslinkable monomer may be introduced as a comonomer component. Moreover, you may bridge | crosslink by an electron beam, a gamma ray, and light irradiation.
Hereinafter, the present invention will be specifically described with reference to examples. In addition, this invention is not restrict | limited by the following Example.

1, Preparation of Sample (1) Sample A: Preparation of poly (2-methoxyethyl acrylate) (PMEA) material 15 g of 2-methoxyethyl acrylate in 60 g of 1,4-dioxane (azobisisobutyronitrile (0.1) The polymerization was carried out at 75 ° C. for 10 hours with nitrogen bubbling. After completion of the polymerization reaction, the product was isolated by dropwise addition to n-hexane. The product was dissolved in tetrahydrofuran and further purified twice using n-hexane. The purified product was dried under reduced pressure overnight. A colorless and transparent syrupy polymer was obtained. The yield (yield) was 12.3 g (82.0%). The obtained polymer structure was confirmed by ¹ H-NMR. From the results of GPC molecular weight analysis, the number average molecular weight (Mn) was 26,000 and the molecular weight distribution (Mw / Mn) was 3.27. A 0.2% by weight solution of the obtained polymer (poly (2-methoxyethyl acrylate) (PMEA)) dissolved in methanol was cast on a polyethylene terephthalate (PET) disc and twice using a spin coater. Coating was performed. It was confirmed that PMEA was coated on the PET surface by static contact angle of water and X-ray photoelectron spectroscopy (XPS) measurement.

(2) Sample B: Preparation of MEA-HEMA copolymer 2-methoxyethyl acrylate (MEA) and 2-hydroxyethyl methacrylate (HEMA) with a HEMA ratio of 10, 20, 30, 40, 50, 100 mol %, And a polymer was obtained by polymerizing in the same manner as in the PMEA material, except that the mixture was mixed so as to be 15 g in total. A 0.2 wt% solution obtained by dissolving the obtained polymer in methanol was cast on a PET disc, and coating was performed twice using a spin coater.

(3) Sample C: Production of poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) material An ethanol solution of 2-methacryloyloxyethyl phosphorylcholine (MPC) was cast on a PET disk and coated using a spin coater. The poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) which was crosslinked by irradiation with 80 kGy (10 kGy / h for 8 hours) of γ-rays using ⁶⁰ Co as a radiation source. ) Was produced.

2, Measurement of intermediate water amount About each of the hydratable composition samples (Samples A to C) prepared above by exposure to a moist atmosphere or by immersing them in water for a maximum of 7 days, respectively. An appropriate amount of water was added. A predetermined amount of each water-containing sample was taken and spread thinly on the bottom of an aluminum oxide pan whose weight was measured in advance. Using a differential scanning calorimeter (DSC-8230, manufactured by Rigaku Corporation), cool from room temperature to -100 ° C, hold for 10 minutes, and then heat from -100 ° C to 50 ° C at a heating rate of 2.5 ° C / min. The amount of heat generated and absorbed during the process was measured. It was confirmed that the PET substrate used for the base material of each sample did not produce a significant amount of water.
About each sample, after DSC measurement, the pinhole was made to the aluminum pan, and it vacuum-dried, and calculated | required the weight loss as water content. The water content (WC) was determined by the following formula (I) after excluding the mass of the PET substrate used for the base material.
Water content (WC) = (W ₁ −W ₀ ) / W ₀ (I)
(W ₀ : dry weight of sample (g), W ₁ : moisture content of sample (g))

Next, for each Sample A to C (each composition for Sample B), the relationship between the calorific value associated with cold crystallization at each water content and the endothermic amount near 0 ° C., The maximum amount of frozen water and intermediate water was obtained and divided by W ₀ to obtain the amount of antifreeze water and intermediate water in each sample. Table 2 shows the amount of intermediate water for each sample. In Sample B, 50 mol% HEMA and 100 mol% HEMA, no significant amount of intermediate water was observed in the DSC measurement, so the intermediate water amount was set to 0 wt%.

3, Platelet adsorption test Saturated water-containing Samples A to C, a glass substrate and a glass substrate as a control material, and a human fresh platelet-rich plasma anticoagulated with sodium citrate and a sample surface at 37 ° C., 60 After contact for a minute, it was rinsed with a phosphate buffer solution and fixed with glutaraldehyde. Thereafter, the number of platelets adhered to the sample surface 1 × 10 ⁴ μm ² was observed with an electron microscope. The suitability with blood was evaluated by classifying into I (normal), II (pseudopod formation), and III (extension) types according to the degree of progression of changes in platelet adhesion.
FIG. 7 shows the relative number of adherent platelets in each sample with respect to the amount of intermediate water in each sample when the total number of platelets adsorbed on PET is 1000.

4. Cancer cell adsorption test
Saturated water-containing Samples A and C, and a glass substrate and a PET substrate as control materials were used to perform an adsorption test for cancer cells. Each sample was brought into contact with human fibrosarcoma cells (HT-1080) cultured in a medium supplemented with fetal calf serum at 37 ° C. for 60 minutes, rinsed with a phosphate buffer solution, and fixed with formaldehyde. The cell nucleus was stained with DAPI, the actin skeleton was stained with phalloidin antibody, and the vinculin adhesion group was stained with anti-vinculin antibody, and the number of adherent cells was measured using a confocal laser microscope.
FIG. 7 shows the relative number of adsorbed cancer cells in each sample with respect to the amount of intermediate water in each sample when the number of cancer cells adsorbed on PET is 1000. In addition, on the surface of PMPC as Sample C, adhesion of cancer cells was weak, and most cancer cells were observed to be detached when rinsed with a phosphate buffer solution.

5. Adsorption test of vascular endothelial cells An adsorption test of human vascular endothelial cells was performed using Samples A and C that had been saturated with water, and a glass substrate and a PET substrate as control materials. Human umbilical vein endothelial cells (HUVEC) used were cultured in a medium supplemented with fetal bovine serum, heparin, and endothelial cell growth supplement. Each sample was contacted with a culture solution of vascular endothelial cells at 37 ° C. for 60 minutes, rinsed with a phosphate buffer solution, and fixed with formaldehyde. The cell nucleus was stained with DAPI, the actin skeleton was stained with phalloidin antibody, and the vinculin adhesion group was stained with anti-vinculin antibody, and the number of adherent cells was measured using a confocal laser microscope.
FIG. 7 shows the relative number of adsorbed vascular endothelial cells in each sample with respect to the amount of intermediate water in each sample when the number of vascular endothelial cells adsorbed on PET is 1000. Since the adhesion of vascular endothelial cells was weak on the surface of PMPC, which was Sample C, and most cells were detached when rinsed with a phosphate buffer solution, approximate numbers were entered in FIG.

6. Adsorption test of periodontal ligament-derived (stem) cells An adsorption test of human periodontal ligament-derived cells was performed using saturated samples A and C, a glass substrate and a PET substrate as control materials. Human periodontal ligament-derived cells (PDL) were cultured in a medium supplemented with fetal calf serum. Each sample was contacted with a culture solution of vascular endothelial cells at 37 ° C. for 60 minutes, rinsed with a phosphate buffer solution, and fixed with formaldehyde. The cell nucleus was stained with DAPI, the actin skeleton was stained with phalloidin antibody, and the vinculin adhesion group was stained with anti-vinculin antibody, and the number of adherent cells was measured using a confocal laser microscope.
FIG. 7 shows the relative number of adsorbed periodontal ligament-derived cells in each sample with respect to the amount of intermediate water in each sample when the number of periodontal ligament-derived cells adsorbed on PET is 1000. In addition, on the surface of PMPC as Sample C, adhesion of periodontal ligament-derived cells was weak, and most cells were observed to be detached when rinsed with a phosphate buffer solution.

7. Plasma protein adsorption test Saturated water-containing Samples A and C, a glass substrate and a PET substrate as control materials, were immersed in plasma separated from fresh human blood by a centrifuge at 37 ° C for 60 minutes. After immersion, the plasma protein was rinsed with a phosphate buffer solution, and the amount of plasma protein adsorbed on the surface of each substrate was determined by a colloidal gold dot plot method. The results are shown in Table 3.

  The amount of plasma protein adsorbed on the surfaces of PMEA and PMPC was small enough to correspond to the level at which plasma protein was adsorbed on a monolayer. On the other hand, protein adsorption was observed on the glass as well as on the PET surface in such an amount that plasma protein was thought to be adsorbed in multiple layers.

8. Intermediate water amount of each sample and cell adsorption frequency In FIG. 7, for the samples other than the PET substrate, the number of cells adsorbed is shown by the relative number when the number of cells adsorbed to the PET substrate is 1000. Yes. For this reason, considering that the number of times each cell contacts the surface of each sample and the number of times the cell contacts the PET substrate are equal, each value in FIG. It shows the frequency (probability) that occurs.

  Regarding platelets, the adsorption frequency decreased sharply as the amount of intermediate water increased, even on the MEA-HEMA copolymer, in which intermediate water was hardly observed by DSC measurement. On the other hand, in cancer cells, vascular endothelial cells, and stem cells, it was observed that the adsorption frequency hardly decreased to about 10 wt% of the intermediate water volume, but hardly adsorbed on the surface of the PMPC having the intermediate water volume of 45 wt%. It was. Thus, according to the present invention, by using a hydratable composition having an appropriate amount of intermediate water, it is possible to utilize the fact that a large difference in the tendency of adsorption frequency occurs between platelets and other cells. Thus, it becomes possible to sort out desired cells from the blood. In particular, for vascular endothelial cells, a high adsorption frequency can be maintained even in the range of intermediate water that hardly adsorbs platelets. A blood vessel can be obtained.

Claims (6)

  A cell separation method comprising contacting a hydratable composition having an intermediate water amount of 30 wt% or less with a solution, and adsorbing cells in the solution to the surface of the hydratable composition to separate the solution from the solution. .   The method for separating cells according to claim 1, wherein the amount of intermediate water in the hydratable composition is 1 wt% or more.   The cell separation method according to claim 1 or 2, wherein the hydratable composition is a polymer containing 60 mol% or more of poly (2-methoxyethyl acrylate).   A hydrating composition for cancer cell sorting, wherein the amount of intermediate water is 30 wt% or less.   A hydrating composition for fractionating stem cells, wherein the amount of intermediate water is 30 wt% or less.   A tube for circulating blood, wherein at least a part of a surface in contact with blood is made of a hydrating composition having an amount of intermediate water of 1 to 30 wt%.