Aqueous solution for separating blood cells, blood collection container, and method for separating monocytes

Abstract

Provided is an aqueous solution for separating blood cells, the aqueous solution being capable of suppressing a change in properties even when sterilized by radiation.　This aqueous solution for separating blood cells comprises: polysucrose (A); diatrizoic acid and / or a metal salt thereof (B); and carboxylic acid different from diatrizoic acid and / or a metal salt thereof (C).

Description

Aqueous solution for blood cell separation, blood collection container, and method for separating mononuclear cells

[0001] The present invention relates to an aqueous solution for separating blood cells. The present invention also relates to a blood collection container comprising the aqueous solution for separating blood cells. The present invention also relates to a method for separating mononuclear cells using the blood collection container.

[0002] In clinical testing, blood collection containers such as blood collection tubes are widely used to collect blood. As an example of a blood collection container, a blood collection container capable of separating mononuclear cells from blood is also known.

[0003] For example, Patent Document 1 listed below describes a method for separating mononuclear cells from blood by performing the following steps (a) to (d): (a) preparing a container having a closed end and an open end; (b) placing a gel-like substance and a water-soluble density gradient substance having a larger specific gravity than the gel-like substance in the container; (c) placing a liquid sample in the container; and (d) placing the container under centrifugal force to separate the liquid sample into a heavy phase and a light phase, and establishing a barrier layer consisting of separate layers of the gel-like substance and the water-soluble density gradient substance between the heavy phase and the light phase.

[0004] Japanese Unexamined Patent Publication No. 61-84557

[0005] To separate mononuclear cells from blood, a blood collection container containing an aqueous solution for blood cell separation, sometimes called a specific gravity solution or density gradient solution, is sometimes used. By centrifuging the blood collection container containing the collected blood, mononuclear cells can be separated from blood cell components other than mononuclear cells.

[0006] Sterilized blood collection containers are sometimes used. For example, blood collection containers used as medical devices must be sterilized. However, it is difficult to increase the yield of mononuclear cells using conventional sterilized blood collection containers. In particular, it is difficult to increase the yield of mononuclear cells using conventional blood collection containers sterilized by radiation.

[0007] The present inventors have conducted extensive research and found that the difficulty in recovering a large amount of mononuclear cells using conventional sterilized blood collection containers is due to changes in the properties of the aqueous solution for blood cell separation during sterilization. Furthermore, the present inventors have found that the properties of the aqueous solution for blood cell separation are particularly susceptible to change during sterilization by radiation.

[0008] An object of the present invention is to provide an aqueous solution for blood cell separation that can suppress changes in properties even when sterilized by radiation. Another object of the present invention is to provide a blood collection container containing the aqueous solution for blood cell separation. A further object of the present invention is to provide a method for separating mononuclear cells using the blood collection container.

[0009] This specification discloses the following aqueous solution for blood cell separation, blood collection container, and method for separating mononuclear cells.

[0010] Item 1. An aqueous solution for separating blood cells, comprising polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid other than diatrizoic acid and / or a metal salt thereof (C).

[0011] Item 2. The aqueous solution for separating blood cells according to Item 1, wherein the content of the carboxylic acid and / or metal salt thereof (C) is 0.01% by weight or more and 5% by weight or less in 100% by weight of the aqueous solution for separating blood cells.

[0012] Item 3. The aqueous solution for separating blood cells according to Item 1 or 2, wherein the carboxylic acid and / or metal salt thereof (C) comprises a carboxylic acid having from 1 to 10 carbon atoms and / or a metal salt thereof.

[0013] Item 4. The aqueous solution for separating blood cells according to any one of Items 1 to 3, wherein the carboxylic acid in the carboxylic acid and / or metal salt thereof (C) includes at least one selected from the group consisting of citric acid, acetic acid, lactic acid, succinic acid, and malic acid.

[0014] Item 5. The aqueous solution for separating blood cells according to any one of Items 1 to 4, wherein the aqueous solution for separating blood cells has a specific gravity at 25° C. of 1.060 or more and 1.100 or less.

[0015] Item 6. A blood collection container comprising a blood collection container body, an aqueous solution for blood cell separation contained within the blood collection container body, and a blood separation material contained within the blood collection container body, wherein the aqueous solution for blood cell separation is the aqueous solution for blood cell separation according to any one of Items 1 to 5.

[0016] Item 7. The blood collection container according to Item 6, which is a blood collection container that has been sterilized by radiation.

[0017] Item 8. The blood collection container according to Item 6, which is a blood collection container sterilized by radiation, and wherein the aqueous solution for blood cell separation has a pH of 3.5 or more and 6.4 or less.

[0018] Item 9. The blood collection container according to any one of Items 6 to 8, wherein the blood collection container body has an open end and a closed end, and the aqueous solution for blood cell separation is contained within the blood collection container body closer to the closed end of the blood collection container body than the position where the blood separation material is contained.

[0019] Item 10. The blood collection container according to any one of Items 6 to 9, wherein the blood separation material is a blood separation composition.

[0020] Item 11. The blood collection container according to Item 10, wherein the blood separating composition comprises an organic component that has flowability at 25°C and an inorganic fine powder, the organic component comprising a resin, and the inorganic fine powder comprising finely powdered silica.

[0021] Item 12. The blood collection container according to any one of Items 6 to 11, further comprising an anticoagulant contained within the blood collection container body, the blood collection container body having an open end and a closed end, and the anticoagulant contained within the blood collection container body closer to the open end of the blood collection container body than the position where the aqueous solution for blood cell separation is contained.

[0022] Item 13. The blood collection container according to any one of Items 6 to 12, wherein the blood collection container body is a polyethylene terephthalate container.

[0023] Item 14. A method for separating mononuclear cells, comprising the steps of collecting blood in the blood collection container according to any one of Items 6 to 13, and centrifuging the blood collection container into which the blood has been collected.

[0024] The aqueous solution for separating blood cells according to the present invention contains polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid other than diatrizoic acid and / or a metal salt thereof (C). Because the aqueous solution for separating blood cells according to the present invention has the above-mentioned composition, it is possible to suppress changes in properties even when sterilized by radiation.

[0025] Fig. 1 is a front cross-sectional view schematically showing a blood collection container according to a first embodiment of the present invention, Fig. 2 is a front cross-sectional view schematically showing a blood collection container according to a second embodiment of the present invention, and Fig. 3 is a front cross-sectional view schematically showing a blood collection container according to a third embodiment of the present invention.

[0026] The present invention will be described in detail below.

[0027] The aqueous solution for blood cell separation according to the present invention (hereinafter sometimes abbreviated as "aqueous solution" in this specification) contains polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid different from diatrizoic acid and / or a metal salt thereof (C).

[0028] The aqueous solution according to the present invention has the above-mentioned constitution, and therefore, even if it is sterilized by radiation, it can be prevented from changing in properties.

[0029] Blood collection containers containing an aqueous solution (specific gravity solution) containing polysucrose and sodium diatrizoate are sometimes used as blood collection containers for separating mononuclear cells from blood. Sterilized blood collection containers are also sometimes used. For example, blood collection containers used as medical devices must be sterilized. The present inventors have found that it is difficult to increase the recovery of mononuclear cells using conventional sterilized blood collection containers containing an aqueous solution (specific gravity solution) containing polysucrose and sodium diatrizoate, and that it is particularly difficult to increase the recovery of mononuclear cells using conventional blood collection containers sterilized by radiation. After extensive research, the present inventors have found the following (1) and (2).

[0030] (1) The difficulty in recovering a large amount of mononuclear cells using conventional sterilized blood collection containers is due to changes in the properties of the aqueous solution (specific gravity solution) during sterilization. For example, precipitates may form in the aqueous solution during sterilization. Furthermore, the aqueous solution after sterilization may become discolored (yellowing) over time, which also indicates changes in the properties of the aqueous solution during sterilization.

[0031] (2) By using an aqueous solution (specific gravity liquid) containing polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid other than diatrizoic acid and / or a metal salt thereof (C), it is possible to suppress changes in the properties of the aqueous solution even when sterilization treatment is performed.

[0032] The reasons why the combination of the above components (A) to (C) can suppress changes in the properties of the aqueous solution are presumed to be as follows: 1) The carboxylic acid and / or its metal salt (C) itself may play a role in suppressing changes in the properties of the aqueous solution during sterilization. 2) The carboxylic acid and / or its metal salt (C) may be able to maintain the pH of the aqueous solution after sterilization within a suitable range. However, the reasons why the combination of the above components (A) to (C) can suppress changes in the properties of the aqueous solution are not limited to 1) and 2) above.

[0033] Therefore, a blood collection container containing the aqueous solution according to the present invention can increase the amount of mononuclear cells recovered not only before sterilization but also after sterilization.

[0034] Furthermore, a blood collection container containing the aqueous solution of the present invention allows for easy separation of mononuclear cells from blood. For example, mononuclear cells can be separated from blood by collecting blood in the blood collection container and then centrifuging the blood collection container. By appropriately adjusting the specific gravity of the aqueous solution in the blood collection container, mononuclear cells can be suspended in the aqueous solution or a layer containing mononuclear cells can be formed above the aqueous solution after centrifugation.

[0035] The aqueous solution and blood collection container according to the present invention will be described in detail below. In this specification, "(meth)acrylic" refers to either or both of "acrylic" and "methacrylic." Furthermore, in this specification, the content (concentration) of each component in the aqueous solution refers to the content (concentration) in an anhydrous state.

[0036] [Aqueous Solution for Blood Cell Separation] The aqueous solution contains polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid other than diatrizoic acid and / or a metal salt thereof (C). The aqueous solution preferably contains water. The aqueous solution is also called a specific gravity solution or a density gradient solution.

[0037] <Polysucrose (A)> The aqueous solution contains polysucrose (sometimes referred to as polysucrose (A) in this specification). As the polysucrose (A), a conventionally known polysucrose used in a specific gravity solution (density gradient solution) can be used. Only one type of polysucrose (A) may be used, or two or more types may be used in combination.

[0038] The number-average molecular weight of polysucrose (A) is preferably at least 150,000, more preferably at least 300,000, and preferably at most 700,000, more preferably at most 550,000. When the number-average molecular weight of polysucrose (A) is at least the above-mentioned lower limit and at most the above-mentioned upper limit, the amount of mononuclear cells recovered can be further increased.

[0039] The number average molecular weight means a pullulan-equivalent number average molecular weight measured by gel permeation chromatography (GPC).

[0040] The content of polysucrose (A) in the aqueous solution (100% by weight) is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 3% by weight or more, particularly preferably 5% by weight or more, preferably 30% by weight or less, more preferably 25% by weight or less, and even more preferably 15% by weight or less. When the content of polysucrose (A) is equal to or more than the above lower limit and equal to or less than the above upper limit, the recovery amount of mononuclear cells can be further increased.

[0041] <Diatrizoic acid and / or metal salt thereof (B)> The aqueous solution contains diatrizoic acid and / or a metal salt thereof (sometimes referred to herein as diatrizoic acid and / or a metal salt thereof (B)). As the diatrizoic acid and / or a metal salt thereof (B), a conventionally known diatrizoic acid and / or a metal salt thereof used in a specific gravity solution (density gradient solution) can be used. The diatrizoic acid and / or a metal salt thereof (B) may be diatrizoic acid, a metal salt of diatrizoic acid, or both diatrizoic acid and a metal salt of diatrizoic acid. Only one type of diatrizoic acid and / or a metal salt thereof (B) may be used, or two or more types may be used in combination.

[0042] Examples of the metal salt in diatrizoic acid and / or its metal salt (B) include sodium salt, potassium salt, and lithium salt, etc. Therefore, examples of the metal salt of diatrizoic acid include sodium diatrizoate, potassium diatrizoate, and lithium diatrizoate.

[0043] From the viewpoint of further increasing the recovery amount of mononuclear cells, the diatrizoic acid and / or metal salt thereof (B) preferably contains a metal salt of diatrizoic acid, more preferably sodium diatrizoate.

[0044] The content of diatrizoic acid and / or its metal salt (B) in the aqueous solution (100% by weight) is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 3% by weight or more, and preferably 20% by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less. When the content of diatrizoic acid and / or its metal salt (B) is equal to or more than the above lower limit and equal to or less than the above upper limit, the recovery amount of mononuclear cells can be further increased.

[0045] The content of sodium diatrizoate in 100% by weight of the aqueous solution is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 3% by weight or more, and preferably 20% by weight or less, more preferably 15% by weight or less, and even more preferably 10% by weight or less. When the content of sodium diatrizoate is equal to or more than the above lower limit and equal to or less than the above upper limit, the recovery amount of mononuclear cells can be further increased.

[0046] In the aqueous solution, the weight ratio ((A) / (B)) of the content of polysucrose (A) to the content of diatrizoic acid and / or a metal salt thereof (B) is preferably 0.1 or more, more preferably 0.5 or more, even more preferably 1 or more, preferably 200 or less, more preferably 100 or less, even more preferably 50 or less, still more preferably 10 or less, and particularly preferably 5 or less. When the weight ratio ((A) / (B)) is equal to or more than the above lower limit and equal to or less than the above upper limit, the amount of mononuclear cells recovered can be further increased.

[0047] In the aqueous solution (100% by weight), the total content of polysucrose (A) and diatrizoic acid and / or its metal salt (B) is preferably 10% by weight or more, more preferably 13% by weight or more, even more preferably 15% by weight or more, and preferably 30% by weight or less, more preferably 25% by weight or less, and even more preferably 20% by weight or less. When the total is equal to or more than the lower limit and equal to or less than the upper limit, the recovery amount of mononuclear cells can be further increased.

[0048] <Carboxylic Acid and / or Metal Salt Thereof (C)> The aqueous solution contains a carboxylic acid and / or a metal salt thereof other than diatrizoic acid (sometimes referred to herein as a carboxylic acid and / or a metal salt thereof (C)). The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is other than diatrizoic acid. The carboxylic acid and / or metal salt thereof (C) is a carboxylic acid other than diatrizoic acid and / or a metal salt of a carboxylic acid other than diatrizoic acid. The carboxylic acid and / or metal salt thereof (C) may be a carboxylic acid other than diatrizoic acid, a metal salt of a carboxylic acid other than diatrizoic acid, or both a carboxylic acid other than diatrizoic acid and a metal salt of a carboxylic acid other than diatrizoic acid. Only one type of carboxylic acid and / or a metal salt thereof (C) may be used, or two or more types may be used in combination.

[0049] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) has a carboxy group. The carboxylic acid may be a monocarboxylic acid, a dicarboxylic acid, a tricarboxylic acid, or a tetracarboxylic acid.

[0050] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) has a carboxy group. The number of carboxy groups in the carboxylic acid may be one, one or more, two, two or more, six or less, or four or less.

[0051] The number of carboxy groups possessed by the carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is preferably 1 or more, preferably 6 or less, more preferably 4 or less, and even more preferably 3 or less. When the number of carboxy groups possessed by the carboxylic acid is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0052] The number of carbon atoms in the carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is preferably 1 or more, more preferably 2 or more, and preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less. When the number of carbon atoms in the carboxylic acid is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0053] The carboxylic acid and / or metal salt thereof (C) preferably contains a carboxylic acid and / or metal salt thereof having from 1 to 10 carbon atoms, more preferably a carboxylic acid and / or metal salt thereof having from 2 to 10 carbon atoms, even more preferably a carboxylic acid and / or metal salt thereof having from 2 to 8 carbon atoms, and particularly preferably a carboxylic acid and / or metal salt thereof having from 2 to 6 carbon atoms. In this case, the effects of the present invention can be exerted even more effectively. Therefore, the recovery amount of mononuclear cells can be increased even in a blood collection container that has been sterilized.

[0054] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) may be an aliphatic carboxylic acid or an aromatic carboxylic acid.

[0055] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is preferably an aliphatic carboxylic acid. In this case, the effects of the present invention can be more effectively exhibited. Therefore, even if the blood collection container is sterilized, the amount of mononuclear cells recovered can be increased.

[0056] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) may be a saturated carboxylic acid or an unsaturated carboxylic acid.

[0057] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is preferably a saturated carboxylic acid. In this case, the effects of the present invention can be more effectively exhibited. Therefore, even if the blood collection container is sterilized, the amount of mononuclear cells recovered can be increased.

[0058] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) may be a linear carboxylic acid, a branched carboxylic acid, or a cyclic carboxylic acid.

[0059] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) is preferably a straight-chain carboxylic acid. In this case, the effects of the present invention can be more effectively exhibited. Therefore, even if the blood collection container is sterilized, the amount of mononuclear cells recovered can be increased.

[0060] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) may or may not have a hydroxy group different from the hydroxy group contained in the carboxy group. The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) preferably has a hydroxy group different from the hydroxy group contained in the carboxy group.

[0061] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) preferably does not have an iodine atom and / or an amino group, more preferably does not have both an iodine atom and an amino group. The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) preferably does not have an iodine atom and preferably does not have an amino group.

[0062] Examples of the carboxylic acid in the carboxylic acid and / or metal salt thereof (C) include citric acid, acetic acid, lactic acid, succinic acid, malic acid, formic acid, oxalic acid, benzoic acid, phthalic acid, fumaric acid, maleic acid, trimellitic acid, pyromellitic acid, and shikimic acid.

[0063] Examples of the metal salt in the carboxylic acid and / or metal salt thereof (C) include sodium salt, lithium salt, potassium salt, magnesium salt, and the like.

[0064] Examples of the carboxylic acid and / or metal salt thereof (C) include citric acid, sodium citrate, acetic acid, lithium acetate, potassium acetate, lactic acid, lithium lactate, succinic acid, sodium succinate, malic acid, sodium malate, formic acid, sodium formate, oxalic acid, sodium oxalate, benzoic acid, sodium benzoate, phthalic acid, sodium phthalate, fumaric acid, sodium fumarate, maleic acid, sodium maleate, trimellitic acid, pyromellitic acid, and shikimic acid.

[0065] The carboxylic acid in the carboxylic acid and / or metal salt thereof (C) preferably includes at least one selected from the group consisting of citric acid, acetic acid, lactic acid, succinic acid, and malic acid, more preferably includes at least one selected from the group consisting of citric acid, lactic acid, and malic acid, and even more preferably includes citric acid. In this case, the effects of the present invention can be more effectively exerted. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0066] The carboxylic acid and / or metal salt thereof (C) preferably contains at least one selected from the group consisting of citric acid, sodium citrate, acetic acid, lithium acetate, potassium acetate, lactic acid, lithium lactate, succinic acid, sodium succinate, malic acid, and sodium malate, more preferably contains at least one selected from the group consisting of citric acid, sodium citrate, lactic acid, lithium lactate, malic acid, and sodium malate, and even more preferably contains at least one selected from the group consisting of citric acid and sodium citrate. In this case, the effects of the present invention can be more effectively exerted. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0067] The content of the carboxylic acid and / or its metal salt (C) in the aqueous solution (100% by weight) is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, even more preferably 0.2% by weight or more, and preferably 5% by weight or less, more preferably 2% by weight or less, and even more preferably 1% by weight or less. When the content of the carboxylic acid and / or its metal salt (C) is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exhibited. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0068] In the aqueous solution, the weight ratio ((C) / (A)) of the content of carboxylic acid and / or its metal salt (C) to the content of polysucrose (A) is preferably 0.001 or more, more preferably 0.01 or more, even more preferably 0.02 or more, preferably 0.2 or less, more preferably 0.15 or less, and even more preferably 0.1 or less. When the weight ratio ((C) / (A)) is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exerted. Therefore, even in a blood collection container after sterilization, the recovery amount of mononuclear cells can be increased.

[0069] In the aqueous solution, the weight ratio ((C) / (B)) of the content of carboxylic acid and / or its metal salt (C) to diatrizoic acid and / or its metal salt (B) is preferably 0.001 or more, more preferably 0.01 or more, even more preferably 0.05 or more, preferably 30 or less, more preferably 20 or less, even more preferably 10 or less, still more preferably 3 or less, and particularly preferably 1 or less. When the weight ratio ((C) / (B)) is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the effects of the present invention can be more effectively exerted. Therefore, even in a blood collection container after sterilization, a large amount of mononuclear cells can be recovered.

[0070] <Water> The aqueous solution preferably contains water, which serves as a solvent.

[0071] In 100% by weight of the aqueous solution, the content of the water is preferably 50% by weight or more, more preferably 60% by weight or more, even more preferably 65% ​​by weight or more, even more preferably 70% by weight or more, particularly preferably 75% by weight or more, and preferably 99% by weight or less, more preferably 95% by weight or less, even more preferably 90% by weight or less.

[0072] <Silica microparticles> The aqueous solution preferably contains silica microparticles. Conventionally known silica microparticles used in specific gravity solutions (density gradient solutions) can be used as the silica microparticles. The silica microparticles are preferably silica microparticles coated with polyvinylpyrrolidone. Percoll (Cytiva) is a commercially available aqueous suspension of silica microparticles coated with polyvinylpyrrolidone and is easily available. Only one type of silica microparticle may be used, or two or more types may be used in combination.

[0073] The average particle size of the silica fine particles is preferably 5 nm or more, more preferably 10 nm or more, even more preferably 15 nm or more, and is preferably 50 nm or less, more preferably 40 nm or less, even more preferably 30 nm or less. The average particle size of the silica fine particles can be measured by electron microscopy.

[0074] The content of the silica microparticles in 100% by weight of the aqueous solution is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 10% by weight or more, and preferably 65% ​​by weight or less, more preferably 45% by weight or less, and even more preferably 25% by weight or less. When the content of the silica microparticles is equal to or more than the above lower limit and equal to or less than the above upper limit, the recovery amount of mononuclear cells can be further increased.

[0075] <Other Components> The aqueous solution may contain other components in addition to the above-mentioned components (polysucrose (A), diatrizoic acid and / or its metal salt (B), carboxylic acid and / or its metal salt (C), water, and silica fine particles). Examples of the other components include hydroxyethyl starch, sucrose, dextran, iodixanol, and glycerol. Only one type of each of the other components may be used, or two or more types may be used in combination.

[0076] <Other Details of the Aqueous Solution> The specific gravity of the aqueous solution at 25° C. is preferably 1.060 or more, more preferably 1.065 or more, even more preferably 1.070 or more, particularly preferably 1.080 or more, and preferably 1.100 or less, more preferably 1.098 or less, and even more preferably 1.095 or less. When the specific gravity of the aqueous solution at 25° C. is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the recovery amount of mononuclear cells can be further increased.

[0077] The specific gravity of the aqueous solution at 25° C. is measured using a hydrometer (for example, "DA-130N" manufactured by Kyoto Electronics Manufacturing Co., Ltd.).

[0078] The aqueous solution is preferably sterilized before use, more preferably sterilized by radiation, and even more preferably sterilized by gamma rays before use.

[0079] [Blood collection container] The blood collection container according to the present invention comprises a blood collection container body, an aqueous solution for blood cell separation contained in the blood collection container body, and a blood separation material contained in the blood collection container body. In the blood collection container according to the present invention, the aqueous solution for blood cell separation is the aqueous solution for blood cell separation described above.

[0080] The blood collection container according to the present invention, having the above-described configuration, can increase the amount of mononuclear cells collected. The blood collection container according to the present invention can increase the amount of mononuclear cells collected not only before sterilization but also after sterilization. In particular, the blood collection container according to the present invention can increase the amount of mononuclear cells collected even after sterilization by radiation.

[0081] (Blood Separation Material) The blood collection container comprises a blood separation material housed within the blood collection container body. A conventionally known blood separation material can be used as the blood separation material. Examples of the blood separation material include a blood separation composition and a blood separation tool. The blood separation material is preferably a blood separation composition or a blood separation tool. Because the blood separation material is easy to prepare, the blood separation material is preferably the blood separation composition.

[0082] The specific gravity of the blood separation material at 25° C. is preferably 1.060 or more, more preferably 1.065 or more, even more preferably 1.070 or more, and preferably 1.095 or less, more preferably 1.090 or less, and even more preferably 1.085 or less. When the specific gravity of the blood separation material at 25° C. is not less than the above-mentioned lower limit and not more than the above-mentioned upper limit, the amount of mononuclear cells recovered can be further increased.

[0083] The specific gravity of the blood separation material at 25°C is preferably smaller than the specific gravity of the aqueous solution at 25°C.

[0084] <Blood Separation Composition> The blood separation composition is preferably a composition that migrates between the aqueous solution layer and the blood cell layer during centrifugation to form a partition. The blood separation composition is used for the purpose of preventing component migration between the aqueous solution layer and the blood cell layer after centrifugation. The blood separation composition is preferably thixotropic.

[0085] As the blood separation composition, a conventionally known blood separation composition can be used.

[0086] The blood separating composition preferably contains an organic component that has fluidity at 25° C. and an inorganic fine powder. In this case, the fluidity of the blood separating composition is increased, and the strength of the partition wall can be increased. The organic component that has fluidity at 25° C. and the inorganic fine powder may each be used alone or in combination of two or more types.

[0087] Organic component having fluidity at 25°C: The phrase "having fluidity at 25°C" means that the viscosity at 25°C is 500 Pa·s or less.

[0088] The viscosity of the organic component at 25° C. is preferably 30 Pa s or more, more preferably 50 Pa s or more, and preferably 200 Pa s or less, more preferably 100 Pa s or less. When the viscosity is equal to or greater than the above lower limit and equal to or less than the above upper limit, the fluidity of the blood separating composition is further increased, and the strength of the partition wall can be further increased.

[0089] The viscosity of the organic component at 25°C was measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 sec. －１ It is measured under the following conditions.

[0090] Examples of the organic component include a resin and a mixture of a resin and an organic compound such as a plasticizer. Therefore, the organic component preferably contains the resin, and more preferably contains the resin and the organic compound. When the organic component is a mixture of the resin and the organic compound, it is sufficient that the mixture (the organic component) has fluidity, and the resin or the organic compound does not have to have fluidity. When the organic component is a mixture of the resin and the organic compound, the resin may be, for example, a resin that is solid at 25°C. Only one type of the resin and the organic compound may be used, or two or more types may be used in combination.

[0091] Examples of the resin include petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, polyurethane resins, (meth)acrylic resins, silicone resins, α-olefin-fumaric acid ester copolymers, copolymers of sebacic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane-based resins, polyether polyester-based resins, etc. Only one type of the resins may be used, or two or more types may be used in combination.

[0092] The resin preferably contains at least one selected from the group consisting of petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, and (meth)acrylic resins, and more preferably contains a (meth)acrylic resin, which increases the fluidity of the blood separation composition and increases the strength of the partition wall.

[0093] Commercially available petroleum-based resins include "Regalite S5090" manufactured by Eastman Chemical Company.

[0094] Examples of the cyclopentadiene resin include a polymer of a cyclopentadiene monomer, a copolymer of a cyclopentadiene monomer and an aromatic monomer, and a dicyclopentadiene resin. The cyclopentadiene resin may be hydrogenated. The polymer of a cyclopentadiene monomer and the copolymer of a cyclopentadiene monomer and an aromatic monomer may be an oligomer.

[0095] Examples of the cyclopentadiene-based monomer include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0096] Examples of the aromatic monomer include styrene, methylstyrene, indene, and methylindene.

[0097] Commercially available dicyclopentadiene resins include "Scoretz SU500" and "Scoretz SU90" manufactured by Colon Co., Ltd.

[0098] Examples of the polyester resin include polyalkylene terephthalate resins and polyalkylene naphthalate resins, etc. Examples of the polyalkylene terephthalate resin include polyethylene terephthalate, polybutylene terephthalate, and poly-1,4-cyclohexanedimethylene terephthalate.

[0099] Examples of the polyurethane resin include a reaction product of a polyol compound and an isocyanate compound.

[0100] Examples of the (meth)acrylic resin include a resin obtained by polymerizing at least one type of (meth)acrylic acid ester monomer, and a resin obtained by polymerizing at least one type of (meth)acrylic acid ester monomer and at least one type of monomer other than the (meth)acrylic acid ester monomer.

[0101] Examples of the (meth)acrylic acid ester monomer include (meth)acrylic acid alkyl esters having an alkyl group having from 1 to 20 carbon atoms, (meth)acrylic acid polyalkylene glycol esters, (meth)acrylic acid alkoxyalkyl esters, (meth)acrylic acid hydroxyalkyl esters, (meth)acrylic acid glycidyl esters, (meth)acrylic acid dialkylaminoalkyl esters, (meth)acrylic acid benzyl esters, (meth)acrylic acid phenoxyalkyl esters, (meth)acrylic acid cyclohexyl esters, (meth)acrylic acid isobornyl esters, and (meth)acrylic acid alkoxysilyl alkyl esters. The (meth)acrylic acid ester monomers may be used alone or in combination of two or more.

[0102] The organic compound may be a benzene polycarboxylic acid alkyl ester derivative. The organic compound is preferably a benzene polycarboxylic acid alkyl ester derivative. Therefore, the organic component is preferably a mixture of the resin and the benzene polycarboxylic acid alkyl ester derivative.

[0103] Examples of the benzene polycarboxylic acid alkyl ester derivatives include phthalates, trimellitates, pyromellitates, etc. The benzene polycarboxylic acid alkyl ester derivatives may be used alone or in combination of two or more.

[0104] Examples of the trimellitic acid ester include trimellitic acid tri-n-octyl, triisooctyl, and triisodecyl trimellitate.

[0105] Examples of the pyromellitic acid ester include tetraisooctyl pyromellitic acid.

[0106] Commercially available trimellitic acid esters include "Monocizer W700" and "Monocizer W-750" manufactured by DIC Corporation, and "Sansocizer TOTM" and "Sansocizer TITM" manufactured by New Japan Chemical Co., Ltd.

[0107] Commercially available pyromellitic esters include "Monocizer W-7010" manufactured by DIC Corporation.

[0108] The benzenepolycarboxylic acid alkyl ester derivative is preferably at least one selected from the group consisting of phthalic acid esters, trimellitic acid esters, and pyromellitic acid esters, and more preferably trimellitic acid esters.

[0109] The content of the organic component in 100% by weight of the blood separating composition is preferably 80% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, and preferably 97% by weight or less.

[0110] Inorganic fine powder: Examples of the inorganic fine powder include fine silica powder, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, and zirconium powder.

[0111] The inorganic fine powder is preferably at least one selected from the group consisting of fine silica powder, titanium oxide powder, calcium carbonate powder, zinc oxide powder, alumina powder, glass fine powder, talc powder, kaolin powder, bentonite powder, titania powder, and zirconium powder.

[0112] From the viewpoint of effectively increasing the specific gravity of the blood separating composition, the inorganic fine powder preferably contains finely powdered silica, and more preferably contains finely powdered silica and an inorganic fine powder other than finely powdered silica (a second inorganic fine powder). The inorganic fine powder, the finely powdered silica, and the second inorganic fine powder may each be used alone or in combination of two or more.

[0113] The finely powdered silica includes natural silica and synthetic silica. Synthetic silica includes hydrophilic silica and hydrophobic silica. Hydrophilic silica, for example, imparts thixotropy to the blood separation composition by hydrogen bonding between hydroxyl groups on the particle surface, and also has the effect of adjusting the specific gravity. On the other hand, hydrophobic silica has a smaller effect of imparting thixotropy than hydrophilic silica.

[0114] From the viewpoint of maintaining both the specific gravity and thixotropy of the blood separating composition within a suitable range, the finely powdered silica preferably contains hydrophilic silica, and more preferably contains hydrophilic silica and hydrophobic silica. The finely powdered silica preferably contains at least hydrophilic silica.

[0115] The second inorganic fine powder is preferably an inorganic fine powder having a specific gravity greater than that of fine silica powder, and more preferably an inorganic fine powder having a specific gravity of 2.5 or more. Examples of inorganic fine powders having a specific gravity of 2.5 or more include zinc oxide powder, titanium oxide powder, alumina powder, and calcium carbonate powder. The specific gravity of the second inorganic fine powder may be 10 or less, 7 or less, or 5 or less.

[0116] The average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are not particularly limited, and may be 1 nm or more, 10 nm or more, 500 nm or less, or 100 nm or less.

[0117] The average particle diameters of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder are average diameters measured on a volume basis (volume average particle diameter), and are the 50% median diameter (D50) values. The volume average particle diameter (D50) can be measured by laser diffraction / scattering method, image analysis method, Coulter method, centrifugal sedimentation method, etc. The volume average particle diameter (D50) is preferably determined by laser diffraction / scattering method or image analysis method.

[0118] The specific surface area of ​​the finely powdered silica is not particularly limited. ２ / g or more, and ２ / g or more, and ２ / g or less, and ２ / g or less.

[0119] The specific surface area of ​​the finely powdered silica is measured by the BET method.

[0120] The content of the hydrophilic silica in 100% by weight of the blood separating composition is preferably 0.01% by weight or more, more preferably 0.10% by weight or more, even more preferably 0.30% by weight or more, and preferably 2.50% by weight or less, more preferably 2.00% by weight or less. When the content of the hydrophilic silica is equal to or more than the lower limit and equal to or less than the upper limit, both the specific gravity and thixotropy of the blood separating composition can be maintained within more suitable ranges.

[0121] The content of the finely powdered silica in 100% by weight of the blood separating composition is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the finely powdered silica is equal to or more than the above lower limit and equal to or less than the above upper limit, both the specific gravity and thixotropy of the blood separating composition can be maintained within more suitable ranges.

[0122] The content of the second inorganic fine powder in 100% by weight of the blood separating composition is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the second inorganic fine powder is equal to or more than the above lower limit and equal to or less than the above upper limit, the specific gravity of the blood separating composition can be effectively increased.

[0123] The content of the inorganic fine powder in 100% by weight of the blood separating composition is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, and preferably 10% by weight or less, more preferably 7% by weight or less. When the content of the inorganic fine powder is equal to or more than the above lower limit and equal to or less than the above upper limit, the specific gravity of the blood separating composition can be effectively increased.

[0124] Other Components: The blood separation composition may contain components other than those described above, as long as they can migrate between the aqueous solution layer and the blood cell layer during centrifugation to form a partition. Examples of such components include organic gelling agents, thermoplastic elastomers, polyalkylene glycols, silicone oils, cosolvents, antioxidants, colorants, and water. Each of the above other components may be used alone or in combination of two or more.

[0125] The specific gravity of the blood separation composition at 25° C. is preferably 1.060 or more, more preferably 1.065 or more, even more preferably 1.070 or more, and preferably 1.095 or less, more preferably 1.090 or less, and even more preferably 1.085 or less. When the specific gravity of the blood separation composition at 25° C. is equal to or more than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the amount of mononuclear cells recovered can be further increased.

[0126] The specific gravity of the blood separating composition at 25°C is measured by dropping one drop of the blood separating composition into successively adjusted saline solutions at 25°C with specific gravities of 0.002 each, and observing the floating and sinking of the composition in the saline solution. The specific gravity of the saline solution at 25°C is measured using a hydrometer (for example, "DA-130N" manufactured by Kyoto Electronics Manufacturing Co., Ltd.).

[0127] The viscosity of the blood separation composition at 25° C. is preferably 100 Pa s or more, more preferably 150 Pa s or more, and preferably 500 Pa s or less, more preferably 400 Pa s or less. When the viscosity is equal to or greater than the lower limit and equal to or less than the upper limit, the amount of mononuclear cells recovered can be further increased.

[0128] The viscosity of the blood separating composition at 25°C was measured using an E-type viscometer (for example, "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 sec. －１ It is measured under the following conditions.

[0129] <Blood Separation Device> The blood separation device is a device that moves between an aqueous solution layer and a blood cell layer during centrifugation to form a partition wall. The blood separation device is used for the purpose of preventing component migration between the aqueous solution layer and the blood cell layer.

[0130] The blood separation jig may be a conventionally known jig, such as the mechanical separator described in WO2010 / 132783A1.

[0131] Examples of materials for the blood separation jig include elastomers.

[0132] (Anticoagulant) The blood collection container preferably includes an anticoagulant contained in the blood collection container body. Conventionally known anticoagulants can be used as the anticoagulant. Only one type of anticoagulant may be used, or two or more types may be used in combination.

[0133] Examples of the anticoagulant include heparin, metal salts of heparin, ethylenediaminetetraacetic acid (EDTA), metal salts of EDTA, citric acid, and sodium citrate.

[0134] From the viewpoint of exhibiting good anticoagulant performance, the anticoagulant is preferably at least one selected from the group consisting of EDTA, metal salts of EDTA, heparin, metal salts of heparin, and sodium citrate.

[0135] The anticoagulant may be contained in the blood collection container body in the form of a powder or in the form of a solution in a liquid (anticoagulant-containing liquid). Note that the anticoagulant may be present in the blood collection container body in both the form of a powder and the form of a solution in a liquid.

[0136] The liquid may be water, alcohol, etc. The liquid is preferably water.

[0137] The amount of the anticoagulant contained in the blood collection container body is not particularly limited as long as it can exert anticoagulant properties.

[0138] In addition, when the blood collected in the blood collection container is blood to which an anticoagulant has been added, the blood collection container does not need to be provided with an anticoagulant.

[0139] (Blood collection container body) The shape of the blood collection container body is not particularly limited. The blood collection container body is preferably a tubular container with a bottom. The blood collection container body preferably has an open end and a closed end. The open end of the blood collection container body is one end in the length direction of the blood collection container body, and the closed end of the blood collection container body is the other end in the length direction of the blood collection container body. In the blood collection container body, the distance between the open end and the closed end is the length of the blood collection container body.

[0140] The material of the blood collection container body is not particularly limited. Examples of materials for the blood collection container body include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; thermosetting resins such as unsaturated polyester resin, epoxy resin, and epoxy-acrylate resin; modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, and ethyl chitin; and glass such as silicate glass, such as soda-lime glass, phosphosilicate glass, and borosilicate glass, and quartz glass. The blood collection container body may be made of one material or two or more materials.

[0141] The blood collection container body is preferably a resin container, more preferably a thermoplastic resin container, and even more preferably a polyethylene terephthalate container. When the blood collection container body is a resin container (particularly a polyethylene terephthalate container), sterilization using radiation such as gamma rays is often employed as a blood collection container sterilization method. Conventional blood collection containers tend to undergo sterilization using radiation, resulting in a decrease in the amount of mononuclear cells recovered. However, the present invention can prevent this change in the aqueous solution, allowing resin containers to be used as blood collection container bodies without any problems. Therefore, when the blood collection container body is a resin container (particularly a polyethylene terephthalate container), the above-mentioned effects are even more effectively achieved. Furthermore, because resin containers are less likely to break than glass containers, when the blood collection container body is a resin container (particularly a polyethylene terephthalate container), the risk of collected blood scattering due to breakage or other factors is reduced.

[0142] (Stopper) The blood collection container preferably includes a stopper. The stopper is preferably attached to the open end of the blood collection container body. A conventionally known stopper can be used as the stopper. The stopper is preferably made of a material and has a shape that allows it to be attached to the open end of the blood collection container body in an airtight and liquid-tight manner. The stopper is preferably configured so that it can be pierced by a blood collection needle.

[0143] Examples of the stopper include a stopper having a shape that fits into the open end of the blood collection container body, a sheet-like seal stopper, and the like.

[0144] The stopper may be a stopper comprising a stopper body such as a rubber stopper and a cap member made of plastic, etc. In this case, the risk of blood coming into contact with the human body can be reduced when the stopper body is pulled out from the open end of the blood collection container body after blood collection.

[0145] Examples of materials for the stopper (or the stopper main body) include synthetic resin, elastomer, rubber, and metal foil. Examples of the rubber include butyl rubber and halogenated butyl rubber. Examples of the metal foil include aluminum foil. From the viewpoint of improving sealing performance, the stopper (or the stopper main body) is preferably made of butyl rubber. The stopper (or the stopper main body) is preferably a butyl rubber stopper.

[0146] (Other Details of the Blood Collection Container) The blood collection container is preferably used for separating mononuclear cells from blood, and more preferably for separating peripheral blood mononuclear cells (PBMCs) from blood.

[0147] The aqueous solution is preferably contained within the blood collection container body closer to the closed end of the blood collection container body than the location where the blood separation material is contained. The location where the aqueous solution is contained within the blood collection container body is preferably closer to the closed end of the blood collection container body than the location where the blood separation material is contained. That is, the aqueous solution and the blood separation material are preferably contained side by side in this order within the blood collection container body, from the closed end toward the open end of the blood collection container body. In this case, mononuclear cells can be effectively separated from blood.

[0148] The position where the aqueous solution is contained within the blood collection container body is preferably such that the aqueous solution does not come into contact with the blood when the blood is collected into the blood collection container. It is also preferable that the blood does not come into contact with the aqueous solution when the blood is collected into the blood collection container. In this case, mononuclear cells can be effectively separated from the blood. Note that "when the blood is collected into the blood collection container" means after the blood has been collected into the blood collection container and before the blood collection container containing the collected blood is centrifuged.

[0149] When the blood collection container containing the collected blood is centrifuged, it is preferable that the blood and the aqueous solution come into contact with each other, and it is more preferable that the liquid containing the blood and the anticoagulant (a mixture of blood and anticoagulant) come into contact with the aqueous solution.

[0150] The anticoagulant is preferably contained within the blood collection container body closer to the open end of the blood collection container body than the aqueous solution. The anticoagulant is preferably contained within the blood collection container body closer to the open end of the blood collection container body than the aqueous solution. The anticoagulant is more preferably contained within the blood collection container body closer to the open end of the blood collection container body than the blood separation material. The anticoagulant is preferably contained within the blood collection container body closer to the open end of the blood collection container body than the blood separation material. The aqueous solution, blood separation material, and anticoagulant are preferably contained within the blood collection container body in this order from the closed end toward the open end of the blood collection container body.

[0151] The anticoagulant is preferably contained in a position within the blood collection container body where the anticoagulant comes into contact with the blood when the blood is collected in the blood collection container.

[0152] The anticoagulant is preferably disposed on the inner wall surface of the blood collection container body or on the upper surface of the blood separation material. The anticoagulant may be disposed on the inner wall surface of the blood collection container body, on the upper surface of the blood separation material, or on both the inner wall surface of the blood collection container body and the upper surface of the blood separation material. The upper surface of the blood separation material refers to the surface of the blood separation material on the side of the open end of the blood collection container body.

[0153] When the anticoagulant is contained in a powdered state, the powdered anticoagulant is preferably attached to the inner wall surface of the blood collection container body or disposed on the upper surface of the blood separation material. For example, a mixture of anticoagulant and water can be sprayed onto the inner wall surface of the blood collection container body and then dried, thereby adhering the anticoagulant in a powdered state to the inner wall surface of the blood collection container body.

[0154] When the anticoagulant is contained in a dissolved state in liquid (anticoagulant-containing liquid state), it is preferable that the anticoagulant-containing liquid is placed on the upper surface of the blood separation material.

[0155] The amount of the aqueous solution contained in the blood collection container body is preferably 0.1 mL or more, more preferably 0.15 mL or more, even more preferably 0.2 mL or more, preferably 0.4 mL or less, more preferably 0.35 mL or less, and even more preferably 0.3 mL or less per mL of blood collected in the blood collection container, which allows for an even greater recovery of mononuclear cells.

[0156] The amount of blood collected in the blood collection container varies depending on the size and internal pressure of the blood collection container, etc. The amount of blood collected in the blood collection container may be 1 mL or more, 2 mL or more, 4 mL or more, 12 mL or less, 11 mL or less, or 10 mL or less.

[0157] The blood collection container is preferably a blood collection tube, and the blood collection container body is preferably a blood collection tube body.

[0158] The internal pressure of the blood collection container is not particularly limited. The internal pressure of the blood collection container is preferably reduced. When the blood collection container is a reduced-pressure container, a predetermined amount of blood can be easily collected into the blood collection container. The blood collection container can also be used as a vacuum blood collection tube, which is evacuated and then sealed with the stopper. When it is a vacuum blood collection tube, a predetermined amount of blood can be easily collected regardless of the skill level of the blood collector.

[0159] From the viewpoint of preventing bacterial infection, the interior of the blood collection container is preferably sterilized in accordance with ISO or JIS standards. Conventional sterilization methods can be used. Examples of sterilization methods include radiation sterilization such as gamma ray sterilization and electron beam sterilization, and high-pressure steam sterilization.

[0160] The blood collection container is preferably a sterilized blood collection container, more preferably a blood collection container sterilized by radiation, and even more preferably a blood collection container sterilized by gamma rays. In conventional blood collection containers, sterilization by radiation easily changes the properties of the aqueous solution, resulting in a further decrease in the amount of mononuclear cells recovered. However, the present invention effectively prevents changes in the properties of the aqueous solution even when sterilized by radiation. Therefore, when the blood collection container is a blood collection container sterilized by radiation (particularly gamma rays), the effects of the present invention are even more effectively exhibited, and the effect of increasing the amount of mononuclear cells recovered is even more effectively exhibited.

[0161] The blood collection container sterilized by radiation is preferably irradiated with a dose of 1 kGy to 40 kGy, more preferably 5 kGy to 30 kGy. The blood collection container sterilized by gamma rays is preferably irradiated with gamma rays at a dose of 1 kGy to 40 kGy, more preferably 5 kGy to 30 kGy.

[0162] When the blood collection container is a blood collection container sterilized by radiation, the pH of the aqueous solution (the pH of the aqueous solution after sterilization by radiation) is preferably 3.5 or more, more preferably 4.0 or more, even more preferably 4.5 or more, and preferably 6.4 or less, more preferably 6.0 or less, even more preferably 5.5 or less. When the pH is equal to or more than the above lower limit and equal to or less than the above upper limit, coloration (yellowing) of the aqueous solution over time can be more effectively suppressed, and changes in the properties of the aqueous solution can be suppressed.

[0163] The pH of the aqueous solution is measured at 20°C using a pH meter.

[0164] The blood collection container can be manufactured, for example, as follows.

[0165] An aqueous solution is obtained by mixing polysucrose (A), diatrizoic acid and / or its metal salt (B), carboxylic acid and / or its metal salt (C), and water. The obtained aqueous solution is placed in a blood collection container body. Next, a blood separation material is placed in the blood collection container body. An anticoagulant is also placed in the blood collection container body. In this manner, a blood collection container can be obtained. Furthermore, by sterilizing the obtained blood collection container with radiation, a radiation-sterilized blood collection container can be obtained.

[0166] Specific embodiments of the present invention will be described below with reference to the drawings. Note that in the drawings, the size, thickness, shape, etc. may differ from the actual size, thickness, shape, etc. for the sake of convenience of illustration.

[0167] FIG. 1 is a front cross-sectional view schematically showing a blood collection container according to a first embodiment of the present invention.

[0168] The blood collection container 1 shown in Figure 1 comprises a blood collection container body 2, an aqueous solution 3, a blood separating composition 4, an anticoagulant-containing liquid 5, and a stopper 6. The aqueous solution 3, the blood separating composition 4, and the anticoagulant-containing liquid 5 are each contained within the blood collection container body 2. The blood collection container body 2 has an open end 2a and a closed end 2b. The stopper 6 is attached to the open end 2a of the blood collection container body 2.

[0169] The aqueous solution 3 contains polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), a carboxylic acid and / or a metal salt thereof (C), and water. The anticoagulant-containing solution 5 contains an anticoagulant and water.

[0170] The aqueous solution 3 is contained within the blood collection container body 2 on the closed end 2 b side of the blood collection container body 2 relative to the location where the blood separating composition 4 is contained.

[0171] The anticoagulant-containing liquid 5 is contained within the blood collection container body 2 on the side of the opening end 2a of the blood collection container body 2 relative to the location where the aqueous solution 3 is contained. The anticoagulant-containing liquid 5 is contained within the blood collection container body 2 on the side of the opening end 2a of the blood collection container body 2 relative to the location where the blood separating composition 4 is contained. The anticoagulant-containing liquid 5 is disposed on the upper surface of the blood separating composition 4.

[0172] In the blood collection container 1, an aqueous solution 3, a blood separating composition 4, and an anticoagulant-containing liquid 5 are arranged in this order from the closed end 2b of the blood collection container body 2 toward the open end 2a.

[0173] When blood is collected in the blood collection container 1, the blood does not come into contact with the aqueous solution 3, but comes into contact with the anticoagulant-containing solution 5. When the blood collection container 1 containing the collected blood is centrifuged, the blood (more specifically, the mixture of the blood and the anticoagulant-containing solution 5) comes into contact with the aqueous solution 3.

[0174] FIG. 2 is a front cross-sectional view schematically showing a blood collection container according to a second embodiment of the present invention.

[0175] The blood collection container 1A shown in Fig. 2 comprises a blood collection container body 2, an aqueous solution 3, a blood separating composition 4, a powdered anticoagulant 5A, and a stopper 6. The blood collection container 1 shown in Fig. 1 and the blood collection container 1A shown in Fig. 2 differ in the way the anticoagulant is contained. That is, in the blood collection container 1 shown in Fig. 1, the anticoagulant is contained in a state dissolved in water within the blood collection container body 2, whereas in the blood collection container 1A shown in Fig. 2, the anticoagulant is contained in a powdered state within the blood collection container body 2. More specifically, in the blood collection container 1A shown in Fig. 2, the powdered anticoagulant 5A is disposed on the upper surface of the blood separating composition 4.

[0176] FIG. 3 is a front cross-sectional view schematically showing a blood collection container according to a third embodiment of the present invention.

[0177] The blood collection container 1B shown in Figure 3 comprises a blood collection container body 2, an aqueous solution 3, a blood separating composition 4, a powdered anticoagulant 5B, and a stopper 6. The blood collection container 1 shown in Figure 1 and the blood collection container 1B shown in Figure 3 differ in the way the anticoagulant is contained. That is, in the blood collection container 1 shown in Figure 1, the anticoagulant is contained in a state dissolved in water within the blood collection container body 2, whereas in the blood collection container 1B shown in Figure 3, the anticoagulant is contained in a powdered state on the inner wall surface of the blood collection container body 2. In the blood collection container 1B shown in Figure 3, the anticoagulant 5B is arranged in a layered form.

[0178] [Method for Separating Mononuclear Cells] The blood collection container can be used to separate mononuclear cells from blood. The method for separating mononuclear cells preferably includes a step of collecting blood in the blood collection container, and a step of centrifuging the blood collection container containing the collected blood.

[0179] The centrifugation conditions in the centrifugation step are not particularly limited, and examples of the centrifugation conditions include centrifugation at 400 G or more and 4000 G or less for 5 minutes or more and 120 minutes or less.

[0180] After the centrifugal separation step, for example, red blood cell components are located below the partition formed by the blood separation material, an aqueous solution layer containing mononuclear cells is located above the partition formed by the blood separation material, and plasma is located above the aqueous solution layer containing mononuclear cells. Mononuclear cells can be obtained by recovering the aqueous solution layer containing mononuclear cells.

[0181] The method for isolating mononuclear cells is preferably a method for isolating peripheral blood mononuclear cells (PBMCs).

[0182] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0183] The following materials were prepared for the aqueous solution:

[0184] (Polysucrose (A)) Polysucrose (manufactured by Cytiva, Inc., "Ficoll" ＴＭ PM 400

[0185] (Diatrizoic acid and / or metal salts thereof (B)) Sodium diatrizoate

[0186] (Carboxylic Acid and / or Its Metal Salt (C)) Sodium citrate (trisodium citrate anhydrous) (number of carbon atoms: 6) Lithium acetate dihydrate (number of carbon atoms: 2) Potassium acetate (number of carbon atoms: 2) Lithium L-lactate (number of carbon atoms: 3) Sodium succinate (disodium succinate) (number of carbon atoms: 4) Sodium L-malate (number of carbon atoms: 4)

[0187] (Compounds not corresponding to carboxylic acid and / or its metal salt (C)) Sodium hydrogen carbonate Disodium hydrogen phosphate Tripotassium phosphate Sodium tetraborate decahydrate

[0188] water

[0189] The following materials were prepared as the blood separation composition.

[0190] (Organic component materials having fluidity at 25° C.) (Meth)acrylic resin: 2-Ethylhexyl acrylate and butyl acrylate were radically polymerized by solution polymerization in the presence of an azo polymerization initiator to obtain a (meth)acrylic acid ester polymer having fluidity at 25° C. The specific gravity of the (meth)acrylic resin at 25° C. was 1.033.

[0191] (Inorganic fine powder) Hydrophilic silica (fine powder silica, "200CF" manufactured by Nippon Aerosil Co., Ltd.) Hydrophobic silica (fine powder silica, "RX200" manufactured by Nippon Aerosil Co., Ltd.) Calcium carbonate powder ("Socal UP-G" manufactured by IMERYS)

[0192] (Other ingredients) Silicone oil ("SF8410" manufactured by Toray Dow Corning Co., Ltd.)

[0193] Preparation of blood separation composition A: 91.9 parts by weight of a (meth)acrylic resin, 1.00 parts by weight of hydrophilic silica, 5.0 parts by weight of hydrophobic silica, 1.95 parts by weight of calcium carbonate powder, and 0.15 parts by weight of silicone oil were mixed to prepare blood separation composition A. The specific gravity of the obtained blood separation composition A at 25°C was 1.077.

[0194] Preparation of blood separation composition B: 93.15 parts by weight of a (meth)acrylic resin, 1.00 parts by weight of hydrophilic silica, 4.0 parts by weight of hydrophobic silica, 1.7 parts by weight of calcium carbonate powder, and 0.15 parts by weight of silicone oil were mixed to prepare blood separation composition B. The specific gravity of the obtained blood separation composition B at 25°C was 1.070.

[0195] Preparation of blood separation composition C: 90.65 parts by weight of a (meth)acrylic resin, 1.00 parts by weight of hydrophilic silica, 6.0 parts by weight of hydrophobic silica, 2.2 parts by weight of calcium carbonate powder, and 0.15 parts by weight of silicone oil were mixed to prepare blood separation composition C. The specific gravity of the obtained blood separation composition C at 25°C was 1.085.

[0196] (Example 1) Preparation of aqueous solutions: Aqueous solutions were obtained by blending and mixing the components shown in Table 1 in the blending ratios shown in Table 1. The blend amounts in Table 1 and Tables 2 to 7 described below are pure amounts, and the content (concentration) of each component in the aqueous solution is the content (concentration) in an anhydrous state.

[0197] Preparation of blood collection container: A tubular polyethylene terephthalate container (PET bottomed tube) with a length of 100 mm and an inner diameter of 14 mm at the open end was prepared as the blood collection container body. 1.0 mL of the resulting aqueous solution was placed in the blood collection container body. Next, 1 mL of the resulting blood separation composition was placed in the blood collection container body. Next, 0.5 mL of a 3.2 wt% aqueous solution of anhydrous trisodium citrate (anticoagulant-containing solution) was placed in the blood collection container body. The pressure inside the blood collection container was then reduced to 4 mL of blood, and the container was sealed with a butyl rubber stopper. In this way, a blood collection container containing the aqueous solution, blood separation composition, and anticoagulant was prepared. The blood collection container was then irradiated with gamma rays at a dose of 15 kGy to obtain a gamma-ray-sterilized blood collection container. In the obtained blood collection container, the aqueous solution is contained within the blood collection container body closer to the closed end of the blood collection container body than the location where the blood separating composition is contained, and the anticoagulant is contained within the blood collection container body closer to the open end than the location where the blood separating composition is contained. That is, in the obtained blood collection container, the aqueous solution, the blood separating composition, and the anticoagulant are contained in this order within the blood collection container body from the closed end toward the open end of the blood collection container body.

[0198] Examples 2 to 13 and Comparative Examples 1 to 6 Aqueous solutions and blood collection containers sterilized by gamma rays were prepared in the same manner as in Example 1, except that the formulations of the aqueous solutions were changed as shown in Tables 1 to 7.

[0199] (Evaluation) (1) Specific Gravity of Aqueous Solution (25°C) The specific gravity of the obtained aqueous solution at 25°C was measured using a hydrometer (DA-130N manufactured by Kyoto Electronics Manufacturing Co., Ltd.) In each of the Examples and Comparative Examples, the specific gravity of the aqueous solution before sterilization with gamma rays was the same as the specific gravity of the aqueous solution after sterilization with gamma rays.

[0200] (2) Properties of the aqueous solution (2-1) Presence or absence of precipitates in the aqueous solution The aqueous solutions before and after sterilization with gamma rays were visually observed to confirm whether precipitates had formed in the aqueous solution. The presence or absence of precipitates in the aqueous solution was evaluated according to the following criteria.

[0201] <Criteria for determining whether or not a precipitate is present in an aqueous solution> A: No precipitate is present in the aqueous solution B: Precipitation is present in the aqueous solution

[0202] (2-2) Coloring (yellowing) of aqueous solution A blood collection container before sterilization by gamma rays was stored in a constant temperature bath at 40°C for 7 days. Furthermore, a blood collection container after sterilization by gamma rays was stored in a constant temperature bath at 40°C for 7 days. After storage, the aqueous solution was visually observed to confirm whether or not coloring (yellowing) had occurred in the aqueous solution. Coloring (yellowing) of the aqueous solution was evaluated according to the following criteria.

[0203] <Criteria for determining coloration (yellowing) of aqueous solution> AA: No coloration occurs in the aqueous solution (colorless and transparent) A: The aqueous solution is slightly colored (yellowing), but at a level that does not cause any problems in terms of appearance B: The aqueous solution is deeply colored (yellowing)

[0204] In Examples 1 to 13 and Comparative Examples 1 to 6, the aqueous solutions were colorless and transparent before sterilization with gamma rays and before storage in a constant temperature bath. In Examples 1 to 13 and Comparative Examples 2, 5, and 6, the aqueous solutions were colorless and transparent after sterilization with gamma rays and before storage in a constant temperature bath. In Comparative Examples 3 and 4, the aqueous solutions were colored (yellowing) after sterilization with gamma rays and before storage in a constant temperature bath. In Comparative Example 1, the aqueous solution was cloudy due to the formation of a large amount of precipitates. Therefore, in the table, the evaluation result of Comparative Example 1 after sterilization with gamma rays is marked with "-".

[0205] (3) pH of aqueous solution after sterilization by gamma rays An aqueous solution was collected from a blood collection container sterilized by gamma rays, and the pH of the aqueous solution was measured at 20°C using a pH meter.

[0206] (4) Recovery Amount of Mononuclear Cells (PBMCs) Blood was prepared from three individuals (donor A, donor B, and donor C), and the following procedure was performed on each. 4 mL of blood was collected into the resulting blood collection container (a blood collection container before sterilization with gamma rays or a blood collection container sterilized with gamma rays). The blood collection container containing the collected blood was then centrifuged at 20°C and 1700 x g for 20 minutes. After centrifugation, a red blood cell layer was located below the partition formed by the blood separation composition. Furthermore, an aqueous solution layer was located above the partition formed by the blood separation composition, and a plasma layer was located above the aqueous solution layer. Furthermore, mononuclear cells were floating in the aqueous solution layer.

[0207] The aqueous layer was sucked up with a pipette and transferred to a centrifuge tube. Next, 1 mL of phosphate-buffered saline was added to the centrifuge tube. The centrifuge tube was then centrifuged at room temperature and 500 × g for 10 minutes to precipitate the cells. After removing the supernatant, the precipitated cells were suspended in 1 mL of plasma to obtain a mononuclear cell suspension. The mononuclear cell suspension was analyzed using a multi-parameter automated hematology analyzer (Sysmex Corporation, "XE-5000") to measure the number of mononuclear cells in the mononuclear cell suspension, which was used as the mononuclear cell recovery amount. Furthermore, the number of mononuclear cells contained in 4 mL of whole blood from donors A, B, and C was measured using a multi-parameter automated hematology analyzer (Sysmex Corporation, "XE-5000").

[0208] The recovery rate of mononuclear cells was calculated using the following formula.

[0209] Recovery rate of mononuclear cells (%) = A / B x 100 A: Amount of recovered mononuclear cells (cells) B: Number of mononuclear cells (cells) contained in 4 mL of blood (whole blood)

[0210] The average of the recovery rates of mononuclear cells from donor A, donor B, and donor C was taken as the average recovery rate (Ave).

[0211] <Criteria for determining the amount of recovered mononuclear cells> ○: Average recovery rate (Ave) is 20% or more ×: Average recovery rate (Ave) is less than 20%

[0212] The configurations and results are shown in Tables 1 to 7 below.

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] DESCRIPTION OF SYMBOLS 1, 1A, 1B... Blood collection container 2... Blood collection container body 2a... Open end 2b... Closed end 3... Aqueous solution (aqueous solution for separating blood cells) 4... Blood separation composition 5... Anticoagulant-containing liquid 5A, 5B... Powdered anticoagulant 6... Stopper

Claims

1. An aqueous solution for separating blood cells, comprising polysucrose (A), diatrizoic acid and / or a metal salt thereof (B), and a carboxylic acid other than diatrizoic acid and / or a metal salt thereof (C).

2. The aqueous solution for separating blood cells according to claim 1, wherein the content of the carboxylic acid and / or its metal salt (C) is 0.01% by weight or more and 5% by weight or less in 100% by weight of the aqueous solution for separating blood cells.

3. The aqueous solution for separating blood cells according to claim 1 or 2, wherein the carboxylic acid and / or metal salt thereof (C) comprises a carboxylic acid and / or metal salt thereof having 1 to 10 carbon atoms.

4. The aqueous solution for separating blood cells according to any one of claims 1 to 3, wherein the carboxylic acid in the carboxylic acid and / or metal salt thereof (C) comprises at least one selected from the group consisting of citric acid, acetic acid, lactic acid, succinic acid, and malic acid.

5. The aqueous solution for separating blood cells according to any one of claims 1 to 4, wherein the aqueous solution for separating blood cells has a specific gravity at 25°C of 1.060 or more and 1.100 or less.

6. A blood collection container comprising: a blood collection container body; an aqueous solution for blood cell separation contained within the blood collection container body; and a blood separation material contained within the blood collection container body, wherein the aqueous solution for blood cell separation is the aqueous solution for blood cell separation described in any one of claims 1 to 5.

7. The blood collection container according to claim 6, which is a blood collection container sterilized by radiation.

8. The blood collection container according to claim 6, which is a blood collection container sterilized by radiation, and wherein the pH of the aqueous solution for blood cell separation is 3.5 or more and 6.4 or less.

9. A blood collection container according to any one of claims 6 to 8, wherein the blood collection container body has an open end and a closed end, and the aqueous solution for blood cell separation is contained within the blood collection container body closer to the closed end of the blood collection container body than the position where the blood separation material is contained.

10. The blood collection container according to any one of claims 6 to 9, wherein the blood separation material is a blood separation composition.

11. The blood collection container according to claim 10, wherein the blood separating composition comprises an organic component that is fluid at 25°C and an inorganic fine powder, the organic component comprising a resin, and the inorganic fine powder comprising finely powdered silica.

12. A blood collection container according to any one of claims 6 to 11, further comprising an anticoagulant contained within the blood collection container body, the blood collection container body having an open end and a closed end, and the anticoagulant contained within the blood collection container body closer to the open end of the blood collection container body than the position where the aqueous solution for blood cell separation is contained.

13. The blood collection container according to any one of claims 6 to 12, wherein the blood collection container body is a polyethylene terephthalate container.

14. A method for separating mononuclear cells, comprising the steps of: collecting blood in a blood collection container according to any one of claims 6 to 13; and centrifuging the blood collection container into which the blood has been collected.

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