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

Abstract

This invention provides an aqueous solution for blood cell separation that can suppress changes in properties even after sterilization by radiation. The aqueous solution for blood cell separation according to the present invention comprises polysucrose (A), diatrizoic acid and / or its metal salt (B), and a carboxylic acid and / or its metal salt different from diatrizoic acid (C).

Description

Technical Field

[0001] The present invention relates to an aqueous solution for blood cell separation. The present invention also relates to a blood collection container including the aqueous solution for blood cell separation. The present invention further relates to a method for separating monocytes using the blood collection container.

Background Art

[0002] In clinical examinations, 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 monocytes from blood is also known.

[0003] For example, Patent Document 1 below describes a method for separating monocytes from blood by performing the following steps (a) to (d). (a) Prepare a container having a closed end and an open end. (b) Place a gel-like substance and a water-soluble density gradient substance having a specific gravity greater than that of the substance in the container. (c) Place a liquid sample in the container. (d) Place the container under centrifugal force to separate the liquid sample into a heavy phase and a light phase, and establish a barrier layer composed of separate layers of the gel-like substance and the water-soluble density gradient substance between the heavy phase and the light phase.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In order to separate monocytes from blood, a blood collection container including an aqueous solution for blood cell separation, sometimes referred to as a specific gravity liquid or a density gradient solution, may be used. In this blood collection container, monocytes and blood cell components other than monocytes can be separated by centrifuging the blood collection container after collecting blood.

[0006] Incidentally, sterilized blood collection containers are sometimes used. For example, blood collection containers used as medical device products must be sterilized. However, it is difficult to recover a large amount of mononuclear cells with conventional sterilized blood collection containers. In particular, it is difficult to recover a large amount of mononuclear cells with conventional blood collection containers sterilized by radiation.

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

[0008] The object of the present invention is to provide an aqueous solution for blood cell separation that can suppress changes in properties even after sterilization by radiation. The present invention also aims to provide a blood collection container equipped with the above-mentioned aqueous solution for blood cell separation. Furthermore, the present invention aims to provide a method for separating mononuclear cells using the above-mentioned blood collection container. [Means for solving the problem]

[0009] This specification discloses the following aqueous solutions for blood cell separation, blood collection containers, and methods for separating mononuclear cells.

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

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

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

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

[0014] Item 5. A blood cell separation aqueous solution according to any one of items 1 to 4, wherein the specific gravity of the blood cell separation aqueous solution at 25°C is 1.060 or more and 1.100 or less.

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

[0016] Item 7. The blood collection container described in Item 6, which is a blood collection container sterilized by radiation.

[0017] Item 8. The blood collection container according to Item 6, wherein the blood collection container is sterilized by radiation and the pH of the aqueous solution for blood cell separation is 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 on the closed end side of the blood collection container body, rather than in the position where the blood separation material is contained.

[0019] Item 10. A 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 composition for blood separation contains an organic component having fluidity at 25°C and inorganic fine powder, the organic component contains a resin, and the inorganic fine powder contains fine powder silica.

[0021] Item 12. The blood collection container according to any one of items 6 to 11, further comprising an anticoagulant accommodated in the blood collection container main body, the blood collection container main body having an open end and a closed end, and the anticoagulant being accommodated in the blood collection container main body on the open end side of the blood collection container main body rather than the accommodation position of the blood cell separation aqueous solution.

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

[0023] Item 14. A method for separating monocytes, comprising a step of collecting blood in the blood collection container according to any one of items 6 to 13, and a step of centrifuging the blood collection container in which the blood has been collected.

Advantages of the Invention

[0024] The blood cell separation aqueous solution according to the present invention contains polysucrose (A), diatrizoic acid and / or its metal salt (B), and a carboxylic acid different from diatrizoic acid and / or its metal salt (C). In the blood cell separation aqueous solution according to the present invention, since the above configuration is provided, even if it is sterilized by radiation, a change in properties can be suppressed.

Brief Description of the Drawings

[0025] [Figure 1] FIG. 1 is a front cross-sectional view schematically showing a blood collection container according to a first embodiment of the present invention. [Figure 2] FIG. 2 is a front cross-sectional view schematically showing a blood collection container according to a second embodiment of the present invention. [Figure 3] FIG. 3 is a front cross-sectional view schematically showing a blood collection container according to a third embodiment of the present invention. [Modes for carrying out the invention]

[0026] The details of the present invention will be described below.

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

[0028] Since the aqueous solution according to the present invention has the above-described configuration, changes in its properties can be suppressed even if it is sterilized by radiation.

[0029] As a blood collection container for separating mononuclear cells from blood, a blood collection container equipped with an aqueous solution (specific gravity solution) containing polysucrose and sodium diatrizoate is sometimes used. In addition, sterilized blood collection containers are sometimes used as blood collection containers. For example, blood collection containers used as medical device products must be sterilized. The inventors have found that it is difficult to recover a large amount of mononuclear cells with conventional blood collection containers equipped with an aqueous solution (specific gravity solution) containing polysucrose and sodium diatrizoate and that are sterilized, and that it is particularly difficult to recover a large amount of mononuclear cells with conventional blood collection containers sterilized by radiation. The inventors then conducted diligent research and found the following (1) and (2).

[0030] (1) The reason why it is difficult to recover a large amount of mononuclear cells in conventional sterilized blood collection containers is that the properties of the aqueous solution (specific gravity solution) change during sterilization. For example, precipitates may form in the aqueous solution during sterilization. In addition, the aqueous solution may change color (yellowing) over time after sterilization, which also indicates that the properties of the aqueous solution change during sterilization.

[0031] (2) By using an aqueous solution (specific gravity solution) containing polysucrose (A), diatrizoic acid and / or its metal salt (B), and a carboxylic acid and / or its metal salt other than diatrizoic acid (C), changes in the properties of the aqueous solution can be suppressed even after sterilization.

[0032] The following reasons can be inferred for why combining the above components (A) to (C) can suppress changes in the properties of the aqueous solution: 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 within a suitable range after sterilization. However, the reasons why combining 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, with the blood collection container equipped with the aqueous solution according to the present invention, the amount of mononuclear cells recovered can be increased not only before sterilization but also after sterilization.

[0034] Furthermore, in a blood collection container equipped with the aqueous solution according to the present invention, mononuclear cells can be easily separated from the blood. In the above blood collection container, for example, after collecting blood in the blood collection container, mononuclear cells can be separated by centrifuging the blood collection container. In the above blood collection container, by appropriately adjusting the specific gravity of the aqueous solution, after centrifugation, the mononuclear cells can be suspended in the aqueous solution or a layer containing mononuclear cells can be formed on top of the aqueous solution.

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

[0036] [Aqueous solution for blood cell separation] The above aqueous solution contains polysucrose (A), diatrizoic acid and / or its metal salt (B), and a carboxylic acid and / or its metal salt other than diatrizoic acid (C). The above aqueous solution preferably contains water. The above aqueous solution may also be referred to as a specific gravity solution or a density gradient solution.

[0037] <Polyscrotum (A)> The above aqueous solution contains polysucrose (which may be referred to as polysucrose(A) in this specification). Conventional polysucrose used in specific gravity solutions (density gradient solutions) can be used as polysucrose(A). 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 150,000 or more, more preferably 300,000 or more, more preferably 700,000 or less, and more preferably 550,000 or less. When the number-average molecular weight of polysucrose (A) is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0039] The above number-average molecular weight refers to the number-average molecular weight in pullulan terms, measured by gel permeation chromatography (GPC).

[0040] In the above aqueous solution at 100% by weight, the content of polysucrose (A) 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 above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0041] <Diatrizoic acid and / or its metal salt (B)> The above aqueous solution contains diatrizoic acid and / or its metal salt (which may be referred to as diatrizoic acid and / or its metal salt (B) in this specification). Conventionally known diatrizoic acid and / or its metal salt used in specific gravity solutions (density gradient solutions) can be used as diatrizoic acid and / or its metal salt (B). Diatrizoic acid and / or its metal salt (B) may be diatrizoic acid, a metal salt of diatrizoic acid, or both diatrizoic acid and a metal salt of diatrizoic acid. Diatrizoic acid and / or its metal salt (B) may be used alone or in combination of two or more types.

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

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

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

[0045] In the above aqueous solution at 100% by weight, the content of sodium diatrizoate is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 3% by weight or more, 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 above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0046] In the above aqueous solution, the weight ratio ((A) / (B)) of the polysucrose (A) content to the diatrizoic acid and / or its metal salt (B) content 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, even more preferably 10 or less, and particularly preferably 5 or less. When the above weight ratio ((A) / (B)) is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0047] In 100% by weight of the above aqueous solution, 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, preferably 30% by weight or less, more preferably 25% by weight or less, and even more preferably 20% by weight or less. If the above total is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0048] <Carboxylic acids and / or their metal salts (C)> The above aqueous solution contains a carboxylic acid and / or its metal salt (which may be referred to as carboxylic acid and / or its metal salt (C) in this specification) that is different from diatrizoic acid. The carboxylic acid in carboxylic acid and / or its metal salt (C) is different from diatrizoic acid. Carboxylic acid and / or its metal salt (C) is a carboxylic acid different from diatrizoic acid and / or a metal salt of a carboxylic acid different from diatrizoic acid. Carboxylic acid and / or its metal salt (C) may be a carboxylic acid different from diatrizoic acid, a metal salt of a carboxylic acid different from diatrizoic acid, or both a carboxylic acid different from diatrizoic acid and a metal salt of a carboxylic acid different from diatrizoic acid. Only one type of carboxylic acid and / or its metal salt (C) may be used, or two or more types may be used in combination.

[0049] The carboxylic acid in (C) of the carboxylic acid and / or its metal salt has a carboxyl 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 its metal salt (C) has a carboxyl group. The number of carboxyl groups in the carboxylic acid may be one, one or more, two, two or more, six or fewer, or four or fewer.

[0051] The number of carboxyl groups in the carboxylic acid and / or its metal salt (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 carboxyl groups in the carboxylic acid is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0052] The number of carbon atoms in the carboxylic acid and / or its metal salt (C) is preferably 1 or more, more preferably 2 or more, 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 above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0053] The carboxylic acid and / or its metal salt (C) preferably contains a carboxylic acid and / or its metal salt having 1 to 10 carbon atoms, more preferably a carboxylic acid and / or its metal salt having 2 to 10 carbon atoms, even more preferably a carboxylic acid and / or its metal salt having 2 to 8 carbon atoms, and particularly preferably a carboxylic acid and / or its metal salt having 2 to 6 carbon atoms. In this case, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

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

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

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

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

[0058] The carboxylic acid in (C) of the carboxylic acid and / or its metal salt may be a straight-chain carboxylic acid, a branched-chain carboxylic acid, or a cyclic carboxylic acid.

[0059] In the carboxylic acid and / or its metal salt (C), the carboxylic acid is preferably a linear carboxylic acid. In this case, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0060] In the carboxylic acid and / or its metal salt (C), the carboxylic acid may or may not have a hydroxyl group different from the hydroxyl group contained in the carboxyl group. It is preferable that the carboxylic acid in the carboxylic acid and / or its metal salt (C) has a hydroxyl group different from the hydroxyl group contained in the carboxyl group.

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

[0062] Examples of carboxylic acids in carboxylic acids and / or their metal salts (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 salts in the carboxylic acid and / or its metal salt (C) include sodium salts, lithium salts, potassium salts, and magnesium salts.

[0064] Examples of carboxylic acids and / or their metal salts (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 its metal salt (C) preferably contains at least one selected from the group consisting of citric acid, acetic acid, lactic acid, succinic acid, and malic acid, more preferably contains at least one selected from the group consisting of citric acid, lactic acid, and malic acid, and even more preferably contains citric acid. In this case, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0066] The carboxylic acid and / or its metal salt (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 exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0067] In 100% by weight of the above aqueous solution, the content of carboxylic acid and / or its metal salt (C) 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, 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 carboxylic acid and / or its metal salt (C) is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0068] In the above 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 above weight ratio ((C) / (A)) is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0069] In the aqueous solution described above, the weight ratio ((C) / (B)) of the content of carboxylic acid and / or its metal salt (C) to the content of 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, even more preferably 3 or less, and particularly preferably 1 or less. When the weight ratio ((C) / (B)) is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. Therefore, even in blood collection containers after sterilization, the amount of mononuclear cells recovered can be increased.

[0070] <Water> The above aqueous solution preferably contains water. In the above aqueous solution, water plays the role of a solvent.

[0071] In 100% by weight of the above aqueous solution, the water content 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, preferably 99% by weight or less, more preferably 95% by weight or less, and even more preferably 90% by weight or less.

[0072] <Silica microparticles> The above aqueous solution preferably contains silica microparticles. Conventional known silica microparticles used in specific gravity solutions (density gradient solutions) can be used as the above silica microparticles. The above silica microparticles are preferably silica microparticles coated with polyvinylpyrrolidone. Percoll (Cytiva) is a commercially available and easily obtainable suspension aqueous solution of the above polyvinylpyrrolidone-coated silica microparticles. 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 above silica fine particles is preferably 5 nm or more, more preferably 10 nm or more, even more preferably 15 nm or more, preferably 50 nm or less, more preferably 40 nm or less, and even more preferably 30 nm or less. The average particle size of the above silica fine particles can be measured by electron microscopy.

[0074] In 100% by weight of the above aqueous solution, the content of the above silica fine particles is preferably 0.1% by weight or more, more preferably 1% by weight or more, even more preferably 10% by weight or more, 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 above silica fine particles is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[0075] <Other ingredients> The above aqueous solution may contain other components besides those listed above (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 these other components include hydroxyethyl starch, sucrose, dextran, iodixanol, and glycerol. Each of these other components may be used individually or in combination of two or more.

[0076] <Further details on aqueous solutions> The specific gravity of the above aqueous solution at 25°C is preferably 1.060 or higher, more preferably 1.065 or higher, even more preferably 1.070 or higher, particularly preferably 1.080 or higher, preferably 1.100 or lower, more preferably 1.098 or lower, and even more preferably 1.095 or lower. If the specific gravity of the above aqueous solution at 25°C is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

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

[0078] The above aqueous solution is preferably used after sterilization, more preferably after sterilization by radiation, and even more preferably after sterilization by gamma rays.

[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 within the blood collection container body, and a blood separation material contained within 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 has the above configuration, which allows for a large amount of mononuclear cells to be recovered. The blood collection container according to the present invention allows for a large amount of mononuclear cells to be recovered not only before sterilization but also after sterilization. In particular, the blood collection container according to the present invention allows for a large amount of mononuclear cells to be recovered even after sterilization by radiation.

[0081] (Blood separation material) The blood collection container described above 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 blood separation compositions and blood separation jigs. The blood separation material is preferably a blood separation composition or a blood separation jig. It is preferable that the blood separation material is a blood separation composition because it is easier to manufacture.

[0082] The specific gravity of the blood separation material at 25°C is preferably 1.060 or higher, more preferably 1.065 or higher, even more preferably 1.070 or higher, preferably 1.095 or lower, more preferably 1.090 or lower, and even more preferably 1.085 or lower. If the specific gravity of the blood separation material at 25°C is above the lower limit and below the upper limit, the amount of mononuclear cells recovered can be increased even further.

[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 described above is preferably a composition that moves between the aqueous solution layer and the blood cell layer during centrifugation to form a partition. Furthermore, the blood separation composition is used to prevent component migration between the aqueous solution layer and the blood cell layer after centrifugation. The blood separation composition is preferably thixotropic.

[0085] Conventional blood separation compositions can be used as the blood separation composition described above.

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

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

[0088] The viscosity of the above organic component at 25°C is preferably 30 Pa·s or more, more preferably 50 Pa·s or more, preferably 200 Pa·s or less, and more preferably 100 Pa·s or less. When the viscosity is above the lower limit and below the upper limit, the fluidity of the blood separation composition can be further increased, and the strength of the septum can be further increased.

[0089] The viscosity of the above organic components at 25°C was determined using an E-type viscometer (e.g., "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 seconds. -1 It is measured under these conditions.

[0090] Examples of the above-mentioned organic component include resins and mixtures of resins with organic compounds such as plasticizers. Therefore, it is preferable that the above-mentioned organic component includes the above-mentioned resin, and more preferably that it includes the above-mentioned resin and the above-mentioned organic compound. When the above-mentioned organic component is a mixture of the above-mentioned resin and the above-mentioned organic compound, it is sufficient that the mixture (the above-mentioned organic component) has fluidity, but the resin or the organic compound does not need to have fluidity. When the above-mentioned organic component is a mixture of the above-mentioned resin and the above-mentioned organic compound, the resin may be, for example, a resin that is solid at 25°C. Only one type of the above-mentioned resin and the above-mentioned organic compound may be used, or two or more types may be used in combination.

[0091] Examples of the above resins include petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, polyurethane resins, (meth)acrylic-based resins, silicone resins, α-olefin-fumarate copolymers, copolymers of sebaciic acid, 2,2-dimethyl-1,3-propanediol, and 1,2-propanediol, polyether polyurethane-based resins, and polyether polyester-based resins. Only one of these resins may be used, or two or more may be used in combination.

[0092] The above resin preferably includes at least one selected from the group consisting of petroleum-based resins, cyclopentadiene-based resins, polyester-based resins, and (meth)acrylic-based resins, and more preferably includes a (meth)acrylic-based resin. In this case, the fluidity of the blood separation composition can be increased, and the strength of the septum can be increased.

[0093] Examples of commercially available petroleum-based resins include Eastman Chemical Company's "Rigalite S5090".

[0094] Examples of the cyclopentadiene resins mentioned above include polymers of cyclopentadiene monomers, copolymers of cyclopentadiene monomers and aromatic monomers, and dicyclopentadiene resins. The cyclopentadiene resins may be hydrogenated. The polymers of cyclopentadiene monomers and copolymers of cyclopentadiene monomers and aromatic monomers may be oligomers.

[0095] Examples of the cyclopentadiene monomers mentioned above include cyclopentadiene, dicyclopentadiene, and alkyl-substituted derivatives of cyclopentadiene.

[0096] Examples of the above-mentioned aromatic monomers include styrene, methylstyrene, indene, and methylindene.

[0097] Examples of commercially available dicyclopentadiene resins include "Scolettes SU500" and "Scolettes SU90" manufactured by Colon Co., Ltd.

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

[0099] Examples of the polyurethane resin mentioned above include reaction products of polyol compounds and isocyanate compounds.

[0100] Examples of the (meth)acrylic resins mentioned above include resins obtained by polymerizing at least one (meth)acrylic acid ester monomer, and resins obtained by polymerizing at least one (meth)acrylic acid ester monomer with at least one monomer other than the (meth)acrylic acid ester monomer.

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

[0102] Examples of the above organic compound include benzenepolycarboxylate alkyl ester derivatives. Preferably, the above organic compound is a benzenepolycarboxylate alkyl ester derivative. Therefore, it is preferable that the above organic component is a mixture of the above resin and the above benzenepolycarboxylate alkyl ester derivative.

[0103] Examples of the above-mentioned alkyl benzene polycarboxylate derivatives include phthalate esters, trimellitic acid esters, and pyromellitic acid esters. The above-mentioned alkyl benzene polycarboxylate derivatives may be used individually or in combination of two or more.

[0104] Examples of the trimellitic acid esters mentioned above include tri-n-octyl trimellitic acid, triisooctyl trimellitic acid, and triisodecyl trimellitic acid.

[0105] Examples of the pyromellitic acid esters mentioned above include tetraisooctyl pyromellitic acid.

[0106] Examples of commercially available trimellitic acid esters include DIC Corporation's "Monosizer W700" and "Monosizer W-750," and Shin Nippon Rika Co., Ltd.'s "Sansoizer TOTM" and "Sansoizer TITM."

[0107] Examples of commercially available pyromellitic acid esters include DIC Corporation's "Monosizer W-7010".

[0108] The above-mentioned alkyl benzene polycarboxylate derivative is preferably at least one selected from the group consisting of phthalate esters, trimellitic acid esters, and pyromellitic acid esters, and more preferably a trimellitic acid ester.

[0109] In 100% by weight of the above blood separation composition, the content of the above organic component 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 above-mentioned inorganic fine powders 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 separation composition, the inorganic fine powder preferably contains fine silica powder, and more preferably contains fine silica powder and inorganic fine powder other than fine silica powder (second inorganic fine powder). The inorganic fine powder, the fine silica powder, and the second inorganic fine powder may each be used individually or in combination of two or more.

[0113] Examples of the fine silica powder mentioned above include natural silica and synthetic silica. Examples of synthetic silica include hydrophilic silica and hydrophobic silica. Hydrophilic silica, for example, imparts thixotropy to blood separation compositions and adjusts specific gravity by hydrogen bonding between hydroxyl groups on the particle surface. On the other hand, hydrophobic silica has a smaller effect on imparting thixotropy compared to hydrophilic silica.

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

[0115] The second inorganic fine powder described above is preferably an inorganic fine powder with a specific gravity greater than that of silica fine powder, and more preferably an inorganic fine powder with a specific gravity of 2.5 or higher. Examples of inorganic fine powders with a specific gravity of 2.5 or higher 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 size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder is not particularly limited. The average particle size of the inorganic fine powder, the fine silica powder, and the second inorganic fine powder may be 1 nm or more, 10 nm or more, 500 nm or less, or 100 nm or less.

[0117] The average particle diameter of the above inorganic fine powder, the above fine silica powder, and the above second inorganic fine powder is the average diameter measured on a volume basis (volume-average particle diameter), and is the value of the median diameter (D50) at which 50% is reached. The above volume-average particle diameter (D50) can be measured by laser diffraction / scattering, image analysis, Coulter method, and centrifugal sedimentation method, etc. It is preferable to determine the above volume-average particle diameter (D50) by laser diffraction / scattering or image analysis.

[0118] The specific surface area of ​​the above-mentioned fine silica powder is not particularly limited. 2 It may be more than / g, 100m 2 It may be more than / g, 500m 2 It may be less than / g, and 300m 2 It may be less than / g.

[0119] The specific surface area of ​​the above-mentioned fine silica powder is measured by the BET method.

[0120] In 100% by weight of the above blood separation composition, the content of the hydrophilic silica 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, preferably 2.50% by weight or less, and more preferably 2.00% by weight or less. When the content of the hydrophilic silica is above the lower limit and below the upper limit, both the specific gravity and thixotropy of the blood separation composition can be maintained within a more favorable range.

[0121] In 100% by weight of the above blood separation composition, the content of the above fine silica powder is preferably 0.1% by weight or more, more preferably 0.5% by weight or more, preferably 10% by weight or less, and more preferably 7% by weight or less. When the content of the above fine silica powder is above the lower limit and below the upper limit, both the specific gravity and thixotropy of the blood separation composition can be maintained within a more favorable range.

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

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

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

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

[0126] The specific gravity of the blood separation composition at 25°C is measured by sequentially adding one drop of the blood separation composition to saline solution at 25°C, with the specific gravity adjusted in increments of 0.002, and observing its buoyancy. The specific gravity of the saline solution at 25°C is measured using a hydrometer (for example, a "DA-130N" manufactured by Kyoto Electronics Manufacturing Co., Ltd.).

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

[0128] The viscosity of the above blood separation composition at 25°C was determined using an E-type viscometer (e.g., "TVE-35" manufactured by Toki Sangyo Co., Ltd.) at 25°C and a shear rate of 1.0 seconds. -1 It is measured under these conditions.

[0129] <Blood separation jig> The above-described blood separation jig is a device that moves between the aqueous solution layer and the blood cell layer during centrifugation to form a partition. Furthermore, the above-described blood separation jig is used to prevent the migration of components between the aqueous solution layer and the blood cell layer.

[0130] Conventional known fixtures can be used as the blood separation fixtures described above. Examples of such blood separation fixtures include mechanical separators described in WO2010 / 132783A1, etc.

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

[0132] (Anticoagulant) The blood collection container described above preferably includes an anticoagulant contained within the blood collection container body. Conventional 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 above-mentioned anticoagulants 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, it is preferable that the above anticoagulant is 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 in powder form or in a liquid-dissolved state (anticoagulant-containing solution). The anticoagulant may exist in both powder and liquid-dissolved states within the blood collection container.

[0136] Examples of the above-mentioned liquid include water and alcohol. Water is preferred as the liquid.

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

[0138] Furthermore, if 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 equipped with an anticoagulant.

[0139] (Blood collection container body) The shape of the blood collection container body described above is not particularly limited. Preferably, the blood collection container body is a tubular container with a bottom. Preferably, the blood collection container body has an open end and a closed end. The open end of the blood collection container body is one end in the longitudinal direction of the blood collection container body, and the closed end of the blood collection container body is the other end in the longitudinal 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 described above 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 resins, epoxy resins, and epoxy-acrylate resins; modified natural resins such as cellulose acetate, cellulose propionate, ethylcellulose, and ethyl chitin; silicate glass such as soda-lime glass, phosphate glass, and borosilicate glass; and glass such as quartz glass. The blood collection container body may be made of only one type of material, or two or more types may be used in combination.

[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 (especially a polyethylene terephthalate container), sterilization methods using radiation such as gamma rays are often employed as sterilization methods for the blood collection container. In conventional blood collection containers, sterilization by radiation tends to change the properties of the aqueous solution, reducing the amount of mononuclear cells recovered. However, in the present invention, changes in the properties of the aqueous solution can be suppressed, so a resin container can be used as the blood collection container body without any problems. Therefore, when the blood collection container body is a resin container (especially a polyethylene terephthalate container), the above-mentioned effects are exhibited even more effectively. In addition, since the resin container is less likely to break than a glass container, when the blood collection container body is a resin container (especially a polyethylene terephthalate container), the risk of the collected blood scattering due to breakage can be reduced.

[0142] (stopper) The blood collection container described above 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 liquidtight manner. The stopper is preferably configured so that a blood collection needle can be inserted through it.

[0143] Examples of the above-mentioned stoppers include stoppers having a shape that fits onto the open end of the blood collection container body, and sheet-shaped sealing stoppers.

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

[0145] Examples of materials for the stopper (or stopper body) include synthetic resin, elastomer, rubber, and metal foil. Examples of rubber include butyl rubber and halogenated butyl rubber. Examples of metal foil include aluminum foil. From the viewpoint of improving sealing performance, it is preferable that the material of the stopper (or stopper body) be butyl rubber. It is preferable that the stopper (or stopper body) be a butyl rubber stopper.

[0146] (Other details about the blood collection container) The above blood collection container is suitably used for separating mononuclear cells from blood. The above blood collection container is more suitably used for separating peripheral blood mononuclear cells (PBMCs) from blood.

[0147] It is preferable that the aqueous solution is contained within the blood collection container body on the closed end side of the blood collection container body, rather than at the location where the blood separation material is contained. It is preferable that the location where the aqueous solution is contained within the blood collection container body is on the closed end side of the blood collection container body, rather than at the location where the blood separation material is contained. In other words, it is preferable that the aqueous solution and the blood separation material are contained within the blood collection container body in this order, from the closed end toward the open end. In this case, mononuclear cells can be effectively separated from the blood.

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

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

[0150] It is preferable that the anticoagulant is contained within the blood collection container body on the open end side of the blood collection container body, rather than at the location where the aqueous solution is contained. It is preferable that the location where the anticoagulant is contained within the blood collection container body is on the open end side of the blood collection container body, rather than at the location where the aqueous solution is contained. It is even more preferable that the anticoagulant is contained within the blood collection container body on the open end side of the blood collection container body, rather than at the location where the blood separation material is contained. It is preferable that the location where the anticoagulant is contained within the blood collection container body is on the open end side of the blood collection container body, rather than at the location where the blood separation material is contained. It is preferable that the aqueous solution, the blood separation material, and the anticoagulant are contained in this order, from the closed end to the open end of the blood collection container body.

[0151] The position of the anticoagulant within the blood collection container body is preferably such that it comes into contact with the blood when blood is collected in the blood collection container.

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

[0153] When the anticoagulant is contained in powder form, it is preferable that the powdered anticoagulant adheres to the inner wall surface of the blood collection container body or is placed on the upper surface of the blood separation material. For example, the anticoagulant can be attached to the inner wall surface of the blood collection container body in powder form by spraying a mixture of the anticoagulant and water onto the inner wall surface of the blood collection container body and drying it.

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

[0155] The amount of 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 1 mL of blood collected in the blood collection container. In this case, the amount of mononuclear cells recovered can be increased even further.

[0156] The amount of blood collected in the blood collection container may be adjusted as appropriate depending on the size and internal pressure of the blood collection container. 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. 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. Preferably, the internal pressure of the blood collection container is 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 sealed with the stopper after the inside has been evacuated. When it is a vacuum blood collection tube, a fixed amount of blood can be easily collected regardless of the skill level of the blood collector.

[0159] From the standpoint of preventing bacterial infection, it is preferable that the inside of the blood collection container be sterilized in accordance with ISO or JIS standards. Conventional known methods can be used for sterilization. Examples of sterilization methods include radiation sterilization such as gamma ray sterilization and electron beam sterilization, as well as autoclaving.

[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 tends to change the properties of the aqueous solution, resulting in a further decrease in the amount of mononuclear cells recovered. However, in the present invention, even when sterilization by radiation is performed, the change in the properties of the aqueous solution can be effectively suppressed. Therefore, when the blood collection container is a blood collection container sterilized by radiation (especially gamma rays), the effects of the present invention are exhibited more effectively, and the effect of increasing the amount of mononuclear cells recovered is exhibited more effectively.

[0161] The blood collection container sterilized by radiation is preferably a blood collection container that has been irradiated with a dose of 1 kGy to 40 kGy, and more preferably a blood collection container that has been irradiated with a dose of 5 kGy to 30 kGy. The blood collection container sterilized by gamma rays is preferably a blood collection container that has been irradiated with gamma rays with a dose of 1 kGy to 40 kGy, and more preferably a blood collection container that has been irradiated with gamma rays with a dose of 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 (pH of the aqueous solution after sterilization by radiation) is preferably 3.5 or higher, more preferably 4.0 or higher, even more preferably 4.5 or higher, preferably 6.4 or lower, more preferably 6.0 or lower, and even more preferably 5.5 or lower. When the pH is above the lower limit and below the upper limit, the discoloration (yellowing) of the aqueous solution over time can be suppressed more effectively, and changes in the properties of the aqueous solution can be suppressed.

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

[0164] The blood collection container described above can be manufactured, for example, as follows:

[0165] Polysucrose (A), diatrizoic acid and / or its metal salt (B), carboxylic acid and / or its metal salt (C), and water are mixed to obtain an aqueous solution. The obtained aqueous solution is placed inside the blood collection container body. Next, a blood separation material is placed inside the blood collection container body. An anticoagulant is also placed inside the blood collection container body. In this way, 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 the size, thickness, and shape shown in the following drawings may differ from the actual size, thickness, and shape for illustrative purposes.

[0167] Figure 1 is a schematic front cross-sectional view showing a blood collection container according to the 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 separation composition 4, an anticoagulant-containing solution 5, and a stopper 6. The aqueous solution 3, the blood separation composition 4, and the anticoagulant-containing solution 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] Aqueous solution 3 contains polysucrose (A), diatrizoic acid and / or its metal salt (B), carboxylic acid and / or its metal salt (C), and water. 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 side of the closed end 2b of the blood collection container body 2, rather than at the location where the blood separation composition 4 is contained.

[0171] The anticoagulant-containing solution 5 is contained within the blood collection container body 2, closer to the open end 2a of the blood collection container body 2 than the location where the aqueous solution 3 is contained. The anticoagulant-containing solution 5 is contained within the blood collection container body 2, closer to the open end 2a of the blood collection container body 2 than the location where the blood separation composition 4 is contained. The anticoagulant-containing solution 5 is placed on the upper surface of the blood separation composition 4.

[0172] In the blood collection container 1, the aqueous solution 3, blood separation composition 4, and anticoagulant-containing solution 5 are arranged in the following order from the closed end 2b side to the open end 2a side of the blood collection container body 2.

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

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

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

[0176] Figure 3 is a schematic front cross-sectional view 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 separation 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. Specifically, in the blood collection container 1 shown in Figure 1, the anticoagulant is contained in a dissolved state 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 powder form 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 layers.

[0178] [Method for isolating mononuclear cells] Using the blood collection container described above, mononuclear cells can be separated from the blood. The method for separating mononuclear cells preferably includes the step of collecting blood into the blood collection container, and preferably includes the step of centrifuging the blood collection container from which the blood has been collected.

[0179] The centrifugal separation conditions in the above centrifugal separation process are not particularly limited. Examples of such centrifugal separation conditions include centrifugal separation at 400G to 4000G for 5 minutes to 120 minutes.

[0180] After the centrifugation process described above, for example, the red blood cell component is located below the septum formed by the blood separation material, the aqueous solution layer containing mononuclear cells is located above the septum formed by the blood separation material, and the plasma is located above the aqueous solution layer containing mononuclear cells. By recovering the aqueous solution layer containing mononuclear cells, mononuclear cells can be obtained.

[0181] The above 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. The present invention is not limited to the following examples.

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

[0184] (Polyscrotum (A)) Polyscoat (Cytiva "Ficoll") TM PM 400)

[0185] (Diatrizoic acid and / or its metal salt (B)) Sodium diatrizoate

[0186] (Carboxylic acids and / or their metal salts (C)) Sodium citrate (trisodium citrate anhydrous) (6 carbon atoms) Lithium acetate dihydrate (2 carbon atoms) Potassium acetate (2 carbon atoms) Lithium lactate (3 carbon atoms) Sodium succinate (disodium succinate) (4 carbon atoms) Sodium L-malate (4 carbon atoms)

[0187] (Compounds that do not correspond to carboxylic acids and / or their metal salts (C)) sodium bicarbonate Disodium hydrogen phosphate Tripotassium phosphate Sodium tetraborate decahydrate

[0188] water

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

[0190] (An organic component material that is fluid at 25°C) (Meth)acrylic resin: Ethylhexyl acrylate and butyl acrylate were radically polymerized in solution in the presence of an azo polymerization initiator to obtain a (meth)acrylic ester polymer that is fluid at 25°C. The specific gravity of the (meth)acrylic resin at 25°C was 1.033.

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

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

[0193] Preparation of blood separation composition A: Composition A for blood separation was prepared by mixing 91.9 parts by weight of (meth)acrylic resin, 1.00 part 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. The specific gravity of the obtained blood separation composition A at 25°C was 1.077.

[0194] Preparation of blood separation composition B: Composition B for blood separation was prepared by mixing 93.15 parts by weight of (meth)acrylic resin, 1.00 part 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. The specific gravity of the obtained blood separation composition B at 25°C was 1.070.

[0195] Preparation of blood separation composition C: Composition C for blood separation was prepared by mixing 90.65 parts by weight of (meth)acrylic resin, 1.00 part 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. The specific gravity of the obtained blood separation composition C at 25°C was 1.085.

[0196] (Example 1) Preparation of aqueous solutions: An aqueous solution was obtained by mixing the components shown in Table 1 in the proportions indicated in Table 1. Note that the amounts in Table 1 and Tables 2-7 described below represent the net amounts, and the content (concentration) of each component in the aqueous solution is the content (concentration) in the anhydrous state.

[0197] Preparation of blood collection containers: 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 obtained aqueous solution was placed inside the blood collection container body. Next, 1 mL of the obtained blood separation composition was placed inside the blood collection container body. Then, 0.5 mL of a 3.2 wt% aqueous solution of anhydrous trisodium citrate (anticoagulant-containing solution) was placed inside the blood collection container body. Next, the inside of the blood collection container was reduced in pressure so that the blood collection volume would be 4 mL, and it was sealed with a butyl rubber stopper. In this way, a blood collection container was prepared in which the aqueous solution, blood separation composition, and anticoagulant were placed inside the blood collection container body. Next, the blood collection container was irradiated with gamma rays at a dose of 15 kGy to obtain a blood collection container sterilized by gamma rays. In the resulting blood collection container, the aqueous solution is contained within the blood collection container body on the closed end side of the container body, while the anticoagulant is contained within the open end side of the container body, while the anticoagulant is contained within the container body on the open end side of the container body, while the anticoagulant is contained within the container body on the open end side of the container body. In other words, in the resulting blood collection container, the aqueous solution, the blood separation composition, and the anticoagulant are contained within the blood collection container body in this order, from the closed end to the open end.

[0198] (Examples 2-13 and Comparative Examples 1-6) Except for changing the composition of the aqueous solution as shown in Tables 1 to 7, a blood collection container sterilized with an aqueous solution and gamma rays was prepared in the same manner as in Example 1.

[0199] (evaluation) (1) Specific gravity of aqueous solution (at 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 example and comparative example, 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 aqueous solutions (2-1) Presence or absence of precipitates in aqueous solution The aqueous solution was visually inspected before and after sterilization with gamma rays to check for the presence of precipitates. The presence or absence of precipitates in the aqueous solution was evaluated according to the following criteria.

[0201] <Criteria for determining the presence or absence of precipitates in aqueous solutions> A: No precipitate has formed in the aqueous solution. B: Precipitates have formed in the aqueous solution.

[0202] (2-2) Discoloration (yellowing) of the aqueous solution Blood collection containers were stored in a 40°C constant temperature bath for 7 days before sterilization with gamma rays. Similarly, blood collection containers sterilized with gamma rays were stored in a 40°C constant temperature bath for 7 days. After storage, the aqueous solutions were visually inspected to check for discoloration (yellowing). The discoloration (yellowing) of the aqueous solutions was evaluated according to the following criteria.

[0203] <Criteria for determining discoloration (yellowing) of aqueous solutions> AA: The aqueous solution is colorless and transparent. A: The aqueous solution is slightly discolored (yellowing), but it is at a level that does not affect its appearance. B: The aqueous solution is heavily colored (yellowing).

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

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

[0206] (4) Amount of mononuclear cells (PBMCs) recovered Blood samples were prepared from three donors (Donor A, Donor B, and Donor C), and the following procedure was performed on each. 4 mL of blood was collected in the blood collection container (either before or after gamma ray sterilization). The blood collection containers were then centrifuged at 20°C and 1700 × g for 20 minutes. After centrifugation, the red blood cell layer was located below the septum formed by the blood separation composition. The aqueous solution layer was located above the septum formed by the blood separation composition, and the plasma layer was located above the aqueous solution layer. Mononuclear cells were suspended in the aqueous solution layer.

[0207] The aqueous solution layer was pipetteed and transferred to a centrifuge tube. Next, 1 mL of phosphate-buffered saline was added to the centrifuge tube. Then, the centrifuge tube was 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 number of mononuclear cells in the suspension was measured using a multi-parameter automated hematology analyzer (Sysmex Corporation "XE-5000") and this was used as the amount of mononuclear cells recovered. In addition, 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] Mononuclear cell recovery rate (%) = A / B × 100 A: Amount of mononuclear cells recovered (cells) B: Number of mononuclear cells in 4 mL of blood (whole blood)

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

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

[0212] The configuration and results are shown in Tables 1-7 below.

[0213] [Table 1]

[0214] [Table 2]

[0215] [Table 3]

[0216] [Table 4]

[0217] [Table 5]

[0218] [Table 6]

[0219] [Table 7] [Explanation of Symbols]

[0220] 1,1A,1B…Blood collection container 2…Blood collection container body 2a...Open end 2b...Closed end 3...Aqueous solution (aqueous solution for blood cell separation) 4...Composition for blood separation 5…Anticoagulant-containing solution 5A, 5B... Powdered anticoagulant 6…Bung body

Claims

1. Polyscrotum (A) and, Diatrizoic acid and / or its metal salt (B), An aqueous solution for blood cell separation comprising a carboxylic acid and / or its metal salt (C) different from diatrizoic acid.

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

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

4. The aqueous solution for blood cell separation according to claim 1 or 2, wherein the carboxylic acid in the carboxylic acid and / or its metal salt (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 blood cell separation according to claim 1 or 2, wherein the weight ratio ((C) / (A)) of the content of the carboxylic acid and / or its metal salt (C) to the content of the polysucrose (A) is 0.001 or more and 0.2 or less.

6. The aqueous solution for blood cell separation according to claim 1 or 2, wherein the weight ratio ((C) / (B)) of the content of the carboxylic acid and / or its metal salt (C) to the diatrizoic acid and / or its metal salt (B) is 0.001 or more and 30 or less.

7. The aqueous solution for blood cell separation according to claim 1 or 2, wherein the specific gravity of the aqueous solution for blood cell separation at 25°C is 1.060 or more and 1.100 or less.

8. The blood collection container body, The blood cell separation aqueous solution contained within the blood collection container body, The blood collection container comprises a blood separation material housed within the blood collection container body, A blood collection container wherein the aqueous solution for blood cell separation is the aqueous solution for blood cell separation described in claim 1 or 2.

9. The blood collection container according to claim 8, wherein the blood collection container is sterilized by radiation.

10. These are blood collection containers that have been sterilized by radiation. The blood collection container according to claim 8, wherein the pH of the aqueous solution for blood cell separation is 3.5 or more and 6.4 or less.

11. The blood collection container body has an open end and a closed end, The blood collection container according to claim 8, wherein the aqueous solution for blood cell separation is contained within the blood collection container body on the closed end side of the blood collection container body, rather than at the location where the blood separation material is contained.

12. The blood collection container according to claim 8, wherein the blood separation material is a blood separation composition.

13. The blood separation composition comprises an organic component that is fluid at 25°C and an inorganic fine powder. The aforementioned organic component includes a resin, The blood collection container according to claim 12, wherein the inorganic fine powder contains fine silica powder.

14. The blood collection container body further comprises an anticoagulant contained within the blood collection container body, The blood collection container body has an open end and a closed end, The blood collection container according to claim 8, wherein the anticoagulant is contained within the blood collection container body on the open end side of the blood collection container body, more so than the location where the aqueous solution for blood cell separation is contained.

15. The blood collection container according to claim 8, wherein the blood collection container body is a polyethylene terephthalate container.

16. A step of collecting blood into a blood collection container according to claim 8, A method for separating mononuclear cells, comprising the step of centrifuging the blood collection container from which the blood has been collected.

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