JP3065727B2 - Mononuclear cell separation tube and mononuclear cell separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mononuclear cell separation tube and a method for separating mononuclear cells. More specifically, the present invention relates to a mononuclear cell separation tube and a mononuclear cell capable of easily separating and collecting mononuclear cells (lymphocytes and mononuclear large leukocytes) from anticoagulated blood with high purity. It relates to a separation method.

[0002]

2. Description of the Related Art Investigation of the function of lymphocytes, which play an important role in the immune surveillance mechanism, and the proportion of their subsets (subfractions) is important for the diagnosis of various diseases, particularly immunodeficiency and autoimmune diseases. It is important to know the change of the condition. In addition, when performing blood transfusion or organ transplantation including bone marrow transplantation, it is important to examine the compatibility of HLA (major histocompatibility antigen), which is a type of leukocyte, for suppressing rejection or side effects after transplantation.

[0003] In these tests, furthermore, in order to analyze the killer activity of lymphocytes in tumor immunity and the interaction between cells, the separation of mononuclear cells is an indispensable technique. It is desired to provide a method.

[0004] The most common technique for separating mononuclear cells from human peripheral blood is 5.7% w / v Ficoll 40.
0 and 9.0% w / v aqueous solution of sodium diatrizoate, Ficoll-Paque (registered trademark, Pharmacia Fine Chemicals (Registered trademark) whose specific gravity (density) is adjusted to 1.077 g / ml. Pharmacia FineCh
emicals).

This method comprises the following basic steps (1) to
(4). (1) placing a predetermined amount of Ficoll pack on the bottom of the test tube; (2) carefully pipetting the neat or diluted blood sample onto the Ficoll pack; (3) preparing the Ficoll pack. The Ficoll-packed blood preparation is placed in a 400-mm basin so that blood components having a specific gravity greater than the specific gravity will travel into or pass through the Ficoll-pack.
Centrifuging at ~ 500G for about 30-40 minutes,
(4) A step of collecting a mononuclear cell layer separated above the Ficoll pack with a pipette.

[0006] However, this method has the following disadvantages. That is, when a blood sample is first transferred onto a Ficoll pack using a pipette, if leukocytes spread below the surface of the Ficoll pack,
There is a problem that the specific gravity of Ficoll Pak is locally reduced. Therefore, when transferring blood, the blood must be gently layered so as not to disturb the interface with the Ficoll pack, and the necessary amount of Ficoll pack must be placed in the test tube at the time of use. However, there is a problem that the operation is complicated.

It is also conceivable that a Ficoll pack is stored in a test tube in advance, but since it has high fluidity, the test tube must be allowed to stand until the interface shape is stabilized before blood is supplied. And the operation is inefficient.

In order to solve this problem, a mononuclear cell separation tube having a structure in which a high specific gravity liquid whose specific gravity is adjusted to 1.077 g / ml is sealed with a water-insoluble gel-like substance having thixotropic properties is used. A method is disclosed (U
SP 4.190.535).

However, since the gel-like substance used in this method is water-insoluble, after centrifugation, a part of the erythrocytes and granulocytes remain between the gel-like substance and the inner wall of the test tube, and as a result, When separating and collecting the sphere layer, there is a problem that the possibility that erythrocytes and granulocytes are mixed increases.

[0010]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a simple and efficient method for separating mononuclear cells from human peripheral blood without mixing red blood cells, and recovers mononuclear cells without impairing cell functions. It is an object of the present invention to provide a mononuclear cell separation tube and a mononuclear cell separation method capable of performing the method.

[0011]

The above object is achieved by the present invention described below. (1) The specific gravity in the centrifuge tube is 1.064 to 1.079.
A high specific gravity liquid layer adjusted to g / ml, and an insoluble hydrogel layer containing a hydrogel as a main component, which is laminated so as to enclose the high specific gravity liquid layer and made water insoluble by divalent cations, are housed. A mononuclear cell separation tube, comprising: (2) The mononuclear cell separation tube according to the above (1) containing a binder in the insoluble hydrogel layer. (3) The specific gravity is 1.064 to 1.079 in the centrifuge tube.
a step of housing a high specific gravity liquid layer adjusted to g / ml and further laminating an insoluble hydrogel layer containing a hydrogel made water insoluble by divalent cations as a main component so as to encapsulate the high specific gravity liquid layer. Collecting blood and subjecting the obtained blood to an anticoagulant treatment with an anticoagulant containing a binding component that forms a complex with the divalent cation; and supplying the blood containing the anticoagulant into the centrifuge tube. And performing a centrifugal treatment on the supplied blood to change the hydrogel from water-insoluble to water-soluble by the action of the binding component in the anticoagulant, so that the high specific gravity liquid layer can be brought into contact with blood. At the same time, the supplied blood is
Separating a layer, a second layer composed of mononuclear cells, a third layer composed of red blood cells and granulocytes, and a high specific gravity liquid layer positioned between the second layer and the third layer; Collecting a second layer separated in the above step.

Hereinafter, the present invention will be described in detail with reference to FIGS.

FIG. 1 is a sectional view showing the structure of a mononuclear cell separation tube according to the present invention.

The mononuclear cell separation tube 1 of the present invention has a specific gravity of 1.064 to 1.079 g in the centrifuge tube 2 having one end closed.
/ Ml of a high specific gravity liquid layer 3 and an insoluble hydrogel layer 4 containing a hydrogel as a main component, which is laminated so as to enclose the high specific gravity liquid layer and made water insoluble by divalent cations. It is.

In the present invention, the gel precursor component constituting the water-insoluble hydrogel layer 4 is not particularly limited as long as it can be crosslinked and water-insoluble by divalent cations. And polyanions. Among them, polysaccharides which are hardly changed in oncotic pressure due to their large molecular weight and do not adversely affect cells in blood are preferred.

The polysaccharide is a generic name of a polymer compound obtained by dehydrating and condensing a large number of monosaccharides through glycosidic bonds, and is hydrolyzed by an acid to produce a large and generally indeterminate number of polysaccharides and monosaccharides. Among these polysaccharides, those that bind to divalent cations to form insoluble hydrogels include:
Glycuronane (polyuronic acid) can be mentioned.
Pectic acid, alginic acid, protuberic acid and the like are known as glycuronans. Among them, alginic acid which can easily form an insoluble hydrogel by the action of divalent cations and has a high gel strength is used. Is preferred.

Examples of the divalent cation that forms a water-insoluble hydrogel by binding to glycuronane include organic divalent cations such as diamines and polyamino acids, and inorganic divalent cations such as alkaline earth metals and transition metals. Can be. Among them, the binding speed with glycuronan is fast,
Calcium ions and zinc ions that have strong bonds and are excellent in the transparency of the resulting gel are preferred.

The combination of the gel precursor component and the divalent cation constituting the water-insoluble hydrogel layer 4 is not limited to the above, and is usually water-insoluble, There is no particular limitation as long as it becomes water-soluble by the action.

It is preferable that the water-insoluble hydrogel layer 4 further contains a binder. Due to the action of the binder, the adhesiveness to the inner wall of the container is increased, and the shape of the gel can be suitably maintained even with vibration during transportation. Furthermore, even when blood is layered on the gel, the interface of the gel is less disturbed, so that the mononuclear cell separation operation can be performed efficiently. Examples of the binder include polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, vinyl acetate, crystalline cellulose, hydroxypropyl starch, carboxymethylcellulose, and the like, semi-synthetic polymer compounds, dextrin, gelatin, Arabic Natural polymer compounds such as rubber, tragacanth rubber and starch can be used. Among them, hydroxyethylcellulose and carboxymethylcellulose are particularly preferable from the viewpoint of adhesion to the inner wall of the container and ability to retain gel formation.

Below such a hydrogel layer 4,
The high specific gravity liquid layer 3 is enclosed.

The high specific gravity liquid constituting the high specific gravity liquid layer 3 is as follows:
Dextran, gum arabic, low specific gravity components such as albumin, and viscosity, high specific gravity components composed of inorganic substances such as silica powder, or organic substances such as triiodobenzoic acid derivatives are blended at a predetermined ratio, and the specific gravity is 1.064 to 1.079 g. / Ml.

As the high specific gravity component, a triiodobenzoic acid derivative having a large specific gravity, a low viscosity of the solution even at a high concentration, and a relatively low osmotic pressure is particularly preferable.

As the high specific gravity liquid, the above-mentioned Ficoll pack can be used.

The centrifuge tube 2 constituting the mononuclear cell separation tube 1 of the present invention is not particularly limited as long as it can withstand a centrifugal acceleration of 400 to 3000 G and can be hermetically sealed. Specifically, as shown in FIG. 1, one end is closed, and the other end is aseptically sealed with a piercable plug 5 and sterilized by any one of wet heat sterilization, radiation sterilization, and chemical sterilization. It is desired that internal sterilization is maintained by the operation. As the material, glass, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate and the like can be used, and polypropylene and polycarbonate to which various sterilization methods can be applied are particularly preferable. Further, instead of the stopper 5, a bacteria-impermeable membrane having a certain strength may be used.

Next, the mononuclear cell separation method of the present invention will be described with reference to FIGS. FIG. 2 to FIG.
It is a figure showing the outline of the mononuclear cell separation method of the present invention.

First, a specific gravity of 1.064 is set in a centrifuge tube 2 whose one end is closed and the other end of which is sealed with a pierceable plug 5.
A high specific gravity liquid 3 adjusted to ~ 1.079 g / ml is accommodated, and a plate-like carrier (not shown) serving as a scaffold for forming a water-insoluble hydrogel described later is placed on the high specific gravity liquid 3. As the plate-shaped carrier, one having a structure in which a filter paper in which a divalent cation is supported on a thin film made of a resin such as polypropylene is preferably used.

Next, a solution containing the above-mentioned water-insoluble hydrogel precursor component and a binder is overlaid on the plate-shaped carrier, and a solution containing a divalent cation is added and brought into contact with the plate-like carrier to form an insoluble hydrogel layer 4. To form a mononuclear cell separation tube 1
Is prepared.

On the other hand, blood 6 is collected with a syringe 7 or the like in which an anticoagulant has been previously sealed, and supplied into a mononuclear cell separation tube (FIG. 2). The anticoagulant includes a binding component that forms a complex with a divalent cation added to the insoluble hydrogel, and specifically includes an ACD solution, an ACD-A solution,
CPD solution, EDTA solution and the like can be used.

Next, the mononuclear cell separation tube 1 to which the blood 6 has been supplied as described above is placed at about 15 to 30 ° C. for 4 hours.
The centrifugal operation is performed at a centrifugal acceleration of 00 to 2000 G for about 10 to 40 minutes. By this centrifugation operation, the binding component in the anticoagulant forms a complex with the divalent cation in the hydrogel, whereby the hydrogel changes from water-insoluble to water-soluble. When the hydrogel becomes soluble in this manner, the high specific gravity liquid layer 3 can come into contact with the blood 6, and at the same time, the supplied blood 6 becomes the first layer 8 composed of plasma and platelets and the second layer composed of mononuclear cells. 9, a third layer 10 composed of red blood cells and granulocytes, and a high specific gravity liquid layer 3 located between the second layer and the third layer (FIG. 3).

At this time, most of the water-soluble hydrogel is mixed into the first layer 8. Further, the plate-shaped carrier sinks to the lowest position.

After the centrifugation as described above, the first layer 8 composed of plasma and platelets is suctioned and removed using the suction tube 11 or the like, and is transferred to a Spitz tube (not shown) or the like. 4). Subsequently, the second layer 9 made of mononuclear cells is collected in another Spitz tube (not shown) using the pipette 12 or the like (FIG. 5). Then, the collected mononuclear cells are washed with a phosphate buffered balanced salt solution, a cell culture solution, or the like, and used for an inspection or the like.

Next, the present invention will be described in more detail with reference to examples.

[0033]

【Example】

[Example 1] Ficoll
Pack solution (Ficoll-Paque, registered trademark, Pharmacia
Fine Chemicals) (1 ml) was placed therein, and a circular thin film made of polypropylene and a circular filter paper impregnated with a 0.5 M zinc sulfate solution were placed thereon in this order. In addition, 1% w / v sodium alginate and 1% w / v
0.15 ml of a mixed solution of hydroxyethyl cellulose was overlaid, and 15 μl of a 0.5 M zinc sulfate solution was added thereto, and the mixture was allowed to stand at room temperature until a hydrogel was sufficiently formed.

The thus prepared mononuclear cell separation tube was supplied with 2 ml of peripheral blood collected by adding a CPD solution as an anticoagulant, and centrifuged at 400 G for 30 minutes. Then, after the plasma / platelet layer separated by centrifugation is removed by aspirating with a pipette, the mononuclear cell layer is collected in a Spitz tube, and a phosphate buffered balanced salt solution containing a 1% w / v albumin solution (A -PBS) and centrifuged at 1200G. The supernatant was discarded, the blood cells were suspended in A-PBS, and then centrifuged again at 700 G. One part of the supernatant was discarded to obtain 1 ml of a cell suspension.

The number of each blood cell in the cell suspension was measured by using a multi-item automatic blood analyzer (Sysmex NE manufactured by Toa Medical Electronics Co., Ltd.).
-6000), and compared with the number of blood cells in blood before mononuclear cell separation, thereby calculating the blood cell recovery rate.
Table 1 shows the results.

Example 2 Instead of using a mixed solution of 1% w / v sodium alginate and 1% w / v hydroxyethyl cellulose, a mixed solution of 1% w / v sodium alginate and 1% w / v carboxymethyl cellulose was used. Except for using, mononuclear cells were separated in the same manner as in Example 1, and the recovery rate of each blood cell count was calculated. Table 1 shows the results.

Example 3 Mononuclear cells were separated in the same manner as in Example 1 except that the centrifugation conditions at 400 G for 30 minutes were changed to 1600 G for 15 minutes, and the recovery rate of each blood cell was calculated. . Table 1 shows the results.

Example 4 Mononuclear cells were separated in the same manner as in Example 2 except that the centrifugation conditions at 400 G for 30 minutes were changed to 1600 G for 15 minutes, and the recovery rate of each blood cell was calculated. . Table 1 shows the results.

[Comparative Example 1] 2 ml of a Ficoll-pack solution was placed in a polystyrene spitz tube, and C
2 ml of PD-added peripheral blood was gently overlaid and centrifuged at 400 G for 30 minutes. Subsequent operations were performed in the same manner as in Example 1 to separate mononuclear cells and calculate the recovery rate of each blood cell. Table 1 shows the results.

Comparative Example 2 Mononuclear cells were separated in the same manner as in Comparative Example 1 except that the conditions for centrifugation at 400 G for 30 minutes were changed to 1600 G for 15 minutes, and the recovery rate of each blood cell was calculated. . Table 1 shows the results.

[Table 1]

From Table 1, it was confirmed that by using the mononuclear cell separation tube of the present invention, mononuclear cells having a low mixing ratio of red blood cells and platelets can be collected. Moreover, the mononuclear cell separation tube of the present invention did not have the troublesome operation of gently layering blood, unlike the mononuclear cell separation tube of the comparative example, and could be used easily.

Comparative Example 3 A mononuclear cell separation tube described in US Pat. No. 4.190.535 (trade name: Leukoprep,
Becton Dickinson and Company) and supplied with 2 ml of CPD-added peripheral blood,
G. Centrifuged for 15 minutes. Then, after removing the plasma / platelet layer separated by centrifugation with a pipette and removing the mononuclear cell layer with a pipette, red blood cells and granulocytes were found between the upper part of the thixotropic gel and the inner wall of the separation tube. Since they remained, they were mixed into mononuclear cells, and accurate separation was difficult.

[0042]

As described above in detail, the mononuclear cell separation tube of the present invention has a specific gravity of 1.064 to 1.0 in the centrifuge tube.
A high specific gravity liquid layer adjusted to 79 g / ml and an insoluble hydrogel layer containing a hydrogel as a main component, which is laminated so as to enclose the high specific gravity liquid layer and made water insoluble by divalent cations, are housed. As a result, mononuclear cells can be easily and efficiently separated from human peripheral blood without contaminating red blood cells, and mononuclear cells can be collected without impairing cell functions. It is. Therefore, the mononuclear cell separation tube of the present invention can be used for basic immunological research and HL.
It can be effectively applied in the compatibility test of A, the function test of lymphocytes, and the like.

Further, according to the method for separating mononuclear cells of the present invention, a high specific gravity liquid layer whose specific gravity is adjusted to 1.064 to 1.079 g / ml is accommodated in a centrifuge tube. Laminating an insoluble hydrogel layer containing a hydrogel made water-insoluble by divalent cations as a main component, collecting blood, and bonding the obtained blood to form a complex with the divalent cation. A step of performing anticoagulation treatment with an anticoagulant containing a component, a step of supplying blood containing the anticoagulant into the centrifuge tube, and performing a centrifugal treatment on the supplied blood to bind the binding component in the anticoagulant Changes the hydrogel from water-insoluble to water-soluble by the action of water, thereby enabling the high-density liquid layer to come into contact with blood, and, at the same time, converting the supplied blood into a first layer composed of plasma and platelets, and a mononuclear cell. A second layer consisting of A step of separating into a third layer composed of erythrocytes and granulocytes, a high specific gravity liquid layer positioned between the second layer and the third layer, and collecting the second layer separated in the step Therefore, mononuclear cells can be easily and efficiently separated from human peripheral blood without mixing red blood cells, and mononuclear cells can be collected without impairing cell functions. . Therefore, the mononuclear cell separation method of the present invention can be effectively applied to basic immunological research, HLA compatibility test, lymphocyte function test, and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of a mononuclear cell separation tube of the present invention.

FIG. 2 is a diagram schematically showing a method for separating mononuclear cells according to the present invention.

FIG. 3 is a diagram schematically illustrating a method for separating mononuclear cells according to the present invention.

FIG. 4 is a diagram schematically illustrating a method for separating mononuclear cells according to the present invention.

FIG. 5 is a diagram schematically showing a method for separating mononuclear cells according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Monocyte separation tube 2 Centrifuge tube 3 High specific gravity liquid 4 Water-insoluble hydrogel 5 Plug 6 Blood 7 Syringe 8 Plasma and platelet layer 9 Mononuclear cell layer 10 Red blood cell and granulocyte layer 11 Suction tube 12 Pipette

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-54-126718 (JP, A) JP-A-5-322886 (JP, A) JP-A-2-134564 (JP, A) JP-A-56-126 44056 (JP, A) JP-A-61-84557 (JP, A) JP-A-60-53845 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/48 G01N 33 / 50

Claims (3)

(57) [Claims] 1. A centrifuge tube having a specific gravity of 1.064 to 1.064.
Contains a high specific gravity liquid layer adjusted to 1.079 g / ml and an insoluble hydrogel layer mainly composed of a hydrogel which is laminated so as to enclose the high specific gravity liquid layer and made water insoluble by divalent cations. A mononuclear cell separation tube, comprising: 2. The mononuclear cell separation tube according to claim 1, wherein a binder is contained in the insoluble hydrogel layer. 3. A centrifuge tube having a specific gravity of 1.064 to
Containing a high specific gravity liquid layer adjusted to 1.079 g / ml,
A step of laminating an insoluble hydrogel layer containing a hydrogel made water-insoluble by divalent cations as a main component so as to enclose the liquid layer having a high specific gravity; collecting blood, and obtaining the obtained blood A step of performing anticoagulant treatment with an anticoagulant containing a binding component that forms a complex with a cation; a step of supplying blood containing the anticoagulant into the centrifuge tube; and performing a centrifugal treatment on the supplied blood; By changing the hydrogel from water-insoluble to water-soluble by the action of the binding component in the anticoagulant, the high-density liquid layer can be brought into contact with blood, and at the same time, the supplied blood is made up of plasma and platelets. A step of separating into one layer, a second layer composed of mononuclear cells, a third layer composed of erythrocytes and granulocytes, and a high specific gravity liquid layer positioned between the second layer and the third layer And separated in the above process A method for separating mononuclear cells, comprising a step of collecting the second layer.