JPH0920687A - Lyophilized blood reconstitution - Google Patents

Abstract

(57) Abstract: A freeze-dried blood containing cells and / or a blood component containing cells which does not reduce the survival rate (regeneration rate) of blood cells such as red blood cells in the lyophilized blood returning step. Provide a reconstitution solution. SOLUTION: A solution containing at least one of a saccharide, a biopolymer, or an acid or a salt of an acid,
It is characterized by being obtained by containing a cell activating agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention does not require a special refrigerant or device for storage, requires a small amount of energy supply for storage, and does not need to mix / eliminate an antifreezing agent such as glycerin. The present invention relates to lyophilized blood containing cells and / or blood components containing cells (hereinafter referred to as lyophilized blood), and more particularly to a reconstitution solution for obtaining a high blood cell regeneration rate.

[0002]

2. Description of the Related Art So-called autologous blood transfusion, in which one's own blood is stored and transfused when necessary, rare blood to be stored in case of a person having a rare blood type, or general blood Blood for transfusion and these blood components require preservation. In many cases, the storage period is one month or more.

An electric freezer is typically used for long-term storage of blood and blood components at -80 ° C.
There is a freezing method using liquid nitrogen and a freezing method using liquid nitrogen (stored at about -196 ° C) and a freezing method using liquid helium (stored at -270 ° C). Each of these blood storage methods requires a special refrigerant and a container for cooling, and the supply of energy therefor. Further, in these methods, a cryoprotectant such as glycerin is mixed with blood before cryopreservation in order to prevent cell membrane destruction due to growth of ice crystals or osmotic pressure. However, at the time of use, it was necessary to thaw it to remove the antifreezing agent such as glycerin.

However, such a method requires a special refrigerant and a container for storing blood, and must supply a large amount of energy for that purpose, and the deglycerin operation is complicated and time-consuming, so that it is necessary for emergency use. There was a problem that it could not be applied.

Therefore, in order to solve such problems, a method of freeze-drying blood has been developed. In this method, one liquid selected from the group consisting of blood containing blood cells, bone marrow fluid (hematopoietic stem cells), and plasma platelets is treated with at least one of sugars, biopolymers, or acids or salts thereof. A step of pretreating the pretreated liquid with a solution containing, a step of micronizing the pretreated liquid into a first predetermined size, and a step of rapidly freezing the micronized liquid into a product of a second predetermined size. And a process of sublimating water from the frozen material and drying (see Japanese Patent Application No. 7-32718).

[0006]

The freeze-dried blood thus prepared is stored, if necessary, dissolved in a reconstitution solution before use, washed and then used for blood transfusion. In the above, it is important to maintain the level before lyophilization as much as possible without reducing the viable number of blood cells such as erythrocytes in lyophilized blood. The present invention has been made in view of the above circumstances, and an object thereof is to provide a lyophilized blood reconstitution solution that does not reduce the survival rate (regeneration rate) of blood cells such as red blood cells in the lyophilized blood reconstitution step. To do.

[0007]

According to the present invention, sugars,
A lyophilized blood reconstitution solution obtained by adding a cell activating agent to a solution containing at least one of a biopolymer or an acid or a salt of an acid is provided.

The saccharide may contain at least one saccharide selected from mannose, xylose, glucose, trehalose, sucrose and maltrose. The biopolymer may include at least one biopolymer selected from dextran, phosphate ester salts, polyvinylpyrrolidone (PVP), and albumin.

The above-mentioned acid or acid salt is phytic acid (also known as inositol hexaphosphate: IHP), pyrophosphate, adenosine triphosphate (ATP), 2,3-diphosphoglycerin (2,3-DPG). ), And may contain at least one or more substances selected from The cell activating agent may be composed of a substance containing phosphoenolpyruvate (PEP).

The pH of the lyophilized blood reconstitution solution may be between 4 and 9. Further, the pH of the lyophilized blood reconstitution solution may be between 6 and 8.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. In the following description, blood or blood components will be taken as an example and simply referred to as blood for convenience of description.

Examples of saccharides used in the reconstitution solution of the present invention include those containing at least one saccharide selected from mannose, xylose, glucose, trehalose, sucrose and maltose. The saccharide concentration is preferably about 1 to 2 M, and if it is 3 M or more, the hypertonicity is too high, which is not preferable.

Examples of the biopolymer include those containing at least one biopolymer selected from dextran, phosphate ester salts, polyvinylpyrrolidone (PVP) and albumin. Commercially available products of these biopolymers can be widely used. For example, examples of the phosphoric acid ester salt include phosphorylcholine chloride [Sigma (Sigma)].
a) Reagent # P0378], 5-phosphorylribose-1
- pyrophosphate (Sigma reagent # P8296), _{also, C 3 H 3 NO 6 P} ( Sigma reagent # P5506), C ₃ H
₅ NO ₆ P (Sigma reagent # P0753), C ₃ H ₈ NO ₆ P
(Sigma reagent # P0878) and C ₃ H ₁₀ NO ₆ P (Sigma reagent # P1003), and examples of salts thereof include sodium salt and calcium salt.

Examples of the albumin include those containing at least one albumin selected from human albumin, bovine albumin and ovalbumin.

Here, examples of human albumin include Sigma reagent A-1653, A-1887, and A-951.
1, the same A-8763, the same A-3782, the same A-678
4, the same A-6019, the same A-6909, and dinitriphenyl (the same A-6661).

Further, as an example of bovine albumin, Sigma reagent A-2153, A-3350, A-4503,
A-3425, A-9647, A-8022, A-6003, A-2934, A-9543, A
-4628, same A-7888, same A-4378, same A-
3424, A-751, A-7638, A-0
281, the same A-3059, the same A-3902, the same A-79
06, same A-6793, same A-6918, same A-391
2, the same A-7409, the same A-7534, the same A-728
4, same A-1662, same A-3299, same A-317
4, A-7159, A-7034, A-329
4, the same A-7030, the same A-9430, the same A-380
3, same A-7688, same A-3675, same A-930
There are 6, etc.

Examples of ovalbumin include Sigma Reagents A-5253, A-5378, and A-550.
3, same A-2512, same A-7641, same A-315
There are 4, etc.

Polyvinylpyrrolidone (PVP) having a molecular weight of about 10,000 to 360,000 can be preferably used, and a molecular weight of about 40,000 is more preferable.

The concentration of biopolymer in the reconstitution solution is
It is preferably about 5 to 20%, more preferably about 10 to 20%.

As an example of the above-mentioned acid or acid salt, phytic acid (also known as inositol hexaphosphoric acid: I
HP), pyrophosphate, adenosine triphosphate (AT
P), 2,3-diphosphoglyceric acid (2,3-DP
Examples thereof include those containing at least one kind of substance selected from G). And, as an example of the pyrophosphate,
Calcium salt _{(Ca 2 P 2 O 7)} , iron salts _{[Fe 4 (P 2 O 7} )
₃ ], potassium salt (K ₄ P ₂ O ₇ ), sodium salt (Na ₂
H ₂ P ₂ O _7, or _{Na 4 P 2 O 7 · 10H} 2 O), tin salts _{(Sn 2 P 2 O 7)} , include tributylammonium salt. The concentration of the acid or acid salt in the reconstitution solution is 0.
About 1 to 3% is preferable, and about 1% is more preferable.

Examples of the cell activating agent include phosphoenolpyruvate (PEP, Phosphoenol).
pytuvate). The concentration of the cell activating agent in the reconstituted solution is preferably about 50 to 500 mM, and 5
About 0 to 100 mM is more preferable.

The pH of the return liquid is controlled between 4 and 9. It is preferably between 6.0 and 8.0. pH
As a control method, a commonly used phosphate buffer or the like can be used. For example, disodium hydrogen phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH ₂ P)
O ₄ ) to a phosphate buffer prepared by adding a physiological electrolyte substance to a buffer solution, and adding one or more of any one of the saccharides, biopolymers, acids or acid salts and a cell activating agent,
Finally, its pH is between 4 and 9, preferably 6.0.
It should be between ~ 8.0. Specifically, 1
/ 15 mol Na ₂ HPO ₄ and 1/15 mol KH ₂ PO ₄
Buffer consisting of 0.2 M NaCl and 4 mM KC
Those to which 1 is added can be preferably used.

Here, when the pH of the return liquid is less than 4,
After reconstitution, the freeze-dried blood is converted into meth and functional deterioration such as the oxygen carrying capacity not being recovered is observed, which is not preferable.
On the other hand, if the pH is higher than 9, the freeze-dried blood becomes alkaline, and a tendency of lysis / hemolysis is observed, which is unsuitable.

[0024]

EXAMPLES Next, a method for reconstitution of freeze-dried blood using the reconstitution solution of the present invention will be described. The freeze-dried blood to be mixed with the reconstitution solution of the present invention is prepared by freeze-drying blood or blood components. First, the frozen blood is preferably used after undergoing pretreatment. For example, a hematocrit value of 55 to 5 is collected from the collected blood by a method such as centrifugation.
90% concentrated red blood cells are obtained. As an example of the pretreatment liquid, for example, as described in Japanese Patent Application No. 7-32718,
It may be a solution containing at least one of sugars, biopolymers, or acids or salts of acids.

In the pretreatment, the concentrated red blood cells and the pretreatment liquid are mixed (mixed) in appropriate amounts. As a means of mixing,
A conventional method may be used, such as stirring and shaking. Also,
In the mixing procedure or the method of injecting the blood into the pretreatment liquid,
The reverse method may be used. Preferably, the pretreatment liquid is added to the blood while mixing.

Examples of the saccharides used in the pretreatment liquid include those containing at least one saccharide selected from mannose, xylose, glucose, trehalose, sucrose and maltose.

Examples of biopolymers used in the pretreatment liquid include those containing at least one biopolymer selected from dextran, phosphate ester salts, and polyvinylpyrrolidone (PVP) albumin.

Examples of the acid or acid salt used in the pretreatment solution include phytic acid (also known as inositol hexaphosphate: IHP), pyrophosphate, adenosine triphosphate (ATP), 2, Examples thereof include those containing at least one substance selected from 3-diphosphoglycerin (2,3-DPG).

The pH of the pretreatment liquid is controlled between 4 and 9. It is preferably between 7.0 and 8.0. For example, in a phosphate buffer obtained by adding a physiological electrolyte substance to a buffer solution containing disodium hydrogen phosphate (Na ₂ HPO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ), the sugar, biopolymer, acid or Any one or more of the acid salts is added so that the final pH is between 4 and 9, preferably between 7.0 and 8.0.

Next, the pretreated blood mixed with the pretreatment liquid is microparticulated. As a method of forming fine particles,
Carrier gas such as air and nitrogen gas and blood (mixed liquid)
Is preferably mixed and sprayed using a nozzle. In this atomization method using mixed spray, the mixed and sprayed blood scatters in different particle sizes (cluster size in which a plurality of blood cells are collected) depending on the nozzle diameter. For example,
When a nozzle having a nozzle diameter of 1.2 mm is used, the relationship depends on the pressure of carrier gas and the flow rate of blood. When the nozzle diameter is 1.2 mm, the blood flow rate is in the range of 1-20 cc / min, and the cluster size of several microns to 40 microns can be obtained in the range of carrier gas pressure of 0.5-6 kg / cm ^2. . The larger the nozzle diameter, the larger the cluster size, and it is possible to control the cluster size and the throughput of the blood mixture.

In the method of atomizing by mixing and spraying, it is preferable that the nozzle is composed of a nozzle that generates a jet flow and / or a spiral flow. In particular,
The nozzle preferably has the following functions. That is,
"Patent application publication No. 4-21551" (publication date April 1992)
10 / May / Japanese Patent No. 1730868) "Nozzle for generating ultrafine particles by high-speed swirl gas", "a liquid jet port and an annular swirl chamber formed at a position where the liquid jet port is engaged. Then, it extends spirally into the swirl chamber,
A plurality of swirl guide holes for introducing a high-speed airflow into the swirl chamber, and a tapered conical shape having a focal point in front of the liquid jet port, which is opened toward the front of the liquid jet port on the side of the swirl chamber facing the liquid jet port. And an ultra-fine particle generation nozzle using a high-speed vortex gas, which has an annular gas injection port for forming and forming a high-speed vortex flow. Also, Japanese Patent No. 16896 having the same function.
No. 45, No. 1689660, No. 1757351, or No. 1770496 may be used.
These define various examples of gas and liquid introduction methods.

The point is that "the gas introduced from the gas inlet is formed into a spiral shape by a plurality of swirl guide holes that spirally extend into the swirl chamber and introduce a high-speed air flow into the swirl chamber.
A high-speed swirl is generated by injecting from a gas outlet having a tapered conical shape having a focal point in front of the liquid jet through the swirl chamber, thereby pulverizing and atomizing the liquid. " By using this nozzle, the blood containing the cells can be atomized. Further, the distribution of particle size is narrow, and it becomes possible to make fine particles of uniform size. Further, it may be a nozzle having another configuration utilizing the so-called normal spiral flow principle.

In this way, the blood which has been made into fine particles, preferably fine particles having a diameter of several tens of microns by the mixed spray is attached to the inner wall of the container and is instantaneously frozen (freezing step).
For the freezing step, it is preferable that the container is made to have an extremely low temperature of about -196 ° C by using a refrigerant such as liquid nitrogen. After the freezing process, the blood is evacuated by a vacuum pump and the blood is dehydrated in a so-called sublimation process to obtain freeze-dried blood.

In the freeze-drying method, a method of atomizing and freezing blood by mixing and spraying has been described. As a method different from the above-described methods for atomizing and freezing, at least a low-temperature gas below freezing point is used. A method of freezing as small particles by spraying blood onto the skin may be used. This method, which is also called an ice scriber, includes a method of producing small frozen particles by introducing a liquid into nitrogen gas at the temperature of liquid nitrogen. The atomization can also be performed by a head element driven by ultrasonic waves.

The freeze-dried blood obtained by such a method can be stored in an ordinary refrigerator (storage temperature of about 5 ° C.). Then, when used for actual blood transfusion and the like, the lyophilized blood thus prepared is mixed with the reconstitution solution of the present invention. The freeze-dried blood and the reconstituted solution may be mixed in a conventional manner, for example, by stirring or shaking. Further, the mixing procedure may be a method of injecting the blood into the pretreatment liquid or a reverse method thereof. Preferably, the pretreatment liquid is added to the blood while mixing. Further, after the returning step, it is washed with physiological saline or the like and used for actual blood transfusion.

[0036]

EXAMPLE As a reconstitution solution of the present invention, based on a phosphate buffer consisting of 1/15 mol of disodium hydrogen phosphate and 1/15 mol of potassium dihydrogen phosphate, sugars, biopolymers, acids or Solutions containing at least one kind of acid salt were prepared with various compositions and further containing a cell activator (# 1 to # 7 in Table 1).
Up to)). All were prepared by the same method except for the sugar, biopolymer, type of acid or acid salt, and concentration of cell activating agent.

Lyophilized blood was reconstituted as follows using the reconstitution solutions of # 1 to # 7. The freeze-dried blood was stored in an ordinary refrigerator (storage temperature of about 5 ° C.), stored 6 months later, and then returned, and the blood cell survival rate was measured by a blood cell counter. The survival rate is 2000 rpm of the blood returned, 1
After centrifuging for 0 minutes, the supernatant was collected and the hemoglobin amount was measured with a blood counter (sysmecs NE-8000). A value obtained by dividing (dividing) this by the hemoglobin value of the mixed solution before microparticulation / freeze-drying, which was measured in advance, was taken as the hemolysis rate. The hemolysis rate at this time was approximated to the survival rate after microparticulation and freeze-drying. That is, the survival rate = 100- (hemolysis rate). Table 1 also shows the results.

Here, the concentrations of glucose, xylose, mannose, and disaccharide were set to 1 to 2 mol, and PVP was used.
Mainly used those having a molecular weight of about 40,000. Table 1 shows the results. The results showed that the survival rate of blood cells was high when the type of the saccharide was changed and when the molecular weight of PVP was changed. Regarding the pyrophosphate salt, similar results were obtained with any of sodium salt, potassium salt, calcium salt and iron salt.

[0039]

[Table 1]

The returned blood of the above examples was washed with physiological saline and subjected to actual blood transfusion, and good results were obtained.

[0041]

By using the lyophilized blood reconstitution solution of the present invention, a high regeneration rate of blood cells such as red blood cells can be increased in the lyophilized blood reconstitution step. Therefore, a good blood for transfusion can be obtained by performing the lyophilized blood reconstitution step using the reconstitution solution of the present invention.

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Claims (7)

[Claims] 1. Lyophilized cell-containing blood and / or
Alternatively, it is a reconstitution solution of blood components containing cells, which is obtained by containing a cell activating agent in a solution containing at least one kind of sugar, biopolymer, or acid or acid salt. Freeze-dried blood reconstitution solution. 2. The saccharides are mannose, xylose,
The lyophilized blood reconstitution solution according to claim 1, which contains at least one saccharide selected from glucose, trehalose, sucrose, and maltrose. 3. The biopolymer is dextran,
The lyophilized blood reconstitution solution according to claim 1 or 2, comprising at least one biopolymer selected from a phosphate salt, polyvinylpyrrolidone, and albumin. 4. The acid or acid salt is phytic acid,
The reconstitution of lyophilized blood according to any one of claims 1 to 3, characterized in that it contains at least one substance selected from pyrophosphate, adenosine triphosphate and 2,3-diphosphoglycerin. liquid. 5. The lyophilized blood reconstitution solution according to any one of claims 1 to 4, wherein the cell activating agent is composed of a substance containing phosphoenolpyruvate. 6. The pH of the lyophilized blood reconstitution solution is
Is between 4 and 9, and the lyophilized blood reconstitution solution according to any one of claims 1 to 5. 7. The pH of the lyophilized blood reconstituted solution is
7. The lyophilized blood reconstitution solution according to claim 6, characterized in that