JP5458446B2 - Biomaterial storage method - Google Patents

Description

  The present invention relates to a biomaterial storage method including blood, cells, tissues, and organs.

  Conventionally, as a method for preserving microorganisms and animal-derived materials, an electrostatic field atmosphere is formed by applying an AC or DC voltage of 100 V to 5000 V to the electrode, and the electrostatic field atmosphere is placed at −20 ° C. to −40 ° C. A storage method has been disclosed. This is done by inactivating or inactivating the activity of microorganisms or animal-derived substances by storing them in an electrostatic field atmosphere of AC or DC voltage of 100V to 5000V, preferably 100V to 3000V. It can be stored without any problem (see, for example, Patent Document 1).

Japanese Patent Laid-Open No. 2005-112839

  However, in the conventional storage method, regarding the method of applying the current voltage, the current may be either alternating current or direct current. In addition to each voltage value such as 100V, 500V, 1000V, etc., an effective voltage application method It was not specified. In addition, the above conventional storage method focuses on storing the activity of microorganisms or animal-derived materials without inactivation, inactivation, or death, in a state close to nature for a long period of time. It was. For this reason, it cannot be said that it is possible to sufficiently suppress oxidation and reduce cell damage due to storage.

In order to solve the above problems, the present invention adopts the following means (1) to (2) . That is,
(1) In the biomaterial storage method of the present invention , a voltage application plate is installed in a refrigerator, a container containing the biomaterial is placed on the voltage application plate, and an AC voltage is applied to the biomaterial. And the negative DC voltage are applied simultaneously to the biological material storage method to freeze the biological material while performing the simultaneous voltage application step, wherein the setting value of the DC voltage is higher than the absolute value of the setting value of the AC voltage. The absolute value of is characterized by being larger .

(2) In the biomaterial storage method, in the simultaneous voltage application step, the biomaterial is accommodated in a container made of a conductive metal surrounding at least the periphery of the biomaterial, and a direct current is applied to an outer surface of the container. It is preferable that the voltage is applied to the biomaterial indirectly by directly or indirectly following the voltage application plate on which the voltage and the alternating voltage are superimposed and applied.

  By adopting the above-described configuration, inactivation of dissolved oxygen in the biomaterial O during storage can be achieved, and more effective suppression of oxidation and reduction of cell damage can be achieved.

FIG. 6 is a perspective explanatory view illustrating an example of a state when the application plate, the container, and the electrode are stored in the biomaterial storage method according to the first embodiment. It is front view cross-section explanatory drawing which shows the whole structure at the time of the preservation | save including the storage in the example of a state of Example 1 shown in FIG. It is a perspective explanatory view showing the example of the state at the time of preservation of an application board, a container, and an electrode in a preservation method of biomaterial of Example 2. It is front view sectional explanatory drawing which shows the whole structure at the time of the preservation | save including the storage in the example of a state of Example 2 shown in FIG. It is a conceptual explanatory view showing the entire configuration (including storage) in the biomaterial storage method of Example 3 at the time of storage. It is a figure which shows the data regarding the suppression of the rat serum lipid peroxidation reaction by room temperature preservation | save of Experiment 1. It is a figure which shows the data regarding the predominance of the direct-current alternating current application in the freezing of human erythrocytes of Experiment 2. FIG. It is a figure which shows the data regarding the optimal conditions of the voltage application in the human erythrocyte freezing of the experiment 3. FIG. It is a figure which shows the data regarding the comparison of the cell death of the experiment 4 under the oxidative stress by cell culture.

  Embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 is an explanatory perspective view illustrating an example of a state when the application plate 2, the container 1 and the electrode are stored in the method for storing the biomaterial O according to the first embodiment. FIG. 2 is a front cross-sectional explanatory view showing the entire configuration during storage including the storage 4 in addition to the state example of the embodiment 1 shown in FIG. 1. FIG. 3 is a perspective explanatory view illustrating an example of a state of the application plate 2, the container 1, and the electrode during storage in the method for storing the biomaterial O according to the second embodiment. FIG. 4 is a front view cross-sectional explanatory diagram showing the overall configuration during storage including the storage 4 in addition to the state example of the embodiment 2 shown in FIG. 3. FIG. 5 is a conceptual explanatory diagram showing an overall configuration (including the storage 4) at the time of storage in the biomaterial O storage method of the third embodiment. 6 to 9 are diagrams showing data obtained in Experiments 1 to 4, respectively. Specifically, FIG. 6 is data of Experiment 1 regarding suppression of rat serum lipid peroxidation by storage at room temperature. 7 is data of Experiment 2 regarding the superiority of DC / AC simultaneous application in freezing of human erythrocytes, FIG. 8 is data of Experiment 3 regarding optimal conditions for voltage application in freezing of human erythrocytes, and FIG. The data of Experiment 4 regarding the comparison of cell death under oxidative stress by cell culture.

(Regarding the scope of the term “preservation of blood, cells, tissues, and organs”)
Regarding the scope covered by the term “preservation of biomaterial O”, “biomaterial O” of the present invention includes blood, cells, tissues, and organs. In addition, regarding the range covered by the term “preservation of biomaterial O”, “preservation” of the present invention includes the following three meanings.
(1) Room temperature storage (4 ° C to 20 ° C (room temperature))
(2) Cryopreservation (-20 ° C to -196 ° C (temperature of liquid nitrogen))
(3) Cell culture preservation (37 ° C)
Experiment 1 (FIG. 6), which will be described later, is the above-described 1) room temperature storage of rat serum. Experiments 2 (FIG. 7) and 3 (FIG. 8) are cryopreservation of human erythrocytes. (Experiment 2) and (Experiment 3) are the same experiment, but (Experiment 2) is a comparison between α (alternating current) alone, β (direct current) alone and αβ combined, (experiment 3). Indicates the optimum conditions for simultaneous application. Experiment 4 (FIG. 9) is the culture preservation of (3).

  The biomaterial O storage method according to the first embodiment of the present invention is a voltage simultaneous application step in which a DC voltage and an AC voltage are simultaneously applied to the biomaterial O by the storage device shown in FIGS. It is characterized by including. In this voltage simultaneous application step, the biomaterial O is accommodated in a tube stand type holder as the container 1, and the voltage application plate 2 on which a DC voltage and an AC voltage are superimposed and applied to the outer surface of the container 1 is applied. A voltage is applied to the biomaterial O indirectly by direct contact. A storage state in which the container 1 and the application plate 2 are not brought into contact with each other and the application plate 2 is indirectly aligned may be employed.

  When performing the simultaneous voltage application of the present invention, for example, the indirect as described above rather than the voltage application by directly connecting the electrode to the container 1 or the voltage application by directly placing the biomaterial O on the application plate 2. It was found that a uniform voltage application effect can be obtained efficiently by applying a proper voltage.

  Specifically, in the voltage simultaneous application step, a container 1 in which a biomaterial O is placed on a voltage application plate 2 on which a DC voltage and an AC voltage are superimposed and applied (the container 1 of Example 1 is a tube stand type holder). By placing a container).

(Container 1)
The container 1 is a container that stores the biomaterial O so as to surround at least the periphery of the biomaterial O, and is mainly composed of a conductive metal, particularly a highly conductive metal. In Example 1, it consists of the metal which has aluminum of purity 80% or more as a main component. Specifically, it is a tube stand type holder in which an aluminum plate having a thickness of 60 mm is provided with eight accommodation holes 1h for accommodating the biomaterial O, with a predetermined hole interval.

(Accommodating hole 1h)
The accommodation hole 1h is covered with at least the wall and the bottom (preferably further a lid) of the container 1, and is configured to surround at least the surrounding four directions and the lower surface direction (preferably the upper surface) of the accommodated biomaterial O. The However, the bottom here (or the lower surface direction seen from the biomaterial O) refers to a component plate that deflects the application plate 2 to which a voltage is applied.

  When viewed from the biomaterial O in the accommodation hole 1h, the direction in which the indirect voltage is applied (lower surface direction) is covered with a conductive plate (bottom plate), and at least the direction opposite to the same direction (upper surface direction) is excluded. All side surfaces) are covered with a conductive plate (a wall plate connected in all directions).

  The accommodation hole 1h according to the first embodiment is a cylindrical hole hollowed into a cylindrical shape. When the biomaterial O is blood, the blood contained in the test tube is accommodated in the accommodation hole 1h or the blood is directly accommodated. The housing hole 1h preferably has the same cross section at any depth of the hole.

(Wall thickness and bottom thickness)
Because of the effect of simultaneous application, the thickness of the constituent material of the container 1 is preferably 10 mm or more (at least a bottom thickness of 10 mm and a wall thickness of 15 mm or more, preferably about 15 mm for both the bottom thickness and the wall thickness).

In Example 1, the minimum wall thickness 1a in the vertical direction in plan view (the horizontal minimum distance from the side end of the accommodation hole 1h to the front side surface (or the back side surface) of the container 1) is 15 mm,
The minimum wall thickness 1b in the horizontal direction in plan view (the horizontal minimum distance from the side end of the receiving hole 1h to the right side (or left side) of the container 1) is 30 mm,
Minimum height 1c in the height direction (vertical minimum distance from the bottom of the receiving hole 1h to the bottom of the container 1) is 15 mm,
The representative length 1d of the cross section of the accommodation hole 1h (the diameter of the cross section of the circular accommodation hole 1h, the long side of the cross section of the square accommodation hole 1h) is 14 mm,
The minimum wall thickness 1e between the receiving holes 1h (the minimum horizontal distance from the adjacent first receiving hole 1h side end to the adjacent second receiving hole 1h side end) is 15 mm.

(Apply plate 2)
The application plate 2 is a plate made of a conductive material, and is formed by arranging electrodes of a pair of electric wirings 3 at symmetrical positions. The electrical wiring 3 is a common wiring 3 for both direct current and alternating current, and the electrodes are also arranged to share direct current and alternating current. Thereby, by simultaneous application, a part of the AC voltage is converted into a DC voltage in a superimposed manner, and the actual value of the AC voltage (to the biomaterial O) is lower than the set value of the AC voltage. The effective value of the actual DC voltage (to the biomaterial O) is higher than the set value of the DC voltage.

  The applied voltage preferably does not exceed 5000 V for both direct current and alternating current. Furthermore, it is preferable that the set value of the AC voltage is 500 to 2500 V and the set value of the DC voltage is 200 to 1000 V. The DC voltage is preferably negative, and the set value (absolute value) of the DC voltage is preferably larger than the set value of the AC voltage.

  In addition, it is preferable for the combination of AC and DC voltage values that the AC voltage set value is about 500 to 550 V when the DC voltage set value is about 1000 V or about 3000 V.

  However, the set value referred to above and in the present invention refers to an effective value for the actual biomaterial O when a voltage is applied independently of either alternating current or direct current.

(Storage 4)
The application plate 2 is placed on the storage 4 of the storage shelf month as shown in FIG. 2, and the biomaterial O is stored in this state.

  In Example 2, as a voltage simultaneous application step, a container 1 (both of Example 2 in which the bottom thickness and wall thickness of biomaterial O are placed on a voltage application plate 2 on which a DC voltage and an AC voltage are superimposed and applied) The container 1 is by stacking a cage holder). According to the simultaneous application and storage of voltage using the container 1 of the second embodiment, the other configuration is the same as that of the first embodiment even for the biomaterial O having a relatively large volume.

  In the third embodiment, as compared with the first embodiment, a storage bag having a predetermined bag material thickness or more is used instead of the storage container 1. However, a conductive material is applied or laminated on the inner surface of the storage bag, and the effect of simultaneous application is obtained efficiently by contacting the biomaterial O in almost all directions. The biomaterial O is superimposed on the ion attraction direction by the DC voltage indicated by A in the figure and the ion attraction direction by the AC voltage indicated by the arrow B.

  Each of the above-described embodiments is merely an example for achieving the storage method of the present invention, and is not necessarily limited to the above-described configuration, and various modifications can be made without departing from the spirit of the present invention.

(Experiment 1 “Inhibition of Rat Serum Lipid Peroxidation by Applying Voltage” (FIG. 6))
In Experiment 1, rat serum was stored at room temperature and the amount of lipid peroxide was measured.

(Experiment method of Experiment 1)
Serum was isolated from rat peripheral blood and used as a sample. The sample was dispensed into a tube and left to stand at room temperature (20 degrees) for 12 hours for storage. At this time, voltages of α (alternating current) 2020 V and β (direct current) 3000 V were applied to the application plate, and the sample was stored while being applied on the application plate. After storage for 12 hours, the amount of lipid peroxide was measured using the TBARS method (thiobarbituric acid reactant method) and compared with a sample that had not been applied. In the measurement by the TBARS method, malondialdehyde (MDA) produced by decomposition of lipid peroxide in a sample reacts with thiobarbituric acid to produce a red reactant, and its absorbance is measured with a spectrophotometer. (525 nm) is measured. In the measurement, absorbance was measured using an Ultraspec 3000 manufactured by Pharmacia Biotech, and the absorbance was compared between the groups.

(Result of Experiment 1)
The amount of lipid peroxide in the sample to which an AC / DC voltage was applied was significantly lower in absorbance than the sample to which no voltage was applied, indicating that production of lipid peroxide was suppressed.

  This has shown that the oxidation of the lipid in serum is suppressed by voltage application.

(Experiment 2) “Advantage of AC / DC simultaneous application in human erythrocyte freezing” (see FIG. 7 entitled “Effects of NICE01 Electric Device RBC Cryopreservation”
(Experiment method of Experiment 2)
After adding an equal amount of cryopreservation solution (albumin-added CP-1, Far East Pharmaceutical Co., Ltd.) to human peripheral blood washed erythrocytes obtained by collecting blood from volunteers, the mixture was frozen at −20 ° C. for 3 hours. At this time, an aluminum application plate was installed in the freezer, and the voltage application storage device of Example 1 of the present invention was used, and the AC voltage (α5: 980 V) alone was applied alone, and the DC voltage (β8: 3000 V) alone was used. Electric fields were created for each of the three groups of application and AC / DC simultaneous (superimposed) application (α5β8), and the electric field was frozen on the application plate.

  The frozen sample was then stored for 2 weeks in a -80 degree freezer, thawed in a 37 degree constant temperature bath, centrifuged, and the amount of LDH (lactate dehydrogenase) in the supernatant was measured. The LDH measurement was performed using an LDH release assay kit manufactured by Promega, and the absorbance was measured using a TERMO MAX microplate reader manufactured by Molecular Devices. The measurement results were compared between the three groups of AC voltage (α5: 980V) single application, DC voltage (β8: 3000V) single application, and AC / DC simultaneous (superposition) application (α5β8).

(Result of Experiment 2)
LDH deviated from peripheral blood erythrocytes after freezing and thawing decreased in all groups of alternating current alone, direct current alone, or alternating current direct current combined group as compared with voltage application (-), indicating that cell damage was small. In addition, in AC / DC combination, a significant decrease in cell damage was observed compared to AC alone or DC alone.
From this result, it was confirmed that the reduction of cell damage in voltage application freezing was more noticeable when both α and β were applied simultaneously.

(Experiment 3: “Optimum Conditions for Voltage Application in Freezing Human Red Blood Cells” (FIG. 8))
(Experiment method of Experiment 3)
As in Experiment 2, after adding an equal amount of cryopreservation solution (albumin-added CP-1, Far East Pharmaceutical Co., Ltd.) to human peripheral blood washed erythrocytes obtained by collecting blood from volunteers, it was frozen at −20 ° C. for 3 hours. . At this time, an aluminum application plate was installed in the freezer, and both the AC voltage (α) shown in Table 1 and the DC voltage (β) shown in Table 2 were used using the voltage application storage device of Example 1 of the present invention. Was applied to create an electric field and frozen on the application plate. The voltage application was performed in the combinations shown in FIG. 8 for α and β.

The frozen sample was then stored for 2 weeks in a -80 degree freezer, thawed in a 37 degree constant temperature bath, centrifuged, and the amount of LDH (lactate dehydrogenase) in the supernatant was measured. The LDH measurement was performed using an LDH release assay kit manufactured by Promega, and the absorbance was measured using a TERMO MAX microplate reader manufactured by Molecular Devices. At this time, the absorbance itself varies depending on the experiment depending on the temperature conditions at the time of measurement. Therefore, control, that is, voltage is not applied (left side of each of the two parallel graphs in FIG. 8) and voltage is applied. (The right side of each of the two parallel graphs in FIG. 8) was performed at the same time for each experiment, and it was examined whether there was an application effect by comparing them.

  (Results of Experiment 3) FIG. 8 shows the results of Experiment 3. In FIG. 8, the LDH amount in each combination of α (AC voltage) β (DC voltage) is not applied with voltage (light-colored graph on the left side) and applied with voltage (dark graph on the right-hand side). Two are shown side by side. In FIG. 8, the scale on the Y-axis (vertical axis) indicates the measured value of absorbance. The larger the value, the greater the amount of enzyme (LDH) released from the broken erythrocyte cell, and the greater the degree of cell damage. The absorbance itself varies depending on the experiment depending on the temperature conditions at the time of measurement. Therefore, the control, that is, the voltage is not applied (left side of the two parallel graphs in FIG. 8) and the voltage is applied (FIG. 8). The right side of each of the two parallel graphs was performed simultaneously for each experiment, and by comparing them, it was examined whether there was an application effect. In each of the two parallel graphs in FIG. 8, the larger the difference between NICE (−) (without voltage application) and NICE (+) (with voltage application), the greater the effect of application.

  LDH deviated from peripheral blood erythrocytes after freezing and thawing is decreased compared to the group to which no application is applied in any combination of α (alternating current) and β (direct current). It was shown to be suppressed. In particular, the combination of α5 (980 V) β8 (3000 V), α5 (980 V) β3 (950 V), and α3 (530 V) β8 (3000 V) showed a stronger decrease in free LDH.

(Experiment 4 “Inhibition of cell damage under oxidative stress by applying voltage” (FIG. 9))
As Experiment 4, hydrogen peroxide was added to the cell culture to induce cell damage, and a comparative experiment of cell death was performed.

(Experimental purpose)
In order to investigate the effect of simultaneous voltage application with Nice Zero One on living cells and the effect of reducing participation stress, the effect of voltage application in a state in which oxidative stress due to hydrogen peroxide was applied to cultured cells was examined.

(experimental method)
THP-1 cells, a human macrophage cell line, were cultured in a RPMI1640 culture medium at 37 ° C. in the presence of 5% CO 2 for 12 hours using a 96-well culture plate. At that time, 0 uM to 250 uM hydrogen peroxide was added. Added to induce cell damage due to oxidative stress. The incubator is an incubator manufactured by Napco, an aluminum application plate is installed in the incubator, a 96-well culture plate is placed on the application plate, and AC voltage and DC voltage are simultaneously applied (α5β8). The culture was performed below. The MTT method was used for evaluation of cell injury. 12 hours after the start of cell culture, 20 ul / well of MTT reagent (5 mg / ml) was added to each well of the culture plate, and further cultured for 4 hours. After completion of the entire culture, SDS reagent was added to each well to lyse the cells, and then the formazan reduced by dehydrogenase in mitochondria was produced using an absorbance plate reader (TERMO MAX microplate reader manufactured by Molecular Devices). The absorbance was measured at a wavelength of 490 nm.

  (Results of Experiment 4 (FIG. 9)) The effect of hydrogen peroxide addition on cell viability increases cell viability at low concentrations up to 100 uM, and conversely induces cell death at high concentrations. Also in this experiment, the absorbance value by the MTT method slightly increased at a low concentration and decreased at a high concentration. In the voltage application group, the absorbance value was higher at any hydrogen peroxide concentration than in the application (-), and the viability of the cells was increased or maintained.

  In the experiment, the viability of the cells is measured using the MTT method. However, the greater the absorbance value, the higher the viability, and conversely, the lower the value, the fewer live cells. Here, cells generally use a low concentration of oxidative stress as a stimulus for cell proliferation or the like, but a pathologically high level of oxidative stress induces cell damage or cell death. For this reason, the viability of the cell is slightly increased at a low concentration up to 100 uM, while the cell is damaged and the absorbance is decreased at a high concentration of 250 uM. As a result, it can be said that when a voltage is applied, cells are less likely to die even at a high concentration of oxidative stress.

  As an explanation of this phenomenon, as shown in Experiment 1 (FIG. 6), the neutralization and suppression of the oxidation reaction by applying a voltage can be mentioned, but in addition, a new that electrical energy is converted into biological energy. There seems to be a mechanism. That is, in order to prevent the cells from dying, it is important to increase the energy (ATP) in the cells, but it is considered that the cells are difficult to die by applying a voltage.

(Specific procedure for frozen storage of blood)
Specific examples of procedures for cryopreserving blood are as follows. Approximately 400 ml of peripheral blood is collected by puncture of the donor's elbow vein and stored in a blood storage bag. After the plasma component and blood cell component are separated by a centrifuge, the plasma component is removed, and physiological saline and a cryoprotectant (CP-1, manufactured by Kyokuto Pharmaceutical Co., Ltd.) are added and stored frozen. At this time, freezing is performed while simultaneously applying α (alternating current) and β (direct current).

  Applicability of the simultaneous application technique of the present invention considered in the medical industry includes the following. First, cryopreservation by voltage application: allogeneic peripheral blood cryopreservation for blood transfusion, autologous blood cryopreservation, bone marrow transplant bone marrow cryopreservation, cord blood cryopreservation, islet cell cryopreservation, various cultured cell cryopreservation, ES cell cryopreservation And cryopreservation of various organs for transplantation, and cryopreservation of tissue grafts such as bone, aorta, trachea, heart valve, cornea, and skin.

  Next, examples of cold storage by applying voltage include allogeneic peripheral blood storage for blood transfusion, autologous blood storage, and cold storage of various organs for transplantation.

DESCRIPTION OF SYMBOLS 1 Container 11 Tube stand type | mold container 12 Vertical type container 1h Housing hole 1a The minimum wall thickness of the vertical direction of planar view (horizontal direction minimum distance from a housing hole side end to the front side surface (or back side surface) of a container)
1b Minimum wall thickness in the horizontal direction in plan view (horizontal minimum distance from the side of the receiving hole to the right side (or left side) of the container)
1c Minimum bottom thickness in the height direction (vertical minimum distance from the bottom of the receiving hole to the bottom of the container)
1d Representative length of cross section of receiving hole (diameter of circular receiving hole cross section, long side of rectangular receiving hole cross section)
1e Minimum wall thickness between receiving holes (horizontal minimum distance from adjacent first receiving hole side end to adjacent second receiving hole side end)
2 Voltage application plate 3 Electrical wiring 4 Storage box 41 Storage shelf 5 Storage bag O Biomaterial

Claims (3)

A voltage application plate is installed in the refrigerator, and a container containing a biomaterial is placed on the voltage application plate,
The biological material is frozen while performing the voltage simultaneous application step by simultaneously applying an AC voltage of a set value of 530 V to 980 V and a DC voltage of a set value of -950 V to -3000 V to the voltage application plate. Material storage method. The biomaterial storage method according to claim 1, wherein the absolute value of the set value of the DC voltage is larger than the absolute value of the set value of the AC voltage. In the voltage simultaneous application step, the biomaterial is accommodated in a container made of a conductive metal surrounding at least the periphery of the biomaterial, and a voltage application plate in which a DC voltage and an AC voltage are superimposed and applied to one surface outside the container. The biomaterial storage method according to claim 1 or 2, wherein a voltage is applied to the biomaterial indirectly by direct or indirect alignment.