JP3862085B2 - Preservation method of microorganisms and animal-derived materials - Google Patents

Description

  The present invention relates to a novel method for preserving microorganisms and animal products. More specifically, the present invention relates to a storage method characterized in that microorganisms and animal-derived substances are placed in an electrostatic field atmosphere.

  Liver transplantation from brain-dead donors has been established as a treatment for end-stage liver disease, and more than 8,000 cases have already been performed annually in the United States and Europe. In Japan, the organ transplantation law was finally enforced in 1997, but as of October 2003, six years have passed, only 23 cases of liver transplantation by donating liver from brain dead. On the other hand, in Japan, since partial living donor liver transplantation, which was donated to the liver by relatives or spouses, was first implemented in 1989, more than 2300 cases have been carried out to date, and living donor liver transplantation is now becoming a daily practice.

  Unlike living donor liver transplantation, which can proceed simultaneously with donor surgery and can be stored in the shortest possible cold storage, long-term cold storage (0 to 4 ° C.) is unavoidable in the case of brain dead liver transplantation. The University of Wisconsin (UW) fluid was developed in the late 1980s, and the limit of cold storage time was greatly extended to 24 hours from the previous 7-8 hours, and liver transplantation changed from emergency surgery to quasi-emergency / waiting surgery. Has been achieved. However, even after 5 to 10% of cases, graft dysfunction is observed after transplantation, and in reality, the probability of graft dysfunction is very high when stored for more than 16 hours. In addition, the limit of preservation for heart / lung transplantation is still 6 to 7 hours, and the current situation is that it is not out of emergency surgery. Therefore, if the storage time can be further extended, the global impact on various organ transplants is considered to be so large.

  Focusing on organ preservation temperature, it is known that metabolism is 1/10 at the conventional cold preservation temperature of 4 ° C and 1/17 at -4 ° C, suggesting the usefulness of non-freezing non-freezing preservation. It was. Conventionally, experiments using non-freezing agents have been carried out. However, although non-freezing storage can be performed below freezing point, grafting troubles caused by non-freezing agents have not been avoided.

  Due to the development of transplant technology, various improvements in the preservation method of tissues of animals to be transplanted have been studied. Storage under conditions where the tissue is not frozen is a preferred storage method because the tissue is in a more natural state. For example, blood in a blood vessel is removed and replaced with a first solution containing glucose, and the first solution is replaced with a second solution containing dimethyl sulfoxide or glycerin and mannitol. A method of storing at 0 ° C. is disclosed (Patent Document 1). There is also a proposal of a method for preserving viable microorganisms, cells, or tissues by a combination of preservatives of non-reducing disaccharides and fillers (Patent Document 2). However, any storage method requires addition of a stabilizer and complicated treatment, and early improvement is desired.

  An apparatus for storing food or the like in a supercooled state using an electrostatic field atmosphere has been developed by one of the inventors (Patent Documents 3 to 7). However, they were all used only in the food field.

JP-A-8-325101 Special Table 2003-505024 JP-A-10-136882 JP-A-11-332464 JP 2000-297976 A Japanese Patent Laid-Open No. 2001-241824 International Publication No. WO 98/41115

  It is an object of the present invention to provide a novel method for preserving microorganisms and animal origin.

As a result of intensive studies to solve the above problems, the present inventors have found that microorganisms and animal-derived materials can be preserved in a more natural form by placing microorganisms or animal-derived materials in an electrostatic field atmosphere. Completed the invention.
That is, this invention consists of the following.
1. A storage method in which red blood cells derived from animals are placed in an electrostatic field atmosphere and stored under a temperature condition of 0 ° C to 4 ° C.
2. A preservation method in which an animal-derived heart, liver, or kidney that is immersed in a preservation solution is placed in an electrostatic field atmosphere and preserved under the following conditions.
2. Heart: -5 ° C to 2 ° C temperature condition Liver: -5 ° C to 2 ° C temperature condition Kidney: -5 ° C to 0 ° C temperature condition 3. The storage method according to item 1 or 2, wherein the electrostatic field atmosphere is formed by applying an AC or DC voltage of 100 V to 5000 V to the electrode.

  By storing organs or organs in the electrostatic field atmosphere of the present invention, it is possible to store them for a long time without damaging the tissues or the like. That is, in the method of the present invention, the microorganism or animal-derived material is stored in a state close to nature for a long period of time without inactivating or inactivating the activity of the microorganism or animal-derived material or killing it. be able to.

  In organ preservation, there is a possibility that metabolism is suppressed by non-freezing preservation below freezing and that oxidative stress at the time of reperfusion is suppressed by antioxidative action to reduce ischemia-reperfusion injury. By placing the organ in an electrostatic field atmosphere and applying micro-vibration energy, it is possible to prevent the organ or the organ from freezing at a temperature that would normally freeze, for example, about -5 ° C, and prevent damage for a long time without damage. It can be saved.

  The electrostatic field atmosphere of the present invention refers to, for example, bringing a closed or open container into an electrostatic field state. Various means are known in order to obtain an electric field atmosphere. For example, the electrode plate can be brought into an electrostatic state by simply placing the electrode plate in an insulated state on the bottom of the container. Moreover, a normal household or commercial refrigerator can be easily converted into an electrostatic field refrigerator. For example, it is formed of a horizontal plate made of an insulating material (vinyl chloride plate), side plates that can be assembled to both sides of the horizontal plate via hinges, and a bottom plate that closes the bottom of the electric field box. And when the front and upper surfaces are opened and the refrigerator door is opened, the object can be easily put in and out. A high voltage is applied to one of the metal bars or the like by a connecting line or a high voltage generator to form an electrostatic field.

  As a storage device that can be used in the method for storing a microorganism or animal-derived cell of the present invention, specifically, a container equipped with an electrode for forming an electrostatic field atmosphere, and an AC or DC voltage is applied to the electrode Examples of the apparatus include an electrostatic field generating power source and a cooling device capable of maintaining an organ of an animal at a refrigerated temperature, for example.

  Furthermore, in order to charge the air in the refrigeration room, a conductive curtain may be provided. This curtain is made by attaching a conductive paint to the surface of a flexible cloth or plastic, or the curtain itself is made of a thin aluminum plate or the like. You may form by doing. The curtain is connected to the high voltage generator via a rail or the like.

  The electrostatic field atmosphere of the present invention is formed by applying an AC or DC voltage of 100 V to 5000 V, preferably 100 V to 3000 V, to the electrodes. The voltage to be applied can be appropriately selected depending on the storage object and its storage state. In particular, the voltage to be applied can be selected in the case of storing in a storage solution or depending on the material of the storage container. The current may be either alternating current or direct current.

  The temperature that can be applied to the storage method in the electrostatic field atmosphere of the present invention is -20 to 40 ° C, preferably -20 to 5 ° C, more preferably -12 to -1 ° C, and further preferably -5 to -1 ° C. It is. The temperature to preserve | save can be suitably selected with a preservation | save object and its preservation | save state. In particular, even when the temperature is 0 ° C. or lower, the object to be stored can be stored without freezing due to the supercooling phenomenon. Here, the supercooling phenomenon refers to a phenomenon in which a substance does not freeze even at a temperature below the freezing point, which is the temperature just before the liquid begins to freeze. Even when the temperature is below the freezing point, in the electrostatic field atmosphere of the present invention, it is considered that microvibration energy occurs at the same time that the temperature is transmitted to the substance, the aqueous solution does not freeze, and the microorganisms and animal-derived substances do not freeze. .

  The microorganism or animal-derived material being immersed in the preservation solution means a state where the microorganism or animal-derived material is immersed in the preservation solution in a container such as metal or plastic, and any generally known cells, etc. Can be used as well as future storage solutions. As typical preservation solutions, for example, Ringer's solution, Eurocollins solution, UW solution, SLS solution, H-- exemplified in today's transplantation (Vol.11, No.5, September p.549-557 (1998)). Examples include L solution, HTK solution and the like, and commercially available products such as Lactec (manufactured by Otsuka Pharmaceutical).

The term “microorganism or animal-derived substance is left as it is in an electrostatic field atmosphere” means that the microorganism or animal-derived substance is left in an electrostatic field atmosphere without being placed in an aqueous solution. It may be stored in a container such as plastic. For example, if microorganisms are left as they are in an electrostatic field atmosphere, they can be put into a dead state due to a supercooling phenomenon, and a purified protein can be stored as it is for a long time without worrying about inactivation. In addition, collected blood and its derivatives include CPD solution (including sodium citrate, citrate, glucose, NaH ₂ PO ₄ · 2H ₂ O), MAP solution (including mannitol, adenine, and phosphate). When placed in a blood collection bag made of plastic (pp) or the like and left as it is in an electrostatic field atmosphere, it can be stably stored, for example, at 0 ° C. or higher.

  Thus, the method of the present invention comprises organ / tissue preservation in the transplant area, blood component preservation in the transfusion area, ingredient preservation in the biological preparation area, preservation of the plasma fraction preparation, cell / tissue preservation in the regenerative medicine area. Application in areas such as storage of various cultured cells in the basic experimental area, storage of genes and drugs introduced in the gene therapy area, specimen storage in the clinical laboratory area, and storage of purified protein in the pharmaceutical / reagent area Can do.

  Microorganisms and animal-derived materials applied to the preservation method of the present invention mean that they include microorganisms such as bacteria, fungi, and viruses, and substances derived from humans and animals other than humans. The heart, lung, liver, kidney, pancreas, small intestine, heart valve, skin, blood vessel, cornea, eyeball, dura mater, bone, trachea, ear ossicle, etc. , Umbilical cord blood, plasma components, various factors, etc., in the biological product area, purified proteins derived from blood and urine components such as blood coagulation factor, anticoagulation factor, thrombin, urokinase, ulinastatin, placenta and these Recombinant proteins, other gelatins, heparin, chondroitin, hyaluronic acid, etc. are hematopoietic stem cells, ES cells (embryonic stem cells), bone marrow, various factors However, biochemical samples, endocrine samples, virus samples, bacterial samples, fungal samples, immune serum samples, cellular immune samples, genes, chromosome samples, chromosome samples, hematology samples, microbial samples, Pathological specimens, etc. include microorganisms containing vectors and genes introduced in gene therapy areas, and various cultured cells in basic experimental areas include vascular endothelial cells, blood stem cells, various cells for regenerative medicine, etc. .

  EXAMPLES The present invention will be described below with reference to examples, but these show typical representative examples of the present invention, and the present invention is not limited thereto.

(Example 1) Kidney 1) Removal and preservation of kidney An adult rat was anesthetized, the limbs were fixed with an 18G needle, and the abdomen was opened from the chest of the rat to the abdomen. The aorta was clamped directly under the diaphragm, the inferior vena cava and hepatic vein were clamped together, the distal part thereof was opened, and a paper wiper was inserted. The aorta was identified on the dorsal side of the left renal vein of the rat, and the kidney was slowly perfused with 5 mL of Lactec (manufactured by Otsuka Pharmaceutical).
The right and left kidneys of the rat were removed and placed in a dish containing Lactec (manufactured by Otsuka Pharmaceutical). 4 mL of the preservation solution was injected into the kidney piece using an injection needle and preserved under each preservation condition.
2) Preservation of kidney 1st time: When the voltage was not applied at 4 ° C. and under the condition that each voltage of 500 V and 1000 V was applied at −5 ° C., the kidney was stored for 28 hours.
Second time: The sample was stored for 28.5 hours and 67 hours under the condition that no voltage was applied at 0 ° C and a voltage of 100 V was applied at -3 ° C.
3) Results Lactic acid dehydrogenase (l-lactate dehydrogenase, hereinafter referred to as “LDH”) leaked into the liquid when stored under the above storage conditions was measured. Furthermore, at the second time, kidney tissue sections were stained and observed with an optical microscope. The results are shown in Tables 1 and 2. Although individual differences were observed, the value of LDH leaked into the storage solution was lower when a voltage was applied than when no voltage was applied, and good results were obtained in all cases. In addition, the tissue pieces after the 2nd storage for 28.5 hours were observed to denature when no voltage was applied, but no obvious degeneration was observed when the voltage was applied.

(Example 2) Liver 1) Extraction of Liver An adult rat was anesthetized, the limbs were fixed with an 18G needle, and the abdomen was opened from the chest of the rat to the abdomen. Slowly inject 4 mL of UW fluid from the portal vein (today's transplant: Vol.11, No.5, September p.549-557 (1998)), perfuse and extract the liver, place it in a dish containing UW fluid, and organ saved. 10 mL of a preservative solution was injected into the extracted liver piece using an injection needle and refrigerated under each storage condition.
2) Liver preservation The liver was preserved for 4 hours under the conditions where no voltage was applied at 4 ° C and each voltage of 500 V and 1000 V was applied at -5 ° C.
3) Results Glutamate-oxaloacetic transaminase (hereinafter referred to as “GOT”), glutamic acid-pyruvic transaminase (hereinafter referred to as “GPT”) leaked into the preservation solution when stored under each condition. Table 3 shows the measurement results of LDH. As a result, the best results were obtained when stored under conditions where a voltage of 500 V was applied.

(Example 3) Heart 1) Extraction and preservation of heart An adult rat was anesthetized, the limbs were fixed with an 18G needle, and the thoracotomy was performed from the chest to the abdomen of the rat. The heart was quickly removed together with the large blood vessel, placed in a dish (6 cm) containing Lactec (manufactured by Otsuka Pharmaceutical), and the organ was preserved.
The heart chamber of the preserved heart was replaced with Lactec (manufactured by Otsuka Pharmaceutical Co., Ltd.) by the autonomous heartbeat.
4 mL of the preservation solution was injected into the heart piece using an injection needle and refrigerated under each preservation condition.
2) Preservation of the heart GOT, GPT, LDH in the preservation solution is measured when the heart is stored for 4 hours under the condition that no voltage is applied at 4 ° C and each voltage of 500V and 1000V is applied at -5 ° C. did. In addition, tissue sections were stained and observed with an optical microscope.
3) Results Table 4 shows the measurement results of creatine kinase (creatine kinase, hereinafter referred to as “CK”), GOT, and LDH leaked into the storage solution when stored under each condition. As a result, the values of CK, GOT, and LDH leaked into the storage solution were lower when the voltage was applied than when no voltage was applied, indicating a good result. Myocardial tissue was good with no obvious changes in cytoplasm and nucleus in each condition.

(Example 4) Red blood cells 1) Acquisition of red blood cells Blood was collected by a preoperative blood storage type self-transfusion method for self-transfusion. The collected blood was separated into red blood cells and plasma by a conventional centrifugation, and the red blood cell solution stored in the MAP solution was stored in a pp blood collection bag.
2) Storage of red blood cells The MAP red blood cell solution was dispensed in 2 mL aliquots into plastic test tube tubes, and stored at 4 ° C under the conditions of a non-electric field group to which no voltage was applied and an electric field group to which a voltage of 3000 V was applied.
3) Results The storage state when the MAP erythrocyte solution was stored under each condition was observed, and the results are shown in Tables 5 and 6. Even when the voltage was not applied even on the seventh day after storage, it was confirmed that the change in pH and the change in the degree of leakage of potassium (K) were less and the storage conditions were better (Table 5). Furthermore, even after 15 days of storage, the leakage of potassium (K) and the change in the amount of sodium (Na) are less when the voltage is applied than when no voltage is applied, and the storage conditions are good. It was confirmed (Table 6).

(Example 5) Heart segment 1) Extraction and preservation of heart An adult male rat was anesthetized with ether, the limbs were fixed with an 18G needle, and the abdomen was opened from side to side to the neck. The upper edge of the liver was cut off from the diaphragm and the diaphragm was perforated to open the thoracotomy. After opening the front edge of the diaphragm to the left and right, the ribs on both sides were cut along the sternum. Clamp the descending aorta, inject 5 mL of organ myocardial protective fluid Lactec G (manufactured by Otsuka Pharmaceutical), inject 3 mL of myocardial protective fluid myo- tector (manufactured by Nissin Oil) into the proximal part of the descending aorta, remove the heart, Placed in a dish containing and preserved organs.
2) Preservation of the heart 4 mL of myocardial protective solution was injected into the heart with an 18G needle, shaken gently, and then stored under the conditions where no voltage was applied at 2 ° C. and each voltage of 100 V and 500 V was applied.
3) Results Table 7 shows the measurement results of creatine phosphokinase (creatin phospho kinase, hereinafter referred to as “CPK”), GOT, and LDH leaked into the myocardial protective solution when stored under each condition. As a control, it was also measured after 1 hour of storage. After storage for 24 hours, leakage of CPK, GOT and LDH tended to be kept lower when voltage was applied than when no voltage was applied.

(Example 6) Liver 1) Extraction and preservation of liver After the heart was removed in Example 5, the rat lower aorta was clamped, and the organ preservation solution beer span (from Fujisawa Pharmaceutical Co., Ltd.) as far as possible from the portal vein. 4 mL was slowly injected, the liver was removed, placed in a dish containing beer span, and preserved in organs.
2) Preservation of the liver 10 mL of the preservative solution was injected into the liver with an 18G needle, shaken gently, and stored under the conditions where no voltage was applied at 2 ° C. and each voltage of 100 V and 500 V was applied.
3) Results Table 8 shows the measurement results of GOT, GPT, LDH, γGPT, and alkaline phosphatase (hereinafter referred to as “ALP”) leaked into the storage solution when stored under each condition. As a control, it was also measured after 1 hour of storage. As a result, GOT, GPT, LDH and ALP tended to increase after storage for 24 hours compared to the control. It was recognized that leakage of GOT, GPT and LDH tended to be kept lower when stored under voltage application conditions than when no voltage was applied.

(Example 7) Heart fragment 1) Extraction and preservation of heart An adult rat male was anesthetized with ether, the limbs were fixed with an 18G needle, the skin was cut from the abdomen to the neck, and the abdomen was opened left and right. The upper edge of the liver was cut off from the diaphragm and the diaphragm was perforated to open the thoracotomy. After opening the front edge of the diaphragm to the left and right, the descending aorta is clamped, 5 mL of organ preservation solution Lactec G (manufactured by Otsuka Pharmaceutical) is injected, and 3 mL of myocardial protective fluid myo- tector (manufactured by Nisshin Oil) is injected into the proximal part of the descending aorta. Then, the heart was removed, placed in a dish containing a myocector, and preserved in organs.
2) Preservation of the heart 4 mL of preservative solution was injected into the heart with an 18G needle, and the heart was preserved under the condition where no voltage was applied at 0 ° C. after applying light shaking and under the conditions of applying 100 V and 500 V voltages.
3) Results Table 9 shows the measurement results of CPK leaked into the storage solution when stored under each condition. As a control, measurement was also performed for 30 minutes from the start of storage. As a result, after 4 hours, the CPK value hardly increased compared to the control. After 24 hours of storage, the CPK value increased when no voltage was applied, but a tendency to be kept low when the voltage was stored under the applied conditions was observed.

(Example 8) Liver 1) Extraction and preservation of liver After the heart was removed in Example 7, the rat lower aorta was unclamped, and the organ preservation solution beer span (made by Fujisawa Pharmaceutical Co., Ltd.) from the distal end of the portal vein as much as possible. 4 mL was slowly injected, the liver was removed, placed in a dish containing beer span, and preserved in organs.
2) Preservation of liver 10 mL of the preservative solution was injected into the liver with an 18G needle and shaken gently, and then stored under the conditions where no voltage was applied at 0 ° C. and each voltage of 100 V and 500 V was applied.
3) Results Table 10 shows the measurement results of GOT, GPT, LDH, and ALP leaked into the storage solution when stored under each condition. As a control, measurement was also performed after 30 minutes of storage. As a result, GOT, GPT, LDH, and ALP tended to increase 24 hours after liver preservation, but the leakage of each substance was kept lower when stored under conditions where voltage was applied than when not applied. A tendency was observed.

(Example 9 ) Kidney 1) Extraction and preservation of liver The left and right kidneys were sequentially removed from the rat after the liver was removed in Example 9, placed in a dish with Lactec (manufactured by Otsuka Pharmaceutical) and preserved in organs. .
2) Preservation of kidney 4 mL of the preservative solution was injected into the kidney with an 18G needle, shaken gently, and stored under the conditions where no voltage was applied at 0 ° C. and each voltage of 100 V and 500 V was applied.
3) Results Table 11 shows the measurement results of GOT and LDH leaked into the storage solution when stored under each condition. As a control, measurement was also performed after 30 minutes of storage. As a result, the measured values of GOT and LDH tended to increase after storage for 23 hours and 69 hours, but the leakage of each substance was lower when stored under the condition of applying voltage than when not applied. A tendency to be suppressed was observed.

(Example 10) Heart 1) Extraction and preservation of heart An adult rat male was anesthetized with ether, the limbs were fixed with an 18G needle, and the skin was cut from the abdomen to the neck. The upper edge of the liver was cut off from the diaphragm and the diaphragm was perforated to open the thoracotomy. After opening the front edge of the diaphragm to the left and right, the descending aorta is clamped, 5 mL of organ preservation solution Lactec G (manufactured by Otsuka Pharmaceutical) is injected, and 3 mL of myocardial protective fluid myo- tector (manufactured by Nisshin Oil) is injected into the proximal part of the descending aorta. Then, the heart was removed, placed in a dish containing a myocector, and preserved in organs.

2) Preservation of the heart 4 mL of preservative solution was injected into the heart with an 18G needle, and the heart was preserved under the condition where no voltage was applied at 0 ° C. after applying light shaking and under the conditions of applying 100 V and 500 V voltages.

3) Measurement of CK-MB Measured by immunosuppression-UV method. Specifically, the following method is used. The activity of the CK-B subunit is measured using an antibody that specifically inhibits only the CK-M subunit in the preservation solution when the heart is preserved under the above preservation conditions. By doubling the value, the activity of CK-MB can be determined.

4) Measurement of troponin T Measured by immunochemiluminescence immunoassay (ECLIA). This method is a step sandwich method using an electro-chemiluminescent reactions. The troponin T in the sample is reacted with the biotinylated anti-troponin T antibody and Ru (bpy) ₃ ²⁺ labeled anti-troponin T antibody to produce a labeled antibody-troponin T-biotinylated antibody complex. Next, magnetic microparticles (MP) coated with streptavidin (SA) are added to cause avidin-biotin reaction. After completion of the reaction, the mixed solution is sucked into the measurement cell, and the magnetic microparticles are attracted to the electrode by the magnetic force of the electrode. B / F separation is performed with triproamine (TPA), which is an electrical donor, and the amount of luminescence from the Ru ²⁺ complex generated by the oxidation reaction by the electrode and the reduction action of the TPA radical is measured, and the amount of troponin T is measured. .

5) Results FIG. 1 shows the measurement results of CK-MB leaked into the preservation solution when stored under each condition, and FIG. 2 shows the measurement results of cardiac troponin. As a control, measurement was also performed for 30 minutes from the start of storage. As a result, these measured values were kept lower when the voltage was stored under the applied condition than when no voltage was applied after 24 hours of storage, and a better result was obtained when the applied voltage was 500V than 100V.

(Example 11) Red blood cells 1) Acquisition of red blood cells For self-transfusion, blood was collected by a preoperative blood storage type self-transfusion method. The collected blood was separated into red blood cells and plasma by a conventional centrifugation, and the red blood cell solution stored in the MAP solution was stored in a pp blood collection bag.
2) Storage of erythrocytes The MAP erythrocyte solution was dispensed in 2 mL aliquots into plastic test tube tubes, and stored at 4 ° C. for each of a non-electric field group to which no voltage was applied and an electric field group to which a voltage of 500 V was applied.
3) Results Sodium (Na), potassium (K), free hemoglobin (free Hb), and total haptoglobin (total Hp) when stored under each condition were measured, and the results are shown in Tables 12-15. As the storage time passed, Na decreased, and K and free Hp increased. Regarding Na, K, and total Hp, there was almost no difference between the non-electric field group and the electric field group, but free Hb was more effective to suppress the increase in the electric field group than in the non-electric field group.

(Example 12) Red blood cells 1) Acquisition of red blood cells For self-transfusion, blood was collected by a preoperative blood storage type self-transfusion method. The collected blood was separated into red blood cells and plasma by a conventional centrifugation, and the red blood cell solution stored in the MAP solution was stored in a pp blood collection bag.
2) Storage of erythrocytes The MAP erythrocyte solution was dispensed at 2 mL into plastic test tube tubes, and stored at 4 ° C. for each of a non-electric field group to which no voltage was applied and an electric field group to which a voltage of 3000 V was applied.
3) Results Na, K, free Hb, and total Hp when stored under each condition were measured, and the results are shown in Tables 16-19. In the non-electric field group, Na decreased in the non-electric field group and K and free Hb increased, but the decrease in Na was suppressed in the non-electric field group, and the increase in K and free Hb was also suppressed. It was.

(Example 13) Liver A) Examination of temperature conditions Voltages of 100V, 500V, 1000V, 2000V, 3000V were applied to organ preservation solution (UW solution) at temperatures of -4 ° C, -5 ° C, -6 ° C, and UW Conditions were set so that the liquid did not freeze. As a result, it was confirmed that when the voltage was applied, it did not freeze at -4 ° C.
B) Voltage application conditions (4 ° C 0V, -4 ° C, 100V)
Rat livers were removed, and AST, ALT, and LDH were measured in a group that was stored in UW solution for 24 hours by applying a voltage of 100 V at -4 ° C. A condition where no voltage was applied at 4 ° C. was taken as a control group.
The results are shown in FIG. Compared with the control group, when volt was applied at −4 ° C. with a voltage of 100 V, all of AST, ALT, and LDH showed lower values and were better.
C) Voltage application conditions (4 ° C 0V, 4 ° C, 3000V)
Rat livers were removed, and AST, ALT, and LDH were measured in a group stored at 4 ° C. with a voltage of 3000 V and stored in UW solution for 24 hours. A condition where no voltage was applied at 4 ° C. was taken as a control group.
The results are shown in FIG. There were no effective differences in AST, ALT, and LDH between the control and voltage applied groups.

  As described above, when placed in an electrostatic field atmosphere of the present invention, microorganisms or animal-derived materials such as organs and organs can be stored without freezing even at 0 ° C. or lower, and 0 ° C. or higher. Even in this case, blood and the like could be stored in a good state. In other words, by applying the preservation method under the electrostatic field atmosphere of the present invention, it becomes possible to preserve microorganisms or animal-derived substances in a state close to nature for a longer period of time than before, particularly in transplantation regions and transfusion regions. It can be used for regenerative medicine, basic experiment, gene therapy, clinical testing, pharmaceutical / reagent, etc.

It is a figure which shows the result of having measured CK-MB in a preservation | save liquid when the heart is preserve | saved under voltage application conditions. (Example 10) It is a figure which shows the result of having measured the troponin T in a preservation | save liquid when the heart is preserve | saved under voltage application conditions. (Example 10) It is a figure which shows the result of having measured AST, ALT, and LDH in a preservation | save liquid when the liver was preserve | saved under the voltage application conditions of 100V. (Example 13) It is a figure which shows the result of having measured AST, ALT, and LDH in a preservation | save liquid when the liver was preserve | saved on the voltage application conditions of 3000V. (Example 13)

Claims (3)

A storage method in which red blood cells derived from animals are placed in an electrostatic field atmosphere and stored under a temperature condition of 0 ° C to 4 ° C. A preservation method in which an animal-derived heart, liver, or kidney that is immersed in a preservation solution is placed in an electrostatic field atmosphere and preserved under the following conditions.
Heart: -5 ° C to 2 ° C temperature condition Liver: -5 ° C to 2 ° C temperature condition Kidney: -5 ° C to 0 ° C temperature condition The storage method according to claim 1 or 2, wherein the electrostatic field atmosphere is formed by applying an AC or DC voltage of 100V to 5000V to the electrodes.

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