JP6427649B2 - Method of preserving biomaterial, method of producing biomaterial, biomaterial, implant material, implantation method and preservation device of biomaterial - Google Patents

Description

  The present invention relates to a method of preserving biomaterials, a method of producing biomaterials, biomaterials, implant materials, transplantation methods, and a device for preserving biomaterials.

  In recent years, transplantation treatment of biomaterials such as a part of the living body, organs, cells, etc. has become common, and in Japan, more than 300 transplantation treatments a year are performed, and over 70,000 cases a year in the whole world. Transplantation treatment is being performed. However, with the increase of the transplantation treatment, there is a problem that the biomaterial runs short. For this reason, it is necessary to transport the biomaterial not only from the nearby area of the hospital where the transplant treatment is performed but also from a more distant area. From these things, development of the preservation | save method of the said biomaterial is calculated | required.

  As a storage method of the biomaterial, a method of perfusing the biomaterial using a low temperature storage solution before or after excision, and further immersing the biomaterial after perfusion in the low temperature storage solution (simple immersion method) There are known a method of continuously perfusing the storage solution (continuous perfusion method), a method of storing in a high pressure gas (high pressure gas phase storage method), and the like (patent documents 1 and 2, non-patent document 1). However, the storage period of the biomaterial by these methods was up to 18 hours. For this reason, there has been a problem that it is difficult to transport the biomaterial from a remote area of a hospital where transplantation treatment is performed and to use it for the transplantation treatment. From these things, the new preservation method of a biomaterial with a longer preservation period is called for.

WO 2010/049996 Japanese Patent Application Laid-Open No. 2000-072601

Maathuis MH, et. al. , Transplantation, 2007, vol 83, pp 1289-1298.

  Then, an object of this invention is to provide the preservation | save method of the new biomaterial with a long preservation | save period.

  In order to achieve the above object, the method of storing biomaterials of the present invention is characterized by including a storing step of storing biomaterials under an aerosol atmosphere of a medical gas and a storage solution.

  The method for producing a biomaterial of the present invention includes a material storage step of storing the produced biomaterial, and the material storage step is performed by the storage method of the present invention.

  The biomaterial of the present invention is characterized by being produced by the production method of the present invention.

  The graft material of the present invention is characterized by comprising the biomaterial stored by the storage method of the present invention.

  The implanting method of the present invention is characterized by including an implanting step of implanting at least one of the biomaterial of the present invention and the implant of the present invention in an animal.

  The biomaterial storage apparatus of the present invention includes a storage room, an aerosol supply means, and a medical gas supply means, the aerosol supply means supplies aerosol of a storage solution into the storage room, and the medical gas supply means A medical gas is supplied into the storage chamber.

  According to the storage method of the present invention, long-term storage of biomaterials is possible. Therefore, the present invention is extremely useful in the fields of clinical medicine and clinical research.

FIG. 1 is a transparent perspective view of the storage device of the present invention in the first embodiment. FIG. 2 is a transparent perspective view of the storage device of the present invention in the second embodiment. FIG. 3 is a diagram showing the preservation conditions of the heart in Comparative Examples 1 to 8.

<Storage method of biomaterial>
As described above, the method of storing biomaterials of the present invention is characterized by including the step of storing biomaterials under an aerosol atmosphere of medical gas and storage solution. The storage method of the present invention is characterized in that the biomaterial is stored under an aerosol atmosphere of the storage solution, and the other conditions and steps are not particularly limited. The storage method of the present invention can be conveniently carried out by using the storage device of the present invention described later.

  In the present invention, the atmosphere of the storage solution aerosol refers to "a dispersed state in which fine particles of the storage solution are suspended in a gas".

  The storage method of the present invention can sufficiently suppress, for example, water absorption and dehydration of the biomaterial during storage by storing the biomaterial under the aerosol atmosphere of the storage solution. Thereby, for example, expansion and contraction of the biological material during storage can be suppressed, and damage to the biological sample can be reduced.

  Further, according to the storage method of the present invention, by storing the biomaterial under the atmosphere of the medical gas, for example, the metabolism of the biomaterial during storage is suppressed by the medical gas, whereby the biomaterial is stored. It can protect cells contained in biomaterials. For this reason, according to the storage method of the present invention, for example, storage can be performed while maintaining the function of the biomaterial over a long period of time. In addition, since the medical gas evaporates immediately from the biomaterial after storage, for example, when transplanted, the side effect of the medical gas is suppressed after transplantation.

  Furthermore, the storage method of the present invention can suppress edema of the biomaterial after storage, for example, with respect to the simple immersion method, by storing the biomaterial under an aerosol atmosphere of the storage solution. it can. In the storage method of the present invention, for example, the drying of the biomaterial after storage is suppressed with respect to the high-pressure gas phase storage method by storing the biomaterial under an aerosol atmosphere of the storage solution. Can. From these things, according to the preservation | save method of this invention, when the heart is preserve | saved, long-term preservation | save of 48 hours or more is possible compared with the conventional preservation | save method, for example.

  In the present invention, the origin of the biological material is not particularly limited, and examples thereof include humans and non-human animals excluding humans, and the non-human animals include, for example, mice, rats, guinea pigs, dogs, cats and monkeys. And mammals such as rabbits, sheep, horses and pigs, and non-mammalian animals such as flies.

  In the present invention, the biomaterial is not particularly limited, and examples thereof include a part of an organism, an organ, a cell and the like. The part of the living body is not particularly limited, and examples thereof include limbs, fingers, face, bones, muscles, hair roots, teeth, periodontal membrane, and the like. The four limbs are not particularly limited, and examples thereof include the upper limbs, the upper limbs, the upper arms, the forearms, the hands, the lower limbs, the lower limbs, the thighs, the lower legs, and the legs. The organs are not particularly limited, and for example, eyeball, cornea, lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, small intestine, large intestine, testis, ovary, central nerve, peripheral nerve, blood vessel, skin Etc. The cells are not particularly limited, and examples thereof include sperm, eggs, ES cells, iPS cells, stimulation-induced pluripotency (STAP) cells, cell sheets, red blood cells, white blood cells, platelets, cultured cells and the like. Further, examples of the biomaterial include a part of a living body cultured and / or derived from the cell, an organ, a cell sheet, a cell and the like.

  In the present invention, the preservation solution is not particularly limited, and for example, physiological saline, University of Wisconsin (UW) solution, histidine-tryptophan-ketoglutarate (HTK) solution, Celsoir solution, ET-Kyoto solution, extracellular solution (Infusion), IGL-1 solution, Polysol solution, Euro-Collins solution, etc. may be mentioned. The extracellular fluid is not particularly limited, and examples thereof include Lactec D infusion, vine F, solugen D, sorbacto, lactorin gel and the like.

  In the present invention, the storage solution is not particularly limited, and may be, for example, a hypotonic solution whose osmotic pressure is lower than the osmotic pressure of the biomaterial, or is isotonic with the same osmotic pressure as the osmotic pressure of the biomaterial The solution may be a liquid or a hypertonic solution having an osmotic pressure higher than the osmotic pressure of the biomaterial, but is preferably an isotonic solution because water absorption to the biomaterial in the storage step and dehydration from the biomaterial can be suppressed. It is.

  The preservation solution may contain other components. The other components are not particularly limited, and examples thereof include antifreeze agents and sugars such as glucose.

  In the present invention, the medical gas is not particularly limited, and, for example, any gas manufactured for medical use can be used. The medical gas includes, for example, hydrogen, nitric oxide, carbon monoxide, carbon dioxide, hydrogen sulfide, oxygen, ozone, helium, xenon, argon, krypton, nitrogen, nitrous oxide, air, methane, ethane, propane, Examples include fluoromethane, difluoromethane, carbon tetrafluoride, ethylene oxide and the like. In the medical gas, the medical gas is, for example, nitrous oxide, xenon, argon, helium, krypton, oxygen, carbon dioxide because the danger of explosion, the risk of toxicity and the risk of flammability are low. And nitrogen are preferred. In the medical gas, for example, carbon monoxide, xenon, and hydrogen sulfide are preferable because the medical material can suppress the energy consumption of the biological material and maintain the function of the biological material. The medical gas is preferably carbon monoxide, for example, because the medical gas can suppress the expansion of the injury of the biomaterial and can maintain the function of the biomaterial. The medical gas is preferably, for example, carbon monoxide or xenon because the medical gas can suppress cell death of cells in the biological material and can maintain the function of the biological material. In the medical gas, the peripheral circulation in the biological material after transplantation can be improved by expanding the blood vessel of the biological material, and the survival rate of the biological material can be improved. Nitrogen monoxide is preferred. In the medical gas, the generation of active oxygen in the biological material can be suppressed, and oxidation (aging) of the biological material during storage can be suppressed, so that the medical gas is preferably, for example, hydrogen. In the present invention, one type of medical gas may be used as the medical gas, or two or more types of medical gases may be used in combination. When the two or more types of medical gases are used in combination, for example, they may be used as a mixed gas in which the two or more types of medical gases are mixed.

  The mixed gas is not particularly limited, and for example, a mixed gas containing carbon monoxide, carbon dioxide, oxygen and helium, a mixed gas containing oxygen and nitrous oxide, a mixed gas containing oxygen and xenon, oxygen and argon Examples thereof include mixed gases containing oxygen, mixed gases containing nitrogen and nitric oxide, and mixed gases containing oxygen and hydrogen sulfide. Thus, by using the medical gas as a mixed gas, for example, the effects of the above-described medical gases can be synergistically obtained.

  In the storage step, for example, a part of the biomaterial may be under the atmosphere, or the whole may be under the atmosphere. In the former case, for example, there is a method of disposing the biomaterial in the storage device under the atmosphere. In the latter case, for example, there is a method of suspending the biomaterial in the storage device under the atmosphere.

  In the storage step, the method of supplying the aerosol of the storage solution is not particularly limited, and may be performed by a known method. Examples of the supply method include ultrasonic type, liquid spray type, nozzle spray type, steam type, vaporization type, centrifugal type, and a combination of these, and the like, and heating of the biomaterial by generation of heat at the time of supply. The ultrasonic type is preferable because it can suppress the

  In the storage step, the particle diameter of the aerosol of the storage solution is not particularly limited, and the lower limit thereof is, for example, 1 nm, and the upper limit thereof is, for example, 1 mm, preferably 100 μm, more preferably 10 μm. The range is, for example, 1 nm to 1 mm, preferably 1 nm to 100 μm, and more preferably 1 nm to 10 μm. The particle size of the aerosol of the storage solution can increase the number of particles of the aerosol of the storage solution per unit volume, whereby, for example, necrosis of cells contained in the biological sample due to drying of the biological sample and The thickness is preferably 100 μm or less because the injury due to contraction can be suppressed and the temperature change of the biological sample during storage can be suppressed.

  In the storage step, the concentration of the storage solution (weight of solute / weight of solution) constituting the aerosol is not particularly limited, and the lower limit thereof is, for example, 3 g / kg, preferably 7.8 g / kg. More preferably, the upper limit is, for example, 12 g / kg, preferably 9.8 g / kg, more preferably 9 g / kg, and the range is, for example, 3 to 3 g / kg. It is in the range of 12 g / kg, preferably in the range of 7.8 to 12 g / kg, more preferably in the range of 8 to 9.8 g / kg, and still more preferably in the range of 8 to 9 g / kg.

  In the storage step, the temperature during storage is not particularly limited, and the lower limit is, for example, -20 ° C, preferably -2 ° C, and the upper limit is, for example, 37 ° C, preferably 4 The range is, for example, -20 to 37 ° C, preferably -2 to 4 ° C.

  In the storage step, the partial pressure of the medical gas during storage is not particularly limited, and is, for example, in the range of 0.01 to 0.7 MPa, preferably in the range of 0.07 to 0.35 MPa. The partial pressure of the medical gas may be a partial pressure of one kind of the medical gas, or may be a total partial pressure of two or more kinds of the medical gases. When the medical gas is carbon monoxide, the partial pressure of carbon monoxide is, for example, in the range of 0.01 to 0.7 MPa, preferably in the range of 0.07 to 0.35 MPa. When the medical gas is oxygen, the partial pressure of oxygen is, for example, in the range of 0.01 to 0.7 MPa, preferably in the range of 0.07 to 0.35 MPa.

  In the storage step, the storage period for storing the biomaterial is not particularly limited, and can be appropriately determined according to the type of the biomaterial. The storage period is not particularly limited, and is, for example, 1 year or less, preferably 24 hours or less, based on the time when the biomaterial was produced. When the biomaterial is a part of the living body, the storage period is, for example, 168 hours or less, preferably 72 hours or less, based on the time when a part of the living body is produced. When the biological material is the organ, the storage period is, for example, 120 hours or less, preferably 24 hours or less, based on the time when the organ is produced. When the organ is the heart, the storage period is, for example, 72 hours or less, preferably 24 hours or less, based on the time when the heart is produced. When the organ is the lung, the storage period is, for example, 96 hours or less, preferably 48 hours or less, based on the time when the lung is produced. When the organ is the kidney, the storage period is, for example, 96 hours or less, preferably 48 hours or less, based on the time when the kidney is produced. When the organ is the liver, the storage period is, for example, 96 hours or less, preferably 48 hours or less, based on the time when the liver is produced. When the organ is a cornea, the storage period is, for example, 120 hours or less, preferably 72 hours or less, based on the time when the cornea was produced. When the biological material is the cells, the storage period is, for example, 1 year or less, preferably half a year or less, more preferably 3 months or less, based on the time when the cells are produced.

  Hereinafter, the storage method of the present invention will be described by way of examples, but the present invention is not limited to the following embodiments. Specifically, this embodiment uses the mixed gas of carbon monoxide and oxygen which is the medical gas, extracellular fluid which is the storage solution, and the heart which is the biological material, in the storage step. In an example of suspending the heart under an atmosphere of the mixed gas and an aerosol of the extracellular fluid, the heart is stored for a predetermined storage period.

  First, the heart is removed, and the blood in the heart is removed by the storage solution to prepare the heart before storage. Next, the heart before storage is suspended and exposed using a biomaterial suspending means under an atmosphere of the mixed gas and the aerosol of the extracellular fluid supplied by an ultrasonic method. Then, in this state, the heart before storage is stored for a predetermined storage period.

  The particle diameter of the aerosol of the extracellular fluid during storage is, for example, in the range of 1 nm to 1 mm, preferably in the range of 1 nm to 100 μm, and more preferably in the range of 1 nm to 10 μm.

  The temperature at the time of storage is, for example, in the range of -20 to 37 ° C, preferably in the range of -2 to 4 ° C. The storage period is, for example, 72 hours or less, preferably 24 hours or less.

  In this way, the heart can be stored.

<Method of producing biomaterial>
The method for producing a biomaterial according to the present invention, as described above, includes a material storage step of storing the produced biomaterial, and the material storage step is performed by the storage method according to the present invention. . The production method of the present invention is characterized in that the material storage step is performed by the storage method of the present invention, and the other conditions and steps are not particularly limited. According to the production method of the present invention, the biomaterial of the present invention described later can be produced. The description of the storage method of the present invention can be used for the production method of the present invention.

  In the material storage step, the produced biomaterial is not particularly limited. For example, the illustration of the biomaterial can be used.

  In the material storage step, the method of providing the produced biomaterial is not particularly limited. The provided method of the produced biomaterial is, for example, a method of producing the biomaterial by a medical technique such as a surgical technique, and a method of culturing and / or inducing the cells to produce the biomaterial. , A method of recovering the biological material generated due to an accident or a crime, and the like.

The production method by the medical method is not particularly limited, and can be appropriately determined according to the produced biomaterial. For the production method by the medical method, for example, the following reference 1 can be referred to. When producing a rat heart by the above-mentioned medical method, for example, the following reference 2 can be referred to.
Reference 1: "Organ Transplantation Experiment Manual, Transplantation Using Rat and Mouse, Ischemia-Reperfusion Injury Model", Shujunsha Co., Ltd., supervised by: Nozawa, Y., Editing: Tanaka, Y., 1999, Reference 2: Naoyuki Hatayama et. al. , Cell Transplantation, 2012, Vol. 21, pp. 609-615

  The method for culturing and / or inducing and producing the cells is not particularly limited, and can be appropriately determined depending on the produced biomaterial.

<Biomaterial>
The biomaterial of the present invention is characterized by being produced by the production method of the present invention as described above. The biomaterial of the present invention is characterized by being produced by the production method of the present invention, and the other constitution and conditions are not particularly limited. The biomaterial of the present invention can be used, for example, in the transplantation method of the present invention described later. The description of the storage method and production method of the present invention can be used for the biomaterial of the present invention.

  Since the biomaterial of the present invention preserves the biomaterial under the aerosol atmosphere of the preservation solution, for example, the amount of the preservation solution contained in the biomaterial after the preservation is different from the method of immersing in the preservation solution. Few. In addition, since the biomaterial of the present invention preserves the biomaterial under the aerosol atmosphere of the preservation solution, for example, the preservation solution contained in the biomaterial after preservation with respect to the method of preservation in the high pressure gas There is a large amount of

<Transplant material>
The implant material of the present invention, as described above, is characterized by comprising the biomaterial stored by the storage method of the present invention. The graft material of the present invention is characterized by including the biomaterial stored by the storage method of the present invention, and the other configurations and conditions are not particularly limited. The implant material of the present invention can be used, for example, in the implant method of the present invention described later. In addition, description of the preservation | save method of the said invention, a production method, etc. can be used for the transplant material of this invention.

<Porting method>
As described above, the transplantation method of the present invention is an implanting step of implanting at least one of the biomaterial of the present invention and the graft material of the present invention (hereinafter referred to as "biomaterial after storage") into an animal. It is characterized by including. The transplantation method of the present invention is characterized by using the biomaterial after the storage, and the other conditions and steps are not particularly limited. The description of the storage method, production method and the like of the present invention can be used for the transplantation method of the present invention.

  In the transplantation step, the animal is not particularly limited, and examples thereof include humans and non-human animals excluding humans, and the non-human animals include, for example, mice, rats, guinea pigs, dogs, cats, monkeys and rabbits. And mammals such as sheep, horses and pigs, and non-mammalian animals such as flies. In the transplantation step, the type of the animal and the origin of the biomaterial after storage may be the same or different.

  The transplantation method of the present invention further includes a washing step of washing the stored biomaterial with a washing solution prior to the transplantation step, and the grafting step is a step of transplanting the washed biomaterial to the animal. It may be. The washing step is a step of, for example, reducing the amount of the storage solution contained in the biomaterial after storage by washing the biomaterial after storage with a cleaning solution. The transplantation method of the present invention can further reduce the side effects due to the preservation solution to the transplanted animal, for example, when the biomaterial after preservation is transplanted by including the washing step.

  The washing solution is not particularly limited, and examples thereof include physiological saline, phosphate buffered saline, Ringer's solution and the like.

  In the implanting step, the method for implanting the biomaterial after storage is not particularly limited, and can be appropriately determined according to the biomaterial after storage. For the transplantation method, for example, reference 1 can be referred to. When the biomaterial after storage is a rat heart after storage, for example, reference 2 can be referred to.

<Storage device of biomaterial>
As described above, the biomaterial storage apparatus of the present invention includes a storage room, an aerosol supply means, and a medical gas supply means, and the aerosol supply means supplies aerosol of a storage solution into the storage room, and the medical treatment A medical gas is supplied into the storage chamber by the gas supply means. The storage apparatus of the present invention is characterized by including the aerosol supply means, whereby an aerosol of the storage solution is supplied into the storage room, and the other configurations and conditions are not particularly limited. According to the storage apparatus of the present invention, for example, the storage method and the like of the present invention can be easily performed. In addition, the storage apparatus of the present invention can use the description of the storage method, production method and the like of the present invention.

  Hereinafter, the storage device of the present invention will be described in detail by way of examples with reference to the drawings. However, the present invention is not limited and limited to the following examples. Further, in the drawings, for convenience of explanation, the structure of each part may be appropriately simplified and shown, and the dimensional ratio of each part may be schematically shown differently from the actual one.

(Embodiment 1)
FIG. 1 is a transparent perspective view of the storage device of the present embodiment. In FIG. 1, the solid line represents the outer shape of each part of the present embodiment, and the broken line represents the shape of the storage chamber 1 and the storage chamber 1 inside of the medical gas supply means 3. As shown in FIG. 1, the storage device 10 of the present embodiment includes a storage room 1, an aerosol supply means 2 and a medical gas supply means 3 as main components. The storage chamber 1 includes a storage container 4 and a lid 5, and the lid 5 is detachably mounted on the top of the storage container 4. As shown in FIG. 1, in the present embodiment, the aerosol supply means 2 is installed in the storage room 1, and the medical gas supply means 3 has a lid 5 so as to supply medical gas into the storage room 1. It is connected to the. In addition, the cloud-like description which has come out of the aerosol supply means 2 has shown typically the state to which the aerosol of the said storage liquid is supplied from the aerosol supply means 2, and does not restrict | limit this invention at all.

  The storage chamber 1 may be a detachable type such as the storage container 4 and the lid 5 of the present embodiment, or may be an integral type in which a door that can be opened and closed is installed in the storage container 4. The former is preferable because pressure resistance and sealing can be improved. The volume of the storage chamber 1 is not particularly limited, and can be appropriately determined according to the size of the biomaterial to be stored. The material of the storage chamber 1 is not particularly limited, and can be appropriately determined according to, for example, the partial pressure of the medical gas in the storage chamber 1. The material of the storage chamber 1 may be, for example, a pressure resistant material.

  The aerosol supply means 2 supplies, for example, an aerosol of the storage solution into the storage chamber 1. The aerosol supply means 2 is not particularly limited, and may be, for example, an aerosol generator installed in the storage room 1 or connects the aerosol generator installed outside the storage room 1 and the storage room 1 The connection part for aerosol supply formed in the storage chamber 1 may be used. In the latter case, the aerosol generating device and the aerosol supply connection portion may be integrally formed or may be detachably formed. Further, the aerosol supply connection portion may be connected to the storage chamber 1 so as to be able to supply the aerosol of the storage solution, and may be connected to the storage container 4 or to the lid 5, for example. May be Examples of the aerosol generator include an ultrasonic generator, a liquid spray type, a nozzle spray type, a steam type, a vaporization type, a centrifugal type, and a method of combining the above, and the above-described aerosol generation device An ultrasonic aerosol generator is preferable because it can suppress the heating of the biomaterial. The ultrasonic aerosol generating apparatus is not particularly limited, and examples thereof include an apparatus in which an ultrasonic transducer is immersed in the storage solution.

  The medical gas supply means 3 supplies, for example, medical gas into the storage chamber 1. The medical gas supply means 3 is not particularly limited, and may be a medical gas supply device installed in the storage chamber 1 or a medical gas supply device installed outside the storage chamber 1 and the storage chamber 1 The connection part for medical gas supply formed in the storage chamber 1 which connects with may be sufficient. In the latter case, the medical gas supply device and the medical gas supply connection portion may be integrally formed or may be detachably formed. In the present embodiment, the medical gas supply means 3 is connected to the lid 5, but is not limited thereto. The medical gas supply means 3 may be connected to the storage chamber 1 so as to be able to supply the medical gas, and may be connected to the storage container 4, for example. The medical gas supply apparatus is not particularly limited, and examples thereof include a medical gas cylinder, solid phase gas, liquid phase gas, gas hydrate and the like.

  In the present embodiment, the area for storing the biomaterial is not particularly limited, and can be stored at any place in the storage room 1. The biomaterial can be placed, for example, on the bottom of the storage container 4 and stored.

Second Embodiment
The storage device of the present embodiment further includes a biomaterial suspension unit 6 and a pressure control unit 7. FIG. 2 is a transparent perspective view of the storage device of the present embodiment. In FIG. 2, the solid line represents the outer shape of each part of the present embodiment, and the broken line represents the shape of the storage chamber 1 and the medical gas supply means 3 and the storage chamber 1 inside the pressure control means 7 Are given the same reference numerals. As shown in FIG. 2, the storage device 20 of the present embodiment further includes a biomaterial suspending means 6 and a pressure control means 7, and the biomaterial suspending means 6 is installed in the storage chamber 1 and the pressure control means Reference numeral 7 is connected to the lid 5 so as to control the pressure in the storage chamber 1. In the present embodiment, the storage device 20 includes the biomaterial suspension means 6 and the pressure control means 7, but the storage device 20 is not limited to this, and any of the biomaterial suspension means 6 and the pressure control means 7 Only one may be included. Except for this point, the storage device 20 of the present embodiment has the same configuration as the storage device 10 shown in FIG. The storage device 20 according to the present embodiment includes the biomaterial suspending unit 6 to increase the contact area between the biomaterial during storage and the aerosol of the medical gas and the storage solution in the storage room 1, for example. be able to. Therefore, according to the storage device 20 of the present embodiment, for example, damage due to water absorption and dehydration of the biomaterial during storage can be reduced, thereby maintaining the function of the biomaterial for a longer period of time. It can be saved in a state of being allowed. In addition, the storage device 20 of the present embodiment can control the pressure in the storage chamber 1 during storage, for example, to be constant by providing the pressure control means 7. Therefore, damage due to pressure fluctuation of the biomaterial during storage can be reduced, whereby the biomaterial can be stored in a function-maintained state for a longer period of time.

  The biomaterial suspending means 6 suspends the biomaterial, for example, in the storage chamber 1. The method for suspending the biomaterial to the biomaterial suspending means 6 is not particularly limited. For example, the biomaterial may be suspended directly or indirectly. In the latter case, for example, there is a method of suspending the biomaterial on the biomaterial suspending means 6 through the net or the like using a net, a mesh container for sample, a thread for medical use, a mesh net or the like. The biomaterial suspending means 6 may be any one as long as the biomaterial can be suspended, and known suspending devices can be used. The biomaterial suspending means 6 is not particularly limited, and examples thereof include a hook and the like.

  The pressure control means 7 adjusts, for example, the pressure in the storage chamber 1. The pressure control means 7 is not particularly limited, and may be, for example, a back pressure valve, a pressure reducing valve, a control valve, a safety valve, a pressure control valve, a check valve, a shutoff valve, a flow path changing valve, a flow control valve, a leak valve, etc. . In the present embodiment, the pressure control means 7 is connected to the lid 5 but is not limited thereto. The pressure control means 7 may be connected to be able to control the pressure in the storage chamber 1, for example, may be connected to the storage container 4.

  The storage apparatus of the present invention may further include an aerosol supply control unit, which may control at least one of an aerosol supply amount and a supply time of the aerosol supply unit 2. The aerosol supply control means is not particularly limited, and examples thereof include a timer, a power switch, a controller and the like.

  The storage apparatus of the present invention may further include a cold storage means, and the cold storage means may cool the storage room 1. The cold storage means only needs to cool the cold storage chamber 1 to a predetermined temperature, and the cold storage method is not particularly limited. Examples of the cold storage method include a method of arranging the storage chamber 1 in a cold storage container, a method of immersing the cold storage chamber 1 in a refrigerant, and the like. The refrigerator is not particularly limited, and examples thereof include a refrigerator and the like. The predetermined temperature may be, for example, the temperature during storage described above.

  The storage device of the present invention may further include an observation window capable of observing the inside of the storage room 1, a pressure detection means for detecting the pressure in the storage room 1, and the like.

  Below, the Example of this invention is described. However, the present invention is not limited by the following examples.

Example 1
The rat heart was stored for 48 hours under an atmosphere of a mixed gas of carbon monoxide and oxygen and an aerosol of extracellular fluid, and it was confirmed that the heart after storage was functional.

(1) Preparation of rat heart for storage A 6-week-old LEW / SsN Slc male rat (n = 5) is administered pentobarbital (Kyoritsu Pharmaceutical) at 50 mg / kg (drug / body weight), Deep anesthesia was performed. Next, the heart was removed from the rat. Furthermore, after dissecting the aorta and the pulmonary artery of the heart, an extracellular fluid containing 5% glucose (trade name: Lactec D infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.) is infused to remove the blood before preservation. It was a heart. Then, the weight of the heart before storage was measured.

(2) Storage of Rat Heart The storage device of FIG. 2 was used as the storage device. The storage device previously supplies the aerosol of the storage solution into the storage chamber by the aerosol supply means, and keeps the container cold in a 4 ° C. refrigerator. Next, the heart before storage was suspended to the biomaterial suspension means in the storage container. Furthermore, the storage container was sealed with the lid and stored in the 4 ° C. refrigerator for 48 hours. Further, during the storage, aerosol is supplied into the storage chamber by the aerosol supply unit, and a partial pressure (PCO) of carbon monoxide in the storage chamber is 0.15 MPa by the medical gas supply unit. Carbon monoxide and oxygen were supplied such that the partial pressure of oxygen (PO ₂ ) was 0.2 MPa. Moreover, the humidity in the storage room during storage was 100%.

(3) Function of the heart after storage The heart after storage for 48 hours was recovered from the biomaterial suspending means in the storage container, and the weight was measured. Then, left ventricular pressure (LVP) was measured on the heart after storage using a cardiac function measurement device (Langendorff perfusion device, manufactured by Bioresearch Center). In Comparative Examples 1 to 8, weight and left ventricular pressure were measured in the same manner as in FIG. 3 and conditions described later except that the heart before storage was stored for 48 hours in the 4 ° C. refrigerator. Furthermore, left ventricular pressure was measured in the same manner except that the pre-stored heart was used as a control.

(Comparative example 1)
As shown in FIG. 3A, in Comparative Example 1 (n = 12), in the storage device, a flask containing 50 mL of water (DW) was placed in the storage chamber, instead of the aerosol supply means. . In Comparative Example 1, carbon monoxide and oxygen were added so that the partial pressure of carbon monoxide in the storage chamber was 0.4 MPa and the partial pressure of oxygen was 0.3 MPa by the medical gas supply means. Supplied. In addition, the humidity in the storage room during storage was 96.3%.

(Comparative example 2)
As shown to FIG. 3 (B), the comparative example 2 (n = 5) did not install the said aerosol supply means in the said storage apparatus. In Comparative Example 2, carbon monoxide and oxygen were added so that the partial pressure of carbon monoxide in the storage chamber was 0.4 MPa and the partial pressure of oxygen was 0.3 MPa by the medical gas supply means. Supplied. Moreover, the humidity in the storage room during storage was 61%.

(Comparative example 3)
As shown in FIG. 3C, in Comparative Example 3 (n = 6), a flask containing 50 mL of water was installed in the storage chamber, instead of the aerosol supply means in the storage device. In Comparative Example 3, the carbon monoxide and oxygen were mixed so that the partial pressure of carbon monoxide in the storage chamber was 0.0571 MPa and the partial pressure of oxygen was 0.0429 MPa by the medical gas supply means. Supplied. In addition, the humidity in the storage room during storage was 96.33%.

(Comparative example 4)
As shown in FIG. 3D, in Comparative Example 4 (n = 5), a flask containing 50 mL of water was placed in the storage chamber in place of the aerosol supply means in the storage device. In Comparative Example 4, air was supplied by the medical gas supply means so that the partial pressure (PAy) of air in the storage chamber was 0.1 MPa. In addition, the humidity in the storage room during storage was 96.17%.

(Comparative example 5)
As shown in FIG. 3 (E), in Comparative Example 5 (n = 5), 50 mL of the University of Wisconsin (UW) solution was used instead of the aerosol supply means and the biomaterial suspending means in the storage device. The flask containing it was placed in the storage chamber. In addition, in Comparative Example 5, the heart before the storage is immersed in the UW solution in the flask, and the partial pressure of air in the storage chamber is 0.1 MPa by the medical gas supply unit. Air was supplied. In addition, the humidity in the storage room during storage was 96.33%.

(Comparative example 6)
As shown in FIG. 3 (F), Comparative Example 6 (n = 5) does not use the storage device, and carbon monoxide is generated by bubbling carbon monoxide gas at 4 ° C. for 5 minutes in advance. A flask containing 50 mL of dissolved UW solution (UW with CO) was placed in the refrigerator. And the comparative example 6 immersed the heart before the said preservation | save in the UW liquid in the said flask, and left still under the atmospheric pressure of 0.1 MPa.

(Comparative example 7)
As shown in FIG. 3 (G), in Comparative Example 7 (n = 6), a flask containing 50 mL of UW solution was substituted for the aerosol supply means and the biological material suspending means in the storage device. Installed in storage room. In Comparative Example 7, the heart before the storage is immersed in the UW solution in the flask, and the partial pressure of carbon monoxide in the storage chamber is 0.4 MPa and oxygen by the medical gas supply means. Carbon monoxide and oxygen were supplied so that the partial pressure of H.sub.2 was 0.3 MPa. In addition, the humidity in the storage room during storage was 96.2%.

(Comparative example 8)
As shown in FIG. 3 (H), in Comparative Example 8 (n = 5), in the storage device, extracellular containing 50 mL of the 5% glucose instead of the aerosol supply means and the biomaterial suspending means A flask containing the solution (EF) was placed in the storage chamber. In Comparative Example 8, the heart before storage is immersed in the flask, and the medical gas supply unit supplies air so that the partial pressure of air in the storage chamber is 0.1 MPa. did. Moreover, the humidity in the storage room during storage was 100%.

  The weights of the heart before and after storage of the example and comparative examples 1 to 8 are shown in Table 1 below.

  As shown in Table 1, in Comparative Examples 1 to 8, the weight of the heart after storage was changed by 10% or more with respect to the weight of the heart before storage. Specifically, in Comparative Examples 1 to 7, the heart dehydrated by 10% or more during storage, and in Comparative Example 8, the heart absorbed water by 10% or more during storage. On the other hand, in the embodiment, the weight of the heart after storage is suppressed to less than 5% of the weight of the heart before storage, and the heart is almost dehydrated and absorbed water during storage. It was not.

  Next, the left ventricular pressure of the heart after storage of the example, the comparative examples 1 to 8 and the control is shown in Table 2 below.

  As shown in Table 2, in Comparative Examples 1 to 8, the left ventricular pressure of the heart after storage was lower than the left ventricular pressure of the control (heart before storage). In particular, in Comparative Examples 2, 3 and 5 to 7, the left ventricular pressure was significantly reduced relative to the control left ventricular pressure, and in Comparative Example 4 and Comparative Example 8, no left ventricular pressure was observed. . On the other hand, in the example, the left ventricular pressure of the heart after the storage was the same as the control. From these results, Comparative Examples 1 to 7 are dried and contracted by dehydration of the heart during storage, causing necrosis and contraction of cells of the heart due to drying, and the function of the heart after storage is It was found to decrease or lose. In addition, in Comparative Example 8, it was found that the heart swells due to water absorption during storage, thereby causing injury due to edema and reducing the function of the heart after storage. On the other hand, according to the storage method of the present invention, since the heart hardly absorbs and dehydrates and does not expand and contract during storage, the injury is suppressed, whereby the heart before storage is also used for storage. It turned out that an equivalent function is maintained.

  According to the storage method of the present invention, biomaterials can be stored for a long time. Therefore, the present invention is extremely useful in the fields of clinical medicine and clinical research.

DESCRIPTION OF SYMBOLS 1 Storage room 2 Aerosol supply means 3 Medical gas supply means 4 Storage container 5 Lid 6 Biological material suspension means 7 Pressure control means 10, 20 Storage device

Claims (12)

Under aerosol atmosphere of medical gases and preservation solution, it viewed including a storage step of storing a biological material,
The medical gas contains oxygen,
The partial pressure of oxygen is in the range of 0.07 to 0.35 MPa,
The particle diameter of the aerosol is 100 μm or less . The preservation solution is physiological saline, University of Wisconsin (UW) solution, histidine-tryptophan-ketoglutarate (HTK) solution, Celsoir solution, ET-Kyoto solution, extracellular fluid (infusion), IGL-1 solution, Polysol solution The storage method according to claim 1, comprising at least one selected from the group consisting of and Euro-Collins solution. The aerosol of the storage solution comprises an aerosol supplied by at least one method selected from the group consisting of ultrasonic, liquid spray, nozzle spray, steam, vaporization and centrifugal. 2 Storage method described. The medical gas is hydrogen, nitrogen monoxide, carbon monoxide, carbon dioxide, hydrogen sulfide, ozone, helium, xenon, argon, krypton, nitrogen, nitrous oxide, air, methane, ethane, propane, fluoromethane, difluoro The storage method according to any one of claims 1 to 3, comprising at least one selected from the group consisting of methane, carbon tetrafluoride and ethylene oxide. The medical gas is a mixed gas containing carbon monoxide, carbon dioxide, oxygen and helium, a mixed gas containing oxygen and nitrous oxide, a mixed gas containing oxygen and xenon, a mixed gas containing oxygen and argon, oxygen and The storage method according to any one of claims 1 to 4, which is a mixed gas containing nitric oxide or a mixed gas containing oxygen and hydrogen sulfide. The storage method according to any one of claims 1 to 5, wherein the partial pressure of the medical gas is in the range of 0.01 to 0.7 MPa. The preservation | save method of Claim 4 or 5 whose partial pressure of the said carbon monoxide is the range of 0.01-0.7 Mpa. The storage method according to any one of claims 1 to 7, wherein the biomaterial is at least one selected from the group consisting of a part of an organism, an organ, and a cell. The preservation method according to claim 8, wherein the part of the living body includes at least one selected from the group consisting of limbs, fingers, face, bones, muscles, hair roots, teeth and periodontal membrane. The organ is selected from the group consisting of eyeball, cornea, lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, small intestine, large intestine, testis, ovary, central nerve, peripheral nerve, blood vessel and skin The storage method according to claim 8, comprising at least one. Said cells comprise at least one selected from the group consisting of sperm, ova, ES cells, iPS cells, stimulation-induced pluripotency (STAP) cells, cell sheets, red blood cells, white blood cells, platelets and cultured cells. The storage method according to claim 8. Including a material preservation step of preserving the produced biomaterial,
A method of producing a biomaterial, wherein the material storage step is performed by the storage method according to any one of claims 1 to 11.

Images