JP6242001B2 - Biomaterial storage method, biomaterial production method, biomaterial, transplant material, transplant method, and biomaterial storage apparatus - Google Patents

Description

  The present invention relates to a biomaterial storage method, biomaterial production method, biomaterial, transplant material, transplant method, and biomaterial storage apparatus.

  In recent years, transplantation treatments for biomaterials such as parts of living bodies, organs, cells, etc. have become commonplace. In Japan, more than 300 transplantation treatments are performed annually, and worldwide, more than 70,000 transplantation treatments are performed annually. Transplantation treatment is being performed. However, with the increase in the transplantation treatment, there is a problem that the biomaterial is insufficient. For this reason, it is necessary to transport the biomaterial not only from the vicinity of the hospital where the transplantation treatment is performed, but also from a more remote area. For these reasons, development of a method for preserving the biomaterial has been demanded.

  As a method of preserving the biomaterial, a method of perfusing the biomaterial using a low-temperature preservation solution before or after extraction, and further immersing the biomaterial after perfusion in a low-temperature preservation solution (simple immersion method), Methods for continuously perfusing the preservation solution (continuous perfusion method), methods for preserving in a high-pressure gas (high-pressure gas phase preservation method), and the like are known (Patent Documents 1 and 2, Non-Patent Document 1). However, the maximum storage period of the biomaterial by these methods was 18 hours. For this reason, there has been a problem that it is difficult to transport the biomaterial from a remote area of the hospital where the transplantation treatment is performed and to use it for the transplantation treatment. For these reasons, there is a need for a new method for storing biomaterials that has a longer storage period.

International Publication No. 2010/049996 JP 2000-076021

Maathuis MH, et. al. , Transplantation, 2007, vol83, pp1289-1298

  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 biomaterial storage method of the present invention includes a storage step of storing the biomaterial in an aerosol atmosphere of medical gas and storage solution.

  The biomaterial production method 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 produced by the production method of the present invention.

  The transplant material of the present invention includes a biomaterial stored by the storage method of the present invention.

  The transplantation method of the present invention includes a transplantation step of transplanting at least one of the biomaterial of the present invention and the transplantation material of the present invention into an animal.

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

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

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

<Preservation method of biomaterial>
As described above, the biomaterial storage method of the present invention includes a storage step of storing the biomaterial in an aerosol atmosphere of medical gas and storage solution. The preservation method of the present invention is characterized in that the biomaterial is preserved in an aerosol atmosphere of the preservation solution, and other conditions and steps are not particularly limited. The preservation | save method of this invention can be easily implemented by using the preservation | save apparatus of this invention mentioned later.

  In the present invention, the atmosphere of the preservation solution in an aerosol refers to a “dispersed state in which fine particles of the preservation solution are suspended in a gas”.

  The storage method of the present invention can sufficiently suppress water absorption and dehydration of the biomaterial during storage, for example, by storing the biomaterial in an 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, in the storage method of the present invention, by storing the biomaterial in an atmosphere of the medical gas, for example, the metabolism of the biomaterial during storage is suppressed by the medical gas. Cells contained in biomaterials can be protected. For this reason, according to the preservation | save method of this invention, it can preserve | save in the state which maintained the function of the said biomaterial over a long period of time, for example. In addition, since the medical gas volatilizes immediately from the stored biomaterial, for example, when transplanted, side effects due to the medical gas are suppressed after transplantation.

  Further, the storage method of the present invention can suppress the edema of the biomaterial after storage by storing the biomaterial in an aerosol atmosphere of the storage solution, for example, with respect to the simple immersion method or the like. it can. Further, the storage method of the present invention suppresses drying of the biomaterial after storage by storing the biomaterial in an aerosol atmosphere of the storage solution, for example, with respect to the high-pressure gas phase storage method. Can do. For these reasons, according to the storage method of the present invention, compared to the conventional storage method, for example, when the heart is stored, it can be stored for a long period of 48 hours or more.

  In the present invention, the origin of the biomaterial is not particularly limited, and examples thereof include humans and non-human animals other than humans. Examples of the non-human animals include mice, rats, guinea pigs, dogs, cats, monkeys. 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 a living body, an organ, and a cell. A part of the living body is not particularly limited, and examples thereof include limbs, fingers, faces, bones, muscles, hair roots, teeth, periodontal ligament and the like. The limbs are not particularly limited, and examples thereof include upper limbs, upper limb bands, upper arms, forearms, hands, lower limbs, lower limb bands, thighs, lower legs, legs, and the like. The organ is not particularly limited, for example, eyeball, cornea, lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, small intestine, large intestine, testis, ovary, central nervous system, peripheral nerve, blood vessel, skin Etc. The cell is not particularly limited, and examples thereof include sperm, ovum, ES cell, iPS cell, stimulation-induced pluripotency acquisition (STAP) cell, cell sheet, erythrocyte, leukocyte, platelet, cultured cell and the like. Examples of the biomaterial include a part of a living body cultured and / or derived from the cell, an organ, a cell sheet, and a cell.

  In the present invention, the preservation solution is not particularly limited. For example, physiological saline, University of Wisconsin (UW) solution, histidine-tryptophan-ketoglutarate (HTK) solution, Celsoil solution, ET-Kyoto solution, extracellular solution. (Infusion), IGL-1 solution, Polysol solution, Euro-Collins solution and the like. The extracellular fluid is not particularly limited, and examples thereof include lactec D infusion solution, vin F, solugen D, sollacto, and lactolin gel.

  In the present invention, the preservation solution is not particularly limited, and may be, for example, a hypotonic solution having an osmotic pressure lower than the osmotic pressure of the biomaterial, or an isotonic pressure having the same osmotic pressure as that of the biomaterial. 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 into the biomaterial and dehydration from the biomaterial in the storage step can be suppressed. It is.

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

  In the present invention, the medical gas is not particularly limited, and for example, any gas produced for medical use can be used. The medical gas is, 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, and ethylene oxide. In the medical gas, since the risk of explosion, the risk of toxicity, and the risk of flammability are low, the medical gas is, for example, nitrous oxide, xenon, argon, helium, krypton, oxygen, carbon dioxide Nitrogen is preferred. In the medical gas, since the energy consumption of the biomaterial can be suppressed and the function of the biomaterial can be maintained, the medical gas is preferably, for example, carbon monoxide, xenon, or hydrogen sulfide. In the medical gas, since expansion of injury of the biomaterial can be suppressed and the function of the biomaterial can be maintained, the medical gas is preferably carbon monoxide, for example. In the medical gas, since the cell death of cells in the biomaterial can be suppressed and the function of the biomaterial can be maintained, the medical gas is preferably, for example, carbon monoxide or xenon. In the medical gas, by expanding the blood vessel of the biomaterial, peripheral circulation in the biomaterial after transplantation can be improved, and the survival rate of the biomaterial can be improved. Nitric oxide is preferred. In the medical gas, for example, hydrogen is preferable as the medical gas because generation of active oxygen in the biomaterial can be suppressed and oxidation (aging) of the biomaterial during storage can be suppressed. In the present invention, as the medical gas, one type of medical gas may be used, or two or more types of medical gas may be used in combination. When the two or more types of medical gases are used in combination, for example, the two or more types of medical gases may be used as a mixed gas.

  The mixed gas is not particularly limited. 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 are used. A mixed gas containing oxygen, nitrogen monoxide, a mixed gas containing oxygen and hydrogen sulfide, and the like. Thus, by using the medical gas as a mixed gas, for example, the effects of the above-described medical gases can be obtained synergistically.

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

  In the preservation step, the method for supplying the aerosol of the preservation solution is not particularly limited, and can be performed by a known method. Examples of the supply method include an ultrasonic method, a liquid spray method, a nozzle spray method, a steam method, a vaporization method, a centrifugal method, and a combination thereof, and the heating of the biomaterial by the generation of heat at the time of supply. Therefore, the ultrasonic type is preferable.

  In the preservation step, the aerosol particle size of the preservation 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, 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 particle size of the aerosol of the preservation solution can increase the number of particles of the aerosol of the preservation solution per unit volume, and thereby, for example, necrosis of cells contained in the biological sample due to drying of the biological sample and 100 μm or less is preferable because injury due to contraction can be suppressed and temperature change of the biological sample during storage can be suppressed.

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

  In the storage step, the temperature during storage is not particularly limited, and the lower limit thereof is, for example, −20 ° C., preferably −2 ° C., and the upper limit thereof is, for example, 37 ° C., preferably 4 The range is, for example, a range of -20 to 37 ° C, and preferably a range of -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, and 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 a total partial pressure of two or more kinds of the medical gas. When the medical gas is carbon monoxide, the partial pressure of the carbon monoxide is, for example, in the range of 0.01 to 0.7 MPa, and preferably in the range of 0.07 to 0.35 MPa. When the medical gas is oxygen, the partial pressure of the oxygen is, for example, in the range of 0.01 to 0.7 MPa, and 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 depending on 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 is 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 the part of the living body was produced. When the biomaterial 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 was 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 was 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 was 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 was 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 was produced. When the organ is the 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 biomaterial is the cell, the storage period is, for example, 1 year or less, preferably 6 months or less, and more preferably 3 months or less, based on the time when the cell was produced.

  Hereinafter, the storage method of the present invention will be described with examples, but the present invention is not limited to the following embodiments. Specifically, this embodiment uses a mixed gas of carbon monoxide and oxygen that is the medical gas, an extracellular fluid that is the preservation solution, and a heart that is the biomaterial, and in the preservation step, This is an example in which the heart is stored for a predetermined storage period in a state where the heart is suspended in an atmosphere of aerosol of the mixed gas and the extracellular fluid.

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

  The aerosol particle size of the extracellular fluid during the 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, and 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 preserved.

<Production method of biomaterial>
As described above, the biomaterial production method 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 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 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. In addition, description of the preservation | save method of the said this invention can be used for the production method of this invention.

  In the material storage step, the produced biomaterial is not particularly limited, and examples of the biomaterial can be used.

  In the material storage step, the method for providing the produced biomaterial is not particularly limited. The method for providing the produced biomaterial includes, for example, a method for producing the biomaterial by a medical technique such as a surgical technique, and a method for producing the biomaterial by culturing and / or inducing the cells. And a method for recovering the biomaterial generated by an accident or a criminal case.

The production method by the medical technique is not particularly limited, and can be appropriately determined according to the produced biomaterial. For the production method by the medical technique, for example, the following Reference 1 can be referred to. When the rat heart is produced by the medical technique, for example, the following Reference 2 can be referred to.
Reference 1: “Organ transplantation experiment manual Rat, mouse transplantation, ischemia-reperfusion injury model”, Shujunsha Co., Ltd., supervision: Yasumi Nozawa, Editor: Yayoi Tanaka, 1999 Reference 2: Naoyuki Hatayama et. al. , Cell Transplantation, 2012, Vol. 21, pp. 609-615

  A method for culturing and / or inducing and producing the cells is not particularly limited, and can be appropriately determined according to the produced biomaterial.

<Biomaterial>
The biomaterial of the present invention is 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 other configurations 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. In addition, description of the preservation | save method and the production method of the said invention can be used for the biomaterial of this invention.

  Since the biomaterial of the present invention preserves the biomaterial in an aerosol atmosphere of the preservation solution, for example, the amount of the preservation solution contained in the preserved biomaterial is different from the method of immersing in the preservation solution. Few. Moreover, since the biomaterial of the present invention preserves the biomaterial in an aerosol atmosphere of the preservation solution, for example, the preservation solution contained in the preserved biomaterial in contrast to the method of preservation in the high-pressure gas. There is a lot of.

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

<Transplantation method>
As described above, the transplantation method of the present invention comprises a transplantation step of transplanting at least one of the biomaterial of the present invention and the transplant 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 in that the stored biomaterial is used, and other conditions and steps are not particularly limited. In addition, the description of the preservation | save method of the said this invention, a production method, etc. can be used for the transplant method of this invention.

  In the transplantation step, the animal is not particularly limited, and examples thereof include humans and non-human animals other than humans. Examples of the non-human animals include mice, rats, guinea pigs, dogs, cats, monkeys, rabbits. Mammals such as sheep, horses and pigs, and non-mammalian animals such as flies. In the transplantation step, the type of 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, wherein the transplantation step is a step of transplanting the washed biomaterial into the animal. There may be. The said washing | cleaning process is a process of reducing the quantity of the preservation | save liquid contained in the said biomaterial after the preservation | save, for example by wash | cleaning the preserve | saved biomaterial with a washing | cleaning liquid. By including the washing step, the transplantation method of the present invention can further reduce the side effects caused by the preservation solution to the transplanted animal when the biomaterial after storage is transplanted, for example.

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

  In the transplantation step, the method for transplanting the biomaterial after storage is not particularly limited, and can be appropriately determined according to the biomaterial after storage. For the transplantation method, reference can be made to, for example, the above-mentioned Reference Document 1. In the case where the stored biomaterial is a rat heart after storage, for example, Reference 2 can be referred to.

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

  Hereinafter, the storage device of the present invention will be described in detail with reference to the drawings with examples. However, the present invention is not limited or limited to the following examples. In the drawings, for convenience of explanation, the structure of each part may be simplified as appropriate, and the dimensional ratio of each part may be schematically shown, unlike the actual case.

(Embodiment 1)
FIG. 1 is a 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 medical gas supply means 3 inside the storage chamber 1. As shown in FIG. 1, the storage device 10 of the present embodiment includes a storage chamber 1, an aerosol supply unit 2, and a medical gas supply unit 3 as main components. The storage chamber 1 includes a storage container 4 and a lid 5, and the lid 5 is detachably installed on the upper part of the storage container 4. As shown in FIG. 1, in this embodiment, the aerosol supply means 2 is installed in the storage chamber 1, and the medical gas supply means 3 has a lid 5 so that medical gas can be supplied into the storage chamber 1. It is connected to the. In addition, the cloud-like description which comes out from the aerosol supply means 2 shows the state in which the aerosol of the said preservation | save 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 separation type like the storage container 4 and the lid 5 of this embodiment, or may be an integrated 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 hermeticity 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 the partial pressure of the medical gas in the storage chamber 1, for example. Examples of the material of the storage chamber 1 include a pressure resistant material.

  The aerosol supply unit 2 supplies, for example, an aerosol of the storage solution into the storage chamber 1. The aerosol supply unit 2 is not particularly limited, and may be, for example, an aerosol generation device installed in the storage chamber 1, or connects the aerosol generation device installed outside the storage chamber 1 and the storage chamber 1. It may be an aerosol supply connection formed in the storage chamber 1. In the latter case, the aerosol generating device and the aerosol supply connecting portion may be formed integrally or detachably. Further, the aerosol supply connecting portion only needs to be connected to the storage chamber 1 so as to be able to supply the aerosol of the storage solution. For example, the connection portion may be connected to the storage container 4 or connected to the lid 5. 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 combination thereof, and the above-mentioned aerosol generation device by generating heat during supply An ultrasonic aerosol generator is preferable because heating of the biomaterial can be suppressed. The ultrasonic aerosol generator is not particularly limited, and examples thereof include an apparatus in which an ultrasonic vibrator 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 inside 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 preservation | save chamber 1 which connects may be sufficient. In the latter case, the medical gas supply device and the medical gas supply connection portion may be formed integrally or detachably. 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 unit 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 device is not particularly limited, and examples thereof include a medical gas cylinder, a solid phase gas, a liquid phase gas, and a gas hydrate.

  In this embodiment, the area | region which preserve | saves the said biomaterial is not restrict | limited in particular, It can preserve | save in the arbitrary places in the preservation | save chamber 1. FIG. The said biomaterial can be arrange | positioned and preserve | saved at the bottom face in the storage container 4, for example.

(Embodiment 2)
The storage device of this embodiment further includes a biomaterial suspension means 6 and a pressure control means 7. FIG. 2 is a 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 inside of the storage chamber 1 of the medical gas supply means 3 and the pressure control means 7, and is the same as in FIG. 1. Are denoted by the same reference numerals. As shown in FIG. 2, the storage device 20 of the present embodiment further includes a biomaterial suspension means 6 and a pressure control means 7, and the biomaterial suspension means 6 is installed in the storage chamber 1 and is a pressure control means. 7 is connected to the lid 5 so that the pressure in the storage chamber 1 can be controlled. 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 either the biomaterial suspension means 6 or 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 of the present embodiment includes the biomaterial suspension means 6 to increase, for example, the contact area between the biomaterial during storage and the aerosol of the medical gas and the storage liquid in the storage chamber 1. be able to. For this reason, according to the storage device 20 of the present embodiment, for example, damage due to water absorption and dehydration of the biological material being stored can be reduced, thereby maintaining the function of the biological material for a longer period of time. Can be saved in the Moreover, the storage apparatus 20 of this embodiment can control the pressure in the storage chamber 1 during storage to be constant, for example, by including the pressure control means 7. For this reason, the damage by the fluctuation | variation of the pressure of the said biomaterial during preservation | save can be reduced, and, thereby, the said biomaterial can be preserve | saved in the state with which the function was maintained over a longer period.

  The biomaterial suspension means 6 suspends the biomaterial in the storage chamber 1, for example. The method for suspending the biomaterial on the biomaterial suspending means 6 is not particularly limited, and for example, the biomaterial may be suspended directly or indirectly. In the latter case, for example, a method of suspending the biomaterial on the biomaterial suspending means 6 through the net or the like using a net, a sample mesh container, a medical thread, a mesh net or the like can be mentioned. The biological material suspending means 6 only needs to suspend the biological material, and a known hanging instrument can be used. The biomaterial suspension means 6 is not particularly limited, and examples thereof include a hook.

  The pressure control means 7 adjusts the pressure in the storage chamber 1, for example. The pressure control means 7 is not particularly limited, and examples thereof include 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 rate adjusting valve, and a leak valve. . 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 the storage container 4, for example, as long as the pressure in the storage chamber 1 can be controlled.

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

  The storage device of the present invention may further include a cold insulation unit, and the storage chamber 1 may be cooled by the cold insulation unit. The cold insulation means is not particularly limited as long as the cold insulation chamber 1 can be kept at a predetermined temperature. Examples of the cold insulation method include a method of arranging the storage chamber 1 in a cold storage, a method of immersing the cold storage chamber 1 in a refrigerant, and the like. The cold storage is not particularly limited, and examples thereof include a refrigerator. Examples of the predetermined temperature include the above-described storage temperature.

  In addition to this, the storage device of the present invention may include an observation window through which the inside of the storage chamber 1 can be observed, pressure detection means for detecting the pressure in the storage chamber 1, and the like.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

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

(1) Preparation of rat heart for preservation Pentobarbital (manufactured by Kyoritsu Pharmaceutical Co., Ltd.) was administered to 6-week-old LEW / SsN Slc male rats (n = 5) so as to be 50 mg / kg (drug / body weight). Deep anesthesia was performed. Next, the heart was extracted from the rat. Further, after incising the aorta and pulmonary artery of the heart, an extracellular fluid containing 5% glucose as a preservation solution (trade name: Lactec D infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.) is injected to remove blood, and this is preserved before the preservation. The 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. In the storage device, the aerosol of the storage solution was supplied in advance into the storage chamber by the aerosol supply means, and kept in a refrigerator at 4 ° C. Next, the pre-storage heart was suspended from the biomaterial suspension means in the storage container. Further, the storage container was sealed with the lid and stored in the refrigerator at 4 ° C. for 48 hours. Further, during the storage, the aerosol is supplied into the storage chamber by the aerosol supply means, and the partial pressure (PCO) of carbon monoxide in the storage chamber is 0.15 MPa by the medical gas supply means, Carbon monoxide and oxygen were supplied so that the partial pressure of oxygen (PO ₂ ) was 0.2 MPa. The humidity in the storage room during storage was 100%.

(3) Function of heart after storage The heart after storage for 48 hours was collected from the biomaterial suspension means in the storage container and weighed. And the left ventricular pressure (LVP) was measured about the said heart after the preservation | save using the cardiac function measuring apparatus (Langendorff perfusion apparatus, Bio Research Center company make). Moreover, Comparative Examples 1-8 measured the weight and the left ventricular pressure similarly except having preserve | saved the said heart before the preservation | save in the said 4 degreeC refrigerator for 48 hours on the conditions mentioned later in FIG. Further, as a control, the left ventricular pressure was measured in the same manner except that the unstored heart was used.

(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 installed in the storage chamber instead of the aerosol supply unit. . Further, in Comparative Example 1, carbon monoxide and oxygen are added by the medical gas supply means so that the partial pressure of carbon monoxide is 0.4 MPa and the partial pressure of oxygen is 0.3 MPa. Supplied. The humidity in the storage room during storage was 96.3%.

(Comparative Example 2)
As shown in FIG. 3B, in Comparative Example 2 (n = 5), the aerosol supply unit was not installed in the storage device. Further, in Comparative Example 2, carbon monoxide and oxygen were added by the medical gas supply means so that the partial pressure of carbon monoxide was 0.4 MPa and the partial pressure of oxygen was 0.3 MPa. Supplied. The humidity in the storage room during storage was 61%.

(Comparative Example 3)
As shown in FIG. 3C, in Comparative Example 3 (n = 6), in the storage device, a flask containing 50 mL of water was installed in the storage chamber instead of the aerosol supply unit. Further, in Comparative Example 3, carbon monoxide and oxygen were added by the medical gas supply means so that the partial pressure of carbon monoxide was 0.0571 MPa and the partial pressure of oxygen was 0.0429 MPa. Supplied. Further, 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), in the storage device, instead of the aerosol supply unit, a flask containing 50 mL of water was installed in the storage chamber. In Comparative Example 4, air was supplied by the medical gas supply means so that the partial pressure (PAir) of the air in the storage chamber was 0.1 MPa. The humidity in the storage room during storage was 96.17%.

(Comparative Example 5)
As shown in FIG. 3E, in Comparative Example 5 (n = 5), in the storage device, 50 mL of Universality of Wisconsin (UW) solution was used instead of the aerosol supply unit and the biomaterial suspension unit. The contained flask was placed in the storage chamber. In Comparative Example 5, the heart before storage is immersed in the UW liquid in the flask so that the partial pressure of air in the storage chamber becomes 0.1 MPa by the medical gas supply means. Air was supplied. Further, 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 in addition, carbon monoxide is bubbled by using carbon monoxide gas at 4 ° C. for 5 minutes in advance. A flask containing 50 mL of dissolved UW liquid (UW with CO) was placed in the refrigerator. And the comparative example 6 immersed the said heart before the 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), in the storage device, instead of the aerosol supply unit and the biological material suspension unit, a flask containing 50 mL of UW liquid was used. Installed in the storage room. In Comparative Example 7, the pre-storage heart was immersed in the UW liquid in the flask, and the carbon gas partial pressure of the storage chamber was 0.4 MPa, oxygen by the medical gas supply means. Carbon monoxide and oxygen were supplied so that the partial pressure of was 0.3 MPa. 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, instead of the aerosol supply means and the biological material suspension means, 50 mL of extracellular containing 5% glucose A flask containing the liquid (EF) was placed in the storage chamber. In Comparative Example 8, the heart before storage is immersed in the flask, and further, air is supplied by the medical gas supply means so that the partial pressure of air in the storage chamber is 0.1 MPa. did. The humidity in the storage room during storage was 100%.

  The weights of the hearts before and after storage in Examples 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 changed by 10% or more with respect to the weight of the heart before storage. Specifically, in Comparative Examples 1 to 7, the heart was dehydrated by 10% or more during storage, and in Comparative Example 8, the heart absorbed 10% or more during storage. On the other hand, in the embodiment, the weight of the heart after storage is suppressed to a change of less than 5% with respect to the weight of the heart before storage, and the heart is almost dehydrated and absorbed during storage. There wasn't.

  Next, the left ventricular pressure of the heart after storage of Examples, 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 remarkably reduced with respect to the control left ventricular pressure. Further, in Comparative Examples 4 and 8, no left ventricular pressure was observed. . On the other hand, in the Examples, the left ventricular pressure of the heart after the storage was the same as that of the control. From these results, in Comparative Examples 1 to 7, the heart was dehydrated during storage and dried and contracted, necrosis and contraction of the heart cells due to drying occurred, and the function of the heart after storage was It was found to be reduced or lost. Further, it was found that Comparative Example 8 expands when the heart absorbs water during storage, which causes injury due to edema and decreases the function of the heart after storage. On the other hand, in the preservation method of the present invention, since the heart hardly absorbs water and dehydrates during the preservation, and does not expand and contract, the injury is suppressed. It was found that the equivalent function was maintained.

  According to the storage method of the present invention, the biomaterial can be stored for a long period of time. For this reason, the present invention is extremely useful in clinical medicine and clinical research fields.

DESCRIPTION OF SYMBOLS 1 Storage chamber 2 Aerosol supply means 3 Medical gas supply means 4 Storage container 5 Lid 6 Biomaterial suspension means 7 Pressure control means 10 and 20 Storage apparatus

Claims (21)

A biomaterial storage method comprising a storage step of storing a biomaterial excluding blood vessels in an aerosol atmosphere of a medical gas and a preservative solution. The preservation solution is physiological saline, University of Wisconsin (UW) solution, histine-tryptophan-ketoglutarate (HTK) solution, Celsoir solution, ET-Kyoto solution, extracellular solution (infusion solution), IGL-1 solution, Polysol solution. and Euro-Collins comprising at least one selected from the group consisting of liquid, storage method of claim 1, wherein. Aerosols of the preservation solution, ultrasonic, liquid spray, nozzle spray, steam type, including aerosol supplied by at least one method selected from the group consisting of vaporization and centrifugal claim 1 or 2. The storage method according to 2 . The medical gas is hydrogen, nitric oxide, carbon monoxide, carbon dioxide, hydrogen sulfide, oxygen, ozone, helium, xenon, argon, krypton, nitrogen, nitrous oxide, air, methane, ethane, propane, fluoromethane The preservation | save method as described in any one of Claim 1 to 3 containing at least 1 selected from the group which consists of, difluoromethane, 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 partial pressure of the medical gas is in the range of 0.01～0.7MPa, storage method 5 according to claims 1. The storage method according to claim 4 or 5 , wherein a partial pressure of the carbon monoxide is in a range of 0.01 to 0.7 MPa. The storage method according to claim 4 or 5 , wherein the partial pressure of oxygen is in a range of 0.01 to 0.7 MPa. The storage method according to any one of claims 1 to 8, wherein the biomaterial is at least one selected from the group consisting of a part of an organism, an organ, and a cell. The storage method according to claim 9 , wherein the part of the living body includes at least one selected from the group consisting of limbs, fingers, faces, bones, muscles, hair roots, teeth, and periodontal ligament. The organ has been selected eyeball, cornea, lung, heart, liver, kidney, spleen, pancreas, gall bladder, esophagus, stomach, small intestine, large intestine, testis, ovary, central nervous system, from the group consisting of peripheral nerve you and skin The storage method according to claim 9 , comprising at least one. The cells include at least one selected from the group consisting of sperm, ovum, ES cells, iPS cells, stimulation-induced pluripotency (STAP) cells, cell sheets, erythrocytes, leukocytes, platelets and cultured cells; The storage method according to claim 9 . Including a material storage step for storing the produced biomaterial,
A biomaterial production method, wherein the material storage step is performed by the storage method according to any one of claims 1 to 12. A storage process for storing biomaterials;
The stored biological materials, and a transplant implanting the non-human animal,
A transplanting method, wherein the storing step is performed by the storing method according to any one of claims 1 to 12 . Including a storage room, an aerosol supply means and a medical gas supply means,
By the aerosol supply means, an aerosol of the storage liquid is supplied to the internal space of the storage chamber,
A biomaterial storage apparatus, wherein the medical gas is supplied into the storage chamber by the medical gas supply means. The storage device according to claim 15 , wherein the aerosol supply means includes at least one selected from the group consisting of an ultrasonic type, a liquid spray type, a nozzle spray type, a steam type, a vaporization type, and a centrifugal type. And further includes a biomaterial suspension means,
The biomaterial suspension means is disposed in the storage chamber,
The storage device according to claim 15 or 16 , wherein the biomaterial is suspended in the storage chamber by the biomaterial suspension means. The storage device according to any one of claims 15 to 17 , wherein the aerosol supply unit is disposed in the storage chamber. Furthermore, an aerosol supply control means is included,
The storage device according to any one of claims 15 to 18 , wherein at least one of an aerosol supply amount and a supply time of the aerosol supply unit is controlled by the aerosol supply control unit. And further includes pressure control means,
The storage device according to any one of claims 15 to 19 , wherein the pressure in the storage chamber is controlled by the pressure control means. In addition, including a cold insulation means,
The storage device according to any one of claims 15 to 20 , wherein the storage chamber is cooled by the cold storage means.

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