JPS6155881B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for storing various organs extracted from the human body, etc., and preserving the organs for a long period of time in order to transplant them in a timely manner.

Conventionally, extracted organs have been preserved until transplantation, and the preservation method involves injecting Collins fluid at a temperature of approximately 4°C, which has properties similar to blood, through the organ's artery or portal vein. A so-called perfusion method is known that drains this through the veins.
The organ after such perfusion treatment is stored under the above-mentioned temperature condition of about 4° C., and blood flow is applied to the stored organ at the time of transplantation before use.

However, when using this preservation method, organs can only be preserved for about 12 hours, which makes it difficult to coordinate the time between organ donation and demand, creating a major bottleneck in saving human lives.

Therefore, in order to extend the preservation time, it is possible to freeze the organ by setting the storage temperature to a low temperature, but freezing the organ subjected to the above conventional method will cause cell destruction and cause the organ itself to die. Become.

In view of the above points, the present invention aims to freeze extracted organs without causing cell destruction, thereby enabling semi-permanent preservation.

The present invention will be described in detail with reference to the drawings.The extracted organ 1 is stored in a heat-insulating container 2 as shown, and the opening/closing door 3 is closed. An inflow pipe 6 connected to the supply pipe 5 and extending through the heat insulating container 2 is connected to the organ 1.
connected to artery 1a or portal vein 1b, and further connected to vein 1c
In this case, a discharge pipe 7 extending through the heat insulating container 2 is connected, and an air supply pipe 9 of the device 4 is connected to an air supply nozzle 8 provided inside the container 2.

In the illustrated perfusion freezing device 4, liquid nitrogen LN ₂ is stored between an insulated outer tank 10 and an intermediate tank 11, and between the intermediate tank 11 and an inner tank 13 containing a refrigerant 12 such as fluorocarbon. , a cooling tank 14 filled with helium gas GHe, and first, second, and third containers 15 each containing Collins liquid, dimethyl sulfoxide (DMSO), glycerin, and alcohol in the refrigerant 12. , 16 and 17 are immersed in the water, and under the control of the controller 18, the first, second and third on-off valves 19, 20 and 21 provided on the outflow pipes of the containers 15, 16 and 17 are opened and closed at appropriate times. By operating the pump 22 provided in the liquid supply pipe 5, the Collins liquid,
DMSO, alcohol selectively affects organ 1 artery 1a
Alternatively, it can be supplied to the portal vein 1b.

Furthermore, various members of the temperature control mechanism 23 are immersed in the refrigerant 12, and 24, 25, 2
Reference numeral 6 indicates a temperature sensor, a stirrer, and a heater, and the temperature of the refrigerant 12 can be adjusted to a desired temperature.

Further, LN _{2 is supplied from the liquid nitrogen cylinder 27 to the air supply container 28 immersed in the refrigerant 12, and the LN 2 flowing} out from the container 28 is adjusted to a desired temperature by a heat exchanger 29. The nitrogen gas GN ₂ is supplied to the air supply nozzle 8 via the air supply pipe 9 described above.

Therefore, in the present invention, first, the controller 18 opens the first on-off valve 19, and the pump 22 operates to supply a blood equalization perfusion liquid such as Collins solution from the first container 15, so that the perfusion liquid is supplied to the artery of the organ 1. It flows into the organ 1 through the portal vein 1a or the portal vein 1b and is discharged to the outside through the vein 1c. At this time, the blood perfusion solution is injected while gradually lowering its temperature.

In other words, the extracted organ 1 is initially at approximately body temperature37
℃, the temperature of the blood-equalizing perfusate is gradually lowered from the body temperature, and by injecting the perfusate, the blood in organ 1 is drained and replaced with the same perfusate. , the first like this
The perfusion process continues until the blood homologous perfusate is at a first near-decreased temperature below its freezing point.

Here, the temperature reduction is adjusted by controlling the refrigerant 12, which is cooled from the LN ₂ in the outer tank 10 through the GHe in the intermediate tank 11, by the temperature control mechanism 23. Since the freezing point is approximately 0°C, the first perfusion step is approximately 1 to 2°C.
For example, in order to lower the temperature of the blood perfusate from the body temperature of 37°C to 2°C, the temperature lowering rate should be 3.5°C/min and the perfusion time should be about 10 minutes. can.

By operating the heat exchanger 29, the temperature-controlled GN ₂ is ejected from the air supply nozzle 8. This is because it is desirable to make the temperature equal to that of the perfusate and to avoid a temperature gradient between the internal and external temperatures of the organ 1, and this will continue in the following steps.

Next, by closing the first on-off valve 19 and opening the second on-off valve 20, the above-described antifreeze damage agent such as dimethyl sulfoxide and glycerin in the second container 16 is replaced with the above-mentioned blood equalization perfusate. Then, the process moves on to the second perfusion step, in which perfusion is supplied to the body.

In the same process, the antifreeze agent is first added to the
By controlling this by the temperature control mechanism 23, the temperature is gradually lowered until the temperature of the antifreeze damage agent reaches the second temperature drop below its freezing point. continue.

Since the freezing point of DMSO is approximately -5℃,
The temperature drop in the second neighborhood is preferably about -4℃,
In fact, in order to lower the temperature from 2°C to -4°C, which is the first temperature drop in the first vicinity, the perfusion time can be set at 0.3°C/min for about 20 minutes, and by perfusion of such antifreeze agent, Due to the osmotic pressure difference between the agent and the water in the cells of the organ 1, the water is sufficiently absorbed by the antifreeze agent, and this completes the process according to the first invention of the present application.

Next, in the second invention of the present application, once the second perfusion step is completed, the third perfusion step is started. For this reason, the second on-off valve 20 is closed and the third on-off valve 21 is opened to supply and perfuse the final perfusion liquid, which has a lower freezing point than the anti-freeze agent such as alcohol, instead of the anti-freeze agent.

Therefore, in this step as well, the temperature of the final perfusate is controlled to gradually decrease from the second temperature drop (-4°C), and the perfusion brings the final perfusate to a third temperature below its freezing point. Perfusion continues until the perfusion fluid freezes and perfusion is stopped; in the case of alcohol, the freezing point is approximately -80°C.
For example, -60°C may be set as the third neighborhood drop temperature or -80°C.

In fact, this third perfusion step is 0.1℃/mi to lower the temperature of alcohol from -4℃ to -37℃.
The perfusion time was approximately 330 minutes at a falling rate of n, and the temperature was lowered from -37℃ to -60℃ for 5 minutes.
A fall rate of °C/min and a perfusion time of about 5 minutes can be used.

The above first to second perfusion steps or first to third
Frozen organs obtained through the perfusion process are stored in the frozen state, and in the case of the above examples, freeze-damaged organs are stored at -5°C or -80°C, respectively.
It may be stored in a small freezer or immersed in liquefied gas such as liquid nitrogen.

Now, the frozen organs stored in this way will be thawed and used for transplantation, but the means for thawing is to move the freezing step from the third step to the first step. This can be done by substantially reversing.

That is, the frozen organ is removed from the storage location and set in the state shown in FIG.
At this time, first, by gradually raising the atmospheric temperature in the heat insulating container 2, the final perfusion liquid such as alcohol present in the blood vessels of the organ concerned is raised to a temperature above its freezing point and thawed, and then the third opening/closing step is performed. When the valve 21 is opened, alcohol or the like is perfused by the pump 22.

In this first thawing perfusion step, the temperature of the final perfusate is gradually raised by the temperature control mechanism 23 until the temperature reaches the second neighborhood drop temperature (-4°C).
Continue until .

Next, the process will proceed to the second thawing perfusion step in which the above-mentioned cryoprotectant is perfused in place of the final perfusion solution, but here too, the temperature is gradually raised from the second neighborhood drop temperature (-4°C), Perfusion is continued until the temperature of the antifreeze agent reaches the first temperature drop (1 to 2° C.).

Furthermore, in place of the above-mentioned anti-freezing agent, a blood equalization perfusion solution such as the above-mentioned Collins solution is perfused while gradually raising the temperature from the above-mentioned first neighborhood drop temperature.
The thaw-perfusion step is continued until the blood-isolated perfusate is at about body temperature.

The organ that has undergone the complete thawing and perfusion process can be supplied with the necessary blood and used for transplantation.

As described above, the first invention of the present application is to simply perfuse organs with Collins solution instead of blood as in the conventional method, and to
Rather than trying to preserve the blood at a certain level, in the first perfusion step, the temperature of the blood perfusion solution such as Collins solution is gradually lowered, and the organ is perfused to the first temperature drop below its coagulation point. It is unaffected and is supplied with nutrients equal to blood at a temperature of 1 to 2 degrees Celsius, when the metabolism of organ cells is most active.

In the second perfusion step, the temperature of the cryoprotectant is further lowered to the second temperature drop, which is below the freezing point of the antifreeze agent. Due to the osmotic pressure difference with water, the water in the cells is absorbed, and therefore, when an organ is frozen by lowering its temperature, it can be frozen without causing cell destruction because there is no water.

In the second invention, a third perfusion step is further added, but in this case, a final perfusion liquid with a freezing point lower than that of the above-mentioned antifreeze agent is used as the perfusion liquid, and the temperature ranges from -4°C to -80°C. Since perfusion is carried out while lowering the temperature to a low temperature such as Since there is no water present, no cell destruction occurs.

Therefore, not only in the second invention but also in the first invention, by preserving frozen organs in a frozen state, they can be stored semi-permanently without dying.

Next, the third invention provides a method for preserving frozen organs stored according to the second invention to a state where they can be transplanted.
Based on the idea of implementing the invention reversibly, as described above, the temperature of the preserved frozen organ is gradually raised to thaw the final perfusate in the blood vessels of the organ, and then the final perfusate is gradually raised in temperature. The first thawing perfusion step of perfusing from the artery or portal vein to the vein as described above is continued until the temperature reaches the second drop in temperature, and then the final perfusate is replaced with the cryoprotectant and the third
The second thawing perfusion step of perfusing while gradually raising the temperature from the temperature drop in the vicinity is continued until the temperature reaches the temperature drop in the first vicinity, and then the blood homogenization perfusate is added to the area in the first vicinity instead of the antifreeze agent. The third thawing-perfusion process, in which perfusion is performed while gradually increasing the temperature from the drop temperature, is continued until the organ reaches body temperature, and then the required amount of blood is applied to the organ concerned, so that the frozen organ can be thawed without damaging the organ. Obtained.

[Brief explanation of the drawing]

The figure is an overall explanatory view, partially cut away, showing the state of use of a perfusion device that can be used to carry out the organ preservation method according to the present invention. 1...organ, 1a...artery, 1b...portal vein, 1
c...Vein.

Claims (1)

[Scope of Claims] 1. A first perfusion step in which a blood perfusion solution such as Collins solution is injected from the artery or portal vein of the excised organ while gradually lowering the temperature and discharged from the vein until the perfusion solution reaches its coagulation point. The process continues until the temperature drops to the previous first neighborhood drop, and then, in place of this blood homogenization perfusate, a second antifreeze agent such as dimethyl sulfoxide or glycerin is perfused while gradually lowering the temperature from the first neighborhood drop temperature. A method for preserving an organ, characterized in that the perfusion step is continued until the temperature of the cryoprotectant reaches a second neighborhood drop below its freezing point, and the frozen organ obtained thereby is preserved in a frozen state. 2. The first perfusion step involves injecting a blood perfusate such as Collins solution from the artery or portal vein of the excised organ while gradually lowering the temperature and draining it from the vein until the perfusate drops to the first vicinity below its freezing point. The second perfusion step is continued until the temperature reaches the same temperature, and then a second perfusion step is performed in which a cryoprotective agent such as dimethyl sulfoxide or glycerin is perfused in place of this blood homogeneous perfusate while gradually lowering the temperature from the first temperature drop near the first temperature drop. The temperature is continued until the temperature of the inhibitor reaches a second temperature drop below its freezing point, and then, in place of this frost damage inhibitor, a final perfusate having a freezing point lower than that of the frost damage inhibitor, such as alcohol, is gradually lowered from the second temperature range. The third perfusion step, in which perfusion is performed while perfusing the organ, is continued until the final perfusate reaches a third-degree drop in temperature below its freezing point, or until the perfusate is frozen, and the resulting frozen organ is stored in a frozen state. A method for preserving organs characterized by: 3. The first perfusion step involves injecting a blood perfusate such as Collins solution from the artery or portal vein of the excised organ while gradually lowering the temperature and draining it from the vein until the perfusate drops to the first vicinity below its freezing point. The second perfusion step is continued until the temperature reaches the same temperature, and then a second perfusion step is performed in which a cryoprotective agent such as dimethyl sulfoxide or glycerin is perfused in place of this blood homogeneous perfusate while gradually lowering the temperature from the first temperature drop near the first temperature drop. This is continued until the temperature of the inhibitor reaches a second temperature drop below its freezing point, and then, in place of this frost damage inhibitor, a final irrigation fluid having a freezing point lower than that of the frost damage inhibitor, such as alcohol, is gradually added from the second temperature range below the freezing point. The third perfusion step, in which the temperature is lowered and perfused, is continued until the final perfusate reaches a third drop in temperature below its freezing point, or until the perfusate is frozen, and the frozen organ thus obtained is brought into a frozen state. The stored frozen organ is then gradually heated to thaw the final perfusate in the blood vessels of the organ, and then the final perfusate is gradually heated and transferred from the artery or portal vein to the vein as described above. The first thawing perfusion step of perfusing is continued until the temperature reaches the second neighborhood drop temperature, and then the frost damage preventive agent is perfused in place of the final perfusate while gradually increasing the temperature from the second neighborhood drop temperature. 2. Continuing the thawing perfusion step until the temperature reaches the first neighborhood drop, and then perfusing the blood equalization perfusate instead of the antifreeze agent while gradually increasing the temperature from the first neighborhood drop temperature. A method for preserving an organ, characterized in that the thawing perfusion step is continued until the organ reaches body temperature, and then the required amount of blood is applied to the organ.