JPS6156202B2 - - 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.
Organs subjected to such perfusion treatment are stored under the above-mentioned temperature conditions of about 4° C., and blood flow is applied to the stored organs before transplantation before use.

However, when using this preservation method, organs can only be stored for about 12 hours in liver storage and 96 hours in kidney storage, which makes it difficult to coordinate the time between organ donation and demand, which is a big deal in terms of saving human lives. It has become a bottleneck.

Therefore, in order to extend the storage 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 may 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 long-term 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. The inflow pipe 6 connected to the supply pipe 5 and passed 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 penetrating discharge pipe 7 is connected to the heat insulating container 2, 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 and second containers 15 and 1 containing Collins liquid, dimethyl sulfoxide (DMSO), and glycerin in the refrigerant 12, respectively.
Under the control of the controller 17, the first and second on-off valves 18 and 19 provided on the outflow pipes of the containers 15 and 16 are opened and closed as appropriate, and the pump provided on the flowing liquid supply pipe 5 is immersed. 20, the Collins solution and DMSO can be selectively supplied to the vein 1a or portal vein 1b of the organ 1.

Further, various members of the temperature control mechanism 21 are immersed in the refrigerant 12, and 22, 23, 2
Reference numeral 4 indicates a temperature sensor, a stirrer, and a heater, and the temperature of the refrigerant 12 can be adjusted to a desired value.

Further, LN _{2 is supplied from the liquid nitrogen cylinder 25 to the air supply container 26 immersed in the refrigerant 12, and the LN 2 flowing} out from the container 26 is adjusted to a desired temperature by the heat exchanger 27. 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 17 opens the first on-off valve 18 and the pump 20 is operated to supply a blood-equalizing perfusate such as Collins solution from the first container 15. 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-equal 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 one 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 drop is adjusted by controlling the refrigerant 12, which is cooled from LN ₂ in the outer tank 10 through GHe in the intermediate tank 11, by the temperature control mechanism 21, and the freezing point of the Collins liquid is Since the temperature is approximately 0°C, the temperature in the first perfusion step is approximately 1 to 2°C.
Temperature reduction is controlled so that the temperature drops in the vicinity. For example, in order to lower the temperature of the blood perfusion solution from the above body temperature of 37°C to 2°C, the temperature lowering rate can be set to 3.5°C/min, and the perfusion time can be set to about 10 minutes. .

By operating the heat exchanger 27, 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 creating 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 18 and opening the second on-off valve 19, 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 a second temperature drop below its freezing point. or until the perfusate freezes and perfusion is stopped.

Here, since the freezing point of DMSO is -5°C, the temperature drop in the second neighborhood is about -4°C, but in reality, in order to lower the temperature from 2°C, which is the temperature drop in the first neighborhood, to -4°C, it is 0.3°C. /min, and the perfusion time can be approximately 20 minutes, and by perfusion of such a cryoprotectant, the water is absorbed by the cryoprotectant due to the osmotic pressure difference between the cryoprotectant and the water in the cells of organ 1. It will be fully absorbed.

The frozen organs obtained through the first and second perfusion steps above will be stored in the frozen state.
For this purpose, the frozen organ may be stored in a freezer at about -5° C. in the case of the above embodiment, or it may be stored by immersing it in a liquid 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 carried out by substantially reversing the freezing process described above. can do.

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 ambient temperature inside the heat insulating container 2, the final perfusate, which is an antifreeze agent such as DMSO, existing in the blood vessels of the organ concerned, is removed.
After thawing by raising the temperature above the freezing point, the second on-off valve 19 is opened to perfuse DMSO or the like with the pump 20.

In this first thawing perfusion step, the temperature of the final perfusion liquid is gradually increased by the temperature control mechanism 21 until the temperature reaches the first drop temperature (1 to 2
Continue until the temperature reaches ℃).

Next, in place of the above-mentioned anti-freezing agent, a blood equalization perfusate such as the above-mentioned Collins solution is perfused while gradually increasing 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 present invention does not simply perfuse organs with Collins' solution instead of blood as in the conventional method and store them at around 4°C, but the present invention uses a blood equivalent perfusion solution such as Collins' solution in the first perfusion step. Since the temperature is gradually lowered and perfusion is carried out to the first temperature drop below the freezing point, the organ is not affected by sudden changes in temperature, and even when the organ cells are still metabolically active at a temperature of 1 to 2 degrees Celsius. , supplied with blood and equal nutrients.

In the second perfusion process, the cryoprotectant is further lowered in temperature until it reaches a second temperature drop that is below the freezing point of the antifreeze agent, or until the perfusate is frozen. Due to the osmotic pressure difference between the antifreeze agent and the water inside the organ cells, the water inside the cells will be absorbed.
Therefore, when an organ is frozen by lowering the temperature, it can be frozen without causing cell destruction because it contains less water.

Furthermore, in the present invention, the frozen organs obtained through the above steps are preserved in a frozen state, and are further thawed to a state where they can be transplanted. After gradually raising the temperature of the preserved frozen organ as described above to thaw the cryoprotectant, which is the final perfusate in the blood vessels of the organ, the final perfusate is gradually heated and transferred from the artery or portal vein to the vein as described above. Continue the first thawing perfusion step of perfusing the blood to the first neighborhood drop temperature, and then gradually raise the temperature of the blood equalization perfusate instead of the cryoprotectant from the first neighborhood drop temperature. After the second thawing/perfusion step of perfusing the organ was continued until the organ reached body temperature, the required amount of blood was applied to the organ, so that the frozen organ could be thawed without damaging the organ.

[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)

[Claims] 1. The first perfusion step involves injecting a blood perfusate such as Collins solution from the artery or portal vein of the removed 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 process is continued until the inhibitor reaches a second temperature drop below its freezing point or until the perfusate is frozen, the frozen organ thus obtained is stored in a frozen state, and the stored frozen organ is gradually heated. , after thawing the cryoprotectant as the final perfusion solution in the blood vessels of the organ,
Continue the first thawing perfusion step of perfusing the final perfusion fluid from the artery or portal vein to the vein as described above while gradually raising the temperature until the temperature reaches the first neighborhood drop;
Next, a second thawing perfusion step is performed in which the blood equalization perfusate is perfused in place of the anti-freezing agent while gradually raising the temperature from the first near-decreased temperature until it reaches body temperature, and then the necessary amount is applied to the organ. A method for preserving organs characterized by adding blood.