JPS6156201B2 - - 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, the maximum amount of time an organ can be preserved is about 12 hours for the liver and 96 hours for the kidney, which makes it difficult to coordinate the time between organ donation and demand, making it difficult to save human lives. It has become a big bottleneck.

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 long-term preservation.

The invention will now be described in more detail with reference to the drawings, in which an apparatus such as that shown can be used to carry out the method according to the invention.

In other words, as described later, the extracted organ 1 was kept at -4°C.
The organ 1 is immersed in the perfusion environmental liquid 4 in the perfusion container 3 housed in the refrigerator 2. At this time, the organ 1 is placed on the mesh board 6 of the pedestal 5, and the perfusion device 7 The perfusion fluid supply pipe 8 has its distal end 8' connected to the portal vein 1b by the artery 1a of the organ 1, and the vein 1c opens into the perfusion environmental fluid 4. The inflow pipe 10 and the discharge pipe 11 of the circulation pump 9 are opened into the perfusion container 3 to enable circulation, and the environmental liquid heat exchange section 12 formed in the inflow pipe 10 It is immersed in a refrigerant 14 such as fluorocarbon in a cooling tank 13.

In the cooling tank 13, liquid nitrogen LN ₂ is stored between the insulated outer tank 15 and the intermediate tank 16, and helium gas GHe is stored between the intermediate tank 16 and the inner tank 17 containing the refrigerant 14. It is enclosed.

Not only the above-mentioned environmental liquid heat exchange section 12 is immersed in this refrigerant 14, but also the perfusion liquid heat exchange section 18 provided in the above-mentioned perfusion liquid supply pipe 8 in the above-mentioned perfusion device 7. It is also soaked.

What is illustrated as the perfusion device 7 is a first container 19 containing Collins solution, which is a blood equivalent liquid, and a first container 19 containing dimethyl sulfoxide (DMSO).
A second container 20 containing an antifreeze agent such as glycerin, and a mixture of 1% physiological saline (500 ml), which is a blood equivalent liquid, and an anticoagulant such as heparin (approximately 0.5 ml) are added. Each of these containers 19, 20, 2 is provided with a third container 21 containing
1 are the first, second, and third on-off valves 22, 23, 2, respectively.
4 is connected to a flow pump 25, and the perfusion liquid supply pipe 8 forming the above-mentioned perfusion liquid heat exchange section 18 is connected to the outflow side of the pump 25. 2. Third on-off valve 21,
22 and 23 are controlled to open and close as appropriate by a controller 27 of a refrigerant temperature control mechanism 26.

The refrigerant temperature control mechanism 26 has its temperature sensor 28, stirrer 29, and heater 30 immersed in the refrigerant 14, and by controlling these members with the controller 27, the refrigerant 14 is adjusted to a desired temperature. It has become freely possible.

Therefore, in order to carry out the method according to the present invention using the above-mentioned device, first, the extracted organ 1 is perfused evenly with blood, such as 1% physiological saline or Collins solution, from its artery A or portal vein B as soon as possible. A first perfusion step is performed by injecting a mixture of the liquid and a blood coagulant such as heparin, and replacing the intravascular blood of the organ 1 with the mixture.

To carry out the first perfusion step, immediately after extracting the organ 1, it is administered into a container (not shown) containing 1% physiological saline at about 2°C, and in this state, the mixed liquid is injected from the artery 1a etc. Alternatively, it is possible to use a syringe to inject and perfuse the vein 1c at a temperature of 2° C., that is, at a temperature close to the freezing point of the perfusate mixture.

Although the method described above is preferable because it allows the process to be carried out most quickly, it is also possible to carry out the process using the apparatus shown in the drawings.

That is, the extracted organ 1 is immediately placed in the perfusion environmental liquid 4 of the perfusion container 3 and placed on the mesh board 6 of the pedestal 5, and the third on-off valve 24 is opened by the controller 27, and the flow pump 25 is turned on. In operation,
The mixed liquid in the third container 21 is allowed to flow into the organ 1 through the vein 1a, etc., and is allowed to flow out from the vein c into the perfusion environmental liquid 4.

In the figure, 25' indicates a flow meter.

At this time, the temperature of the refrigerant 14 is controlled by the controller 27 of the refrigerant temperature control mechanism 26, so that both the perfusion environmental liquid 4 and the mixed liquid, which is the blood homogeneous perfusion liquid, are kept at about 2°C. It is.

In addition, in the above process, the perfusion environmental liquid 4 includes:
You may choose to use a DMSO solution or a cryoprotectant such as glycerin for convenience in the next step, which will be detailed later. It is most preferable to prepare a perfusion container and perfuse within the container.

In other words, by selecting the same liquid as the perfusion liquid and the perfusion environmental liquid 4, the same liquid can be used as the perfusion environmental liquid.
This is because it eliminates the possibility that the DMSO solution would seep into the organ through which the perfusate, which is a separate solution, would cause effects such as hemolysis (destruction of red blood cells) on organ 1. .

Next, when the first perfusion step is performed outside the illustrated apparatus as described above, the organ in which the blood and the mixed liquid have been replaced is stored as the DMSO liquid as the perfusion environmental liquid 4. Perfusion container 3
It is placed inside the mesh board 6 of the pedestal 5, and the irrigation fluid supply pipe 8 is connected to the artery 1a or the portal vein 1b.

When the first perfusion step is performed again using the illustrated apparatus, the organ 1 is transferred to the perfusion container 3 containing the DMSO solution.

Then, in the second perfusion step, the controller 27 closes the third on-off valve 24 and opens the second on-off valve 23, so that the DMSO solution is transferred to the second container in which the antifreeze agent such as glycerin is stored. From 20,
The perfusion fluid is sent to the organ 1 by the flow pump 25.

At this time, the temperature of the perfusion environment liquid 4, which is the DMSO liquid, is set to about 2°C in advance, and the temperature of the refrigerant 14 is gradually lowered from this temperature by the refrigerant temperature control mechanism 26 as described above. The temperature of the perfusate is set to about -4°C, that is, the temperature drops near the freezing point of the perfusate, which is an antifreeze agent.

It is best to set the temperature decreasing rate at about 0.5 to 1℃/min at this time, and when the temperature reaches -4℃ mentioned above,
The organ is stored at this temperature using an appropriate thermostatic device.

By using the illustrated device, the perfusion fluid and the perfusion environmental fluid 4 are made isothermal, so that the internal and external temperatures of the organ 1 are equalized and no temperature gradient is created. Therefore, a desirable perfusion process can be carried out, and since the organ 1 is in the environment liquid 4 for perfusion and is under buoyancy as described above, the organ can be placed on a predetermined table in the air and perfused. This eliminates the risk of the organ being pressed against the table by its own weight, as in the case where the organ is pressed against the table, and as a result, the perfusion fluid does not flow sufficiently into the crushed part of the organ due to the pressure contact, resulting in death of the part. Moreover, by placing it on the mesh board 6, the contact surface with the organ 1 is made smaller compared to when it is placed on a board.

Now, the organ 1, which has been maintained at a temperature drop near the freezing point (-4°C) through the first and second perfusion steps, will be used for transplantation if necessary. The means for transplantation can be carried out by substantially reversing the steps for freezing.

That is, in the first retrograde perfusion step, the organ maintained at -4°C is taken out from the storage location, set in the illustrated apparatus as described above, and operated by the controller 27.
By controlling the temperature of the refrigerant 14 and gradually increasing the temperature from -4°C, the temperature of the perfusion environmental liquid 4, which is a DMSO liquid, and the opening of the second opening/closing valve 23 are controlled to increase the temperature of the refrigerant 14, which is sent from the second container 20. as an irrigant
Perfusion is performed while increasing the temperature of the DMSO solution in an isothermal state, and the perfusion continues until the temperature reaches 2° C., which is the temperature in the vicinity described in the perfusion step.

Next, as a second retrograde perfusion step, the second on-off valve 23 is closed, and the first on-off valve 22 is controlled by the controller 27.
When the first container 19 is opened, the Collins fluid in the first container 19 is sent to the organ 1 by the flow pump 25. At this time, the temperature of the refrigerant 14 is gradually increased by the controller 27, and the blood equivalent liquid is sent to the organ 1 by the controller 27. The temperature of the perfusate is raised from 2° C. to body temperature.

At this time, when the perfusate was switched from the DMSO solution to the blood equivalent solution, the temperature of the blood equivalent perfusate was maintained at that temperature for a while at 2°C, and then the temperature was gradually increased as described above. It is preferable to perfuse the organ 1 while it is flowing, and by doing so, the shock to the cells of organ 1 can be reduced.

In this way, the organ that has undergone the retrograde perfusion process may be provided with the necessary blood for transplantation, and at this time it is desirable to add a blood coagulation inhibitor such as heparin to the blood.

According to the first invention of the present application, instead of simply perfusing the organ with Collins solution instead of blood as in the conventional method and storing it at about 4°C, in the first perfusion step, the artery or portal of the excised organ is A mixture of a blood equalizing perfusate such as Collins solution or physiological saline and a blood coagulation inhibitor such as pepperin is injected through the pulse, and the perfusion is discharged from the vein at a temperature near the freezing point of the perfusate. (about 2 degrees Celsius), the organs are not affected by sudden changes in temperature, and when the temperature is still 1 to 2 degrees Celsius, when the metabolism of organ cells is active, nutrients are distributed in equal amounts to blood. It can be supplemented without the risk of blood clotting.

In the second perfusion step, a frost damage inhibitor such as dimethyl sulfoxide or glycerin is used in place of the above-mentioned liquid mixture, while gradually lowering the temperature from the above-mentioned vicinity, so that the temperature of the inhibitor falls below its freezing point (- Since we perfused the organ until it reached a temperature of around -4°C, and then stored it at a temperature around that point, the organ would not freeze even though it was kept at a low temperature of around -4°C. Since the water in the organ cells does not cause problems such as destruction of the cells by freezing, it is possible to store the organ for a long period of time without killing it.

Next, the second invention provides a method for bringing the stored and cooled organs according to the first invention into a transplantable state. Based on this, the first retrograde perfusion step of perfusing the perfusate as the cryoprotectant from the artery or portal vein of the storage organ according to the first invention into the vein while gradually increasing the temperature from the storage temperature is performed at a temperature near the temperature. The second retrograde perfusion step is continued until the perfusion liquid is replaced with the above-mentioned perfusion liquid as a cryoprotectant, and the perfusion liquid as the blood-equal perfusion liquid is perfused into the vein while gradually raising the temperature from the above-mentioned neighborhood temperature. After the procedure continues until the temperature rises, the necessary blood is supplied to the organ, so that the preserved organ can be returned to a transplantable state without damaging it.

Furthermore, in the third invention of the present application, in the second retrograde step that is performed until the temperature rises to body temperature in the second invention, after continuing the perfusion at the temperature in the vicinity for a while, the temperature is gradually raised. With this method, it is possible to obtain more stable and desirable results without subjecting organ cells to sudden shocks due to temperature changes.

[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, a...artery, b...portal vein,
c...Vein.

Claims (1)

[Claims] 1. Injecting a mixture of a blood perfusion solution such as Collins solution or physiological saline and a blood coagulation inhibitor such as heparin into the artery or portal vein of the excised organ,
The first perfusion step, in which the perfusate is drained from the vein, is performed at a temperature near the freezing point of the perfusate, and then a cryoprotectant such as dimethyl sulfoxide or glycerin is added to this mixed solution gradually from the above temperature. The second perfusion step of perfusing while lowering the temperature is continued until the cryoprotectant reaches a temperature drop near its freezing point, so that the organ is stored substantially at a temperature drop near this temperature. How to preserve organs. 2. Inject a mixture of a blood perfusion solution such as Collins solution or physiological saline and a blood clotting inhibitor such as pepperin through the artery or portal vein of the removed organ.
The first perfusion step, in which the perfusate is drained from the vein, is performed at a temperature near the freezing point of the perfusate, and then a cryoprotectant such as dimethyl sulfoxide or glycerin is added to this mixed solution gradually from the above temperature. A second perfusion step of perfusion with cooling is continued until the cryoprotectant is at a temperature near its freezing point, and the organ is stored at substantially this temperature below its freezing point, and the artery or ostium of the storage organ is The first retrograde perfusion step of perfusing the perfusate as the cryoprotectant from the vein into the vein while gradually raising the temperature from the storage temperature is continued until the temperature is around the above, and then the perfusion liquid as the cryoprotectant is perfused into the vein. Instead of liquid,
A second step of perfusing the perfusion liquid as the blood equalization perfusion liquid into the vein while gradually raising the temperature from the above-mentioned vicinity temperature.
After the retrograde perfusion process was continued until the temperature reached body temperature,
A method for preserving an organ, characterized in that the organ is provided with the required amount of blood. 3. Inject a mixture of a blood perfusion solution such as Collins solution or physiological saline and a blood clotting inhibitor such as pepperin through the artery or portal vein of the removed organ, and
The first perfusion step, in which the perfusate is drained from the vein, is performed at a temperature near the freezing point of the perfusate, and then a cryoprotectant such as dimethyl sulfoxide or glycerin is added to this mixed solution gradually from the above temperature. A second perfusion step of cooling and perfusion is continued until the cryoprotectant reaches a temperature drop near its freezing point, and the organ is stored at substantially this temperature drop, and the artery or ostium of the storage organ is The first retrograde perfusion step of perfusing the perfusate as the cryoprotectant from the vein into the vein while gradually raising the temperature from the storage temperature is continued until the temperature is around the above, and then the perfusion liquid as the cryoprotectant is perfused into the vein. Instead of liquid,
A second step of perfusing the perfusion liquid as the blood equalization perfusion liquid into the vein while gradually raising the temperature from the above-mentioned vicinity temperature.
When the retrograde perfusion step is continued until the temperature rises to body temperature, the temperature is raised after the perfusion at the temperature near the above is continued for a while, and the necessary blood is supplied to the organ that has completed the step. How to preserve organs.