JPS6061501A - Method for preserving viscus - Google Patents

Abstract

PURPOSE:To make it possible to preserve an extirpated viscus for a long term, by reducing the temperature of the viscus to a specific temperature while perfusing the viscus with a blood equivalent solution, reducing the temperature of the viscus while perfusing the viscus with a freezing damage inhibitor, and freezing the viscus without causing cytoclasis. CONSTITUTION:A mixture solution of a blood equivalent solution 19, e.g. Collins' solution, and a blood anticoagulant 21 is injected from an artery (1a) or portal vein (1b) of an extirpated viscus 1 while passing the mixture solution through a cooling bath 13 under cooling and then discharged from a vein (1c) to carry out the first perfusion step at a reduced temperature (2 deg.C) near and above the freezing point of the above-mentioned perfusate. A freezing damage inhibitor 20, e.g. dimethyl sulfoxide (DMSO) or glycerol, in place of the above-mentioned mixture solution is used as the perfusate, and the temperture of the viscus 1 is slowly reduced from the above-mentioned temperature near and above the freezing point of the perfusate to a reduced temperature (-4 deg.C) near and above the freezing point of the freezing damage inhibitor 20 in the second perfusion step. Thus, the above-mentioned viscus 1 is preserved at the reduced temperature near the freezing point of the freezing damage inhibitor 20.

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 transfer them at a timely manner.

Traditionally, extracted organs have been preserved until they are transported, but the preservation method involves injecting Collins solution at approximately 4°C, which has properties similar to blood, into the organ's artery or portal vein. A so-called perfusion method is known in which organs are discharged through veins, and organs after such perfusion treatment are stored at a temperature of about 4°C, and blood flow is applied to the stored organs before use before transport. That's what I do.

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, making it difficult to coordinate the time between organ donation and demand9. This has become a major bottleneck for relief efforts.

Therefore, in order to extend the storage time, it may be possible to freeze the organ by setting the storage temperature to a low temperature.If an organ subjected to the above conventional method is frozen, cell destruction will occur and the organ itself will die5. 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.

That is, like the last five organs, the extracted organ 1 is immersed in the blood flow environment fluid 4 in the perfusion container 3 stored in the refrigerator 2 at about -4°C. The perfusion fluid supply pipe 8 of the perfusion device 7 has its tip 8' connected to the artery 1 of the organ 1.
The vein a is connected to the portal vein 1b, and the vein 1c is connected to the perfusion environmental fluid 4.
The perfusion environmental liquid 4 can be circulated by opening the inflow pipe 1o and the discharge pipe 11 of the circulation pump 9 into the bubble flow container 3, respectively. The environmental liquid heat exchange section 12 formed in 1Q is connected to the cooling tank 1.
3 is immersed in a refrigerant 14 made of fluorocarbon or the like.

Now, the cooling tank 13 consists of an insulated outer tank 16 and an intermediate tank 16.
Liquid nitrogen LN2 is stored between the intermediate tank 16 and the inner tank 17 containing the refrigerant 14, and helium gas G
It is encapsulated with He.

In this refrigerant 14, not only the above-mentioned environmental liquid heat exchange part 12 is eliminated, but also the perfusion liquid heat exchange part provided in the perfusion liquid supply vibe 8 in the above-mentioned perfusion device 7. 18 is also immersed.

The perfusion device 7 shown in the figure includes a first container 19 for storing Collins fluid, which is a blood equivalent fluid;
A second container 20 contains a cryoprotectant such as dimethyl sulfoxide (CD-MSO) or glycerin, and a thin liquid anti-coagulant such as heparin (approx. A third container 21 is provided in which a mixed solution containing the agent is stored, and each of these containers 19, 20, 21 is supplied with fluid through first, second, and third on-off valves 22, 23, and 24, respectively. The pump 25 is connected to the pump 25, and to the outflow side of the pump 25, the laminar fluid supply vibe 8, which has the above-mentioned irrigation fluid heat exchange section 18, is also connected.

- The above first, second and third on-off valves 21.22.2
3 is controlled to open and close as appropriate by a controller 27 of a refrigerant temperature control mechanism 26.

and It can be adjusted freely.

Therefore, in order to carry out the method according to the present invention using the above-mentioned device, first, for the extracted organ 1, a 1φ blood perfusion solution such as physiological saline or Collins' solution is poured from the artery Ia or the portal vein 1b as soon as possible. and a liquid coagulant such as -\valine is injected to perform step 1a, which replaces the intravascular blood of the organ 1 with the mixed liquid.

To carry out the first meandering process, immediately after removing organ 1, about 2
℃ into a container (not shown) containing physiological saline, and in this state, the mixture was poured from the artery 1a etc. at a temperature of 2℃, that is, before the freezing point of the perfusate, which is the same mixture. At near temperature, inject vein 1C with a syringe.
It is possible to take measures to bend the flow.

2 is preferable because it can carry out the process most quickly, but it is also possible to carry out the process using the illustrated apparatus described above.

That is, the extracted organ 1 is immediately placed in the blood flow environment liquid 4 of the diversion container 3 and placed on the mesh plate 6'2 of the pedestal 6, and the controller 2 opens the third on-off valve 24 and sends the organ. The flow pump 25 is operated to cause the mixed liquid in the third v vessel 21 to flow into the artery 1a of the organ 1, etc., and into the vein I.
c into the perfusion environmental fluid 4.

In the figure, 25' indicates the flow placement.

At this time, the controller 27 of the refrigerant temperature control mechanism 26
Thus, the temperature of the refrigerant 14 is controlled, and thereby the temperature of both the environmental liquid 4 for dish diversion and the above-mentioned mixed liquid, which is a blood homogeneous perfusion liquid, is about 2°C.

In the above process, it may be possible to use an anti-freezing agent such as DMSO solution or glycerin as the perfusion environmental liquid 4 for convenience in the next process which will be detailed later. It is most desirable to separately prepare a container for perfusion containing a mixed solution of physiological saline and heparin in the same manner as the mixed solution, and perfuse within the container.

That is, by selecting the same solution as the perfusate and the perfusion environmental liquid 4, the DMSO liquid as the perfusion environmental liquid or a separate perfusion liquid permeates into the organ through which it is flowing. This is because it is possible to eliminate the possibility that the organ 1 will be affected by hemolysis (destruction of red blood cells) or the like.

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

Then, when step 1a is carried out using the illustrative apparatus, the organ 1 is transferred to the blood flow container 3 containing DMSOi.

Then, in the second curve process, the controller 27
Next, by closing the third on-off valve 24 and opening the second on-off valve 23, the DMSO liquid is transferred from the second container 20 storing antifreeze agents such as glycerin to the organ 1 by the flow pump 25. The perfusion fluid is sent.

At this time, the temperature of the oil flow environment o, 4 containing the DMSO liquid is set to about 2°C in advance, and from this temperature, the refrigerant 1
By gradually lowering the temperature of 4 using the refrigerant temperature control mechanism 26, the temperature of the environmental liquid 4 and the perfusate is reduced to about 14°C, that is, the near-lowering temperature that is below the freezing point of the perfusate, which is an antifreeze agent. That's why.

The rate of temperature drop at this time is preferably about 0.5 to 1°C, and when the temperature reaches 14°C, the organ is stored at that temperature using an appropriate thermostat.

By using the illustrated device, perfusion fluid and perfusion environmental fluid 4 can be used.
Since these are equalized, the internal and external temperatures of the organ 1 are equalized, and there is no temperature gradient, so a desirable perfusion process can be carried out.
In addition, as mentioned above, since the organ 1 is in the perfusion environmental liquid 4 and is under buoyancy, the organ 1 is placed on a predetermined table in the air and perfused, and the organ 1 is placed on the table due to its own weight. This eliminates the possibility that the perfusion fluid will not flow sufficiently into the crushed part of the organ due to the pressure contact and the part will die.In addition, by placing it on the mesh board 6, The contact surface with the organ 1 is also made smaller compared to when placed on top.

Now, the organ 1, which is maintained at a temperature drop near the freezing point (-4°C) through the first and second perfusion steps, is
This will be used for transplantation if necessary, and the means for transferring the vessel can be carried out by substantially reversing the freezing vessel step described in υII.

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 the temperature of the refrigerant 14 is controlled by the controller 27, and the organ is kept at -4°C. By gradually raising the temperature from the state of
Since the temperature of the perfusion environmental liquid 4 which is dripped with the DMSO liquid and the temperature of the DMSO liquid as the perfusion liquid sent from the second container 2o by opening the second on-off valve 23 are raised in an isothermal state, perfusion is performed. The perfusion continues until the temperature reaches 2°C, which is around the temperature described in the perfusion step.

Next, as a second retrograde perfusion step, the second on-off valve 23 is closed, the first on-off valve 22 is opened by the controller 27, and the blood equivalent liquid, which is the Collins liquid, in the first container 19 is pumped through the flow pump 25. At this time, the controller 27 gradually increases the temperature of the refrigerant 14 to raise the temperature of the perfusate, which is a blood equivalent fluid, from 2° C. to body temperature. .

At this time, when I switched the perfusion fluid from DMSO to blood equivalent fluid, it was time to go! It is preferable to maintain the temperature of the blood homogenizer at 12° C. for a while, and then start perfusion while gradually increasing the temperature as described above.
By doing so, the shock to the cells of organ 1 can be reduced.

In this way, the organ after the retrograde perfusion process can be supplied 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 trying to store Collins solution in a 11-liter vessel at 4°C in the presence of the liquid as in the conventional method, in the first perfusion step, A mixture of a blood equalizing perfusion solution such as Collins solution or physiological saline and a blood coagulation inhibitor such as Peverin is injected from the artery or portal vein of an organ, and the perfusion is discharged from the vein until the coagulation point of the perfusion solution is applied. Since we previously performed the test at a temperature close to a certain level (about 2℃), the organs are not affected by sudden changes in temperature, and at a temperature of 1 to 2℃, when the metabolism of organ cells is active, the temperature is even with blood. nutrients can be supplied without the risk of blood clotting.

In the second oil flow step, instead of the above-mentioned mixed liquid, a freeze damage inhibitor such as dimethyl sulfoxide or glycerin is added, while the temperature is gradually lowered from the above-mentioned vicinity, until the temperature of the inhibitor is near the freezing point. The organs were perfused until the temperature dropped (approximately -4°C), and then the organs were stored at approximately the same temperature drop, so the organs were kept at a low temperature of about -4°C and were not frozen. Since the organ cells are not permanently damaged, there is no problem such as the water inside the organ cells destroying the cells due to freezing, and this makes it possible to store the organ for a long period of time without dying.

Next, the second invention provides a method for bringing stored frozen organs into a transplantable state from the first invention (4), and this invention reversibly implements the first invention. Based on this idea, 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 as described above. A second retrograde irrigation step is continued until the temperature reaches the vicinity, and then, instead of the above-mentioned perfusate as a cryoprotectant, the perfusate as the blood equalizing perfusion liquid is perfused into the vein while gradually raising the temperature from the above-mentioned vicinity temperature. After continuing to raise the temperature to the body temperature, the necessary blood is supplied to the organ concerned, so the preserved organ can be returned to a transplantable state without damage.

Furthermore, in the third invention of the present application, in the second retrograde step in the second invention, which is carried out until the temperature rises to body temperature, after the oil flow is continued for a while at the temperature in the vicinity, Since the method uses ectotherm, it is possible to achieve more stable and desired results without subjecting the organ cells to sudden shock due to temperature changes.

[Brief explanation of drawings]

The figure is an overall explanatory view, partially cut away, showing the state of use of the mixed flow device that can be used to carry out the organ preservation method according to the present invention. 1... Organ Ia... Artery Ib... Portal vein re... Vein Patent applicant's agent Makoto Ito, patent attorney

Claims (2)

[Claims] (1) First perfusion in which a mixture of a blood-equalizing perfusion solution such as Collins solution or physiological saline and a blood clotting inhibitor such as heparin is injected into the artery or portal vein of the removed organ and drained through the vein. The process is carried out at a temperature near the freezing point of the perfusate, and then, in place of this mixed solution, a frost damage inhibitor such as dimethyl sulfochloride, glycerin, etc. is perfused while gradually lowering the temperature from the above temperature. Preservation of an organ, characterized in that the second perfusion step is continued at 1, at which the cryoprotectant has a temperature drop near its freezing point, so that the organ is stored substantially at a temperature drop near this temperature. Method. (2) The first perfusion step is to inject a blood equivalent perfusate such as the artery or portal vein of the excised organ, Collins solution, or physiological saline, and drain it from the vein before the solidification point of the perfusate. The second perfusion step is carried out at a temperature in the vicinity of the above temperature range, and then replaces this mixed solution with a freeze damage inhibitor such as dinotyl sulfoxide or glycerin while gradually lowering the temperature from the above temperature range. The organ is stored at a temperature substantially below the freezing point, and the perfusate as a cryoprotectant is supplied from the artery or portal vein of the stored organ from the storage temperature. The first retrograde flow process, in which blood flows into the vein gradually in parallel,
The process continues until the temperature reaches the above-mentioned vicinity, and then, in place of the above-mentioned perfusate as a cryoprotectant, the perfusate as the blood-equalizing perfusate is perfused into the vein while gradually increasing the temperature from the above-mentioned vicinity. A method for preserving an organ, characterized in that the blood flow process is continued until the temperature reaches body temperature, and then the necessary blood is supplied to the organ. The first perfusion step, in which a mixture of a blood equalizing perfusate such as a nozzle solution or physiological saline and a blood coagulation inhibitor such as pepperin is injected and drained through the veins, is performed at a temperature near the freezing point of the perfusate. Then, in place of this mixed solution, a second perfusion step is performed in which a cryoprotectant such as dimethyl sulfoxide or glycerin is perfused while gradually lowering the temperature from the above-mentioned vicinity, until the cryoprotectant is below its freezing point. The organ is then stored at substantially this near-falling temperature, and the perfusate as the cryoprotectant is gradually raised from the storage temperature through the artery or portal vein of the stored organ. The first retrograde perfusion step of perfusing the vein while warming it is continued until the temperature reaches the above-mentioned vicinity, and then, in place of the above-mentioned perfusate as a cryoprotectant, the perfusate as the blood-equalizing perfusate is gradually added from the above-mentioned temperature to the above-mentioned vicinity. When continuing the second retrograde current step in which perfusion is carried out into the veins while raising the temperature to body temperature, the temperature is raised after the perfusion at the above-mentioned temperature has been continued for a while, and the organ after the step is given the required amount. A method for preserving organs characterized by donating the blood of