JP2017186295A - Organ preservation method and organ transplantation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an organ preservation method and organ transplantation method in which an organ can be properly preserved.SOLUTION: The organ preservation method is characterized by preserving an organ separated from a living body or a dead body within a range of -2.0°C ± 1.0°C. Further, it is preferable to preserve organs in supercooled state. Further, it is preferable to preserve organs by placing organs in a coolant which has been previously adjusted within a range of -2.0°C ± 1.0°C. Further, it is preferable to place organs in a coolant within 1 minute after separating from a living body or a dead body.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an organ preservation method and an organ transplantation method.

  As a method for storing an organ taken out (separated) from a living body or a dead body, for example, a storage method described in Patent Document 1 is disclosed. In Patent Document 1, an organ is stored in a cold storage solution at 4 ° C. to 20 ° C., and the organ is stored by perfusing the organ with a cooling solution.

JP 2013-075888 A

  However, organs cannot be stored properly even if they are stored in a cold storage solution at 4 ° C.

  An object of the present invention is to provide an organ preservation method and an organ transplantation method that can appropriately preserve an organ.

  The organ preservation method of the present invention is characterized in that an organ separated from a living body or a cadaver is preserved within a range of −2.0 ° C. ± 1.0 ° C.

  In the organ preservation method of the present invention, the organ is preferably preserved in a supercooled state.

  In the organ preservation method of the present invention, it is preferable to preserve the organ by placing the organ in a coolant that has been adjusted in the range of −2.0 ° C. ± 1.0 ° C. in advance.

  In the organ preservation method of the present invention, it is preferable that the organ is separated from the living body or the corpse and placed in the coolant within 5 minutes.

  In the organ preservation method of the present invention, the coolant is preferably sherbet-like ice.

  The organ transplantation method of the present invention is characterized in that the organ preserved by the organ preservation method of the present invention is transplanted into a living body.

  According to the present invention, the low temperature of −2.0 ° C. ± 1.0 ° C. reduces the metabolism of the cells of the living body (organ) and contributes to the storage in a state where the biological function is maintained. can do. Therefore, for example, it becomes possible to preserve | save for a long time in the state which can transplant an organ to a biological body. In addition, it is possible to reduce a decrease in organ function after living-body transplantation.

It is a graph which shows the temperature change of the organ at the time of using the coolant of different temperature. It is a figure which shows an extracorporeal lung perfusion apparatus. It is a graph which shows the tidal volume of a donor lung. It is a graph which shows the oxygen concentration of a donor lung. It is a graph which shows the weight of a donor lung. It is a graph which shows the amount of mRNA of a donor lung. It is an image which shows ED1 positive cell. It is a graph which shows the number of ED1 positive cells. It is a graph which shows the survival rate of a rat. FIG. 6 is an image showing a donor lung graft. FIG.

  Hereinafter, the organ preservation method and organ transplantation method of the present invention will be described in detail with reference to the accompanying drawings.

  In recent years, due to the development of supercooling technology, an organ removed (separated) from a donor (living body or cadaver) for organ transplantation has not been frozen at a freezing point (0 ° C.) or less, specifically −6 ° C. to −5 ° C. Research and development have been conducted on the technology for preserving in an uncooled state. According to such a storage method, it is considered that organs can be stored for a long time while being fresh. Therefore, the inventors of the present application have conducted research for further improvement of the above technique, and as a result, have found that there is a storage temperature that is more suitable for storage of organs. This will be described below. In the present specification, the dead body includes not only heart death but also brain death and the like (for example, death determined by the dead donor rule).

  The inventor of the present application preserves an organ extracted from a donor in a supercooled state (in a state where it is kept unfrozen below the freezing point (0 ° C.) within a range of −2.0 ° C. ± 1.0 ° C., It has been found that organs can be stored for a longer period of time while being fresh, and further, it is possible to effectively suppress a decrease in organ function after organ transplantation to a patient. This is considered to be because a low temperature of −2.0 ° C. ± 1.0 ° C. reduces the metabolism of cells in the living body (organ) and contributes to preservation in a state where the biological function is maintained. In addition, storage temperature is so preferable that there are few fluctuations from -2.0 degreeC. Specifically, it is more preferable to store in a supercooled state within a range of −2.0 ° C. ± 0.5 ° C., and store in a supercooled state within a range of −2.0 ° C. ± 0.1 ° C. More preferably.

  The organ to be preserved is not particularly limited, and for example, heart, lung, stomach, small intestine, duodenum, large intestine, rectum, liver, kidney, spleen, etc. (all organs that can be transplanted) and tissues (cornea, Trachea, heart valve, ligament, bone, etc.).

  The organ removed from the donor is, for example, in a coolant (preservation solution) that has been adjusted (maintained) within a range of −2.0 ° C. ± 1.0 ° C. by a cooling device such as a compressor type or a Peltier type. It is preferable to store it by immersing it. Thus, by immersing the organ in a coolant adjusted within the range of −2.0 ° C. ± 1.0 ° C., the organ is removed at −2.0 ° C. ± 1. It can be cooled to within the range of 0 ° C. Therefore, the organ can be preserved while being fresher. In addition, the temperature of the coolant is preferable in that the fluctuation from −2.0 ° C. is less, because the organ can be stably stored at around −2.0 ° C. Specifically, it is more preferable that the temperature is maintained within a range of −2.0 ° C. ± 0.5 ° C., and it is more preferable that the temperature is maintained within a range of −2.0 ° C. ± 0.1 ° C. . However, for example, the organ may be immersed in a normal temperature coolant, and then the coolant may be cooled.

  The coolant is not particularly limited, and for example, a known organ / tissue preservation solution such as physiological saline or ET-Kyoto solution can be used.

  In addition to the above organ / tissue preservation solution, for example, sherbet-like ice (that is, a mixture of water and ice) may be used as the coolant. The sherbet-like ice is manufactured from salt water (physiological saline), and the freezing point is adjusted to about −2.0 ° C. Further, the ice concentration of the sherbet-like ice is not particularly limited, but is preferably about 40% or more and 60% or less, for example. In the organ / tissue preservation solution described above, the temperature must be controlled by a cooling device or the like, and thus, for example, temperature fluctuations are likely to occur. In addition, since a power source for driving the cooling device is required, for example, it is conceivable that restrictions on organ transportation occur. On the other hand, if sherbet-like ice is used as a coolant, a power source is not required and temperature fluctuations can be reduced.

  Further, it is preferable that the time from the removal of the organ from the donor to the immersion of the organ in the coolant is as short as possible. Specifically, it is preferably within 5 minutes, more preferably within 3 minutes, and even more preferably within 1 minute. In this way, the organ can be stored fresher by quickly immersing the organ in the coolant.

  Moreover, it is preferable that the organ extracted from the donor is brought into a supercooled state of 0 ° C. or less in as short a time as possible. Since cell metabolism occurs while the organ is being cooled, this cell metabolism can be suppressed and the deterioration of the organ can be effectively suppressed by making it supercooled in as short a time as possible. . Therefore, the organ can be stored for a longer period of time while being fresher.

  FIG. 1 shows changes in the temperature of an organ when the organ is immersed in a coolant adjusted to −2.0 ° C., and the temperature of the organ when the organ is immersed in a coolant adjusted to 0 ° C. It is a graph which compares with a change. As can be seen from this figure, when the organ is immersed in a coolant adjusted to −2.0 ° C., the organ is cooled to 0 ° C. or less within 20 minutes. Therefore, the organ can be brought into a supercooled state in a shorter time, and the organ can be stored for a longer time while remaining fresher.

  The organs stored in this manner (herein described on behalf of the lungs) are actually used only when, for example, the function is confirmed using the extracorporeal lung perfusion device 100 described later and it is determined that transplantation is possible. Transplanted to the patient. Further, when blood is perfused after transplantation, for example, it is preferable to increase the perfusion rate by 10 ml / min at intervals of 2 minutes. Thereby, for example, damage to blood vessels can be reduced.

  By using such an extracorporeal lung perfusion apparatus 100, lung function can be confirmed and lung function can be recovered. Specifically, for example, water accumulated in the lungs can be reduced, nutrients such as sugar and oxygen can be supplied, and the contracted part can be returned to normal. Therefore, transplantable lungs can be increased and lungs can be transplanted to more patients.

  As mentioned above, although the organ preservation | save method and the organ transplantation method of this invention were demonstrated, this invention is not limited to this.

[Subject]
In order to reduce individual differences, inbred male rats were used as subjects.

Example 1
First, 550 ml of ET Kyoto liquid maintained at −2.0 ° C. was prepared, and then the donor lung and heart (hereinafter also referred to as “donor cardiopulmonary block”) were removed and removed from the male rat of the subject. Donor cardiopulmonary blocks were immediately immersed in ET Kyoto fluid. Then, while maintaining the ET Kyoto solution in the temperature range of −2.0 ° C. ± 0.25 ° C., the supercooled state was stabilized, and the donor cardiopulmonary block was stored for 17 hours.

≪Comparative example 1≫
First, an ET Kyoto solution kept at −5.0 ° C. was prepared, and then a donor cardiopulmonary block was extracted from a male rat of the subject, and the extracted donor cardiopulmonary block was immediately immersed in the ET Kyoto solution. And the supercooled state was stabilized and the donor cardiopulmonary block was preserve | saved for 17 hours, maintaining ET Kyoto liquid in the temperature range of -5.0 degreeC +/- 0.25 degreeC as it was.

≪Comparative example 2≫
First, an ET Kyoto solution kept at + 4.0 ° C. was prepared, and then a donor cardiopulmonary block was extracted from the male rat of the subject, and the removed donor cardiopulmonary block was immediately immersed in the ET Kyoto solution. Then, the temperature range of + 4.0 ° C. ± 0.25 ° C. was maintained as it was, and the donor cardiopulmonary block was stored in ET Kyoto Solution for 17 hours.

«Comparative Example 3»
A donor cardiopulmonary block immediately after removal from the male rat of the subject was prepared. That is, no cold storage is performed in this military donor cardiopulmonary block (rat lung).

[Evaluation]
First, the donor cardiopulmonary block obtained in Example 1 and Comparative Examples 1 and 2 was perfused for 60 minutes using the extracorporeal lung perfusion apparatus 100 (ex vivo) shown in FIG. The extracorporeal lung perfusion apparatus 100 can maintain the blood flow and respiration and maintain the organ in the same state as the living body using an artificial pump artificial respirator with the organ taken out of the body. Device.

  Such an extracorporeal pulmonary perfusion apparatus 100 includes a chamber 110 for housing a cardiopulmonary block, a perfusion line 120 having one end connected to the pulmonary artery (PA) and the other end connected to the left atrium (LA), and a lung. Oxygen removal that removes (reduces) oxygen from the perfusate from the left atrium, air supply line 130 and ventilator 140 for feeding air, pump 150 provided in the middle of the perfusion line 120, and in the middle of the perfusion line 120 Part 160. The perfusate was composed of heparinized rat blood, physiological saline containing 4% bovine serum albumin, and hemoglobin, and the pH was adjusted to 7.25 to 7.35. During perfusion, the temperature of the perfusate was kept at approximately 37 ° C., and the perfusate was supplied to the pulmonary artery at 10 ml / min. At the same time, 21% oxygen was supplied to the lungs.

≪Evaluation of air ventilation to lungs≫
About Example 1 and Comparative Examples 1 and 2, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after the start of perfusion of the perfusate into the donor lung using the extracorporeal lung perfusion apparatus 100 described above At timing, the tidal volume of the donor lung (the amount of air entering the lung) was measured. The result is shown in the graph of FIG. In addition, this measurement was performed using 4 (n = 4) donor cardiopulmonary blocks for each of Comparative Examples 1 and 2. As can be seen from the graph of FIG. 3, the tidal volumes of Comparative Examples 1 and 2 are greatly reduced to substantially the same value after 60 minutes, although the ways of reduction are different from each other. In contrast, in Example 1, the state remained almost unchanged until the lapse of 60 minutes.

≪Evaluation on oxygen concentration≫
About Example 1 and Comparative Examples 1 and 2, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after the start of perfusion of the perfusate into the donor lung using the extracorporeal lung perfusion apparatus 100 described above At the timing, the amount of oxygen in the perfusate excreted from the pulmonary vein of the donor lung was measured. The result is shown in the graph of FIG. In addition, this measurement was performed using 4 (n = 4) donor cardiopulmonary blocks for each of Comparative Examples 1 and 2. As can be seen from the graph of FIG. 4, the amounts of oxygen in the perfusates of Comparative Examples 1 and 2 are greatly reduced to substantially the same value after 60 minutes, although the ways of reduction are different from each other. In contrast, in Example 1, the state remained almost unchanged until the lapse of 60 minutes.

≪Evaluation of donor lung weight≫
About Example 1 and Comparative Examples 1 and 2, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after the start of perfusion of the perfusate into the donor lung using the extracorporeal lung perfusion apparatus 100 described above At the timing, the donor lung was weighed. The results are shown in the graph of FIG. In addition, this measurement was performed using 4 (n = 4) donor cardiopulmonary blocks for each of Comparative Examples 1 and 2. As can be seen from the graph in FIG. 5, the donor lungs of Comparative Examples 1 and 2 are heavier with the passage of time, although the way of increase is different from each other. Thus, it was found that in Comparative Examples 1 and 2, the lungs became heavier with time and the lung function was reduced accordingly. In contrast, in Example 1, the state remained almost unchanged until the lapse of 60 minutes.

≪Evaluation of mRNA expression≫
For Example 1 and Comparative Examples 2 and 3, the amount of mRNA was measured by a two-step real-time RT-PCR method using Cyber Green (registered trademark). The result is shown in FIG. In addition, this measurement was performed using 7 donor cardiopulmonary blocks for Example 1 and Comparative Examples 2 and 3, respectively. As shown in FIG. 6, iNOS (inducible nitric oxide synthase), COX-2 (cyclooxygenase-2), IL-6 (interleukin-6), IL-1beta (interleukin-1 beta), TNFalfa ( For each mRNA amount of tumor necrosis factor α), Example 1 is significantly less than Comparative Example 2 and almost as small as Comparative Example 3.

≪Evaluation of macrophage infiltration≫
For Example 1 and Comparative Examples 2 and 3, immunostaining was performed using an anti-ED1 antibody as a macrophage marker, and macrophage infiltration in the graft was observed. The results are shown in FIGS. As shown in FIGS. 7 and 8, the number of ED1-positive cells in Example 1 is significantly smaller than that in Comparative Example 2 and almost the same as that in Comparative Example 3.

≪ Survival rate after lung transplantation ≫
The donor lung of Example 1 and the donor lung of Comparative Example 2 were transplanted into rats, and the survival rate thereafter was examined. The results are shown in FIG. 9 and FIG. FIG. 9 is a graph showing the survival rate of rats for 7 days, and FIG. 10 is a microscopic image of a lung graft after 7 days. This measurement was carried out using 7 rats for Example 1 and Comparative Example 2, respectively. As shown in FIG. 9, in Example 1, the survival rate after 7 days is 100%, whereas in Comparative Example 2, the survival rate starts to decrease from the third day. Further, as shown in FIG. 10, the graft of Example 1 is almost pathologically normal, whereas in Comparative Example 2, severe fibrosis with alveolar hemorrhage and edema appears. .

  As described above, the experimental results on the lungs have been described as representatives, but the same effect can be exerted on organs other than the lungs (for example, heart, stomach, small intestine, duodenum, large intestine, rectum, liver, kidney, spleen, etc.). Can do. Further, in addition to Example 1, the same applies to those in which the ET Kyoto fluid was maintained in the temperature range of −2.0 ° C. ± 1.0 ° C., the supercooled state was stabilized, and the donor cardiopulmonary block was stored for 17 hours. As a result, almost the same result as in Example 1 was obtained.

DESCRIPTION OF SYMBOLS 100 ... Extracorporeal lung perfusion apparatus 110 ... Chamber 120 ... Perfusion line 130 ... Air supply line 140 ... Ventilator 150 ... Pump 160 ... Oxygen removal part

Claims (6)

  An organ preservation method, wherein an organ separated from a living body or a cadaver is preserved within a range of −2.0 ° C. ± 1.0 ° C.   The organ preservation method according to claim 1, wherein the organ is preserved in a supercooled state.   The organ preservation method according to claim 1 or 2, wherein the organ is preserved by placing the organ in a coolant that has been adjusted in a range of -2.0 ° C ± 1.0 ° C in advance.   The organ preservation method according to claim 3, wherein the organ is separated from the living body or the cadaver and placed in the coolant within 5 minutes.   The organ preservation method according to claim 3 or 4, wherein the coolant is sherbet-like ice.   An organ transplantation method, wherein the organ preserved by the organ preservation method according to any one of claims 1 to 5 is transplanted into a living body.

Images