JP7541216B2 - Method for producing hydrogen-containing organ preservation solution and hydrogen-containing organ preservation solution - Google Patents

Description

The present invention relates to a method for producing a hydrogen-containing organ preservation solution by dissolving hydrogen gas, and to this hydrogen-containing organ preservation solution.

It has been revealed in recent years that hydrogen gas induces various biological reactions. In particular, the mechanism by which it is effective in preventing ischemia-reperfusion injury has been elucidated (e.g., Non-Patent Document 1), and the effectiveness of hydrogen gas has been reported in various organ transplant models using small animals (e.g., Non-Patent Documents 2-4). However, these relate to organ transplant models of small animals such as mice, and do not use pig models. On the other hand, there are papers that have verified the effectiveness of hydrogen gas in preclinical models such as pig models (e.g., Non-Patent Documents 5-9), but these are few in number and do not suggest a consistent view.

In addition, in the prior art, including the disclosures in these non-patent documents, hydrogen gas was generated in an electrolysis device and supplied, or was supplied from a hydrogen cylinder.

Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S, “Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals”, Nat Med. 2007 Jun ;13(6):688-94. Epub 2007 May 7 Abe T, Li XK, Yazawa K, Hatayama N, Xie L, Sato B, Kakuta Y, Tsutahara K, Okumi M, Tsuda H, Kaimori JY, Isaka Y, Natori M, Takahara S, Nonomura N, “Hydrogen-rich University of Wisconsin solution attenuates renal cold ischemia-reperfusion injury”, Transplantation. 2012 Jul 15;94(1):14-21. doi: 10.1097/TP.0b013e318255f8be Lobb I, Jiang J, Lian D, Liu W, Haig A, Saha MN, Torregrossa R, Wood ME, Whiteman M, Sener A. “Hydrogen Sulfide Protects Renal Grafts Against Prolonged Cold Ischemia-Reperfusion Injury via Specific Mitochondrial Actions”, Am J Transplant. 2017 Feb;17(2):341-352. doi: 10.1111/ajt.14080. Epub 2016 Nov 29. Tamaki I, Hata K, Okamura Y, Nigmet Y, Hirao H, Kubota T, Inamoto O, Kusakabe J, Goto T, Tajima T, Yoshikawa J, Tanaka H, Tsuruyama T, Tolba RH, Uemoto S, “Hydrogen Flush After Cold “Storage as a New End-Ischemic Ex Vivo Treatment for Liver Grafts Against Ischemia/Reperfusion Injury”, Liver Transpl. 2018 Nov;24(11):1589-1602. doi: 10.1002/lt.25326 Hosgood SA, Moore T, Qurashi M, Adams T, Nicholson ML, “Hydrogen Gas Does Not Ameliorate Renal Ischemia Reperfusion Injury in a Preclinical Model”, Artif Organs. 2018 Jul;42(7):723-727. doi: 10.1111/ aor.13118. Epub 2018 Apr 2. Sekijima M, Sahara H, Miki K, Villani V, Ariyoshi Y, Iwanaga T, Tomita Y, Yamada K, “Hydrogen sulfide prevents renal ischemia-reperfusion injury in CLAWN miniature swine”, J Surg Res. 2017 Nov;219:165- 172. doi: 10.1016/j.jss.2017.05.123. Epub 2017 Jun 28 Lee G, Hosgood SA, Patel MS, Nicholson ML, “Hydrogen sulphide as a novel therapy to ameliorate cyclosporine nephrotoxicity”, J Surg Res. 2015 Aug;197(2):419-26. doi: 10.1016/j.jss.2015.02 .061. Epub 2015 Mar 17. Haam S, Lee S, Paik HC, Park MS, Song JH, Lim BJ, Nakao A, “The effects of hydrogen gas inhalation during ex vivo lung perfusion on donor lungs obtained after cardiac death”, Eur J Cardiothorac Surg. 2015 Oct; 48(4):542-7. doi: 10.1093/ejcts/ezv057. Epub 2015 Mar 6 Matsuno N, Watanabe R, Kimura M, Iwata S, Fujiyama M, Kono S, Shigeta T, Enosawa S, “Beneficial effects of hydrogen gas on porcine liver reperfusion injury with use of total vascular exclusion and active venous bypass”, Transplant Proc. 2014 May;46(4):1104-6. doi: 10.1016/j.transproceed.2013.11.134.

At the medical site of organ transplant donors, the conventional method of generating hydrogen gas using an electrolysis device and supplying it from a hydrogen cylinder brought to the site and supplying hydrogen gas from this cylinder not only requires a great deal of effort and time in preparation, but also involves risks, making it virtually impossible to carry out quickly at sites where urgency is required.

Therefore, the present invention aims to solve such problems in the prior art, and its object is to provide a method for producing hydrogen-containing organ preservation fluid and a hydrogen-containing organ preservation fluid that can quickly, easily and safely be produced at sites where organs for transplantation are handled.

According to the present invention, a method for producing a hydrogen-containing organ preservation solution involves, at a site where organs for transplant are handled, pressuring hydrogen gas from a hydrogen storage alloy canister into a flexible container containing organ preservation solution, vibrating the flexible container to dissolve the hydrogen gas in the organ preservation solution, and opening the flexible container to the outside air to reduce the internal pressure, thereby producing a hydrogen-containing organ preservation solution containing a predetermined concentration or more of dissolved hydrogen in the flexible container and used for flushing and/or preserving organs for transplant.

Thus, according to the present invention, in a medical field, hydrogen gas from a hydrogen storage alloy canister is used, this hydrogen gas is injected into a flexible container, and the flexible container is vibrated to dissolve the hydrogen gas in the organ preservation liquid, and then the flexible container is opened to the outside air to reduce the internal pressure. Since a hydrogen storage alloy canister is used as a hydrogen gas source, the hydrogen gas source can be easily and safely brought to a medical field, and even in an emergency, hydrogen gas can be quickly injected into the flexible container. In addition, by vibrating the flexible container after hydrogen gas injection, more hydrogen gas can be dissolved in the organ preservation liquid, and the hydrogen gas nanobubbles contained in the organ preservation liquid can be at a higher concentration. Furthermore, by subsequently opening the flexible container to the outside air to reduce the internal pressure, not only can the organ preservation liquid in the flexible container be used to wash and preserve organs for transplantation, but the hydrogen gas nanobubbles contained in the organ preservation liquid can be at a higher concentration. These hydrogen gas nanobubbles contribute greatly to improving the flushing effect of organs for transplantation.

It is also preferable to supply hydrogen gas until the pressure of the hydrogen gas supplied from the hydrogen storage alloy canister to the flexible container reaches a predetermined pressure.

In this case, it is more preferable that the specified pressure is in the range of 0.02 MPa to 0.07 MPa (gauge pressure).

It is also preferable to vibrate the flexible container for at least 30 seconds.

According to the present invention, furthermore, the hydrogen-containing organ preservation solution is a liquid that contains dissolved hydrogen at a predetermined concentration or more and is used to flush and/or preserve organs for transplantation.

It is preferable that this hydrogen-containing organ preservation solution is a liquid used to rinse the extracted organ for transplant at the site of the organ's removal.

It is also preferable that this hydrogen-containing organ preservation solution is an organ preservation liquid used to preserve extracted organs for transplantation and to transport the organs for transplantation to the transplant site.

This hydrogen-containing organ preservation solution is contained in a flexible container, and it is also preferable that a portion of the contained hydrogen-containing organ preservation solution is a liquid used to rinse the extracted organ for transplantation, and the remainder contained in the flexible container is a liquid used to preserve the extracted organ for transplantation.

It is also preferable that the above-mentioned specified concentration is a concentration that results in a dissolved hydrogen concentration of 1.0 mg/L or more after 4 hours.

According to the present invention, since a hydrogen storage alloy canister is used as a hydrogen gas source, the hydrogen gas source can be easily and safely brought to the medical site, and hydrogen gas can be quickly injected into the flexible container even in an emergency. In addition, by vibrating the flexible container after hydrogen gas is injected, more hydrogen gas can be dissolved in the organ preservation liquid, and the hydrogen gas nanobubbles contained in the organ preservation liquid can be at a higher concentration. Furthermore, by subsequently opening the flexible container to the outside air to reduce the internal pressure, not only can the organ preservation liquid in the flexible container be used to wash and preserve organs for transplantation, but the hydrogen gas nanobubbles contained in the organ preservation liquid can be at a higher concentration. These hydrogen gas nanobubbles greatly contribute to the effect of washing organs for transplantation.

FIG. 1 is a flow chart showing a process for producing a hydrogen-containing organ preservation solution and a process for using the hydrogen-containing organ preservation solution, according to one embodiment of the present invention. FIG. 2 is a perspective view showing a schematic configuration of an apparatus for producing a hydrogen-containing organ preservation solution in the embodiment of FIG. 1. 1 is a graph showing the initial and time-dependent changes in the dissolved hydrogen concentration in a hydrogen-containing organ preservation solution. FIG. 1 shows images of kidneys in donor cardiac death (DCD) that have been flushed with hydrogen gas-containing ETK solution and hydrogen gas-free ETK solution. FIG. 1 shows images of DCD kidneys flushed and preserved with ETK solution without hydrogen gas. FIG. 1 shows images of DCD kidneys flushed and preserved with ETK solution containing hydrogen gas. 1 is a graph showing changes in blood BUN during the chronic phase after surgery. 1 is a graph showing the change in blood Cre level over time during the chronic phase after surgery.

Figure 1 shows an outline of the process for producing hydrogen-containing organ preservation solution and the process for using the hydrogen-containing organ preservation solution as one embodiment of the present invention, and Figure 2 shows an outline of the configuration of an apparatus for producing hydrogen-containing organ preservation solution in this embodiment.

In this embodiment, at the site where transplant organs such as kidneys are handled, i.e., at the site where organs are extracted from donors whose hearts have stopped beating, a process of dissolving hydrogen gas in an organ preservation solution is instantaneously performed to generate a hydrogen-containing organ preservation solution with a predetermined dissolved hydrogen concentration, and the extracted organ is flushed out (flushed out) and preserved using this hydrogen-containing organ preservation solution. In the following explanation, ETK (ET-Kyoto) solution is used as the organ preservation solution, but UW (University of Wisconsin) solution or HTK (Histidine-Tryptophan-Ketoglutarate) solution may also be used.

The process for producing hydrogen-containing ETK solution at the organ harvesting site is described below.

First, as shown in Fig. 2, a plastic soft bag 10 (flexible container made of a film such as polyethylene, polypropylene, or polyvinyl chloride) containing ETK liquid 10a is prepared, and a hydrogen storage alloy canister 21 is connected to this plastic soft bag 10 (step S1 in Fig. 1). Specifically, as shown in Fig. 2, a bottle needle 12 is inserted through a rubber stopper 11 of the plastic soft bag 10, and the hydrogen storage alloy canister 21 is connected to the plastic soft bag 10 via a tube 13 connected to the bottle needle 12, a one-touch coupler 14 (socket 14a and plug 14b), a tube 15, a joint 16, a tube 18, a one-touch coupler 19 (socket 19a and plug 19b), and a tube 20.

With this connection, hydrogen gas from the hydrogen storage alloy canister 21 is pressurized into the plastic soft bag 10 through a supply path consisting of the tube 20, one-touch coupler 19, tube 18, fitting 16, tube 15, one-touch coupler 14, tube 13, and bottle needle 12 (step S2 in Figure 1).

Because a relief valve 17 is connected to the flow path of the fitting 16, the internal pressure in the flow path, i.e., the pressure inside the plastic soft bag 10, is maintained at a predetermined pressure, for example in the range of 0.02 MPa to 0.07 MPa (gauge pressure).

The hydrogen storage alloy canister 21 is a cylinder-shaped container that contains a hydrogen storage alloy that can reversibly absorb and release hydrogen at room temperature and low pressure through exothermic and endothermic reactions, and is generally available commercially (for example, manufactured by Japan Steel Works, Ltd. (JSW)). This hydrogen storage alloy canister 21 does not fall under the category of high-pressure gas as defined by the High Pressure Gas Safety Act, since the internal pressure of the hydrogen storage alloy can be no more than 1 MPa. Therefore, it can be easily used at organ removal sites such as hospitals. In addition, the size of the container can be designed to be as small as a few centimeters in diameter and about 10 cm in height. Furthermore, since the hydrogen storage alloy can be reused by reabsorbing hydrogen even after it has been consumed, there is an advantage in that costs can be reduced by repeated use. In this way, the hydrogen storage alloy canister 21 is lightweight and compact, yet can safely supply high-purity hydrogen gas.

Next, the one-touch coupler 14 is put into a disconnected state, and the plastic soft bag 10 is separated from the hydrogen storage alloy canister 21. Even in the disconnected state, the stop valve (valve) provided in the plug 14b of the one-touch coupler 14 is closed, so hydrogen gas does not leak from the plastic soft bag 10, and similarly, the stop valve (valve) provided in the plug 14a is closed, so hydrogen gas is not released from the hydrogen storage alloy canister 21 to the atmosphere.

In this state, the plug 14b of the one-touch coupler 14, the tube 13, the bottle needle 12, and the plastic soft bag 10 are vigorously shaken, for example, by hand, to dissolve more hydrogen gas into the liquid 10a in the plastic soft bag 10 (step S3 in FIG. 1). It is desirable to vibrate for 30 seconds or more.

Next, the plastic soft bag 10 is opened to the atmosphere (step S4 in FIG. 1). This opening is performed, for example, by connecting a socket (not shown) without a shutoff valve to the plug 14b.

Through the above process, a hydrogen-containing ETK solution containing hydrogen at the desired dissolved hydrogen concentration is generated in a very short time at the organ removal site inside the plastic soft bag 10. In order to suppress the expansion of the plastic soft bag 10, a cylindrical expansion suppression member may be provided on its outer periphery.

The extracted organs are then flushed out with the hydrogen-containing ETK solution thus produced (step S5 in FIG. 1). This flushing is performed with a portion of the hydrogen-containing ETK solution in the plastic soft bag 10.

Next, the rubber stopper side of the plastic soft bag 10 containing the remaining hydrogen-containing ETK liquid is placed on top, the top of the plastic soft bag is cut open with scissors, and the washed, removed organ is stored in the remaining hydrogen-containing ETK liquid inside the plastic soft bag, and the organ is transported to the transplant site (step S6 in Figure 1).

Next, at the transplant site, the excised organ is removed from the hydrogen-containing ETK solution and transplanted (step S7 in Figure 1).

As described above, according to this embodiment, the hydrogen gas from the hydrogen storage alloy canister 21, which stores hydrogen in the storage alloy, is dissolved in the ETK liquid, so that the hydrogen gas supply source can be safely and easily transported anywhere. Moreover, since the hydrogen storage alloy canister 21 is configured to be easily connected to the plastic soft bag 10, hydrogen gas can be easily and quickly injected into the plastic soft bag 10 at the organ removal site in an emergency. In addition, by vibrating the plastic soft bag 10 after hydrogen gas is injected, more hydrogen gas can be dissolved in the ETK liquid, and the hydrogen gas nanobubbles contained in the ETK liquid become more concentrated. Furthermore, by subsequently opening the plastic soft bag 10 to the outside air to reduce the internal pressure, not only can the ETK liquid in the plastic soft bag 10 be used to wash and preserve the organ for transplantation, but the hydrogen gas nanobubbles contained in the ETK liquid can be made to have a higher concentration. These hydrogen gas nanobubbles greatly contribute to improving the washing effect of the organ for transplantation.

Next, we will explain why it is desirable for the pressure (gauge pressure) of the hydrogen gas supplied to the plastic soft bag 10 to be a specified pressure in the range of 0.02 MPa to 0.07 MPa.

ヘンリーの法則によって計算した最大溶解量は、表２のようになる。
全内容量 ： １，８５０ ｍＬ
医療用液体量 ： １，０００ ｍＬ
初期空気容量 ： １５０ ｍＬ
注入ガス全体容量 ： ８５０ ｍＬ
液温 ： １５ ℃
振る時間 ： ３０ 秒間

The following test was conducted to determine the relationship between the hydrogen gas pressure and the dissolved hydrogen concentration inside a plastic soft bag, which is a flexible container that expands and contracts.
1. Test method 1 L of medical liquid (e.g., ETK solution) was placed into a plastic soft bag (made of a multi-layer film of polyethylene film and polypropylene film) with a total volume of 1,850 mL (space volume: 150 mL), and hydrogen gas was injected until the internal pressure (gauge pressure) reached 0.04 MPa, 0.05 MPa, and 0.06 MPa, and the dissolved hydrogen concentration of the solution was measured using a BIH-50D measuring device manufactured by Bionics Equipment Co., Ltd.
2. Test Results The following Table 1 shows the test results.

The maximum dissolution amount calculated by Henry's law is shown in Table 2.
Total content: 1,850 mL
Medical fluid volume: 1,000 mL
Initial air volume: 150 mL
Total gas volume: 850 mL
Liquid temperature: 15℃
Shaking time: 30 seconds

As shown in Table 2, when dissolving hydrogen gas in a medical liquid (e.g., ETK liquid), in order to obtain a dissolved hydrogen concentration of about 1.7 mg/L, it is desirable for the pressure of the injected hydrogen gas to be 0.02 MPa or more, and if the flexibility of the plastic soft bag is reduced to improve pressure resistance, for example, the maximum is 0.07 MPa. Therefore, it is desirable for the pressure of the hydrogen gas supplied to the plastic soft bag to be a predetermined pressure in the range of 0.02 MPa to 0.07 MPa.

Next, we will explain why it is desirable for the vibration time of the plastic soft bag to be 30 seconds or more.

ヘンリーの法則に従って演算すると、液体への水素ガスへの最大溶解量は２．４０ｍｇ／Ｌであることが別途求められ、この最大溶解量から、振った時間の効率差を求めると、次の表４のようになる。

この表４より、３０秒より多くの時間振り混ぜることで６９．６％以上の溶解効率が得られることが分かり、従って、プラスチックソフトバック１０の振動（振り混ぜ）を、３０秒間以上行うことが望ましい。 The following test was carried out to determine the desirable vibration time for a plastic soft bag, which is a flexible container capable of expanding and contracting.
1. Test method 1 L of tap water was placed in a plastic soft bag with a total volume of 1,550 mL, and the plastic soft bag was crushed until the volume of the remaining air was 50 mL. Hydrogen gas was then injected until the pressure inside the bag (gauge pressure) was 0.05 MPa, and the bag was sealed. The dissolved hydrogen concentration of the contents of the following items was measured using a BIH-50D measuring device manufactured by Bionics Instruments Co., Ltd.: one that was left unshaken for 1 hour, one that was shaken and mixed for 10 seconds, one that was shaken and mixed for 30 seconds, one that was shaken and mixed for 1 minute, and one that was shaken and mixed for 2 minutes. The water temperature was 10°C.
2. Test Results The test results are shown in Table 3.

When calculated according to Henry's law, the maximum dissolution amount of hydrogen gas into the liquid is separately determined to be 2.40 mg/L. When the efficiency difference depending on the shaking time is calculated from this maximum dissolution amount, it is as shown in Table 4 below.

From Table 4, it is clear that a dissolution efficiency of 69.6% or more can be obtained by shaking for more than 30 seconds, and therefore it is desirable to vibrate (shake) the plastic soft bag 10 for 30 seconds or more.

We examined the initial (when the bag was released) dissolved hydrogen concentration in the hydrogen-containing ETK liquid produced in this way and its change over time.

First, hydrogen gas dissolution was carried out on the UW solution, the HTK solution, and the ETK solution under the following conditions.
Total content: 1,850 mL
Contents: 1,000 mL
Initial air volume: 50 mL
Total gas volume: 850 mL
Liquid temperature: 1℃
Hydrogen gas pressure: 0.06 MPa
Vibration time: 1 minute Measurements were made by gas chromatography due to the low temperature. A Taiyo Trilyzer mBA-3000 gas chromatograph was used. The dissolved hydrogen concentration was then measured when the plastic soft bag containing the liquid was opened to the atmosphere, and the change over time in the dissolved hydrogen concentration when cooled was also measured. The measurement results are shown in Table 5 and Figure 3.

表５及び図３に示すように、同等の加圧下ではＵＷ液、ＨＴＫ液が高濃度水素を含有した。また、ＵＷ液、ＨＴＫ液、及びＥＴＫ液のいずれも常圧下、１℃保存で時間的な減衰は極めて緩徐であった。４時間の範囲であれば、１ｍｇ／Ｌ以上の溶存水素濃度は確保できた。

As shown in Table 5 and Figure 3, the UW solution and the HTK solution contained high concentrations of hydrogen under the same pressure. In addition, the UW solution, the HTK solution, and the ETK solution all showed a very slow decrease in hydrogen concentration over time when stored at normal pressure and 1°C. A dissolved hydrogen concentration of 1 mg/L or more could be ensured within a 4-hour period.

The effectiveness of using hydrogen-containing ETK solution produced by the method of this embodiment (hydrogen gas pressure 0.02 to 0.07 MPa, vibration time 30 seconds or more) at an organ harvesting site was investigated in a pig model of kidney transplantation from a non-circulating donor.

As a result, in a 30-minute DCD (donor cardiac arrest) model, this hydrogen-containing ETK solution was highly effective in flushing blood from the DCD kidney. Furthermore, in short-term observations (up to 5 days after surgery) after transplantation of DCD kidneys, transplanted organs stored in the conventional cold organ preservation solution had become non-functional (primary non-function) but after washing and preservation with this hydrogen-containing ETK solution, urinary excretion was observed. This method was therefore found to be a new technique for resuscitating damaged organs into transplantable organs that can be used safely and easily in clinical settings.

Next, the method and results of testing the efficacy and safety of the hydrogen-containing cold ETK solution produced by this embodiment in a kidney transplant model from DCD pigs will be described. Here, micro mini pigs (MMPs), which do not grow larger with age, were used to more closely reflect clinical practice. A kidney was created in an ischemic state with 30 minutes of circulatory arrest, and extracted as a donor kidney. It was then preserved using either a hydrogen-containing or non-hydrogen-containing ETK solution, and the recipient's renal function in the acute phase after transplantation was compared.

(Micro mini pigs used)
The MMP used was a miniature pig developed for laboratory use, weighing no more than 30 kg at most, specifically, female, aged 25-40 months and weighing 20-26 kg.

(Production of hydrogen-containing ETK solution and measurement of hydrogen concentration)
The hydrogen-containing ETK solution was prepared by cooling the ETK to 4°C and then filling it with hydrogen gas using the method described above immediately before perfusion. As a control, cold saline was placed on ice and the preservation solution was exposed to the atmosphere, and the hydrogen gas concentration in the hydrogen-containing ETK solution was measured over time by gas chromatography (using a Trilyzer mBA-3000 manufactured by Taiyo Co., Ltd.).

(Verification of the effectiveness of hydrogen content in DCD kidney flushing effect)
The donor was subjected to a midline abdominal incision under general anesthesia, and the left and right kidneys were freed. The chest was then opened, the thoracic aorta was blocked, and ischemia of all abdominal organs was induced. After creating a warm ischemic state for 30 minutes, the left and right kidneys were removed together to be used as donor kidneys. During this time, no anticoagulants such as heparin were added to the donor. The excised kidney was immediately placed on the back table and separated into left and right kidneys. One was perfused for 5 minutes with a hydrogen-containing cold ETK solution (temperature 4°C) produced by the method of this embodiment (hydrogen gas pressure 0.02 to 0.07 MPa, vibration time 30 seconds or more) and the other was perfused with a hydrogen-free cold ETK solution (temperature 4°C) produced without adding hydrogen gas, at a natural drip drop of 1 m. During the perfusion, the dripping speed of the hydrogen-containing cold ETK solution and the hydrogen-free cold ETK solution was counted. Table 6 shows the perfusion rates of cold ETK solution containing hydrogen and cold ETK solution not containing hydrogen, expressed as counts per minute.

また、図４は、Ａは水素含有冷ＥＴＫ液を灌流した摘出ＤＣＤ腎臓、Ｂは水素非含有冷ＥＴＫ液を灌流した摘出ＤＣＤ腎臓をそれぞれ示している。

FIG. 4A shows an isolated DCD kidney perfused with cold hydrogen-containing ETK solution, and FIG. 4B shows an isolated DCD kidney perfused with cold hydrogen-free ETK solution.

As can be seen from Table 6, when the isolated DCD kidney was perfused with hydrogen-containing cold ETK solution, the natural drip perfusion rate was faster from the beginning of the perfusion compared to the case of hydrogen-free cold ETK solution. Also, as can be seen from Figure 4, macroscopic findings of the perfusion area at the end of the perfusion showed that a larger amount of hydrogen-containing cold ETK solution was perfused than that of hydrogen-free cold ETK solution.

(Evaluation in a kidney transplant model)
As the donor kidney, a kidney that had been stopped for 30 minutes was used, as in the above-mentioned case. The kidney separated into the left and right sides was simply immersed and preserved in hydrogen-containing cold ETK solution and normal hydrogen-free cold ETK solution, and was preserved for the time until it was put into the recipient (preserved for 1 to 4 hours). The hydrogen-containing cold ETK solution was produced by the production method of this embodiment, and had a dissolved hydrogen concentration of 1 mg/L or more. The recipient was first subjected to a midline abdominal incision after general anesthesia, and the area around the left renal artery and vein of the recipient was exposed. After intravenous administration of 1 cc of heparin, the abdominal aorta around the renal vein was totally clamped. A bulldog clamp was placed on the recipient renal vein, and the left kidney of the recipient was excised in a Carrel patch shape from the base of the renal artery. The donor kidney that had been preserved was put in, and the renal vein at the Carrel patch part was anastomosed end-to-side with a continuous 5-0 nylon. After the arterial anastomosis, the peripheral renal vein was clipped again, and the total blockage of the abdominal aorta was released. The total occlusion time of the abdominal aorta was set to 30 minutes. The renal vein was then anastomosed end-to-end in succession using 6-0 nylon, and finally the ureter was sutured with 6-0 interrupted sutures. After reflow, the blood flow of the transplanted kidney was confirmed, the right kidney was removed, and the abdomen was closed. Antibiotics were administered on the day of surgery, and thereafter, drinking water and eating were allowed ad libitum. The animals were observed for 6 days after surgery, sacrificed, and peripheral blood, bladder urine, and transplanted kidney were sampled. BUN and Cre in peripheral blood and urine were measured by the GLDH method and the enzyme method, respectively. Urinary TP was measured by the Pigallolette method, electrolytes by the ion-selective electrode method, IP by the enzyme method, and GLU by the HK-G6PDH method. The transplanted kidney was fixed in 10% neutral buffered formaldehyde solution and cut longitudinally to include the papilla with the cortex at the center. The tissue was embedded in a conventional manner, thin sections were prepared, and stained with Hematoxylin Eosin (H.E.) and Elastica van Gieson (EVG) for pathological examination. The transplanted kidney was scored according to the Banff classification. The results are shown in Table 7.

All transplanted minipigs survived until the observation period (6 days after the transplant). At the time of sacrifice, in all cases of the group treated with non-hydrogen-containing ETK solution, no urine was observed in the bladder, and no blood flow was observed in the transplanted kidney. In contrast, in the group treated with hydrogen-containing ETK solution, blood flow was observed in the transplanted kidney and urine was observed in the bladder, even in the case of the group treated with hydrogen-containing ETK solution, which had been preserved for 4 hours. As can be seen from Table 7, the average BUN value in the peripheral blood collected was 143 (mg/dL) in the hydrogen-containing ETK solution group and 270 (mg/dL) in the non-hydrogen-containing ETK solution group, and the average Cre value was 15.5 (mg/dL) in the hydrogen-containing ETK solution group and 24 (mg/dL) in the non-hydrogen-containing ETK solution group. Analysis of the urine collected from the hydrogen group showed acute glomerular damage.

Further pathological analysis revealed that kidneys preserved with the non-hydrogen-containing ETK solution group had renal cortical necrosis due to acute renal failure. Figure 5 shows images of DCD kidneys from the non-hydrogen-containing ETK solution group, with (A) and (B) showing an example of subcapsular necrosis, and (C) and (D) showing an example of necrosis near the corticomedullary junction. However, (A) and (C) are low-magnification (40x) images, while (B) and (D) are high-magnification (400x) images.

As shown in Figure 5 (A), most of the observed area was necrotic, and pan-renal cortical necrosis was diagnosed. As shown in Figure 5 (B), the capsule and the cell nuclei of the tubules directly below were preserved in some cases, but the cytoplasm of these tubules had undergone degeneration and necrosis. Furthermore, as shown in Figures (C) and (D), the interstitium contained a large number of lymphocytes, monocytes, and neutrophils as infiltrated cellular debris.

In contrast, kidneys preserved with hydrogen-containing ETK solution showed acute tissue damage but maintained blood flow. Figure 6 shows images of DCD kidneys in the hydrogen-containing ETK solution group, with (A) and (B), (C) and (D), and (E) and (F) showing three examples of subcapsular lesions. However, (A), (C), and (E) are low-magnification (40x) images, while (B), (D), and (F) are high-magnification (400x) images.

In the examples of Figures 6 (A) and (B), tubular dilation and cellular infiltration were observed at low magnification (A). At high magnification (B), infiltration of mononuclear cells and blockage of the loop were observed in the glomeruli. In addition, tubulitis due to infiltration of numerous mononuclear cells and lymphocytes was observed in the interstitium of the tubules. In the examples of Figures 6 (C) and (D), bleeding and tubular dilation were observed at low magnification (C). At high magnification (D), cellular infiltration of the tubular interstitium was less than that in the example of Figure 6 (B). In the examples of Figures 6 (E) and (F), many tubules were atrophied and dilated at low magnification (E), and some tubules had hyaline casts. At high magnification (F), infiltration of numerous mononuclear cells and lymphocytes was observed in the interstitium of the tubules.

(Evaluation of the chronic phase of kidney transplantation model)
The kidney transplant was performed in the same manner as the kidney transplantation described above. However, as the donor kidney, a kidney that had been subjected to 20 minutes of circulatory arrest was used. The kidney was washed with hydrogen-free cold ETK solution and hydrogen-containing cold ETK solution, respectively, and then simply immersed and preserved in hydrogen-free cold ETK solution and hydrogen-containing cold ETK solution, respectively. A kidney preserved in hydrogen-free cold ETK solution for 1 hour (without hydrogen), a kidney preserved in hydrogen-containing cold ETK solution for 1 hour (with hydrogen 1h), and a kidney preserved in hydrogen-containing cold ETK solution for 4 hours (with hydrogen 4h) were put into the recipient, respectively. The hydrogen-containing cold ETK solution was produced by the production method of the present invention and has a dissolved hydrogen concentration of 1 mg/L or more. An immunosuppressant was administered after surgery. The immunosuppressant and its dosage and administration were as follows. Tacrolimus (Prograf): 0.15-0.30 mg/kg, oral, every 12 hours, twice a day, mycophenolate mofetil: 500 mg/head, oral, twice a day, prednisolone: 0.5-2 mg/kg, intravenous or oral, twice a day. Thereafter, water and food were allowed ad libitum. Peripheral blood BUN and Cre were measured by the GLDH method and the enzyme method, respectively, before surgery, immediately after surgery, and 1-14 days after surgery. The results are shown in Table 8 and Figure 7, and Table 9 and Figure 8.

As can be seen from Table 8 and Figure 7, as well as Table 9 and Figure 8, in the miniature pigs transplanted with kidneys preserved in normal ETK solution without hydrogen (non-containing), the BUN and Cre increased simply with the number of days after surgery, but in the kidneys preserved in cold hydrogen-containing ETK solution with a dissolved hydrogen concentration of 1 mg/L or more that can be produced by the production method of the present invention, both the BUN and Cre began to decrease after 4 days after surgery, and renal function was restored. In other words, it was found that kidneys preserved in cold hydrogen-containing ETK solution with a dissolved hydrogen concentration of 1 mg/L or more produced by the production method of the present invention significantly recovered renal function in the chronic phase after transplantation when immunosuppressants were administered.

(summary)
It has been known that hydrogen gas is effective in preventing ischemia-reperfusion injury, but high-concentration hydrogen gas is flammable, and its clinical use is problematic in terms of safety. The inventors of the present application have demonstrated that inhalation of hydrogen gas is effective in ischemia-reperfusion injury after coronary artery intervention in a physician-initiated clinical trial at a well-equipped hospital. However, in clinical settings where donors suddenly occur, it is very difficult to prepare organ preservation solutions filled with hydrogen in advance or to bring hydrogen gas cylinders to the site. In addition, the method of generating hydrogen gas by electrolysis in organ preservation solutions lacks convenience in clinical settings. In contrast, according to the present invention, focusing on hydrogen storage alloy equipment, hydrogen gas can be easily and quickly filled in clinical settings to a gauge pressure of about 0.06 MPa in flexible containers for existing organ preservation solutions.

To verify the present invention, hydrogen gas was filled into an existing organ preservation solution, and the initial dissolved hydrogen gas concentration and its change over time were measured. It was found that the organ preservation solution is very easy to contain hydrogen at low temperatures and can maintain hydrogen gas for a long time. In addition, the flush-out effect in DCD kidneys was verified using ETK solution, which is an extracellular fluid type organ preservation solution. It was found that ETK solution has a lower viscosity than intracellular fluid type UW solution, but is perfused at a higher flow rate from the initial perfusion compared to when it does not contain hydrogen gas. From the tissue analysis of the perfusion area, it was estimated that the expansion of the capillary system such as glomeruli and the washing out effect of microthrombi were observed. As a mechanism, the effect of hydrogen gas on ischemia-reperfusion injury assuming pig kidney transplantation was verified in an actual kidney transplant. In a DCD 30-minute model with aged MMP used in this model, all cases with non-hydrogen-containing ETK solution were PNF, whereas blood flow was maintained and urine was produced with hydrogen-containing ETK solution. As mentioned above, it was also verified that the transplanted kidney functions extremely well not only during the acute postoperative period, but also during the chronic period under the administration of immunosuppressants. The effects of the hydrogen-containing organ preservation solution of the present invention are expected to make kidneys that were previously untransplantable possible to transplant, by combining the rinsing effect during surgery, the administration of immunosuppressants during and after surgery, and auxiliary cell therapy such as MSC.

The above-described embodiments and examples are merely illustrative of the present invention and are not limiting, and the present invention can be implemented in various other modified and altered forms. Therefore, the scope of the present invention is defined only by the claims and their equivalents.

10 Plastic soft bag 10a ETK liquid 11 Rubber stopper 12 Bottle needle 13, 15, 18, 20 Tube 14, 19 One-touch coupler 14a, 19a Socket 14b, 19b Plug 16 Joint 17 Relief valve 21 Hydrogen storage alloy canister

Claims (4)

At the site of removal of an organ for transplantation, hydrogen gas is injected from a hydrogen storage alloy canister into a flexible container containing an organ preservation solution, the flexible container is vibrated to dissolve the hydrogen gas in the organ preservation solution, and the flexible container is opened to the outside air to reduce the internal pressure, thereby producing a hydrogen-containing organ preservation solution containing a predetermined concentration of dissolved hydrogen in the flexible container and used for washing and/or preserving an organ for transplantation ,
The method for producing a hydrogen-containing organ preservation solution , wherein the predetermined concentration is a concentration at which the dissolved hydrogen concentration after 4 hours is 1.0 mg/L or more . The method for producing hydrogen-containing organ preservation solution according to claim 1, characterized in that the hydrogen gas is supplied from the hydrogen storage alloy canister to the flexible container until the pressure of the hydrogen gas reaches a predetermined pressure. The method for producing hydrogen-containing organ preservation solution according to claim 2, characterized in that the predetermined pressure is in the range of 0.02 MPa to 0.07 MPa. The method for producing hydrogen-containing organ preservation solution according to any one of claims 1 to 3, characterized in that the flexible container is vibrated for 30 seconds or more.