CN111357737B

CN111357737B - Normal-temperature mechanical organ perfusate and application thereof

Info

Publication number: CN111357737B
Application number: CN202010164242.XA
Authority: CN
Inventors: 游可为; 史国营; 陈浩源
Original assignee: Runfang Beijing Biotechnology Co ltd; Redpharm Beijing Biomedical Research Institute Co ltd
Current assignee: Redpharm Beijing Biomedical Research Institute Co ltd; Runfang Beijing Biotechnology Co ltd
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2021-10-15
Anticipated expiration: 2040-03-11
Also published as: CN111357737A

Abstract

The invention relates to a normal-temperature mechanical organ perfusate and application thereof, and particularly provides application of an isolated organ perfusate in preparation of a medicament for preserving an isolated organ for transplantation, wherein the isolated organ perfusate consists of the following substances based on the total volume of each 1000 mL: the isolated organ perfusate comprises, per 10g of polymeric hemoglobin: 10g of high molecular polymer hemoglobin, 2.5-4g of glucose, 7500-10000u of heparin sodium, 2-3g of sodium chloride, 0.5-0.75g of cefoxitin sodium, 0.37-0.5g of sodium bicarbonate, 15-20mL of 10% compound amino acid injection, 0.05-0.08mL of 12 kinds of compound vitamin for injection, 50-80u of insulin and the balance of water. The perfusate can perform the functions of oxygen carrying and releasing for a longer time, thereby obviously prolonging the perfusion preservation time of isolated organs.

Description

Normal-temperature mechanical organ perfusate and application thereof

Technical Field

The invention relates to the field of medical instruments, in particular to a normal-temperature mechanical organ perfusate and application thereof.

Background

Many human diseases are caused by necrosis of cells, tissues and organs, thereby causing functional loss and causing disability and even death of human bodies, and with the development of modern medicine, organ transplantation technology is mature day by day, and the following diseases are caused only from the surgical view: the organ transplantation technology is perfect, and head replacement surgery is not a big problem. In fact, organ transplantation still faces various difficulties, and preservation of isolated organs is one of them. Organ transplantation requires the transplantation of a living organ, and therefore, it is a prerequisite for successful transplantation that the organ for transplantation maintains intact anatomy and viability from the time of excision from the donor until its principal blood vessel is connected to the recipient's blood vessel. However, once any organ loses its blood supply, the cells do not receive the necessary oxygen and nutrients and die in a short period of time at normal temperature. The tolerable time is very short, such as 10-15 minutes for heart and liver and 45-60 minutes for kidney, and if the time is exceeded, it is difficult to restore function after transplantation. In clinical practice, it is required that the ischemia time at normal temperature is as short as possible, preferably not longer than 3-5 minutes, and at most not longer than 7-8 minutes. However, it is not possible at all to complete the transplantation procedure in such a short time. Therefore, it is necessary to try to maintain the activity of the isolated organ for a long time.

In order to prolong the preservation time of isolated organs, scientists have studied various methods, wherein cryopreservation is a method which is commonly used at present, but the cryopreservation can effectively prolong the preservation time of organs, and the damage to cells can be obvious when the cryopreservation is carried out. When metabolism is inhibited, ATP supply is not available to Na pump, and intracellular proteins produce oncotic pressure, which leads to cell edema. Intracellular proteins and non-permeable anions generate osmotic tension of about 110-140 mOsm/kg. When the Na pump activity is inhibited by low-temperature ischemia, the cell membrane potential is lowered. Na and Cl enter the cell due to concentration gradient, and in order to maintain water balance inside and outside the semipermeable membrane, intracellular water is accumulated to cause cell edema.

In addition, the preservation of isolated organs is prolonged by adopting a perfusion mode, and the currently commonly used perfusion solutions comprise Krebs-Henseleit solution, St Thomas II solution, UW solution, Celsior solution and the like, but the perfusion solutions lack oxygen carrying agents, so that the perfused organs are always in an anoxic state. If the in vitro perfusate can not effectively supply oxygen to the isolated organ, the tissues and cells can be subjected to anaerobic glycolysis increased due to oxygen deficiency, and the adverse effects of acidosis and lysosomal enzyme activation are caused. Therefore, ensuring oxygen supply, maintaining minimal energy metabolism, and removing metabolic waste products are issues that need to be addressed in organ preservation in vitro.

Meanwhile, organs preserved under the condition of hypoxia cause significant secondary damage in the process of transplantation and reperfusion, the mechanism of restoring blood flow to aggravate the damage of the preserved organs is not completely understood, and is generally considered to be related to the damage effect of oxygen free radicals, and under the normal condition, the metabolite hypoxanthine of ATP is rapidly converted into purine by Xanthine Oxidase (XOD) so as to be converted into the intermediate product uric acid. However, in severe hypoxia, the high-energy bonds are quickly depleted, the hypoxanthine content increases, and XOD is converted into an enzyme that produces peroxide. At the same time, hypoxia inactivates or depletes endogenous antioxidants such as superoxide dismutase (SOD), which can produce excessive free radicals to damage cells when the blood circulation reestablishes oxygen supply suddenly.

Therefore, it is important to add an oxygen-carrying agent to the organ perfusate. Erythrocytes are natural oxygen carriers in animals and have been used directly by humans for organ perfusion preservation in vitro. However, erythrocytes are subject to rupture and extravasation of intramembranous aminophospholipids, endotoxins, glycophorins and intracellular enzymes can damage vascular endothelium and myocardial cells and cause capillary obstruction.

The adoption of stroma-free hemoglobin to replace red blood cells is a new idea. Research and development of artificial polymerized hemoglobin has been made abroad around the 60 s of the 20 th century. Hemoglobin is subjected to chemical modification means such as intramolecular cross-linking, polymerization, covalent connection of macromolecules and the like to form various hemoglobin derivatives. In the former patent (201910846580.9) of this company, high-activity oxygen-carrying hemoglobin is obtained by glutaraldehyde polymerization, and added into organ perfusate to satisfy the oxygen required for normal temperature metabolism of organs, so as to solve the problem that organs cannot be preserved at normal temperature for a long time due to hypoxia, improve the normal temperature preservation time of organs, but sometimes still cannot satisfy the requirement of actual clinical preservation time, for example, when donors and patients who accept the donors are in different cities far away, the donor and the patient who accept the donors are inconsistent in time, and the like, organs are likely to be unable to be transplanted due to inactivation, and this time limit becomes a big reason of donor organ shortage crisis, and many patients are difficult to wait for transplantation.

Therefore, there is a need to further increase the time of cold storage of isolated organs.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

on the other hand, the inventors have found that when the molecular weight distribution of the high molecular weight polymerized hemoglobin is different, the high molecular weight polymerized hemoglobin contains different molecular weights The perfusate of the invention of distributed high molecular polymer hemoglobin has obvious and unpredictable preservation effect difference on isolated organs, specifically, the method comprises the following steps: the inventors found that the molecular weight of the hemoglobin is higher than that of the high molecular weight polymerized hemoglobin of the invention with the molecular weight of 320kD to 1024kD If the molecular weight of the molecular polymerized hemoglobin is less than 320kD (such as 32 kD-256 kD or 128 kD-256 kD), the perfusate is dissociated The preservation safety time limit of the body organ is reduced by more than one time, and the safety time limit is obviously shortened; and for polymerized blood larger than 1024kD The amount of the red egg albumin which can be obtained in the actual production is very small, and the red egg albumin has no research significance basically. Thus, the invention encompasses molecules The perfusate of high molecular polymerized hemoglobin with the weight of 320kD-1024kD has obvious perfusion preservation effect on isolated organs Safety time limit is advantageous.

On the other hand, the inventors have unexpectedly found that the perfusion fluid of the present invention contains a polymer having a molecular weight of 320kD to 1024kD When the purity of the polymerized hemoglobin is different, the preservation effect of the perfusate on isolated organs is also obviously different and unpredictable, and the perfusate has In terms of a body, the method comprises the following steps: the inventors have surprisingly found that the perfusate of the invention has a safe time limit for preserving isolated organs with molecular weight The purity of the high molecular polymerized hemoglobin with the purity of 320kD-1024kD is increased in a fluctuating way. In particular, the molecular weight is 320kD When the purity of the 1024kD macromolecular polymerized hemoglobin is increased from 65 percent to 80 percent and is further increased to 90 percent, the perfusate The preservation safety time limit of the isolated organ is basically kept unchanged or even reduced; however, the purity of the high-molecular polymerized hemoglobin When the temperature is increased to 95 percent, the safe time limit of the perfusate for preserving the isolated organ is obviously prolonged by about 50 percent, and the macromolecule When the purity of the polymerized hemoglobin is increased from 95 percent to 97 percent, 99 percent or even higher, the perfusate protects the isolated organ The safe time period remains substantially constant and does not change. Therefore, the invention contains high molecular weight of 320-1024kD ≥ 95% The perfusion fluid of the molecular polymerization hemoglobin has obvious safety time limit advantages for perfusion preservation of isolated organs.

In conclusion, the high molecular weight polymerized hemoglobin (the molecular weight is 320kD-1024kD is more than or equal to 95 percent) prepared by the invention The perfusate can exert the functions of oxygen carrying and oxygen releasing for a longer time, obviously prolong the perfusion preservation time of isolated organs, and obtain the expectation The technical effect which cannot be achieved makes a substantial contribution to the prior art.

To this end, in a first aspect of the invention, the invention provides the use of an ex vivo organ perfusate in the preparation of a medicament for preserving an ex vivo organ for transplantation, wherein the ex vivo organ perfusate consists of, on a total volume per 1000 mL:

the isolated organ perfusate comprises, per 10g of polymeric hemoglobin:

10g of high-molecular polymer hemoglobin is obtained,

glucose 2.5-4g (such as 3g or 3.5g),

7500u-10000u (8000u, 8333u, 8500u, 9000u or 9500u) of heparin sodium,

2g to 3g (such as 2.3g, 2.5g or 2.7g) of sodium chloride,

0.5g-0.75g (such as 0.55g, 0.6g, 0.65g, 0.66g or 0.7g) of cefoxitin sodium,

sodium bicarbonate 0.37-0.5g (such as 0.375g, 0.4g, 0.42g, 0.45g, 0.47g or 0.49g),

15mL-20mL (such as 16.5mL, 16.7mL, 17mL, 18mL, 19mL or 19.5mL) of 10% compound amino acid injection,

0.05mL-0.08mL (such as 0.055mL, 0.06mL, 0.065mL, 0.066mL, 0.07mL or 0.075mL) of the 12 kinds of compound vitamins for injection,

insulin 50u-80u (e.g., 55u, 60u, 65u, 70u, or 75u), and

the balance of water;

wherein the content of polymerized hemoglobin with the molecular weight of 320kD-1024kD in the high-molecular polymerized hemoglobin is not less than 95%, and the high-molecular polymerized hemoglobin is obtained by the following method:

collecting 1L of fresh blood, and diluting with 6g/L sodium citrate solution with the volume of 0.5-1 time of the blood;

filtering the diluted blood with a 60 μm depth filter and washing the residue on the 60 μm depth filter with 6g/L sodium citrate solution until the hemoglobin passing through the filter reaches 95% or more;

placing the substances passing through the 60 μm depth filter on a 0.65 μm hollow fiber membrane, and washing the substances on the 0.65 μm hollow fiber membrane with 6g/L sodium citrate solution 5-8 times the original blood volume so that the substances smaller than 0.65 μm pass through the 0.65 μm hollow fiber membrane;

adding injection water into a substance intercepted by a 0.65-micron hollow fiber membrane according to the initial blood volume of 1:1-2 to crack red blood cells, carrying out ultrafiltration on the cracked red blood cells by using a 100KD filter membrane, wherein the permeation end of the red blood cells is required hemoglobin, simultaneously adding the injection water according to the permeation rate to keep constant ultrafiltration volume until the yield of the hemoglobin is more than or equal to 95%, and stopping 100KD ultrafiltration;

concentrating the hemoglobin purified by the 100KD filter membrane by using a 30KD membrane package until the concentration of the hemoglobin is 10-14 g/dL;

purifying the purified hemoglobin with the concentration of 10-14g/dL by anion chromatography (20mM tris solution equilibrium column → 40min up sample → 3 times of purified hemoglobin volume of 10-14g/dL 29mM tris solution elution → 4-5 times of purified hemoglobin volume of 10-14g/dL 50mM tris solution elution) to obtain purified hemoglobin;

placing the purified hemoglobin on a 30KD filter membrane, and replacing the solution with 50mM disodium hydrogen phosphate buffer solution with the volume 3 times that of the purified hemoglobin;

then introducing inert gas to deoxidize until the oxyhemoglobin is less than 5%, adding glutaraldehyde (see the prior patent: 201910846580.9 specifically) in an atomization method according to the proportion of 1g of hemoglobin to 35-45mg of glutaraldehyde, and terminating the polymerization reaction according to 1g of sodium borohydride with 13-18mg of hemoglobin;

concentrating the obtained polymerized hemoglobin to 6-7g/dL, placing the polymerized hemoglobin concentrated to 6-7g/dL on an ultrafiltration membrane bag with 300KD, changing the solution by using lactated ringer's solution (6.73 g/L sodium chloride, 0.3g/L potassium chloride, 0.2g/L calcium chloride dihydrate and 3.07g/L, N-acetyl-L-cysteine 22g/L) with the volume 5-10 times that of the polymerized hemoglobin with 6-7g/dL to obtain the polymerized hemoglobin, collecting the protein solution when the protein content with the molecular weight of 320-1024kD reaches an index of more than or equal to 95 percent in the solution changing process, introducing inert gas to deoxidize until the oxygenated hemoglobin content is less than or equal to 5 percent, and filtering and sterilizing by 0.2 mu m to obtain the high molecular polymerized hemoglobin.

In some embodiments, the polymeric hemoglobin having a molecular weight of 320kD to 1024kD has a polymeric hemoglobin content of 95% to 99% (e.g., 95%, 97%, or 99%); correspondingly, the index of the protein content of 320kD-1024kD is adaptively changed in the preparation method.

In some embodiments, the ex vivo organ perfusate consists of, based on a total volume per 1000 mL:

30g of high-molecular polymer hemoglobin,

9g of glucose is added into the mixture,

the content of the heparin sodium is 25000u,

7g of sodium chloride, namely 7g of sodium chloride,

2g of cefoxitin sodium, namely 2g,

sodium hydrogen carbonate (1.25 g),

50mL of 10 percent compound amino acid injection,

0.2mL of 12 kinds of compound vitamin for injection,

insulin 180u, and

the balance of water.

40g of high-molecular polymer hemoglobin is prepared,

10g of glucose is added into the mixture,

the heparin sodium is 30000u,

8g of sodium chloride, namely 8g of sodium chloride,

3g of cefoxitin sodium, namely 3g of cefoxitin sodium,

1.5g of sodium bicarbonate is added,

60mL of 10 percent compound amino acid injection,

0.3mL of 12 kinds of compound vitamin for injection,

insulin 200u, and

the balance of water.

20g of high-molecular polymer Hemoglobin (HMP),

the glucose content of the mixture is 8g,

the heparin sodium is 20000u,

6g of sodium chloride, namely 6g of sodium chloride,

1g of cefoxitin sodium, namely 1g,

1g of sodium bicarbonate, namely sodium bicarbonate,

40mL of 10 percent compound amino acid injection,

0.1mL of 12 kinds of compound vitamin for injection,

insulin 160u, and

the balance of water.

In some embodiments, the preservation is performed at 2-40 ℃ (e.g., 2 ℃, 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃ or 40 ℃).

In some embodiments, the preservation is performed at 4-37 deg.C (e.g., 4 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C).

In some embodiments, the preservation is performed at 10-30 ℃ (e.g., 10 ℃, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 25 ℃, or 30 ℃).

In some embodiments, the storing is performed at 16-20 ℃ (e.g., 18 ℃).

In some embodiments, the storing is performed at 18 ℃.

In some embodiments, the cefoxitin sodium is cefoxitin sodium for injection.

In some embodiments, the water is injection water.

In some embodiments, the 10% compound amino acid injection is a 10% compound amino acid injection produced by chenxin pharmaceutical industry.

In some embodiments, the 12 multivitamins for injection are "12 multivitamins for injection" manufactured by shanxi prader pharmaceuticals, inc.

In some embodiments, the isolated organ is a heart, liver, kidney, or lung.

In some embodiments, the isolated organ is a human, dog, rat, or pig isolated organ.

In a second aspect of the invention, the invention provides an ex vivo organ perfusate consisting of, based on a total volume per 1000 mL:

the isolated organ perfusate comprises, per 10g of polymeric hemoglobin:

10g of high-molecular polymer hemoglobin is obtained,

glucose 2.5-4g (such as 3g or 3.5g),

7500u-10000u (8000u, 8333u, 8500u, 9000u or 9500u) of heparin sodium,

2g to 3g (such as 2.3g, 2.5g or 2.7g) of sodium chloride,

0.5g-0.75g (such as 0.55g, 0.6g, 0.65g, 0.66g or 0.7g) of cefoxitin sodium,

insulin 50u-80u (e.g., 55u, 60u, 65u, 70u, or 75u), and

the balance of water;

filtering blood with 60 μm deep filter to obtain erythrocytes; washing the red blood cells with a 0.65 μm hollow fiber membrane; lysing the washed erythrocytes with a hypotonic solution to obtain a lysate; ultrafiltering the cleavage product by 100KD to obtain crude pure hemoglobin; purifying the crude hemoglobin by anion exchange chromatography to obtain pure hemoglobin; carrying out polymerization reaction on glutaraldehyde and the purified hemoglobin in an atomized manner; terminating the polymerization reaction by using sodium borohydride, and carrying out 300KD ultrafiltration liquid exchange to obtain the polymerized hemoglobin, wherein the index requirements of the polymerized hemoglobin are as follows: the molecular weight of 320kD-1024kD is more than or equal to 95 percent, the methemoglobin is less than 5 percent, and the oxyhemoglobin is less than 5 percent.

In some embodiments, the high molecular weight polymerized hemoglobin is obtained by:

In some embodiments, the ex vivo organ perfusate consists of, based on a total volume per 1000 mL: 20g to 40g (such as 25g, 30g or 35g) of high molecular weight polymerized hemoglobin, 8g to 10g (such as 8.5g, 9g or 9.5g) of glucose, 20000u to 30000u (23000u, 25000u or 27000u) of heparin sodium, 6g to 8g (such as 6.5g, 7g or 7.5g) of sodium chloride, 1g to 3g (such as 1.5g, 2g or 2.5g) of cefoxitin sodium, 1g to 1.5g (such as 1.0g, 1.25g or 1.5g) of sodium bicarbonate, 40mL to 60mL (such as 45mL, 50mL or 55mL) of 10% compound amino acid injection, 0.1mL to 0.3mL (such as 0.15mL, 0.2mL or 0.25mL) of 12 multivitamins for injection, 160u to 200u (such as 170u, 180u or 190u) of insulin and the balance of water.

30g of high-molecular polymer hemoglobin,

9g of glucose is added into the mixture,

the content of the heparin sodium is 25000u,

7g of sodium chloride, namely 7g of sodium chloride,

2g of cefoxitin sodium, namely 2g,

sodium hydrogen carbonate (1.25 g),

50mL of 10 percent compound amino acid injection,

0.2mL of 12 kinds of compound vitamin for injection,

insulin 180u, and

the balance of water.

40g of high-molecular polymer hemoglobin is prepared,

10g of glucose is added into the mixture,

the heparin sodium is 30000u,

8g of sodium chloride, namely 8g of sodium chloride,

3g of cefoxitin sodium, namely 3g of cefoxitin sodium,

1.5g of sodium bicarbonate is added,

60mL of 10 percent compound amino acid injection,

0.3mL of 12 kinds of compound vitamin for injection,

insulin 200u, and

the balance of water.

20g of high-molecular polymer Hemoglobin (HMP),

the glucose content of the mixture is 8g,

the heparin sodium is 20000u,

6g of sodium chloride, namely 6g of sodium chloride,

1g of cefoxitin sodium, namely 1g,

1g of sodium bicarbonate, namely sodium bicarbonate,

40mL of 10 percent compound amino acid injection,

0.1mL of 12 kinds of compound vitamin for injection,

insulin 160u, and

the balance of water.

In some embodiments, the cefoxitin sodium is cefoxitin sodium for injection.

In some embodiments, the water is injection water.

In some embodiments, the isolated organ is a heart, liver, kidney, or lung.

In a third aspect of the invention, the invention provides an ex vivo organ perfusate consisting of, per 1000mL of total volume: 20g to 40g (such as 25g, 30g or 35g) of high molecular weight polymerized hemoglobin, 8g to 10g (such as 8.5g, 9g or 9.5g) of glucose, 20000u to 30000u (23000u, 25000u or 27000u) of heparin sodium, 6g to 8g (such as 6.5g, 7g or 7.5g) of sodium chloride, 1g to 3g (such as 1.5g, 2g or 2.5g) of cefoxitin sodium, 1g to 1.5g (such as 1.0g, 1.25g or 1.5g) of sodium bicarbonate, 40mL to 60mL (such as 45mL, 50mL or 55mL) of 10% compound amino acid injection, 0.1mL to 0.3mL (such as 0.15mL, 0.2mL or 0.25mL) of 12 complex vitamins, 160u to 200u (such as 170u, 180u or 190u) of insulin and the balance of water;

wherein the content of polymerized hemoglobin with the molecular weight of 320kD-1024kD in the high-molecular polymerized hemoglobin is not less than 95 percent, and the high-molecular polymerized hemoglobin is obtained by the following method:

In some embodiments, the ex vivo organ perfusate includes, per 10g of high molecular weight polymerized hemoglobin:

10g of high-molecular polymer hemoglobin is obtained,

glucose 2.5-4g (such as 3g or 3.5g),

7500u-10000u (8000u, 8333u, 8500u, 9000u or 9500u) of heparin sodium,

2g to 3g (such as 2.3g, 2.5g or 2.7g) of sodium chloride,

0.5g-0.75g (such as 0.55g, 0.6g, 0.65g, 0.66g or 0.7g) of cefoxitin sodium,

insulin 50u-80u (e.g., 55u, 60u, 65u, 70u, or 75u), and

the balance of water.

30g of high-molecular polymer hemoglobin,

9g of glucose is added into the mixture,

the content of the heparin sodium is 25000u,

7g of sodium chloride, namely 7g of sodium chloride,

2g of cefoxitin sodium, namely 2g,

sodium hydrogen carbonate (1.25 g),

50mL of 10 percent compound amino acid injection,

0.2mL of 12 kinds of compound vitamin for injection,

insulin 180u, and

the balance of water.

40g of high-molecular polymer hemoglobin is prepared,

10g of glucose is added into the mixture,

the heparin sodium is 30000u,

8g of sodium chloride, namely 8g of sodium chloride,

3g of cefoxitin sodium, namely 3g of cefoxitin sodium,

1.5g of sodium bicarbonate is added,

60mL of 10 percent compound amino acid injection,

0.3mL of 12 kinds of compound vitamin for injection,

insulin 200u, and

the balance of water.

20g of high-molecular polymer Hemoglobin (HMP),

the glucose content of the mixture is 8g,

the heparin sodium is 20000u,

6g of sodium chloride, namely 6g of sodium chloride,

1g of cefoxitin sodium, namely 1g,

1g of sodium bicarbonate, namely sodium bicarbonate,

40mL of 10 percent compound amino acid injection,

0.1mL of 12 kinds of compound vitamin for injection,

insulin 160u, and

the balance of water.

In some embodiments, the cefoxitin sodium is cefoxitin sodium for injection.

In some embodiments, the water is injection water.

In some embodiments, the isolated organ is a heart, liver, kidney, or lung.

In a fourth aspect of the invention, the invention provides the use of the ex vivo organ perfusate described above in the preparation of a medicament for preserving an ex vivo organ for transplantation.

In some embodiments, the storing is performed at 16-20 ℃ (e.g., 18 ℃).

In some embodiments, the storing is performed at 18 ℃.

In some embodiments, the isolated organ is a heart, liver, kidney, or lung.

In a fifth aspect of the present invention, there is provided a method for preserving an isolated organ for transplantation, the method comprising: perfusing the organ with the isolated organ perfusate.

In some embodiments, the method further comprises: immersing the organ after perfusion in the isolated organ perfusion solution.

In some embodiments, the perfusion and/or the immersion is performed at a temperature of 2-40 ℃ (e.g., 2 ℃, 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃ or 40 ℃).

In some embodiments, the perfusion and/or the immersion is performed at a temperature of 4-37 ℃ (e.g., 4 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃).

In some embodiments, the perfusion and/or the immersion is performed at a temperature of 10-30 ℃ (e.g., 10 ℃, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 25 ℃, or 30 ℃).

In some embodiments, the perfusion and/or the submerging is performed at a temperature of 16-20 ℃ (e.g., 18 ℃).

In some embodiments, the perfusion and/or the immersion is performed at a temperature of 18 ℃.

In some embodiments, the isolated organ is a heart, liver, kidney, or lung.

In the present invention, the ordinary temperature means 2 to 40 ℃, preferably 4 to 37 ℃, further preferably 10 to 30 ℃, more preferably 16 to 20 ℃, and most preferably 18 ℃.

Advantageous effects

1. The prepared macromolecular polymerized hemoglobin (the molecular weight is 320-1024kD and is more than or equal to 95 percent) can exert the functions of oxygen carrying and oxygen release for a longer time, and effectively prolong the perfusion preservation time of organs;

2. by adding the high-molecular polymerized hemoglobin, the in-vitro organ preservation time is greatly prolonged, sufficient time is provided for solving the problems of mismatch time difference between a donor and a receptor, remote transplantation and the like, the organ waste phenomenon caused by short preservation time is reduced, and the utilization rate of transplanted organs is improved;

3. the oxygen-carrying perfusate at normal temperature solves the problem that organs stored under the anoxic condition can cause obvious secondary damage in the transplantation and reperfusion process, and simultaneously meets the condition of normal-temperature perfusion;

4. with the increasing year by year of organ transplantation and the unpredictable requirement on long-time organ preservation, the invention has important clinical application value and popularization.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.

The invention aims to provide a normal-temperature mechanical perfusate which can meet the requirement of perfusion preservation of isolated organs at normal temperature (4-37 ℃) and can obviously prolong the preservation time of safe organs.

The invention also aims to provide a preparation method of the normal-temperature mechanical organ perfusate.

The invention also aims to provide a preparation method of the self-made high-molecular polymerized hemoglobin in the normal-temperature organ perfusate.

The purpose of the invention is realized as follows: an organ perfusion solution mainly comprises 20-40 g of polymerized hemoglobin (the molecular weight is 320kD-1024kD and is more than or equal to 95%), 20000-30000U of heparin sodium, 8-10 g of glucose, 6-8 g of sodium chloride, 1-3 g of cefoxitin sodium for injection, 1-1.5 g of sodium bicarbonate, 40-60 mL of 10% compound amino acid injection, 0.1-0.3 mL of 12 compound vitamins for injection and 160-200U of insulin, the injection water is added to 1000mL, inert gas is introduced to perform deoxidation until the content of oxygenated hemoglobin is less than 5%, and the sealing and oxygen-isolating preservation are performed.

The content of each component is preferably: 30g of polymerized hemoglobin (the molecular weight is 320-1024kD and is more than or equal to 95 percent), 25000U of heparin sodium, 9g of glucose, 7g of sodium chloride, 2g of cefoxitin sodium for injection, 1.25g of sodium bicarbonate, 50mL of 10 percent compound amino acid injection, 0.2mL of 12 compound vitamins for injection and 180U of insulin, and the injection water is added to 1000 mL.

Another object of the invention is achieved by: the preparation method of the normal-temperature mechanical organ perfusate is characterized by comprising the following steps of: under the aseptic condition, adding injection water into the polymerized hemoglobin (the molecular weight is 320kD-1024kD and is more than or equal to 95 percent), heparin sodium, glucose, sodium chloride, cefoxitin sodium for injection, sodium bicarbonate, 10 percent compound amino acid injection, 12 compound vitamins for injection and insulin to 1000mL, uniformly mixing, deoxidizing, and sealing and storing in an oxygen-isolated plastic bag.

A further object of the invention is achieved by: the preparation method of the self-made high-molecular polymerized hemoglobin is characterized by comprising the following steps: 60 mu m deep layer blood filtration, 0.65um hollow fiber membrane washing erythrocyte, adding hypotonic solution to crack erythrocyte, obtaining crude and pure hemoglobin through 100KD ultrafiltration, obtaining pure hemoglobin through sample loading anion exchange chromatography, glutaraldehyde crosslinking hemoglobin, sodium borohydride terminating polymerization reaction, 300KD ultrafiltration liquid exchange to obtain self-made polymerized hemoglobin, the index requirement is as follows: the molecular weight of 320kD-1024kD is more than or equal to 95 percent, the methemoglobin is less than 5 percent, and the oxyhemoglobin is less than 5 percent.

After the product is prepared, the product is used immediately; the product of the invention has stable performance after sterilization and can be stored for 1-2 years. The pH value of the product is 7.0-8.0.

The product can be applied to normal-temperature organ perfusion preservation of isolated organs, can effectively ensure the biological activity of the organs within a safe preservation period, and is used in an amount of 100-1000 mL each time according to individual and organ differences.

The specific using method comprises the following steps:

liver: trimming to obtain a liver, trimming redundant tissues in a hepatic portal area, removing and cleaning hepatic portal lymph nodes, perfusing portal veins in a constant flow mode, setting the flow to be 0.5mL/min/g (liver weight), perfusing hepatic arteries in a pressure control mode, and starting normal-temperature perfusion, wherein the pressure is 80/60 mmHg; heart: the obtained heart was trimmed and after examination the ex vivo heart perfusion system was switched on at a flow rate of about 15 mL/min. Lifting the aorta by the micro-instrument, inserting the perfusion pipeline, placing the perfusion pipeline above the aortic valve and the coronary artery opening, fixing the noninvasive vascular clamp, knotting and fixing the silk threads, taking down the vascular clamp, and beginning normal-temperature perfusion; kidney: and (5) correcting the obtained kidney, connecting the kidney with a perfusion instrument, and setting the renal artery pressure to be 50-70 mmHg for perfusion.

The invention will be further explained with reference to specific examples.

EXAMPLE 1 preparation of bovine blood polymeric hemoglobin

1. Preparation method

Collecting 1L of fresh bovine blood (self-raising cattle), and diluting with 6g/L sodium citrate solution with the volume of 0.5-1 time of that of the blood;

then concentrating the obtained polymerized hemoglobin to 6-7g/dL, placing the polymerized hemoglobin concentrated to 6-7g/dL on an ultrafiltration membrane bag with 300KD, using lactated ringer's solution (sodium chloride 6.73g/L, potassium chloride 0.3g/L, calcium chloride dihydrate 0.2g/L, 40% sodium lactate 3.07g/L, N-acetyl-L-cysteine 22g/L) with the volume 5-10 times of the volume of the polymerized hemoglobin with 6-7g/dL for changing the solution, wherein the remaining end is the macromolecular polymerized hemoglobin of the invention, the volume of the changed solution is determined by the molecular weight content detection index in the following 2 (namely the following molecular weight content detection part), when the protein content with the molecular weight of 320-1024kD reaches 65%, 80%, 90%, 95%, 97%, 99% index in the solution changing process, respectively collecting corresponding protein solutions, introducing inert gas to perform deoxygenation until the content of oxyhemoglobin is less than 5%, filtering and sterilizing at 0.2 μm, sealing, and storing in oxygen-isolated manner. Meanwhile, collecting the permeate, concentrating the permeate by using a 30kD ultrafiltration membrane, replacing the permeate by using 5 times of the ringer's lactate solution by volume to obtain polymeric hemoglobin with the molecular weight of 32 kD-256 kD which is more than or equal to 95 percent, introducing inert gas to deoxidize until the content of oxygenated hemoglobin is less than 5 percent, filtering and sterilizing the mixture by 0.2 mu m, sealing and storing the filtered hemoglobin in an oxygen-isolated manner. And in addition, further ultrafiltering the polymerized hemoglobin with the molecular weight of 128 kD-256 kD of 95% or more with 100kD ultrafiltering membrane coated with ringer's lactate solution, stopping ultrafiltration liquid exchange when the molecular weight of 128 kD-256 kD of 95% or more is reached, introducing inert gas to deoxidize until the content of oxygenated hemoglobin is less than 5%, filtering and sterilizing at 0.2 μm, sealing and isolating oxygen for storage.

2. Molecular weight content detection

2.1 detection of the main materials to be prepared:

2.2 detection of the main devices to be prepared:

2.3 preparation of buffers required for detection:

buffer 750mM MgCl was prepared as follows₂，50mM tris，0.1mM EDTA pH 6.5。

2.3.1A 1000mL measuring cylinder was used to measure 800mL of ultrapure water and transferred to a 1L beaker.

2.3.2 to a 1L beaker, 10.46. + -. 0.05g of Bis-Tris was added and gently stirred with a magnetic stir bar until complete dissolution.

2.3.3 the pH of the solution was adjusted to 6.7. + -. 0.1 with concentrated HCl and gently stirred.

2.3.4 to a 1L beaker 152.48. + -. 0.05g of MgCl was added₂·6H₂O。

2.3.5 to a 1L beaker was added 0.030. + -. 0.001g of EDTA and gently stirred with a magnetic stir bar until complete dissolution.

2.3.6 the pH of the solution was adjusted to 6.5. + -. 0.1 with concentrated hydrochloric acid and gently stirred.

2.3.7 adjust the pH to the desired range, use a 1000ml graduated cylinder and increase the volume to 1000ml with ultra pure water.

2.3.8 the solution was filtered using a 0.1 μm PES filter.

2.4 preparation of samples

2.4.1 Standard preparation (Bio Rad Gel Filtration Standard)

2.4.1.1 Add 500. mu.L of ultrapure water to the standard and spin gently.

2.4.1.2 were allowed to stand on ice for 2-3 minutes and the solution was transferred to a 1.5 ml EP tube.

2.4.1.313000 rpm, and centrifuging at 4 ℃ for 10 minutes.

2.4.1.4 transfer the supernatant to a high performance liquid chromatography vial.

2.4.2 preparation of test samples

2.4.2.1 the diluted sample was transferred to a 1.5 ml EP tube.

2.4.2.213000 rpm, and centrifuging at 4 ℃ for 10 minutes.

2.4.2.3 transfer the supernatant to a high performance liquid chromatography vial.

2.5 detection method

2.5.1 chromatography column used: agilent advanced Bio SEC 2.7 μm,

7.8×300mm

2.5.2 parameter settings

Parameter(s)	Is provided with
		Run time	30min
Flow rate of flow	0.5mL/min
		Column temperature	25℃
Temperature of the sample	25℃
		Basic operation instruction program	100% mobile phase
VWD detector	Absorption at 280nm
		MALLS (Multi-angle laser scattering instrument)	DAWN EOS,λ＝690nm

2.5.3, storing and analyzing the detection data, and determining that the detection data is qualified when the molecular weight is 320kD-1024kD which is more than or equal to 95%;

3. blood gas detection

The sample is loaded with 60um of the deoxidized polymerized hemoglobin by an ABL90FLEX blood gas analyzer for blood gas value detection, and the high-iron hemoglobin is less than 5 percent, and the oxygenated hemoglobin is less than 5 percent, which is qualified.

EXAMPLE 2 preparation of porcine blood high-molecular polymerized hemoglobin

1L of fresh pig blood (self-raising pig) was collected, and the rest of the preparation process and the detection method were the same as in example 1.

EXAMPLE 3 preparation of organ perfusate

Weighing the components according to the preparation proportion of each 1000mL of organ perfusate:

30g of high molecular weight polymerized hemoglobin (prepared by the method of example 1, molecular weight of 320kD to 1024kD, content: 95%, 97%, 99%, respectively)

Glucose (alpha-D-glucose, Aladdin reagent Co.) 9g

Heparin sodium (Alantin reagent company) 25000u

Sodium chloride (sodium chloride, national reagents Co.) 7g

Cefoxitin Sodium (Cefoxitin Sodium for Injection, Xin Li Tai medicine) 2g

Sodium bicarbonate (sodium bicarbonate, national reagents) 1.25g

50mL of 10% compound amino acid injection (Chenxin pharmaceutical industry)

0.2mL of 12 kinds of multivitamins for injection (Shanxi Pude pharmaceutical Co., Ltd.)

Insulin (Wanbang Jinqiao pharmaceutical Co., Ltd.) 180U

Under aseptic conditions, sequentially adding glucose, sodium chloride, heparin sodium, cefoxitin sodium for injection and sodium bicarbonate according to the above amount, adding injection water to 200mL, stirring until the mixture is dissolved, then adding 10% compound amino acid injection, 12 kinds of compound vitamins for injection, insulin and polymerized hemoglobin, fixing the volume of the injection water to 1000mL, introducing nitrogen for deoxidation until the content of the oxygenated hemoglobin is less than 5%, filtering and sterilizing by 0.2 mu m, filling into an oxygen-isolated plastic bag under an aseptic and anaerobic environment, and carrying out thermoplastic sealing to obtain the perfusate of the embodiment 3.

EXAMPLE 4 preparation of organ perfusate

40g of high molecular weight polymerized hemoglobin (prepared by the method of example 2, molecular weight of 320kD-1024kD, content 95%, 97%, 99%, respectively)

Glucose (. alpha. -D-glucose, Aladdin reagent Co.) 10g

Heparin sodium (Alantin reagent) 30000u

Sodium chloride (sodium chloride, national reagent Co.) 8g

Cefoxitin Sodium (Cefoxitin Sodium for Injection, Xin Li Tai medicine) 3g for Injection

Sodium bicarbonate (sodium bicarbonate, national reagents) 1.5g

60mL of 10% compound amino acid injection (Chenxin pharmaceutical industry)

0.3mL of 12 kinds of multivitamins for injection (Shanxi Pude pharmaceutical Co., Ltd.)

Insulin (Wanbang Jinqiao pharmaceutical Co., Ltd.) 200U

Under aseptic conditions, sequentially adding glucose, sodium chloride, heparin sodium, cefoxitin sodium for injection and sodium bicarbonate according to the above amount, adding injection water to 200mL, stirring until the mixture is dissolved, then adding 10% compound amino acid injection, 12 kinds of compound vitamins for injection, insulin and polymerized hemoglobin, fixing the volume of the injection water to 1000mL, introducing nitrogen for deoxidation until the content of the oxygenated hemoglobin is less than 5%, filtering and sterilizing by 0.2 mu m, filling into an oxygen-isolated plastic bag under an aseptic and anaerobic environment, and carrying out thermoplastic sealing to obtain the perfusate of the embodiment 4.

EXAMPLE 5 preparation of organ perfusate

20g of high molecular weight polymerized hemoglobin (prepared by the method of example 1, molecular weight of 320kD to 1024kD, content of 95%, 97%, 99%, respectively)

Glucose (alpha-D-glucose, Aladdin reagent Co.) 8g

Heparin sodium (Alantin sodium, Allantin reagent Co.) 20000u

Sodium chloride (sodium chloride, national reagent Co.) 6g

Cefoxitin Sodium (Cefoxitin Sodium for Injection, Xin Li Tai pharmaceutical) for Injection 1g

Sodium bicarbonate (sodium bicarbonate, national reagents) 1.0g

10% Compound amino acid injection (Chenxin pharmaceutical industry) 40mL

0.1mL of 12 kinds of multivitamins for injection (Shanxi Pude pharmaceutical Co., Ltd.)

Insulin (Wanbang Jinqiao pharmaceutical Co., Ltd.) 160U

Under aseptic conditions, sequentially adding glucose, sodium chloride, heparin sodium, cefoxitin sodium for injection and sodium bicarbonate according to the above amount, adding injection water to 200mL, stirring until the mixture is dissolved, then adding 10% compound amino acid injection, 12 kinds of compound vitamins for injection, insulin and polymerized hemoglobin, fixing the volume of the injection water to 1000mL, introducing nitrogen for deoxidation until the content of the oxygenated hemoglobin is less than 5%, filtering and sterilizing by 0.2 mu m, filling into an oxygen-isolated plastic bag under an aseptic and anaerobic environment, and carrying out thermoplastic sealing to obtain the perfusate of the embodiment 5.

Example 6 perfusion Effect experiment of isolated pig liver under different concentration formulas of the invention

Experimental pigs (small Bama pigs, 10-12 months old, 20-28kg, purchased from Beijing Ministry pig raising base) are fasted for 12h before operation, water is forbidden for 6h, anaesthesia is performed, the experimental pigs are fixed on an operating table in a supine position, an abdominal midline incision enters the abdomen, sternal xiphoid process is resisted on the incision, pubic symphysis is issued, left and right triangular ligaments under the diaphragm are loosened, common bile duct and pancreatic branch are ligated, liver is dissociated and obtained under the condition of protecting the first hepatic portal, ligaments around the liver are dissociated, thoracic aorta is cut off above the thoracic aorta ligation position, superior and inferior vena cava of the liver is cut off, the cut thoracic aorta is used as traction, the liver is dissociated from the posterior abdominal wall, the liver is upwards closely attached to the spine, perihepatic abdominal tissues and diaphragm muscle are dissociated, the isolated aorta is cut off to the position below the hepatic artery branch, the abdominal aorta is cut off at the position 1cm below the hepatic artery branch, the liver is obtained, the liver is trimmed, and the redundant tissues in the hepatic portal area are obtained, and removing liver and portal lymph nodes. The perfusion machine was connected, the portal vein was perfused with a constant flow rate set at 0.5mL/min/g (liver weight), the hepatic artery was perfused with a pressure control mode at 80/60mmHg, and perfusion was started at 18 ℃. The livers obtained were divided into four groups: the first group is lactic acid ringer's solution; the second group is the formulation of example 3 of the present invention with a polymerized hemoglobin content of 95%; the third group is the formulation of example 4 of the present invention with a polymerized hemoglobin content of 95%; the fourth group was the formulation of example 5 of the present invention having a polymerized hemoglobin content of 95%.

The perfusate is collected every 20min for 0h-1h, every 1h for 1h-72h and the perfusate is collected at the safe time limit point for observing the following indexes:

half-life variation levels of AST (aspartate aminotransferase), ALT (glutamate pyruvate transaminase), TBIL (total bilirubin), ET-1 (endothelin 1), and polymerized hemoglobin in the perfusate;

altered levels of NO (nitric oxide) in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the perfusion maintenance safety time limit is that the AST content is not more than 40U/L, ALT, the AST content is not more than 40U/L, TBIL, the AST content is not more than 20.5umol/L, ET-1, the AST content is not more than 20ng/L, and the safety time limits of the perfused organs on the indexes are 40min, 26h, 28h and 23h respectively. The half-life of the polymerized hemoglobin in the second group, the third group and the fourth group is 29h, 30h and 25h respectively. Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 1.80 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4, washed for 3 times for 1 hour in phosphate buffer solution with the pH value of 7.38, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by using an ultrathin slicer, double electron staining is carried out by using uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to observe the change of the cell morphology and further judge the change of the organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the outline of the hepatic lobule is clear, hepatic sinusoids in the lobules are arranged in a radial shape and are inosculated into a net, adjacent hepatic lobules are widely inosculated, interlobular arteries and interlobular veins in a junction area can be seen, and terminal branches of the hepatic arteries continuously converge into the hepatic sinusoids along the periphery of the hepatic lobules.

In summary, under mechanical perfusion at 18 ℃, the safety time limit of the first group, the second group, the third group and the fourth group of the indexes is respectively 40min, 26h, 28h and 23h, which shows that the perfusion preservation time of the isolated liver can be effectively prolonged within the concentration range of the invention.

Example 7 perfusion Effect experiment of isolated rat Heart under perfusates of different concentrations

Rats (Wistar, 9-11 weeks, female 220-. The skin of the abdomen was cut open along the white line of the abdomen to the xiphoid process. Putting the intestinal tract on one side, exposing the inferior vena cava, injecting heparin normal saline through the inferior vena cava, fully heparinizing after 1min, and cutting the abdominal blood vessel to bleed. The chest is opened rapidly, the thymus tissue is cut off, and the heart and the great vessels are fully exposed. The distal end of the innominate aorta is free and cut off, the rest great vessels are cut off rapidly, the heart is taken out, and the simple pruning is carried out. The isolated heart perfusion system was turned on with a flow of about 15 mL/min. The micro-instrument lifts the aorta, inserts the perfusion tube, places the perfusion tube above the aortic valve and coronary artery opening, fixes the non-invasive vascular clamp, ties the silk thread, takes off the vascular clamp, and starts perfusion at 18 ℃. The hearts obtained were divided into four groups: the first group is lactic acid ringer's solution; the second group is the formulation of example 3 of the present invention with a polymerized hemoglobin content of 95%; the third group is the formulation of example 4 of the present invention with a polymerized hemoglobin content of 95%; the fourth group was the formulation of example 5 of the present invention having a polymerized hemoglobin content of 95%.

The perfusate is collected every 10min for 0h-1h, every 1h for 1h-72h and the perfusate is collected at the safe time limit point for observing the following indexes:

varying levels of half-life of perfusate CTN (troponin), CK-MB (creatine kinase isozyme), ANP (angiocardin), and polymerized hemoglobin;

altered levels of NO (nitric oxide) in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: by the above-mentioned different perfusion solutions to perfuse the coreThe viscera have protection effect, perfusate is taken at different perfusion time points for detection, the content of CTN is not more than 0.13ug/L, CK-MB and not more than 16U/L, ANP and not more than 54pmol/L are taken as perfusion maintenance safety time limit, and the safety time limit of each group of perfused organs for maintaining each index is respectively 50min, 27h, 30h and 25 h. The half-lives of the polymerized hemoglobin in the second group, the third group and the fourth group are respectively 30h, 34h and 26 h. Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 2.30 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4, washed for 3 times for 1 hour in phosphate buffer solution with the pH value of 7.38, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by using an ultrathin slicer, double electron staining is carried out by using uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to observe the change of the cell morphology and further judge the change of the organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the myocardial interstitial network is in a dense honeycomb shape, the myocytes are positioned in the myocyte sheath, the myocyte sheath is in a circular or elliptical structure, the collagen fibers on the outer surface of the sheath are arranged in a net shape, the directions of the collagen fibers on the outer surface of the myocyte sheath are not consistent, the myocyte sheath is in a net shape, the collagen fibers are in different diameters, and the collagen fibers are in different diameters and are in a normal state.

In summary, under mechanical perfusion at 18 ℃, the safety time limit of the first group, the second group, the third group and the fourth group of the indexes is respectively 50min, 27h, 30h and 25h, which shows that the isolated heart perfusion preservation time can be effectively prolonged within the concentration range of the invention.

Example 8 experiment of perfusion Effect of isolated dog Kidney in perfusate of different concentrations according to the present invention

Experimental dogs (adult beagle dog, 7-10kg, purchased from beijing experimental animals center) were anesthetized, fixed in the supine position, tissues were isolated after laparotomy, kidneys were exposed and excised, trimmed, connected to a perfusion apparatus, and perfused at 18 ℃ with renal artery pressure set at 55 mmHg. The kidneys obtained were divided into four groups: the first group is lactic acid ringer's solution; the second group was the formulation of example 3 of the present invention with a polymerized hemoglobin content of 95%; the third group is the formulation of example 4 of the present invention with a polymerized hemoglobin content of 95%; the fourth group was the formulation of example 5 of the present invention having a polymerized hemoglobin content of 95%.

Perfusate is collected from each group of perfused organs every 1 hour at 0h-120h and perfused tissues are collected at safe time limit points to observe the following indexes:

the level of change in half-life of Scr (creatinine), BUN (urea nitrogen), ET-1 (endothelin 1), and polymerized hemoglobin in the perfusate;

altered levels of NO (nitric oxide) in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the safe perfusion maintaining time limit is that the content of Scr is not more than 120 mu mol/L, BUN and the content of Scr is not more than 7.0mmol/L, ET-1 and the content of Scr is not more than 20ng/L, and the safe perfusion maintaining time limits of the perfused organs for the indexes are respectively 2h, 89h, 97h and 82 h. The half-lives of the polymerized hemoglobin in the second group, the third group and the fourth group are respectively 51h, 58h and 50 h. Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured results are all lower limit standards of not less than 3.40 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue, pre-fixing with 2.5% glutaraldehyde (0.1M) phosphate buffer solution of pH7.4 at 4 deg.C for 2 hr, washing in phosphate buffer solution of pH7.38 for 3 times for 1 hr, dehydrating with 50%, 70%, 80%, 90%, 95% acetone for 15 min, embedding in 60 deg.C incubator, polymerizing for 48 hr, preparing ultrathin section with microtome, and performing polymerization with uranyl acetate and lead citrateDouble electron staining, JEM-1200EX electron microscope observation, to observe the change of cell morphology, and further judge the change of organ activity. The electron microscope results show that in the safety time limit of each perfusate, the karyotype is approximately normal, the nucleoplasm distribution is uniform, the mitochondria are slightly swollen, but the cristae arrangement is good, and the individual cristae membrana gaps are slightly widened. There is also a slight swelling of the endoplasmic reticulum. The proximal tubular epithelial cells have microvilli edema, thickening and falling off individually. The capillary endothelium and the endothelial cells of the filtration membrane are slightly edematous, have clear structures and are in a normal state.

In summary, under mechanical perfusion at 18 ℃, the safety time limit of the first group, the second group, the third group and the fourth group of the indexes is respectively 2h, 89h, 97h and 82h, which shows that the perfusion preservation time of the isolated kidney can be effectively prolonged within the concentration range of the invention.

Example 9 perfusion effect experiment of waste human liver under perfusate of different concentrations

Waste human livers are obtained from hospitals, the livers are trimmed, redundant tissues in the hepatic portal area are trimmed, and hepatic portal lymph nodes are removed and cleaned. The perfusion apparatus was connected, the portal vein was perfused with a constant flow rate set at 0.5mL/min/g (liver weight), the hepatic artery was perfused with a pressure control mode at 80/60mmHg, and perfusion was started at 18 ℃. The livers obtained were divided into four groups: the first group is lactic acid ringer's solution; the second group is the formulation of example 3 of the present invention with a polymerized hemoglobin content of 95%; the third group is the formulation of example 4 of the present invention with a polymerized hemoglobin content of 95%; the fourth group was the formulation of example 5 of the present invention having a polymerized hemoglobin content of 95%.

altered levels of NO (nitric oxide) in tissue;

observing the change condition of the tissue by an electron microscope;

In summary, under mechanical perfusion at 18 ℃, the safety time limit of the first group, the second group, the third group and the fourth group of the indexes is respectively 40min, 26h, 28h and 23h, which shows that the perfusion preservation time of the liver of the isolated human can be effectively prolonged within the concentration range of the invention.

Example 10 perfusion Effect test of isolated pig liver at different temperatures according to the invention

Experimental pigs (small Bama pigs, 10-12 months old, 20-28kg, purchased from Beijing Ministry pig raising base) are fasted for 12h before operation, water is forbidden for 6h, anaesthesia is performed, the experimental pigs are fixed on an operating table in a supine position, an abdominal midline incision enters the abdomen, sternal xiphoid process is resisted on the incision, pubic symphysis is issued, left and right triangular ligaments under the diaphragm are loosened, common bile duct and pancreatic branch are ligated, liver is dissociated and obtained under the condition of protecting the first hepatic portal, ligaments around the liver are dissociated, thoracic aorta is cut off above the thoracic aorta ligation position, superior and inferior vena cava of the liver is cut off, the cut thoracic aorta is used as traction, the liver is dissociated from the posterior abdominal wall, the liver is upwards closely attached to the spine, perihepatic abdominal tissues and diaphragm muscle are dissociated, the isolated aorta is cut off to the position below the hepatic artery branch, the abdominal aorta is cut off at the position 1cm below the hepatic artery branch, the liver is obtained, the liver is trimmed, and the redundant tissues in the hepatic portal area are obtained, and removing liver and portal lymph nodes. The perfusion machine was connected, the portal vein was perfused with a constant flow rate set at 0.5mL/min/g (liver weight), the hepatic artery was perfused with a pressure control mode at 80/60mmHg, and the perfusion was started at different temperatures. The obtained livers were divided into three groups: the first group was a 4 ℃ infusion of the formulation of example 3 of the invention with a polymerized hemoglobin content of 95%; the second group was 18 ℃ infusion of the formulation of example 3 of the invention with 95% polymerized hemoglobin; the third group was infused at 37 ℃ for the formulation of example 3 of the present invention having a polymerized hemoglobin content of 95%.

(ii) altered levels of half-life of AST, ALT, TBIL, ET-1, and polymerized hemoglobin in the perfusate;

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the different perfusates have protective effect on perfused liver, the perfusates are taken at different perfusion time points for detection, the safe perfusion maintaining time limit is that the content of AST is not more than 40U/L, ALT, the content of AST is not more than 40U/L, TBIL, the content of AST is not more than 20.5umol/L, ET-1 and the content of AST is not more than 20ng/L, and the safe time limit for maintaining the indexes of the perfused organs of each group is respectively 30h, 26h and 21 h. The half-life period of the polymerized hemoglobin of each group of perfusate is 35h, 29h and 22h in sequence. Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 1.80 umol/L. Meanwhile, 0.8 multiplied by 0.2cm3 tissue is taken at the safety time limit point of each group of perfusate, 2.5 percent glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4 is used for pre-fixing for 2 hours at 4 ℃, the tissue is washed for 3 times in phosphate buffer solution with the pH value of 7.38 for 1 hour totally, then 50 percent, 70 percent, 80 percent, 90 percent and 95 percent acetone are used for dehydration step by step, each time is 15 minutes, and finally the tissue is embedded in a 60-degree incubator for polymerization for 48 hours, an ultrathin section is prepared by an ultrathin slicer, double electronic staining is carried out by uranyl acetate and lead citrate, and the change of the cell morphology is observed by a JEM-1200EX electron microscope so as to judge the change of the organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the outline of the hepatic lobule is clear, hepatic sinusoids in the lobules are arranged in a radial shape and are inosculated into a net, adjacent hepatic lobules are widely inosculated, interlobular arteries and interlobular veins in a junction area can be seen, and terminal branches of the hepatic arteries continuously converge into the hepatic sinusoids along the periphery of the hepatic lobules.

In summary, under mechanical perfusion at different temperatures, the safety time limit of the first group, the second group and the third group for maintaining the indexes is respectively 30h, 26h and 21h, which shows that the perfusion preservation time of the isolated liver can be effectively prolonged in different temperature perfusion ranges.

Example 11 perfusion Effect test of isolated rat Heart at different temperatures according to the invention

Rats (Wistar, 9-11 weeks, female 220-. The skin of the abdomen was cut open along the white line of the abdomen to the xiphoid process. Putting the intestinal tract on one side, exposing the inferior vena cava, injecting heparin normal saline through the inferior vena cava, fully heparinizing after 1min, and cutting the abdominal blood vessel to bleed. The chest is opened rapidly, the thymus tissue is cut off, and the heart and the great vessels are fully exposed. The distal end of the innominate aorta is free and cut off, the rest great vessels are cut off rapidly, the heart is taken out, and the simple pruning is carried out. The isolated heart perfusion system was turned on with a flow of about 15 mL/min. The micro-instrument lifts the aorta, inserts the perfusion tube, places the perfusion tube above the aortic valve and coronary artery opening, fixes the non-invasive vascular clamp, ties the silk thread for fixation, takes down the vascular clamp, and starts perfusion at different temperatures. The obtained hearts were divided into three groups: the first group was a 4 ℃ infusion of the formulation of example 3 of the invention with a polymerized hemoglobin content of 95%; the second group was 18 ℃ infusion of the formulation of example 3 of the invention with 95% polymerized hemoglobin; the third group was infused at 37 ℃ for the formulation of example 3 of the present invention having a polymerized hemoglobin content of 95%.

the half-life change levels of CTN, CK-MB, ANP and polymerized hemoglobin in the perfusate;

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the different perfusates have protective effect on perfused heart, the perfusates are taken at different perfusion time points for detection, the safe perfusion maintaining time limit is that the content of CTN is not more than 0.13ug/L, CK-MB and not more than 16U/L, ANP and not more than 54pmol/L, and the safe perfusion maintaining time limit of each group of perfused organs to each index is 32h, 27h and 22h respectively. The half-life period of the polymerized hemoglobin of each group of perfusate is 38h, 30h and 26h in sequence. Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 2.30 umol/L. Meanwhile, 0.8 multiplied by 0.2cm3 tissue is taken at the safety time limit point of each group of perfusate, 2.5 percent glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4 is used for pre-fixing for 2 hours at 4 ℃, the tissue is washed for 3 times in phosphate buffer solution with the pH value of 7.38 for 1 hour totally, then 50 percent, 70 percent, 80 percent, 90 percent and 95 percent acetone are used for dehydration step by step, each time is 15 minutes, and finally the tissue is embedded in a 60-degree incubator for polymerization for 48 hours, an ultrathin section is prepared by an ultrathin slicer, double electronic staining is carried out by uranyl acetate and lead citrate, and the change of the cell morphology is observed by a JEM-1200EX electron microscope so as to judge the change of the organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the myocardial interstitial network is in a dense honeycomb shape, the myocytes are positioned in the myocyte sheath, the myocyte sheath is in a circular or elliptical structure, the collagen fibers on the outer surface of the sheath are arranged in a net shape, the directions of the collagen fibers on the outer surface of the myocyte sheath are not consistent, the myocyte sheath is in a net shape, the collagen fibers are in different diameters, and the collagen fibers are in different diameters and are in a normal state.

In summary, under mechanical perfusion at different temperatures, the safety time limit of the first group, the second group and the third group for maintaining the indexes is respectively 32h, 27h and 22h, which shows that the isolated heart perfusion preservation time can be effectively prolonged under different perfusion temperatures.

Comparative example 1 comparison of perfusion experiment effects of isolated pig liver under different formulas

Experimental pigs (small Bama pigs, 10-12 months old, 20-28kg, purchased from Beijing Ministry pig raising base) are fasted for 12h before operation, water is forbidden for 6h, anaesthesia is performed, the experimental pigs are fixed on an operating table in a supine position, an abdominal midline incision enters the abdomen, sternal xiphoid process is resisted on the incision, pubic symphysis is issued, left and right triangular ligaments under the diaphragm are loosened, common bile duct and pancreatic branch are ligated, liver is dissociated and obtained under the condition of protecting the first hepatic portal, ligaments around the liver are dissociated, thoracic aorta is cut off above the thoracic aorta ligation position, superior and inferior vena cava of the liver is cut off, the cut thoracic aorta is used as traction, the liver is dissociated from the posterior abdominal wall, the liver is upwards closely attached to the spine, perihepatic abdominal tissues and diaphragm muscle are dissociated, the isolated aorta is cut off to the position below the hepatic artery branch, the abdominal aorta is cut off at the position 1cm below the hepatic artery branch, the liver is obtained, the liver is trimmed, and the redundant tissues in the hepatic portal area are obtained, and removing liver and portal lymph nodes. The perfusion machine was connected, the portal vein was perfused with a constant flow rate set at 0.5mL/min/g (liver weight), the hepatic artery was perfused with a pressure control mode at 80/60mmHg, and perfusion was started at 18 ℃. The livers obtained were divided into ten groups: the first group is lactic acid ringer's solution; the second group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 32 kD-256 kD and the content of more than or equal to 95 percent; the third group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 128 kD-256 kD and the content of more than or equal to 95 percent; the fourth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 65 percent; the fifth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 80 percent; the sixth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 90 percent; the seventh group is the formula of the embodiment 3 of the invention, namely the hemoglobin with the molecular weight of 320kD-1024kD and the content of 95 percent; the eighth group is the formulation of example 3 of the present invention, i.e., hemoglobin with molecular weight of 320kD to 1024kD and 97% content; the ninth group is the formulation of the embodiment 3 of the invention, namely hemoglobin with molecular weight of 320kD-1024kD and content of 99 percent; the tenth group is polymerized hemoglobin (molecular weight 320kD-1024kD < 45%) prepared by the method of the company patent No. 201910846580.9.

(ii) the level of change in half-life of AST, ALT, TBIL, ET-1 and polymerized hemoglobin in the perfusate;

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the perfusion maintenance safety time limit is that the AST content is not more than 40U/L, ALT, the AST content is not more than 40U/L, TBIL, the AST content is not more than 20.5umol/L, ET-1, and the contents of the AST content is not more than 20ng/L, and the safety time limits of the perfused organs on the indexes are respectively 40min, 10h, 12h, 19h, 17h, 18h, 26h, 27h and 18 h. The half-lives of the polymerized hemoglobin in the second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups were 12h, 15h, 22h, 21h, 29h, 30h, and 20h, respectively (as shown in table 1 below). Then perfusing each group with the liquidShearing small pieces of tissue at a safety time limit, weighing, shearing, adding a small amount of quartz sand and cold normal saline, homogenizing, centrifuging the homogenate at a temperature of below 4 ℃ at 10000r/min for 20min, taking supernatant to determine the NO content of the tissue, wherein the determination result is not lower than the lower limit standard of 1.80 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4, washed for 3 times for 1 hour in phosphate buffer solution with the pH value of 7.38, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by using an ultrathin slicer, double electron staining is carried out by using uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to observe the change of the cell morphology and further judge the change of the organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the outline of the hepatic lobule is clear, hepatic sinusoids in the lobules are arranged in a radial shape and are inosculated into a net, adjacent hepatic lobules are widely inosculated, interlobular arteries and interlobular veins in a junction area can be seen, and terminal branches of the hepatic arteries continuously converge into the hepatic sinusoids along the periphery of the hepatic lobules.

In summary, the safety time periods maintained by the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups under mechanical infusion at 18 ℃ were 40min, 10h, 12h, 19h, 17h, 18h, 26h, 27h, and 18h, respectively (as shown in table 1 below). The second and third groups use different sections of polymerized hemoglobin with a lower than 320kD, which have slightly different perfusion safety intervals, but shorter perfusion safety intervals. The tenth group used polymerized hemoglobin without separate mixing, which was generally not selectively perfused for a long period of time. The polymeric hemoglobin with the content of 320-1024kD is used in the fourth group, the fifth group, the sixth group and the seventh group, which is fluctuated with the content increase (65%, 80%, 90% and 95%) to prolong the perfusion safety time limit, but the perfusion safety time limit of the hemoglobin with the content of 320-1024kD of 95%, 97% and 99% used in the seventh group, the eighth group and the ninth group is not fluctuated any more, and has obvious perfusion safety time limit advantages.

Table 1: comparison of perfusion experiment effects of in-vitro pig liver under different formulas

Comparative example 2 comparison of perfusion experiment results of isolated rat hearts under different formulations

Rats (Wistar, 9-11 weeks, female 220-. The skin of the abdomen was cut open along the white line of the abdomen to the xiphoid process. Putting the intestinal tract on one side, exposing the inferior vena cava, injecting heparin normal saline through the inferior vena cava, fully heparinizing after 1min, and cutting the abdominal blood vessel to bleed. The chest is opened rapidly, the thymus tissue is cut off, and the heart and the great vessels are fully exposed. The distal end of the innominate aorta is free and cut off, the rest great vessels are cut off rapidly, the heart is taken out, and the simple pruning is carried out. The isolated heart perfusion system was turned on with a flow of about 15 mL/min. The micro-instrument lifts the aorta, inserts the perfusion tube, places the perfusion tube above the aortic valve and coronary artery opening, fixes the non-invasive vascular clamp, ties the silk thread, takes off the vascular clamp, and starts perfusion at 18 ℃. The obtained hearts were divided into ten groups: the first group is lactic acid ringer's solution; the second group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 32 kD-256 kD and the content of more than or equal to 95 percent; the third group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 128 kD-256 kD and the content of more than or equal to 95 percent; the fourth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 65 percent; the fifth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 80 percent; the sixth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 90 percent; the seventh group is the formula of the embodiment 3 of the invention, namely the hemoglobin with the molecular weight of 320kD-1024kD and the content of 95 percent; the eighth group is the formulation of example 3 of the present invention, i.e., hemoglobin with molecular weight of 320kD to 1024kD and 97% content; the ninth group is the formulation of the embodiment 3 of the invention, namely hemoglobin with molecular weight of 320kD-1024kD and content of 99 percent; the tenth group is polymerized hemoglobin (molecular weight 320kD-1024kD < 45%) prepared by the method of the company patent No. 201910846580.9.

the half-life change levels of the perfusates CTN, CK-MB, ANP and polymerized hemoglobin;

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the content of CTN is not more than 0.13ug/L, CK-MB and not more than 16U/L, ANP is not more than 54pmol/L, the perfusion maintenance safety time limit is set, and the safety time limit of each group of perfused organs for maintaining the indexes is respectively 50min, 12h, 13h, 20h, 17h, 19h, 27h, 28h and 20 h. The half-lives of the polymerized hemoglobin in the second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups were 14h, 15h, 22h, 20h, 22h, 30h, 31h, and 24h, respectively (as shown in table 2 below). Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 2.30 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue, pre-fixing with 2.5% glutaraldehyde (0.1M) phosphate buffer solution of pH7.4 at 4 deg.C for 2 hr, washing in phosphate buffer solution of pH7.38 for 3 times for 1 hr, dehydrating with 50%, 70%, 80%, 90%, 95% acetone for 15 min, embedding in 60 deg.C incubator, polymerizing for 48 hr, preparing ultrathin section with ultrathin microtome, and performing double electron with uranyl acetate and lead citrateStaining, JEM-1200EX electron microscope observation, to observe the change of cell morphology, and further judge the change of organ activity. The electron microscope result shows that in the safe time limit of each perfusate, the myocardial interstitial network is in a dense honeycomb shape, the myocytes are positioned in the myocyte sheath, the myocyte sheath is in a circular or elliptical structure, the collagen fibers on the outer surface of the sheath are arranged in a net shape, the directions of the collagen fibers on the outer surface of the myocyte sheath are not consistent, the myocyte sheath is in a net shape, the diameters of the collagen fibers are not equal, and the result shows a normal state.

In summary, the safety time limit maintained by the indexes of the first group, the second group, the third group, the fourth group, the fifth group, the sixth group, the seventh group, the eighth group, the ninth group and the tenth group under mechanical perfusion at 18 ℃ is respectively 50min, 12h, 13h, 20h, 17h, 19h, 27h, 28h and 20h (as shown in the following table 2). The second and third groups use different sections of polymerized hemoglobin with a lower than 320kD, which have slightly different perfusion safety intervals, but shorter perfusion safety intervals. The tenth group used polymerized hemoglobin without separate mixing, which was generally not selectively perfused for a long period of time. The polymeric hemoglobin with the content of 320-1024kD is used in the fourth group, the fifth group, the sixth group and the seventh group, which is fluctuated with the content increase (65%, 80%, 90% and 95%) to prolong the perfusion safety time limit, but the perfusion safety time limit of the hemoglobin with the content of 320-1024kD of 95%, 97% and 99% used in the seventh group, the eighth group and the ninth group is not fluctuated any more, and has obvious perfusion safety time limit advantages.

Table 2: comparison of perfusion experiment effects of isolated rat hearts under different formulas

Comparative example 3 comparison of perfusion Experimental Effect of isolated dog Kidney under different formulations

Experimental dogs (adult beagle dog, 7-10kg, purchased from beijing experimental animals center) were anesthetized, fixed in the supine position, tissues were isolated after laparotomy, kidneys were exposed and excised, trimmed, connected to a perfusion apparatus, and perfused at 18 ℃ with renal artery pressure set at 55 mmHg. The obtained kidneys were divided into ten groups: the first group is lactic acid ringer's solution; the second group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 32 kD-256 kD and the content of more than or equal to 95 percent; the third group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 128 kD-256 kD and the content of more than or equal to 95 percent; the fourth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 65 percent; the fifth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 80 percent; the sixth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 90 percent; the seventh group is the formula of the embodiment 3 of the invention, namely the hemoglobin with the molecular weight of 320kD-1024kD and the content of 95 percent; the eighth group is the formulation of example 3 of the present invention, i.e., hemoglobin with molecular weight of 320kD to 1024kD and 97% content; the ninth group is the formulation of the embodiment 3 of the invention, namely hemoglobin with molecular weight of 320kD-1024kD and content of 99 percent; the tenth group is polymerized hemoglobin (molecular weight 320kD-1024kD < 45%) prepared by the method of the company patent No. 201910846580.9.

the half-life variation levels of Scr, BUN, ET-1 and polymerized hemoglobin in the perfusate;

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the safe time limit for perfusion maintenance is that the content of Scr is not more than 120 mu mol/L, BUN and the content of Scr is not more than 7.0mmol/L, ET-1 and the content of Scr is not more than 20ng/L, and the safe time limit for the perfusion organs to maintain the indexes is respectively 2h, 40h, 43h, 73h, 68h, 71h, 89h, 91h, 92h and 72 h. The second mentionedThe half-lives of the polymerized hemoglobin in the groups, the third group, the fourth group, the fifth group, the sixth group and the seventh group are respectively 30h, 33h, 42h, 40h, 42h, 51h, 53h and 36h (shown in the following table 3). Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 3.40 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4, washed for 3 times for 1 hour in phosphate buffer solution with the pH value of 7.38, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by using an ultrathin slicer, double electron staining is carried out by using uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to observe the change of the cell morphology and further judge the change of the organ activity. The electron microscope results show that in the safety time limit of each perfusate, the karyotype is approximately normal, the nucleoplasm distribution is uniform, the mitochondria are slightly swollen, but the cristae arrangement is good, and the individual cristae membrana gaps are slightly widened. There is also a slight swelling of the endoplasmic reticulum. The proximal tubular epithelial cells have microvilli edema, thickening and falling off individually. The capillary endothelium and the endothelial cells of the filtration membrane are slightly edematous, have clear structures and are in a normal state.

In summary, the safety time periods maintained by the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups under mechanical infusion at 18 ℃ were 2h, 40h, 43h, 73h, 68h, 71h, 89h, 91h, 92h, and 72h, respectively (as shown in table 3 below). The second and third groups use different sections of polymerized hemoglobin with a lower than 320kD, which have slightly different perfusion safety intervals, but shorter perfusion safety intervals. The tenth group used polymerized hemoglobin without separate mixing, which was generally not selectively perfused for a long period of time. The polymeric hemoglobin with the content of 320-1024kD is used in the fourth group, the fifth group, the sixth group and the seventh group, which is fluctuated with the content increase (65%, 80%, 90% and 95%) to prolong the perfusion safety time limit, but the perfusion safety time limit of the hemoglobin with the content of 320-1024kD of 95%, 97% and 99% used in the seventh group, the eighth group and the ninth group is not fluctuated any more, and has obvious perfusion safety time limit advantages.

Table 3: comparison of perfusion experiment effects of in vitro dog kidney under different formulas

Comparative example 4 comparison of perfusion experiment effects of waste human livers under different formulas

Waste human livers are obtained from hospitals, the livers are trimmed, redundant tissues in the hepatic portal area are trimmed, and hepatic portal lymph nodes are removed and cleaned. Connecting with perfusion apparatus, perfusing portal vein with flow constant mode, flow setting to 0.5mL/min/g (liver weight), perfusing hepatic artery with pressure control mode at 18 deg.C, pressure 80/60mmHg, and beginning perfusion at 18 deg.C. The livers obtained were divided into ten groups: the first group is lactic acid ringer's solution; the second group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 32 kD-256 kD and the content of more than or equal to 95 percent; the third group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 128 kD-256 kD and the content of more than or equal to 95 percent; the fourth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 65 percent; the fifth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 80 percent; the sixth group is that the hemoglobin in the embodiment 3 is replaced by the hemoglobin with the molecular weight of 320kD-1024kD and the content of 90 percent; the seventh group is the formula of the embodiment 3 of the invention, namely the hemoglobin with the molecular weight of 320kD-1024kD and the content of 95 percent; the eighth group is the formulation of example 3 of the present invention, i.e., hemoglobin with molecular weight of 320kD to 1024kD and 97% content; the ninth group is the formulation of the embodiment 3 of the invention, namely hemoglobin with molecular weight of 320kD-1024kD and content of 99 percent; the tenth group is polymerized hemoglobin (molecular weight 320kD-1024kD < 45%) prepared by the method of the company patent No. 201910846580.9.

altered levels of NO in tissue;

observing the change condition of the tissue by an electron microscope;

the results show that: the perfusate is taken at different perfusion time points for detection, the perfusion maintenance safety time limit is that the AST content is not more than 40U/L, ALT, the AST content is not more than 40U/L, TBIL, the AST content is not more than 20.5umol/L, ET-1, and the contents of the AST content is not more than 20ng/L, and the safety time limits of the perfused organs on the indexes are respectively 40min, 10h, 12h, 19h, 17h, 18h, 26h, 27h and 18 h. The half-lives of the polymerized hemoglobins in the second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth groups were 12h, 15h, 22h, 21h, 29h, 30h and 20h, respectively (see table 4 below). Then cutting small tissues at the safe time limit of each group of perfusate, weighing, cutting into pieces, adding a small amount of quartz sand and cold normal saline to homogenate, centrifuging the homogenate for 20min at 10000r/min below 4 ℃, taking supernatant to measure the NO content of the tissues, wherein the measured result is not lower than the lower limit standard of 1.80 umol/L. Simultaneously, the safety time limit point of each group of perfusate is 0.8 multiplied by 0.2cm³Tissue is pre-fixed for 2 hours at 4 ℃ by using 2.5% glutaraldehyde (0.1M) phosphate buffer solution with the pH value of 7.4, washed for 3 times for 1 hour in phosphate buffer solution with the pH value of 7.38, dehydrated step by using 50%, 70%, 80%, 90% and 95% acetone for 15 minutes each time, finally embedded in a 60-DEG incubator for polymerization for 48 hours, an ultrathin section is prepared by using an ultrathin slicer, double electron staining is carried out by using uranyl acetate and lead citrate, and JEM-1200EX electron microscope observation is carried out to observe the change of the cell morphology and further judge the change of the organ activity. The results of electron microscopy show that the liver lobular profile is clear within the safe time limit of each perfusateClearly, hepatic sinusoids within the lobules are radially arranged and anastomosed into a mesh, with extensive anastomosis between adjacent hepatic lobules, and visible interlobular arteries and interlobular veins in the area of the junction, with terminal branches of hepatic arteries constantly joining the hepatic sinusoids along the periphery of the hepatic lobules.

In summary, the safety time periods maintained by the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth groups under mechanical infusion at 18 ℃ were 40min, 10h, 12h, 19h, 17h, 18h, 26h, 27h, and 18h, respectively (as shown in table 4 below). The second and third groups use different sections of polymerized hemoglobin with a lower than 320kD, which have slightly different perfusion safety intervals, but shorter perfusion safety intervals. The tenth group used polymerized hemoglobin without separate mixing, which was generally not selectively perfused for a long period of time. The polymeric hemoglobin with the content of 320-1024kD is used in the fourth group, the fifth group, the sixth group and the seventh group, which is fluctuated with the content increase (65%, 80%, 90% and 95%) to prolong the perfusion safety time limit, but the perfusion safety time limit of the hemoglobin with the content of 320-1024kD of 95%, 97% and 99% used in the seventh group, the eighth group and the ninth group is not fluctuated any more, and has obvious perfusion safety time limit advantages.

Table 4: comparison of perfusion experiment effects of waste human liver under different formulas

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. Use of an ex vivo organ perfusate in the preparation of a medicament for preserving an ex vivo organ for transplantation, wherein the ex vivo organ perfusate consists of, on a total volume per 1000 mL:

the isolated organ perfusate comprises, per 10g of polymeric hemoglobin:

10g of high-molecular polymer hemoglobin is obtained,

2.5g to 4g of glucose,

7500u-10000u of heparin sodium,

2g-3g of sodium chloride,

0.5g-0.75g of cefoxitin sodium,

0.37g-0.5g of sodium bicarbonate,

15mL-20mL of 10% compound amino acid injection,

0.05mL-0.08mL of 12 kinds of compound vitamin for injection,

insulin 50u-80u, and

the balance of water;

collecting 1L of fresh blood, and diluting with 6g/L sodium citrate solution with volume 0.5-1 times of blood;

placing the above-mentioned material passed through the 60 μm depth filter on a 0.65 μm hollow fiber membrane, washing the material on the 0.65 μm hollow fiber membrane with 6g/L sodium citrate solution of 5-8 times the original blood volume so that the material less than 0.65 μm passes through the 0.65 μm hollow fiber membrane;

adding injection water into the substance retained by the 0.65 μm hollow fiber membrane according to the ratio of 1:1-2 in the initial blood volume to lyse red blood cells, ultrafiltering the lysed red blood cells with a 100KD filter membrane, wherein the permeation end is the desired hemoglobin, and simultaneously adding injection water according to the permeation rate to maintain the constant ultrafiltration volume until the yield of hemoglobin is more than or equal to 95%, and stopping 100KD ultrafiltration;

concentrating the hemoglobin purified by the 100KD filter membrane to a concentration of 10-14g/dL with a 30KD membrane module;

purifying the purified hemoglobin with the concentration of 10-14g/dL by anion chromatography to obtain pure hemoglobin;

then introducing inert gas to deoxidize until oxyhemoglobin is less than 5%, adding glutaraldehyde by an atomization method according to the proportion of 1g of hemoglobin to 35-45mg of glutaraldehyde, and terminating the polymerization reaction according to 1g of sodium borohydride with 13-18mg of hemoglobin;

then concentrating the obtained polymerized hemoglobin to 6-7g/dL, placing the polymerized hemoglobin concentrated to 6-7g/dL on an ultrafiltration membrane bag with 300KD, using lactated ringer's solution with the volume 5-10 times that of the polymerized hemoglobin with 6-7g/dL to exchange the solution to obtain the polymerized hemoglobin, collecting the protein solution when the content of the polymerized hemoglobin with the molecular weight of 320-1024kD reaches an index of more than or equal to 95 percent in the solution exchange process, introducing inert gas to deoxidize until the content of oxygenated hemoglobin is less than or equal to 5 percent, and filtering and sterilizing by 0.2 mu m to obtain the high molecular polymerized hemoglobin.

2. The use of claim 1, wherein the anionic chromatographic purification is carried out by:

equilibrating the column with a solution of 20mM tris; finishing the sample in 40min later; followed by a wash with 29Mmtris solution at 3 times the volume of purified hemoglobin at a concentration of 10-14 g/dL; followed by elution with a 50mM tris solution at 4-5 times the volume of purified hemoglobin at a concentration of 10-14 g/dL.

3. The use of claim 1, wherein the composition of ringer's lactate is: 6.73g/L of sodium chloride, 0.3g/L of potassium chloride, 0.2g/L of calcium chloride dihydrate and 22g/L of 40% sodium lactate, 3.07g/L, N-acetyl-L-cysteine.

4. The use of claim 1, said ex vivo organ perfusate consisting of, per 1000mL of total volume:

30g of high-molecular polymer hemoglobin,

9g of glucose is added into the mixture,

the content of the heparin sodium is 25000u,

7g of sodium chloride, namely 7g of sodium chloride,

2g of cefoxitin sodium, namely 2g,

sodium hydrogen carbonate (1.25 g),

50mL of 10 percent compound amino acid injection,

0.2mL of 12 kinds of compound vitamin for injection,

insulin 180u, and

the balance of water.

5. The use of claim 1, said ex vivo organ perfusate consisting of, per 1000mL of total volume:

40g of high-molecular polymer hemoglobin is prepared,

10g of glucose is added into the mixture,

the heparin sodium is 30000u,

8g of sodium chloride, namely 8g of sodium chloride,

3g of cefoxitin sodium, namely 3g of cefoxitin sodium,

1.5g of sodium bicarbonate is added,

60mL of 10 percent compound amino acid injection,

0.3mL of 12 kinds of compound vitamin for injection,

insulin 200u, and

the balance of water.

6. The use of claim 1, said ex vivo organ perfusate consisting of, per 1000mL of total volume:

20g of high-molecular polymer Hemoglobin (HMP),

the glucose content of the mixture is 8g,

the heparin sodium is 20000u,

6g of sodium chloride, namely 6g of sodium chloride,

1g of cefoxitin sodium, namely 1g,

1g of sodium bicarbonate, namely sodium bicarbonate,

40mL of 10 percent compound amino acid injection,

0.1mL of 12 kinds of compound vitamin for injection,

insulin 160u, and

the balance of water.

7. The use according to any one of claims 1 to 6, wherein the preservation is carried out at 2 to 40 ℃.

8. The use according to any one of claims 1 to 6, wherein the storage is carried out at 4 to 37 ℃.

9. The use according to any one of claims 1 to 6, wherein the storage is carried out at 10 to 30 ℃.

10. The use according to any one of claims 1 to 6, wherein the storage is carried out at 16 to 20 ℃.

11. The use according to any one of claims 1 to 6, wherein the storage is carried out at 18 ℃.

12. The use of any one of claims 1-6, wherein the cefoxitin sodium is cefoxitin sodium for injection; or

The water is injection water; or

The 10% compound amino acid injection is a 10% compound amino acid injection produced by Chenxin pharmaceutical industry; or

The 12 kinds of compound vitamins for injection are '12 kinds of compound vitamins for injection' produced by Shanxi Pude pharmaceutical industry Limited.

13. The use of any one of claims 1-6, wherein the isolated organ is a heart, liver, kidney or lung.

14. The use of any one of claims 1-6, wherein the isolated organ is a human, dog, rat or pig isolated organ.