阅读说明：本技术 一种灌注保存液 (Perfusion preservation liquid ) 是由 何惠仪 加文·杰尔 巴里·富勒 陈焕伟 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种灌注保存液,包括以下浓度的组分：柠檬酸钾8.5-10g/L；柠檬酸钠8-9g/L；甘露醇30-35g/L；硫酸镁8-12g/L；HIF-PH抑制剂25-1000μM,所述灌注保存液用于在保存温度发生变化条件下细胞、组织或器官的动态保存。该灌注保存液能用于器官或细胞的动态保存应用,保证器官或细胞的活性,减轻移植细胞、组织或器官的冷缺血再灌注损伤,从而在保存温度变化的环境中提高细胞、组织或器官存活率,提高移植效果质量。(The invention discloses a perfusion preservation solution which comprises the following components in concentration: 8.5-10g/L potassium citrate; 8-9g/L of sodium citrate; 30-35g/L of mannitol; 8-12g/L magnesium sulfate; HIF-PH inhibitor 25-1000 mu M, the perfusion preserving fluid is used for dynamic preservation of cells, tissues or organs under the condition of changing preserving temperature. The perfusion preservation solution can be used for dynamically preserving and applying organs or cells, the activity of the organs or cells is ensured, and the cold ischemia reperfusion injury of transplanted cells, tissues or organs is relieved, so that the survival rate of the cells, the tissues or the organs is improved in the environment with the change of preservation temperature, and the quality of the transplanting effect is improved.)

1. A perfusion preservation solution is characterized in that: comprising the following components in concentration:

8.5-10g/L potassium citrate;

8-9g/L of sodium citrate;

30-35g/L of mannitol;

8-12g/L magnesium sulfate;

HIF-PH inhibitor 25-1000. mu.M;

the perfusion preservation solution is used for dynamically preserving cells, tissues or organs under the condition that the preservation temperature changes.

2. The perfusion preservation solution according to claim 1, wherein: the HIF-PH inhibitor is a cobalt salt compound.

3. The perfusion preservation solution according to claim 2, wherein: the HIF-PH inhibitor is cobalt chloride hydrate, and the concentration of the cobalt chloride hydrate in the perfusion preservation solution is 25-200 mu M.

4. The perfusion preservation solution according to claim 1, wherein: the HIF-PH inhibitor is dimethyloxalyl glycine, and the concentration of the dimethyloxalyl glycine in the perfusion preservation solution is 125-1000 mu M.

5. The perfusion preservation solution according to claim 1, wherein: the pH value of the perfusion preservation solution is 7.0-8.0.

6. The perfusion preservation solution according to claim 1, wherein: the osmotic pressure of the perfusion preservation solution is 450-500 mOsmol/kg.

7. Use of the perfusion preservation solution of any one of claims 1-6 in cell, tissue or organ transplantation, wherein the perfusion preservation solution can alleviate stress injury of cells, tissues or organs during preservation due to preservation temperature change.

8. A method for preserving cells, comprising the steps of:

(1) inoculating the cells into a cell culture plate at a certain inoculation density, and culturing for 20-30 hours in an aerobic incubator;

(2) refrigerating the cultured cells in the perfusion preservation solution of any one of claims 1-6 for 20-30 hours at a refrigerating temperature of 3-5 ℃;

(3) the cells are returned to fresh medium, slowly warmed to 35-40 ℃ and cultured for 5-8 days for cell survival, recovery and recovery.

9. A method of preserving cells according to claim 8, wherein: the seeding density of the cells in step (1) was 7000-9000 cells/cm.

Technical Field

The invention belongs to the technical field of organ transplantation, and particularly relates to a perfusion preservation solution.

Background

At present, in addition to HCA preservation solution which is self-developed in China, other organ preservation solution such as University of Wisconsin (UW) is expensive and basically depends on import. Therefore, the development of the perfusion preservation solution which has excellent preservation effect on multiple organs or single organ and is low in price is urgent. In addition, the current organ perfusion preservation solution has the defects that the static refrigeration application can be only carried out on organs or cells, the organ or cell storage time is limited, when the preservation temperature is increased, the vitality and the proliferation capacity of the organs or cells are rapidly reduced, the organ damage or the cell apoptosis is very easy to occur, and the condition of being not beneficial to transplantation is caused.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a perfusion preservation solution which can be used for dynamic preservation application of organs or cells, ensures the activity of the organs or cells, and relieves cold ischemia reperfusion injury of transplanted cells, tissues or organs, thereby improving the survival rate of the cells, tissues or organs in an environment with temperature change and improving the quality of a transplanting effect.

The technical purpose of the invention is realized by the following technical scheme:

a perfusion preservation solution comprising the following components in concentration: 8.5-10g/L potassium citrate; 8-9g/L of sodium citrate; 30-35g/L of mannitol; 8-12g/L magnesium sulfate; HIF-PH inhibitor 25-1000. mu.M; the perfusion preservation solution is used for dynamically preserving cells, tissues or organs under the condition that the preservation temperature changes.

Preferably, the HIF-PH inhibitor is a cobalt salt.

Preferably, the HIF-PH inhibitor is cobalt chloride hydrate, and the concentration of the cobalt chloride hydrate in the perfusion preservation solution is 25-200 μ M.

Further preferably, the HIF-PH inhibitor is cobalt chloride hydrate, and the concentration of the cobalt chloride hydrate in the perfusion preservation solution is 100 μ M or 200 μ M.

Preferably, the HIF-PH inhibitor is dimethyloxalglycine, and the concentration of dimethyloxalglycine in the perfusion stock solution is 125-1000. mu.M.

Further preferably, the HIF-PH inhibitor is dimethyloxalglycine, and the concentration of dimethyloxalglycine in the perfusion stock solution is 500 or 1000 μ M.

Preferably, the pH of the perfusion preservation solution is 7.0-8.0.

Preferably, the osmotic pressure of the perfusion preservation solution is 450-500 mOsmol/kg.

The perfusion preservation solution can relieve stress injury of cells, tissues or organs caused by preservation temperature change in the preservation process.

A method of preserving cells comprising the steps of:

(1) inoculating the cells into a cell culture plate at a certain inoculation density, and culturing for 20-30 hours in an aerobic incubator;

(2) refrigerating the cultured cells in the perfusion preservation solution for 20-30 hours at the temperature of 3-5 ℃;

(3) the cells are returned to fresh medium, slowly warmed to 35-40 ℃ and cultured for 5-8 days for cell survival, recovery and recovery.

Preferably, the seeding density of the cells in step (1) is 7000-9000 cells/cm.

The invention has the beneficial effects that:

(1) the perfusion preservation liquid system contains a specific HIF-PH inhibitor, which can effectively inhibit the decomposition of HIF-1 alpha, prolong the service life of the HIF-1 alpha in isolated cells, improve the adaptability of the isolated cells to the environment, be used for dynamically preserving the cells, effectively reduce stress injury, and still have higher cell survival rate and cell activity after being preserved at low temperature (4 ℃) for 24 hours and then being reheated to 37 ℃;

(2) the applicant discovers for the first time that the perfusion preservation solution system can promote HepG2 spheres to form a simulated liver natural tissue;

(3) after the perfusion preservation liquid system treats the isolated cells, the daughter cells of the isolated cells still have stronger recovery capability and survival rate during isolated culture.

Drawings

In FIG. 1, A is the cell metabolic activity of the ADMSCs after 1 day, 3 days and 7 days of culture, and B in FIG. 1 is the DNA content of the cell culture solution of the ADMSCs after 1 day, 3 days and 7 days of culture;

in FIG. 2, A represents the cell metabolic activity of HepG2 cells after being refrigerated for 24 hours (4 ℃) in perfusion preservation solution, and B in FIG. 2 represents the DNA content of HepG2 cells in cell culture solution after being cultured for 1 day, 3 days and 7 days;

FIG. 3 is a fluorescent plot of HepG2 cells after 7 days of culture, stained and encapsulated in 3D hydrogel about 100 μm thick in HepG2 cells;

FIG. 4 is a fluorescent graph of HIF-1. alpha. expression levels in HepG2 cells 30 minutes after rewarming (37 ℃, 21% normoxia);

FIG. 5 shows HepG2 daughter cells obtained after the HepG2 cells were cultured by the method of test example, which were cultured again by the method of test example, and the perfusion preservation solutions of comparative example 1 were used during the culture again by the method of test example, wherein A in FIG. 5 represents the amount of metabolic activity of the HepG2 daughter cells after 1 day, 3 days, and 7 days of culture, and B in FIG. 5 represents the amount of DNA in the cell culture solution of the HepG2 daughter cells after 1 day, 3 days, and 7 days of culture.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; 25 mu M of hydrated cobalt chloride, the pH value of the perfusion preservation solution is 7.1, and the osmotic pressure of the perfusion preservation solution is 486 mOsmol/kg.

Example 2:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; 50 mu M of hydrated cobalt chloride, the pH value of the perfusion preservation solution is 7.1, and the osmotic pressure of the perfusion preservation solution is 486 mOsmol/kg.

Example 3:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; 100 mu M of hydrated cobalt chloride, the pH value of the perfusion preservation solution is 7.1, and the osmotic pressure of the perfusion preservation solution is 486 mOsmol/kg.

Example 4:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; cobalt chloride hydrate of 200 μ M, pH of the perfusion preservation solution of 7.1, and osmotic pressure of the perfusion preservation solution of 486 mOsmol/kg.

Example 5:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; 125 mu M of dimethyloxalglycine, the pH value of the perfusion preservation solution is 7.1, and the osmotic pressure of the perfusion preservation solution is 486 mOsmol/kg.

Example 6:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; 250 mu M of dimethyloxalglycine, 7.1 of pH of the perfusion preservation solution and 486mOsmol/kg of osmotic pressure of the perfusion preservation solution.

Example 7:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; dimethyloxalglycine 500. mu.M, pH of the perfusion preservation solution 7.1, osmolality of the perfusion preservation solution 486 mOsmol/kg.

Example 8:

a perfusion preservation solution comprising the following components in concentration: 8.6g/L of potassium citrate; 8.2g/L of sodium citrate; mannitol 33.8 g/L; magnesium sulfate is 10 g/L; dimethyloxalglycine 1000. mu.M, pH of the perfusion preservation solution 7.1, and osmotic pressure of the perfusion preservation solution 486 mOsmol/kg.

Example 9:

a perfusion preservation solution comprising the following components in concentration: 10g/L of potassium citrate; 9g/L of sodium citrate; 35g/L of mannitol; magnesium sulfate is 8 g/L; 50 mu M of hydrated cobalt chloride, the pH value of the perfusion preservation solution is 7.0, and the osmotic pressure of the perfusion preservation solution is 450 mOsmol/kg.

Example 10:

a perfusion preservation solution comprising the following components in concentration: 8.5g/L of potassium citrate; 8g/L of sodium citrate; 30g/L of mannitol; 12g/L magnesium sulfate; dimethyloxalglycine 500. mu.M, pH of the perfusion preservation solution 8.0, osmolality of the perfusion preservation solution 500 mOsmol/kg.

Comparative example 1:

a perfusion preservation solution is different from the perfusion preservation solution in example 1 only in that the perfusion preservation solution does not contain HIF-PH inhibitor, and the rest formulas and the concentrations of the formulas are completely the same as the perfusion preservation solution in example 1.

Test example:

the test was carried out according to the following procedure:

(1) inoculating the ADMSCs (human adipose-derived mesenchymal stem cells) and the HepG2 cells (human hepatocytes) into a 96-well plate at a density of 8000 cells/cm, respectively, and culturing in an normoxic incubator for 24 hours;

(2) the cultured cells were refrigerated in the perfusion preservation solutions of examples 1 to 8 and comparative example 1 for 24 hours at 4 ℃;

(3) the cells were returned to fresh medium, warmed slowly to 37 ℃ and cultured for 7 days for cell survival, recovery and recovery.

Analyzing the metabolic activity of the cells and analyzing the proliferation of the cells through the DNA content.

The test results are shown in fig. 1-5.

As can be seen from fig. 1, after the perfusion preservation solution of the present invention is used to treat the ADMSCs cells, the metabolic activity and proliferation of the ADMSCs cells are greatly affected, and after the treatment with the perfusion preservation solution of the present invention and the rewarming (37 ℃) stress, the ADMSCs cells still have strong resuscitation and recovery capabilities, thereby indicating that the perfusion preservation solution of the present invention can significantly improve the resuscitation capability and cell survival rate of the ADMSCs cells.

It can be seen from a in fig. 2 that after the perfusion preservation solution of the present invention is used to treat HepG2 cells, HepG2 cells still have strong metabolic capacity when being refrigerated at 4 ℃, and it can be seen from B in fig. 2 that after HepG2 cells are treated by the perfusion preservation solution of the present invention, the proliferation of HepG2 cells is greatly affected, and after the treatment by the perfusion preservation solution of the present invention and then the rewarming (37 ℃) stress, HepG2 cells still have strong resuscitation and recovery capacities, and it can be seen that the perfusion preservation solution of the present invention can significantly improve the cell resuscitation capacity and cell survival rate of HepG2 cells.

As can be seen from fig. 3, the perfusion preservation solution of the present application can promote the formation of HepG2 spheroids mimicking natural tissues in the liver when the concentration of cobalt chloride hydrate in the perfusion preservation solution of the present application is 100 μ M or 200 μ M, or when the concentration of dimethyloxalyl glycine in the perfusion preservation solution of the present application is 500 μ M or 1000 μ M.

As is clear from FIG. 4, the half-life of HIF-1. alpha. can be stabilized and prolonged from less than 5 minutes to 30 minutes in the case where the concentration of cobalt chloride hydrate in the perfusion preservation solution of the present application is 100. mu.M or 200. mu.M, or the concentration of dimethyloxalylglycine in the perfusion preservation solution of the present application is 500. mu.M or 1000. mu.M.

As can be seen from fig. 5, when the concentration of cobalt chloride hydrate in the perfusion preservation solution of the present application is 100 μ M or 200 μ M, or when the concentration of dimethyloxalyl glycine in the perfusion preservation solution of the present application is 500 μ M or 1000 μ M, HepG2 cells were cultured by the method of the test example to obtain HepG2 daughter cells, which were cultured again by the method of the test example, and the perfusion preservation solution of comparative example 1 was used in the culture process again by the method of the test example, HepG2 daughter cells still have strong metabolic capability, and HepG2 daughter cells still have strong recovery and recovery capability after rewarming (37 ℃) stress, which indicates that the perfusion preservation solution of the present invention can make the daughter cells of the isolated cells still have strong recovery and survival capability during the isolated culture after treating the isolated cells.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.