CN112425602A - Isolated lung mechanical perfusion liquid and preparation method and application thereof - Google Patents
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Abstract
The invention relates to an isolated lung mechanical perfusate and a preparation method and application thereof, wherein the isolated lung mechanical perfusate comprises a basic culture medium, human serum albumin, polysucrose, dextran, mannitol and nitroglycerin; in the isolated lung mechanical perfusate, the concentration of human serum albumin is 5-20 g/L, the concentration of polysucrose is 25-70 g/L, the concentration of dextran is 1-50 g/L, the concentration of mannitol is 1-15 g/L, and the concentration of nitroglycerin is 1-20 g/L. The mechanical perfusate for the isolated lung can keep the isolated lung stable in function for a long time, relieve pulmonary edema, repair the lung, expand the utilization rate of the supplied lung, reduce the use amount of albumin and greatly reduce the cost.
Description
Technical Field
The invention relates to the technical field of organ preservation, in particular to an isolated lung mechanical perfusate and a preparation method and application thereof.
Background
Since the inception of Lung transplantation technology (Lung Transplantations), clinical Lung transplantation has been largely developed and with great success over the past few decades. Lung transplantation is an effective way to treat end-stage pulmonary parenchymal and pulmonary vascular diseases, and the number of lung transplants is still rapidly increasing every year. Lung transplantation is the ultimate solution for patients with end-stage respiratory failure, however the rate of obtaining lung organs from deceased donors is much lower than other solid organs. The proportion of lungs available for transplantation is low due to the fact that donor lungs are subject to various injury mechanisms during brain death and organ donation (e.g., ventilator acquired pneumonia, neurogenic and resting pulmonary edema, barotrauma, etc.). Thus, most donor lungs are not used for transplantation. Although there have been some improvements in surgical techniques and perioperative treatments, ischemia-reperfusion-induced lung injury (ischemia/reperfusion-induced lung injury) remains a significant cause of early death following lung transplantation, mainly characterized by nonspecific alveolar injury, pulmonary edema, and hypoxemia. In addition, severe reperfusion injury increases the incidence of acute rejection of the donor lung and long-term lung failure. And the effect of relieving ischemia reperfusion lung injury is positively correlated with the preservation effect of the donor lung, so that only by improving the preservation perfusion technology of the donor lung, each donor lung can be more effectively utilized, and the early mortality rate after transplantation is reduced. Compared with the traditional static cold preservation, the mechanical perfusion can better preserve the isolated lung and even repair the lung organ of the marginal donor, provides a new idea for solving the shortage of the lung, and is expected to further expand the source of the supplied lung. In the 90 s of the 20 th century, mechanical lung perfusion techniques were used to study lung physiology. In the field of lung transplantation, Steen, et al, 2003 proposed Ex Vivo Lung Perfusion (EVLP) and further improved in the toronto lung transplantation group of canada.
At present, the perfusion preservation solution used in lung transplantation is mainly Steen solution, is expensive and single in product, and the maximum time of mechanical perfusion of the lung by the Steen solution can only be maintained for 12 hours. This limits the ability of EVLP to perform perfusion repair, for example, for bacterial pneumonia, which is difficult to treat effectively within 12 hours. If the EVLP maintenance time is prolonged, the marginal lung supply can be better evaluated and repaired, for example, some gene therapy and stem cell therapy can be applied to the repair of the marginal lung, so that the use rate of the supply lung is expanded.
Disclosure of Invention
In view of the above, there is a need for an ex vivo pulmonary mechanical perfusate that can improve the duration of stable pulmonary function.
An in vitro lung mechanical perfusion solution comprises a basic culture medium, human serum albumin, polysucrose, dextran, mannitol and nitroglycerin; in the in-vitro lung mechanical perfusion solution, the concentration of human serum albumin is 5-20 g/L, the concentration of ficoll is 25-70 g/L, the concentration of dextran is 1-50 g/L, the concentration of mannitol is 1-15 g/L, and the concentration of nitroglycerin is 1-20 g/L.
In one embodiment, in the in-vitro lung mechanical perfusion solution, the concentration of human serum albumin is 8g/L to 15g/L, the concentration of ficoll is 55g/L to 65g/L, the concentration of dextran is 5g/L to 15g/L, the concentration of mannitol is 4g/L to 10g/L, and the concentration of nitroglycerin is 5g/L to 15 g/L.
In one embodiment, in the in-vitro lung mechanical perfusion solution, the concentration of human serum albumin is 8g/L to 12g/L, the concentration of ficoll is 58g/L to 62g/L, the concentration of dextran is 8g/L to 12g/L, the concentration of mannitol is 6g/L to 8g/L, and the concentration of nitroglycerin is 9g/L to 11 g/L.
In one embodiment, the basal medium is one or more of DMEM medium, F12 medium, RPMI-1640 medium, and McCoy's 5A medium.
In one embodiment, the basal medium is a DMEM medium and a F12 medium, and the volume ratio of the DMEM medium to the F12 medium is 1 (0.9-1.1).
In one embodiment, the ficoll is ficoll 70.
In one embodiment, the dextran is dextran 40.
In one embodiment, the isolated lung mechanical perfusion solution has a pH of 7.2-7.6 and an osmotic pressure of 290-350 mOsm/kg.
The invention also provides a preparation method of the in vitro lung mechanical perfusion solution, which comprises the following steps: dissolving the human serum albumin, the polysucrose, the dextran, the mannitol and the nitroglycerin in the basic culture medium, and then adjusting the pH value to 7.2-7.6.
The invention also provides application of the isolated lung mechanical perfusion fluid in isolated lung preservation.
The human serum albumin can provide colloid osmotic pressure, prevent the pulmonary edema produced in the process of perfusion, and the decomposition product amino acid of the human serum albumin can provide nutrient substances for cells, and the human serum albumin can also combine other nutrient substances such as trace elements and the like to carry out nutrient substance transportation, and can also play an important role in maintaining the stability of other enzymes in the cells. Dextran, as a mild free radical scavenger, can reduce free radicals generated during ischemia-reperfusion during perfusion, thereby reducing ischemia-reperfusion injury, and in addition, can protect lung endothelial cells from injury caused by inflammatory reaction or blood coagulation, and can also provide partial oncotic pressure. The in vitro lung mechanical perfusion solution is prepared by adding polysucrose, mannitol, nitroglycerin and the like into a basic culture medium for matching on the basis of adding human serum albumin and dextran, and further adjusting and optimizing the concentration of each component, so that the in vitro lung function can be kept stable for a long time, pulmonary edema is reduced, lung repair is carried out, the utilization rate of the supplied lung is increased, the use amount of albumin is reduced, and the cost is greatly reduced.
Drawings
Fig. 1 is a graph of oxygenation index changes after ex vivo perfusion repair of the lung using ex vivo pulmonary mechanical perfusion fluid of example 1;
fig. 2 is a graph of the change in compliance after ex vivo perfusion repair of the lung using the ex vivo pulmonary mechanical perfusion fluid of example 1;
fig. 3 is a graph of tidal volume and peak airway pressure changes after ex vivo lung perfusion repair using the ex vivo lung mechanical perfusion fluid of example 1.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The in-vitro lung mechanical perfusion solution provided by the embodiment of the invention comprises a basic culture medium, human serum albumin, ficoll, dextran, mannitol and nitroglycerin. In the isolated lung mechanical perfusate, the concentration of human serum albumin is 5-20 g/L, the concentration of polysucrose is 25-70 g/L, the concentration of dextran is 1-50 g/L, the concentration of mannitol is 1-15 g/L, and the concentration of nitroglycerin is 1-20 g/L.
The human serum albumin can provide colloid osmotic pressure, prevent the pulmonary edema produced in the process of perfusion, and the decomposition product amino acid of the human serum albumin can provide nutrient substances for cells, and the human serum albumin can also combine other nutrient substances such as trace elements and the like to carry out nutrient substance transportation, and can also play an important role in maintaining the stability of other enzymes in the cells. Dextran, as a mild free radical scavenger, can reduce free radicals generated during ischemia-reperfusion during perfusion, thereby reducing ischemia-reperfusion injury, and in addition, can protect lung endothelial cells from injury caused by inflammatory reaction or blood coagulation, and can also provide partial oncotic pressure. The in vitro lung mechanical perfusion solution is prepared by adding polysucrose, mannitol, nitroglycerin and the like into a basic culture medium for matching on the basis of adding human serum albumin and dextran, and further adjusting and optimizing the concentration of each component, so that the in vitro lung function can be kept stable for a long time, pulmonary edema is reduced, lung repair is carried out, the utilization rate of the supplied lung is increased, the use amount of albumin is reduced, and the cost is greatly reduced.
In a specific example, in the isolated lung mechanical perfusion solution, the concentration of human serum albumin is 8 g/L-15 g/L, the concentration of polysucrose is 55 g/L-65 g/L, the concentration of dextran is 5 g/L-15 g/L, the concentration of mannitol is 4 g/L-10 g/L, and the concentration of nitroglycerin is 5 g/L-15 g/L.
In a specific example, in the isolated lung mechanical perfusion solution, the concentration of human serum albumin is 8 g/L-12 g/L, the concentration of polysucrose is 58 g/L-62 g/L, the concentration of dextran is 8 g/L-12 g/L, the concentration of mannitol is 6 g/L-8 g/L, and the concentration of nitroglycerin is 9 g/L-11 g/L.
In a specific example, the basal medium is one or more of DMEM medium, F12 medium, RPMI-1640 medium, and McCoy's 5A medium. Preferably, the basal medium is a DMEM medium and an F12 medium, and the volume ratio of the DMEM medium to the F12 medium is 1 (0.9-1.1).
In one embodiment, the basal medium is prepared by mixing DMEM medium and F12 medium at a volume ratio of 1:1, and the specific components are shown in the following table.
Preferably, the ficoll is ficoll 70. Preferably, the dextran is dextran 40.
In a specific example, the isolated lung mechanical perfusate has a pH of 7.2-7.6 and an osmolality of 290-350 mOsm/kg.
The preparation method of the in-vitro lung mechanical perfusion fluid provided by the embodiment of the invention comprises the following steps: human serum albumin, polysucrose, dextran, mannitol and nitroglycerin are dissolved in a basal medium according to concentration, and then the pH is adjusted to 7.2-7.6 with a pH adjusting substance such as sodium hydroxide.
The following are specific examples.
Example 1
Human serum albumin, polysucrose 70, dextran 40, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
10 |
| Polysucrose 70 | 60 |
| Dextran 40 | 10 |
| Mannitol | 7.2 |
| Nitroglycerin | 10 |
The isolated lung mechanical perfusate prepared in example 1 was used for animal experiments.
Anesthesia and weighing: pigs for experiment were injected intramuscularly with 2ml atropine, 15min later with 2ml shutitai, and after stabilization the pigs were weighed to a weight of 30.0 kg. Establishing a venous channel and continuously administering propofol intravenously.
Opening the chest: after intubation of the trachea and respiratory support, the chest was opened.
Lung in vivo perfusion: no. 10X 12 special-shaped joints are adopted in the body forward irrigation, 2L cold LPD (low potassium dextran) liquid flows in from pulmonary artery, and left atrial appendage flows out. After opening the chest, the patient can fill the chest in the body quickly.
Cardiopulmonary harvest: the pericardium and the lung ligament were separated blunt, the superior and inferior vena cava were cut, the aorta was cut, the trachea and the esophagus were separated, and the cardiopulmonary module was carefully removed.
In vitro reverse perfusion: the in vitro reverse perfusion adopts an inverted funnel, and 1L of LPD liquid is perfused.
The isolated lung mechanical perfusion fluid prepared in example 1 is then used for isolated lung perfusion repair, and the isolated lung mechanical perfusion fluid can be maintained for 24 hours, and the lung does not have edema.
During the perfusion process, the perfusion is started and is performed every 4 hoursThe perfusate flowing out through pulmonary vein is subjected to blood gas detection, and the oxygen concentration fraction in the inhaled air is set as 100% before detection to obtain the pulmonary vein oxygen pressure (PaO) at each time point2) Data, calculate oxygenation index (PO)2/FiO2) As shown in fig. 1. As can be seen in fig. 1, the slow decline occurred during the first 8 hours, followed by a sustained increase to 255mmHg, which was 4.4% higher than the increase at the beginning of perfusion, indicating that the oxygenation capacity of the lung was enhanced and lung function was restored during perfusion.
During the perfusion process, the ventilator performs dynamic real-time detection on the compliance of the lung, and data of perfusion for 0 hour and every 2 hours are recorded from real-time data on the ventilator, as shown in fig. 2. As can be seen from FIG. 2, the lung compliance generally increased throughout the perfusion process, from 14.4ml/cmH at 0 hours of perfusion, except for the decrease in compliance at 6-8 hours, 14-16 hours, and 18-20 hours2The O content increased to 23.1ml/cmH2O, rise by 60.4%, indicating increased lung tissue elasticity, decreased airway resistance, and decreased pulmonary edema.
During the perfusion process, the ventilator performs dynamic real-time detection on the tidal volume of the lung and the peak airway pressure, and data of perfusion for 0 hour and every 2 hours are recorded from real-time data on the ventilator, as shown in fig. 3. As can be seen from fig. 3, the tidal volume rises significantly during the perfusion process from 2 hours to 6 hours, and then remains relatively stable during the perfusion process. Throughout the perfusion, tidal volume increased from 170ml at 0 hours to 188ml at the end of perfusion, an increase of 10.6%, indicating improved lung function. It can also be seen from fig. 3 that peak airway pressure remained stable and decreased throughout the perfusion, except for fluctuations that decreased and then increased between 8 hours and 14 hours. Airway peak pressure from 0 hour perfusion at 16cmH2O drop to 14cmH at end of perfusion2A 12.5% drop in O indicates a decrease in airway resistance. During the entire perfusion process, both the rise in tidal volume and the decrease in peak airway pressure indicate a reduction in pulmonary edema and an improvement in pulmonary function.
Example 2
Human serum albumin, polysucrose 70, dextran 40, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
The isolated lung mechanical perfusion fluid prepared in the example 2 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 8 hours, and edema does not appear in the lung.
Example 3
Human serum albumin, polysucrose 70, dextran 40, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| Human serum albumin | 8 |
| Polysucrose 70 | 65 |
| |
15 |
| |
10 |
| |
15 |
The isolated lung mechanical perfusion fluid prepared in example 3 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 18 hours, and edema does not appear in the lung.
Example 4
Human serum albumin, polysucrose 70, dextran 40, mannitol and nitroglycerin were dissolved in RPMI-1640 medium according to the concentrations in the table below, and then the pH was adjusted to 8.
The isolated lung mechanical perfusion fluid prepared in example 4 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 20 hours, and edema does not appear in the lung.
Example 5
Human serum albumin, polysucrose 400, dextran 70, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
10 |
| Polysucrose 400 | 60 |
| Dextran 70 | 10 |
| Mannitol | 7.2 |
| |
10 |
The isolated lung mechanical perfusion fluid prepared in example 5 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 6 hours, and edema does not appear in the lung.
Comparative example 1
Human serum albumin, dextran 40 and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
10 |
| |
10 |
| |
10 |
The isolated lung mechanical perfusion fluid prepared in the comparative example 1 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 2 hours, and edema does not appear in the lung.
Comparative example 2
Human serum albumin, polysucrose 70 and dextran 40 were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
10 |
| Polysucrose 70 | 60 |
| |
10 |
The isolated lung mechanical perfusion fluid prepared in the comparative example 2 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 1 hour, and edema does not appear in the lung.
Comparative example 3
Human serum albumin, sucrose, dextran 40, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
10 |
| |
60 |
| |
10 |
| Mannitol | 7.2 |
| |
10 |
The isolated lung mechanical perfusion fluid prepared in the comparative example 3 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 2 hours, and edema does not appear in the lung.
Comparative example 4
Human serum albumin, polysucrose 70, dextran 40, mannitol and nitroglycerin were dissolved in DMEM/F12(1:1) medium according to the concentrations in the table below, and then the pH was adjusted to 7.4 with sodium hydroxide.
| Components | Concentration (g/L) |
| |
40 |
| Polysucrose 70 | 10 |
| |
60 |
| Mannitol | 0.5 |
| Nitroglycerin | 0.5 |
The isolated lung mechanical perfusion fluid prepared in the comparative example 4 is used for isolated perfusion repair of the lung, the isolated lung mechanical perfusion fluid can be maintained for 4 hours, and edema does not appear in the lung.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The in-vitro lung mechanical perfusion solution is characterized by comprising a basic culture medium, human serum albumin, ficoll, dextran, mannitol and nitroglycerin; in the in-vitro lung mechanical perfusion solution, the concentration of human serum albumin is 5-20 g/L, the concentration of ficoll is 25-70 g/L, the concentration of dextran is 1-50 g/L, the concentration of mannitol is 1-15 g/L, and the concentration of nitroglycerin is 1-20 g/L.
2. The ex-vivo lung mechanical perfusion solution of claim 1, wherein the concentration of human serum albumin in the ex-vivo lung mechanical perfusion solution is 8g/L to 15g/L, the concentration of ficoll is 55g/L to 65g/L, the concentration of dextran is 5g/L to 15g/L, the concentration of mannitol is 4g/L to 10g/L, and the concentration of nitroglycerin is 5g/L to 15 g/L.
3. The ex-vivo lung mechanical perfusion solution of claim 1, wherein the concentration of human serum albumin in the ex-vivo lung mechanical perfusion solution is 8g/L to 12g/L, the concentration of ficoll is 58g/L to 62g/L, the concentration of dextran is 8g/L to 12g/L, the concentration of mannitol is 6g/L to 8g/L, and the concentration of nitroglycerin is 9g/L to 11 g/L.
4. The ex vivo lung mechanical perfusion solution of claim 1, wherein the basal medium is one or more of DMEM medium, F12 medium, RPMI-1640 medium, and McCoy's 5A medium.
5. The ex-vivo lung mechanical perfusion solution as claimed in claim 4, wherein the basal medium is a DMEM medium and a F12 medium, and the volume ratio of the DMEM medium to the F12 medium is 1 (0.9-1.1).
6. The ex vivo lung mechanical perfusion fluid of any one of claims 1-5, wherein the ficoll is ficoll 70.
7. The ex-vivo lung mechanical perfusion solution as claimed in any one of claims 1 to 5, wherein the dextran is dextran 40.
8. The isolated lung mechanical perfusate of any one of claims 1 to 5, wherein the isolated lung mechanical perfusate has a pH of 7.2 to 7.6 and an osmolality of 290 to 350 mOsm/kg.
9. The preparation method of the isolated lung mechanical perfusion fluid of any one of claims 1-8, comprising the following steps: dissolving the human serum albumin, the polysucrose, the dextran, the mannitol and the nitroglycerin in the basic culture medium, and then adjusting the pH value to 7.2-7.6.
10. Use of the ex vivo pulmonary mechanical perfusion fluid of any one of claims 1-8 for preserving an ex vivo lung.
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