CN112138232A

CN112138232A - In-vitro plasma purification system and application thereof

Info

Publication number: CN112138232A
Application number: CN202011113645.8A
Authority: CN
Inventors: 黄媛; 张文健; 娄晋宁
Original assignee: Suzhou Rui Lai Biotechnology Co ltd
Current assignee: Suzhou Rui Lai Biotechnology Co ltd
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2020-12-29

Abstract

The invention provides an in vitro plasma purification system and application thereof. The purification system comprises a plasma bag, an oxygenation device, a peristaltic pump, a human hepatocyte plasma perfusion device, a fixed support, a fixed clamp, a cell filter and a circulation pipeline, wherein the plasma bag is provided with a sampling port, a liquid outlet and a liquid inlet; the fixing bracket and the fixing clamp fix the human hepatocyte plasma perfusion device; the liquid outlet of the plasma bag is connected with one end of the oxygenator and one end of the human hepatocyte plasma perfusion device in turn through a circulating pipeline, and the other end of the human hepatocyte plasma perfusion device is connected with the cell filter and the liquid inlet of the plasma bag through the circulating pipeline, thereby forming a closed loop. When in use, the peristaltic pump is clamped on the circulating pipeline to drive the plasma to circulate through the human hepatocyte plasma perfusion device, and the plasma purification purpose is achieved by means of the detoxification, synthesis and metabolism functions of the loaded hepatocytes. The invention is suitable for in vitro purification of the blood plasma of liver failure. Its advantages are simple structure, easy operation and easy control of in-vitro purifying condition and effect.

Description

In-vitro plasma purification system and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an in-vitro plasma purification system and application thereof.

Background

The biological artificial liver system is a set of devices for treating liver failure, the core part of the system is that liver cells cultured and proliferated in vitro are placed in a special bioreactor, plasma of a patient with liver failure is introduced into the bioreactor by using an extracorporeal circulation device such as a dialysis machine, the action mechanism is to exchange substances between the plasma of the patient and the liver cells in the bioreactor, the liver cells play roles of metabolism, detoxification, synthesis, secretion, biotransformation and the like, and can metabolize, convert bilirubin, synthesize and secrete blood coagulation factors, albumin and the like in the plasma of the patient, so that the effect of replacing the damaged liver function of the patient by the liver cells in the bioreactor is realized.

The bioartificial liver is a real-time treatment through extracorporeal circulation, is a set of very complex systems, and has certain safety risk in the treatment, so that the requirements on the bioartificial liver per se and the treatment process are high. Meanwhile, anticoagulant, supplementary liquid and the like are used in the treatment process, so that the treatment effect can be evaluated after a certain time is finished. The invention aims to provide an in-vitro plasma purification system, which can change the purification process of the plasma of a liver failure patient into in-vitro purification, is safe and controllable, can detect the plasma purification effect in time, can use the purified plasma for the plasma replacement therapy of the patient himself (namely the plasma in the patient with the same volume is replaced by the purified plasma) after the required purification effect is achieved, can continuously purify the plasma in vitro, then carries out plasma replacement again, and is repeated for multiple times until the patient is separated from the liver failure state.

Disclosure of Invention

In order to simplify the use method of the bioartificial liver and improve the safety, the invention aims to provide the in vitro plasma purification system which has simple composition, convenient operation, controllable in vitro plasma purification effect and low use safety risk.

It is another object of the present invention to provide the use of such ex vivo plasma purification system for ex vivo purification of liver failure plasma.

In order to achieve the purpose, the technical scheme adopted by the method is as follows:

the in vitro plasma purification system is characterized by comprising a plasma bag 1, an oxygenation device 2, a peristaltic pump 3, a human hepatocyte plasma perfusion device 4, a fixing bracket 5, a fixing clamp 6, a cell filter 7 and a circulating pipeline 8, wherein: the plasma bag is provided with a sampling port 9, a liquid outlet 10 and a liquid inlet 11; the fixing bracket and the fixing clamp fix the human hepatocyte plasma perfusion device; the liquid outlet of the plasma bag is connected with one end of the oxygenator and one end of the human hepatocyte plasma perfusion device in turn through a circulating pipeline, and the other end of the human hepatocyte plasma perfusion device is connected with the cell filter and the liquid inlet of the plasma bag through the circulating pipeline, thereby forming a closed loop.

In the in vitro plasma purification system, a closed circulation passage is formed between the plasma bag and the human hepatocyte plasma perfusion device, the plasma with purification is purified when passing through the human hepatocyte plasma perfusion device and then returns to the plasma bag, and the plasma can reach the required purification standard after circulating for a certain time.

In the in-vitro plasma purification system, the peristaltic pump is used for providing power for plasma circulation, and in order to be matched with the peristaltic pump, the circulating pipelines in the system all use silicone tubes, and the peristaltic pump can be placed on any section of circulating pipeline.

In order to ensure the activity and the function of the liver cells in the human liver cell plasma perfusion device, the in-vitro plasma purification system is provided with the oxygenation device, and the dissolved oxygen concentration is increased when the plasma flows through the oxygenation device, so that the oxygenated plasma can supply oxygen for the liver cells after entering the human liver cell plasma perfusion device.

Since the purified plasma can be used for the treatment of the patient himself, the purified plasma is free from insoluble particles such as exfoliated cells and cell debris. Usually the diameter of the hepatocytes is about 30 μm, but cell debris is considered to be intercepted, and with reference to the requirement of insoluble particles in the injection in the "chinese pharmacopoeia", a cell filter with an effective interception pore size of 5-10 μm is used in the ex vivo plasma purification system of the present invention. The cell filter may be a filter of a filtration membrane type or a disc filter.

In the in-vitro plasma purification system, the plasma bag is provided with the sampling port, so that the plasma in the plasma bag can be conveniently sampled in the in-vitro purification process of the plasma, the purification effect is judged by detecting relevant indexes in the plasma, such as total bilirubin, transaminase and the like, and the purification time is controlled, wherein the purification time is generally 2-5h, and the bilirubin can be reduced by over 40 percent.

In the in vitro plasma purification system, the human hepatocyte plasma perfusion device is the core playing a purification role, the device is a hollow container with two ports, and hepatocytes growing on microcarriers are filled in the device, wherein the hepatocytes are cells with hepatocyte functions, including but not limited to human hepatoma cell lines and immortalized human hepatocytes. When the hepatocyte growing on the microcarrier is filled in the human hepatocyte plasma perfusion device, the microcarrier hepatocyte is suspended in a culture medium for filling, before the human hepatocyte plasma perfusion device is connected with the in vitro plasma purification system of the invention for use, the liquid in the human hepatocyte plasma perfusion device needs to be discharged, sterile normal saline or ringer's solution is used for pre-flushing, and the system of the invention is connected after the pre-flushing.

In the in-vitro plasma purification system, the human hepatocyte plasma perfusion device is fixed on the fixed support through the fixed clamp, preferably vertically fixed, and enables plasma to enter from the lower port and exit from the upper port.

Another object of the method of the invention is to provide the use of said one ex vivo plasma purification system for ex vivo purification of liver failure plasma. In the application, the plasma replacement treatment is firstly used once, the liver failure plasma is obtained by replacing fresh plasma, then the liver failure plasma is put into a plasma bag, and the in vitro plasma purification system provided by the invention is used for purification.

The method for purifying the blood plasma in vitro by using the invention comprises the following steps:

fully pre-flushing the human hepatocyte plasma perfusion apparatus with ringer's solution under aseptic operation, removing the original solution inside, and fixing the human hepatocyte plasma perfusion apparatus 4 on a fixed bracket 5 by a fixing clamp 6; the plasma bag 1 is filled with the plasma of the liver failure patient, and the liquid outlet 10 of the plasma bag 1 is connected with the oxygenation device 2; the oxygenation device 2 is connected with the lower end of a human hepatocyte plasma perfusion device 4, and the upper end of the human hepatocyte plasma perfusion device 4 is connected with a cell filter 7; the cell filter 7 is connected with the liquid inlet 11 of the plasma bag 1 to form a circulation passage; the peristaltic pump 3 is clamped on a silicone tube, and the system device is placed in a constant temperature incubator at 37 ℃; starting the peristaltic pump 3, setting the rotating speed, and allowing the plasma to enter the human hepatocyte plasma perfusion device 4 at a slow speed for purification; the plasma after purifying is monitored through the sampling port 9, and the purifying process is completed after the monitoring index meets the requirement.

The invention has the beneficial effects that:

1. the operation under the in vitro condition not only reduces the safety risk, but also achieves the plasma purification effect of the bioartificial liver;

2. the change of key indexes in the purified plasma can be detected at any time, and the purification effect is judged;

3. the structure is simple, the operation is convenient, and the whole body can be placed in a constant temperature incubator at 37 ℃ to provide constant temperature guarantee for blood plasma and liver cells;

4. the purified plasma can be used for carrying out plasma replacement for patients without using fresh plasma, so that the trouble of insufficient fresh plasma is solved, and the cross contamination risk of other plasma is avoided;

5. one separation plasma clean system can carry out purification many times to same patient's plasma, practices thrift the cost.

Description of the drawings:

FIG. 1: an ex vivo plasma purification system schematic.

In the figure: 1. a plasma bag; 2. an oxygenation device; 3. a peristaltic pump; 4. a human hepatocyte plasma perfusion device; 5. fixing a bracket; 6. a fixing clip; 7. a cell filter; 8. a circulation line; 9. a sampling port; 10. a liquid outlet; 11. and (4) a liquid inlet.

Detailed Description

The present invention will now be further described with reference to the accompanying drawings, it being understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1: separation plasma clean system

The invention relates to an in vitro plasma purification system, which is shown in figure 1 and comprises a plasma bag 1, an oxygenation device 2, a peristaltic pump 3, a human hepatocyte plasma perfusion device 4, a fixed bracket 5, a fixed clamp 6, a cell filter 7 and a circulating pipeline 8, wherein: the plasma bag is provided with a sampling port 9, a liquid outlet 10 and a liquid inlet 11; the fixing bracket and the fixing clamp fix the human hepatocyte plasma perfusion device; the liquid outlet of the plasma bag is connected with one end of the oxygenator and one end of the human hepatocyte plasma perfusion device in turn through a circulating pipeline, and the other end of the human hepatocyte plasma perfusion device is connected with the cell filter and the liquid inlet of the plasma bag through the circulating pipeline, thereby forming a closed loop.

In the embodiment, all the circulating pipelines use silicone tubes, the peristaltic pump is arranged on the circulating pipeline between the oxygenator and the human hepatocyte plasma perfusion device, and the rotation direction of the peristaltic pump is to enable the plasma to flow from the oxygenator to the human hepatocyte plasma perfusion device; the liver cells loaded by the human liver cell plasma perfusion device are immortalized human liver cells; and the cell filter used a disk filter having a cut-off diameter of 10 μm.

Example 2: separation plasma clean system

In the embodiment, all the circulating pipelines use silicone tubes, the peristaltic pump is arranged on the circulating pipeline between the cell filter and the human hepatocyte plasma perfusion device, and the rotation direction of the peristaltic pump is to enable plasma to flow from the human hepatocyte plasma perfusion device to the cell filter; the liver cells loaded by the human liver cell plasma perfusion device are human liver cancer cell lines HepG 2; and a 5 μm pore size filter membrane type filter was used as the cell filter.

Example 3: application of in-vitro plasma purification system in-vitro purification of liver failure plasma

Ex vivo purification of liver failure plasma using the ex vivo plasma purification system described in example 1, the specific steps:

fully pre-flushing a human hepatocyte plasma perfusion device loaded with immortalized artificial hepatocytes with ringer's solution under aseptic operation, removing the original solution inside, and fixing the human hepatocyte plasma perfusion device 4 on a fixed bracket 5 by using a fixing clamp 6; 1000mL of plasma of a patient with liver failure is filled in the plasma bag 1, and the liquid outlet 10 of the plasma bag 1 is connected with the oxygenation device 2; the oxygenation device 2 is connected with the lower end of a human hepatocyte plasma perfusion device 4, the upper end of the human hepatocyte plasma perfusion device 4 is connected with a cell filter 7, the aperture of the cell filter is 5 mu m, and the cell filter 7 is connected with a liquid inlet 11 of a plasma bag 1 to form a circulation passage; the peristaltic pump 3 is arranged on a circulating pipeline between the oxygenator and the human hepatocyte plasma perfusion device, and the system device is put into a 5 percent carbon dioxide incubator at 37 ℃; starting a peristaltic pump 3, setting the rotating speed to be 10rpm, and allowing the plasma to enter a human hepatocyte plasma perfusion device 4 at a slow speed for purification; after 5 hours of extracorporeal circulation, the bilirubin level in the blood plasma is sampled and detected from a sampling port and compared with that before purification, and the result shows that after 5 hours of purification, the total bilirubin and the direct bilirubin in the blood plasma are respectively reduced by 40.0 percent and 39.4 percent, and the purification effect is obvious.

Example 4: application of in-vitro plasma purification system in-vitro purification of liver failure plasma

Ex vivo purification of liver failure plasma using the ex vivo plasma purification system described in example 2, the specific steps:

fully pre-flushing a human hepatocyte plasma perfusion device loaded with a human hepatocyte cell line HepG2 with a ringer's solution under aseptic operation, removing a preservation solution, and fixing the human hepatocyte plasma perfusion device 4 on a fixed bracket 5 with a fixing clamp 6; 500mL of plasma of a patient with liver failure is filled in the plasma bag 1, and the liquid outlet 10 of the plasma bag 1 is connected with the oxygenation device 2; the oxygenation device 2 is connected with the lower end of a human hepatocyte plasma perfusion device 4, the upper end of the human hepatocyte plasma perfusion device 4 is connected with a cell filter 7, and the cell filter 7 is connected with a liquid inlet 11 of a plasma bag 1 to form a circulation passage; the peristaltic pump is arranged on a circulating pipeline between the cell filter and the human hepatocyte plasma perfusion device, and the rotation direction of the peristaltic pump is to ensure that the plasma flows from the human hepatocyte plasma perfusion device to the cell filter; the system device is placed in a 5% carbon dioxide incubator at 37 ℃; starting the peristaltic pump 3, setting the rotating speed to be 20rpm, and allowing the plasma to enter the human hepatocyte plasma perfusion device 4 at a slow speed for purification; after extracorporeal circulation is carried out for 2h, the total bilirubin, direct bilirubin, indirect bilirubin, alkaline phosphatase and glutamic-oxaloacetic transaminase in the blood plasma of the patient with liver failure are respectively reduced by 47.70%, 26.31%, 51.93%, 46.89% and 47.58%, and the purification effect is obvious.

Claims

1. The in vitro plasma purification system is characterized by comprising a plasma bag (1), an oxygenation device (2), a peristaltic pump (3), a human hepatocyte plasma perfusion device (4), a fixed support (5), a fixed clamp (6), a cell filter (7) and a circulating pipeline (8), wherein: the plasma bag is provided with a sampling port (9), a liquid outlet (10) and a liquid inlet (11); the fixing bracket (5) and the fixing clamp (6) are used for fixing the human hepatocyte plasma perfusion device (4); the liquid outlet (11) of the plasma bag (1) is sequentially connected with the oxygenator (2) and one port of the human hepatocyte plasma perfusion device (4) through a circulating pipeline, and the other port of the human hepatocyte plasma perfusion device (4) is connected with the cell filter (7) and the liquid inlet (11) of the plasma bag through the circulating pipeline, thereby forming a closed loop.

2. The ex vivo plasma purification system of claim 1, wherein the circulation line is a silicone tube.

3. An ex vivo plasma purification system according to claim 1 wherein the peristaltic pump can be clipped to any one of the circulation lines.

4. An ex vivo plasma purification system according to claim 1, wherein the cell filter is any device capable of trapping cells and cell debris with an effective trapping pore size of 5-10 μm.

5. An ex vivo plasma purification system as claimed in claim 1, wherein the human hepatocyte plasma perfusion device is a hollow container with two ports, and the inside of the hollow container is filled with hepatocytes growing on microcarriers, wherein the hepatocytes are cells with hepatocyte functions, including but not limited to human hepatoma cell lines and immortalized human hepatocytes.

6. Use of any one of the ex vivo plasma purification systems according to claims 1-5 for ex vivo purification of liver failure plasma.