CN112826997A

CN112826997A - Bioartificial liver support system based on cell contact bioreactor

Info

Publication number: CN112826997A
Application number: CN202011620276.1A
Authority: CN
Inventors: 高毅; 冯磊; 王毅; 李阳; 易笑
Original assignee: Guangdong Qianhui Biotechnology Co ltd
Current assignee: Guangdong Qianhui Biotechnology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-05-25

Abstract

The application discloses biological artificial liver support system based on cell contact bioreactor includes following branch pipeline that connects gradually at least: the blood input branch pipeline with the upstream end set as a blood input end, the first plasma branch pipeline comprising the first plasma separator, the biological purification branch pipeline comprising the cell contact bioreactor, and the plasma return branch pipeline with the downstream end set as a blood output end. In the bioartificial liver support system based on the cell contact bioreactor, the cell contact bioreactor can provide a good in vitro growth environment for seed cells, maintain the functional form of the seed cells in vitro, and provide the decomposition, detoxification, biosynthesis and other effects of in vitro liver cells for patients with liver failure. And the support system has high material exchange efficiency, stable operation and easy maintenance.

Description

Bioartificial liver support system based on cell contact bioreactor

Technical Field

The application relates to the field of bioartificial liver, in particular to a bioartificial liver support system based on a cell contact bioreactor.

Background

The artificial liver support system mainly comprises an abiotic artificial liver and a biotype artificial liver support system, a large number of researches show that the abiotic artificial liver support system can not obviously improve the survival rate of patients with liver failure, and the biotype artificial liver and the mixed type biotype artificial liver support system are hot spots of the current researches. At present, commonly used bioreactors include hollow fiber bioreactors, multilayer flat plate bioreactors, microcapsule-wrapped bioreactors, and the like. However, the existing bioreactors have different advantages and disadvantages, cannot better simulate the in-vivo growth environment of seed cells in vitro, and cannot better maintain the functional morphology of the seed cells in vitro. The research group of our subjects constructs a cell contact bioreactor in the early stage, which can better simulate the in vivo growth environment in vitro, provide a good in vitro growth environment for seed cells and maintain the functional form of the seed cells in vitro.

Disclosure of Invention

The present application has an object to provide a bioartificial liver system capable of overcoming biocompatibility and sufficiently supplying oxygen.

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

a bioartificial liver support system based on a cell contact bioreactor at least comprises the following branch pipelines which are connected in sequence: the blood input branch pipeline with the upstream end set as a blood input end, the first plasma branch pipeline comprising the first plasma separator, the biological purification branch pipeline comprising the cell contact bioreactor, and the plasma return branch pipeline with the downstream end set as a blood output end.

Specifically, the cell contact bioreactor comprises an outer tank body, an upper cover covering the top of the outer tank body and a reaction column arranged inside the outer tank body; the reaction column comprises a reaction cavity limited by an annular column wall, an inflow disc covering the bottom of the reaction cavity and an outflow disc covering the top of the reaction cavity; and a magnetic stirring device is arranged at the bottom of the outer tank body.

Furthermore, the upper cover is provided with a perfusion inflow guide pipe communicated with the first slurry dividing pipeline and a perfusion outflow guide pipe communicated with the slurry returning sub-pipeline, and the lower ends of the perfusion inflow guide pipe and the perfusion outflow guide pipe extend to the bottom of the outer tank body.

Preferably, the biological purification branch pipeline further comprises a second slurry branch pipeline: the plasma bioreactor comprises a second plasma separator provided with a second plasma inlet, a second plasma outlet and a cell outlet, wherein the second plasma inlet is communicated with the perfusion outflow conduit, the second plasma outlet is communicated with the plasma return branch pipeline, and the cell outlet is communicated with the cell contact type bioreactor.

Further, the upper cover of the cell contact bioreactor is also provided with a cell inflow conduit connected with the cell outlet, and the lower end of the cell inflow conduit extends to be close to the outflow disc.

Preferably, the upper cover is provided with an air duct, and the lower end of the air duct extends to the bottom of the outer tank body.

Further preferably, the airway tube is located remotely from the perfusion inflow and outflow tubes.

More preferably, an adsorption branch pipeline comprising an adsorption column is further arranged between the first slurry branch pipeline and the biological purification branch pipeline.

Optionally, the adsorption column comprises a resin adsorption column and/or a bilirubin adsorption column.

Preferably, the blood input branch pipeline, the first plasma separation branch pipeline, the biological purification branch pipeline and the plasma return branch pipeline are respectively provided with a peristaltic pump.

Compared with the prior art, the method has the following advantages:

in the bioartificial liver support system based on the cell contact bioreactor, the cell contact bioreactor can provide a good in vitro growth environment for seed cells, maintain the functional form of the seed cells in vitro, provide the in vitro decomposition, detoxification, biosynthesis and other effects of the liver cells for patients with liver failure, provide the liver regeneration opportunity for the patients with liver failure or strive for sufficient time for the bridging liver transplantation of the patients with liver failure,

the cell contact bioreactor can realize direct contact between cells and patient plasma, and solves the problem of low material exchange efficiency caused by the use of an isolation membrane in the existing bioreactor during immune rejection reaction.

Meanwhile, the method of secondary plasma separation avoids the immune reaction caused by the direct contact of blood cells and in-vitro seed cells when in-vitro seeds flow into a human body, and ensures the life safety of a patient.

Drawings

Fig. 1 is a schematic structural diagram of an exemplary embodiment of a bioartificial liver support system based on a cell contact bioreactor according to the present application.

Detailed Description

The present application is described in further detail below with reference to the attached drawings and the detailed description.

Referring to fig. 1, the bioartificial liver support system based on cell contact bioreactor of the present application comprises the following branch pipes:

the blood is input into the branch pipeline 1, and the blood input branch pipeline 1 comprises a blood input end, a medicine input device which is connected into the blood input branch pipeline 1 in parallel, and an arterial pot which is connected in the blood input branch pipeline 1 in series. In other possible embodiments, the drug injector may be a syringe or other device that is quantitative or timed to inject the drug into the blood inlet branch 1. In this embodiment, the drug input from the drug input device may be an anticoagulant drug for preventing blood coagulation. More specifically, heparin is used as the anticoagulant in this embodiment, and in other possible embodiments, heparin, a natural anticoagulant such as hirudin, or at least one of potassium salts such as sodium citrate and potassium fluoride, and calcium ion chelating agents such as EDTA, may be used as the anticoagulant.

The first plasma separator is used for separating human blood into plasma and blood cells, so that the plasma can further participate in subsequent extracorporeal circulation, and the blood cells can be timely returned to the human body. The first plasma separator also isolates immune cells in the blood and prevents immune factors from entering the subsequent bioreactor 31.

The plasma returning branch pipeline 4 at least comprises a vein kettle which is connected with a blood cell outlet of the first plasma separator and is provided with a whole blood outlet, and a bubble monitor which is connected with the whole blood outlet, the bubble monitor is also connected with the blood output end, and the upstream of the plasma returning branch pipeline 4 is used for flowing purified plasma.

A biological purification branch pipeline 3, wherein the biological purification branch pipeline 3 comprises a cell contact type bioreactor 31 connected with the upstream first slurry branch pipeline 2 and a second slurry branch pipeline 32 connected with the downstream slurry return branch pipeline 4.

Specifically, the cell contact bioreactor 31 comprises an outer tank 311, an upper cover 312 closely covering the top of the outer tank 311, and a reaction column 313 arranged inside the outer tank 311.

Further, the outer container 311 provides a storage space and a flow space for plasma during extracorporeal circulation, and also limits the total amount of extracorporeal circulation. The reaction column 313 includes a reaction chamber defined by an annular column wall, an inflow plate 3131 fixedly fitted to the bottom of the reaction chamber, and an outflow plate 3132 fixedly fitted to the top of the reaction chamber. A magnetic stirring device 314 is arranged at the bottom of the outer tank 311 and below the reaction column 313, and the rotating speed of the magnetic stirring device 314 is controlled within the range of 0-1200 rpm. The resulting liquid vortex is initiated by the magnetic stirring device 314, driving the plasma flow in the cell-contact bioreactor 31.

The plasma flowing in from the first plasma separation pipeline 2 is fully contacted with oxygen in the external space of the reaction column 313, so as to ensure the oxygen supply of the seed cells in the reaction column 313; the reaction column 313 is filled with a biological scaffold material, and the seed cells are inoculated on the biological scaffold material; the magnetic stirring device 314 can provide power to the plasma, drive the plasma to enter the interior of the reaction column 313 through the inflow disk 3131 and then flow out through the outflow disk 3132, and the plasma continues to flow along the outer surface of the reaction column 313 toward the bottom of the outer tank 311. The plasma flows from the lower part to the upper part inside the reaction column 313 because of the local pressure difference of the liquid, so that the plasma can be in full contact with the seed cells inside the reaction column 313 to perform detoxification, biosynthesis and decomposition, and the dissolved oxygen of the plasma can be increased to improve the dissolved oxygen efficiency.

Further, for the purpose of plasma perfusion through the reaction column 313, the

inflow disks

3131 and 3131 may be provided with hollowed holes, or gaps for liquid to flow through may be provided between the inflow disks 3131 and 3132 and the annular column wall of the reaction column 313, as an alternative to the hollowed holes. From this, whole cell contact bioreactor 31 need not use the barrier film to separate seed cell and plasma in the process of carrying out biological purification to plasma, has not only practiced thrift the barrier film as the consumptive material, and owing to can not take place the condition that the barrier film blockked up, has improved the material exchange efficiency between plasma and the seed cell greatly, has also improved bioreactor's operating stability, avoids carrying out frequent maintenance.

The outer tank 311 and the reaction column 313 are preferably made of polycarbonate material, and the biological scaffold material for culturing animal cells is also preferably made of polycarbonate material. The polycarbonate material has high transparency, and is convenient for operators to observe the change of the liquid height and the color in the bioreactor 31 during the cell culture process.

Further, the upper cover 312 is provided with a perfusion inflow conduit 315 communicated with the first slurry dividing pipeline 2 and a perfusion outflow conduit 316 communicated with the slurry returning dividing pipeline 4, and the lower ends of the perfusion inflow conduit 315 and the perfusion outflow conduit 316 extend to the bottom of the outer tank 311. Specifically, the extension of the perfusion inflow conduit 315 to the bottom of the outer tank 311 is beneficial to the fact that the plasma can contact with the seed cells in the reaction column 313 more quickly, and the biological reaction process can be completed as soon as possible; the extension of the perfusion outflow conduit 316 to the bottom of the outer tank 311 is beneficial to prolonging the dissolved oxygen path of the plasma completing the biological reaction process and improving the dissolved oxygen amount of the plasma. The perfusion inflow conduit 315 and perfusion outflow conduit 316 should be spaced appropriately to avoid the influx of plasma out of the cell-contact bioreactor 31 without participating in the bioreaction process.

The upper cover 312 is further provided with an air duct 318, the lower end of the air duct 318 extends to the bottom of the outer tank 311 to achieve sufficient oxygen dissolution of the plasma, and the upper end of the air duct 318 is connected with an external oxygen supply device to ensure stable oxygen supply conditions. Preferably, the ventilation conduit 318 is disposed away from the perfusion inflow conduit 315 and the perfusion outflow conduit 316, so as to avoid small bubbles formed by the air filled into the ventilation conduit 318 directly flowing into the subsequent circulation pipeline, which increases the formation of the pipeline air plug.

Further preferably, the upper cover 312 may be further provided with other gas-liquid exchange ports, probe sockets and sampling ports penetrating through the cover surface, so as to adjust and optimize the culture conditions of the cell contact bioreactor 31. For example, a plurality of probe sockets may be provided, in which a temperature detector, a dissolved oxygen detector, a ph detector, a glucose concentration detector, a lactate concentration detector, a urea concentration detector, and the like are respectively inserted into the outer tank 311, and the above-mentioned detectors are fixed to the upper cover 312. The sample connection is provided in the middle of the upper cover 312, and the sample connection is provided with a thread, which may be an external thread or an internal thread, but no matter what kind of thread the sample connection is provided with, a thread cover matched with the sample connection is provided.

The second plasma separation line at least comprises a second plasma separator 32 provided with a second plasma inlet 321, a second plasma outlet 322 and a cell outlet 323, the second plasma inlet 321 is connected with the perfusion outflow conduit 316, and the second plasma outlet 322 is connected with the plasma return line 4. The cell outlet 323 is connected to the cell-contacting bioreactor 31 through a cell inflow guide 317 provided at the upper cover 312, and the lower end of the cell inflow guide 317 extends to be adjacent to the outflow tray 3132 so that the seed cells escaping from the reaction column 313 are returned to the reactor 31, thereby preventing the seed cells from entering the human body and causing an allergic reaction.

The branch lines are connected in sequence to form a circulation path for extracorporeal purification of blood.

Preferably, the bioartificial liver support system based on cell contact bioreactor of this application still includes the absorption branch line 5, the absorption branch line 5 concatenates between first plasma branch line 2 and biological purification branch line 3, specifically, the absorption branch line 5 comprises at least one adsorption column, the adsorption column can be resin adsorption column 51 for non-selective adsorption of toxin in the plasma, inflammation medium, or bilirubin adsorption column 52, specifically adsorbs bilirubin in the plasma. The adsorption branch pipe 5 performs prepositive purification on the plasma, is beneficial to maintaining longer activity of the seed cells in the cell contact bioreactor 31, and is beneficial to benign operation of the support system.

Further preferably, the blood input branch pipeline 1, the first plasma separation branch pipeline, the biological purification branch pipeline 3 and the plasma return branch pipeline 4 are all provided with peristaltic pumps 6, so that the flow rate and the hydraulic pressure of each branch pipeline in the circulation passage can be adjusted in time.

In other possible embodiments, the support system is further provided with a temperature regulator. One way to achieve this is to place the entire biological decontamination branch 3 in a 37 ℃ incubator to provide the seed cells with the optimal growth temperature. Other alternative realization is to connect a temperature regulator in series on at least any branch pipeline, and the temperature regulator can realize the refinement and regionalization temperature regulation of all or part of the area of the support system through the change of the arrangement number and the position.

Furthermore, in the support system, a blood flow detection device and a blood flow pressure detection device may be introduced into each branch line to monitor the state of the support system in real time and monitor the condition of the patient in real time. Pinch valves should also be provided in each branch line to control the make and break of the branch lines.

Further, the blood circulation process in the bioartificial liver support system based on the cell contact bioreactor of the present application is as follows:

under the power provided by each peristaltic pump 6, the blood enters the first plasma separation line 1: through the first plasma separator, plasma components and blood cells in the blood of a patient are respectively separated by the first plasma separator in a mode plasma separation method, wherein the separated plasma flows out from the first plasma outlet, if the adsorption branch pipeline 5 is arranged, the plasma firstly flows through the adsorption branch pipeline 5 for primary purification, otherwise, the plasma directly enters the biological purification branch pipeline 3.

In the biological purification branch pipeline 3, the cell contact bioreactor 31 performs functions of detoxification, conversion, synthesis and the like on toxic plasma, and the detoxified plasma flows out through the second plasma separator 32 and flows to the plasma return branch pipeline 4 through the second plasma outlet 322.

After entering the plasma return branch pipeline 4, the plasma after biological purification is mixed with blood cells from a blood cell outlet in the venous kettle to form whole blood, and the whole blood is detected by the bubble monitor and then returns to the body of a patient through the blood output end. The blood is subjected to the above process to complete the biological purification process.

In summary, in the bioartificial liver support system based on the cell contact bioreactor, the cell contact bioreactor can provide a good in vitro growth environment for the seed cells, maintain the functional morphology of the seed cells in vitro, and provide the in vitro decomposition, detoxification, biosynthesis and other effects of the liver cells for the patients with liver failure. And the support system has high material exchange efficiency, stable operation and easy maintenance.

The above embodiments are only preferred embodiments of the present application, but 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 application should be construed as equivalents and are included in the scope of the present application.

Claims

1. A bioartificial liver support system based on a cell contact bioreactor is characterized by at least comprising the following branch pipelines which are connected in sequence: the blood input branch pipeline with the upstream end set as a blood input end, the first plasma branch pipeline comprising the first plasma separator, the biological purification branch pipeline comprising the cell contact bioreactor, and the plasma return branch pipeline with the downstream end set as a blood output end.

2. The system of claim 1, wherein the cell-contacting bioreactor comprises an outer tank, an upper cover covering the top of the outer tank, and a reaction column disposed inside the outer tank; the reaction column comprises a reaction cavity limited by an annular column wall, an inflow disc covering the bottom of the reaction cavity and an outflow disc covering the top of the reaction cavity; and a magnetic stirring device is arranged at the bottom of the outer tank body.

3. The system of claim 2, wherein the upper cover is provided with a perfusion inflow conduit communicated with the first slurry branch pipeline and a perfusion outflow conduit communicated with the slurry return branch pipeline, and the lower ends of the perfusion inflow conduit and the perfusion outflow conduit extend to the bottom of the outer tank body.

4. The system of claim 3, wherein the biological decontamination branch line further comprises a second slurry branch line: the plasma bioreactor comprises a second plasma separator provided with a second plasma inlet, a second plasma outlet and a cell outlet, wherein the second plasma inlet is communicated with the perfusion outflow conduit, the second plasma outlet is communicated with the plasma return branch pipeline, and the cell outlet is communicated with the cell contact type bioreactor.

5. The system of claim 4, wherein the upper cover of the cell-contacting bioreactor is further provided with a cell inflow conduit connected to the cell outlet, and a lower end of the cell inflow conduit extends to be adjacent to the outflow tray.

6. The system of claim 3, wherein the upper lid is provided with an air duct, the lower end of which extends to the bottom of the outer tank.

7. The system of claim 6, wherein the airway tube is disposed distal to the perfusion inflow tube and perfusion outflow tube.

8. The system of claim 1, wherein an adsorption branch line comprising an adsorption column is further disposed between the first slurry branch line and the biological purification branch line.

9. The system of claim 8, wherein the adsorption column comprises a resin adsorption column and/or a bilirubin adsorption column.

10. The system of claim 1, wherein the blood inlet branch line, the first plasma branch line, the biological purification branch line, and the plasma return branch line are each provided with a peristaltic pump.