CN112798393A

CN112798393A - Large-volume biological cell sap concentration and washing system and method thereof

Info

Publication number: CN112798393A
Application number: CN202110136697.5A
Authority: CN
Inventors: 魏东兵
Original assignee: Zhongbo Ruikang Beijing Biotechnology Co ltd
Current assignee: Zhongbo Ruikang (Beijing) Biomedical Technology Co.,Ltd.
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-05-14

Abstract

The invention provides a large-volume biological cell sap concentration washing system and a method thereof, and the system comprises a centrifugal component and a concentration washing component, wherein the centrifugal component comprises a centrifuge, the concentration washing component comprises a centrifugal cup, the centrifugal cup is arranged on the centrifuge, and the centrifuge drives the centrifugal cup to rotate at a high speed; the centrifugal cup comprises a static part and a rotating part, the static part is in rotary sealing fit with the rotating part, the rotating part comprises a separation cavity, the separation cavity is a cavity formed by a cup body and an inner core, and when the centrifugal cup rotates at a high speed, cells in a cell sap sample are separated from liquid in the separation cavity. The large-volume biological cell sap concentration and washing system and the method thereof have the advantages that the cell sap movement path is long, and the centrifugal efficiency is high; meanwhile, by adding the inner core design in the separation cup, the dynamic volume of the separation cup can be adjusted, the minimum centrifugal force is ensured, the separation effect is further ensured, and the cell recovery rate is improved.

Description

Large-volume biological cell sap concentration and washing system and method thereof

Technical Field

The invention belongs to the field of automatic treatment and separation of biological cells, and particularly relates to a large-volume biological cell sap concentration and washing system and a method thereof.

Background

Cell therapy refers to a therapeutic method for repairing tissues or organs by using autologous (or allogeneic) adult cells (or stem cells) of a patient. Can be widely used for bone marrow transplantation, late liver cirrhosis, femoral head necrosis and malignant tumorTumor, myocardial infarction, etc. The current treatment cells are generally derived from umbilical cord blood, bone marrow, peripheral blood, fat and the like. Since their content is small, it is generally necessary to culture cells so that the number of the cells is expanded to a certain amount (for example, the number of the cells obtained is 1X 10)⁹The quantity of the active carbon is required to be increased by 10 times to reach 1 multiplied by 10¹⁰One). During the cell culture process, a culture medium containing cell growth factors and nutrients, oxygen, air, carbon dioxide and the like are continuously added to the cells to be cultured to promote the growth. When the cell culture is completed, the cultured cell fluid needs to be subjected to treatments such as "concentration" and "washing" to remove excess medium, metabolic waste liquid, and the like from the cell fluid. Under different culture requirements and conditions, the difference of the cell fluid volume after culture is large, and is generally different from 200ml to 50L.

For the concentration of cell sap, there are currently some automated equipments for concentrating hematopoietic stem cells, such as one proposed in patent No. CN1331610, which has an axial biological fluid inlet/outlet. The treatment chamber has a movable piston which can introduce a measured amount of biological fluid and extrude the treated biological fluid component through an outlet. The optical device monitors the piston position to control the expression of the imbibed fluid and the components. In the independent mode of the system, the liquid to be concentrated is sucked into the processing chamber, the target cells with higher density are reserved outside the processing chamber under the action of high-speed centrifugation through a density gradient centrifugation principle, the waste liquid with lower density is extruded in the middle of the processing chamber, when the piston runs, the waste liquid is discharged along the axial outlet, and the quantitative volume can be reserved by controlling the position of the piston to obtain the cell liquid with the target concentration. The equipment has the advantages of good sealing performance, high automation degree and good result consistency; it also has the following drawbacks:

(1) the separation device has a small process chamber volume (about 220ml) and can process up to 220ml of cells per cycle. Each concentration cycle (for example, the volume of 220ml is concentrated to 22ml) comprises the steps of sucking cell sap, centrifuging at a high speed, removing redundant waste liquid, discharging the concentrated cell sap into a collection bag, sucking the cell sap into liquid …, and the like, when the amount of target cell sap to be concentrated is large, such as 2L, 5L or more, a plurality of cycles are required, and the treatment efficiency is very low;

(2) the liquid inlet and the liquid outlet of the processing chamber are arranged at the top, and the top is a conical surface. In a single circulation, the shorter the moving path of the cells in the cell fluid sucked later, the shorter the centrifugation time; the centrifugal radius of the cells closer to the conical surface at the top of the processing chamber is smaller, the centrifugal force is smaller, and the centrifugal effect is poorer;

(3) the same outlet is used for the inlet and the outlet, and when the cell sap is sucked in each circulation, a part of the cell sap is remained in the inlet and the pipeline near the inlet; when the waste solution is discharged, the remaining cell sap is also discharged into the waste solution bag.

In summary, the current treatment apparatus has a low recovery rate of cells and is inefficient in concentrated washing with a large amount of cell liquid. In addition to the above methods, when the amount of cell fluid to be treated is large, the operator may use a manual treatment method, but the manual treatment method is complicated, time-consuming and labor-consuming, results are not consistent well, the recovery efficiency is different greatly, and contamination may occur.

Disclosure of Invention

In order to solve at least one of the above-mentioned technical problems, the technical scheme adopted by the invention is to provide a large-volume biological cell sap concentration washing system and a method thereof, the cell sap movement path is long, the centrifugal efficiency is high, the effect is good, and the cells and the waste liquid are separated thoroughly; meanwhile, by adding the inner core design in the separation cup, the dynamic volume of the separation cup can be adjusted, the minimum centrifugal force is ensured, the separation effect is further ensured, and the cell recovery rate is improved.

In order to at least achieve one of the above purposes, the invention adopts the technical scheme that:

the invention provides a large-volume biological cell sap concentrated washing system, which comprises a centrifugal assembly and a concentrated washing assembly, wherein the centrifugal assembly comprises a centrifuge, the concentrated washing assembly comprises a centrifugal cup, the centrifugal cup is arranged on the centrifuge, and the centrifuge drives the centrifugal cup to rotate at a high speed; the centrifugal cup comprises a static part and a rotating part, the static part is in rotary sealing fit with the rotating part, the rotating part comprises a separation cavity, the separation cavity is a cavity formed by a cup body and an inner core, and when the centrifugal cup rotates at a high speed, cells in a cell sap sample are separated from liquid in the separation cavity.

Further, the centrifugal assembly further comprises a leakage sensor, the leakage sensor is installed in the centrifugal machine, a humidity sensitive resistor is installed on the leakage sensor, and the humidity sensitive resistor is used for judging whether leakage occurs during centrifugal separation.

Furthermore, the concentrated washing assembly also comprises a sample puncture outfit, a washing puncture outfit, a product bag, a pump pipe pipeline, a pump pipe clamp, a pressure monitoring connector, a pressure monitoring pipeline and a waste liquid bag; the sample puncture device, the washing puncture device, the product bag, the pump pipe pipeline, the pressure monitoring connector, the centrifugal cup and the waste liquid bag are connected with each other through pipelines, and two pump pipe clamps are bonded on the pump pipe pipeline.

Further, the static part of the centrifugal cup comprises a liquid inlet, a central pipe, a liquid outlet and a rotary sealing pair, wherein the liquid inlet and the liquid outlet are both arranged at the upper part of the centrifugal cup, the liquid inlet is communicated with the central pipe and leads to the bottommost part of the centrifugal cup, and the rotary sealing pair comprises a bearing and a sealing ring; a small opening is formed in the uppermost part of the inner side of the separation cavity of the rotating part and communicated with the liquid outlet; the static part and the rotating part are in interference fit through the bearing, the static part is in interference fit with an outer ring of the bearing, and the rotating part is in interference fit with an inner ring of the bearing.

Further, concentrated washing system of cell sap still includes the subassembly of weighing, the subassembly of weighing contains sample scale, washing scale, product scale and waste liquid balance, sample scale, washing scale, product scale and waste liquid balance are weighing sensor, the sample scale is used for hanging the sample bag, the washing scale is used for hanging the washing liquid bag, the product scale is used for hanging the product bag, the waste liquid scale is used for hanging the waste liquid bag, the subassembly of weighing is used for weighing in real time the liquid in sample bag, washing liquid bag, product bag and the waste liquid bag that hang above that.

Further, the cell sap concentration washing system also comprises a peristaltic pump, wherein the peristaltic pump is used as a power source, liquid in the sample bag or the washing liquid bag is pumped into the centrifuge cup rotating at a high speed for centrifugal separation, and the concentrated product is returned into the product bag.

Furthermore, the cell sap concentration washing system also comprises a pipeline detection assembly, wherein the pipeline detection assembly comprises a pipeline pressure detector, an air detector and a pipeline component detector, the pipeline pressure detector is internally provided with a pressure sensor, and can detect the pressure in the pipeline in real time and send the pipeline pressure to the control module; the air detector is used for detecting whether the pipeline is installed correctly or not and whether liquid exists in the pipeline or not in real time; the pipeline component detector is used for detecting the component of the liquid in the pipeline.

Furthermore, the cell sap concentration and washing system also comprises a human-computer interaction module, wherein the human-computer interaction module comprises a touch display screen, an emergency stop button and an audible and visual alarm, and the touch display screen is used for inputting parameters and displaying the current state; the emergency stop button is a trigger switch; the audible and visual alarm is used for prompting that the system is abnormal or needs an operator to participate in processing.

Further, the cell sap concentration washing system further comprises a pipe clamp valve, wherein the pipe clamp valve comprises a sample valve, a washing valve and a product valve, and the pipe clamp valve is used for controlling the flow of liquid in a pipeline.

The method of the large-volume biological cell sap concentration washing system comprises the following steps:

1) preliminary self-checking: after the system is started, automatically detecting whether each component is abnormal or not;

2) selecting a program, inputting parameters: selecting a program and inputting related parameters on the touch display screen;

3) hanging a cell sap sample bag on a sample scale, and hanging a washing solution bag on a washing scale;

4) installing a concentrated washing component: mounting the centrifugal cup on a centrifugal machine, hanging a waste liquid bag on a waste liquid scale, and hanging a product bag on a product scale;

5) diagnosis before use: the control module judges whether the current sample bag, the washing liquid bag and the concentrated washing component are installed correctly according to the input parameters and the data fed back by each weighing sensor and the pipeline detection component;

6) and (3) concentration process: when the concentration is started, the sample valve is opened, the washing valve and the product valve are closed, the peristaltic pump and the centrifuge start to work, the cell fluid in the sample bag enters the centrifuge cup under the action of the peristaltic pump, and the centrifuge cup rotates at a high speed to separate the cells from the waste fluid; when the sample scale detects that the weight of the cell sap in the sample bag is half of the initial weight, the sample valve is closed, and the peristaltic pump stops rotating; finishing concentration;

7) washing process: opening a washing valve, starting a peristaltic pump, pumping the washing liquid into the centrifugal cup, and completely extruding the waste liquid into a waste liquid bag; when the washing scale detects that the weight of the washing liquid is reduced to the set value of the program, the washing valve is closed, the peristaltic pump stops rotating, and the centrifuge stops rotating slowly; finishing washing;

8) and (3) collecting flow: when the centrifugal machine is completely stopped, cell sap in the separation cavity flows into the inner core of the centrifugal cup under the action of gravity, the product valve is opened, the sample valve and the washing valve are closed, the peristaltic pump is reversely rotated, the concentrated and washed cell sap in the centrifugal cup is pumped into the product bag through the central tube of the centrifugal cup, and when the air detector detects that the pipeline is emptied, the peristaltic pump stops rotating, and the product valve is closed; finishing the collection;

9) repeating the steps, inputting the residual cell sap in the sample bag into a centrifuge cup for concentration and washing, and then collecting the concentrated and washed cell sap;

10) a cleaning process: the washing valve is opened, the sample valve and the product valve are closed, the centrifuge rotates at a low speed, the peristaltic pump pumps a proper amount of washing liquid into the centrifuge cup, then the washing valve is closed, the product valve is opened, the centrifuge stops rotating, the peristaltic pump rotates reversely, the washing liquid in the centrifuge cup is pumped into the product bag, and when the air detector detects that the pipeline is emptied, the product valve is closed, and the peristaltic pump stops rotating.

Compared with the prior art, the large-volume biological cell sap concentration and washing system and the method thereof have the beneficial effects that:

1) the cell sap concentration and washing system adopted by the invention can continuously concentrate and wash the cell sap, has incomparable advantages for the concentration and washing of large-volume (500ml-50L) sample cell sap, and greatly improves the working efficiency;

2) the unique separation cup structure in the concentrate wash assembly of the present invention: the liquid inlet and the liquid outlet are separated, liquid is fed from the bottom through the central tube, liquid is discharged from the top, and meanwhile, the inner core is increased, the centrifugal efficiency is improved, and the cell recovery rate is increased;

3) the cell sap concentration and washing system disclosed by the invention is matched with a plurality of sensors and execution elements, so that the intelligentization and automation degree is high, the labor intensity of operation is reduced, the consistency of products is improved, and the reliability of equipment and the quality of final products are guaranteed.

In a word, the invention provides a concentration washing system and a method thereof, which have the advantages of good cell liquid separation effect, high centrifugal efficiency and capability of effectively recovering large-volume cell liquid, and have wide application prospects.

Drawings

FIG. 1 is a schematic diagram of a large-volume concentrated washing system for biological cell sap according to the present invention;

FIG. 2 is a schematic diagram of the construction of the concentrated scrubbing assembly of the present invention;

FIG. 3 is a schematic diagram of the construction of the centrifuge cup of the present invention;

FIG. 4 is a schematic view of a centrifuge cup seal configuration of the present invention;

FIG. 5 is a schematic view of the concentrated wash module installation of the present invention;

FIG. 6 is a schematic view of the initial state of the concentration process of the present invention (when the separation chamber is not filled with sample liquid);

FIG. 7 is a schematic view of the concentration process of the present invention;

FIG. 8 is a schematic view of the washing process of the present invention;

FIG. 9 is a schematic view of the collection flow state of the present invention;

wherein, 1B-a sample scale, 2B-a washing scale, 3B-a product scale, 4B-a waste liquid scale, 5B-a sample valve, 6B-a washing valve, 7B-a product valve, 8B-a peristaltic pump, 9B-a pipeline pressure detector, 10B-an air detector, 11B-a pipeline component detector, 12B-a centrifuge, 13B-a liquid leakage sensor, 14B-a touch display screen, 15B-an emergency stop button, 16B-an audible and visual alarm,

1A-a sample puncture outfit, 2A-a sample pipeline, 3A-a washing puncture outfit, 4A-a washing pipeline, 5A-a first tee joint, 6A-a liquid inlet pipeline, 7A-a second tee joint, 8A-a product pipeline, 9A-a product bag, 10A-a pump pipeline, 11A-a pump pipe clamp, 12A-a third tee joint, 13A-a pressure monitoring pipeline, 14A-a pressure monitoring connector, 15A-a centrifugal cup inlet pipeline, 16A-a centrifugal cup, 17A-a waste liquid pipeline, 18A-a waste liquid bag, 19A-a sample bag and 20A-a washing liquid bag,

16A 1-liquid inlet, 16A 2-central tube, 16A 3-separation cavity, 16A 31-cup body, 16A 32-inner core, 16A 33-small opening, 16A 4-liquid outlet, 16A 5-rotary sealing pair, 16A 51-bearing and 16A 52-sealing ring.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to specific examples. Note that the following described embodiments are illustrative only for explaining the present invention, and are not to be construed as limiting the present invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; the connection can be mechanical connection or electrical connection; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Hereinafter, the present invention will be described in detail with reference to the following embodiments:

"sample" as described hereinafter refers to "cell culture" comprising expanded cells, culture medium, metabolic waste cell culture fluid, after completion of the cell culture; the "product" refers to "cell sap" which has completed the "concentration", "washing" treatment.

As shown in the attached figures 1-4, the large-volume biological cell sap concentrated washing system provided by the invention comprises a weighing component, a pipe clamp valve, a peristaltic pump, a centrifugal component, a concentrated washing component, a pipeline detection component, a human-computer interaction module, a control module and the like.

The weighing assembly according to the invention consists of a plurality of "scales" comprising weighing sensors, including at least a sample scale 1B, a wash scale 2B, a product scale 3B, and a waste scale 4B, which function to weigh in real time the liquid suspended thereon. The pipe clamp valve is used for controlling the liquid flow in each section of pipeline, and directly acts on the hose without contacting with the liquid in the hose; the system at least comprises a sample valve 5B, a washing valve 6B and a product valve 7B. The peristaltic pump 8B is the source of fluid flow within the tubing and functions to pump fluid from the sample bag 19A or the wash solution bag 20A into the high speed spinning centrifuge cup 16A for centrifugation and to return the concentrated "product" to the product bag 9A. The pipeline detection assembly comprises a pipeline pressure detector 9B, an air detector 10B and a pipeline composition detector 11B. The pipeline pressure detector 9B is internally provided with a pressure sensor, can detect the pressure in a pipeline in real time and send data to the control module, and sends out an alarm to allow an operator to process the pressure when the pressure is abnormal due to pipeline blockage or other accidents; the air detector 10B is preferably an ultrasonic air detector which can detect whether the pipeline is correctly installed and whether liquid exists in the pipeline in real time; the principle of the pipeline component detector 11B is to detect the components of the liquid in the outlet of the waste liquid pipeline 17A by the light transmittance difference of the different components of the liquid (for example, the light transmittance of the cells, the waste liquid and the washing liquid has obvious difference), so as to prevent the cells from being discharged into the waste liquid bag 18A accidentally.

The centrifuge assembly according to the present invention comprises a centrifuge 12B, a leakage sensor 13B. The centrifuge 12B is used for driving the centrifuge cup 16A arranged on the centrifuge cup to rotate at a high speed, and the components in the sample are separated by adopting a gradient density centrifugation principle; the leakage sensor 13B is installed in the centrifuge 12B, and has a moisture sensitive resistor, and when liquid contacts the moisture sensitive resistor, the resistance value changes, so as to determine whether leakage occurs during centrifugal separation.

The human-computer interaction module comprises a touch display screen 14B, an emergency stop button 15B, an audible and visual alarm 16B and the like, wherein the touch display screen 14B is used for inputting parameters and displaying the current system state; the emergency stop button 15B is a trigger switch, which functions as an emergency stop, when it is pressed, the actuators of the whole system, such as the pinch valve, the peristaltic pump 8B, and the centrifuge 12B, will be turned off or stopped to avoid causing unexpected loss; when the system is abnormal or needs operators to participate in processing, the audible and visual alarm 16B reminds an operator through sound, light and other forms, for example, when the pipeline pressure is too high, the peristaltic pump 8B can stop immediately, the audible and visual alarm 16B can flash a red light, and meanwhile continuously emits dripping sound to remind the operator to process faults.

The concentrated washing component comprises a sample puncture outfit 1A, a washing puncture outfit 3A, a sample pipeline 2A, a washing pipeline 4A, a first tee joint 5A, a liquid inlet pipeline 6A, a product pipeline 8A, a product bag 9A, a second tee joint 7A, a pump pipeline 10A, a pump pipe clamp 11A, a pressure monitoring connector 14A, a pressure monitoring pipeline 13A, a third tee joint 12A, a centrifugal cup inlet pipeline 15A, a centrifugal cup 16A, a waste liquid pipeline 17A, a waste liquid bag 18A and the like. The sample puncture outfit 1A and the washing puncture outfit 3A are connected with a sample bag 19A and a washing liquid bag 20A in a sterile manner in a puncture manner, the sample puncture outfit 1A is connected with a sample pipeline 2A, and the washing puncture outfit 3A is connected with a washing pipeline 4A; the sample pipeline 2A and the washing pipeline 4A are communicated with a liquid inlet pipeline 6A through the first tee joint 5A, the liquid inlet pipeline 6A and the product pipeline 8A are connected to the upper part of a pump pipeline 10A through the second tee joint 7A, the product pipeline 8A is connected with a product bag 9A, and the product bag 9A is used for collecting 'final products' (namely concentrated and washed cell sap); the pump pipe line 10A is bonded with two pump pipe clamps 11A, the distance between the two pump pipe clamps 11A is just matched with a peristaltic pump 8B of the system, and the pump pipe clamps 11A are used for preventing the pump pipe line 10A from sliding when the peristaltic pump 8B rotates; the pump pipeline 10A, the pressure monitoring pipeline 13A and the centrifugal cup inlet pipeline 15A are connected through the third tee joint 12A, the pressure monitoring connector 14A is connected to the pressure monitoring pipeline 13A, and a layer of breathable hydrophobic membrane is arranged in the pressure monitoring connector 14A and allows air molecules to pass through but not water molecules to pass through, so that when pressure is monitored, liquid in the pipeline does not need to worry about flowing into the pipeline pressure detector 9B; the centrifuge cup inlet line 15A is connected with the liquid inlet 16A1 of the centrifuge cup 16A.

The centrifugal cup 16A according to the present invention comprises a liquid inlet 16A1, a central tube 16A2, a separation chamber 16A3, a liquid outlet 16A4, a rotary seal pair 16A5, etc., wherein the liquid inlet 16A1 is communicated with the central tube 16A2 and leads to the bottommost part of the centrifugal cup 16A, the separation chamber 16A3 is a cavity formed by the centrifugal cup body 16A31 and an inner core 16A32, and when the centrifugal cup 16A rotates at a high speed, cells in a sample are separated from liquid (waste liquid). At the upper part of the inner side of the separation chamber 16A3, there is a small opening 16A33, and the small opening 16A33 is communicated with the liquid outlet 16A4, when the separated cells and waste liquid fill the separation chamber 16A3, the waste liquid will flow along the small opening 16A33 to the liquid outlet 16A4 as the sample liquid continues to enter. The advantages of the connection of the inlet port 16a1 to the central tube 16a2 leading to the bottom are: the 'sample cell sap' enters from the bottom of the centrifugal cup 16A and moves upwards under the action of the pressure of the peristaltic pump and the centrifugal force, the moving path of the 'sample cell sap' from the inlet to the outlet is longer, the centrifugal time is correspondingly longer, and the cell centrifugation is more sufficient; furthermore, due to the presence of the bowl core 16A32, it is possible to control not only the "dynamic volume" of the bowl 16A (i.e., the maximum volume that the bowl can hold at high speed), but also the minimum centrifugal force of the cells in the separation chamber 16A 3. The rotary sealing pair 16A5 is composed of a bearing 16A51 and a sealing ring 16A52, the centrifugal cup 16A is divided into a static part and a rotary part, the static part is in interference fit with an outer ring of the bearing 16A51, the rotary part is in fit with an inner ring of the bearing 16A51, and the sealing ring 16A52 is generally made of a fluororubber ring with good heat resistance and friction performance, so that the sealing ring can rotate at a relatively high speed, and has a certain sealing capacity. As shown in fig. 4, when the centrifuge cup 16A rotates at a high speed, the part above the outer ring of the bearing 16A51, such as the inlet 16A1, the center tube 16A2, the seal ring 16A52 and other structural members, are stationary, and the part below the inner ring of the bearing 16A51, such as the cup 16A31 and the inner core 16A32, which form the separation chamber 16A3, rotates at a high speed. The waste liquid pipeline 17A is connected to a liquid outlet 16A4 of the centrifugal cup 16A, and the other side of the waste liquid pipeline 17A is connected with a waste liquid bag 18A.

The concentrate wash module is preferably a single use module.

As shown in FIGS. 5-9, when concentration and washing of cells are required, for example, the volume is 5L and the cell concentration is 1X 10⁶One cell/ml (total number of cells 5X 10)⁹Respectively) was concentrated to 500ml (cell concentration in this case 1X 10)⁷Pieces/ml), a method for concentrating a washing system according to the above-described large-volume biological cell liquid, comprising the steps of:

(1) preliminary self-checking of the system: the system automatically detects whether each sensor and each actuator are abnormal. Detecting whether the feedback values of the sensors of the weighing scales are within a set range, detecting whether the actions of the pipe clamping valves are abnormal, detecting whether the peristaltic pumps 8B and the centrifugal machines 12B are abnormal, detecting whether the feedback values of the air detectors 10B and the pipeline component detectors 11B are abnormal, and detecting whether the feedback values of the pipeline pressure detectors 9B are abnormal;

(2) selecting a program, inputting parameters: selecting a program 'concentration and washing' on a touch display screen 14B of the system, and inputting related parameters, such as a volume before concentration of 5L, a volume after concentration of 500ml, a cycle number of 2.. once and the like;

(3) the volume was 5L and the number of cells was 1X 10⁶Hanging a cell culture solution sample bag 19A with the volume of 500ml on a sample scale 1B, and hanging a washing solution bag 20A with the volume of 500ml on a washing scale 2B;

(4) the disposable concentrated washing component is installed in a cell sap concentrated washing system, and comprises the following specific steps: mounting a centrifuge cup 16A on a centrifuge 12B, mounting a waste liquid pipeline 17A on a pipeline component detector 11B, and hanging a waste liquid bag 18A on a waste liquid scale 4B; hanging a product bag 9A on a product scale 3B, and installing a product pipeline 8A on a product valve 7B; a pipeline pressure detector 9B is arranged on the pipeline pressure sensor in a luer connection mode, a pump pipeline 10A between two pump pipeline clamps 11A is arranged on a peristaltic pump 8B, the front section of the pump pipeline 10A (a pipeline between a second tee joint 7A and the pump pipeline 10A) is arranged on an air detector 10B, a sample pipeline 2A is arranged on a sample valve 5B, and a washing pipeline 4A is arranged on a washing valve 6B, as shown in figure 5;

(5) diagnosis before use: the system can judge whether the current sample bag 19A, the washing liquid bag 20A and the concentrated washing component are installed correctly according to the input parameters and the data fed back from each sensor;

(6) and (3) concentration process: when concentration is started, the sample valve 5B is opened, the washing valve 6B and the product valve 7B are closed, the peristaltic pump 8B and the centrifuge 12B start to operate, cell sap in the sample bag 19A is continuously input into the centrifuge cup 16A under the action of the peristaltic pump 8B, as shown in the attached figure 6-7, cells with higher density are attached to the outermost cup wall under the action of centrifugal force generated by high-speed rotation, waste liquid needing to be removed, such as culture medium with lower density, metabolites and the like, is extruded to the innermost side, and the innermost waste liquid is extruded to the liquid outlet 16A4 and flows into the waste liquid bag 18A through the waste liquid pipeline 17A along with continuous input of the peristaltic pump 8B; when sample scale 1B detects that the weight of the cellular fluid in sample bag 19A is half of the initial weight, sample valve 5B closes and peristaltic pump 8B stalls. At this time, the liquid component in the centrifuge cup 16A is "cells + residual waste liquid";

(7) washing process: when the system is automatically switched to the washing process, the washing valve 6B is opened, and then the peristaltic pump 8B pumps the washing solution into the centrifuge cup 16A, since the density of the washing solution is less than that of the cells and is approximately equal to the waste liquid, the washing solution can completely extrude the waste liquid in the washing solution into the waste liquid bag 18A under the action of centrifugal force, as shown in fig. 8. When the system detects that the weight of the washing liquid is reduced to the programmed amount through the washing scale 2B, the washing valve 6B is closed, the peristaltic pump 8B stops rotating, the centrifugal machine 12B stops rotating slowly, and the liquid component in the centrifugal cup 16A is a mixture of cells and the washing liquid;

(8) and (3) collecting flow: as shown in FIG. 9, when the centrifuge 12B is completely stopped, the cellular fluids in the separation chamber 16A3 flow into the inner core 16A32 of the centrifuge cup under the force of gravity, and the system automatically switches to the collection process. At the moment, the product valve 7B is opened, the sample valve 5B and the washing valve 6B are closed, the peristaltic pump 8B is reversed, the concentrated and washed cell sap in the centrifugal cup 16A is pumped into the product bag 9A through the central tube 16A2 of the centrifugal cup 16A, when the air detector 10B detects that the pipeline is emptied, the peristaltic pump 8B stops rotating, and the product valve 7B is closed;

(9) repeating the above process, inputting the residual liquid in the sample bag 19A into the centrifuge cup 16A for concentration and washing, and then collecting the washed cell sap;

(10) a cleaning process: the washing valve 6B is opened, the sample valve 5B and the product valve 7B are closed, the centrifuge 12B rotates at a low speed (about 500rpm), the peristaltic pump 8B pumps a proper amount of washing liquid into the centrifuge cup 16A, then the washing valve 6B is closed, the product valve 7B is opened, the centrifuge 12B stops rotating, the peristaltic pump 8B reverses to pump the washing liquid in the centrifuge cup 16A into the product bag 9A, and when the ultrasonic air detector detects that the pipeline is air, the product valve 7B is closed, and the peristaltic pump 8B stops rotating. The purpose of this step is to recover the cells remaining in the centrifuge cup 16A and the tube into the product bag 9A, thereby increasing the recovery rate of the cells.

the large-volume biological cell sap concentration and washing system adopts a disposable concentration and washing component to be installed in the system, and is matched with a unique separation cup structure: the liquid inlet and the liquid outlet are separated, so that when cell sap with large volume (500ml-50L) is concentrated and washed, the cell sap can be continuously concentrated, washed and discharged. Moreover, because the liquid inlet is arranged at the bottom of the centrifugal cup and the liquid outlet is arranged at the top, in the process that the sample cell liquid moves from the bottom to the top of the centrifugal cup, the movement path is long, the centrifugal efficiency is high, the efficiency is good, and the cells and the waste liquid are thoroughly separated; in addition, through the design of adding the inner core in the separation cup, the dynamic volume of the separation cup can be adjusted, the minimum centrifugal force is ensured, the separation effect is further ensured, and the cell recovery rate is improved.

1) The cell sap can be continuously concentrated and washed, and the cell sap concentration and washing of large-volume (500ml-50L) sample cell sap have incomparable advantages, thereby greatly improving the efficiency;

2) the unique separation cup structure design has the advantages that liquid is fed from the bottom, liquid is discharged from the top, and meanwhile, the inner core is increased, the centrifugal efficiency is improved, and the cell recovery rate is increased;

3) the intelligent and automatic control system has the advantages that the intelligent and automatic control system is matched with a plurality of sensors and execution elements on the system, the intelligent and automatic degrees are high, the labor intensity of operation is reduced, the consistency of products is improved, and the reliability of equipment and the quality of final products are guaranteed.

In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Claims

1. A concentrated washing system for large-volume biological cell sap is characterized by comprising a centrifugal assembly and a concentrated washing assembly, wherein the centrifugal assembly comprises a centrifuge, the concentrated washing assembly comprises a centrifugal cup, the centrifugal cup is mounted on the centrifuge, and the centrifuge drives the centrifugal cup to rotate at a high speed; the centrifugal cup comprises a static part and a rotating part, the static part is in rotary sealing fit with the rotating part, the rotating part comprises a separation cavity, the separation cavity is a cavity formed by a cup body and an inner core, and when the centrifugal cup rotates at a high speed, cells in a cell sap sample are separated from liquid in the separation cavity.

2. The concentrated washing system for large-volume biological cell sap as claimed in claim 1, wherein said centrifuge assembly further comprises a leakage sensor installed in said centrifuge, said leakage sensor having a moisture sensitive resistor installed thereon, said moisture sensitive resistor being used for determining whether leakage occurs during centrifugation.

3. The concentrated bulk biological cell sap washing system of claim 2, wherein the concentrated washing assembly further comprises a sample piercer, a washing piercer, a product bag, a pump tubing clamp, a pressure monitoring connector, a pressure monitoring tubing, and a waste bag; the sample puncture device, the washing puncture device, the product bag, the pump pipe pipeline, the pressure monitoring connector, the centrifugal cup and the waste liquid bag are connected with each other through pipelines, and two pump pipe clamps are bonded on the pump pipe pipeline.

4. The large volume biological cell sap concentrating and washing system of claim 3, wherein the stationary portion of the centrifuge cup comprises a liquid inlet, a central tube, a liquid outlet and a rotating seal pair, wherein the liquid inlet and the liquid outlet are both disposed at the upper portion of the centrifuge cup, the liquid inlet is communicated with the central tube and is communicated with the bottommost portion of the centrifuge cup, and the rotating seal pair comprises a bearing and a sealing ring; a small opening is formed in the uppermost part of the inner side of the separation cavity of the rotating part and communicated with the liquid outlet; the static part and the rotating part are in interference fit through the bearing, the static part is in interference fit with an outer ring of the bearing, and the rotating part is in interference fit with an inner ring of the bearing.

5. The concentrated washing system for large volume of biological cell sap as claimed in claim 4, wherein the concentrated washing system for cell sap further comprises a weighing assembly, the weighing assembly comprises a sample scale, a washing scale, a product scale and a waste liquid scale, the sample scale, the washing scale, the product scale and the waste liquid scale are all weighing sensors, the sample scale is used for hanging a sample bag, the washing scale is used for hanging a washing liquid bag, the product scale is used for hanging a product bag, the waste liquid scale is used for hanging a waste liquid bag, and the weighing assembly is used for weighing the liquid in the sample bag, the washing liquid bag, the product bag and the waste liquid bag hung on the weighing assembly in real time.

6. The concentrated washing system for large volume of biological cell sap as claimed in claim 5, wherein the concentrated washing system for cell sap further comprises a peristaltic pump as a power source for pumping the liquid in the sample bag or the washing liquid bag into the centrifuge cup rotating at high speed for centrifugal separation and for returning the concentrated product to the product bag.

7. The concentrated washing system for large-volume biological cell sap as claimed in claim 6, wherein the concentrated washing system for large-volume biological cell sap further comprises a pipeline detection assembly, the pipeline detection assembly comprises a pipeline pressure detector, an air detector and a pipeline composition detector, the pipeline pressure detector is internally provided with a pressure sensor, and can detect the pressure in the pipeline in real time and send the pipeline pressure to the control module; the air detector is used for detecting whether the pipeline is installed correctly or not and whether liquid exists in the pipeline or not in real time; the pipeline component detector is used for detecting the component of the liquid in the pipeline.

8. The concentrated washing system for large-volume biological cell sap as claimed in claim 7, wherein the concentrated washing system for large-volume biological cell sap further comprises a human-computer interaction module, the human-computer interaction module comprises a touch display screen, an emergency stop button and an audible and visual alarm, the touch display screen is used for inputting parameters and displaying current state; the emergency stop button is a trigger switch; the audible and visual alarm is used for prompting that the system is abnormal or needs an operator to participate in processing.

9. The concentrated washing system for large volume of biological cell sap as claimed in claim 8, further comprising a pinch valve comprising a sample valve, a wash valve and a product valve, the pinch valve being used to control the flow of liquid in the pipeline.

10. The method for a high volume biological cell liquid concentrated washing system of claim 9, comprising the steps of: