Cell separation and harvesting system and method

Abstract

This specification provides a cell separation and harvesting system and method, which can be used in the field of cell separation technology. Based on the above-mentioned cell separation and harvesting system, when it is implemented, a stable continuous flow can be formed by introducing and utilizing a two-way pump, and then the centrifuge and refrigerated centrifugal chamber can be used to transport the relevant fluid in the form of a stable continuous flow, thereby effectively reducing the loss and damage of cells during the operation and processing, avoiding cell inactivation, and efficiently and automatically completing the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain a high-quality cell product.

Description

Technical Field

[0001] The present invention relates to the field of cell separation technology, and in particular to a cell separation and harvesting system and method. Background Art

[0002] With the development of biotechnology, the production of cell products has received more and more attention.

[0003] Based on the existing system equipment, in the process of separating, preparing and packaging cell products, especially in the cell separation and concentration stage, cells are easily discharged along with the supernatant, resulting in serious cell loss; and, during specific operations, cells are easily damaged and inactivated, affecting the quality of the final cell product.

[0004] To address the above problems, no effective solution has been proposed yet. Summary of the invention

[0005] The present specification provides a cell separation and harvesting system and method, which can effectively reduce the loss and damage of cells during the operation and processing process, avoid cell inactivation, and efficiently and automatically complete the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain high-quality cell products.

[0006] This specification provides a cell separation and harvesting system, which at least includes: a two-way pump, a centrifuge cup, a centrifuge, a refrigerated centrifuge chamber, a quantitative tube, and a gas filter valve; wherein:

[0007] The two-way pump comprises a first channel and a second channel;

[0008] The first end of the first channel is connected to the upper opening of the centrifuge cup, and the second end of the first channel is connected to at least the first pipeline, the second pipeline, and the third pipeline; wherein the first pipeline is used to connect to the cell stock solution bag, the second pipeline is used to connect to the cleaning solution bag, and the third pipeline is connected to the quantitative tube;

[0009] The first end of the second channel is connected to the lower opening of the centrifuge cup, and the second end of the second channel is connected to at least the fourth pipeline, the fifth pipeline, the sixth pipeline, and the seventh pipeline; wherein the fourth pipeline is connected to the quantitative tube, the fifth pipeline is used to connect the sub-packaging bag, the sixth pipeline is used to connect the resuspension liquid bag, and the seventh pipeline is used to connect the waste liquid bag; the distance between the upper opening of the centrifuge cup and the adjacent cup wall is smaller than the distance between the lower opening of the centrifuge cup and the adjacent cup wall;

[0010] The quantitative tube is also connected to the air filter valve through an eighth pipeline;

[0011] The centrifugal cup is connected to the centrifuge, and the centrifugal cup is placed in the refrigerated centrifugal chamber, and the refrigerated centrifugal chamber is also connected to a refrigeration module;

[0012] In addition, a first solenoid valve is arranged on the first pipeline, a second solenoid valve is arranged on the second pipeline, a third solenoid valve is arranged on the third pipeline, a fourth solenoid valve is arranged on the fourth pipeline, a fifth solenoid valve is arranged on the fifth pipeline, a sixth solenoid valve is arranged on the sixth pipeline, and a seventh solenoid valve is arranged on the seventh pipeline.

[0013] In one embodiment, the centrifugal cup at least includes: a cup body, and a connecting component disposed above the cup body;

[0014] Wherein, the connecting component is provided with at least a first interface, a second interface, a first flow channel, and a second flow channel;

[0015] The cup body is at least provided with an upper flow channel, a lower flow channel, a central axis flow channel, an upper opening and a lower opening;

[0016] Wherein, the first interface is connected to the first flow channel, the first interface is used to connect to the first channel, and the first flow channel is connected to the upper port through the upper flow channel;

[0017] The second interface is connected to the second flow channel, the second interface is used to connect to the second channel, and the second flow channel is connected to the lower port through the central axis flow channel and the lower flow channel.

[0018] In one embodiment, the size of the hose disposed in the first channel is the same as the size of the hose disposed in the second channel.

[0019] This specification also provides a cell separation and harvesting method based on the cell separation and harvesting system, comprising:

[0020] According to the preset calibration rules, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve and the two-way pump to determine the hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle;

[0021] According to the preset separation and concentration rules and based on the hose coefficient of the two-way pump, the first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow by controlling the first solenoid valve and the two-way pump; at the same time, the cell stock solution in the centrifuge cup is separated into cells and supernatant by controlling the centrifuge, the two-way pump and the seventh solenoid valve, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow;

[0022] According to the preset cleaning rules, the cleaning solution is pumped into the centrifuge cup in the form of a continuous flow by controlling the second solenoid valve and the two-way pump to clean the supernatant on the cells; at the same time, the cleaning solution is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve;

[0023] According to the preset resuspension rule and based on the hose coefficient of the two-way pump, the washed cells in the centrifuge cup are resuspended by controlling the sixth solenoid valve, the two-way pump and the third solenoid valve;

[0024] According to the preset sub-packaging rules and based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifugal cup is transported to the sub-packaging bag by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve.

[0025] In one embodiment, according to a preset calibration rule, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine the hose coefficient of the two-way pump, including:

[0026] According to the preset calibration rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0027] Controlling the two-way pump to rotate in a first direction to pump a second volume of cleaning solution into the centrifuge cup along the second pipeline as a calibration solution; and stopping the centrifuge; wherein the second volume is greater than the volume of the quantitative tube;

[0028] Controlling the two-way pump to rotate in a first direction to transfer the calibration liquid in the centrifugal cup along the seventh pipeline toward the waste liquid bag; and monitoring whether the calibration liquid reaches the seventh solenoid valve;

[0029] When it is detected that the calibration liquid reaches the seventh solenoid valve, the seventh solenoid valve is closed and the fourth solenoid valve is opened;

[0030] Controlling the two-way pump to rotate in a first direction to transfer the calibration liquid in the centrifugal cup to the quantitative tube along the fourth pipeline; and monitoring the detection value of the third bubble sensor;

[0031] When the detection value of the third bubble sensor indicates that liquid is detected, the operation of the two-way pump is stopped; and the number of rotations of the two-way pump is obtained;

[0032] The hose coefficient of the two-way pump is determined based on the volume of the metering tube and the number of revolutions of the two-way pump.

[0033] In one embodiment, according to a preset separation and concentration rule, based on the hose coefficient of the two-way pump, by controlling the first solenoid valve and the two-way pump, a first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow, including:

[0034] According to the preset separation and concentration rules, the first solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0035] Controlling the two-way pump to rotate in a first direction based on a first speed, so as to input the cell stock solution in the cell stock solution bag into the centrifuge cup through the upper opening of the centrifuge cup in the form of a continuous flow along the first pipeline;

[0036] Controlling the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cells of the cell stock solution entering the centrifuge cup enter the wall-adherent state under the action of centrifugal force, so as to separate the cell stock solution into cells and supernatant; and outputting the supernatant from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and inputting it into a waste liquid bag along the seventh pipeline;

[0037] When the volume of the cell stock solution input into the centrifuge cup reaches the first volume based on the hose coefficient of the two-way pump, the two-way pump is stopped, the centrifuge is stopped, and the first solenoid valve and the seventh solenoid valve are closed.

[0038] In one embodiment, according to a preset cleaning rule, by controlling the second solenoid valve and the two-way pump, a cleaning solution is pumped into the centrifuge cup in the form of a continuous flow to clean the supernatant on the cells; at the same time, by controlling the two-way pump and the seventh solenoid valve, the cleaning solution after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow, including:

[0039] According to the preset cleaning rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0040] Controlling the two-way pump to rotate in a first direction based on a second speed, so that the cleaning liquid in the cleaning liquid bag is input into the centrifugal cup through the upper opening of the centrifugal cup in the form of a continuous flow along the second pipeline;

[0041] Controlling the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cleaning solution entering the centrifuge cup washes the cells in the adherent state; and outputting the cleaning solution from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and inputting it into a waste liquid bag along the seventh pipeline;

[0042] When it is determined that the cleaning end condition is met, the second solenoid valve and the seventh solenoid valve are closed, and the first solenoid valve and the fourth solenoid valve are opened;

[0043] The two-way pump is controlled to rotate in the second direction based on the third speed, and the remaining cleaning solution in the centrifuge cup is input into the cell stock solution bag along the first pipeline through the upper opening of the centrifuge cup.

[0044] In one embodiment, according to a preset resuspension rule, based on the hose coefficient of the two-way pump, the sixth solenoid valve, the two-way pump, and the third solenoid valve are controlled to resuspend the washed cells in the centrifuge cup, including:

[0045] According to the preset resuspension rules, the third solenoid valve and the sixth solenoid valve are opened; and the two-way pump is started;

[0046] Controlling the two-way pump to rotate in a second direction based on a fourth speed, so as to input the resuspension in the resuspension bag into the centrifuge cup through a lower opening of the centrifuge cup along a sixth pipeline;

[0047] When the volume of the resuspension liquid input into the centrifuge cup reaches the third volume based on the hose coefficient of the two-way pump, the two-way pump is stopped; and the centrifuge is started;

[0048] The centrifuge is controlled to rotate based on the second rotation speed so that the cells in the adherent state are dissolved in the resuspension solution to obtain a target solution containing cells.

[0049] In one embodiment, according to a preset packaging rule, based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifuge cup is transported to the packaging bag by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve, including:

[0050] According to the preset packaging rules, the third solenoid valve and the fifth solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0051] controlling the centrifuge to rotate based on a third rotation speed;

[0052] Based on the hose coefficient of the two-way pump, the two-way pump is controlled to rotate in the second direction based on the fifth speed, and the target liquid of a specified volume is input into the sub-packaging bag along the fifth pipeline through the lower opening of the centrifugal cup.

[0053] The present specification also provides a computer-readable storage medium having computer instructions stored thereon, and when the instructions are executed by a processor, the relevant steps of the cell separation and harvesting method are implemented.

[0054] Based on the cell separation and harvesting system and method provided in this specification, the system at least includes: a two-way pump, a centrifugal cup, a centrifuge, a refrigerated centrifugal chamber, a quantitative tube, and an air filter valve; wherein the two-way pump includes a first channel and a second channel; the first end of the first channel is connected to the upper opening of the centrifugal cup, and the second end of the first channel is connected to at least the first pipeline, the second pipeline, and the third pipeline; wherein the first pipeline is used to connect the cell stock solution bag, the second pipeline is used to connect the cleaning solution bag, and the third pipeline is connected to the quantitative tube; the first end of the second channel is connected to the lower opening of the centrifugal cup, and the second end of the second channel is connected to at least the fourth pipeline, the fifth pipeline, the sixth pipeline, and the seventh pipeline; wherein the fourth pipeline is connected to the quantitative tube, the fifth pipeline is connected to the quantitative tube, and the sixth pipeline is connected to the seventh pipeline. The sixth pipeline is used to connect the sub-packaging bag, the sixth pipeline is used to connect the resuspension bag, and the seventh pipeline is used to connect the waste liquid bag; the distance between the upper opening of the centrifuge cup and the adjacent cup wall is smaller than the distance between the lower opening of the centrifuge cup and the adjacent cup wall; the quantitative tube is also connected to the air filter valve through the eighth pipeline; the centrifuge cup is connected to the centrifuge, and the centrifuge cup is placed in the refrigerated centrifugal cabin, and the refrigerated centrifugal cabin is also connected to a refrigeration module; and a first solenoid valve is provided on the first pipeline, a second solenoid valve is provided on the second pipeline, a third solenoid valve is provided on the third pipeline, a fourth solenoid valve is provided on the fourth pipeline, a fifth solenoid valve is provided on the fifth pipeline, a sixth solenoid valve is provided on the sixth pipeline, and a seventh solenoid valve is provided on the seventh pipeline. The cell separation and harvesting system based on the above structure can be implemented by using the characteristics of the two-way pump to form a stable continuous flow, and then the stable continuous flow is used to cooperate with the centrifuge, refrigerated centrifugal cabin and other structures to transport the relevant fluids. Specifically, for example, a cell stock solution can be pumped into a centrifuge cup through a stable continuous flow; at the same time, the cell stock solution pumped into the centrifuge cup is separated into cells and supernatant through a stable continuous flow to achieve a separation operation, and the supernatant is discharged from the centrifuge cup through a stable continuous flow to retain the cells to achieve a concentration operation. This can effectively reduce the loss and damage of cells during the operation and processing process, avoid cell inactivation, and efficiently and automatically complete the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain a high-quality cell product. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the embodiments of this specification, the drawings required for use in the embodiments will be briefly introduced below. The drawings described below are only some embodiments recorded in this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0056] Figure 1 It is a schematic diagram of the structure of a cell separation and harvesting system provided in one embodiment of the present specification;

[0057] Figure 2It is a schematic diagram of the structure of a two-way pump in a cell separation and harvesting system provided in an embodiment of the present specification;

[0058] Figure 3 It is a schematic diagram of the structural composition when a two-way pump in a cell separation and harvesting system provided by an embodiment of the present specification is connected to a centrifuge cup;

[0059] Figure 4 It is a schematic diagram of the structure of a centrifuge cup in a cell separation and harvesting system provided in one embodiment of the present specification;

[0060] Figure 5 It is a schematic diagram of the structure of a refrigerated centrifugal chamber in a cell separation and harvesting system provided in one embodiment of the present specification;

[0061] Figure 6 It is a schematic diagram of the structure and composition of a specific product of a cell separation and harvesting system provided in one embodiment of this specification;

[0062] Figure 7 is a schematic diagram of a cell separation and harvesting method provided in one embodiment of the present specification;

[0063] Figure 8 It is a schematic diagram of an embodiment of a calibration operation using the cell separation and harvesting method provided in an embodiment of this specification in a scenario example;

[0064] Fig. 9 It is a schematic diagram of an embodiment of a scenario example, in which a cell separation and harvesting system provided by an embodiment of this specification is used to perform a calibration operation;

[0065] Fig.10 It is a schematic diagram of an embodiment of a separation and concentration operation using the cell separation and harvesting method provided in an embodiment of this specification in a scenario example;

[0066] Fig.11 It is a schematic diagram of an embodiment of a cleaning operation using the cell separation and harvesting method provided in an embodiment of this specification in a scenario example;

[0067] Fig.12 It is a schematic diagram of an embodiment of a resuspension operation using the cell separation and harvesting method provided in the embodiments of this specification in a scenario example;

[0068] Fig.13 is a schematic diagram of the structure of an electronic device provided by an embodiment of this specification;

[0069] Fig.14 It is a schematic diagram of the structure of a cell separation and harvesting device provided in one embodiment of the present specification;

[0070] Fig.15 It is a schematic diagram of a process of fully automatic cell separation and harvesting using the cell separation and harvesting system provided in an embodiment of this specification in a scenario example;

[0071] Fig.16 It is a schematic diagram of an improved structure of a centrifuge cup applied to a cell separation and harvesting system provided in an embodiment of this specification in a scenario example;

[0072] Description of the drawings: 0, solenoid valve; 1, first solenoid valve; 2, second solenoid valve; 3, third solenoid valve; 4, fourth solenoid valve; 5, fifth solenoid valve; 6, sixth solenoid valve; 7, seventh solenoid valve; 10, liquid bag; 11, cell stock liquid bag; 12, cleaning liquid bag; 13, subpackaging bag; 14, resuspension liquid bag; 15, waste liquid bag; 20, pipeline; 21, first pipeline; 22, second pipeline; 23, third pipeline; 24, fourth pipeline; 25, fifth pipeline; 26, sixth pipeline; 27, seventh pipeline; 28, eighth pipeline; 30, two-way pump; 31, first channel; 32, second channel; 33, motor; 34, roller; 40, centrifugal cup; 401, connecting component; 402 , cup body; 403, base; 41, upper opening; 42, lower opening; 43, cup wall; 44, first interface; 45, second interface; 46, bearing; 47, large sealing ring; 48, small sealing ring; 49, flow guide platform; 4-1, first flow channel; 4-2, second flow channel; 4-3, upper flow channel; 4-4, lower flow channel; 4-5, central axis flow channel; 50, refrigeration centrifugal chamber; 51, refrigeration module; 52, temperature sensor; 60, centrifuge; 70, quantitative tube; 80, air filter valve; 91, operation panel; 92, power switch; 93, liquid bag hook; 94, liquid bag bracket; P, bubble sensor; P1, first bubble sensor; P2, second bubble sensor; P3, third bubble sensor. DETAILED DESCRIPTION

[0073] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this specification, not all of the embodiments. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this specification.

[0074] It should be noted that the user-related information and data involved in the embodiments of this specification are all information and data authorized by the user or fully authorized by relevant parties, and the collection, storage, use, processing, transmission, provision, disclosure and application of the relevant data comply with relevant laws, regulations and standards, take necessary confidentiality measures, do not violate public order and good morals, and provide corresponding operation entrances for users or relevant parties to choose to authorize or refuse.

[0075] It should also be noted that in the embodiments of this specification, certain software, components, models and other existing solutions in the industry may be mentioned. They should be regarded as exemplary. Their purpose is only to illustrate the feasibility of implementing the technical solution of this application, but it does not mean that the applicant has or will necessarily use the solution.

[0076] Considering that based on the existing system equipment, when separating and packaging cells, most of them use conventional peristaltic pumps to form negative pressure in the centrifuge cup to generate a pressure difference with the outside world; then the cell stock solution containing cells is sucked into the centrifuge cup through the pressure difference for subsequent cell separation. However, the fluid movement generated by the pressure difference is often unstable, and it is easy to cause sudden interruption or sudden change of flow rate due to factors such as changes in liquid volume or pressure, affecting separation and packaging. In addition, based on the above method, when the cell stock solution is pumped into the centrifuge cup, since the inside of the centrifuge cup is in a negative pressure state, the cell stock solution will suddenly accelerate the flow rate due to the pressure difference at the moment of entering the centrifuge cup, resulting in the cells in the cell stock solution not having time to separate and adhere to the wall, and being discharged from the centrifuge cup together with the cell stock solution, resulting in cell loss. In addition, based on the above method, it is necessary to maintain a large negative pressure in the centrifuge cup for a long time, which will cause the cells in the centrifuge cup to be subject to a large surface tension, thereby affecting the cell activity and even causing cell inactivation.

[0077] Aiming at the root cause of the above problems, the applicant, through creative work, considered introducing and using a dual-channel dual-pass pump to replace the conventional peristaltic pump, and making corresponding improvements to the relevant structure of the system to cooperate with the operation of the dual-pass pump in the process of cell separation and packaging; further, a refrigerated centrifugal chamber was introduced and used to cool the cells in the centrifugal cup to avoid damage to cell activity caused by excessive temperature during the centrifugal concentration stage. In this way, the dual-pass pump can be used to make the liquid inlet speed and liquid outlet speed of the centrifugal cup relatively consistent, so that the pressure in the centrifugal cup is relatively constant, so that a stable continuous flow can be formed. Furthermore, the above-mentioned stable continuous flow can be used to complete operations such as separation, concentration, washing, resuspension, and packaging in the process of cell separation and packaging. On the one hand, the liquid flow rate when entering the centrifugal cup can be stable and reliable, so that the cells in the cell stock solution have sufficient time to enter the wall-attached state under the action of centrifugal force after entering the centrifugal cup, so as to achieve separation and effectively reduce the loss of cells during the separation operation; on the other hand, it can also effectively avoid the cells in the centrifugal cup from being inactivated due to the negative pressure. Thus, the separation and packaging of cells can be completed relatively efficiently, and a cell product with higher quality can be obtained.

[0078] See also Figure 1 As shown, the embodiment of this specification provides a cell separation and harvesting system, which may at least include: a two-way pump 30, a centrifuge cup 40, a centrifuge 60, a refrigerated centrifuge chamber 50, a quantitative tube 70, a gas filter valve 80 and other structures; wherein,

[0079] The two-way pump 30 may specifically include a first channel 31 and a second channel 32;

[0080] The first end of the first channel 31 is connected to the upper opening of the centrifuge cup 40, and the second end of the first channel 31 is connected to at least the first pipeline 21, the second pipeline 22, and the third pipeline 23; wherein the first pipeline 21 is used to connect the cell stock solution bag 11, the second pipeline 22 is used to connect the cleaning solution bag 12, and the third pipeline 23 is connected to the quantitative tube 70;

[0081] The first end of the second channel 32 is connected to the lower opening 42 of the centrifuge cup 40, and the second end of the second channel 32 is connected to at least the fourth pipeline 24, the fifth pipeline 25, the sixth pipeline 26, and the seventh pipeline 27; wherein the fourth pipeline 24 is connected to the quantitative tube 70, the fifth pipeline 25 is used to connect the sub-packaging bag 13, the sixth pipeline 26 is used to connect the resuspension liquid bag 14, and the seventh pipeline 27 is used to connect the waste liquid bag 15; the distance between the upper opening 41 of the centrifuge cup 40 and the adjacent cup wall 43 is smaller than the distance between the lower opening 42 of the centrifuge cup 40 and the adjacent cup wall 43;

[0082] The quantitative pipe 70 is also connected to the air filter valve 80 through the eighth pipeline 28;

[0083] The centrifugal cup 40 is connected to the centrifuge 60, and the centrifugal cup 40 is placed in the refrigerated centrifugal chamber 50, and the refrigerated centrifugal chamber 50 is also connected to a refrigeration module 51;

[0084] In addition, a first solenoid valve 1 (which can be expressed as ①) is arranged on the first pipeline 21, a second solenoid valve 2 (which can be expressed as ②) is arranged on the second pipeline 22, a third solenoid valve 3 (which can be expressed as ③) is arranged on the third pipeline 23, a fourth solenoid valve 4 (which can be expressed as ④) is arranged on the fourth pipeline 24, a fifth solenoid valve 5 (which can be expressed as ⑤) is arranged on the fifth pipeline 25, a sixth solenoid valve 6 (which can be expressed as ⑥) is arranged on the sixth pipeline 26, and a seventh solenoid valve 7 (which can be expressed as ⑦) is arranged on the seventh pipeline 27.

[0085] The above-mentioned pipelines may be collectively referred to as pipelines 20 ; the above-mentioned solenoid valves may be collectively referred to as solenoid valves 0 ; and the above-mentioned liquid bags may be collectively referred to as liquid bags 10 .

[0086] Specifically, the first solenoid valve 1 , the second solenoid valve 2 , the third solenoid valve 3 , the fourth solenoid valve 4 , the fifth solenoid valve 5 , the sixth solenoid valve 6 , and the seventh solenoid valve 7 can be used to control the on-off of the pipelines in which they are located.

[0087] Specifically, the above-mentioned two-way pump 30 (or two-way peristaltic pump) can be a two-channel peristaltic pump. Figure 2 As shown, the two-way pump 30 at least includes: a motor 33, a roller 34, a first channel 31, a second channel 32 and other structures. Further, the first channel 31 and the second channel 32 can be set to use hoses of the same size (for example, the same diameter). Since the first channel 31 and the second channel 32 are driven by the same motor and the same roller, and the two channels have the same diameter, the fluid velocity based on the first channel 31 is the same as the fluid velocity based on the second channel 32.

[0088] Accordingly, the introduction and use of the two-way pump 30 in the cell separation and harvesting system can make the liquid inlet and outlet speeds of the centrifuge cup 40 relatively consistent, maintain the pressure in the centrifuge cup 40 constant, and thus form a stable continuous flow.

[0089] Specifically, the cell stock solution bag 11 can store a cell stock solution containing cells to be separated and concentrated. The cleaning solution bag 12 can store a cleaning solution for cleaning residual liquid on cells. The resuspension solution bag 14 can store a resuspension solution for resuspending the separated cells.

[0090] The waste liquid bag 15 can be an empty liquid bag, used to store waste liquid generated during the operation of the system. The subpackaging bag 13 can be an empty liquid bag, used to store the target liquid containing cells (or cell liquid product, cell product, etc.) obtained by final separation and concentration during the operation of the system.

[0091] Furthermore, in addition to the first pipeline 21, the second pipeline 22, and the third pipeline 23, the second end of the first channel 31 can also be extended to connect other pipelines according to specific application scenarios and processing requirements; wherein other pipelines can be connected to other liquid bags such as other cell stock liquid bags. Similarly, in addition to the fourth pipeline 24, the fifth pipeline 25, the sixth pipeline 26, and the seventh pipeline 27, the second end of the second channel 32 can also be extended to connect other pipelines.

[0092] Specifically, the air filter valve 80 may be connected to the external environment to inhale and filter air fluid from the external environment; or, to discharge air fluid to the external environment.

[0093] Specifically, the centrifuge 60 is connected to the centrifuge cup 40, and can drive the centrifuge cup 40 to rotate through the operation of the internal motor, so that the cells in the solution in the centrifuge cup 40 can be closely attached to the cup wall 43 of the centrifuge cup 40 due to the effect of centrifugal force and enter a wall-adherent state.

[0094] Specifically, the refrigerated centrifugal chamber 50 is connected to a refrigeration module 51. In a specific implementation, the centrifugal cup 40 is placed in the refrigerated centrifugal chamber 50, and the refrigeration module 51 can intelligently control the internal temperature of the refrigerated centrifugal chamber 50, so that the cells in the centrifugal cup 40 are in a stable and appropriate threshold temperature range, thereby preventing the cells from being inactivated due to excessive temperature.

[0095] Specifically, the threshold temperature range may be greater than or equal to 2 degrees Celsius and less than or equal to 8 degrees Celsius.

[0096] Specifically, the quantitative tube 70 can be understood as a communication structure with a known and fixed volume. For example, the volume of the quantitative tube 70 can be recorded as V.

[0097] For details, see Figure 1 As shown, the first pipeline 21, the second pipeline 22, and the third pipeline 23 may first converge at a node; and then the node is connected to the first channel 31 of the two-way pump 30 through corresponding pipelines.

[0098] Similarly, the fourth pipeline 24 , the fifth pipeline 25 , the sixth pipeline 26 , and the seventh pipeline 27 may first converge at a node; and then the node may be connected to the second channel 32 of the two-way pump 30 through corresponding pipelines.

[0099] For details, see Figure 3As shown, the first channel 31 may be connected to the upper opening 41 of the centrifuge cup 40 , and the second channel 32 may be connected to the lower opening 42 of the centrifuge cup 40 .

[0100] For further information, see Figure 4 As shown, the distance between the upper opening 41 and its adjacent cup wall 43 (eg, the right cup wall) is smaller than the distance between the lower opening 42 and its adjacent cup wall 43 (eg, the left cup wall).

[0101] When implementing it, Figure 3 and Figure 4 It can be seen that when the two-way pump 30 rotates in the first direction, liquid can be pumped into the centrifugal cup 40 through the upper opening 41 of the centrifugal cup 40 through the first channel 31; at the same time, liquid is discharged from the centrifugal cup 40 through the lower opening 42 of the centrifugal cup 40 through the second channel 32. Since the liquid flow rates of the first channel 31 and the second channel 32 are the same, the pressure in the centrifugal cup 40 can be maintained constant, and no pressure difference will be generated, thereby forming a stable continuous flow. Further, in the form of the above-mentioned stable continuous flow, liquid can be pumped into the centrifugal cup 40 stably and uniformly, and liquid can be discharged from the centrifugal cup 40 synchronously.

[0102] Specifically, for example, in the cell separation and concentration stage, while the cell stock solution is pumped into the centrifuge cup based on the first channel of the two-way pump, the supernatant remaining after the cells in the cell stock solution are separated by centrifugal force is discharged from the centrifuge cup at the same flow rate based on the second channel of the two-way pump, thereby forming a stable continuous flow in the centrifuge cup and related pipelines.

[0103] Furthermore, the cell stock solution can be pumped into the centrifuge cup in a stable and uniform manner in the form of continuous flow, and the supernatant after the cells are separated can be discharged from the centrifuge cup synchronously. It is not necessary to rely on the pressure difference in the centrifuge cup to pump the cell stock solution into the centrifuge cup like the existing system equipment.

[0104] In this way, on the one hand, the cell stock solution can enter the centrifuge cup continuously, stably and at a uniform speed, so that the cells in the cell stock solution have sufficient time to be pressed against the cup wall under the action of centrifugal force in the centrifuge cup, thereby achieving separation of cells from the supernatant; and avoid the cell stock solution from being directly flushed out of the centrifuge cup due to the unstable flow rate of the cell stock solution when entering the centrifuge cup, and the flow rate is suddenly too high, causing the cells to be lost. On the other hand, it can also effectively prevent the cell stock solution from being damaged or even inactivated due to the squeezing force generated by the pressure difference after entering the centrifuge cup.

[0105] In addition, because the distance between the upper opening of the centrifuge cup and the cup wall of the centrifuge cup is relatively close, when the centrifuge drives the centrifuge cup to rotate, the cells in the cell stock solution entering from the upper opening are more likely to stick to the cup wall (enter the wall-adherent state) under the action of centrifugal force, and it is relatively more difficult to directly discharge the centrifuge cup with the cell liquid, thereby effectively reducing the loss of cells and better achieving the separation of cells and supernatant (which can be simply referred to as cell separation). At the same time, after the centrifuge cup undergoes the above-mentioned cell separation, the supernatant separated from the cell stock solution can refer to Figure 4 The liquid path shown in the figure discharges the centrifuge cup through the lower opening of the centrifuge cup to achieve concentration of the cell fluid (which can be simply referred to as cell concentration). Since the distance between the lower opening of the centrifuge cup and the cup wall of the centrifuge cup is relatively far, the distance between the cells in the centrifuge cup in the wall state and the discharge port is long, which increases the difficulty of the adherent cells to be discharged from the centrifuge cup with the liquid, thereby further reducing the loss of cells.

[0106] In addition, when the two-way pump rotates in the second direction, gas can be pumped into the centrifugal cup through the second channel via the lower opening of the centrifugal cup; at the same time, liquid can be discharged from the centrifugal cup through the upper opening of the centrifugal cup through the first channel. Moreover, since the liquid is discharged to the outside of the centrifugal cup through the upper opening of the centrifugal cup, and the upper opening of the centrifugal cup is relatively close to the cup wall, relatively more liquid can be discharged from the centrifugal cup.

[0107] In addition, by forming a stable continuous flow, a more effective water cooling system can also be formed. The heat generated by the rotational motion in the centrifuge cup can be promptly conducted away through the stable continuous flow, thereby also achieving auxiliary cooling of the centrifuge cup and further reducing cell inactivation caused by excessive temperature.

[0108] In some embodiments, see Figure 1 As shown, a first bubble sensor (which can be represented as P1) is arranged near the second end of the first channel 31, a second bubble sensor (which can be represented as P2) is arranged near the first end of the second channel 32, and a third bubble sensor (which can be represented as P3) is arranged near the quantitative tube 70 on the eighth pipeline 28.

[0109] The first bubble sensor P1 can be used to detect whether the type of the fluid passing through the first channel 31 of the two-way pump 30 is gas or liquid. The second bubble sensor P2 can be used to detect whether the type of the fluid passing through the second channel 32 of the two-way pump 30 is gas or liquid. The third bubble sensor P3 can be used to detect whether the type of the fluid flowing out through the quantitative tube 70 is gas or liquid.

[0110] In some embodiments, the size of the hose disposed in the first channel is the same as that of the hose disposed in the second channel, so as to ensure that the flow rate of the fluid based on the first channel is the same as that of the fluid based on the second channel.

[0111] In some embodiments, a flow rate detector may be provided on the pipeline between the second channel of the two-way pump and the lower opening of the centrifuge cup to detect the velocity of the fluid flowing into the centrifuge cup. The flow rate detector may be electrically connected to the two-way pump.

[0112] Accordingly, the two-way pump can receive and dynamically adjust the rotation speed of the two-way pump according to the flow rate collected by the flow rate detector, so that the fluid can be pumped into the centrifuge cup at a suitable speed.

[0113] In some embodiments, an angular velocity detector may be provided on the centrifuge cup to detect the angular velocity of the centrifuge cup when it rotates with the centrifuge, wherein the angular velocity detector may be electrically connected to the centrifuge.

[0114] Accordingly, the centrifuge can receive and determine the actual rotation speed of the centrifuge cup according to the angular velocity collected by the angular velocity detector, and then adjust the rotation speed of the centrifuge in a targeted manner.

[0115] In some embodiments, in order to cooperate with the above-mentioned cell separation and harvesting system to better complete operations such as separation, concentration, and washing, the structure of the centrifuge cup 40 is further improved. For details, please refer to Fig.16 shown.

[0116] Specifically, the centrifugal cup 40 may at least include: a cup body 402, and a connecting component 401 disposed above the cup body 402;

[0117] The connecting component 401 may at least be provided with a first interface 44, a second interface 45, a first flow channel 4-1, a second flow channel 4-2, etc.;

[0118] The cup body 402 may be provided with at least an upper flow channel 4-3, a lower flow channel 4-4, a central axis flow channel 4-5, an upper opening 41, a lower opening 42, etc.;

[0119] The first interface 44 may be connected to the first flow channel 4-1. The first interface 44 may be specifically used to connect to the first channel 31. The first flow channel 4-1 may be connected to the upper port 41 through the upper flow channel 4-3.

[0120] The second interface 45 can be connected to the second flow channel 4-2. The second interface 45 can be specifically used to connect to the second channel 32. The second flow channel 4-2 can be connected to the lower port 42 through the central axis flow channel 4-5 and the lower flow channel 4-4 in sequence.

[0121] For details, please refer to Fig.16 As shown, the above-mentioned connecting component 401 can be specifically provided with a dual-channel F-head, in which one connector serves as a first interface 44 connected to a first flow channel 4-1 provided inside the connecting component 401, and the other connector serves as a second interface 45 connected to a second flow channel 4-2 provided inside the connecting component 401.

[0122] For details, please refer to Fig.16 As described above, an upper flow channel 4-3 may be provided near the top of the cup body 402 of the centrifugal cup 40; wherein, the upper flow channel 4-3 is connected to the first flow channel 4-1. An upper opening 41 is provided on the upper flow channel 4-3 at a position close to the cup wall 43. In a specific implementation, the fluid outside the centrifugal cup 40 may enter the centrifugal cup 40 through the first interface 44, through the first flow channel 4-1, and then through the upper flow channel 4-3, and flow from the upper opening 41 along the cup wall 43 to the bottom of the cup.

[0123] A lower flow channel 4-4 may be provided near the bottom of the cup body 402 of the centrifugal cup 40; wherein a lower opening 42 is provided on the lower flow channel 4-4 at a position far from the cup wall 43. The distance between the lower opening 42 and the cup wall 43 (for example, can be recorded as) is greater than the distance between the upper opening 41 and the cup wall 43 (for example, can be recorded as). In addition, close to the bottom of the cup body 402, a central axis flow channel 4-5 is also provided along the central axis of the cup body. The central axis flow channel 4-5 is connected to the lower flow channel 4-4 and is also connected to the second flow channel 4-2. In a specific implementation, the fluid at the bottom of the centrifugal cup 40 can flow into the lower flow channel 4-4 through the lower opening 42, and then along the central axis flow channel 4-5, enter the second flow channel 4-2, and flow out of the centrifugal cup 40 from the second interface 45.

[0124] Specifically, a guide platform 49 (for example, a T-shaped guide platform) may be provided at the intersection of the first flow channel 4-1 and the upper flow channel 4-3; further, a large sealing ring 47 may be provided at an adjacent position above the guide platform 49; a small sealing ring 48 may be provided at an adjacent position below the guide platform 49; in addition, a bearing 46 and other structures may be provided at an upper position of the large sealing ring 47 to cooperate with the cell separation and harvesting system for high-speed centrifugal rotation.

[0125] Specifically, the centrifugal cup may further include a base 403 , wherein the base 403 may be fixedly disposed on the centrifuge 60 , and the cup body 402 may be disposed on the base 403 .

[0126] In specific operation, based on the centrifugal cup, when the cell separation and harvesting system rotates, when fluid enters the centrifugal cup from the first interface, it will flow from the upper opening along the cup wall to the bottom of the cup through the upper flow channel. Since the distance between the upper opening and the cup wall is relatively close, in the separation and concentration stage, it is relatively easier for cells to enter the wall-attached state due to the action of centrifugal force, and more cells are separated; at the same time, the separated supernatant flows along the cup wall to the cup body, enters the lower flow channel from the lower opening, and then passes through the central axis flow channel and the second flow channel in turn to be discharged from the centrifugal cup from the second interface.

[0127] When fluid is discharged from the centrifuge cup from the upper opening, through the upper flow channel and the first flow channel in sequence, from the first interface, since the distance between the upper opening and the cup wall is relatively close, relatively more cleaning liquid can be discharged from the centrifuge cup during the cleaning stage.

[0128] Specifically, for example, assuming that the radius of the cup is R, the height is H, and the distance between the top and the cup wall is , the distance between the lower opening and the cup wall is .

[0129] If the liquid is discharged through the lower opening, the remaining liquid volume in the cup can be expressed as: .

[0130] On the contrary, if the liquid is discharged through the upper mouth, the volume of the remaining liquid in the cup can be expressed as: .

[0131] because Less than , therefore, V 下 >V 上 .

[0132] It can be seen that based on the above centrifuge cup, the degree of liquid discharge from the centrifuge cup can be flexibly adjusted by adjusting the interface used when discharging the liquid in cooperation with the cell separation and harvesting system according to specific circumstances and processing requirements.

[0133] In some embodiments, see Figure 5 As shown, a temperature sensor 52 may also be provided in the refrigerated centrifugal chamber 50 for detecting the temperature in the refrigerated centrifugal chamber 50 in real time.

[0134] The temperature sensor 52 may be electrically connected to the refrigeration module 51 .

[0135] In specific implementation, the refrigeration module can intelligently and dynamically adjust the refrigeration power according to the temperature data collected by the temperature sensor, and effectively maintain the temperature of the centrifuge cup in the refrigerated centrifuge chamber within a suitable threshold temperature range.

[0136] In some embodiments, according to specific application scenarios and processing requirements, the second end of the first channel may be connected to other extension pipelines; wherein the other extension pipelines may be connected to other suitable liquid bags, for example, other cell stock liquid bags, or empty liquid bags, etc.

[0137] Similarly, the second end of the second channel may be connected to other extension pipelines; wherein, the other extension pipelines may be connected to other suitable liquid bags, for example, other filling bags, or other waste liquid bags, etc.

[0138] In some embodiments, the cell separation and harvesting system may further include a processor. The processor may be electrically connected to the solenoid valve, bubble sensor, temperature sensor, refrigeration module, two-way pump, and centrifuge in the system. In specific implementation, the processor may receive and intelligently control the corresponding solenoid valve, refrigeration module, two-way pump, centrifuge, etc. according to the relevant data collected by the bubble sensor and the temperature sensor, and automatically complete the cell separation and packaging.

[0139] In some embodiments, during specific implementation, the processor can first complete the calibration operation of the two-way pump by controlling the corresponding solenoid valve, using the quantitative tube, two-way pump, centrifuge and other structures. Then, by controlling the corresponding solenoid valve, the two-way pump is used to pump the specified volume of cell stock solution from the cell stock solution bag into the centrifuge cup in the form of a continuous flow to realize the liquid inlet operation; at the same time, the centrifuge is used to rotate the centrifuge cup so that the cells in the cell stock solution enter the wall-adhering state under the action of centrifugal force, cling to the cup wall, and separate from the supernatant to realize the separation operation; and the two-way pump is also used to synchronously discharge the separated supernatant from the centrifuge cup and transport it to the waste liquid bag to realize the concentration operation. Then, by controlling the corresponding solenoid valve, the two-way pump is used to pump the cleaning liquid from the cleaning liquid bag into the centrifuge cup in the form of a continuous flow to rinse the separated cells to realize the cleaning operation. Then, by controlling the corresponding solenoid valve, the two-way pump is used to pump the corresponding volume of the resuspension liquid from the resuspension liquid bag into the centrifuge cup to mix and dissolve the separated cells to realize the resuspension operation. Finally, by controlling the corresponding solenoid valve, a two-way pump is used to transport the target liquid containing cells in the centrifugal cup to the sub-packaging bag to realize the sub-packaging operation and complete the separation and sub-packaging of the cells.

[0140] In some embodiments, the physical structure of the cell separation and harvesting system can be referred to as Figure 6 As shown. The cell stock liquid bag 11 and the cleaning liquid bag 12 can be mounted on the liquid bag bracket 94 on the right; the sub-packaging bag 13, the resuspension liquid bag 14, and the waste liquid bag 15 can be mounted on the liquid bag bracket 94 on the left. The relevant spare liquid bags can be mounted on the liquid bag hooks 93 on the side wall.

[0141] In specific implementation, the user can control the above-mentioned cell separation and harvesting system through the operation panel 91, and perform operations such as calibration, separation, concentration, cleaning, and packaging in sequence to complete the separation and packaging of cells.

[0142] It should also be pointed out that, compared with conventional system equipment, the cell separation and harvesting system of the above structure can not only realize a more comprehensive variety of functional operations such as calibration, liquid filling, separation, concentration, cleaning, and packaging; it also effectively simplifies the overall structural layout, making the structural layout relatively more concise and clear, which is easier for users to understand and operate. While reducing the difficulty of user operation, it helps to reduce the equipment cost of the system.

[0143] Specifically, compared with conventional system equipment, the total number of solenoid valves in the cell separation and harvesting system has been reduced from 14 to 7, the air filter valves have been reduced from 2 to 1, and the total length of the pipeline has been reduced from 600 cm to 370 cm; the liquid circuit logic is relatively clearer, which is convenient for observation and monitoring during operation; at the same time, it also effectively simplifies the installation of sterile consumables pipelines, greatly reduces the difficulty of operation, reduces human errors caused by complex pipelines, and improves the efficiency and stability of automated cell processing.

[0144] Based on the cell separation and harvesting system and method provided in this specification, the system at least includes: a two-way pump, a centrifugal cup, a centrifuge, a refrigerated centrifugal chamber, a quantitative tube, and an air filter valve; wherein the two-way pump includes a first channel and a second channel; the first end of the first channel is connected to the upper opening of the centrifugal cup, and the second end of the first channel is connected to at least the first pipeline, the second pipeline, and the third pipeline; wherein the first pipeline is used to connect the cell stock solution bag, the second pipeline is used to connect the cleaning solution bag, and the third pipeline is connected to the quantitative tube; the first end of the second channel is connected to the lower opening of the centrifugal cup, and the second end of the second channel is connected to at least the fourth pipeline, the fifth pipeline, the sixth pipeline, and the seventh pipeline; wherein the fourth pipeline is connected to the quantitative tube, the fifth pipeline is connected to the quantitative tube, and the sixth pipeline is connected to the seventh pipeline. Used to connect the filling bag, the sixth pipeline is used to connect the resuspension liquid bag, and the seventh pipeline is used to connect the waste liquid bag; the distance between the upper opening of the centrifuge cup and the adjacent cup wall is smaller than the distance between the lower opening of the centrifuge cup and the adjacent cup wall; the quantitative tube is also connected to the air filter valve through the eighth pipeline; the centrifuge cup is connected to the centrifuge, and the centrifuge cup is placed in the refrigerated centrifugal cabin, and the refrigerated centrifugal cabin is also connected to a refrigeration module; and, a first solenoid valve is arranged on the first pipeline, a second solenoid valve is arranged on the second pipeline, a third solenoid valve is arranged on the third pipeline, a fourth solenoid valve is arranged on the fourth pipeline, a fifth solenoid valve is arranged on the fifth pipeline, a sixth solenoid valve is arranged on the sixth pipeline, and a seventh solenoid valve is arranged on the seventh pipeline. The cell separation and harvesting system based on the above structure can, when implemented, utilize the characteristics of a two-way pump to form a stable continuous flow, and then cooperate with a centrifuge and a refrigerated centrifuge chamber to transport relevant fluids in the form of a stable continuous flow, thereby effectively reducing the loss and damage of cells during the operation and processing, avoiding cell inactivation, and efficiently and automatically completing the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain a higher quality cell product.

[0145] See also Figure 7 As shown, the embodiment of this specification also provides a cell separation and harvesting method based on the above-mentioned cell separation and harvesting system, which may include the following contents during specific implementation:

[0146] S701: According to a preset calibration rule, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine a hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle;

[0147] S702: According to the preset separation and concentration rules and based on the hose coefficient of the two-way pump, the first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow by controlling the first solenoid valve and the two-way pump; at the same time, the cell stock solution in the centrifuge cup is separated into cells and supernatant by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow;

[0148] S703: According to the preset cleaning rule, the cleaning solution is pumped into the centrifuge cup in the form of a continuous flow by controlling the second solenoid valve and the two-way pump to clean the supernatant on the cells; at the same time, the cleaning solution is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve;

[0149] S704: According to a preset resuspension rule and based on the hose coefficient of the two-way pump, the sixth solenoid valve, the two-way pump, and the third solenoid valve are controlled to resuspend the washed cells in the centrifuge cup;

[0150] S705: According to the preset filling rule and based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifugal cup is transported to the filling bag by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve.

[0151] Based on the above embodiments, the above cell separation and harvesting system can be used to perform calibration, separation, concentration, washing, resuspension, packaging and other operations in sequence according to corresponding rules, so as to efficiently complete the separation and packaging of cells and obtain high-quality cell products.

[0152] In some embodiments, before specific implementation, you can refer to Figure 1 The structural diagram of the cell separation and harvesting system shown in the figure determines the configuration parameters of the relevant liquid bags, pipelines, solenoid valves, etc. according to the specific application scenarios and separation and packaging requirements. Then, according to the above system configuration parameters, fully enclosed disposable consumables are used in conjunction with sterile pipe connection machines, heat sealers and other equipment to connect and construct a cell separation and harvesting system that meets the requirements.

[0153] Specifically, you can first determine the required cell stock liquid bag, cleaning liquid bag, resuspension liquid bag, waste liquid bag, and sub-packaging bag and other liquid bags. Then connect the above liquid bags to the corresponding sterile fully enclosed disposable consumables pipelines in turn, and install the above fully enclosed disposable consumables pipelines according to the installation prompt information and the corresponding solenoid valve opening sequence to obtain the required cell separation and harvesting system. Specifically, you can first open the first solenoid valve to combine the cell stock liquid bag and the corresponding pipeline; open the second solenoid valve to combine the cleaning liquid bag and the corresponding pipeline; open the third solenoid valve and the fourth solenoid valve to combine the quantitative tube, the air filter valve and the corresponding pipeline; open the fifth solenoid valve to combine the sub-packaging bag and the corresponding pipeline; open the sixth solenoid valve to combine the resuspension liquid bag and the corresponding pipeline; open the seventh solenoid valve to combine the waste liquid bag and the corresponding pipeline. Then start the two-way pump, install the hose of the first channel and the hose of the second channel according to the installation label; and fix the centrifuge cup to the centrifuge, and ensure that the centrifuge cup is installed stably. Then use a sterile pipe connection machine to connect each liquid bag to the corresponding consumable pipeline in the specified order. From right to left, the liquid bags corresponding to the solenoid valves are: cell stock liquid bag, cleaning liquid bag, filling bag (empty), resuspension liquid bag, and waste liquid bag (empty).

[0154] Among them, the consumable pipeline can be a fully enclosed disposable consumable pipeline sterilized by ethylene oxide. In addition, the related consumables also include centrifuge cups. The above-mentioned entire installation process is isolated from the outside world during implementation, and the liquid bag is connected through a sterile pipe connection machine to avoid contact between the pipeline and the outside world.

[0155] In some embodiments, after the cell separation and harvesting system is connected, the pipeline air tightness test of the cell separation and harvesting system can be performed according to the preset self-test rules to implement the self-test operation. The specific implementation can include the following contents:

[0156] S1: According to the preset self-checking rules, the second solenoid valve is opened, and the other solenoid valves are kept closed to detect whether the cleaning liquid in the cleaning liquid bag flows down along the second pipeline;

[0157] S2: When it is determined that the cleaning liquid in the cleaning liquid bag will not flow down along the second pipeline, it is determined that the air tightness of the first pipeline, the second pipeline, and the third pipeline meets the requirements;

[0158] S3: Start the centrifuge and pump a certain volume of cleaning solution into the centrifuge cup through the cleaning solution bag as the detection solution;

[0159] S4: Stop the centrifuge, open the third solenoid valve, close the second solenoid valve; and start the two-way pump to rotate in the first direction to pump the test liquid in the centrifuge cup toward the seventh solenoid valve, the sixth solenoid valve, the fifth solenoid valve, and the fourth solenoid valve for a specified time, then stop the two-way pump, keep the pipeline in a high-pressure state, and detect whether there is overflow of the test liquid from the seventh solenoid valve, the sixth solenoid valve, the fifth solenoid valve, and the fourth solenoid valve;

[0160] S5: When it is determined that there is no overflow of the detection liquid from the seventh solenoid valve, the sixth solenoid valve, the fifth solenoid valve, and the fourth solenoid valve, it is determined that the air tightness of the seventh pipeline, the sixth pipeline, the fifth pipeline, and the fourth pipeline meets the requirements.

[0161] Based on the above embodiments, there is no need to introduce other professional testing equipment. By utilizing the existing structure of the cell separation and harvesting system, the air tightness test results of the relevant pipelines can be completed accurately and conveniently.

[0162] In specific implementation, according to the air tightness test result, when it is determined that the air tightness of at least one pipeline in the above-mentioned cell separation and harvesting system does not meet the requirements, the above-mentioned method can be used to reconnect and construct a new cell separation and harvesting system.

[0163] In specific implementation, the corresponding solenoid valves can be controlled to perform at least two opening and closing operations in succession according to preset self-checking rules to detect whether the solenoid valves are normal and effective.

[0164] In some embodiments, see Figure 8 As shown, according to the preset calibration rules, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine the hose coefficient of the two-way pump. The specific implementation may include the following contents:

[0165] S1: According to the preset calibration rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0166] S2: Control the two-way pump to rotate in the first direction to pump the second volume of cleaning solution into the centrifuge cup along the second pipeline as the calibration solution; and stop the centrifuge; wherein the second volume is greater than the volume of the quantitative tube;

[0167] S3: Control the two-way pump to rotate in the first direction to transfer the calibration liquid in the centrifuge cup along the seventh pipeline to the waste liquid bag; and monitor whether the calibration liquid reaches the seventh solenoid valve;

[0168] S4: When it is detected that the calibration liquid reaches the seventh solenoid valve, the seventh solenoid valve is closed and the fourth solenoid valve is opened;

[0169] S5: controlling the two-way pump to rotate in a first direction to transfer the calibration liquid in the centrifugal cup to the quantitative tube along the fourth pipeline; and monitoring the detection value of the third bubble sensor;

[0170] S6: when the detection value of the third bubble sensor indicates that liquid is detected, stop the operation of the two-way pump; and obtain the number of rotations of the two-way pump;

[0171] S7: Determine the hose coefficient of the two-way pump based on the volume of the metering tube and the number of revolutions of the two-way pump.

[0172] The first direction may specifically be a direction in which the liquid flows in from the first channel and out from the second channel.

[0173] Based on the above embodiment, the calibration operation for the two-way pump can be conveniently and accurately implemented according to the preset calibration rules without the need to introduce and use additional professional testing equipment.

[0174] For specific implementation, please refer to Fig. 9 As shown, the two-way pump can be controlled to rotate in the first direction, and the centrifuge can be started at the same time to drive the centrifuge cup to rotate, so as to pump the cleaning liquid into the centrifuge cup. When the cleaning liquid is pumped into the centrifuge cup, the cleaning liquid is pressed against the wall of the centrifuge cup by the centrifugal force, so that the centrifuge cup can be cleaned during the calibration process.

[0175] When the accumulated cleaning liquid pumped into the centrifugal cup is greater than or equal to the second volume (for example, V1), the centrifuge can be stopped and the second solenoid valve can be closed. At this time, the cleaning liquid still attached to the cup wall will naturally fall to the bottom of the cup as a calibration liquid. The second volume is at least greater than the volume of the quantitative tube (for example, V).

[0176] Furthermore, the third solenoid valve can be opened to control the two-way pump to rotate in the first direction, and the calibration liquid can be transferred along the seventh pipeline toward the waste liquid bag; when the calibration liquid reaches the seventh solenoid valve, the seventh solenoid valve is closed to implement the pre-filling operation of the relevant pipelines, so that the pipelines between the seventh solenoid valve, the sixth solenoid valve, the fifth solenoid valve and the nodes (for example, node S) where the seventh pipeline, the sixth pipeline, the fifth pipeline, and the fourth pipeline converge are filled with the calibration liquid, so as to avoid the above pipelines from causing errors in subsequent calibration. The above node S can also be used as the calibration starting point during the calibration operation.

[0177] Next, the number of rotations of the two-way pump can be initialized, for example, the current number of rotations of the two-way pump can be recorded as the initial number of rotations. Then the fourth solenoid valve is opened, and the two-way pump is controlled to rotate in the first direction to transfer the calibration liquid along the fourth pipeline to the quantitative tube; when the detection value of the third bubble sensor indicates that liquid is detected, it can be determined that the quantitative tube is full of calibration liquid; at this time, the two-way pump can be stopped and the current number of rotations of the two-way pump can be read; then the difference between the current number of rotations and the initial number of rotations is calculated to obtain the number of rotations of the two-way pump (for example, N).

[0178] Furthermore, the volume of the fluid actually transported by the two-way pump for one rotation can be calculated based on the volume of the metering tube (e.g., V), combined with the pipeline volume offset (e.g., △V), and the number of rotations of the two-way pump, and used as the hose coefficient of the two-way pump to implement the calibration operation of the two-way pump.

[0179] Specifically, the hose coefficient of the two-way pump can be calculated according to the following formula: P=(V+△V) / N.

[0180] The above pipeline volume offset may specifically include the volume sum of the pipeline between the node S and the quantitative tube, and the pipeline between the quantitative tube and the third bubble sensor. The value of V is known.

[0181] Specifically, the above pipeline volume offset can be calculated according to the following formula: .

[0182] Wherein, R represents the diameter of the pipeline, and L represents the sum of the lengths of the pipeline between the node S and the quantitative tube and the pipeline between the quantitative tube and the third bubble sensor.

[0183] Considering that the value of L is relatively small and the variation is relatively small. Before specific implementation, multiple tests can be performed to determine the length of the pipeline between the node S and the quantitative tube, and the length of the pipeline between the quantitative tube and the third bubble sensor as a reference length; then the reference length can be used to calculate the pipeline volume offset; and then the hose coefficient of the two-way pump can be calculated based on the volume of the quantitative tube and the above pipeline volume offset.

[0184] Subsequently, based on the hose coefficient of the two-way pump, the quantitative pumping and discharge of the relevant fluid into and out of the centrifugal cup can be accurately achieved by controlling the number of rotations of the two-way pump.

[0185] In some embodiments, see Fig.10 As shown, according to the preset separation and concentration rules, based on the hose coefficient of the two-way pump, the first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow by controlling the first solenoid valve and the two-way pump; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow. When implemented specifically, the following contents may be included:

[0186] S1: According to the preset separation and concentration rules, the first solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0187] S2: controlling the two-way pump to rotate in a first direction based on a first speed, so as to input the cell stock solution in the cell stock solution bag into the centrifuge cup through the upper opening of the centrifuge cup in the form of a continuous flow along the first pipeline;

[0188] S3: Control the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cells of the cell stock solution entering the centrifuge cup enter the wall-adherent state under the action of centrifugal force, so as to separate the cell stock solution into cells and supernatant; and output the supernatant from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and input it into the waste liquid bag along the seventh pipeline;

[0189] S4: When it is monitored based on the hose coefficient of the two-way pump that the volume of the cell stock solution input into the centrifuge cup reaches the first volume, the two-way pump is stopped, the centrifuge is stopped, and the first solenoid valve and the seventh solenoid valve are closed.

[0190] Based on the above embodiment, according to the preset separation and concentration rules, the first volume of cell stock solution can be controlled to be quantitatively pumped into the centrifuge cup in the form of a stable continuous flow to achieve the liquid filling operation; at the same time, the centrifuge is controlled to drive the centrifuge cup to rotate, so that the cells in the cell stock solution enter the wall adhesion state and are separated from the supernatant to achieve the separation operation; in addition, the separated supernatant is also controlled to be discharged from the centrifuge cup in the form of a stable continuous flow to achieve the concentration operation.

[0191] In specific implementation, when controlling the two-way pump to rotate in the first direction based on the first speed, the flow rate of the cell stock solution in the pipeline should be less than or equal to the upper limit of the flow rate and greater than or equal to the lower limit of the flow rate.

[0192] Specifically, when the speed of the two-way pump is too fast, the flow rate of the cell concentrate entering the centrifuge cup is too large, greater than the upper limit of the flow rate, the cells in the cell concentrate entering the centrifuge cup will not have sufficient time to enter the wall-adherent state due to the centrifugal force, but will directly rush out of the centrifuge cup, causing cell loss.

[0193] When the speed of the two-way pump is too slow, the flow rate of the cell stock solution in the pipeline is too small, less than the upper limit of the flow rate, the cell stock solution will need to be transported in the pipeline outside the centrifuge cup for a relatively long time before it can finally enter the centrifuge cup in the refrigerated centrifuge chamber. If the pipeline outside the centrifuge cup is not refrigerated, when the cells stay in the above pipeline for a long time, it is very likely that the temperature is too high, affecting the cell activity, or even causing cell inactivation, resulting in cell loss.

[0194] Therefore, by controlling the two-way pump to rotate based on the first speed, it can be effectively ensured that the flow rate of the cell stock solution in the pipeline is less than or equal to the upper limit of the flow rate, and greater than or equal to the lower limit of the flow rate. In this way, on the one hand, the cell stock solution outside the centrifuge cup can enter the centrifuge cup in a shorter time to avoid inactivation due to temperature; on the other hand, the flow rate of the cell stock solution that has just entered the centrifuge cup is not too high to avoid the cells from being directly flushed out of the centrifuge cup with the cell stock solution, causing cell loss.

[0195] The upper limit value of the flow rate and the lower limit value of the flow rate can be obtained by performing multiple tests using a cell separation and harvesting system and by statistics of the test data.

[0196] When pumping the cell stock solution into the centrifuge cup in a steady, continuous flow, refer to Figure 4 As shown, since the first channel is connected to the upper opening disposed at the upper position of the centrifuge cup. Therefore, the entrance position of the cell stock solution when entering the centrifuge cup is relatively close to the cup wall. Also, since when the cell stock solution enters the centrifuge cup, the centrifuge simultaneously drives the centrifuge cup to rotate at the first rotation speed, the cells in the cell stock solution are easily attached to the cup wall by the centrifugal force and enter the wall-attached state, thereby achieving the separation of the cells and the supernatant in the cell stock solution, and realizing the separation operation, that is, the cells are attached to the cup wall, and the supernatant flows along the cup wall to the bottom of the cup. Furthermore, the supernatant will be discharged from the centrifuge cup in the form of a stable continuous flow through the lower opening disposed at the lower position of the centrifuge cup, thereby realizing the concentration operation.

[0197] In a specific implementation, when controlling the centrifuge to drive the centrifuge cup to rotate based on the first rotation speed, the rotation speed of the centrifuge cup should be less than or equal to the upper limit of the rotation speed, and greater than or equal to the lower limit of the rotation speed.

[0198] Specifically, when the rotation speed of the centrifuge is too fast, greater than the upper limit of the rotation speed, the centrifugal force on the cells in the adherent state will be too large, which can easily cause a large squeezing effect on the cells, affect cell activity, and even cause cell inactivation.

[0199] When the rotation speed of the centrifuge is too slow, less than the lower limit of the rotation speed, the centrifugal force on the cells will be too small, and the friction between the cells and the cup wall will be less than the cell gravity, making it impossible for the cells to stick to the cup wall stably. They can easily slide to the bottom of the cup and be discharged from the centrifuge cup through the lower port together with the supernatant into the waste liquid bag, causing cell loss.

[0200] Therefore, by controlling the centrifuge to rotate based on the first rotation speed, it can be effectively ensured that the rotation speed of the cells in the centrifuge cup is less than or equal to the upper limit of the rotation speed, and greater than or equal to the lower limit of the rotation speed. In this way, on the one hand, the cells can be kept close to the cup wall, preventing the cells from sliding to the bottom of the cup and being discharged with the supernatant, thereby reducing cell loss; on the other hand, it can also prevent the rotation process from causing excessive squeezing force on the cells, resulting in cell damage or even inactivation.

[0201] In specific implementation, the first volume can be divided by the hose coefficient of the two-way pump to obtain a first number of rotations for the two-way pump; then, by monitoring whether the cumulative number of rotations of the two-way pump reaches the first number of rotations, it is determined whether the volume of the cell concentrate input into the centrifuge cup reaches the first volume.

[0202] When it is determined that the volume of the cell stock solution input into the centrifuge cup has reached the first volume, stop the two-way pump, stop the centrifuge, and close the first solenoid valve and the seventh solenoid valve. At this time, although the centrifuge cup has stopped rotating, due to the relatively light mass of the cells, cells that have been in an adherent state for a long time often do not automatically slide off the cup wall due to the cessation of centrifugation, but continue to adhere to the cup wall. Subsequently, it is necessary to use another resuspension solution for resuspension operation in order to smoothly mix and dissolve the cells adhering to the cup wall.

[0203] In some embodiments, when the method is implemented, it may also include: when the detection value of the first bubble sensor indicates that gas is detected, stopping the operation of the two-way pump and the operation of the centrifuge; and closing the first solenoid valve and the seventh solenoid valve.

[0204] In some cases, it is sometimes necessary to pump the entire cell stock solution bag into the centrifuge cup. In this case, the first bubble sensor can be used. When it is detected that the type of fluid flowing through the first bubble sensor changes from liquid to gas, it can be determined that all the cell stock solution in the cell stock solution bag has been pumped into the centrifuge cup. It can then be determined that the volume of the cell stock solution input into the centrifuge cup has reached the first volume, and the operation of the two-way pump can be stopped.

[0205] In some embodiments, when the rotation of the centrifuge is specifically controlled during the cell separation and concentration stage, the rotation speed of the centrifuge can also be dynamically adjusted.

[0206] In a specific implementation, the cumulative thickness of cells in an adherent state in the centrifuge cup can be monitored by a microscope camera arranged at the cup wall of the centrifuge cup; when it is monitored that the cumulative thickness of cells in an adherent state is greater than a preset thickness threshold, the first rotation speed of the centrifuge is adjusted; wherein the adjusted rotation speed is less than the first rotation speed.

[0207] This is because, as the cell separation and concentration stage continues, the newly separated cells that enter the adherent state will continue to cover the previous cells, resulting in more and more cells in the adherent state and the cumulative thickness of the cells in the adherent state becoming thicker and thicker.

[0208] When the cumulative thickness of cells in the adherent state is greater than the preset thickness threshold, the distance between the newly separated cells in the adherent state and the rotation axis is relatively close. At this time, based on the lower rotation speed, the newly entered cells can also achieve adhesion by clinging to the previous cells. At the same time, due to the squeezing effect of the newly entered cells, the previous cells can stably remain in the adherent state even if the centrifugal force they are subjected to is reduced, and will not fall due to gravity. At the same time, due to the reduced rotation speed, the centrifugal force on the cells closest to the cup wall and the squeezing force from other cells are reduced to a certain extent, thereby avoiding the cells closest to the cup wall from being squeezed and damaged or even inactivated due to excessive combined force.

[0209] In some embodiments, during specific implementation, the order of magnitude of the separated cells can be estimated based on the first volume, the cell concentration in the cell stock solution; and based on the order of magnitude of the cells, a centrifuge cup with a matching cup wall surface area is selected from a plurality of centrifuge cups to connect and construct the above-mentioned cell separation and harvesting system.

[0210] Specifically, when the estimated amount of separated cells is large, a centrifuge cup with a relatively large cup wall surface area can be selected as a matching centrifuge cup. In this way, during the centrifugal concentration stage, the separated cells can be as dispersed as possible and attached to the cup wall, avoiding excessive squeezing of cells closest to the cup wall due to the superposition of multiple layers of cells, resulting in cell damage and inactivation.

[0211] In some embodiments, see Fig.11 As shown, according to the preset cleaning rules, the second solenoid valve and the two-way pump are controlled to pump the cleaning liquid into the centrifuge cup in the form of a continuous flow to clean the supernatant on the cells; at the same time, the two-way pump and the seventh solenoid valve are controlled to transport the cleaning liquid from the centrifuge cup to the waste liquid bag in the form of a continuous flow. When implemented specifically, the following contents may be included:

[0212] S1: According to the preset cleaning rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0213] S2: Control the two-way pump to rotate in the first direction based on the second speed, so that the cleaning liquid in the cleaning liquid bag is input into the centrifuge cup through the upper opening of the centrifuge cup along the second pipeline in the form of a continuous flow;

[0214] S3: Control the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cleaning solution entering the centrifuge cup washes the cells in the adherent state; and outputs the cleaning solution from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and inputs it into the waste liquid bag along the seventh pipeline;

[0215] S4: When it is determined that the cleaning end condition is met, the second solenoid valve and the seventh solenoid valve are closed, and the first solenoid valve and the fourth solenoid valve are opened;

[0216] S5: Control the two-way pump to rotate in the second direction based on the third speed, and input the remaining cleaning solution in the centrifuge cup into the cell stock solution bag through the upper opening of the centrifuge cup along the first pipeline.

[0217] The second direction may specifically be a direction in which the liquid flows in from the second channel and out from the first channel.

[0218] The second rotation speed may be greater than the first rotation speed, so as to use a relatively fast flow rate and a relatively high pressure cleaning liquid flow to wash the cells in the centrifuge cup and obtain a better cleaning effect. The third rotation speed may be less than the second rotation speed.

[0219] The above-mentioned cleaning end condition can be specifically that the volume of the cleaning liquid pumped into the centrifugal cup reaches a specified volume. In specific implementation, it can be determined whether the volume of the cleaning liquid pumped into the centrifugal cup reaches the specified volume by monitoring the number of rotations of the two-way pump based on the hose coefficient of the two-way pump.

[0220] In specific implementation, similarly, the second solenoid valve can be opened instead of the first solenoid valve, and the two-way pump can be controlled to rotate in the second direction based on the third speed, and the remaining cleaning liquid in the centrifuge cup can be input into the cleaning liquid bag along the second pipeline through the upper opening of the centrifuge cup.

[0221] In this way, as much cleaning solution as possible can be discharged from the centrifuge cup, and at the same time, the cell stock solution bag or the cleaning solution bag that is no longer used in the system can be effectively used to store the cleaning solution after cleaning.

[0222] In specific implementation, in the initial stage of cleaning (which lasts for a relatively long time), when the centrifuge drives the centrifuge cup to rotate violently at a relatively fast rotation speed, by pumping cleaning liquid into the centrifuge cup through the upper opening of the centrifuge cup, and then discharging the cleaning liquid after cleaning through the lower opening of the centrifuge cup, it is possible to effectively avoid the cells being discharged from the centrifuge cup along with the cleaning liquid after cleaning during the cleaning process, thereby reducing cell loss; in the later stage of cleaning (which lasts for a relatively short time), the centrifuge continues to drive the centrifuge cup to rotate, and the cells are still in a wall-attached state. By utilizing the air filtered by the air filter valve pumped in through the lower opening of the centrifuge cup, the cleaning liquid after cleaning is pushed out of the centrifuge cup through the upper opening of the centrifuge cup, thereby discharging as much residual cleaning liquid in the centrifuge cup as possible.

[0223] Based on the above embodiment, according to the preset cleaning rules, the cleaning liquid can be controlled to be pumped into the centrifuge cup in the form of a stable continuous flow to effectively clean the cells in the centrifuge cup, wash away the residual supernatant attached to the cells, and effectively discharge the cleaning liquid after cleaning, so as to better realize the cleaning operation and obtain a better cleaning effect.

[0224] In specific implementation, a designated volume of cleaning solution may be pumped into the centrifuge cup multiple times to perform multiple rounds of cleaning on the cells in the centrifuge cup.

[0225] When each round of cleaning is carried out, the centrifuge can be started in the above manner, and the centrifuge can be controlled to drive the centrifuge cup to rotate based on the first rotation speed, while a specified volume of cleaning liquid can be pumped into the centrifuge cup to flush the cells in the adherent state; when the volume of the pumped cleaning liquid reaches the specified volume, the corresponding solenoid valve can be closed and the centrifuge can be stopped at the same time. However, since the cells are relatively light, due to the effect of adhesion, the cells will not automatically slide down to the cup body due to gravity. At this time, the centrifuge can be controlled to drive the centrifuge cup to rotate alternately in the forward and reverse directions, so that the cleaning liquid in the centrifuge cup ripples, so as to dissolve the cells in the adherent state into the cleaning liquid, so that all surfaces of the cells can be fully in contact with the cleaning liquid (i.e., mixing is achieved), and further cleaning is carried out to obtain a relatively better cleaning effect, thereby completing a round of cleaning.

[0226] After completing one round of washing and before the next round of washing, the centrifuge can be started and controlled to rotate the centrifuge cup at a higher rotation speed (for example, the first rotation speed) so that the cells mixed in the cup cleaning liquid are in an adherent state again; then the corresponding solenoid valve is opened, and the two-way pump is controlled to pump the cleaning liquid into the centrifuge cup to form and utilize a stable continuous flow to perform the next round of washing on the cells in the adherent state.

[0227] During the last round of cleaning, when it is determined that the cleaning end conditions are met, the two-way pump can be controlled to rotate in the second direction based on the third speed to input the remaining cleaning solution in the centrifuge cup into the cell stock solution bag along the first pipeline through the upper opening of the centrifuge cup.

[0228] Drain the remaining cleaning solution in the centrifuge cup as much as possible to complete the cleaning operation.

[0229] In some embodiments, see Fig.12 As shown, the above-mentioned resuspension treatment is performed on the washed cells in the centrifuge cup according to the preset resuspension rule and based on the hose coefficient of the two-way pump by controlling the sixth solenoid valve, the two-way pump and the third solenoid valve. When implemented specifically, the following contents may be included:

[0230] S1: According to the preset resuspension rules, open the third solenoid valve and the sixth solenoid valve; and start the two-way pump;

[0231] S2: controlling the two-way pump to rotate in a second direction based on a fourth speed, so as to input the resuspension in the resuspension bag into the centrifuge cup along a sixth pipeline through a lower opening of the centrifuge cup;

[0232] S3: When the volume of the resuspension input into the centrifuge cup reaches the third volume based on the hose coefficient of the two-way pump, the two-way pump is stopped and the centrifuge is started;

[0233] S4: Controlling the centrifuge to rotate based on the second rotation speed, so that the cells in the adherent state are dissolved in the resuspension solution to obtain a target solution containing cells.

[0234] Wherein, the second rotation speed is lower than the first rotation speed.

[0235] In specific implementation, based on the above second rotation speed, the cells in the centrifuge cup cannot enter the wall-adherent state due to the centrifugal force, but the centrifuge cup can be rotated and shaken to fully mix the cells and the resuspension solution to obtain a target solution with higher quality.

[0236] In specific implementation, the centrifuge can also be controlled to rotate alternately in two opposite directions, the first direction and the second direction based on the second rotation speed, so that more and faster cells in the adherent state can break away from the cup wall and dissolve into the resuspension solution.

[0237] In specific implementation, it can be determined whether the volume of the resuspension liquid input into the centrifugal cup reaches the third volume by monitoring the number of rotations of the two-way pump based on the hose coefficient of the two-way pump.

[0238] Based on the above embodiment, according to the preset resuspension rule, the corresponding resuspension liquid can be controlled to be pumped into the centrifuge cup to resuspend and mix the cells in the centrifuge cup, thereby achieving a resuspension operation and obtaining a target liquid with higher quality.

[0239] In some embodiments, according to the preset packaging rules, based on the hose coefficient of the two-way pump, by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve, the target liquid containing cells in the centrifuge cup is transported to the packaging bag. When implemented specifically, the following contents may be included:

[0240] S1: According to the preset dispensing rules, the third solenoid valve and the fifth solenoid valve are opened; and the two-way pump and the centrifuge are started;

[0241] S2: controlling the centrifuge to rotate based on a third rotation speed;

[0242] S3: Based on the hose coefficient of the two-way pump, the two-way pump is controlled to rotate in the first direction based on the fifth speed, and the target liquid of the specified volume is input into the sub-packaging bag along the fifth pipeline through the lower opening of the centrifugal cup.

[0243] The third rotation speed is lower than the first rotation speed.

[0244] In specific implementation, based on the third rotation speed, the cells in the centrifuge cup cannot be made to adhere to the wall, but the target liquid can be mixed evenly by rotating and shaking the centrifuge cup before being sent to the sub-packaging bag, thereby obtaining a cell product with relatively better effect.

[0245] In specific implementation, the specified volume can be divided by the hose coefficient of the two-way pump to obtain the corresponding specified number of rotations of the two-way pump; then, by monitoring whether the cumulative number of rotations of the two-way pump reaches the specified number of rotations, it is determined whether the specified volume of target liquid is input into the filling bag.

[0246] Based on the above embodiments, according to the preset subpackaging rules, the target liquid can be discharged from the centrifugal cup and transported to the subpackaging bag to realize the subpackaging operation, and the required cell products can be collected efficiently and automatically.

[0247] In some embodiments, during the process of cell separation and packaging using a cell separation and harvesting system in the manner described above, the refrigeration module can also be dynamically adjusted. Specifically, the adjustment may include: before pumping the cell concentrate into the centrifuge cup, starting the refrigeration module in advance to pre-cool the centrifuge cup in the refrigerated centrifuge chamber; after the cell concentrate begins to enter the centrifuge cup, dynamically adjusting the refrigeration power of the refrigeration module to maintain the temperature of the centrifuge cup in the refrigerated centrifuge chamber within a threshold temperature range.

[0248] Specifically, considering that during the cell separation and concentration stage, the centrifuge will rotate relatively violently, causing a large temperature rise in the refrigerated centrifuge chamber, at this time, the refrigeration module can be dynamically adjusted according to the matching first power adjustment rule to better maintain the temperature of the centrifuge cup in the refrigerated centrifuge chamber within the threshold temperature range.

[0249] In the early stage of the cell washing stage, when the centrifuge rotates violently, the refrigeration module can continue to be dynamically adjusted according to the matching first power adjustment rule; in the later stage of the cell washing stage, when the centrifuge no longer rotates as violently as before, or in the packaging stage, the refrigeration module can be replaced and dynamically adjusted according to the matching second power adjustment rule to better maintain the temperature of the centrifugal cup in the refrigerated centrifuge chamber within the threshold temperature range.

[0250] The first power adjustment rule and the second power adjustment rule may be generated by performing a large number of experimental tests in advance, sorting out a large number of adjustment records of refrigeration modules according to the experimental test results, and then clustering the large number of adjustment records of refrigeration modules.

[0251] In addition, during the entire cell separation and packaging process, the refrigeration module can be individually adjusted according to the temperature collected by the temperature sensor.

[0252] As can be seen from the above, based on the cell separation and harvesting method provided in the embodiments of this specification, the characteristics of the two-way pump can be used to form a stable continuous flow, and then the centrifuge and the refrigerated centrifuge chamber are used to transport the relevant fluids in the form of a stable continuous flow, thereby effectively reducing the loss and damage of cells during the operation and processing, avoiding cell inactivation, and efficiently and automatically completing the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain a higher quality cell product.

[0253] This specification embodiment provides an electronic device, referring to Fig.13 The electronic device includes a network communication port 1301, a processor 1302 and a memory 1303, and the above structures are connected through internal cables so that each structure can perform specific data interaction.

[0254] The network communication port 1301 can be specifically used to receive a trigger instruction.

[0255] The processor 1302 may be specifically configured to respond to a trigger instruction and, according to a preset calibration rule, calibrate the two-way pump by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine a hose coefficient of the two-way pump;

[0256] The hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle; according to the preset separation and concentration rules, based on the hose coefficient of the two-way pump, by controlling the first solenoid valve and the two-way pump, the first volume of the cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow; according to the preset cleaning rules, by controlling the second solenoid valve and the two-way pump, the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow. The cleaning liquid is pumped into the centrifuge cup in a form to clean the supernatant on the cells; at the same time, the cleaning liquid after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve; according to the preset resuspension rule, based on the hose coefficient of the two-way pump, the sixth solenoid valve, the two-way pump and the third solenoid valve are controlled to resuspend the washed cells in the centrifuge cup; according to the preset subpackaging rule, based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifuge cup is transported to the subpackaging bag by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve.

[0257] The memory 1303 may be specifically used to store corresponding instruction programs and related data such as hose coefficients.

[0258] Based on the above method, the relevant structural performance of the electronic device can be effectively utilized, the data processing speed of the electronic device can be improved, and the data processing related to cell separation and packaging can be efficiently realized.

[0259] In this embodiment, the network communication port 1301 can be a virtual port that is bound to different communication protocols so that different data can be sent or received. For example, the network communication port can be a port responsible for web data communication, a port responsible for FTP data communication, or a port responsible for email data communication. In addition, the network communication port can also be a physical communication interface or communication chip. For example, it can be a wireless mobile network communication chip, such as GSM, CDMA, etc.; it can also be a Wifi chip; it can also be a Bluetooth chip.

[0260] In this embodiment, the processor 1302 may be implemented in any appropriate manner. For example, the processor may take the form of a microprocessor or processor and a computer-readable medium storing a computer-readable program code (such as software or firmware) executable by the (micro)processor, a logic gate, a switch, an application specific integrated circuit (ASIC), a programmable logic controller, and an embedded microcontroller, etc. This specification does not limit this.

[0261] In this embodiment, the memory 1303 may include multiple levels. In a digital system, anything that can store binary data can be a memory; in an integrated circuit, a circuit with a storage function that has no physical form is also called a memory, such as RAM, FIFO, etc.; in a system, a storage device with a physical form is also called a memory, such as a memory stick, TF card, etc.

[0262] The embodiment of the present specification also provides a computer-readable storage medium based on the above-mentioned cell separation and harvesting method, wherein the computer-readable storage medium stores computer program instructions, and when the computer program instructions are executed, the following are achieved: according to a preset calibration rule, by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump, the two-way pump is calibrated to determine the hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle; according to a preset separation and concentration rule, based on the hose coefficient of the two-way pump, by controlling the first solenoid valve and the two-way pump, a first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is pumped into the centrifuge cup. The cell stock solution is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of continuous flow; according to the preset cleaning rule, by controlling the second solenoid valve and the two-way pump, the cleaning liquid is pumped into the centrifuge cup in the form of continuous flow to clean the supernatant on the cells; at the same time, by controlling the two-way pump and the seventh solenoid valve, the cleaning liquid after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of continuous flow; according to the preset resuspension rule, based on the hose coefficient of the two-way pump, by controlling the sixth solenoid valve, the two-way pump, and the third solenoid valve, the washed cells in the centrifuge cup are resuspended; according to the preset subpackaging rule, based on the hose coefficient of the two-way pump, by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve, the target liquid containing cells in the centrifuge cup is transported to the subpackaging bag.

[0263] In this embodiment, the storage medium includes but is not limited to a random access memory (RAM), a read-only memory (ROM), a cache, a hard disk (HDD) or a memory card. The memory may be used to store computer program instructions. The network communication unit may be an interface for network connection communication set in accordance with the standard specified by the communication protocol.

[0264] In this embodiment, the functions and effects specifically implemented by the program instructions stored in the computer-readable storage medium can be explained in comparison with other implementations and will not be described in detail here.

[0265] The embodiment of the present specification also provides a computer program product, which at least includes a computer program, and when the computer program is executed by a processor, the following method steps are implemented: according to a preset calibration rule, by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump, the two-way pump is calibrated to determine the hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle; according to a preset separation and concentration rule, based on the hose coefficient of the two-way pump, by controlling the first solenoid valve and the two-way pump, a first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow; according to the preset cleaning rule, by controlling the second solenoid valve and the two-way pump, the cleaning liquid is pumped into the centrifuge cup in the form of a continuous flow to clean the supernatant on the cells; at the same time, by controlling the two-way pump and the seventh solenoid valve, the cleaning liquid after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow; according to the preset resuspension rule, based on the hose coefficient of the two-way pump, by controlling the sixth solenoid valve, the two-way pump, and the third solenoid valve, the washed cells in the centrifuge cup are resuspended; according to the preset subpackaging rule, based on the hose coefficient of the two-way pump, by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve, the target liquid containing cells in the centrifuge cup is transported to the subpackaging bag.

[0266] See also Fig.14 As shown, the embodiment of this specification also provides a cell separation and harvesting device, which may specifically include the following structural modules:

[0267] The calibration module 1401 may be specifically used to calibrate the two-way pump according to a preset calibration rule by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine the hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle;

[0268] The separation and concentration module 1402 can be specifically used to pump the first volume of cell stock solution from the cell stock solution bag into the centrifuge cup in the form of continuous flow by controlling the first solenoid valve and the two-way pump according to the preset separation and concentration rules and based on the hose coefficient of the two-way pump; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of continuous flow;

[0269] The cleaning module 1403 can be specifically used to pump the cleaning solution into the centrifuge cup in the form of a continuous flow according to a preset cleaning rule by controlling the second solenoid valve and the two-way pump to clean the supernatant on the cells; at the same time, the cleaning solution after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve;

[0270] The resuspension module 1404 may be specifically used to resuspend the washed cells in the centrifuge cup according to a preset resuspension rule and based on the hose coefficient of the two-way pump by controlling the sixth solenoid valve, the two-way pump, and the third solenoid valve;

[0271] The filling module 1405 can be specifically used to transport the target liquid containing cells in the centrifuge cup to the filling bag according to the preset filling rules and based on the hose coefficient of the two-way pump by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve.

[0272] In some embodiments, when the calibration module 1401 is specifically implemented, the two-way pump can be calibrated according to the preset calibration rules by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine the hose coefficient of the two-way pump: according to the preset calibration rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started; the two-way pump is controlled to rotate in a first direction to pump the second volume of cleaning liquid into the centrifuge cup along the second pipeline as the calibration liquid; and the centrifuge is stopped; wherein the second volume is greater than the volume of the quantitative tube; the two-way pump is controlled to rotate in a first direction The two-way pump is controlled to rotate in the first direction to transfer the calibration liquid in the centrifugal cup along the seventh pipeline to the waste liquid bag; and the calibration liquid is monitored to see whether it reaches the seventh solenoid valve; when it is monitored that the calibration liquid reaches the seventh solenoid valve, the seventh solenoid valve is closed and the fourth solenoid valve is opened; the two-way pump is controlled to rotate in the first direction to transfer the calibration liquid in the centrifugal cup along the fourth pipeline to the quantitative tube; and the detection value of the third bubble sensor is monitored; when it is monitored that the detection value of the third bubble sensor indicates that liquid is detected, the two-way pump is stopped; and the number of rotations of the two-way pump is obtained; the hose coefficient of the two-way pump is determined according to the volume of the quantitative tube and the number of rotations of the two-way pump.

[0273] In some embodiments, when the above-mentioned separation and concentration module 1402 is implemented, it can be implemented in the following manner according to the preset separation and concentration rules, based on the hose coefficient of the two-way pump, by controlling the first solenoid valve and the two-way pump, so as to pump the first volume of cell stock solution from the cell stock solution bag into the centrifuge cup in the form of a continuous flow; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow: according to the preset separation and concentration rules, open the first solenoid valve and the seventh solenoid valve; and start the two-way pump and the centrifuge; control the two-way pump to rotate according to the first speed according to the seventh solenoid valve; The centrifuge is controlled to rotate in one direction to input the cell stock solution in the cell stock solution bag into the centrifuge cup through the upper opening of the centrifuge cup in the form of continuous flow along the first pipeline; the centrifuge is controlled to drive the centrifuge cup to rotate based on the first rotation speed, so that the cells of the cell stock solution entering the centrifuge cup enter the wall-adhering state under the action of centrifugal force, so as to separate the cell stock solution into cells and supernatant; and the supernatant is output from the centrifuge cup through the lower opening of the centrifuge cup in the form of continuous flow, and input into the waste liquid bag along the seventh pipeline; when it is monitored based on the hose coefficient of the two-way pump that the volume of the cell stock solution input into the centrifuge cup reaches the first volume, the two-way pump is stopped, the centrifuge is stopped; and the first solenoid valve and the seventh solenoid valve are closed.

[0274] In some embodiments, when the separation and concentration module 1402 is implemented, it can also be used to: when the detection value of the first bubble sensor indicates that gas is detected, stop the operation of the two-way pump and the centrifuge; and close the first solenoid valve and the seventh solenoid valve.

[0275] In some embodiments, when the above-mentioned cleaning module 1403 is specifically implemented, the cleaning solution can be pumped into the centrifuge cup in the form of a continuous flow by controlling the second solenoid valve and the two-way pump according to the preset cleaning rules to clean the supernatant on the cells; at the same time, the cleaning solution after cleaning is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve: according to the preset cleaning rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started; the two-way pump is controlled to rotate in the first direction based on the second speed to pump the cleaning solution in the cleaning solution bag in the form of a continuous flow. The cleaning fluid is input into the centrifuge cup through the upper opening of the centrifuge cup along the second pipeline; the centrifuge is controlled to rotate the centrifuge cup based on the first rotation speed, so that the cleaning fluid entering the centrifuge cup can rinse the cells in the wall-adherent state; and the cleaning fluid after cleaning is output from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and input into the waste liquid bag along the seventh pipeline; when it is determined that the cleaning end condition is met, the second solenoid valve and the seventh solenoid valve are closed, and the first solenoid valve and the fourth solenoid valve are opened; the two-way pump is controlled to rotate in the second direction based on the third speed, and the remaining cleaning fluid in the centrifuge cup is input into the cell stock liquid bag through the upper opening of the centrifuge cup along the first pipeline.

[0276] In some embodiments, when the above-mentioned resuspension module 1404 is implemented, the washed cells in the centrifuge cup can be resuspended in the following manner according to the preset resuspension rules and based on the hose coefficient of the two-way pump by controlling the sixth solenoid valve, the two-way pump, and the third solenoid valve: according to the preset resuspension rules, open the third solenoid valve and the sixth solenoid valve; and start the two-way pump; control the two-way pump to rotate in the second direction based on the fourth speed, and input the resuspension liquid in the resuspension liquid bag along the sixth pipeline into the centrifuge cup through the lower port of the centrifuge cup; when it is monitored based on the hose coefficient of the two-way pump that the volume of the resuspension liquid input into the centrifuge cup reaches the third volume, stop the two-way pump; and start the centrifuge; control the centrifuge to rotate based on the second rotation speed so that the cells in the adherent state are dissolved in the resuspension liquid to obtain a target liquid containing cells.

[0277] In some embodiments, when the above-mentioned resuspension module 1405 is implemented, the target liquid containing cells in the centrifuge cup can be transported to the sub-packaging bag by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve according to the preset sub-packaging rules and based on the hose coefficient of the two-way pump: according to the preset sub-packaging rules, the third solenoid valve and the fifth solenoid valve are opened; and the two-way pump and the centrifuge are started; the centrifuge is controlled to rotate based on the third rotation speed; based on the hose coefficient of the two-way pump, the two-way pump is controlled to rotate in the first direction based on the fifth speed, and the specified volume of the target liquid is input into the sub-packaging bag along the fifth pipeline through the lower opening of the centrifuge cup.

[0278] It should be noted that the units, devices or modules described in the above embodiments can be implemented by computer chips or entities, or by products with certain functions. For the convenience of description, the above devices are described separately by functions divided into various modules. Of course, when implementing this specification, the functions of each module can be implemented in the same or more software and / or hardware, or the modules that implement the same function can be implemented by a combination of multiple sub-modules or sub-units. The device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0279] As can be seen from the above, the cell separation and harvesting device provided in the embodiment of this specification can utilize the characteristics of the two-way pump to form a stable continuous flow, and then cooperate with the centrifuge and the refrigerated centrifuge chamber to transport the relevant fluid in the form of a continuous flow, thereby effectively reducing the loss and damage of cells during the operation and processing, avoiding cell inactivation, and efficiently and automatically completing the separation, concentration, washing, resuspension, and packaging of cells in the cell stock solution to obtain a higher quality cell product.

[0280] In a specific scenario example, the cell separation and harvesting system and method provided in this specification can be used to achieve fully automatic cell separation and harvesting. The specific implementation process can be found in the following content.

[0281] In this scenario example, considering that the cell separation and harvesting process based on existing conventional system equipment is complex and the product homogeneity requirements are high, and with the development of the industry, the demand for general large-volume cell culture and harvesting is becoming more and more urgent, if cell harvesting is done solely by manpower, the risk of cell contamination will increase and the efficiency will be very low, which is completely unable to meet the growing cell culture needs of the cell therapy industry.

[0282] Furthermore, considering that several automatic cell harvesting devices currently available on the market can replace manual labor and realize the automation of cell separation and harvesting, the following problems still exist: (1) They can only culture and harvest individual cells with small liquid volumes, and cannot meet the high throughput and high efficiency characteristics required for general cell culture; (2) They generally do not have a refrigeration function, and the activity of cells will be affected if the cells are exposed to high temperature conditions for a long time; (3) They basically use the negative pressure formed in the centrifuge cup to suck in the cell stock solution to form a continuous flow. The continuous flow formed by this method is unstable and can be easily interrupted due to changes in liquid volume or pressure. In addition, the long-term large negative pressure in the centrifuge cup will cause the cells to be subjected to great surface tension, which will affect the cell activity; (4) The pipeline design and process flow are complicated. Although the automation of cell harvesting can be basically realized, the preliminary preparation work and consumables installation processes are too complicated, and the requirements for operators are high, which is not conducive to the promotion and application of cell harvesting automation technology.

[0283] In order to solve the above problems existing in the existing system equipment, a set of fully automatic cell separation and harvesting system is provided in this scenario example. This is a device that integrates fully closed, automated, and continuous flow centrifugal cleaning technology. By matching centrifuge cups and peristaltic pump hoses of different sizes, it supports high-throughput cell separation and harvesting; by introducing a centrifuge chamber (for example, a refrigerated centrifuge chamber), rapid refrigeration can be achieved; by introducing and utilizing a dual-channel peristaltic pump (or a two-way pump), the flow rate of the inlet and outlet liquids can be made consistent, so that a stable and efficient continuous flow can be formed without changing the pressure in the centrifuge cup, and then the cell harvesting task can be completed with an extremely low cell loss rate under a constant temperature and pressure state. In a closed environment, high-speed centrifugation, concentration, cleaning, resuspension, and packaging of cells can be fully automatically realized, and the homogeneity and standardization of cell products of different batches can be better guaranteed while reducing human intervention.

[0284] For details, see Figure 6 and Figure 1 As shown, the above-mentioned fully automatic cell separation and harvesting system includes: solenoid valve, liquid bag, liquid bag bracket, hook, dual-channel peristaltic pump, bubble sensor, centrifuge, refrigerated centrifugal chamber, air filter valve, quantitative tube and other structures. This is a device that integrates fully enclosed, automated, and continuous flow centrifugal cleaning technology. It can match centrifugal cups and peristaltic pump hoses of different sizes to achieve high-throughput cell separation and harvesting. Among them, the refrigerated centrifugal chamber can effectively protect the cells, and the dual-channel peristaltic pump is used to make the flow rate of the inlet and outlet liquid consistent. Without changing the pressure in the centrifugal cup, a stable and efficient continuous flow can be formed. Under constant temperature and pressure, the cell harvesting task can be completed with an extremely low cell loss rate. In a closed environment, the high-speed centrifugation, concentration, washing, resuspension and packaging of cells can be fully automatically realized. While reducing human intervention, it can also better ensure the homogeneity and standardization of different batches of cell products.

[0285] In this scenario example, in order to achieve a stable and efficient continuous flow, refer to Figure 2 As shown, a two-way peristaltic pump is introduced and used. Specifically, the two channels of the two-way peristaltic pump are used for liquid inlet and liquid outlet, respectively. When the centrifuge cup rotates at high speed, channel one (for example, the first channel) is used to pump the cell stock solution into the centrifuge cup from the upper port, and channel two (for example, the second channel) is used to pump the supernatant out of the centrifuge cup from the lower port. Channel one and channel two of the peristaltic pump are driven by the same motor and the same peristaltic pump roller. When used with hoses of the same diameter, the pumping speeds of channel one and channel two are consistent, so that the liquid flow rate pumped into the centrifuge cup is consistent with the liquid flow rate pumped out of the centrifuge cup. The liquid path flow rate can reach a stable maximum of 500 ml / min, thereby ensuring that a stable and efficient continuous flow can be formed in the centrifuge cup during the entire cell processing process for cell concentration and cleaning.

[0286] During specific implementation, during the process of injecting cell fluid (for example, the original cell fluid) (such as the liquid injection operation), the double-channel peristaltic pump will select an appropriate pump speed V0 according to the total amount of the cell sample and the size of the centrifuge cup. Among them, V0 is between two critical speeds (V1 < V0 < V2), where V1 is the minimum speed for cell injection. A speed lower than V1 will cause the cell injection cycle to be prolonged, which not only affects the viability and state of the cells but also reduces the efficiency of cell harvesting; V2 is the maximum speed for cell injection. If the speed exceeds V2, some cells will be discharged before they have time to adhere under the inherent centrifugal force, resulting in a decrease in the cell harvesting rate.

[0287] During the cell cleaning process (such as the cleaning operation), the double-channel peristaltic pump will select an appropriate pump speed V3 according to the cell concentration and the set number of cleaning rounds. Among them, V3 is between two critical speeds (V4 < V3 < V5). V4 is the minimum speed for cell cleaning. A speed lower than V4 will result in insufficient cleaning force and incomplete cleaning, and it is impossible to completely clean substances such as the culture medium in the centrifuge cup, and it will also reduce the cleaning efficiency; the speed V5 is the maximum speed for cell cleaning. If the speed exceeds V5, the powerful fluid impact will not only carry away the adherent cells but may even kill the cells.

[0288] It should be noted that in the traditional injection method based on conventional system equipment, a single-channel peristaltic pump is used to draw the centrifuge cup into a negative pressure. When the negative pressure in the centrifuge cup is large enough, the negative pressure will be transmitted to the sample bag through the liquid in the pipeline, so as to suck the liquid in the sample bag into the centrifuge cup. Through experiments, it has been proved that during the implementation of this method, at the moment when the negative pressure in the centrifuge cup reaches the critical value, there will be a relatively large pressure difference between the centrifuge cup and the liquid bag. This pressure difference will cause the cell fluid in the sample bag to rush into the centrifuge cup at an extremely fast speed. Because the speed is too fast, some cells will be discharged before they have time to adhere, resulting in cell loss. It is not until the liquid in the centrifuge cup gradually increases and the pressure difference between the centrifuge cup and the sample bag gradually decreases and tends to balance that a relatively stable continuous flow can be formed. The introduction and use of the double-channel peristaltic pump can well avoid the above problems. The double-channel peristaltic pump does not rely on the pressure difference. Once it is started, the liquid in the centrifuge cup enters and exits at the same speed, directly entering the stable continuous flow state, and well avoiding the cell loss caused by the unstable pressure in the early stage of the continuous flow in the traditional method.

[0289] In addition, in addition to providing a stable continuous flow, the above-mentioned two-way peristaltic pump is also more advantageous in the process flow that requires precise control of the liquid inlet and outlet during the resuspension and packaging stages. During the resuspension stage (for example, the resuspension operation), the centrifuge cup remains stationary, channel one is connected to the air filter valve, and channel two is connected to the resuspension liquid bag. While channel two of the two-way peristaltic pump pumps a quantitative resuspension liquid from the lower opening of the centrifuge cup, channel one pumps out an equal volume of air from the upper opening of the centrifuge cup. The entire process is to replace the air in the centrifuge cup with the resuspension liquid while maintaining a constant pressure in the centrifuge cup, which is smooth and precise; rather than forcibly pumping in liquid in the traditional process and then squeezing out the air by increasing the pressure in the cup. During the filling stage (for example, filling operation), the centrifuge cup remains stationary, the rotation direction of the two-way peristaltic pump is opposite to that of the resuspension, channel one is connected to the air filter valve, channel two is connected to the filling bag, and channel two of the two-way peristaltic pump pumps out a quantitative cell fluid from the lower opening of the centrifuge cup while channel one pumps an equal volume of air from the upper opening of the centrifuge cup. While keeping the pressure in the centrifuge cup constant, the cell fluid is replaced by air and pumped into the filling bag. The whole process is smooth and precise, effectively ensuring the high-precision requirements of filling.

[0290] In addition, in some special cases, in addition to the above dual-channel isokinetic applications, the two-way peristaltic pump can also install hoses of different sizes in the two channels to form a fixed flow rate difference. The flow rate difference can continuously increase or decrease the pressure of the centrifuge cup, which is used for certain cell preparation processes that require specific pressures.

[0291] For specific implementation, see Fig. 9 As shown, based on the system of the above structure, the two-way peristaltic pump can be calibrated with high precision (for example, calibration operation) through the quantitative tube. It includes: starting the centrifuge, maintaining the set speed, opening the solenoid valves 2 and 7, and the two-way peristaltic pump pumping a set volume V1 of cleaning liquid (V1 is greater than the volume V of the quantitative tube) into the centrifugal cup. The cleaning liquid V1 is tightly attached to the wall of the centrifugal cup under the action of centrifugal force, and is retained in the cup to be used as the calibration liquid of the two-way peristaltic pump; the centrifuge stops rotating, the liquid in the cup is released from the centrifugal state and is placed at the bottom of the cup, the solenoid valve No. 7 is opened, and the two-way peristaltic pump pumps a set dose of liquid into the waste liquid bag according to the set number of turns, so that the pipeline between the valve No. 7 and the centrifugal cup is filled, the solenoid valve No. 7 is closed, and the solenoid valve No. 4 is opened. Starting from the calibration starting point S, the two-way peristaltic pump rotates in the set direction and speed, pumping the calibration liquid into the quantitative tube until the quantitative tube is full, and stops when the bubble sensor P3 detects the liquid, and records the number of turns N of the two-way peristaltic pump. The volume V of the quantitative tube and the volume offset of the pipeline are known. ( , R is the pipe radius, L is the total length of the pipe from the calibration starting point S to the bubble sensor), then the two-way peristaltic pump hose coefficient , that is, the amount of liquid passed by the two-way peristaltic pump for each rotation is P, and the calibration is completed. Therefore, by controlling the number of rotations and the direction of rotation of the two-way peristaltic pump, the amount of liquid in and out of each liquid bag and centrifuge cup can be accurately controlled. For example, assuming that liquid M needs to be added to the target container, the two-way peristaltic pump needs to rotate n=M / P in the specified direction. In addition, the number of rotations N of the two-way peristaltic pump here can be further converted into the number of pulses Q of the motor rotation (assuming that the motor is set to rotate 40,000 pulses per rotation, then Q=40,000*N), and the amount of liquid in each pulse can be calculated. By controlling the number of pulses of the double-channel peristaltic pump motor, the liquid inlet volume of the double-channel peristaltic pump can be controlled, thereby improving the accuracy of the double-channel peristaltic pump.

[0292] For specific implementation, see Figure 4 As shown, the centrifuge cup is mainly used to provide a place for cell fluid processing. The centrifuge is responsible for providing high-speed rotation power to make the centrifuge cup reach a preset speed, thereby providing sufficient centrifugal force for the cell fluid in the centrifuge cup. Under high-speed centrifugation, the cells and the supernatant are separated, and then the cell fluid is concentrated and cleaned by continuous flow (the upper port pumps in the cell stock solution and the lower port pumps out the supernatant). Finally, the cells on the wall of the centrifuge cup are mixed with a heavy suspension and packaged into the final production bag (for example, a packaging bag).

[0293] For specific implementation, see Fig.16 As shown, another improved centrifugal cup can also be used. Specifically, the centrifugal cup can include: a centrifugal cup base, a centrifugal cup cavity (or cup body), a centrifugal cup central axis (for example, a central axis flow channel), and a double-channel F-head. Among them, the centrifugal cup cavity includes an upper flow channel and an upper opening of the centrifugal cup, a lower flow channel and a lower opening of the centrifugal cup, and the central axis is connected to the lower flow channel; the central axis contains the bearings, large sealing rings, small sealing rings, and T-shaped guide platform required for high-speed centrifugation of the centrifugal cup.

[0294] like Fig.16 As shown, the upper opening and upper flow channel of the centrifugal cup are connected to flow channel 1 (for example, the first flow channel) of the F head through the T-shaped guide platform, and the lower opening and lower flow channel of the centrifugal cup are connected to flow channel 2 (for example, the second flow channel) through the central axis.

[0295] Among them, the distance between the upper mouth of the centrifugal cup and the cup wall is , the distance between the bottom of the centrifugal cup and the cup wall is , > , the radius of the centrifuge cup is R, and the height is H. When the centrifuge cup is centrifuged at high speed, the liquid forms a liquid column along the wall of the centrifuge cup. When the liquid is completely discharged from the lower port, the remaining liquid in the cup is ; When the liquid is completely drained from the top, the remaining liquid in the cup is Obviously, V 下 >V 上In this scenario embodiment, during specific implementation, the upper or lower outlet for liquid discharge can be selected according to actual needs and the amount of liquid remaining in the centrifuge cup.

[0296] For specific implementation, see Figure 5 As shown, a high-precision temperature sensor in the refrigerated centrifuge chamber can be used to monitor the temperature in the chamber in real time and intelligently adjust the temperature so that the temperature in the centrifuge chamber is always maintained at 2-8°C, effectively ensuring the activity of the cells throughout the entire process and greatly reducing the impact of temperature on the cells.

[0297] It should be added that the fully automatic cell separation and harvesting system proposed in this scenario example is a set of separation and harvesting liquid circuit systems with a minimalist design. Specifically, the liquid circuit system is suitable for cell harvesting processes, and supports continuous flow injection of cell fluids, high-speed centrifugation, cell concentration, cell washing, resuspension, and packaging. Its components include closed pipelines, liquid bags, solenoid valves, peristaltic pumps, centrifugal cups, bubble sensors, air filter valves, drop buckets and other components. The layout of the liquid circuit system is simple. Compared with an existing automated cell harvesting device on the market, the total number of solenoid valves has been reduced from 14 to 7, the air filter valves have been reduced from 2 to 1, and the total length of the pipeline has been reduced from 600cm to 370cm. The liquid circuit logic is clear, which is convenient for observation and monitoring during the experiment. At the same time, the installation of sterile consumables pipelines has also been simplified, which greatly reduces the difficulty of operation, reduces human errors caused by complex pipelines, and improves the efficiency and stability of automated cell harvesting.

[0298] Moreover, the fully automatic cell separation and harvesting system is a complete cell separation and harvesting process with adjustable parameters. Among them, the process includes: consumables installation, system self-test, high-speed centrifugation, continuous flow injection, continuous flow cleaning, cell resuspension, cell packaging, process recording and other processes; the numerous processes of the entire harvesting process are all controlled by computer programs, and all steps are automatically completed according to the pre-configured parameters, without the need for human intervention in the middle, saving manpower and greatly avoiding the risks brought by human intervention. For cell harvesting with different cell original liquid volumes, the cell harvesting efficiency of different liquid volumes can be adapted by adjusting parameters. For example, for a 50L sample solution, the peristaltic pump injection speed can be configured to be 350ml / min, that is, it only takes (50000 / 350=143) minutes to complete the injection and concentration of 50L cell stock solution. At present, the harvesting equipment on the market takes at least 3 hours to process the injection of 50L cell stock solution. This system can support a maximum of 1000L of cell original liquid for fully automatic cell separation and harvesting.

[0299] In addition, the above-mentioned fully automatic cell separation and harvesting system is constructed using fully enclosed pipeline consumables. It is mainly used to isolate the cell fluid from the outside world and prevent the cells from being contaminated by the outside world during the entire process of cell harvesting. The pipeline consumables include: pipelines, peristaltic pump hoses, centrifuge cups, liquid bags, air filter valves, drip buckets and other components, which are operated in conjunction with heat sealers, sterile pipe connection machines and other equipment to ensure that the cell fluid is always in a fully enclosed environment during the processing process to ensure the safety of cell products.

[0300] For specific implementation, please refer to Fig.15 As shown, follow the steps below to build and utilize a fully automated cell separation and harvesting system to achieve fully automated cell separation and harvesting.

[0301] S01: Install consumables according to the system prompts.

[0302] In the specific implementation, before officially starting the automated cell harvesting, some preliminary preparation work needs to be done. First, turn on the equipment, select the cell harvesting process, and automatically start pre-cooling the centrifuge chamber to keep the temperature of the centrifuge chamber at 2-8°C, so as to fully guarantee the viability of the cells during the entire cell harvesting process. Secondly, install the consumables, and connect the prepared cell liquid bags, cleaning liquid bags, resuspension liquid bags, final production bags, and waste liquid bags to the sterile consumables pipelines through the sterile pipe connection machine, and install the fully enclosed disposable consumables on the cell harvesting device according to the system prompts and the solenoid valve opening sequence: open valve No. 1, install the cell bag and corresponding pipelines; open valve No. 2, install the cleaning liquid bag and corresponding pipelines; open valves No. 3-4, install the quantitative tube filter valve and its pipelines; open valve No. 5, install the final production bag and its pipelines 6, install the resuspension liquid and its pipeline; open valve No. 7, install the waste liquid bag and its pipeline; turn on the peristaltic pump, install the hoses of channel 1 and channel 2 of the two-way peristaltic pump according to the label, and fix the centrifuge cup to the centrifuge to ensure that the centrifuge cup is installed stably, and then use the sterile pipe connection machine to connect each liquid bag to the consumable pipeline in the specified order. From right to left, the liquid bags corresponding to the solenoid valves are: 1-cell stock solution, 2-cleaning solution, 5-final bag (empty), 6-resuspension liquid, 7-waste liquid bag (empty). The consumables are fully enclosed disposable consumable pipelines sterilized with ethylene oxide, including centrifuge cups. The entire installation process is isolated from the outside world. When connecting the liquid bag, it is also connected through a sterile pipe connection machine to prevent the pipeline from contacting the outside world.

[0303] S02: The system performs self-test, performs airtightness test on consumables, and completes calibration of the two-way peristaltic pump (i.e., self-test operation and calibration operation).

[0304] During the specific implementation, you can first click Start on the operation panel, and the system will automatically start the self-test program. The solenoid valves will be switched on and off twice in sequence to ensure that each solenoid valve can be opened and closed normally, and detect whether the consumables are installed normally. It is also necessary to detect whether the air tightness of all consumable pipelines is good. Valve No. 2 is opened, and the other solenoid valves are closed to observe whether the cleaning liquid will flow down. If the cleaning liquid does not flow down, it proves that the air tightness of the right pipeline is good; the centrifuge is started, and valve No. 2 is opened under high-speed centrifugation, and an appropriate amount of cleaning liquid is pumped into the centrifugal cup to monitor the air tightness of the left pipeline; valve No. 2 is closed, valve No. 3 is opened, and the centrifugal cup is in a stationary state, and the two-way peristaltic pump rotates, pumping a certain amount of liquid into the direction of valves 4-5-6-7 and then stops, maintaining the high pressure in the pipeline for a period of time, and observing whether there is liquid overflow from valves 4-5-6-7 and the middle T-type three-way. If there is no liquid overflow, it proves that the air tightness of the left pipeline is good and the self-test is completed.

[0305] After confirming that the air tightness of the entire pipeline is normal, the peristaltic pump calibration begins: the centrifuge starts and maintains the set speed. The No. 2 and No. 7 solenoid valves are opened, and the two-way peristaltic pump pumps a set volume of cleaning liquid V1 into the centrifugal cup (V1 is greater than the volume V of the quantitative tube). The cleaning liquid V1 is pressed against the wall of the centrifugal cup under the action of centrifugal force, and is retained in the cup to be used as the calibration liquid for the two-way peristaltic pump; the centrifuge stops, the liquid in the cup is released from the centrifugal state and stands still at the bottom of the cup, the No. 2-7 solenoid valves are opened, and the two-way peristaltic pump pumps a set dose of liquid into the waste liquid bag according to the set number of turns, so that the pipeline between the No. 7 valve and the centrifugal cup is filled, the No. 7 solenoid valve is closed, and the No. 2-4 solenoid valves are opened. Starting from the calibration starting point S, the two-way peristaltic pump rotates in the set direction and speed, pumping the calibration liquid into the quantitative tube until the quantitative tube is full. The bubble sensor P3 stops when it detects the liquid, and the number of pulses Q of the two-way peristaltic pump motor rotation is recorded. The quantitative tube volume V and the pipeline volume offset are known. ( , R is the pipe radius, L is the total length of the pipe from the calibration starting point S to the bubble sensor), then the two-way peristaltic pump hose coefficient , that is, the amount of liquid passed by the double-pass peristaltic pump motor for each pulse is P Q , calibration is complete. Therefore, the amount of liquid in and out of each liquid bag and centrifugal cup can be accurately controlled by controlling the number of pulses and the direction of rotation of the two-way peristaltic pump motor. For example, assuming that liquid M needs to be added to the target container, the two-way peristaltic pump motor needs to rotate in the specified direction for the number of pulses m=M / P Q , calibration is completed. Open valves 3-7, and pump the calibration liquid in the quantitative tube into the centrifuge cup through channel 1 of the two-way peristaltic pump, and then pump it into the waste liquid bag through channel 2.

[0306] S03: Continuous flow injection, pumping in the cell stock solution, pumping out the supernatant, and completing cell concentration (e.g., injection operation, separation operation, and concentration operation).

[0307] In specific implementation, assume there is 50 L of cell stock solution, and the feeding speed of the peristaltic pump is set at 350 ml / min. That is, it only takes (50000 / 350 = 143) minutes to complete the feeding and concentration of all 50 L of cell stock solution. Start the centrifuge, keep the centrifuge cup rotating at a specified speed, open solenoid valves 1 and 7, and the peristaltic pump rotates at the set feeding speed V0 and in the set direction. V0 is between two critical speeds (V1 < V0 < V2). V1 is the minimum speed for cell feeding. A speed lower than V1 will cause the cell feeding cycle to be prolonged, which not only affects the viability and state of the cells but also reduces the cell harvesting efficiency. V2 is the maximum speed for cell feeding. When the speed exceeds V2, some cells are discharged before they have time to adhere under the inherent centrifugal force, resulting in a decrease in the cell harvesting rate. The dual-channel peristaltic pump pumps the cell stock solution into the centrifuge cup. After the cell stock solution enters the centrifuge cup, since the density of the cells is greater than that of the supernatant, under the action of centrifugal force, the cells (adhere to the wall) and the supernatant are separated. When the liquid in the centrifuge cup gradually increases to a total volume greater than the critical volume V of the lower opening of the centrifuge cup 下 when, the excess supernatant is discharged from the lower opening into the waste liquid bag. Both channel 1 and channel 2 of the dual-channel peristaltic pump are driven by the same motor and the same peristaltic pump roller. With hoses of the same diameter, the liquid pumping speeds of channel 1 and channel 2 are the same, so that the liquid flow rate pumped into the centrifuge cup is the same as the liquid flow rate pumped out of the centrifuge cup. Thus, the cell stock solution is pumped in from the upper opening, and the supernatant is discharged from the lower opening at the same flow rate, thereby forming a fast and stable continuous cell concentration flow. This continues until all the cell stock solution feeding is completed. When the air bubble sensor P1 detects air, the peristaltic pump stops, and solenoid valves 1 and 7 are closed, and the continuous flow feeding is completed. The supernatant in the cell stock solution is discharged into the waste liquid bag, and only the adhered cells and a small amount of supernatant remain in the centrifuge cup, with a total volume of about 100 ml.至此,细胞液从50L浓缩成100ml。

[0308] S04: Continuous flow cleaning, pump in the cleaning liquid and pump out the cleaning waste liquid (the cleaning liquid after cleaning) to complete cell cleaning (for example, the cleaning operation).

[0309] During specific implementation, after the continuous flow injection is completed, the high-speed centrifugation state of the centrifuge cup is maintained. Solenoid valves 2-7 are opened, and the peristaltic pump rotates at the set cleaning speed V3 and in the set direction. V3 is between two critical speeds (V4 < V3 < V5). V4 is the minimum speed for cell cleaning. If the speed is lower than V4, the cleaning intensity will be insufficient, the cleaning will be incomplete, and substances such as the culture medium in the centrifuge cup cannot be completely cleaned, and the cleaning efficiency will also be reduced. The speed V5 is the maximum speed for cell cleaning. If the speed exceeds V5, the strong fluid impact will not only carry away the adherent cells but may even kill the cells. The double-channel peristaltic pump pumps in the cleaning solution from the upper port and discharges the waste liquid from the lower port, thus forming a stable continuous cleaning flow. The centrifuge cup is carefully designed. The distance from the upper port of the centrifuge cup to the axis of the centrifuge cup is R1, and the distance from the lower port of the centrifuge cup to the axis of the centrifuge cup is R2. During the continuous flow cleaning process, the centrifuge cup is in a high-speed centrifugation state. The thickness of the supernatant between the upper and lower ports of the centrifuge cup is ΔR = R1 - R2. The cleaning solution enters from the upper port of the centrifuge cup and exits from the lower port, passing through the supernatant with a thickness of ΔR. The residual supernatant in the centrifuge cup is displaced by the cleaning solution. To ensure that the supernatant (other components except cells) in the centrifuge cup can be completely displaced, the liquid volume and the number of cleaning rounds of the continuous flow cleaning can be set according to the actual situation. After the continuous flow cleaning is completed, only the adherent cells and a little cleaning solution remain in the centrifuge cup. Keep the centrifuge cup centrifuging at the specified speed, open valves 1-4, and the peristaltic pump rotates at the set speed and direction to discharge the remaining cleaning solution in the centrifuge cup from the upper port to the cell stock solution bag. The upper port of the centrifuge cup is close to the cup wall. Under high-speed centrifugation, the cleaning solution in the cup can be discharged as much as possible.至此,离心杯中只剩下贴壁的细胞。

[0310] S05: Pump in the resuspension solution to mix the cells on the centrifuge cup wall into the resuspension solution (for example, resuspension operation).

[0311] During specific implementation, after the cell cleaning is completed, all cells are in an adherent state in the centrifuge cup. The cells need to be mixed down by the resuspension solution to make the cells in a resuspended state for the next step of processing: The centrifuge stops rotating, solenoid valves 3-6 are opened, and the double-channel peristaltic pump rotates at the set speed and direction. The second channel of the double-channel peristaltic pump pumps a specified dose of resuspension solution into the centrifuge cup from the lower port of the centrifuge cup, and at the same time, the first channel of the double-channel peristaltic pump pumps out the same volume of air from the upper port of the centrifuge cup at the same speed. Under the condition of constant pressure, the specified dose of air in the centrifuge cup is accurately displaced by the resuspension solution. Then the solenoid valves and the peristaltic pump are closed, and the centrifuge starts to mix and rotates back and forth at the set speed and cycle, so that all the adherent cells in the centrifuge cup are dissolved and evenly distributed in the resuspension solution.至此,重悬完成,离心机停止转动。

[0312] S06: Dispense the resuspended liquid in the centrifuge cup into the final production bag according to the set dosage (eg, dispensing operation).

[0313] During specific implementation, after the cells are resuspended, the cell fluid in the centrifuge cup needs to be quantitatively packaged into the final production bag for subsequent reinfusion or cryopreservation. Valves 3-5 are opened, and the peristaltic pump rotates at the set speed and direction. The two-way peristaltic pump channel 2 pumps the resuspended solution filled with cells in the centrifuge cup through the lower mouth of the centrifuge cup and into the final production bag according to the set dosage. At the same time, the two-way peristaltic pump channel 1 passes through the upper mouth of the centrifuge cup at the same speed, and pumps the same volume of clean air after bacteria filtration into the centrifuge cup. Under constant pressure, the cell fluid in the centrifuge cup is replaced by air and pumped into the final production bag. The packaging program will automatically cycle the packaging process according to the cell volume and the number of final production bags to complete the packaging of the cell resuspension. At this point, the entire cell harvesting process is completed, and the final product of the cell fluid is in the final production bag, which can be used for clinical reinfusion or long-term cryopreservation.

[0314] Through the above scenario examples, it is verified that the cell separation and harvesting system and method provided in this specification can effectively reduce the loss and damage of cells during the operation and processing, avoid cell inactivation, and efficiently and automatically complete the separation, concentration, washing, resuspension, packaging and other processes of cells in the cell stock solution by utilizing the characteristics of the two-way pump to form a stable continuous flow, and then cooperate with the centrifuge and refrigerated centrifuge chamber to transport the relevant fluids in the form of a stable continuous flow.

[0315] Although the present specification provides method operation steps as described in the embodiments or flow charts, more or fewer operation steps may be included based on conventional or non-creative means. The order of steps listed in the embodiments is only one way of executing the order of many steps and does not represent the only order of execution. When the device or client product in practice is executed, it can be executed in sequence or in parallel according to the method shown in the embodiments or the drawings (for example, a parallel processor or a multi-threaded processing environment, or even a distributed data processing environment). The terms "include", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, product or device including a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements inherent to such a process, method, product or device. In the absence of more restrictions, it is not excluded that there are other identical or equivalent elements in the process, method, product or device including the elements. The words first, second, etc. are used to represent the name, and do not represent any particular order.

[0316] Those skilled in the art also know that, in addition to implementing the controller in a purely computer-readable program code, the controller can be made to implement the same function in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, and embedded microcontrollers by logically programming the method steps. Therefore, such a controller can be considered as a hardware component, and the devices for implementing various functions included therein can also be considered as structures within the hardware component. Or even, the devices for implementing various functions can be considered as both software modules for implementing the method and structures within the hardware component.

[0317] This specification may be described in the general context of computer-executable instructions executed by a computer, such as program modules. Generally, program modules include routines, programs, objects, components, data structures, classes, etc. that perform specific tasks or implement specific abstract data types. This specification may also be practiced in distributed computing environments where tasks are performed by remote processing devices connected through a communication network. In a distributed computing environment, program modules may be located in local and remote computer-readable storage media including storage devices.

[0318] Through the description of the above embodiments, it can be known that those skilled in the art can clearly understand that the present specification can be implemented by means of software plus a necessary general hardware platform. Based on such an understanding, the technical solution of the present specification can essentially be embodied in the form of a software product, which can be stored in a storage medium such as ROM / RAM, a magnetic disk, an optical disk, etc., and includes a number of instructions for enabling a computer device (which can be a personal computer, a mobile terminal, a server, or a network device, etc.) to execute the methods described in each embodiment of the present specification or some parts of the embodiments.

[0319] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. This specification can be used in many general or special computer system environments or configurations. For example: personal computers, server computers, handheld devices or portable devices, tablet devices, multi-processor systems, microprocessor-based systems, set-top boxes, programmable electronic devices, network PCs, minicomputers, mainframe computers, distributed computing environments including any of the above systems or devices, etc.

[0320] Although the present specification is described through embodiments, those skilled in the art will appreciate that there are many modifications and changes to the present specification without departing from the spirit of the present specification, and it is intended that the appended claims include these modifications and changes without departing from the spirit of the present specification.

Claims

1. A cell separation and harvesting system, characterized in that: At least: Two-way pump, centrifugal cup, centrifuge, refrigerated centrifugal chamber, quantitative tube, air filter valve; among them, The two-way pump comprises a first channel and a second channel; The first end of the first channel is connected to the upper opening of the centrifuge cup, and the second end of the first channel is connected to at least the first pipeline, the second pipeline, and the third pipeline; wherein the first pipeline is used to connect the cell stock solution bag, the second pipeline is used to connect the cleaning solution bag, and the third pipeline is connected to the quantitative tube; the quantitative tube is used to cooperate with the two-way pump to realize the calibration of the two-way pump; The first end of the second channel is connected to the lower opening of the centrifuge cup, and the second end of the second channel is connected to at least the fourth pipeline, the fifth pipeline, the sixth pipeline, and the seventh pipeline; wherein the fourth pipeline is connected to the quantitative tube, the fifth pipeline is used to connect the sub-packaging bag, the sixth pipeline is used to connect the resuspension liquid bag, and the seventh pipeline is used to connect the waste liquid bag; the distance between the upper opening of the centrifuge cup and the adjacent cup wall is smaller than the distance between the lower opening of the centrifuge cup and the adjacent cup wall; The quantitative tube is also connected to the air filter valve through an eighth pipeline; The centrifugal cup is connected to the centrifuge, and the centrifugal cup is placed in the refrigerated centrifugal chamber, and the refrigerated centrifugal chamber is also connected to a refrigeration module; And, a first solenoid valve is provided on the first pipeline, a second solenoid valve is provided on the second pipeline, a third solenoid valve is provided on the third pipeline, a fourth solenoid valve is provided on the fourth pipeline, a fifth solenoid valve is provided on the fifth pipeline, a sixth solenoid valve is provided on the sixth pipeline, and a seventh solenoid valve is provided on the seventh pipeline; The fluid speeds of the first channel and the second channel are the same, and the two-way pump is used to make the liquid inlet speed and the liquid outlet speed of the centrifugal cup relatively consistent to form a stable continuous flow; the centrifugal cup also includes an upper flow channel and a lower flow channel, the first channel is connected to the upper opening of the centrifugal cup through the upper flow channel, and the second channel is connected to the lower opening of the centrifugal cup through the lower flow channel; Correspondingly, based on the dual-pass pump and the centrifugal cup, in the cell separation and concentration stage, the cells in the cell stock solution entering the centrifugal cup from the upper opening are subjected to the centrifugal force and enter the wall-adherent state to achieve separation of the cells and the supernatant; at the same time, the supernatant separated from the cell stock solution is discharged from the centrifugal cup through the lower opening of the centrifugal cup to achieve concentration of the cell fluid.

2. The cell separation and harvesting system according to claim 1, characterized in that: The centrifugal cup at least comprises: a cup body, and a connecting component arranged above the cup body; Wherein, the connecting component is provided with at least a first interface, a second interface, a first flow channel, and a second flow channel; The cup body is at least provided with an upper flow channel, a lower flow channel, a central axis flow channel, an upper opening and a lower opening; Wherein, the first interface is connected to the first flow channel, the first interface is used to connect to the first channel, and the first flow channel is connected to the upper port through the upper flow channel; The second interface is connected to the second flow channel, the second interface is used to connect to the second channel, and the second flow channel is connected to the lower port through the central axis flow channel and the lower flow channel.

3. The cell separation and harvesting system according to claim 1, characterized in that: The size of the rubber hose arranged in the first channel is the same as that of the rubber hose arranged in the second channel.

4. A cell separation and harvesting method based on the cell separation and harvesting system according to any one of claims 1 to 3, characterized in that: include: According to the preset calibration rules, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve and the two-way pump to determine the hose coefficient of the two-way pump; wherein the hose coefficient is used to characterize the volume of the fluid transported by the two-way pump when it rotates one circle; According to the preset separation and concentration rules and based on the hose coefficient of the two-way pump, the first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of a continuous flow by controlling the first solenoid valve and the two-way pump; at the same time, the cell stock solution in the centrifuge cup is separated into cells and supernatant by controlling the centrifuge, the two-way pump and the seventh solenoid valve, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow; According to the preset cleaning rules, the cleaning solution is pumped into the centrifuge cup in the form of a continuous flow by controlling the second solenoid valve and the two-way pump to clean the supernatant on the cells; at the same time, the cleaning solution is transported from the centrifuge cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve; According to the preset resuspension rule and based on the hose coefficient of the two-way pump, the washed cells in the centrifuge cup are resuspended by controlling the sixth solenoid valve, the two-way pump and the third solenoid valve; According to the preset sub-packaging rules and based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifugal cup is transported to the sub-packaging bag by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve.

5. The method according to claim 4, characterized in that According to the preset calibration rules, the two-way pump is calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve, and the two-way pump to determine the hose coefficient of the two-way pump, including: According to the preset calibration rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started; Controlling the two-way pump to rotate in a first direction to pump a second volume of cleaning solution into the centrifuge cup along the second pipeline as a calibration solution; and stopping the centrifuge; wherein the second volume is greater than the volume of the quantitative tube; Controlling the two-way pump to rotate in a first direction to transfer the calibration liquid in the centrifugal cup along the seventh pipeline toward the waste liquid bag; and monitoring whether the calibration liquid reaches the seventh solenoid valve; When it is detected that the calibration liquid reaches the seventh solenoid valve, the seventh solenoid valve is closed and the fourth solenoid valve is opened; Controlling the two-way pump to rotate in a first direction to transfer the calibration liquid in the centrifugal cup to the quantitative tube along the fourth pipeline; and monitoring the detection value of the third bubble sensor; When the detection value of the third bubble sensor indicates that liquid is detected, the operation of the two-way pump is stopped; and the number of rotations of the two-way pump is obtained; The hose coefficient of the two-way pump is determined based on the volume of the metering tube and the number of revolutions of the two-way pump.

6. The method according to claim 4, characterized in that According to the preset separation and concentration rules, based on the hose coefficient of the two-way pump, the first volume of cell stock solution is pumped from the cell stock solution bag into the centrifuge cup in the form of continuous flow by controlling the first solenoid valve and the two-way pump; at the same time, by controlling the centrifuge, the two-way pump, and the seventh solenoid valve, the cell stock solution in the centrifuge cup is separated into cells and supernatant, and the supernatant is transported from the centrifuge cup to the waste liquid bag in the form of continuous flow, including: According to the preset separation and concentration rules, the first solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started; Controlling the two-way pump to rotate in a first direction based on a first speed, so as to input the cell stock solution in the cell stock solution bag into the centrifuge cup through the upper opening of the centrifuge cup in the form of a continuous flow along the first pipeline; Controlling the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cells of the cell stock solution entering the centrifuge cup enter the wall-adherent state under the action of centrifugal force, so as to separate the cell stock solution into cells and supernatant; and outputting the supernatant from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and inputting it into a waste liquid bag along the seventh pipeline; When the volume of the cell stock solution input into the centrifuge cup reaches the first volume based on the hose coefficient of the two-way pump, the two-way pump is stopped, the centrifuge is stopped, and the first solenoid valve and the seventh solenoid valve are closed.

7. The method according to claim 4, characterized in that According to the preset cleaning rules, the cleaning solution is pumped into the centrifugal cup in the form of a continuous flow by controlling the second solenoid valve and the two-way pump to clean the supernatant on the cells; at the same time, the cleaning solution is transported from the centrifugal cup to the waste liquid bag in the form of a continuous flow by controlling the two-way pump and the seventh solenoid valve, including: According to the preset cleaning rules, the second solenoid valve and the seventh solenoid valve are opened; and the two-way pump and the centrifuge are started; Controlling the two-way pump to rotate in a first direction based on a second speed, so that the cleaning liquid in the cleaning liquid bag is input into the centrifugal cup through the upper opening of the centrifugal cup in the form of a continuous flow along the second pipeline; Controlling the centrifuge to rotate the centrifuge cup based on the first rotation speed, so that the cleaning solution entering the centrifuge cup washes the cells in the adherent state; and outputting the cleaning solution from the centrifuge cup through the lower opening of the centrifuge cup in the form of a continuous flow, and inputting it into a waste liquid bag along the seventh pipeline; When it is determined that the cleaning end condition is met, the second solenoid valve and the seventh solenoid valve are closed, and the first solenoid valve and the fourth solenoid valve are opened; The two-way pump is controlled to rotate in the second direction based on the third speed, and the remaining cleaning solution in the centrifuge cup is input into the cell stock solution bag along the first pipeline through the upper opening of the centrifuge cup.

8. The method according to claim 4, characterized in that According to the preset resuspension rule and based on the hose coefficient of the two-way pump, the sixth solenoid valve, the two-way pump and the third solenoid valve are controlled to resuspend the washed cells in the centrifuge cup, including: According to the preset resuspension rules, the third solenoid valve and the sixth solenoid valve are opened; and the two-way pump is started; Controlling the two-way pump to rotate in a second direction based on a fourth speed, so as to input the resuspension in the resuspension bag into the centrifuge cup through a lower opening of the centrifuge cup along a sixth pipeline; When the volume of the resuspension liquid input into the centrifuge cup reaches the third volume based on the hose coefficient of the two-way pump, the two-way pump is stopped; and the centrifuge is started; The centrifuge is controlled to rotate based on the second rotation speed so that the cells in the adherent state are dissolved in the resuspension solution to obtain a target solution containing cells.

9. The method according to claim 4, characterized in that According to the preset filling rules and based on the hose coefficient of the two-way pump, the target liquid containing cells in the centrifugal cup is transported to the filling bag by controlling the fifth solenoid valve, the two-way pump and the third solenoid valve, including: According to the preset packaging rules, the third solenoid valve and the fifth solenoid valve are opened; and the two-way pump and the centrifuge are started; controlling the centrifuge to rotate based on a third rotation speed; Based on the hose coefficient of the two-way pump, the two-way pump is controlled to rotate in the second direction based on the fifth speed, and the target liquid of a specified volume is input into the sub-packaging bag along the fifth pipeline through the lower opening of the centrifugal cup.

10. A computer-readable storage medium, characterized in that: Computer instructions are stored thereon, and when the instructions are executed by a processor, the steps of the method according to any one of claims 4 to 9 are implemented.

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