Detailed Description
In order to make the technical solutions in the present specification better understood by those skilled in the art, the technical solutions in the embodiments of the present specification will be clearly and completely described below with reference to the drawings in the embodiments of the present specification, and it is obvious that the described embodiments are only some embodiments of the present specification, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are intended to be within the scope of the present disclosure.
It should be noted that, the information and data related to the user in the embodiments of the present disclosure are information and data authorized by the user or fully authorized by the related parties, and the processes of collecting, storing, using, processing, transmitting, providing, disclosing and applying the related data all comply with relevant laws and regulations and standards, take necessary security measures, do not violate the public welcome, and provide corresponding operation entries for the user or the related parties to select authorization or rejection.
It should also be noted that in the embodiments of the present disclosure, some existing solutions in the industry such as software, components, models, etc. may be mentioned, and they should be considered as exemplary, only for illustrating the feasibility of implementing the technical solution of the present disclosure, but not meant to imply that the applicant has or must not use the solution.
In view of the existing system equipment, in the process of specifically separating and sub-packaging cells, a conventional peristaltic pump is used for forming negative pressure in a centrifugal cup so as to generate a pressure difference with the outside, and then cell stock solution containing the cells is sucked into the centrifugal cup through the pressure difference for subsequent cell separation. However, the fluid movement generated by the pressure difference is often unstable, and the separation and split charging are easily affected by sudden interruption or abrupt change of the flow rate caused by factors such as liquid amount change or pressure change. Moreover, when the cell stock solution is pumped into the centrifugal cup based on the mode, the pressure difference acts to suddenly accelerate the flow speed at the moment of entering the centrifugal cup because the inside of the centrifugal cup is in a negative pressure state, so that cells in the cell stock solution are not separated and attached, and are discharged out of the centrifugal cup along with the cell stock solution, and the cells are lost. Furthermore, the need to maintain a large negative pressure in the centrifuge cup over a long period of time based on the above approach can result in the cells in the centrifuge cup being subjected to a large surface tension, which in turn can affect the activity of the cells and even lead to inactivation of the cells.
Aiming at the root cause of the problems, after creative labor, the applicant considers that a double-channel double-pass pump can be introduced and used for replacing a conventional peristaltic pump, meanwhile, the related structure of the system is correspondingly improved to match the operation of the double-pass pump in the cell separation and split charging process, and further, a refrigeration centrifugal cabin is also introduced and used for cooling cells in a centrifugal cup, so that the damage to the activity of the cells caused by the overhigh temperature in the centrifugal concentration stage is avoided. In this way, the liquid inlet speed and the liquid outlet speed of the centrifugal cup are relatively consistent by using the two-way pump, so that the pressure in the centrifugal cup is relatively constant, and a stable continuous flow can be formed. Furthermore, the stable continuous flow can be used for completing operations such as separation, concentration, cleaning, resuspension, split charging and the like in the cell separation split charging process, so that on one hand, the flow rate of liquid can be stable and reliable when the liquid enters the centrifugal cup, cells in the cell stock solution enter an attached state after entering the centrifugal cup for a sufficient time under the action of centrifugal force, separation is realized, loss of the cells during the separation operation is effectively reduced, and on the other hand, the cells in the centrifugal cup can be effectively prevented from being inactivated due to the action of negative pressure. Thus, the separation and split charging of the cells can be relatively and efficiently finished, and the cell products with higher quality can be obtained.
Referring to fig. 1, the embodiment of the present disclosure provides a cell separation and harvesting system, which may include at least a structure of a bi-pass pump 30, a centrifugal cup 40, a centrifuge 60, a refrigerated centrifuge chamber 50, a dosing tube 70, a filter valve 80, etc., wherein,
The two-way pump 30 may specifically include a first channel 31, a second channel 32;
the first end of the first channel 31 is connected with the upper opening of the centrifugal cup 40, the second end of the first channel 31 is at least connected with a first pipeline 21, a second pipeline 22 and a third pipeline 23, wherein the first pipeline 21 is used for connecting a cell stock solution bag 11, the second pipeline 22 is used for connecting a cleaning solution bag 12, and the third pipeline 23 is connected with a quantitative pipe 70;
the first end of the second channel 32 is connected with the lower opening 42 of the centrifugal cup 40, the second end of the second channel 32 is at least connected with a fourth pipeline 24, a fifth pipeline 25, a sixth pipeline 26 and a seventh pipeline 27, wherein the fourth pipeline 24 is connected with a quantitative pipe 70, the fifth pipeline 25 is used for connecting a sub-bag 13, the sixth pipeline 26 is used for connecting a heavy suspension bag 14, and the seventh pipeline 27 is used for connecting a waste liquid bag 15;
the metering tube 70 is also connected to a filter valve 80 via an eighth line 28;
the centrifugal cup 40 is connected with a centrifugal machine 60, the centrifugal cup 40 is arranged in the refrigeration centrifugal cabin 50, and the refrigeration centrifugal cabin 50 is also connected with a refrigeration module 51;
And, a first solenoid valve 1 (may be denoted by ①) is provided on the first pipe 21, a second solenoid valve 2 (may be denoted by ②) is provided on the second pipe 22, a third solenoid valve 3 (may be denoted by ③) is provided on the third pipe 23, a fourth solenoid valve 4 (may be denoted by ④) is provided on the fourth pipe 24, a fifth solenoid valve 5 (may be denoted by ⑤) is provided on the fifth pipe 25, a sixth solenoid valve 6 (may be denoted by ⑥) is provided on the sixth pipe 26, and a seventh solenoid valve 7 (may be denoted by ⑦) is provided on the seventh pipe 27.
The above-mentioned piping may be collectively referred to as piping 20, the above-mentioned solenoid valve may be collectively referred to as solenoid valve 0, and the above-mentioned liquid bag may be collectively referred to as liquid bag 10.
Specifically, the first electromagnetic valve 1, the second electromagnetic valve 2, the third electromagnetic valve 3, the fourth electromagnetic valve 4, the fifth electromagnetic valve 5, the sixth electromagnetic valve 6 and the seventh electromagnetic valve 7 can be specifically used for controlling the on-off of a pipeline where the electromagnetic valves are located.
Specifically, the dual-pass pump 30 (or called dual-pass peristaltic pump) may be a dual-channel peristaltic pump. Referring to fig. 2, the dual pump 30 includes at least a motor 33, a roller 34, a first channel 31, a second channel 32, and so on. Further, the first and second passages 31 and 32 may be provided with rubber pipes having the same size (e.g., 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 diameters of the two channels are the same, the fluid velocity in the first channel 31 and the fluid velocity in the second channel 32 are the same.
Accordingly, the introduction and use of the two-way pump 30 in a cell separation harvesting system allows the liquid inlet and outlet speeds of the centrifuge cup 40 to be relatively uniform, maintaining a constant pressure in the centrifuge cup 40, and thus allowing a stable continuous flow.
Specifically, the cell stock bag 11 may store a cell stock containing cells to be separated and concentrated. The washing liquid bag 12 may store washing liquid for washing residual liquid on cells. The resuspension bag 14 may store a resuspension for resuspension of the isolated cells.
The waste liquid bag 15 may be an empty liquid bag, and is used for storing waste liquid generated during the operation of the system. The sub-packaging bag 13 may be an empty liquid bag, and is used for separating and concentrating the target liquid (or called a cell liquid product, a cell product, etc.) containing cells during the operation process of the storage system.
Further, in addition to the first pipeline 21, the second pipeline 22, and the third pipeline 23, the second end of the first channel 31 may be further connected with other pipelines in an expanding manner according to specific application scenarios and processing requirements, where other pipelines may be connected with other liquid bags such as other cell stock liquid bags. Similarly, the second end of the second channel 32 may be connected to other lines in an expanded manner, in addition to the fourth line 24, the fifth line 25, the sixth line 26, and the seventh line 27.
In particular, the filter valve 80 may be connected to the external environment for drawing in and filtering air fluid from the external environment, or for exhausting air fluid to the external environment.
Specifically, the centrifuge 60 is connected to the centrifugal cup 40, and the centrifugal cup 40 is driven to rotate by the operation of an internal motor, so that cells in the solution in the centrifugal cup 40 are clung to the wall 43 of the centrifugal cup 40 due to the centrifugal force, and are in an adhering state.
Specifically, the refrigerating centrifugal chamber 50 is connected to a refrigerating module 51. In specific implementation, the centrifugal cup 40 is placed in the refrigeration centrifugal cabin 50, and the refrigeration module 51 can intelligently control the temperature inside the refrigeration centrifugal cabin 50, so that the cells in the centrifugal cup 40 are in a stable and proper threshold temperature range, and the cells are prevented from being deactivated due to overhigh temperature.
The threshold temperature range may specifically be 2 degrees celsius or more and 8 degrees celsius or less.
In particular, the metering tube 70 is understood to be a known and fixed volume communication structure. For example, the volume of the metering tube 70 may be denoted as V.
Specifically, referring to fig. 1, the first pipeline 21, the second pipeline 22, and the third pipeline 23 may be first assembled at a node, and then connected to the first channel 31 of the two-way pump 30 through corresponding pipelines by the node.
Similarly, the fourth, fifth, sixth and seventh lines 24, 25, 26, 27 may be assembled together at a node, and then connected to the second channel 32 of the two-way pump 30 via the corresponding lines.
Specifically, referring to fig. 3, the first passage 31 may be connected to an upper port 41 of the centrifugal cup 40, and the second passage 32 may be connected to a lower port 42 of the centrifugal cup 40.
Further, as can be seen in FIG. 4, the distance between the upper port 41 and its adjacent cup wall 43 (e.g., the right cup wall) is less than the distance between the lower port 42 and its adjacent cup wall 43 (e.g., the left cup wall).
In particular, as can be seen in fig. 3 and 4, when the two-way pump 30 is rotated in a first direction, liquid can be pumped into the centrifugal cup 40 through the first passageway 31 via the upper port 41 of the centrifugal cup 40, while liquid can be removed from the centrifugal cup 40 through the second passageway 32 via the lower port 42 of the centrifugal cup 40. Since the flow rates of the liquid in the first and second channels 31 and 32 are the same, the pressure in the centrifugal cup 40 can be maintained constant, no pressure difference is generated, and a stable continuous flow can be formed. Further, the liquid can be pumped into the centrifugal cup 40 stably and uniformly in the form of the stable continuous flow, and the liquid can be discharged from the centrifugal cup 40 synchronously.
Specifically, for example, in the cell separation and concentration stage, the cell stock solution is pumped into the centrifugal cup based on the first channel of the two-way pump, and the supernatant liquid which is remained after the cells in the cell stock solution are separated under the centrifugal force action is discharged from the centrifugal cup based on the second channel of the two-way pump at the same flow rate, so that a stable continuous flow is formed in the centrifugal cup and related pipelines.
And then the cell stock solution can be pumped into the centrifugal cup in a stable and uniform manner in the form of the continuous flow, and the supernatant liquid after separating the cells is synchronously discharged from the centrifugal cup. The cell stock solution does not need to be pumped into the centrifuge cup by means of creating a pressure differential in the centrifuge cup as in prior system devices.
Therefore, on one hand, the cell stock solution can continuously, stably and uniformly enter the centrifugal cup, so that cells in the cell stock solution are tightly attached to the wall of the centrifugal cup under the action of centrifugal force for a sufficient time in the centrifugal cup, separation of the cells and the supernatant is realized, and the phenomenon that the cells are lost due to the fact that the flow velocity of the cell stock solution is unstable and suddenly excessive when the cell stock solution enters the centrifugal cup, and the cells are directly flushed out of the centrifugal cup along with the cell stock solution is avoided. On the other hand, the cell damage and even inactivation caused by the extrusion force action generated by the pressure difference after the cell stock solution enters the centrifugal cup can be effectively avoided.
In addition, as the distance between the upper opening of the centrifugal cup and the cup wall of the centrifugal cup is relatively short, cells in the cell stock solution entering from the upper opening are more easily clung to the cup wall (enter an adherence state) under the action of centrifugal force in the process of driving the centrifugal cup to rotate by the centrifugal machine, and are relatively more difficult to directly discharge the centrifugal cup along with the cell solution, thereby effectively reducing the loss of the cells and better realizing the separation of the cells and the supernatant (can be simply called cell separation). Meanwhile, the supernatant liquid separated from the cell stock solution after the cell separation in the centrifugal cup can be referred to as a liquid path shown in fig. 4, and the concentration of the cell liquid (which can be abbreviated as cell concentration) is realized by discharging the centrifugal cup through the lower opening of the centrifugal cup. Because the distance between the lower opening of the centrifugal cup and the cup wall of the centrifugal cup is relatively far, the distance between the cells in the attached state in the centrifugal cup and the discharge opening is longer, the difficulty that the attached cells are discharged out of the centrifugal cup along with liquid is increased, and therefore the loss of the cells can be further reduced.
In addition, when the two-way pump rotates in the second direction, gas can be pumped into the centrifugal cup through the lower opening of the centrifugal cup through the second channel, and meanwhile, liquid is discharged from the centrifugal cup through the upper opening of the centrifugal cup through the first channel. In addition, 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, so that relatively more liquid can be discharged from the centrifugal cup.
In addition, by forming a stable continuous flow, a more efficient water cooling system can also be formed. The heat generated by the rotary motion in the centrifugal cup can be timely conducted out through the stable continuous flow, so that the auxiliary cooling of the centrifugal cup can be further realized, and the cell inactivation caused by overhigh temperature is further reduced.
In some embodiments, referring to fig. 1, a first bubble sensor (may be denoted as P1) is disposed adjacent to the second end of the first channel 31, a second bubble sensor (may be denoted as P2) is disposed adjacent to the first end of the second channel 32, and a third bubble sensor (may be denoted as P3) is disposed adjacent to the dosing tube 70 on the eighth conduit 28.
The first bubble sensor P1 may be used to detect whether the type of fluid passing through the first channel 31 of the two-way pump 30 is gas or liquid. The second bubble sensor P2 described above may be used in particular to detect whether the type of fluid passing through the second channel 32 of the two-way pump 30 is a gas or a liquid. The third bubble sensor P3 described above may be used to detect whether the type of fluid flowing out through the metering tube 70 is a gas or a liquid.
In some embodiments, the hose disposed in the first channel is the same size as the hose disposed in the second channel. Thus, it can be ensured that the fluid flow rate based on the first channel is the same as the fluid flow rate based on the second channel.
In some embodiments, a flow rate detector may also be provided in the line between the second channel of the two-way pump and the lower port of the centrifugal cup for detecting the velocity of the fluid flowing into the centrifugal cup. Wherein, this velocity of flow detector can be with bi-pass pump electric connection.
Accordingly, the two-way pump can receive and dynamically adjust the rotational speed of the two-way pump according to the flow rate acquired by the flow rate detector, so that fluid can be pumped into the centrifugal cup at a proper speed.
In some embodiments, an angular velocity detector may also be provided on the centrifuge cup for detecting the angular velocity of the centrifuge cup as it rotates with the centrifuge. Wherein, this angular velocity detector can be with centrifuge electric connection.
Accordingly, the centrifugal machine can receive and determine the actual rotation speed of the centrifugal cup according to the angular speed acquired by the angular speed detector, and then the rotation speed of the centrifugal machine is adjusted in a targeted manner.
In some embodiments, the structure of the centrifuge cup 40 is further modified to better perform operations such as separation, concentration, washing, etc. in conjunction with the cell separation harvesting system described above, and reference is made specifically to fig. 16.
Specifically, the centrifugal cup 40 may at least include a cup body 402, and a connecting member 401 disposed above the cup body 402;
Wherein the connecting part 401 may be provided with at least a first port 44, a second port 45, a first flow channel 4-1, a second flow channel 4-2, etc.;
the cup body 402 can be provided with at least an upper runner 4-3, a lower runner 4-4, a middle shaft runner 4-5, an upper port 41, a lower port 42 and the like;
Wherein, the first interface 44 may be connected to the first flow channel 4-1, the first interface 44 may be specifically used to connect the first channel 31, and the first flow channel 4-1 may be connected to the upper port 41 through the upper flow channel 4-3;
the second port 45 may be connected to the second flow channel 4-2, and the second port 45 may be specifically used to connect to the second flow channel 32, where the second flow channel 4-2 may be connected to the lower port 42 sequentially through the bottom bracket bearing flow channel 4-5 and the lower flow channel 4-4.
Specifically, as shown in fig. 16, the connection member 401 may be provided with a dual-channel F-head, in which one of the connectors is used as a first port 44 to communicate with a first flow channel 4-1 provided inside the connection member 401, and the other connector is used as a second port 45 to communicate with a second flow channel 4-2 provided inside the connection member 401.
Specifically, as described with reference to FIG. 16, an upper flow passage 4-3 may be provided near the top of the cup body 402 of the centrifugal cup 40, wherein the upper flow passage 4-3 communicates with the first flow passage 4-1. An upper opening 41 is provided in the upper flow passage 4-3 at a position closer to the cup wall 43. In particular, fluid outside the centrifugal cup 40 may enter the centrifugal cup 40 through the first port 44, pass through the first flow channel 4-1, and then pass through the upper flow channel 4-3, and flow from the upper port 41 to the bottom of the cup along the cup wall 43.
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 port 42 is provided in the lower flow channel 4-4 at a position further from the cup wall 43. The distance between the lower port 42 and the cup wall 43 (e.g., may be referred to as) is greater than the distance between the upper port 41 and the cup wall 43 (e.g., may be referred to as). In addition, a center axis flow channel 4-5 is provided along the center axis of the cup, proximate the bottom of the cup 402. Wherein the central shaft runner 4-5 is communicated with the lower runner 4-4 and simultaneously communicated with the second runner 4-2. In particular, fluid at the bottom of the centrifugal cup 40 may flow into the lower flow channel 4-4 through the lower port 42, then along the central axis flow channel 4-5, into the second flow channel 4-2, and out of the centrifugal cup 40 through the second port 45.
Specifically, a diversion platform 49 (for example, a T-shaped diversion platform) can be further arranged at the intersection position of the first runner 4-1 and the upper runner 4-3, a large sealing ring 47 can be further arranged at the position adjacent to the upper side of the diversion platform 49, a small sealing ring 48 can be arranged at the position adjacent to the lower side of the diversion platform 49, and a bearing 46 and other structures can be further arranged at the position above the large sealing ring 47 so as to cooperate with a cell separation harvesting system to perform high-speed centrifugal rotation.
Specifically, the centrifugal cup may further include a base 403. The base 403 may be fixedly disposed on the centrifuge 60, and the cup 402 may be disposed on the base 403.
When the centrifugal cup is matched with the cell separation and harvesting system to rotate, when fluid enters the centrifugal cup from the first interface, the fluid flows to the bottom of the cup along the cup wall from the upper port through the upper flow channel. The separation and concentration stage can make the cells relatively easy to enter the adherence state due to the action of centrifugal force to separate and obtain more cells, and meanwhile, the separated supernatant flows to the cup body along the cup wall, enters the lower runner from the lower runner, and then sequentially passes through the middle shaft runner and the second runner to discharge the centrifugal cup from the second interface.
When fluid is discharged from the centrifugal cup from the first interface through the upper port, the upper flow channel and the first flow channel in sequence, relatively more cleaned cleaning fluid can be discharged from the centrifugal cup in the cleaning stage due to the relatively close distance between the upper port and the cup wall.
Specifically, for example, assume that the radius of the cup is R, the height is H, and the distance between the upper opening and the cup wall isThe distance between the lower opening and the cup wall is。
If the liquid is discharged through the lower port, the remaining liquid volume in the cup can be expressed as:。
Conversely, if the liquid is discharged through the upper port, the volume of the remaining liquid in the cup can be expressed as:。
Due toLess thanThus, V Lower part(s)>V Upper part.
Therefore, based on the centrifugal cup, the liquid draining degree of the centrifugal cup can be flexibly adjusted by adjusting the interface used when the liquid is drained according to specific conditions and processing requirements and matching with a cell separation and harvesting system.
In some embodiments, referring to fig. 5, a temperature sensor 52 may be further disposed in the refrigerated centrifuge chamber 50, for detecting the temperature in the refrigerated centrifuge chamber 50 in real time.
The temperature sensor 52 may be electrically connected to the refrigeration module 51.
In the implementation, the refrigeration module can intelligently and dynamically adjust the refrigeration power according to the temperature data acquired by the temperature sensor, so that the temperature of the centrifugal cup in the refrigeration centrifugal cabin is effectively maintained within a proper threshold temperature range.
In some embodiments, according to specific application scenarios and processing requirements, the second end of the first channel may be further connected to another expansion pipeline, where the other expansion pipeline may be connected to another suitable fluid bag, for example, another cell stock solution bag, or an empty fluid bag, etc.
Similarly, other expansion pipelines can be connected to the second end of the second channel, wherein other suitable liquid bags can be connected to the other expansion pipelines, such as other sub-bags, or other waste liquid bags, etc.
In some embodiments, the cell separation harvesting system described above may specifically further comprise a processor. The processor can be electrically connected with an electromagnetic valve, a bubble sensor, a temperature sensor, a refrigerating module, a two-way pump and a centrifugal machine in the system. In the specific implementation, the processor can receive and intelligently control the corresponding electromagnetic valve, the refrigeration module, the double-pass pump, the centrifugal machine and the like according to the related data acquired by the bubble sensor and the temperature sensor, and automatically complete cell separation and split charging.
In some embodiments, the processor may first perform calibration operations with respect to the two-way pump by controlling the corresponding solenoid valves using a metering tube, two-way pump, centrifuge, etc. configuration. The method comprises the steps of controlling corresponding electromagnetic valves, pumping cell stock solution with a specified volume into a centrifugal cup from a cell stock solution bag in a continuous flow mode by utilizing a double-pass pump to realize liquid feeding operation, simultaneously, rotating the centrifugal cup by utilizing a centrifugal machine to enable cells in the cell stock solution to enter an adherence state under the action of centrifugal force, clinging to the wall of the cup to be separated from supernatant to realize separation operation, and synchronously discharging the separated supernatant out of the centrifugal cup by utilizing the double-pass pump to be conveyed to a waste liquid bag to realize concentration operation. Then, by controlling the corresponding solenoid valves, the washing liquid is pumped from the washing liquid bag into the centrifugal cup in a continuous flow form by using the two-way pump, and the separated cells are washed, so that the washing operation is realized. Then, by controlling the corresponding solenoid valves, the two-way pump is utilized to pump the heavy suspension with corresponding volume from the heavy suspension bag into the centrifugal cup, and the separated cells are mixed and dissolved, so that the heavy suspension operation is realized. And finally, by controlling the corresponding electromagnetic valve, the target liquid containing cells in the centrifugal cup is conveyed into the sub-packaging bags by utilizing the double-way pump, so that sub-packaging operation is realized. And (5) separating and split charging the cells.
In some embodiments, the physical structure of the cell separation harvesting system described above may be as described with reference to fig. 6. The cell stock solution bag 11 and the cleaning solution bag 12 can be mounted on a right side solution bag bracket 94, and the sub-packaging bag 13, the heavy suspension bag 14 and the waste solution bag 15 can be mounted on a left side solution bag bracket 94. The associated spare bag may be mounted on a side wall bag hanger 93.
In specific implementation, the user can control the cell separation and harvesting system through the operation panel 91, and perform operations such as calibration, separation, concentration, cleaning, split charging and the like in sequence, so as to complete separation and split charging of cells.
It should be noted that, compared with the conventional system equipment, the cell separation and harvesting system with the structure not only can realize comprehensive and multifunctional operations such as calibration, liquid feeding, separation, concentration, cleaning, split charging and the like, but also effectively simplifies the overall structural layout, so that the structural layout is relatively simple and clear, the user can understand the operation conveniently, and the equipment cost of the system is reduced while the operation difficulty of the user is reduced.
Specifically, compared with conventional system equipment, the total number of electromagnetic valves of the cell separation and harvesting system is reduced from 14 to 7, the total length of the air filtering valves is reduced from 2 to 1, the total length of the pipeline is reduced from 600cm to 370cm, the liquid path logic is relatively clear, the observation and monitoring in operation are convenient, meanwhile, the installation of a sterile consumable pipeline is effectively simplified, the operation difficulty is greatly reduced, the human error caused by a complex pipeline is reduced, and the efficiency and the stability of automatic cell processing are improved.
The cell separation and harvesting system and the cell separation and harvesting method are based on the specification, the system at least comprises a two-way pump, a centrifugal cup, a centrifugal machine, a refrigeration centrifugal cabin, a quantitative pipe and a filter valve, wherein the two-way pump comprises a first channel and a second channel, the first end of the first channel is connected with an upper opening of the centrifugal cup, the second end of the first channel is at least connected with a first pipeline, a second pipeline and a third pipeline, the first pipeline is used for connecting a cell stock solution bag, the second pipeline is used for connecting a cleaning solution bag, the third pipeline is connected with the quantitative pipe, the first end of the second channel is connected with a lower opening of the centrifugal cup, the second end of the second channel is at least connected with a fourth pipeline, a fifth pipeline, a sixth pipeline and a seventh pipeline, the fourth pipeline is connected with the quantitative pipe, the fifth pipeline is used for connecting a separation bag, the sixth pipeline is used for connecting a heavy suspension bag, the distance between the upper opening of the centrifugal cup and a lower opening of the centrifugal cup is smaller than that between the lower opening of the centrifugal cup and a wall, the quantitative pipe is also connected with the filter valve through a eighth pipeline, the centrifugal cup is connected with the centrifugal cup, the first pipeline is further connected with the centrifugal valve, the fifth pipeline is arranged on the centrifugal cabin, the refrigeration electromagnetic valve is further arranged on the fifth pipeline, the refrigeration electromagnetic valve is arranged on the fifth pipeline, the refrigeration electromagnetic valve is arranged on the refrigeration electromagnetic valve, the refrigeration pipeline and the refrigerating electromagnetic valve is. According to the cell separation and harvesting system based on the structure, during implementation, stable continuous flow can be formed by utilizing the characteristics of the double-pass pump, and then the centrifugal machine and the refrigeration centrifugal cabin are matched in a stable continuous flow mode to convey relevant fluid, so that loss and damage of cells in the operation treatment process can be effectively reduced, cell inactivation is avoided, and the treatments of separating, concentrating, cleaning, resuspension, split charging and the like of the cells in the cell stock solution are automatically and efficiently completed, and cell products with higher quality are obtained.
Referring to fig. 7, the embodiment of the present disclosure further provides a cell separation and harvesting method based on the cell separation and harvesting system, which may include the following steps:
s701, calibrating the double-pass pump by controlling the second electromagnetic valve, the seventh electromagnetic valve, the fourth electromagnetic valve and the double-pass pump according to a preset calibration rule to determine a rubber pipe coefficient of the double-pass pump, wherein the rubber pipe coefficient is used for representing the volume of fluid transported by the double-pass pump in one circle of rotation;
S702, pumping a first volume of cell stock solution from a cell stock solution bag into a centrifugal cup in a continuous flow mode by controlling a first electromagnetic valve and a double-pass pump according to a preset separation concentration rule and based on a rubber tube coefficient of the double-pass pump, and meanwhile, separating the cell stock solution in the centrifugal cup into cells and supernatant by controlling a centrifugal machine, the double-pass pump and a seventh electromagnetic valve, and conveying the supernatant from the centrifugal cup to a waste liquid bag in a continuous flow mode;
S703, pumping cleaning liquid into the centrifugal cup in a continuous flow mode by controlling a second electromagnetic valve and a double-pass pump according to a preset cleaning rule to clean supernatant on cells;
S704, carrying out resuspension treatment on the cleaned cells in the centrifugal cup by controlling a sixth electromagnetic valve, a double-pass pump and a third electromagnetic valve based on the rubber tube coefficient of the double-pass pump according to a preset resuspension rule;
And S705, according to a preset split charging rule, based on the rubber tube coefficient of the two-way pump, conveying the target liquid containing cells in the centrifugal cup to the split charging bag by controlling the fifth electromagnetic valve, the two-way pump and the third electromagnetic valve.
Based on the above embodiment, the operations of calibration, separation, concentration, cleaning, resuspension, split charging and the like can be sequentially performed by using the cell separation and harvesting system according to the corresponding rules, so that the separation and split charging of cells can be efficiently completed, and cell products with higher quality can be obtained.
In some embodiments, before implementation, the configuration parameters of the related liquid bags, pipelines, electromagnetic valves and the like can be determined according to the specific application scene and the separation and split charging requirements by referring to the structure diagram of the cell separation and harvest system shown in fig. 1, and then the cell separation and harvest system meeting the requirements is sequentially connected and constructed by using fully-closed disposable consumables and equipment such as a sterile pipe connecting machine, a heat sealing instrument and the like according to the configuration parameters of the system.
Specifically, the required cell stock solution bags, cleaning solution bags, heavy suspension bags, waste solution bags, sub-packaging bags and other liquid bags can be determined. And sequentially connecting the liquid bags to corresponding sterile fully-closed disposable consumable pipelines, and installing the fully-closed disposable consumable pipelines according to the installation prompt information and the corresponding solenoid valve opening sequence to obtain the required cell separation and harvesting system. Specifically, the method comprises the steps of firstly opening a first electromagnetic valve, combining a cell stock solution bag and a corresponding pipeline, opening a second electromagnetic valve, combining a cleaning solution bag and a corresponding pipeline, opening a third electromagnetic valve, a fourth electromagnetic valve, combining a quantitative pipe, a filter valve and a corresponding pipeline, opening a fifth electromagnetic valve, combining a split-filling bag and a corresponding pipeline, opening a sixth electromagnetic valve, combining a heavy suspension bag and a corresponding pipeline, and opening a seventh electromagnetic valve, and combining a waste liquid bag and a corresponding pipeline. And then starting the two-way pump, installing the rubber pipe of the first channel and the rubber pipe of the second channel according to the installation label, fixedly connecting the centrifugal cup on the centrifugal machine, and ensuring the stable installation of the centrifugal cup. And then using a sterile pipe connecting machine to connect each liquid bag to a corresponding consumable pipeline according to a designated sequence, wherein the liquid bags sequentially corresponding to the electromagnetic valves from right to left are respectively a cell stock liquid bag, a cleaning liquid bag, a split charging bag (empty), a resuspension bag and a waste liquid bag (empty).
Wherein, the consumable pipeline can be the totally enclosed disposable consumable pipeline through ethylene oxide sterilization, and relevant consumptive material still contains the centrifugation cup in addition. The whole installation process is isolated from the outside when in implementation, and the liquid bags are connected through a sterile pipe connecting machine when connected, so that the pipeline is prevented from being in contact with the outside.
In some embodiments, after the cell separation and harvesting system is obtained by connection, the self-checking operation may be implemented by performing pipeline air tightness detection on the cell separation and harvesting system according to a preset self-checking rule, and the following may be included in the specific implementation:
S1, opening a second electromagnetic valve according to a preset self-checking rule, keeping other electromagnetic valves closed, and detecting whether the cleaning liquid in the cleaning liquid bag flows downwards along a second pipeline;
S2, when the fact that the cleaning fluid in the cleaning fluid bag cannot flow down along the second pipeline is determined, the air tightness of the first pipeline, the second pipeline and the third pipeline is determined to meet the requirements;
S3, starting the centrifugal machine, and pumping a certain volume of cleaning liquid into the centrifugal cup through the cleaning liquid bag to serve as detection liquid;
S4, stopping the operation of the centrifugal machine, opening the third electromagnetic valve, closing the second electromagnetic valve, starting the double-pass pump to rotate according to the first direction, pumping the detection liquid in the centrifugal cup to the seventh electromagnetic valve, the sixth electromagnetic valve, the fifth electromagnetic valve and the fourth electromagnetic valve for a specified period of time, stopping the operation of the double-pass pump, maintaining the high-pressure state of the pipeline, and detecting whether the detection liquid overflows or not;
And S5, when the seventh electromagnetic valve, the sixth electromagnetic valve, the fifth electromagnetic valve and the fourth electromagnetic valve are determined to have no overflow of the detection liquid, determining that the air tightness of the seventh pipeline, the sixth pipeline, the fifth pipeline and the fourth pipeline meets the requirement.
Based on the above embodiment, the air tightness detection result of the related pipeline can be accurately and conveniently finished by using the existing structure of the cell separation and harvesting system without additionally introducing other special detection equipment.
In the implementation, according to the air tightness detection result, when the air tightness of at least one pipeline of the cell separation and harvest system is determined to be unsatisfactory, a new cell separation and harvest system can be reconnected and constructed according to the mode.
In specific implementation, the corresponding electromagnetic valves can be controlled to continuously perform at least two times of opening and closing operations according to preset self-checking rules, so as to detect whether the electromagnetic valves are normal and effective.
In some embodiments, referring to fig. 8, the calibrating the two-way pump by controlling the second electromagnetic valve, the seventh electromagnetic valve, the fourth electromagnetic valve and the two-way pump according to the preset calibration rule to determine the rubber pipe coefficient of the two-way pump may include the following steps:
s1, opening a second electromagnetic valve and a seventh electromagnetic valve according to a preset calibration rule, and starting a two-way pump and a centrifugal machine;
S2, controlling the two-way pump to rotate in a first direction, pumping a second volume of cleaning liquid into the centrifugal cup along a second pipeline to serve as a calibration liquid, and stopping the operation of the centrifugal machine, wherein the second volume is larger than the volume of the quantitative pipe;
S3, controlling the two-way pump to rotate in a first direction, transmitting the calibration liquid in the centrifugal cup to the direction of the waste liquid bag along a seventh pipeline, and monitoring whether the calibration liquid reaches a seventh electromagnetic valve or not;
S4, when the calibration liquid is monitored to reach the seventh electromagnetic valve, closing the seventh electromagnetic valve and opening the fourth electromagnetic valve;
s5, controlling the two-way pump to rotate in a first direction, transmitting the calibration liquid in the centrifugal cup to the quantitative pipe along a fourth pipeline, and monitoring the detection value of the third bubble sensor;
S6, stopping the operation of the double-pass pump when the detection value of the third bubble sensor is detected to indicate that liquid is detected, and obtaining the rotation number of the double-pass pump;
and S7, determining the rubber tube coefficient of the bi-pass pump according to the volume of the metering tube and the rotation number of the bi-pass pump.
The first direction may specifically be a direction in which the first channel flows in and the second channel flows out.
Based on the above embodiment, the calibration operation for the two-way pump can be conveniently and accurately realized according to the preset calibration rule without additionally introducing and using professional detection equipment.
In specific implementation, as shown in fig. 9, the bi-pass pump may be controlled to rotate in the first direction, and the centrifuge is started to drive the centrifugal cup to rotate, so as to pump the cleaning solution into the centrifugal cup. When the cleaning liquid is pumped into the centrifugal cup, the cleaning liquid clings to the wall of the centrifugal cup under the action of centrifugal force, so that the centrifugal cup can be cleaned in the calibration process.
When the accumulated cleaning fluid pumped into the centrifuge cup is greater than or equal to a second volume (e.g., V1), the centrifuge operation may be stopped while the second solenoid valve is closed. At this time, the cleaning liquid still adhering to the cup wall naturally falls to the bottom of the cup as the calibration liquid. Wherein the second volume is at least greater than the volume of the dosing tube (e.g., V).
And when the fact that the calibration liquid reaches the seventh electromagnetic valve is monitored, the seventh electromagnetic valve is closed, prefilling operation of related pipelines is achieved, so that the pipelines among the seventh electromagnetic valve, the sixth electromagnetic valve, the fifth electromagnetic valve, the seventh pipeline, the sixth pipeline, the fifth pipeline and the fourth pipeline are filled with the calibration liquid, and errors of the pipelines on subsequent calibration are avoided. The node S may be used as a calibration start point in the calibration operation.
The number of turns of the bi-pass pump may then be initialized, e.g., the current number of turns of the bi-pass pump may be recorded as the initial number of turns. The method comprises the steps of starting a fourth electromagnetic valve, controlling the double-pass pump to rotate in a first direction to transmit calibration liquid to the quantitative tube along a fourth pipeline, judging that the quantitative tube is full of the calibration liquid when the detection value of a third bubble sensor indicates that the liquid is detected, stopping the double-pass pump at the moment, reading the current rotation number of the double-pass pump, and calculating the difference between the current rotation number of the double-pass pump and the initial rotation number of the double-pass pump to obtain the rotation number (for example, N) of the double-pass pump.
Further, according to the volume (e.g., V) of the metering tube, the volume offset (e.g., deltaV) of the pipeline and the rotation number of the double-pass pump, the volume of the fluid actually transported by one rotation of the double-pass pump can be calculated and obtained as the rubber tube coefficient of the double-pass pump, and the calibration operation of the double-pass pump is realized.
Specifically, the hose coefficient of the two-way pump, P= (V+ [ delta ] V)/N, can be calculated according to the following equation.
The above-mentioned displacement amount of the pipe volume may specifically include the volume of the pipe between the node S and the dosing pipe, and the volume of the pipe between the dosing pipe and the third bubble sensor. The value of V is known.
Specifically, the above-mentioned line volume offset may be calculated according to the following equation:。
Wherein R represents the pipe diameter, and L represents the sum of the lengths of the pipe between the node S and the dosing pipe, and the pipe between the dosing pipe and the third bubble sensor.
Considering that the value of L is relatively small and the amplitude of the variation is relatively small. Before the implementation, the pipeline between the node S and the quantitative pipe and the length of the pipeline between the quantitative pipe and the third bubble sensor can be determined through multiple tests, the length of the pipeline between the quantitative pipe and the third bubble sensor is used as a reference length, the reference length can be used for calculating the volume offset of the pipeline, and then the rubber pipe coefficient of the two-way pump is calculated according to the volume of the quantitative pipe and the volume offset of the pipeline.
And then the quantitative pumping and quantitative discharging of the related fluid relative to the centrifugal cup can be accurately realized by controlling the rotation number of the double-pass pump based on the rubber tube coefficient of the double-pass pump.
In some embodiments, referring to fig. 10, according to the preset separation and concentration rule, based on the rubber tube coefficient of the two-way pump, the first electromagnetic valve and the two-way pump are controlled to pump the first volume of cell stock solution from the cell stock solution bag into the centrifugal cup in a continuous flow mode, and meanwhile, the centrifugal machine, the two-way pump and the seventh electromagnetic valve are controlled to separate the cell stock solution in the centrifugal cup into cells and supernatant, and the supernatant is conveyed from the centrifugal cup to the waste liquid bag in a continuous flow mode, so that the following steps can be included in the implementation:
S1, opening a first electromagnetic valve and a seventh electromagnetic valve according to a preset separation and concentration rule, and starting a two-way pump and a centrifugal machine;
s2, controlling a two-way pump to rotate according to a first direction based on a first rotation speed, and inputting the cell stock solution in the cell stock solution bag into a centrifugal cup through an upper opening of the centrifugal cup along a first pipeline in a continuous flow mode;
s3, controlling the centrifugal machine to drive the centrifugal cup to rotate based on the first rotation speed so that cells of the cell stock solution entering the centrifugal cup enter an adherence state under the action of centrifugal force to separate the cell stock solution into cells and supernatant;
and S4, when the volume of the cell stock solution input into the centrifugal cup reaches the first volume based on the rubber pipe coefficient of the double-pass pump, stopping the centrifugal machine, and closing the first electromagnetic valve and the seventh electromagnetic valve.
Based on the embodiment, the method can control to quantitatively pump the cell stock solution with the first volume into the centrifugal cup in a stable continuous flow mode according to a preset separation and concentration rule to realize liquid feeding operation, meanwhile, control the centrifugal machine to drive the centrifugal cup to rotate so that cells in the cell stock solution enter an adherence state to be separated from the supernatant to realize separation operation, and control the separated supernatant to be discharged out of the centrifugal cup in a stable continuous flow mode to realize concentration operation.
In particular, when the two-way pump is controlled to rotate in a first direction based on a first rotation speed, the flow rate of the cell stock solution in the pipeline is made to be smaller than or equal to an upper limit value of the flow rate, and is made to be larger than or equal to a lower limit value of the flow rate.
Specifically, when the rotational speed of the two-way pump is too high, the flow rate of the cell stock solution entering the centrifugal cup is too high, and the flow rate is larger than the upper limit value of the flow rate, cells of the cell stock solution entering the centrifugal cup cannot enter an adherence state for enough time under the action of centrifugal force, but can directly rush out of the centrifugal cup, so that cell loss is caused.
When the rotation speed of the two-way pump is too low and the flow speed of the cell stock solution in the pipeline is smaller than the upper limit value of the flow speed, the cell stock solution can be led to enter the centrifugal cup in the refrigeration centrifugal cabin finally after being conveyed for a relatively long time in the pipeline outside the centrifugal cup. And the pipelines outside the centrifugal cup are not subjected to refrigeration treatment, so that when the cells stay in the pipelines for a long time, the cell activity is influenced, even the cells are inactivated, and the cell loss is caused most probably due to the overhigh temperature.
Therefore, by controlling the two-way pump to rotate based on the first rotational speed, the flow rate of the cell stock solution in the pipeline can be effectively ensured to be less than or equal to the upper limit value of the flow rate and to be greater than or equal to the lower limit value of the flow rate. Therefore, on one hand, the cell stock solution outside the centrifugal cup can enter the centrifugal cup in a short time to avoid inactivation caused by temperature influence, and on the other hand, the flow rate of the cell stock solution just entering the centrifugal cup is not too high to avoid cell loss caused by direct flushing of the cell stock solution out of the centrifugal cup.
The upper limit value of the flow rate and the lower limit value of the flow rate can be obtained by carrying out a plurality of test tests by using a cell separation and harvesting system according to test data statistics.
When the cell stock is pumped into the centrifuge cup in a stable continuous flow, as shown in FIG. 4, the first channel is connected to the upper port provided at a position above the centrifuge cup. Thus, the inlet location for the cell stock solution as it enters the centrifuge cup is relatively close to the wall of the cup. When the cell stock solution enters the centrifugal cup, the centrifugal machine drives the centrifugal cup to rotate according to the first rotation speed, so that cells in the cell stock solution are very easy to cling to the wall of the cup under the action of centrifugal force and enter an adherence state, separation of cells in the cell stock solution and supernatant is realized, separation operation is realized, namely, the cells are adhered to the wall of the cup, and the supernatant flows to the bottom of the cup along the wall of the cup. Further, the supernatant is discharged from the centrifugal cup in a stable continuous flow through a lower port provided at a position below the centrifugal cup, thereby achieving a concentration operation.
When the centrifugal machine is controlled to drive the centrifugal cup to rotate based on the first rotation speed, the rotation speed of the centrifugal cup is smaller than or equal to the upper limit value of the rotation speed and larger than or equal to the lower limit value of the rotation speed.
Specifically, when the rotation speed of the centrifuge is too fast and is larger than the upper limit value of the rotation speed, the centrifugal force applied to the cells in the adherent state is too large, so that the cells are easy to be subjected to larger extrusion, the cell activity is influenced, and even the cells are inactivated.
When the rotation speed of the centrifugal machine is too slow and is smaller than the lower limit value of the rotation speed, the centrifugal force applied to the cells is too small, so that the friction force between the cells and the cup wall is smaller than the gravity of the cells, the cells cannot stably cling to the cup wall and easily slide down to the bottom of the cup, and the cells are discharged out of the centrifugal cup along with the supernatant through the lower port to be flushed into a waste liquid bag, so that the cells are lost.
Therefore, by controlling the centrifuge to rotate based on the first rotation speed, it is possible to effectively ensure that the rotation speed of the cells in the centrifuge cup is equal to or less than the upper limit value of the rotation speed and equal to or greater than the lower limit value of the rotation speed. Therefore, on one hand, the cell can be tightly attached to the cup wall, the cell is prevented from sliding down the cup bottom and being discharged together with the supernatant, the loss of the cell is reduced, and on the other hand, the cell is prevented from being damaged or even inactivated due to excessive extrusion acting force on the cell in the rotating process.
In specific implementation, the first volume is divided by the rubber tube coefficient of the double-pass pump to obtain the first rotation number of the double-pass pump, and then whether the volume of the cell stock solution input into the centrifugal cup reaches the first volume is judged by monitoring whether the accumulated rotation number of the double-pass pump reaches the first rotation number.
And stopping the operation of the double-pass pump when the volume of the cell stock solution input into the centrifugal cup reaches the first volume, stopping the operation of the centrifugal machine, and closing the first electromagnetic valve and the seventh electromagnetic valve. At this time, although the centrifugal cup has stopped rotating, cells in an adherent state for a long time tend not to automatically slide off the cup wall due to stopping centrifugation, but to continue adhering to the cup wall because of the relatively light cell mass. Subsequently, a resuspension operation is performed by using the suspension to smoothly mix and dissolve the cells adhered to the wall of the cup.
In some embodiments, the method may further comprise stopping the two-way pump when the detection value of the first bubble sensor indicates that gas is detected, stopping the centrifuge, and closing the first solenoid valve and the seventh solenoid valve.
In some cases, it is sometimes desirable to pump the entire cell stock bag entirely into the centrifuge cup. At this time, the first bubble sensor may be utilized, and when it is detected that the type of fluid flowing through the first bubble sensor is changed from liquid to gas, it may be judged that all of the cell stock in the cell stock bag has been currently pumped into the centrifugal cup. And then can confirm that the volume of the cell stock solution that is input to the centrifugal cup reaches first volume, can stop the bi-pass pump operation.
In some embodiments, the rotational speed of the centrifuge may also be dynamically adjusted as the centrifuge is specifically controlled to rotate during the cell separation and concentration stage.
In the implementation, the accumulated thickness of cells in an adherent state in the centrifugal cup can be monitored through a microscopic camera arranged at the cup wall of the centrifugal cup, and when the accumulated thickness of the cells in the adherent state is monitored to be larger than a preset thickness threshold value, the first rotation speed of the centrifugal machine is adjusted, wherein the adjusted rotation speed is smaller than the first rotation speed.
This is because, as the cell separation and concentration stage continues, the cells newly separated in the adherent state will be covered on the cells before, resulting in more and more cells in the adherent state, and the accumulated thickness of the cells in the adherent state will be thicker.
When the accumulated thickness of the cells in the adherent state is larger than a preset thickness threshold, the distance between the newly-entered cells separated in the adherent state and the rotating shaft is relatively short, and at the moment, the newly-entered cells can be adhered by the cells before being adhered due to the small rotating speed. Meanwhile, the former cells can be stably in an adherent state even if the centrifugal force is reduced due to the extrusion action of the newly entered cells, and cannot fall down due to the gravity action. Meanwhile, the rotation speed is reduced, so that the centrifugal force suffered by the cells closest to the cup wall and the extrusion acting force from other cells are reduced to a certain extent, and the damage to extrusion and even the inactivation of the cells caused by the overlarge comprehensive acting force suffered by the cells closest to the cup wall can be avoided.
In some embodiments, the cell separation and harvest system can be constructed by estimating the order of magnitude of the separated cells according to the first volume and the concentration of the cells in the cell stock solution, and selecting a centrifugal cup with the matched cup wall surface area from a plurality of centrifugal cups according to the order of magnitude of the cells.
Specifically, when the data magnitude of the estimated separated cells is large, a centrifugal cup with relatively large surface area of the cup wall can be selected as a matched centrifugal cup. Therefore, the separated cells are attached to the cup wall as dispersedly as possible in the centrifugal concentration stage, and the phenomenon that the cells closest to the cup wall are damaged and inactivated due to overlarge extrusion caused by the superposition of multiple layers of cells is avoided.
In some embodiments, referring to fig. 11, according to the preset cleaning rule, the cleaning solution is pumped into the centrifugal cup in a continuous flow mode by controlling the second electromagnetic valve and the two-way pump to clean the supernatant on the cells, and meanwhile, the cleaned cleaning solution is conveyed from the centrifugal cup to the waste liquid bag in a continuous flow mode by controlling the two-way pump and the seventh electromagnetic valve, and when the method is implemented, the method can comprise the following steps:
s1, opening a second electromagnetic valve and a seventh electromagnetic valve according to a preset cleaning rule, and starting a two-way pump and a centrifugal machine;
s2, controlling the two-way pump to rotate in a first direction based on a second rotating speed, and inputting the cleaning liquid in the cleaning liquid bag into the centrifugal cup through the upper opening of the centrifugal cup along a second pipeline in a continuous flow mode;
S3, controlling the centrifugal machine to drive the centrifugal cup to rotate based on the first rotation speed so as to enable the cleaning liquid entering the centrifugal cup to wash cells in an adherent state, outputting the cleaned cleaning liquid from the centrifugal cup in a continuous flow mode through a lower opening of the centrifugal cup, and inputting the cleaning liquid into a waste liquid bag along a seventh pipeline;
s4, when the cleaning end condition is determined to be met, closing the second electromagnetic valve and the seventh electromagnetic valve, and opening the first electromagnetic valve and the fourth electromagnetic valve;
And S5, controlling the two-way pump to rotate in a second direction based on a third rotating speed, and inputting the residual cleaning liquid in the centrifugal cup into the cell stock solution bag along the first pipeline through the upper opening of the centrifugal cup.
The second direction may specifically be a direction in which the second channel flows in and the first channel flows out.
The second rotating speed can be larger than the first rotating speed, so that the cells in the centrifugal cup can be washed by using the washing liquid flow with relatively high flow speed and high pressure, and a good washing effect can be obtained. The third rotational speed may be less than the second rotational speed.
The above-described satisfaction of the cleaning end condition may specifically be that the volume of the cleaning liquid pumped into the centrifugal cup reaches a specified volume. In the specific implementation, whether the volume of the cleaning liquid pumped into the centrifugal cup reaches the specified volume can be judged by monitoring the rotation number of the double-pass pump based on the rubber pipe coefficient of the double-pass pump.
When the centrifugal cup is in specific implementation, the first electromagnetic valve can be replaced by opening the second electromagnetic valve, so that the two-way pump can be controlled to rotate in a second direction based on the third rotating speed, and the residual cleaning fluid in the centrifugal cup is input into the cleaning fluid bag along the second pipeline through the upper opening of the centrifugal cup.
Therefore, the cleaning liquid in the centrifugal cup can be discharged as much as possible, and meanwhile, the cleaned cleaning liquid can be stored by using the cell stock solution bag or the cleaning liquid bag which is not used later in the system.
In the specific implementation, in the initial stage of cleaning (the duration is relatively longer), when the centrifugal machine drives the centrifugal cup to rotate violently at a higher rotating speed, cleaning liquid is pumped into the centrifugal cup through the upper opening of the centrifugal cup and then the cleaned cleaning liquid is discharged through the lower opening of the centrifugal cup, so that cells can be effectively prevented from being discharged out of the centrifugal cup along with the cleaned cleaning liquid in the cleaning process, cell loss is reduced, in the later stage of cleaning (the duration is relatively shorter), the centrifugal machine continues to drive the centrifugal cup to rotate, the cells are still in an adherent state, and the cleaned cleaning liquid is pushed to be discharged out of the centrifugal cup through the upper opening of the centrifugal cup by utilizing air pumped into the lower opening of the centrifugal cup through the air filtering valve, so that the residual cleaning liquid in the centrifugal cup can be discharged as much as possible.
Based on the above embodiment, according to a preset cleaning rule, the pumping of the cleaning liquid into the centrifugal cup in a stable continuous flow manner can be controlled, so that cells in the centrifugal cup can be effectively cleaned, residual supernatant attached to the cells is washed away, the cleaned cleaning liquid is effectively discharged, the cleaning operation is well realized, and a good cleaning effect is obtained.
In particular, the specified volume of cleaning solution can be pumped into the centrifugal cup for a plurality of times so as to carry out multi-round cleaning on the cells in the centrifugal cup.
When each round of cleaning is carried out, the centrifugal machine can be started in the mode, the centrifugal machine is controlled to drive the centrifugal cup to rotate based on the first rotation speed, meanwhile, a specified volume of cleaning liquid is pumped into the centrifugal cup to flush cells in an adherence state, and when the volume of the pumped cleaning liquid reaches the specified volume, the corresponding electromagnetic valve can be closed, and meanwhile, the operation of the centrifugal machine is stopped. However, the cells are lighter and can not automatically slide down to the cup body due to gravity under the action of adhesion force. At this time, the centrifugal machine can be controlled to drive the centrifugal cup to rotate in turn along the positive and negative directions, so that the cleaning liquid in the centrifugal cup is rippled up to dissolve cells in an adherence state into the cleaning liquid, and then each surface of the cells can be fully contacted with the cleaning liquid (namely, uniform mixing is realized), further cleaning is carried out, and a relatively better cleaning effect is obtained, so that one round of cleaning is completed.
After one round of cleaning is completed, before the next round of cleaning is performed, the centrifugal machine can be started, the centrifugal machine is controlled to drive the centrifugal cup to rotate at a higher rotating speed (for example, a first rotating speed) so that cells mixed in the cup cleaning liquid are in an adherence state again, the corresponding electromagnetic valve is opened, and the two-way pump is controlled to pump the cleaning liquid into the centrifugal cup so as to form and utilize a stable continuous flow to perform the next round of cleaning on the cells in the adherence state.
When the last round of cleaning is carried out, and the cleaning end condition is met, the two-way pump can be controlled to rotate in the second direction based on the third rotating speed, and the residual cleaning liquid in the centrifugal cup is input into the cell stock solution bag along the first pipeline through the upper opening of the centrifugal cup.
So as to drain the residual cleaning liquid in the centrifugal cup as much as possible, thereby completing the cleaning operation.
In some embodiments, referring to fig. 12, according to the above-mentioned preset resuspension rule, based on the rubber tube coefficient of the bi-pass pump, by controlling the sixth electromagnetic valve, the bi-pass pump, and the third electromagnetic valve, the method for performing resuspension treatment on the cleaned cells in the centrifugal cup may include the following steps:
s1, opening a third electromagnetic valve and a sixth electromagnetic valve according to a preset resuspension rule, and starting a two-way pump;
S2, controlling the two-way pump to rotate in a second direction based on a fourth rotating speed, and inputting heavy suspension in the heavy suspension bag into the centrifugal cup along a sixth pipeline through a lower opening of the centrifugal cup;
s3, stopping the operation of the double-pass pump and starting the centrifugal machine when the volume of the heavy suspension input into the centrifugal cup is monitored to reach a third volume based on the rubber pipe coefficient of the double-pass pump;
and S4, controlling the centrifugal machine to rotate based on the second rotation speed so as to enable the cells in the attached state to be dissolved in the heavy suspension, and obtaining target liquid containing the cells.
Wherein the second rotation speed is smaller than the first rotation speed.
In the specific implementation, based on the second rotation speed, cells in the centrifugal cup cannot enter an adherence state under the action of centrifugal force, but the centrifugal cup can be rotated and rocked, so that the cells and the heavy suspension are fully mixed, and a target liquid with higher quality is obtained.
In the specific implementation, the centrifugal machine can be controlled to rotate in turn in the first direction and the second direction based on the second rotation speed, so that cells in an adherence state can be separated from the wall of the cup more quickly and dissolved into the heavy suspension.
In the implementation process, whether the volume of the heavy suspension input into the centrifugal cup reaches the third volume can be judged by monitoring the rotation number of the two-way pump based on the rubber pipe coefficient of the two-way pump.
Based on the above embodiment, according to a preset resuspension rule, the pumping of the corresponding resuspension to the centrifugal cup can be controlled to resuspension and uniformly mix the cells in the centrifugal cup, so as to realize the resuspension operation and obtain the target liquid with higher quality.
In some embodiments, according to the preset dispensing rule, based on the rubber tube coefficient of the bi-pass pump, the method for controlling the fifth electromagnetic valve, the bi-pass pump and the third electromagnetic valve to convey the target liquid containing the cells in the centrifugal cup to the dispensing bag may include the following steps when the method is implemented:
S1, opening a third electromagnetic valve and a fifth electromagnetic valve according to a preset split charging rule, and starting a two-way pump and a centrifugal machine;
s2, controlling the centrifugal machine to rotate based on the third rotation speed;
and S3, controlling the bi-pass pump to rotate in a first direction based on a fifth rotating speed based on the rubber pipe coefficient of the bi-pass pump, and inputting the target liquid with the designated volume into the split packaging bags along a fifth pipeline through the lower opening of the centrifugal cup.
Wherein the third rotational speed is less than the first rotational speed.
During implementation, based on the third rotation speed, cells in the centrifugal cup cannot enter an adherence state, but the centrifugal cup can be rotated and rocked, so that target liquid is uniformly mixed and then sent into the separate bags, and a cell product with relatively better effect is obtained.
When the method is implemented, the specified volume can be divided by the rubber tube coefficient of the double-pass pump to obtain the corresponding specified rotation number of the double-pass pump, and whether the target liquid with the specified volume is input into the sub-packaging bags is judged by monitoring whether the accumulated rotation number of the double-pass pump reaches the specified rotation number.
Based on the above embodiment, according to the preset dispensing rule, the target liquid can be controlled to be discharged from the centrifugal cup and conveyed into the dispensing bag, so that the dispensing operation is realized, and the required cell product is efficiently and automatically collected.
In some embodiments, in the process of performing cell separation and split charging by using the cell separation and harvesting system in the above manner, the refrigerating module may be dynamically adjusted, and in a specific implementation, the method may include starting the refrigerating module in advance to perform precooling treatment on the centrifugal cup in the refrigeration centrifugal cabin before the cell stock solution is pumped into the centrifugal cup, and dynamically adjusting the refrigerating power of the refrigerating module after the cell stock solution starts to enter the centrifugal cup, so as to maintain the temperature of the centrifugal cup in the refrigeration centrifugal cabin within a threshold temperature range.
Specifically, in consideration of the fact that in the cell separation and concentration stage, the centrifugal machine can rotate relatively severely, so that the temperature rise in the refrigeration centrifugal cabin is relatively large, at this time, the refrigeration module can be dynamically adjusted according to the matched first power adjustment rule, and therefore the temperature of the centrifugal cup in the refrigeration centrifugal cabin can be well maintained within the threshold temperature range.
The cooling module can be dynamically adjusted continuously according to the matched first power adjustment rule at the initial stage of the cell cleaning stage when the centrifugal machine is rotated violently, or can be replaced and dynamically adjusted according to the matched second power adjustment rule at the later stage of the cell cleaning stage when the centrifugal machine is not rotated violently as before or at the sub-packaging stage, so as to better maintain the temperature of the centrifugal cup in the cooling centrifugal cabin within the threshold temperature range.
Wherein, the first power adjustment rule is described above. The second power adjustment rule can be specifically obtained by performing a large number of experimental tests in advance, sorting out a large number of adjustment records of the refrigeration modules according to the experimental test results, and performing clustering processing on the adjustment records of the large number of refrigeration modules.
In addition, in the whole cell separation and split charging process, the refrigeration module can be adjusted independently in a targeted manner according to the temperature acquired by the temperature sensor.
From the above, according to the cell separation and harvesting method provided by the embodiment of the present disclosure, a stable continuous flow can be formed by using the characteristics of the bi-pass pump, and then the relevant fluid is transported by matching with the centrifuge and the refrigeration centrifugal cabin in a stable continuous flow manner, so that the loss and damage of cells in the operation and treatment process can be effectively reduced, the inactivation of cells is avoided, and the treatments of separation, concentration, cleaning, resuspension, split charging, etc. of cells in the cell stock solution are automatically and efficiently completed, thereby obtaining a cell product with higher quality.
The embodiment of the present disclosure provides an electronic device, and is shown in fig. 13. The electronic device includes a network communication port 1301, a processor 1302, and a memory 1303, where the above structures are connected by an internal cable, so that each structure may perform specific data interaction.
The network communication port 1301 may be specifically configured to receive a trigger instruction.
The processor 1302 may be specifically configured to respond to the trigger instruction, and calibrate the two-way pump by controlling the second electromagnetic valve, the seventh electromagnetic valve, the fourth electromagnetic valve, and the two-way pump according to a preset calibration rule, so as to determine a rubber pipe coefficient of the two-way pump;
The rubber pipe coefficient is used for representing the volume of fluid transported by the two-way pump in one circle of rotation; the method comprises the steps of pumping a first volume of cell stock solution from a cell stock solution bag into a centrifugal cup in a continuous flow mode by controlling a first electromagnetic valve and a double-pass pump according to a rubber tube coefficient of the double-pass pump, separating the cell stock solution in the centrifugal cup into cells and supernatant by controlling a centrifugal machine, the double-pass pump and a seventh electromagnetic valve, conveying the supernatant from the centrifugal cup to a waste liquid bag in a continuous flow mode, pumping cleaning liquid into the centrifugal cup in a continuous flow mode by controlling a second electromagnetic valve and the double-pass pump according to a preset cleaning rule, cleaning the supernatant on the cells, simultaneously conveying the cleaned cleaning liquid from the centrifugal cup to the waste liquid bag in a continuous flow mode by controlling the double-pass pump and the seventh electromagnetic valve, carrying out resuspension treatment on the cleaned cells in the centrifugal cup by controlling a sixth electromagnetic valve, the double-pass pump and the third electromagnetic valve according to a preset resuspension rule, and subpackaging the cleaned cells in the centrifugal cup into the target cell bag by controlling a fifth electromagnetic valve, the double-pass pump and the third electromagnetic valve according to a preset subpackaging rule.
The memory 1303 may be specifically configured to store a corresponding instruction program and related data such as a hose coefficient.
Based on the method, the related structural performance of the electronic equipment can be effectively utilized, the data processing speed of the electronic equipment is improved, and the data processing about cell separation and split charging is effectively realized.
In this embodiment, the network communication port 1301 may be a virtual port that binds with different communication protocols, so that different data may be sent or received. For example, the network communication port may be a port responsible for performing web data communication, a port responsible for performing FTP data communication, or a port responsible for performing mail data communication. The network communication port may also be an entity's communication interface or a communication chip. For example, it may be a wireless mobile network communication chip such as GSM, CDMA, etc., it may also be a Wifi chip, it may also be a bluetooth chip.
In this embodiment, the processor 1302 may be implemented in any suitable manner. For example, a processor may take the form of, for example, a microprocessor or processor, and a computer-readable medium storing computer-readable program code (e.g., software or firmware) executable by the (micro) processor, logic gates, switches, application SPECIFIC INTEGRATED Circuits (ASICs), programmable logic controllers, and embedded microcontrollers, among others. The description is not intended to be limiting.
In this embodiment, the memory 1303 may include multiple levels, and in a digital system, the memory may be any memory as long as binary data can be stored, in an integrated circuit, a circuit with a memory function without a physical form, such as a RAM, a FIFO, etc., and in a system, a memory device with a physical form, such as a memory bank, a TF card, etc.
The embodiment of the specification also provides a computer readable storage medium based on the cell separation and harvesting method, wherein the computer readable storage medium stores computer program instructions, when the computer program instructions are executed, the computer readable storage medium is realized by controlling a second electromagnetic valve, a seventh electromagnetic valve, a fourth electromagnetic valve and a double-pass pump according to preset calibration rules to calibrate the double-pass pump so as to determine the rubber tube coefficient of the double-pass pump, the rubber tube coefficient is used for representing the volume of fluid transported by the double-pass pump in a circle, the cell stock solution of the first volume is pumped into a centrifugal cup in a continuous flow mode from a cell stock solution bag according to the rubber tube coefficient of the double-pass pump by controlling the first electromagnetic valve and the double-pass pump, simultaneously, the cell stock solution in the centrifugal cup is separated into cells and supernatant in a continuous flow mode by controlling the centrifugal machine, the double-pass pump and the seventh electromagnetic valve, the supernatant is transported to the centrifugal cup in a continuous flow mode from the centrifugal cup according to preset washing rules, the second electromagnetic valve and the double-pass pump is controlled to the centrifugal cup in a continuous flow mode, the supernatant is transported to the centrifugal cup in a continuous flow mode from the vacuum cup, and the supernatant is suspended from the centrifugal cup to the centrifugal cup is transported to the centrifugal cup according to the continuous flow mode by controlling the electromagnetic valve and the double-pass pump, and the double-pass pump is suspended from the vacuum pump to the centrifugal cup to the vacuum cup according to the continuous flow mode is controlled and the continuous flow is transported to the continuous flow mode and the vacuum pump is transported to the vacuum pump is separated from the cell stock solution to the centrifugal cup to the cell stock solution is in a continuous liquid is separated into the cell stock solution.
In the present embodiment, the storage medium includes, but is not limited to, a random access Memory (Random Access Memory, RAM), a Read-Only Memory (ROM), a Cache (Cache), a hard disk (HARD DISK DRIVE, HDD), or a Memory Card (Memory Card). The memory may be used to store computer program instructions. The network communication unit may be an interface for performing network connection communication, which is set in accordance with a standard prescribed by a communication protocol.
In this embodiment, the functions and effects of the program instructions stored in the computer readable storage medium may be explained in comparison with other embodiments, and are not described herein.
The embodiment of the specification also provides a computer program product, which at least comprises a computer program, and when the computer program is executed by a processor, the method comprises the following steps of calibrating the two-way pump by controlling a second electromagnetic valve, a seventh electromagnetic valve, a fourth electromagnetic valve and a two-way pump according to a preset calibration rule to determine the hose coefficient of the two-way pump, wherein the hose coefficient is used for representing the volume of a fluid transported by the two-way pump in a circle, according to the preset separation concentration rule, based on the hose coefficient of the two-way pump, pumping the cell stock solution with the first volume from a cell stock solution bag into a centrifugal cup in a continuous flow mode, simultaneously separating the cell stock solution in the centrifugal cup into cells and supernatant in a continuous flow mode by controlling the centrifugal cup, the two-way pump and the seventh electromagnetic valve, conveying the supernatant from the centrifugal cup to a waste liquid bag in a continuous flow mode, controlling the second electromagnetic valve and the two-way pump according to the preset cleaning rule, pumping the cleaning liquid into the centrifugal cup in a continuous flow mode to the centrifugal cup in a cleaning liquid bag, simultaneously, controlling the two-way pump from the seven-way pump to the three-way pump according to the preset continuous flow, and the three-way pump, and the two-way pump according to the preset transfer coefficient of the hose, and the three-way pump according to the preset rules.
Referring to fig. 14, the embodiment of the present disclosure further provides a cell separation and harvesting device, which may specifically include the following structural modules:
The calibration module 1401 is specifically configured to calibrate the two-way pump by controlling the second electromagnetic valve, the seventh electromagnetic valve, the fourth electromagnetic valve, and the two-way pump according to a preset calibration rule, so as to determine a rubber tube coefficient of the two-way pump, where the rubber tube coefficient is used to characterize a volume of a transport fluid that the two-way pump rotates for one circle;
The separation and concentration module 1402 specifically can be used for pumping a first volume of cell stock solution from a cell stock solution bag into a centrifugal cup in a continuous flow mode by controlling a first electromagnetic valve and a double-pass pump based on a rubber tube coefficient of the double-pass pump according to a preset separation and concentration rule;
The cleaning module 1403 specifically may be configured to pump a cleaning solution into the centrifugal cup in a continuous flow manner by controlling the second electromagnetic valve and the two-way pump according to a preset cleaning rule to clean the supernatant on the cells;
the resuspension module 1404 may be specifically configured to perform resuspension treatment on the cells cleaned in the centrifugal cup by controlling the sixth electromagnetic valve, the bi-pass pump, and the third electromagnetic valve based on the coefficient of the rubber tube of the bi-pass pump according to a preset resuspension rule;
The split charging module 1405 may be specifically configured to convey the target solution containing the cells in the centrifugal cup to the split charging bag by controlling the fifth solenoid valve, the two-way pump, and the third solenoid valve based on the rubber tube coefficient of the two-way pump according to a preset split charging rule.
In some embodiments, when the calibration module 1401 is specifically implemented, the two-way pump may be calibrated by controlling the second solenoid valve, the seventh solenoid valve, the fourth solenoid valve and the two-way pump according to a preset calibration rule to determine a hose coefficient of the two-way pump, opening the second solenoid valve and the seventh solenoid valve according to the preset calibration rule, starting the two-way pump and the centrifuge, controlling the two-way pump to rotate in a first direction, pumping a second volume of cleaning solution into the centrifugal cup along the second pipeline as a calibration solution, stopping the centrifuge, wherein the second volume is larger than the volume of the quantifying pipe, controlling the two-way pump to rotate in the first direction, transmitting the calibration solution in the centrifugal cup along the seventh pipeline to the direction of the waste liquid bag, monitoring whether the calibration solution reaches the seventh solenoid valve, closing the seventh solenoid valve when the calibration solution reaches the seventh solenoid valve, controlling the two-way pump to rotate in the first direction, transmitting the calibration solution along the fourth pipeline to the quantifying pipe, monitoring a detection value of the three-bubble sensor, and acquiring the number of turns of the two-way pump according to the detected detection value of the third bubble sensor, and acquiring the number of turns of the two-way pump.
In some embodiments, the separation and concentration module 1402 may be implemented by controlling the first solenoid valve and the bi-pass pump to pump the first volume of the cell stock solution from the cell stock solution bag into the centrifuge cup in a continuous flow mode based on a hose coefficient of the bi-pass pump according to a preset separation and concentration rule, simultaneously, controlling the centrifuge, the bi-pass pump and the seventh solenoid valve to separate the cell stock solution in the centrifuge cup into cells and supernatant liquid, and conveying the supernatant liquid from the centrifuge cup to the waste liquid bag in a continuous flow mode, opening the first solenoid valve and the seventh solenoid valve according to a preset separation and concentration rule, starting the bi-pass pump and the centrifuge, controlling the bi-pass pump to rotate in a first direction based on a first rotation speed, inputting the cell stock solution in the cell stock solution bag in a continuous flow mode along a first pipeline through an upper port of the centrifuge cup, controlling the centrifuge to drive the centrifuge cup to rotate based on the first rotation speed, enabling cells of the cell stock solution entering the centrifuge cup to enter an adherence state under the action of the centrifuge cup to separate the cell stock solution into the cells and supernatant liquid, and the supernatant liquid in a continuous flow mode, stopping the bi-pass pump to operate the first pipeline, and stopping the bi-pass pump from running along the first pipeline based on the hose coefficient when the first pipeline is stopped.
In some embodiments, the separation and concentration module 1402 may be further configured to stop the two-way pump and the centrifuge when the detection value of the first bubble sensor indicates that gas is detected, and to close the first solenoid valve and the seventh solenoid valve.
In some embodiments, the above-mentioned washing module 1403 may be implemented by controlling the second electromagnetic valve and the bi-pass pump to pump the washing liquid into the centrifugal cup in a continuous flow manner to wash the supernatant on the cells according to a preset washing rule, simultaneously, controlling the bi-pass pump and the seventh electromagnetic valve to transfer the washed washing liquid from the centrifugal cup to the waste liquid bag in a continuous flow manner, opening the second electromagnetic valve and the seventh electromagnetic valve according to the preset washing rule, starting the bi-pass pump and the centrifugal machine, controlling the bi-pass pump to rotate in a first direction based on a second rotation speed, inputting the washing liquid in the washing liquid bag in a continuous flow manner into the centrifugal cup through an upper port of the centrifugal cup, controlling the centrifugal machine to rotate the centrifugal cup based on the first rotation speed to enable the washing liquid entering into the centrifugal cup to wash the cells in an adherent state, outputting the washed washing liquid from the centrifugal cup in a continuous flow manner through a lower port of the centrifugal cup, inputting the washing liquid into the waste liquid bag along the seventh pipeline, closing the second electromagnetic valve and the seventh electromagnetic valve when it is determined that the washing end condition is satisfied, opening the first electromagnetic valve and the fourth electromagnetic valve, controlling the bi-pass pump to rotate the washing liquid in a first direction along the remaining rotation direction of the centrifugal cup.
In some embodiments, when the above-mentioned resuspension module 1404 is implemented specifically, the suspension treatment can be performed on the cleaned cells in the centrifugal cup by controlling the sixth solenoid valve, the bi-pass pump and the third solenoid valve according to a preset resuspension rule and based on a rubber tube coefficient of the bi-pass pump, by opening the third solenoid valve and the sixth solenoid valve according to the preset resuspension rule and starting the bi-pass pump, by controlling the bi-pass pump to rotate in a second direction based on a fourth rotation speed, the suspension in the suspension bag is input into the centrifugal cup along the sixth pipeline through a lower opening of the centrifugal cup, when the volume of the suspension input into the centrifugal cup is detected to reach the third volume based on the rubber tube coefficient of the bi-pass pump, the operation of the bi-pass pump is stopped, and the centrifuge is started, and by controlling the centrifuge to rotate based on the second rotation speed, so that the cells in an adherent state are dissolved in the suspension to obtain the target solution containing the cells.
In some embodiments, when the above-mentioned resuspension module 1405 is implemented specifically, the target liquid containing the cells in the centrifugal cup can be transported to the sub-packaging bag by controlling the fifth electromagnetic valve, the bi-pass pump and the third electromagnetic valve according to a preset sub-packaging rule and based on the rubber tube coefficient of the bi-pass pump, the third electromagnetic valve and the fifth electromagnetic valve are opened according to the preset sub-packaging rule, the bi-pass pump and the centrifugal machine are started, the centrifugal machine is controlled to rotate based on the third rotation speed, the bi-pass pump is controlled to rotate in the first direction based on the fifth rotation speed based on the rubber tube coefficient of the bi-pass pump, and the target liquid with a specified volume is input into the sub-packaging bag along the fifth pipeline through the lower opening of the centrifugal cup.
It should be noted that, the units, devices, or modules described in the above embodiments may be implemented by a computer chip or entity, or may be implemented by a product having a certain function. For convenience of description, the above devices are described as being functionally divided into various modules, respectively. Of course, when the present description is implemented, the functions of each module may be implemented in the same piece or pieces of software and/or hardware, or a module that implements the same function may be implemented by a plurality of sub-modules or a combination of sub-units, or the like. The above-described apparatus embodiments are merely illustrative, for example, the division of the units is merely a logical function division, and there may be additional divisions when actually implemented, for example, multiple units or components may be combined or integrated into another system, or some features may be omitted or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed with each other may be an indirect coupling or communication connection via some interfaces, devices or units, which may be in electrical, mechanical or other form.
From the above, based on the cell separation and harvesting device provided in the embodiments of the present disclosure, a stable continuous flow can be formed by using the characteristics of the bi-pass pump, and then the continuous flow is used to cooperate with the centrifuge and the refrigeration centrifugal cabin to transport related fluids, so that the loss and damage of cells in the operation and treatment process can be effectively reduced, the inactivation of cells is avoided, and the treatments of separating, concentrating, cleaning, resuspension, split charging and the like of cells in the cell stock solution are automatically and efficiently completed, thereby obtaining a cell product with higher quality.
In one specific example of a scenario, the cell separation harvesting system and method provided herein may be applied to achieve fully automated cell separation harvesting. The specific implementation process can be referred to as follows.
In the example of the scene, considering that the cell separation and harvesting process based on the conventional system equipment is complex, the product homogenization requirement is high, and along with the development of industry, the cell culture and harvesting requirements of the general large liquid volume are more and more urgent, if the cell is harvested by manpower only, the efficiency is low while the pollution risk of the cell is increased, and the growing cell culture requirements of the cell therapy industry cannot be completely met.
Further, considering that the automatic cell harvesting equipment in the market at present can replace manual work to realize automation of cell separation and harvesting, the method still has the following problems that (1) the method can only aim at cell culture and harvesting of small liquid amount of an individual, cannot meet the characteristics of high flux, high efficiency and the like required by general cell culture, (2) the method generally does not have a refrigeration function, the activity of cells can be influenced when the cells are exposed under high temperature conditions for a long time, (3) the method basically utilizes negative pressure formed in a centrifugal cup to suck cell stock solution, so that continuous flow is formed, continuous flow formed by the method is unstable, continuous flow interruption is easily caused due to liquid amount change or pressure change, and the cells are influenced by great surface tension in the centrifugal cup for a long time, and the pipeline design and the process flow are complex, although the automatic cell harvesting can be basically realized, the process of preliminary preparation work, consumable installation and the like is excessively complex, the requirement on operators is higher, and the promotion and application of automatic cell harvesting technology are not facilitated.
In order to solve the problems of the prior system equipment, a set of full-automatic cell separation and harvesting system is provided in the scene example, which is equipment integrating the full-closed, automatic and continuous flow centrifugal cleaning technology, supports high-flux cell separation and harvesting by matching centrifugal cups and peristaltic pump rubber pipes with different sizes, can realize rapid refrigeration by introducing a centrifugal cabin (such as a refrigeration centrifugal cabin), and can ensure uniform flow rate of inlet and outlet liquid by introducing and utilizing a double-channel peristaltic pump (or called a double-pass pump), so that stable and efficient continuous flow can be formed under the condition of not changing the pressure in the centrifugal cup, further, the cell harvesting task can be completed with extremely low cell loss rate under the condition of constant temperature and constant pressure, and operations such as high-speed centrifugation, concentration, cleaning, resuspension and split charging of cells can be realized fully automatically under the closed environment, and the condition of reducing human intervention can also better ensure the homogeneity and standardization of cell products of different batches.
Specifically, referring to fig. 6 and 1, the fully automatic cell separation and harvesting system comprises a solenoid valve, a liquid bag bracket, a hook, a double-channel peristaltic pump, a bubble sensor, a centrifuge, a refrigeration centrifugal cabin, a filter valve, a metering tube and other structures. The device integrates the totally-enclosed, automatic and continuous flow centrifugal cleaning technology, can be matched with centrifugal cups and peristaltic pump rubber tubes with different sizes, and realizes high-flux cell separation and harvesting. The refrigerating centrifugal cabin can effectively protect cells, the flow rate of inlet and outlet liquid is consistent by using the double-channel peristaltic pump, stable and efficient continuous flow can be formed under the condition that the pressure in the centrifugal cup is not changed, the cell harvesting task can be completed with extremely low cell loss rate under the constant temperature and constant pressure state, the operations such as high-speed centrifugation, concentration, cleaning, resuspension and split charging of the cells can be fully automatically realized under the closed environment, and the homogenization and standardization of cell products in different batches can be well ensured under the condition of reducing human intervention.
In this example of the scenario, in order to enable a stable and efficient continuous flow, a bi-pass peristaltic pump is introduced and used, as shown in fig. 2. Specifically, the two channels of the dual peristaltic pump are used for liquid inlet and liquid outlet respectively, and when the centrifugal cup rotates at a high speed, the first channel (for example, the first channel) is used for pumping the cell stock solution into the centrifugal cup from the upper port, and the second channel (for example, the second channel) is used for pumping the supernatant out of the centrifugal cup from the lower port. The first channel and the second channel of the peristaltic pump are driven by the same motor and the same peristaltic pump roller and are matched with rubber pipes with the same diameter, so that the liquid speeds of the first channel and the second channel are consistent, the liquid flow rate pumped into the centrifugal cup and the liquid flow rate pumped out of the centrifugal cup are consistent, and the liquid flow rate can reach 500ml/min stably, so that stable and efficient continuous flow can be formed in the centrifugal cup in the whole cell treatment process, and the peristaltic pump is used for concentrating and cleaning cells.
In practice, during the injection of a cell fluid (e.g., cell stock) the bi-pass peristaltic pump will select the appropriate pump speed V0 based on the total cell sample size and the centrifuge cup size. Wherein V0 is between two critical speeds (V1 < V0< V2), V1 is the lowest speed of cell sample injection, the speed lower than V1 can lead to the lengthening of the cell sample injection period, not only affecting the activity rate and state of cells, but also reducing the efficiency of cell harvest, V2 is the highest speed of cell sample injection, the speed exceeds V2, part of cells are discharged under the inherent centrifugal force without adherence, and the cell harvest rate is reduced.
During a cell washing process (e.g., a washing operation), the bi-pass peristaltic pump will select the appropriate pump speed V3 based on the cell concentration and the set washing cycle. Wherein V3 is between two critical speeds (V4 < V3< V5), V4 is the lowest speed of cell cleaning, and a speed lower than V4 can lead to insufficient cleaning force and insufficient cleaning, can not completely clean substances such as culture medium in a centrifugal cup, and can reduce cleaning efficiency, and a speed V5 is the maximum speed of cell cleaning, and exceeds V5, so that strong fluid impact can not only take away adherent cells, but also possibly kill the cells.
It should be noted that, in the conventional sample feeding mode based on conventional system equipment, the centrifugal cup is pumped into negative pressure by the single-channel peristaltic pump, and when the negative pressure in the centrifugal cup is large enough, the negative pressure is conducted into the sample bag by the liquid in the pipeline, so that the liquid in the sample bag is sucked and enters the centrifugal cup. Experiments prove that in the implementation process of the method, at the moment when the negative pressure of the centrifugal cup reaches a critical value, the centrifugal cup and the liquid bag have a higher pressure difference, and the pressure difference can cause cell liquid in the earlier-stage sample bag to be flushed into the centrifugal cup at a very high speed, so that part of cells are discharged without being adhered to the wall, and cell loss is caused. A relatively stable continuous flow cannot be established until the fluid in the centrifuge cup increases and the pressure differential between the sample bags decreases toward equilibrium. The problem can be well avoided by introducing and using the double-way peristaltic pump, the double-way peristaltic pump does not depend on pressure difference, once the double-way peristaltic pump is started, liquid in the centrifugal cup enters and is discharged at the same speed, the liquid directly enters a stable continuous flow state, and cell loss caused by unstable pressure in the front stage of continuous flow in the traditional mode is well avoided.
In addition, the dual peristaltic pump is more advantageous in the process flow requiring precise control of the liquid inlet and outlet amounts during the re-suspension and dispensing stages, in addition to providing a stable continuous flow. During the re-suspension stage (e.g., re-suspension operation), the centrifugal cup remains stationary, the first channel is connected with the air filter valve, the second channel is connected with the re-suspension bag, the second channel of the dual-channel peristaltic pump pumps the quantitative re-suspension from the lower opening of the centrifugal cup, and the first channel pumps the air with the same volume from the upper opening of the centrifugal cup. In the split charging stage (for example, split charging operation), the centrifugal cup is kept static, the rotation direction of the double-pass peristaltic pump is opposite to that of heavy suspension, the first passage is connected with the air filtering valve, the second passage is connected with the split charging bag, the second passage of the double-pass peristaltic pump pumps quantitative cell liquid out of the lower outlet of the centrifugal cup, the first passage pumps air with the same volume from the upper outlet of the centrifugal cup, the cell liquid is replaced by the air under the condition that the pressure in the centrifugal cup is kept constant, and the cell liquid is pumped into the split charging bag, so that the whole process is stable and accurate, and the high-precision requirement of split charging is effectively ensured.
In addition, in some special cases, besides the two-channel constant-speed application, the two-way peristaltic pump can also respectively install rubber tubes with different sizes on the two channels, so that a fixed flow speed difference is formed, and the flow speed difference can continuously pressurize or depressurize the centrifugal cup for certain cell preparation treatment processes under specific pressure.
In particular, referring to fig. 9, a system based on the above-described structure may perform high-precision calibration (e.g., calibration operation) of a dual-pass peristaltic pump by a metering tube. The method comprises the steps of starting a centrifugal machine, keeping a set rotating speed, opening a No. 2 electromagnetic valve and a No. 7 electromagnetic valve, pumping a cleaning liquid with a set volume V1 into a centrifugal cup by a double-way peristaltic pump (V1 is larger than the volume V of a quantitative tube), tightly attaching the cleaning liquid with the V1 to the wall of the centrifugal cup under the action of centrifugal force so as to be remained in the cup and used for making a calibration liquid of the double-way peristaltic pump, stopping rotating the centrifugal machine, standing the centrifugal machine at the bottom of the cup when the liquid in the cup is in a centrifugal state, opening the No. 7 electromagnetic valve, pumping a set dosage liquid into a waste liquid bag by the double-way peristaltic pump according to the set number of turns, closing the No. 7 electromagnetic valve, opening the No. 4 electromagnetic valve, starting from the calibration starting point S, pumping the calibration liquid into the quantitative tube until the quantitative tube is full, stopping when the bubble sensor P3 detects the liquid, and recording the rotation number N of the double-way peristaltic pump, and knowing the volume V of the quantitative tube and the volume offset of the pipeline volume(R is the radius of the pipeline, L is the total length of the pipeline between the calibration starting point S and the bubble sensor), the rubber pipe coefficient of the bi-pass peristaltic pumpThe liquid passing through each turn of the bi-pass peristaltic pump is P, and the calibration is completed. Therefore, the liquid inlet and outlet amount in each liquid bag and the centrifugal cup can be accurately controlled by controlling the rotation number and the rotation direction of the double-way peristaltic pump. For example, assuming that liquid M is to be fed into the target container, the two-way peristaltic pump is to be rotated in a specified direction for a number of turns n=m/P. In addition, the number of turns N of the bi-pass peristaltic pump can be further converted into the number of pulses Q of the motor (assuming that the motor is 40000 pulses rotate one turn, q=40000×n), so as to calculate the liquid amount of each pulseThe liquid inlet amount of the double-way peristaltic pump can be controlled by controlling the pulse number of the double-way peristaltic pump motor, so that the accuracy of the double-way peristaltic pump can be improved.
In specific implementation, referring to fig. 4, the centrifugal cup is mainly used for providing a place for treating cell liquid, the centrifugal machine is responsible for providing high-speed rotation power, so that the centrifugal cup reaches a preset rotation speed, and therefore, sufficient centrifugal force is provided for cell liquid in the centrifugal cup, cells and supernatant are separated in a high-speed centrifugal state, then the purpose of concentrating and cleaning the cell liquid is achieved in a continuous flow (a cell stock solution is pumped into an upper port and the supernatant is pumped out of a lower port), finally, cells on the wall of the centrifugal cup are uniformly mixed by heavy suspension, and are packaged into final-product bags (for example, packaging bags).
In particular, and with reference to FIG. 16, another modified centrifuge cup may also be used. Specifically, the centrifugal cup can comprise a centrifugal cup base, a centrifugal cup cavity (or called cup body), a centrifugal cup center shaft (such as a center shaft runner) and a double-channel F head. The cavity of the centrifugal cup comprises an upper runner, an upper opening of the centrifugal cup, a lower runner and a lower opening of the centrifugal cup, a central shaft is communicated with the lower runner, and the central shaft comprises a bearing, a large sealing ring, a small sealing ring and a T-shaped diversion platform which are required by high-speed centrifugation of the centrifugal cup.
As shown in fig. 16, the upper opening and the upper flow channel of the centrifugal cup are communicated with the first flow channel (for example, the first flow channel) of the F-head through the T-shaped flow guiding platform, and the lower opening and the lower flow channel of the centrifugal cup are communicated with the second flow channel (for example, the second flow channel) through the middle shaft.
Wherein the distance between the upper opening of the centrifugal cup and the cup wall isThe distance between the lower opening of the centrifugal cup and the cup wall is,>The radius of the centrifugal cup is R, and the height is H. When the centrifugal cup is centrifuged at high speed, liquid forms a liquid column along the wall of the centrifugal cup, and when the liquid is completely discharged from the lower opening, the residual liquid in the cup isWhen the liquid is completely discharged from the upper opening, the residual liquid in the cup is. Obviously, V Lower part(s)>V Upper part. In this embodiment of the present disclosure, during implementation, the upper port liquid discharge or the lower port liquid discharge may be selected according to the actual requirement and the amount of the liquid required to remain in the centrifugal cup.
In specific implementation, referring to fig. 5, the temperature sensor with high precision in the refrigeration centrifugal cabin can be used to monitor the temperature in the cabin in real time and intelligently regulate the temperature, so that the temperature in the centrifugal cabin is always kept at 2-8 ℃, the activity of cells is effectively ensured in the whole process engineering, and the influence of the temperature on the cells is greatly reduced.
It should be added that the fully automatic cell separation and harvest system provided in the present example is a set of separation and harvest liquid path system with extremely simple design. Specifically, the liquid path system is suitable for a cell harvesting process, supports functions of continuous flow sample injection, high-speed centrifugation, cell concentration, cell cleaning, resuspension, split charging and the like of cell liquid, and comprises a closed pipeline, a liquid bag, an electromagnetic valve, a peristaltic pump, a centrifugal cup, a bubble sensor, a filter valve, a dropping funnel and the like. The liquid way system layout is simple, compares with some current automatic cell harvesting device on the market, and the total number of solenoid valve has reduced 7 from 14, and the gas filter valve has reduced 1 from 2, and the pipeline total length has reduced 370cm from 600cm, and liquid way logic is clear, and observation and the monitoring in the experimental process of being convenient for also have simplified the installation of aseptic consumptive material pipeline simultaneously, and the very big degree has reduced the operation degree of difficulty, has reduced the human error that the complicated pipeline arouses, has promoted automatic cell harvesting's efficiency and stability.
And, based on the above-mentioned fully automatic cell separation and harvest system is a parameter-adjustable whole cell separation and harvest process. The process comprises the steps of consumable installation, system self-detection, high-speed centrifugation, continuous flow sample introduction, continuous flow cleaning, cell resuspension, cell split charging, process recording and the like, and a plurality of processes of the whole harvesting process are controlled by a computer program, all steps are automatically completed according to parameters configured in advance, manual intervention is not needed in the middle, and the risk brought by manual intervention is greatly avoided while the manpower is saved. For harvesting cells with different cell primary liquid volumes, the cell harvesting efficiency with different liquid volumes can be adapted through parameter adjustment, for example, 50L of sample solution can be obtained, the sample injection speed of a peristaltic pump can be configured to be 350ml/min, namely, the whole sample injection concentration of 50L of cell primary liquid can be completed only by (50000/350=143) minutes, and at least 3 hours are needed for processing the sample injection of 50L of cell primary liquid by the harvesting equipment on the market at present. The system can support the original liquid amount of 1000L of cells to perform full-automatic cell separation and harvesting treatment.
In addition, the fully automatic cell separation and harvesting system is constructed by using fully closed pipeline consumable connection. The method is mainly used for isolating cell sap from the outside, and the pollution of the cells to the outside is avoided in the whole process of cell harvesting. The consumable material for the pipeline comprises components such as a pipeline, a peristaltic pump rubber tube, a centrifugal cup, a liquid bag, a filter valve, a drip chamber and the like, and is matched with equipment such as a heat sealing instrument, a sterile pipe connecting machine and the like to operate, so that the cell liquid is always in a totally-enclosed environment in the treatment process of the cell liquid, and the safety of cell products is ensured.
In practice, as shown in FIG. 15, a fully automated cell separation harvesting system can be constructed and utilized to achieve fully automated cell separation harvesting according to the following steps.
And S01, performing consumable installation according to system prompt.
In the specific implementation, before formally starting automatic cell harvesting, some preliminary preparation work is needed, firstly, the equipment is started, a cell harvesting process is selected, and the centrifugal cabin is automatically started for pre-refrigeration, so that the temperature of the centrifugal cabin is always kept at 2-8 ℃, and the cell viability is fully ensured in the whole cell harvesting process. Secondly, performing consumable installation, namely connecting all prepared cell liquid bags, cleaning liquid bags, heavy suspension bags, final product bags and waste liquid bags to a sterile consumable pipeline through a sterile pipe connecting machine, installing fully-closed disposable consumables to a cell harvesting device according to a system prompt and solenoid valve opening sequence, wherein a valve No. 1 is opened, a cell bag and a corresponding pipeline are installed, a valve No. 2 is opened, a cleaning liquid bag and a corresponding pipeline are installed, a valve No. 3-4 is opened, a quantitative pipe air filtering valve and a pipeline thereof are installed, a valve No. 5 is opened, a final product bag and a pipeline thereof are installed, a valve No. 6 is opened, a heavy suspension and a pipeline thereof are installed, a valve No. 7 is opened, a waste liquid bag and a pipeline thereof are installed, a peristaltic pump is opened, a rubber pipe of a two-way peristaltic pump channel I and a two-way peristaltic pump channel II are installed according to a label, a centrifugal cup is fixed to a centrifugal machine, the installation stability of the centrifugal cup is ensured, and the liquid bags are connected to the consumable pipeline according to a designated sequence through the sterile pipe connecting machine, and the liquid bags corresponding to the solenoid valve in sequence from right to left are respectively 1-cell stock solution, 2-cleaning liquid, a valve 5-final product bag (empty), and a waste liquid bag (empty). The consumable is a totally-enclosed disposable consumable pipeline sterilized by ethylene oxide, and comprises a centrifugal cup, the whole installation process is isolated from the outside, and the consumable pipeline is connected through a sterile pipe connecting machine when a liquid bag is connected, so that the pipeline is prevented from contacting with the outside.
S02, performing system self-checking, performing air tightness test on consumable materials, and completing dual-channel peristaltic pump calibration (for example, self-checking operation and calibration operation).
When the method is implemented, the system can automatically start to execute a self-checking program by clicking on an operation panel, the electromagnetic valves are sequentially opened and closed for two times, each electromagnetic valve can be normally opened and closed, whether consumable installation is normal or not is also detected, whether the air tightness of all consumable pipelines is good or not is also detected, the valve No.2 is opened, other electromagnetic valves are closed, whether the cleaning liquid flows downwards or not is observed, if the cleaning liquid does not flow downwards, the air tightness of a right pipeline is proved to be good, the centrifuge is started, the valve No.2 is opened in a high-speed centrifugal state, a proper amount of cleaning liquid is pumped into a centrifugal cup for monitoring the air tightness of a left pipeline, the valve No.2 is closed, the valve No.3 is opened, the centrifugal cup is rotated in a static state, a certain liquid is pumped into the valve No. 4-5-6-7, the high pressure in the pipeline is kept, whether the valve No. 4-5-6-7 and a T-shaped tee in the middle are overflowed or not is observed for a period of time, if no liquid overflows, the air tightness of the left pipeline is proved to be good, and the self-checking is finished.
Starting the centrifugal machine, keeping a set rotating speed, opening a No. 2 electromagnetic valve and a No. 7 electromagnetic valve, pumping a cleaning liquid with a set volume V1 into a centrifugal cup by a double-pass peristaltic pump (V1 is larger than a fixed-volume pipe volume V), tightly attaching the cleaning liquid with the V1 to the wall of the centrifugal cup under the action of centrifugal force so as to remain in the cup and be used as a calibration liquid of the double-pass peristaltic pump, stopping rotating the centrifugal machine, standing the liquid in the cup at the bottom of the cup under the state of releasing the centrifugal effect, opening the No. 2-7 electromagnetic valve, pumping a set dosage of liquid into a waste liquid bag by the double-pass peristaltic pump according to the set number of turns, filling the pipeline between the No. 7 valve and the centrifugal cup by the filling the No. 7 electromagnetic valve, opening the No. 2-4 electromagnetic valve, starting from a calibration starting point S, rotating the double-pass peristaltic pump according to the set direction and the speed, pumping the calibration liquid into the fixed-volume pipe until the fixed-volume pipe is fully filled, stopping when a bubble sensor P3 detects the liquid, and recording the rotating pulse number Q of a motor of the double-pass peristaltic pump, and knowing the fixed-volume V and the displacement of the pipeline volume(R is the radius of the pipeline, L is the total length of the pipeline between the calibration starting point S and the bubble sensor), the rubber pipe coefficient of the bi-pass peristaltic pumpThe liquid passing through each pulse of the double-pass peristaltic pump motor is PQ, and the calibration is completed. Therefore, the liquid inlet and outlet amount in each liquid bag and the centrifugal cup can be accurately controlled by controlling the number and the rotating direction of the rotating pulses of the motor of the double-way peristaltic pump. For example, if the liquid M needs to be fed into the target container, the motor of the dual-channel peristaltic pump needs to rotate the pulse number m=m/PQ towards the designated direction, and the calibration is completed. And opening the valve No. 3-7, pumping the calibration liquid in the metering tube into the centrifugal cup through the first channel of the double-way peristaltic pump, and then pumping the calibration liquid into the waste liquid bag through the second channel.
S03, continuous flow injection, pumping cell stock solution, pumping out supernatant fluid, and completing cell concentration (for example, liquid feeding operation, separation operation and concentration operation).
In specific implementation, assuming 50L of cell stock solution, the peristaltic pump sample injection speed is set to be 350ml/min, namely, the whole sample injection concentration of 50L of cell stock solution can be completed only by 50000/350=143 minutes. Starting a centrifugal machine to enable a centrifugal cup to keep a specified rotation speed for centrifugation, opening electromagnetic valves No. 1 and No. 7, enabling a peristaltic pump to rotate according to a set sampling speed V0 and a set direction, wherein V0 is between two critical speeds (V1 is less than V0 is less than V2), V1 is the lowest speed of cell sampling, the speed is lower than V1, the cell sampling period is prolonged, the living rate and the state of cells are affected, the efficiency of cell harvesting is reduced, V2 is the maximum speed of cell sampling, the speed exceeds V2, and part of cells are discharged under the inherent centrifugal force without adhering to the wall, so that the cell harvesting rate is reduced. After the cell stock solution enters the centrifugal cup, the cell stock solution is pumped into the centrifugal cup by the double-channel peristaltic pump, and because the density of the cell is larger than that of the supernatant, the cell (wall-attached) is separated from the supernatant under the action of the centrifugal force, when the total volume of the liquid in the centrifugal cup is gradually increased to be larger than the critical volume V Lower part(s) of the lower opening of the centrifugal cup, the redundant supernatant is discharged into a waste liquid bag from the lower opening, the first channel and the second channel of the double-channel peristaltic pump are driven by the same motor and the same peristaltic pump roller, and are matched with rubber pipes with the same diameter, so that the liquid speeds of the first channel and the second channel are consistent, the liquid flow rate pumped into the centrifugal cup and the liquid flow rate pumped out of the centrifugal cup are consistent, so that the upper opening pumps the cell stock solution, the lower opening discharges the supernatant at the same flow rate, and a rapid and stable cell concentration continuous flow is formed until all the cell stock solution is completed, the peristaltic pump stops, the No. 1 electromagnetic valves and No. 7 electromagnetic valves are closed, and continuous flow sample injection is completed when the bubble sensor P1 detects air. The supernatant in the cell stock was discharged into a waste bag, and only adherent cells and a small amount of supernatant remained in a centrifuge cup, and the total volume was about 100ml, so far, the cell stock was concentrated from 50L to 100ml.
And S04, continuous flow cleaning, pumping in cleaning liquid, pumping out waste cleaning liquid (cleaned cleaning liquid) to finish cell cleaning (e.g. cleaning operation).
In specific implementation, after continuous flow sample injection is finished, the high-speed centrifugal state of the centrifugal cup is kept continuously, the No. 2-7 electromagnetic valve is opened, the peristaltic pump rotates according to the set cleaning speed V3 and the set direction, V3 is between two critical speeds (V4 is less than V3 and less than V5), V4 is the lowest speed of cell cleaning, the speed is lower than V4, the cleaning force is insufficient, the cleaning is insufficient, substances such as culture mediums in the centrifugal cup cannot be completely cleaned, the cleaning efficiency is reduced, the speed V5 is the maximum speed of cell cleaning, the speed exceeds V5, and strong fluid impact not only can take away adherent cells, but also can kill the cells. The method comprises the steps that a cleaning liquid is pumped in from an upper opening of a double-way peristaltic pump, waste liquid is discharged from a lower opening of the double-way peristaltic pump, so that stable cleaning continuous flow is formed, the distance between the upper opening of the centrifugal cup and the axis of the centrifugal cup is R1, the distance between the lower opening of the centrifugal cup and the axis of the centrifugal cup is R2, the centrifugal cup is in a high-speed centrifugal state in the continuous flow cleaning process, the thickness of supernatant between the upper opening of the centrifugal cup and the lower opening of the centrifugal cup is DeltaR=R1-R2, the cleaning liquid enters from the upper opening of the centrifugal cup, the lower opening of the centrifugal cup is discharged, the supernatant with the thickness DeltaR can pass through, residual supernatant in the centrifugal cup is replaced by the cleaning liquid, and in order to ensure that the supernatant (other components except cells) in the centrifugal cup can be replaced completely, and the liquid quantity and the cleaning cycle of continuous flow cleaning can be set according to practical conditions. After continuous flow cleaning is finished, only cells in an adherent state and a little cleaning solution are left in the centrifugal cup, the centrifugal cup is kept to be centrifuged at a specified rotating speed, the No. 1-4 valve is opened, the peristaltic pump rotates at a set speed and in a set direction, the residual cleaning solution in the centrifugal cup is discharged into a cell stock solution bag from an upper opening, the upper opening of the centrifugal cup is close to the wall of the centrifugal cup, and the cleaning solution in the cup can be discharged as much as possible under high-speed centrifugation, so that only adherent cells are left in the centrifugal cup.
S05, pumping the heavy suspension, and uniformly mixing the cells on the wall of the centrifugal cup into the heavy suspension (e.g. a heavy suspension operation).
When the method is implemented, after the cell cleaning is finished, all cells are in an adherence state in a centrifugal cup, the cells are required to be uniformly mixed by heavy suspension, so that the cells are in a heavy suspension state, the next step is convenient to process, a centrifugal machine stops rotating, a No. 3-6 electromagnetic valve is opened, a double-way peristaltic pump rotates according to a set speed and a set direction, a heavy suspension solution with a set dosage is pumped into the centrifugal cup from the lower opening of the centrifugal cup through a second channel of the double-way peristaltic pump, meanwhile, the air with the same volume is pumped out from the upper opening of the centrifugal cup through a first channel of the double-way peristaltic pump at the same speed, under the condition of constant pressure, the air with the set dosage in the centrifugal cup is accurately replaced by the heavy suspension, then the electromagnetic valve and the peristaltic pump are closed, the centrifugal machine starts to uniformly mix, the adherent cells in the centrifugal cup are completely dissolved and uniformly distributed in the heavy suspension solution according to the set speed and the period, and the centrifugal machine stops rotating.
And S06, sub-packaging the heavy suspension in the centrifugal cup into final product bags according to set doses (for example, sub-packaging operation).
In the specific implementation, after the cell is resuspended, the cell liquid in the centrifugal cup is required to be quantitatively packaged into a final-production bag for subsequent reinfusion or frozen storage. The valve 3-5 is opened, the peristaltic pump rotates according to the set speed and direction, the two-way peristaltic pump channel II pumps the heavy suspension solution filled with cells in the centrifugal cup into the final-product bag according to the set dosage through the lower opening of the centrifugal cup, meanwhile, the two-way peristaltic pump channel I pumps clean air with the same volume after bacteria filtration into the centrifugal cup at the same speed through the upper opening of the centrifugal cup, under the condition of constant pressure, the cell liquid in the centrifugal cup is replaced by the air and is pumped into the final-product bag, and the split charging procedure can automatically circulate and split the split charging procedure according to the cell volume and the quantity of the final-product bags, so that split charging of the cell heavy suspension is completed. The whole cell harvesting process is finished, and the cell sap end product is obtained in the end production bag and can be used for clinical reinfusion or long-term freezing.
Through the scene examples, the system and the method for separating and harvesting the cells, which are provided by the specification, are verified to form a stable continuous flow by utilizing the characteristics of the double-pass pump, and then the stable continuous flow is matched with a centrifugal machine and a refrigerating centrifugal cabin to transport related fluid, so that the loss and damage of the cells in the operation treatment process can be effectively reduced, the inactivation of the cells is avoided, and the treatments of separating, concentrating, cleaning, resuspension, split charging and the like of the cells in the cell stock solution are automatically and efficiently completed, so that the cell products with higher quality are obtained.
Although the present description provides method operational steps as described in the examples or flowcharts, more or fewer operational steps may be included based on conventional or non-inventive means. The order of steps recited in the embodiments is merely one way of performing the order of steps and does not represent a unique order of execution. When implemented by an apparatus or client product in practice, the methods illustrated in the embodiments or figures may be performed sequentially or in parallel (e.g., in a parallel processor or multi-threaded processing environment, or even in a distributed data processing environment). The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, it is not excluded that additional identical or equivalent elements may be present in a process, method, article, or apparatus that comprises a described element. The terms first, second, etc. are used to denote a name, but not any particular order.
Those skilled in the art will also appreciate that, in addition to implementing the controller in a pure computer readable program code, it is well possible to implement the same functionality by logically programming the method steps such that the controller is in the form of logic gates, switches, application specific integrated circuits, programmable logic controllers, embedded microcontrollers, etc. Such a controller can be regarded as a hardware component, and means for implementing various functions included therein can also be regarded as a structure within the hardware component. Or even means for achieving the various functions may be regarded as either software modules implementing the methods or structures within hardware components.
The description may be described in the general context of computer-executable instructions, such as program modules, being executed by a computer. Generally, program modules include routines, programs, objects, components, data structures, classes, etc. that perform particular tasks or implement particular abstract data types. The specification may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer-readable storage media including memory storage devices.
From the above description of embodiments, it will be apparent to those skilled in the art that the present description may be implemented in software plus a necessary general hardware platform. Based on such understanding, the technical solutions of the present specification may be embodied essentially in the form of a software product, which may be stored in a storage medium, such as a ROM/RAM, a magnetic disk, an optical disk, etc., and include several instructions to cause a computer device (which may be a personal computer, a mobile terminal, a server, or a network device, etc.) to perform the methods described in the various embodiments or portions of the embodiments of the present specification.
Various embodiments in this specification are described in a progressive manner, and identical or similar parts are all provided for each embodiment, each embodiment focusing on differences from other embodiments. The specification is operational with numerous general purpose or special purpose computer system environments or configurations. Such as a personal computer, a server computer, a hand-held or portable device, a tablet device, a multiprocessor system, a microprocessor-based system, a set top box, a programmable electronic device, a network PC, a minicomputer, a mainframe computer, a distributed computing environment that includes any of the above systems or devices, and the like.
Although the present specification has been described by way of example, it will be appreciated by those skilled in the art that there are many variations and modifications to the specification without departing from the spirit of the specification, and it is intended that the appended claims encompass such variations and modifications as do not depart from the spirit of the specification.