CN115161164A - Low-damage continuous flow cell harvesting system and method - Google Patents
Low-damage continuous flow cell harvesting system and method Download PDFInfo
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- CN115161164A CN115161164A CN202210786452.1A CN202210786452A CN115161164A CN 115161164 A CN115161164 A CN 115161164A CN 202210786452 A CN202210786452 A CN 202210786452A CN 115161164 A CN115161164 A CN 115161164A
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Abstract
The invention relates to a low-damage continuous-flow cell harvesting system and a method, wherein the cell harvesting system comprises a centrifugal unit, an electromechanical unit, a liquid path unit and a human-computer interaction unit; the centrifugal unit is used for realizing the centrifugation of cell fluid; the electromechanical unit comprises a base, a centrifugal bin, a centrifugal motor and a liquid hanging frame; the centrifugal bin is used for accommodating the centrifugal unit and is provided with a temperature control module; the centrifugal motor is used for driving the centrifugal unit to rotate; the liquid path unit comprises a peristaltic pump, an electromagnetic pinch valve, a sterile catheter, a plurality of liquid bags and an air filter; the man-machine interaction unit comprises a control panel and a touch screen display for inputting/outputting information. The cell harvesting system has the characteristics of low damage, high flux, high efficiency, high recovery rate and full sealing, can obviously improve the defect of low cell biological activity in the prior art, and meets the requirement of harvesting a large-volume cell sample solution.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a low-damage continuous-flow cell harvesting system and a method.
Background
Cell therapy is a therapeutic method in which a cell-based drug is injected into a patient to treat a disease, for example, cancer cells can be combated by cell-mediated immunity after T-lymphocytes (T-lymphocytes) as a cell-based drug are transplanted into a patient in cellular immunotherapy; in addition, for example, transplantation of stem cells as a cell-based drug into a patient can promote tissue regeneration at the damaged site. However, the cell medicine production process has many preparation links and complex processes, and the complex process operations and the cell in vitro environment have obvious influence on the cell biological activity. Especially after completing the in vitro cell expansion culture, a step of final product harvest of suspension cells is required.
Two schemes commonly adopted in the existing equipment are a centrifugal device harvesting scheme and a filtering device harvesting scheme respectively. The centrifugal device harvesting scheme is used for carrying out centrifugal operation on a cultured sample, and after a cell sample is separated from a supernatant, removing the supernatant, cleaning cells, and concentrating and subpackaging the cells, but the fluid shearing force in a centrifugal cup can cause higher shearing damage to the cells in the centrifugal process, so that the biological activity of the cells is reduced. In addition, the cell sample containing the supernatant is filtered by the filter screen according to the harvesting scheme of the filtering device, the supernatant can be filtered while the cells are retained, however, the flow resistance of the filter screen is large, so that the separation efficiency is low, and the biological activity of the cells can be obviously reduced by long-time treatment.
Therefore, the prior art has the defects of low efficiency and high damage, and also has the defect of low flux, namely the requirement of harvesting a large-volume cell sample solution is difficult to meet.
Disclosure of Invention
The invention provides a low-damage continuous-flow cell harvesting system and a method, the cell harvesting system has the characteristics of low damage, high flux, high efficiency, high recovery rate and full sealing, can obviously improve the defect of low cell bioactivity in the prior art, and meets the requirement of harvesting a large-volume cell sample solution.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a low-damage continuous-flow cell harvesting system, which comprises a centrifugal unit, an electromechanical unit, a liquid path unit and a human-computer interaction unit, wherein the centrifugal unit is connected with the electromechanical unit;
the centrifugal unit is used for realizing the centrifugation of cell fluid;
the electromechanical unit comprises a base, a centrifugal bin, a centrifugal motor and a liquid hanging frame; the centrifugal bin is arranged in the base, is used for accommodating the centrifugal unit and is provided with a temperature control module; the centrifugal motor is used for driving the centrifugal unit to rotate; the liquid hanging frame is arranged at the top of the base;
the liquid path unit comprises a plurality of liquid bags, an air filter, a peristaltic pump, a sterile catheter and an electromagnetic pinch valve; the air filter is communicated with the centrifugal unit, the peristaltic pump is communicated with the centrifugal unit, and the peristaltic pump is communicated with the liquid bag through sterile conduits, and electromagnetic pinch valves for controlling the on-off are arranged in the sterile conduits; the liquid bags comprise a waste liquid bag, and a cell sample bag, a cleaning solution bag, a formula solution bag and a product bag which are hung on the liquid hanging rack;
the liquid hanging rack is also provided with weighing sensors which correspond to the liquid bags one by one, and the weighing sensors are used for measuring the weight of the corresponding liquid bags;
the human-computer interaction unit comprises a control panel and a touch screen display; according to the information input by the touch screen display and the weighing sensor, the control panel controls the centrifugal motor, the temperature control module, the peristaltic pump and the electromagnetic pipe clamping valve to act.
Furthermore, the centrifugal unit comprises a centrifugal cup and a liquid pushing plate fixedly arranged in the centrifugal cup;
a gap for balancing the liquid pressure on two sides of the liquid pushing plate is arranged between the liquid pushing plate and the inner peripheral wall of the centrifugal cup;
the centrifugal motor is in transmission connection with the centrifugal cup and is used for driving the centrifugal cup to rotate.
Furthermore, the centrifugal unit further comprises a first flow channel and a second flow channel;
the first flow channel is used for communicating a first port arranged at the top of the centrifugal cup with a second port arranged at the bottom of the side wall of the centrifugal cup;
the second flow passage is used for communicating a third port arranged at the top of the centrifugal cup with a fourth port arranged at the top of the side wall of the centrifugal cup;
the air filter and the plurality of fluid bags are each in communication with the first port and the second port via the peristaltic pump.
Furthermore, the centrifugal unit further comprises a rotary adapter arranged at the bottom of the centrifugal cup, a first sealing ring arranged between the first flow passage and the centrifugal cup, and a second sealing ring arranged between the second flow passage and the centrifugal cup;
the rotary adapter is in transmission connection between the centrifugal cup and the centrifugal motor;
the bottom of the centrifugal cup is provided with a chamfer angle of 5-50 degrees.
Furthermore, the liquid path unit further comprises a first bubble sensor, a second bubble sensor and a third bubble sensor;
the inlet of the first bubble sensor is communicated with the cell sample bag, the cleaning solution bag and the formula solution bag, and the outlet of the first bubble sensor is communicated with the first port of the peristaltic pump through a first branch;
the inlet of the second bubble sensor is communicated with the first interface of the peristaltic pump, and the outlet of the second bubble sensor is communicated with the product bag;
one end of the third bubble sensor is communicated with the second port of the peristaltic pump, and the other end of the third bubble sensor is communicated with the first port through a third branch;
the first interface of the peristaltic pump is also communicated with the waste liquid bag.
Furthermore, the liquid hanging rack also comprises a bracket arranged at the top of the base, a liquid hanging rod fixedly arranged at the top of the bracket and hooks hung on each weighing sensor; each weighing sensor is arranged on the liquid hanging rod;
and one liquid bag is hung at the bottom of each hook.
Furthermore, the temperature control module is a semiconductor thermoelectric cooling device.
In addition, the invention also provides a cell harvesting method adopting the cell harvesting system, which generates a fluid driving force by the driving of a peristaltic pump so as to separate, concentrate, wash and harvest cell liquid containing supernatant, and comprises the following steps:
installing a centrifugal unit, a liquid bag and a sterile catheter, and keeping each electromagnetic pinch valve in a normally closed state;
removing the cell supernatant;
washing cell sap;
concentrating the cell sap;
and (6) subpackaging the cell sap.
Further, the step of removing the cell supernatant specifically comprises: opening electromagnetic pinch valves between the peristaltic pump and the cell sample bag and between the peristaltic pump and the first port, opening the peristaltic pump, introducing the cell sample solution in the cell sample bag into the centrifugal unit through the first flow channel, and closing all the electromagnetic pinch valves after introducing a predetermined amount of the cell sample solution; opening an electromagnetic pinch valve between the cell sample bag and the centrifugal unit to lead the cell sample solution in the cell sample bag into the second flow channel through self gravity; starting a centrifugal motor, controlling the rotating speed of the motor to be a first rotating speed (one rotating speed within the range of 1500RPM to 2000 RPM), completing the separation of cells and supernatant under the pushing of a liquid pushing plate, and connecting a first flow channel and a second flow channel through a vertical centrifugal solution in a centrifugal unit; keeping the opening state of an electromagnetic pinch valve between the cell sample bag and the centrifugal unit, opening a peristaltic pump, the electromagnetic pinch valve between the peristaltic pump and the first flow channel and the electromagnetic pinch valve between the peristaltic pump and the waste liquid bag, increasing the rotating speed of the centrifugal motor to a second rotating speed (one rotating speed in the range of 2000RPM to 3000 RPM), guiding the supernatant to the waste liquid bag from the first flow channel by fluid flow power provided by the peristaltic pump, attaching the cells separated by the supernatant to the inner wall surface of the centrifugal cup until the cell sample solution in the cell sample bag is completely guided to the centrifugal unit and guiding all the supernatant to the waste liquid bag;
the step of washing the cell sap specifically comprises: guiding the cell cleaning solution in the cleaning solution bag to a centrifuge cup, cleaning the centrifuge cup through the cell cleaning solution, and guiding the cleaning waste liquid to a waste liquid bag through a first flow channel by a peristaltic pump;
the cell sap concentration step specifically comprises: reducing the rotation speed of the centrifugal motor from the second rotation speed to a third rotation speed (one rotation speed in the range of 500RPM to 100 RPM) so as to guide the waste liquid to a waste liquid bag under the action of a peristaltic pump to finish the concentration of the cell liquid;
the step of subpackaging the cell sap specifically comprises the following steps: closing the peristaltic pump and all electromagnetic pinch valves, opening the electromagnetic pinch valves between the formula solution bag and the peristaltic pump and between the peristaltic pump and the first port, opening the peristaltic pump, guiding the final product formula solution in the formula solution bag to the centrifugal cup, closing the peristaltic pump and all electromagnetic pinch valves, and performing multiple mixing operations through the centrifugal motor to fully mix the adherent cells with the final product formula solution; closing the peristaltic pump and all solenoid pinch valves; and opening electromagnetic pinch valves between the peristaltic pump and the first port and between the peristaltic pump and the product bag to perform sub-packaging of the product bag.
Furthermore, in the step of subpackaging the cell sap, a centrifugal motor is used for carrying out multiple uniform mixing operations of forward rotation, sudden stop and reverse rotation in sequence;
the step of washing the cell sap is accomplished by a uniform washing method or a continuous flow washing method.
Compared with the prior art, the invention has the following beneficial effects:
in the cell harvesting system, the centrifugal unit is fixedly provided with the liquid pushing plate in the centrifugal cup, the centrifugal cup is driven to rotate by the centrifugal motor, in the autorotation and centrifugation process of the centrifugal cup, cell sap in the centrifugal cup is pushed by the liquid pushing plate to rotate synchronously with the centrifugal cup, and the liquid pushing plate can provide fluid driving force for the centrifugal unit, so that cells in a sample solution are prevented from being sheared in the centrifugal motion process, shearing damage to the cells in the centrifugation process is remarkably reduced, the biological activity of cell harvesting is remarkably improved, the separation efficiency is improved, and the time required by separation is shortened; by adopting the liquid path unit structure and the cell harvesting method, the supernatant can be continuously separated (similar to the siphoning effect), and the efficient removal of the continuous flow cell supernatant is completed by a small-volume cup. Therefore, the cell harvesting system and the cell harvesting method avoid shearing damage of cells through a simple structure, solve the problems of long separation time, low harvesting efficiency and the like in the prior art, and realize cell harvesting with low damage, high flux, high efficiency and high recovery rate.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the cell harvesting system of the present invention;
FIG. 2 is a cross-sectional view of a portion of the centrifuge unit of the cell harvesting system of FIG. 1;
FIG. 3 is a schematic diagram of the structure of the liquid path unit of the cell harvesting system of FIG. 1;
FIG. 4 is a schematic diagram of the overall structure of the centrifugal unit of the cell harvesting system of FIG. 2;
FIG. 5 is a cross-sectional view of the centrifuge unit of FIG. 4;
FIG. 6 is a diagram showing the temperature control scheme of the temperature control module and the heat transfer relationship of the centrifugal chamber;
FIG. 7 is a flow chart of a cell harvesting method of the present invention.
Wherein, 1-centrifugal unit, 2-centrifugal cup, 3-liquid pushing plate, 4-first flow channel, 5-second flow channel, 6-first port, 7-second port, 8-third port, 9-fourth port, 10-machine base, 11-centrifugal cabin, 12-centrifugal motor, 13-support, 14-hanging liquid rod, 15-peristaltic pump, 16-control panel, 17-touch screen display, 18-gap, 19-first electromagnetic pinch valve, 20-second electromagnetic pinch valve, 21-third electromagnetic pinch valve, 22-fourth electromagnetic pinch valve, 23-fifth electromagnetic pinch valve, 24-sixth electromagnetic pinch valve, 25-seventh electromagnetic pinch valve, 26-eighth electromagnetic pinch valve, 27-ninth electromagnetic pinch valve, 28-tenth electromagnetic pinch valve, 29-eleventh electromagnetic pinch valve, 30-twelfth electromagnetic pinch valve, 31-thirteenth electromagnetic pinch valve, 32-thirteenth product bag, 33-waste liquid bag, 34-cell sample bag, 35-cleaning solution bag, 36-solution bag, 37-bubble solution bag, 38-bubble sensor, 39-bubble sensor, 38-bubble formulation sensor, 35-second bubble formulation sensor, and the third electromagnetic pinch valve
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The present embodiment provides a low-damage continuous flow cell harvesting system, which includes a centrifugation unit 1, an electromechanical unit, a fluid path unit, and a human-computer interaction unit, with reference to fig. 1, 2, 3, and 4;
as shown in the structures of fig. 4 and 5, the centrifugal unit 1 is used for realizing the centrifugation of cell fluid and comprises a centrifugal cup 2, a liquid pushing plate 3, a first flow channel 4 and a second flow channel 5; the liquid pushing plates 3 are fixedly arranged in the centrifugal cup 2, and the number of the liquid pushing plates 3 can be one or more; when a plurality of liquid pushing plates 3 are arranged in the centrifugal cup 2, the plurality of liquid pushing plates 3 are uniformly distributed along the circumferential direction of the centrifugal cup 2; the first flow channel 4 is used for communicating a first port 6 arranged at the top of the centrifugal cup 2 with a second port 7 arranged at the bottom of the side wall of the centrifugal cup 2; the second flow passage 5 is used for communicating a third port 8 arranged at the top of the centrifugal cup 2 with a fourth port 9 arranged at the top of the side wall of the centrifugal cup 2;
as shown in the structure of fig. 1, the electromechanical unit includes a machine base 10, a centrifugal bin 11, a centrifugal motor 12 and a liquid hanging rack; the centrifugal bin 11 is arranged in the machine base 10 and used for accommodating the centrifugal unit 1, the centrifugal bin is provided with a temperature control module, the temperature control module can adopt a semiconductor thermoelectric refrigerating device to realize temperature control of 4-40 ℃ in the centrifugal bin 11, the control error is less than 1 ℃, the biological activity of cells placed in the environment for a long time can be improved through the temperature control module, and particularly in the links of frozen stock solution containing DMSO (Dimethyl sulfoxide) and split charging preparation, the low-temperature non-toxic effect of the frozen stock solution containing DMSO in the state of 4 ℃ is kept, so that the influence of the frozen stock solution containing DMSO on the cell activity is reduced. Wherein, the temperature control scheme of the temperature control module and the heat transfer relationship of the centrifugal bin 11 are shown in fig. 6;
the centrifugal bin 11 can also be provided with a hatch cover which can be opened and closed and is used for covering the centrifugal unit 1 in the centrifugal bin 11; the centrifugal motor 12 is used for driving the centrifugal unit 1 to rotate; the centrifugal motor 12 is in transmission connection with the centrifugal cup 2 and is used for driving the centrifugal cup 2 to rotate, so that the centrifugal cup 2 drives the liquid pushing plate 3 to rotate; the liquid hanging frame comprises a support 13 arranged at the top of the machine base 10, a liquid hanging rod 14 fixedly arranged at the top of the support 13, a plurality of weighing sensors arranged on the liquid hanging rod 14, and hooks hung on the weighing sensors; the weighing sensor is used for monitoring the weight of the liquid bag hung on the hook in real time, and the measurement error is less than 1g; one hook on the lower side of the weighing sensor can be vertically arranged, and two hooks can also be symmetrically arranged;
as shown in fig. 1, 2 and 3, the liquid path unit includes a plurality of liquid bags, an air filter for sterile filtering air, a sterile conduit, a peristaltic pump 15 for powering fluid in the sterile conduit, and an electromagnetic pinch valve for controlling the on-off of the corresponding sterile conduit; sterile conduits are communicated between the air filter and the centrifugal unit 1, between the peristaltic pump 15 and the centrifugal unit 1 and between the peristaltic pump 15 and the liquid bag, and electromagnetic pinch valves for controlling the on-off are arranged in the sterile conduits; the air filter and a plurality of liquid bags are communicated with the first port 6 at the top of the first flow channel 4 and the third port 8 of the second flow channel 5 through the peristaltic pump 15; electromagnetic pinch valves are arranged between the peristaltic pump 15 and each liquid bag and between the peristaltic pump 15 and the first port 6; the liquid bags comprise at least one waste liquid bag 33, at least one cell sample bag 34, at least one cleaning solution bag 35, at least one formula solution bag 36 and at least one product bag 32, wherein the cell sample bag 34, the cleaning solution bag 35, the formula solution bag 36 and the product bag 32 are hung on a plurality of hooks at the bottom of the liquid hanging rod 14, as shown in the structures of fig. 1 and 2, the two waste liquid bags 33 are positioned at the bottom end of the liquid path unit and hung on the machine base 10, so that waste liquid can flow in under the action of the peristaltic pump 15 and gravity; three bubble sensors, a first bubble sensor 37, a second bubble sensor 38, and a third bubble sensor 39: the inlet of the first bubble sensor 37 is communicated with the cell sample bag 34, the cleaning solution bag 35 and the formula solution bag 36, and the outlet of the first bubble sensor 37 is communicated with the first port of the peristaltic pump 15 through a first branch; the inlet of the second bubble sensor 38 is communicated with the first interface of the peristaltic pump 15, the outlet of the second bubble sensor 38 is communicated with the product bag 32, and cell sap is filled in the product bag 32 through the second bubble sensor 38; two or more than two filtering devices 40 can be arranged between the inlet of the second bubble sensor 38 and the first interface of the peristaltic pump 15, and cell clusters and fragments in a final product can be filtered and removed through the filtering devices 40, so that the needle blockage phenomenon is avoided, the accuracy of liquid preparation concentration is improved, and cell reinfusion and cell freezing are facilitated;
one end of the third bubble sensor 39 is communicated with the second interface of the peristaltic pump 15, the other end is communicated with the first port 6 of the first flow channel 4 through a third branch, and a sampling joint can be arranged at the other end of the third bubble sensor 39;
meanwhile, the first port of the peristaltic pump 15 is also communicated with the waste liquid bag 32;
as shown in fig. 3, for convenience of explanation, the solenoid pinch valves include a first solenoid pinch valve 19, a second solenoid pinch valve 20, a third solenoid pinch valve 21, a fourth solenoid pinch valve 22, a fifth solenoid pinch valve 23, a sixth solenoid pinch valve 24, a seventh solenoid pinch valve 25, an eighth solenoid pinch valve 26, a ninth solenoid pinch valve 27, a tenth solenoid pinch valve 28, an eleventh solenoid pinch valve 29, a twelfth solenoid pinch valve 30, and a thirteenth solenoid pinch valve 31; the first electromagnetic pinch valve 19 is arranged in a sterile conduit between the air filter and the third port 8 of the second flow channel 5, and the air pressure in the centrifugal cup 2 can be balanced through the air filter, so that the phenomenon that the liquid pumping effect is poor and the sample outlet error of the peristaltic pump is increased due to the fact that the air pressure in the centrifugal cup 2 is too low (negative pressure) during liquid pumping is prevented; the second electromagnetic pinch valve 20 is installed in the sterile conduit that communicates the cleaning solution bag 35 with the first bubble sensor 37; the third electromagnetic pinch valve 21 is installed in the sterile conduit that communicates the formula solution bag 36 with the first bubble sensor 37; the fourth electromagnetic pinch valve 22 is mounted in a sterile conduit that communicates the cell sample bag 34 with the first bubble sensor 37; the three product bags 32 are respectively communicated with the second bubble sensor 38 through a fifth electromagnetic pinch valve 23, a sixth electromagnetic pinch valve 24 and a seventh electromagnetic pinch valve 25; the eighth solenoid pinch valve 26 is mounted in the sterile conduit of the first bubble sensor 37 and the third port 8 of the second flow channel 5; the ninth electromagnetic pinch valve 27 is mounted in a sterile conduit communicating the first bubble sensor 37 with the first interface of the peristaltic pump 15; the on-off between the peristaltic pump 15 and the cleaning solution bag 35, between the formula solution bag 36 and between the peristaltic pump and the cell sample bag 34 are controlled simultaneously through the eighth electromagnetic pinch valve 26 and the ninth electromagnetic pinch valve 27; the tenth electromagnetic pinch valve 28 is installed in the sterile conduit that communicates the third bubble sensor 39 with the first port 6 of the first flow channel 4; the eleventh electromagnetic pinch valve 29 is arranged in a sterile conduit which communicates the first interface of the peristaltic pump 15 with the waste liquid bag 33; the twelfth electromagnetic pinch valve 30 is mounted in the sterile conduit that communicates the first interface of the peristaltic pump 15 with the inlet of the second bubble sensor 38; a thirteenth pinch valve 31 is installed in the sterile conduit between the tenth pinch valve 28 and the sampling connection;
as shown in fig. 1 and 2, the man-machine interaction unit includes a control panel 16 and a touch screen display 17 for inputting/outputting information; the control panel 16 is in signal connection with the centrifugal motor 12, the temperature control module, the weighing sensor, the peristaltic pump 15, the electromagnetic pinch valve, the bubble sensor and the touch screen display 17, and is used for controlling the actions of the centrifugal motor 12, the temperature control module, the peristaltic pump 15 and the electromagnetic pinch valve according to information input by the touch screen display 17, the weighing sensor and the bubble sensor.
In the working process of the cell harvesting system, the centrifugal unit 1 is arranged in the centrifugal bin 11, the centrifugal motor 12 is in transmission connection with the centrifugal cup 2, the liquid pushing plate 3 is fixedly arranged in the centrifugal cup 2, the centrifugal cup 2 is driven to rotate through the centrifugal motor 12, so that the liquid pushing plate 3 is driven to rotate along with the centrifugal cup 2, in the self-rotation and centrifugation process of the centrifugal cup 2, cell sap in the centrifugal cup 2 is pushed by the liquid pushing plate 3 to rotate synchronously with the centrifugal cup 2, and the liquid pushing plate 3 can provide fluid driving force for the cell sap in the centrifugal cup 2, so that cells in a sample solution are prevented from being subjected to shearing action in the centrifugal motion process, the shearing damage to the cells in the centrifugal process is remarkably reduced, the biological activity of cell harvesting is remarkably improved, the separation efficiency is also improved, the time required by separation is reduced, and the biological activity and efficiency of cell harvesting are remarkably improved; since the peristaltic pump 15 and the electromagnetic pinch valve are arranged in the liquid path unit of the cell harvesting system, and the first flow channel 4 and the second flow channel 5 are arranged in the centrifuge cup 2, the supernatant can be continuously separated (similar to the siphon action) through the control of the peristaltic pump 15 and the electromagnetic pinch valve, for example, the cell supernatant with 3L continuous flow can be efficiently removed in a small-volume cup of 300 mL. Therefore, the cell harvesting system and the cell harvesting method avoid shearing damage of the cells through a simple structure, solve the problems of long separation time, low harvesting efficiency and the like in the prior art, and realize the totally-enclosed cell harvesting with low damage, high flux, high efficiency and high recovery rate.
As shown in the structure of FIG. 5, a gap 18 for balancing the liquid pressure on both sides of the liquid pushing plate 3 is arranged between the liquid pushing plate 3 and the inner peripheral wall of the centrifuge cup 2. The bottom of the centrifugal cup 2 is provided with a chamfer angle of 5-50 degrees, such as: 5 degrees, 10 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees and 50 degrees, and the liquid is convenient to discharge through a chamfer arranged at the bottom of the centrifugal cup 2, so that the volume of the residual liquid in the centrifugal cup 2 is 0.1 mL-10 mL; when the chamfer angle is 20 °, it is possible to achieve a residual liquid volume in the centrifuge cup 2 of only 0.2mL. The centrifugal unit 1 further comprises a first sealing ring arranged between the first flow passage 4 and the centrifugal cup 2, and a second sealing ring arranged between the second flow passage 5 and the centrifugal cup 2, and the centrifugal cup 2 can be sealed through the first sealing ring and the second sealing ring.
On the basis of the various embodiments described above, the centrifugal unit 1 further comprises a rotary adapter mounted at the bottom of the centrifugal cup 2, which is drivingly connected between the centrifugal cup 2 and the centrifugal motor 12. The power transmission between the centrifugal cup 2 and the outer centrifugal motor 12 is realized through the rotary adapter, so that the liquid pushing plate 3 rotates, and the centrifugal liquid pumping function with high efficiency and low damage is realized.
Example two
This example provides a cell harvesting method using the above cell harvesting system, as shown in fig. 7, which generates a fluid driving force by the driving of a peristaltic pump 15 to separate, concentrate, wash and harvest cell fluid containing supernatant, comprising the following steps:
step S10, installing a centrifugal unit 1, a liquid bag and a sterile catheter, and keeping each electromagnetic pinch valve in a normally closed state;
step S20, removing cell supernatant: opening the electromagnetic pinch valves 19, between the peristaltic pump 15 and the cell sample bag 34, and between the peristaltic pump 15 and the first port 6, that is, opening the fourth electromagnetic pinch valve 22, the ninth electromagnetic pinch valve 27, and the tenth electromagnetic pinch valve 28 in fig. 3, opening the peristaltic pump 15, introducing the cell sample solution in the cell sample bag 34 into the centrifugal unit 1 through the first flow channel 4, and closing all the electromagnetic pinch valves after introducing a predetermined amount (150 mL) of the cell sample solution; opening the electromagnetic pinch valves between the cell sample bag 34 and the centrifuge unit 1, i.e., opening the fourth electromagnetic pinch valve 22 and the eighth electromagnetic pinch valve 26 in fig. 3, so that the cell sample solution in the cell sample bag 34 is introduced into the second flow channel 5 by its own weight, at which time the pressure in the centrifuge cup 2 is higher than the atmospheric pressure, and the first flow channel 4 and the second flow channel 5 are both completely filled with gas; starting the centrifugal motor 12, controlling the rotation speed of the motor to be a first rotation speed (one rotation speed within the range of 1500RPM to 2500 RPM), starting the liquid pushing plate 3 of the centrifugal unit 1 to rotate and centrifuge along the central shaft, under the pushing of the liquid pushing plate 3, because the density of cells in a cell sample solution is maximum, the cells can be attached to the inner wall surface of the centrifugal cup 2, and under the pushing of the liquid pushing plate 3, the separation of the cells and a supernatant can be completed within 3min, at the moment, the first flow channel 4 and the second flow channel 5 are connected through a vertical centrifugal solution in the centrifugal unit 1; keeping the electromagnetic pinch valves between the cell sample bag 34 and the centrifuge unit 1 in an open state, that is, keeping the fourth electromagnetic pinch valve 22 and the eighth electromagnetic pinch valve 26 in fig. 3 in an open state, and opening the peristaltic pump 15, the electromagnetic pinch valve between the peristaltic pump 15 and the first flow channel 4, and the electromagnetic pinch valve between the peristaltic pump 15 and the waste liquid bag 33, that is, opening the peristaltic pump 15, the tenth electromagnetic pinch valve 28, and the eleventh electromagnetic pinch valve 29 in fig. 3, increasing the rotation speed of the centrifuge motor 12 to a second rotation speed (one rotation speed in the range of 2000RPM to 3000 RPM), wherein the supernatant is continuously drained from the first flow channel 4 to the waste liquid bag 33 by the fluid flow force provided by the peristaltic pump 15, and the supernatant is drained from the first flow channel 4 to the waste liquid bag 33 by the fluid flow force provided by the peristaltic pump 15 due to the height difference of the peristaltic pump, and the liquid pressure of the waste liquid bag 33 is lower than the liquid pressure of the cell sample bag 34, the liquid pressure generates a driving force which continuously drains the cell sample solution in the cell sample bag 34 into the centrifuge cup 2, and the supernatant is completely drained from the first flow force provided by the peristaltic pump 15 to the centrifuge unit, and the flow force of the supernatant is removed to the centrifuge unit 1, and the continuous flow of the supernatant is completed; to further reduce the residual amount of supernatant, the rotation speed of the centrifugal motor 12 may be reduced from the second rotation speed to a third rotation speed (one rotation speed in the range of 500RPM to 1000 RPM) to divert more supernatant to the waste liquid bag 33 under the action of the peristaltic pump and gravity;
step S30, washing cell sap: guiding the cell cleaning solution in the cleaning solution bag 35 to the centrifuge cup 2, cleaning the centrifuge cup 2 by the cell cleaning solution, and guiding the cleaning waste liquid to the waste liquid bag 33 through the peristaltic pump 15 and the first flow channel 4; the cell sap can be cleaned by a uniform mixing cleaning method or a continuous flow cleaning method in the process of cleaning the cell sap; wherein:
the cleaning steps of the uniform mixing cleaning method are as follows: closing the peristaltic pump 15 and all electromagnetic pinch valves, opening the second electromagnetic pinch valve 20, the ninth electromagnetic pinch valve 27, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15, guiding the cell cleaning solution in the cleaning solution bag 35 to the centrifuge cup 2, closing the peristaltic pump 15 and all electromagnetic pinch valves, fully mixing the adherent cells and the cleaning solution through multiple mixing operations of forward rotation, sudden stop and reverse rotation of the centrifugal motor 12 in sequence, then controlling the rotation speed of the centrifugal motor 12 to be a centrifugal rotation speed (one rotation speed in the range of 2500RPM to 3000 RPM), guiding the waste liquid from the first flow channel 4 to the waste liquid bag 33 through the adherent centrifugal force by the fluid flow force provided by the peristaltic pump 15;
the continuous flow cleaning method comprises the following cleaning steps: closing the peristaltic pump 15 and all electromagnetic pinch valves, controlling the rotation speed of the centrifugal motor 12 to be a centrifugal rotation speed (one rotation speed within the range of 2500RPM to 3000 RPM), opening the second electromagnetic pinch valve 20, the eighth electromagnetic pinch valve 26, the tenth electromagnetic pinch valve 28, the eleventh electromagnetic pinch valve 29 and the peristaltic pump 15 after the cells are attached to the wall in a centrifugal manner, enabling the liquid pressure of the waste liquid bag 33 to be lower than the liquid pressure of the cleaning solution bag 35 under the action of height difference, enabling the liquid pressure in the cleaning solution bag 35 to generate a driving force, enabling the cell cleaning solution in the cleaning solution bag 35 to be continuously guided into the centrifugal cup 2 to flush the cells attached to the inner wall surface of the centrifugal cup 2, and enabling the waste liquid to be guided to the waste liquid bag 33 through the first flow channel 4 by the fluid flow force provided by the peristaltic pump 15 to achieve continuous cell flow cleaning;
the two cleaning methods can be matched with each other and carried out for multiple times so as to clean the waste liquid in the cell solution to the required index; step S40, concentrating cell sap: reducing the rotation speed of the centrifugal motor 12 from the second rotation speed to a third rotation speed so as to guide the waste liquid to the waste liquid bag 33 under the action of the peristaltic pump 15 and gravity, completing the concentration of the cell liquid and further reducing the residual quantity of the waste liquid;
step S50, cell sap subpackaging: closing the peristaltic pump 15 and all electromagnetic pinch valves, opening electromagnetic pinch valves between the formula solution bag 36 and the peristaltic pump 15 and between the peristaltic pump 15 and the first port 6, namely, opening a third electromagnetic pinch valve 21, a ninth electromagnetic pinch valve 27 and a tenth electromagnetic pinch valve 28 in fig. 3, opening the peristaltic pump 15, guiding the final product formula solution in the formula solution bag 36 to the centrifugal cup 2 of the centrifugal unit 1, closing the peristaltic pump 15 and all electromagnetic pinch valves, and performing multiple blending operations of forward rotation, sudden stop and reverse rotation through the centrifugal motor 12 to fully blend the adherent cells and the final product formula solution; closing the peristaltic pump 15 and all the electromagnetic pinch valves; electromagnetic pinch valves between the peristaltic pump 15 and the first port 6 and between the peristaltic pump 15 and the product bag 32 are opened to dispense the product bag 32. When three product bags 32 are provided as shown in fig. 3, the three product bags 32 may be dispensed by the following steps: the fifth electromagnetic pinch valve 23, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 are opened to complete the split charging of the first product bag 32; then the peristaltic pump 15 and all the electromagnetic pinch valves are closed, and the sixth electromagnetic pinch valve 24, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 are opened to complete the split charging of the second product bag 32; and then the peristaltic pump 15 and all the electromagnetic pinch valves are closed, and the seventh electromagnetic pinch valve 25, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 are opened to complete the split charging of the third product bag 32.
The cell harvesting system and the cell harvesting method adopt the simple centrifugal unit 1 and liquid path unit structures to avoid shearing damage of cells, solve the problems of long separation time (15 min) and low harvesting efficiency in the prior art, only need 3min for separation time when adopting the cell harvesting system, and realize cell harvesting with low damage, high flux, high efficiency and high recovery rate.
It will be apparent to those skilled in the art that various changes and modifications may be made in the embodiments of the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A low-damage continuous-flow cell harvesting system is characterized by comprising a centrifugal unit, an electromechanical unit, a liquid path unit and a human-computer interaction unit;
the centrifugal unit is used for realizing the centrifugation of cell fluid;
the electromechanical unit comprises a base, a centrifugal bin, a centrifugal motor and a liquid hanging frame; the centrifugal bin is arranged in the base, is used for accommodating the centrifugal unit and is provided with a temperature control module; the centrifugal motor is used for driving the centrifugal unit to rotate; the liquid hanging rack is arranged at the top of the base;
the liquid path unit comprises a plurality of liquid bags, an air filter, a peristaltic pump, a sterile catheter and an electromagnetic pinch valve; the air filter is communicated with the centrifugal unit, the peristaltic pump is communicated with the centrifugal unit, and the peristaltic pump is communicated with the liquid bag through sterile conduits, and electromagnetic pinch valves for controlling the on-off are arranged in the sterile conduits; the liquid bag comprises a waste liquid bag, a cell sample bag, a cleaning solution bag, a formula solution bag and a product bag, wherein the cell sample bag, the cleaning solution bag, the formula solution bag and the product bag are hung on the liquid hanging rack;
the liquid hanging frame is also provided with weighing sensors which are in one-to-one correspondence with the liquid bags, and the weighing sensors are used for measuring the weight of the corresponding liquid bags;
the human-computer interaction unit comprises a control panel and a touch screen display; according to the information input by the touch screen display and the weighing sensor, the control panel controls the centrifugal motor, the temperature control module, the peristaltic pump and the electromagnetic pipe clamping valve to act.
2. The cell harvesting system of claim 1, wherein the centrifuge unit comprises a centrifuge cup and a push plate fixedly mounted within the centrifuge cup;
a gap for balancing the liquid pressure at two sides of the liquid pushing plate is arranged between the liquid pushing plate and the inner peripheral wall of the centrifugal cup;
the centrifugal motor is in transmission connection with the centrifugal cup and is used for driving the centrifugal cup to rotate.
3. The cell harvesting system of claim 2, wherein the centrifugation unit further comprises a first flow channel and a second flow channel;
the first flow channel is used for communicating a first port arranged at the top of the centrifugal cup with a second port arranged at the bottom of the side wall of the centrifugal cup;
the second flow passage is used for communicating a third port arranged at the top of the centrifugal cup with a fourth port arranged at the top of the side wall of the centrifugal cup;
the air filter and the plurality of liquid bags are each in communication with the first port and the second port via the peristaltic pump.
4. The cell harvesting system of claim 3, wherein the centrifuge unit further comprises a rotary adapter mounted to the bottom of the centrifuge cup, a first seal mounted between the first flow channel and the centrifuge cup, and a second seal mounted between the second flow channel and the centrifuge cup;
the rotary adapter is in transmission connection between the centrifugal cup and the centrifugal motor;
the bottom of the centrifugal cup is provided with a chamfer angle of 5-50 degrees.
5. The cell harvesting system of claim 3, wherein the fluid path unit further comprises a first bubble sensor, a second bubble sensor, and a third bubble sensor;
the inlet of the first bubble sensor is communicated with the cell sample bag, the cleaning solution bag and the formula solution bag, and the outlet of the first bubble sensor is communicated with the first port of the peristaltic pump through a first branch;
the inlet of the second bubble sensor is communicated with the first interface of the peristaltic pump, and the outlet of the second bubble sensor is communicated with the product bag;
one end of the third bubble sensor is communicated with the second port of the peristaltic pump, and the other end of the third bubble sensor is communicated with the first port through a third branch;
the first interface of the peristaltic pump is also communicated with the waste liquid bag.
6. The cell harvesting system of claim 5, wherein the rack further comprises a bracket mounted on top of the base, a rack fixedly mounted on top of the bracket, and a hook attached to each of the load cells; each weighing sensor is arranged on the liquid hanging rod;
and one liquid bag is hung at the bottom of each hook.
7. The cell harvesting system of any one of claims 1-6, wherein the temperature control module is a semiconductor thermoelectric cooling device.
8. A cell harvesting method using the cell harvesting system according to any one of claims 3 to 7, wherein the cell harvesting method generates a fluid driving force by a peristaltic pump drive to separate, concentrate, wash and harvest cell fluid containing supernatant, comprising the steps of:
installing a centrifugal unit, a liquid bag and a sterile catheter, and keeping each electromagnetic pinch valve in a normally closed state;
removing the cell supernatant;
washing cell sap;
concentrating the cell sap;
and (6) subpackaging the cell sap.
9. A method for harvesting cells according to claim 8, wherein the step of removing the cell supernatant comprises in particular: opening electromagnetic pinch valves between the peristaltic pump and the cell sample bag and between the peristaltic pump and the first port, opening the peristaltic pump, introducing the cell sample solution in the cell sample bag into the centrifugal unit through the first flow channel, and closing all the electromagnetic pinch valves after introducing a predetermined amount of the cell sample solution; opening an electromagnetic pinch valve between the cell sample bag and the centrifugal unit, so that the cell sample solution in the cell sample bag is introduced into the second flow channel through the self-gravity; starting a centrifugal motor, controlling the rotating speed of the motor to be a first rotating speed, completing the separation of cells and supernatant under the pushing of a liquid pushing plate, and connecting a first flow channel and a second flow channel through a vertical centrifugal solution in a centrifugal unit; keeping the opening state of an electromagnetic pinch valve between the cell sample bag and the centrifugal unit, opening a peristaltic pump, the electromagnetic pinch valve between the peristaltic pump and the first flow channel and the electromagnetic pinch valve between the peristaltic pump and the waste liquid bag, increasing the rotating speed of the centrifugal motor to a second rotating speed, guiding the supernatant to the waste liquid bag from the first flow channel by virtue of fluid flow power provided by the peristaltic pump, attaching the cells separated by the supernatant to the inner wall surface of the centrifugal cup until the cell sample solution in the cell sample bag is completely guided to the centrifugal unit, and guiding all the supernatant to the waste liquid bag;
the step of washing the cell sap specifically comprises: guiding the cell cleaning solution in the cleaning solution bag to a centrifuge cup, cleaning the centrifuge cup through the cell cleaning solution, and guiding the cleaning waste liquid to a waste liquid bag through a first flow channel by a peristaltic pump;
the cell sap concentration step specifically comprises: reducing the rotating speed of the centrifugal motor from the second rotating speed to a third rotating speed so as to guide the waste liquid to a waste liquid bag under the action of a peristaltic pump to finish the concentration of the cell liquid;
the step of subpackaging the cell sap specifically comprises the following steps: closing the peristaltic pump and all electromagnetic pinch valves, opening the electromagnetic pinch valves between the formula solution bag and the peristaltic pump and between the peristaltic pump and the first port, opening the peristaltic pump, guiding the final product formula solution in the formula solution bag to the centrifugal cup, closing the peristaltic pump and all electromagnetic pinch valves, and performing multiple mixing operations through the centrifugal motor to fully mix the adherent cells with the final product formula solution; closing the peristaltic pump and all solenoid pinch valves; and opening electromagnetic pinch valves between the peristaltic pump and the first port and between the peristaltic pump and the product bag to subpackage the product bag.
10. A cell harvesting method according to claim 9, wherein in the step of sub-packaging the cell sap, a plurality of blending operations of forward rotation, sudden stop and reverse rotation are sequentially performed by a centrifugal motor;
the step of washing the cell sap is completed by a uniform washing method or a continuous flow washing method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116392857A (en) * | 2023-06-08 | 2023-07-07 | 深圳赛桥生物创新技术有限公司 | Sample collection method and device |
| CN117089458A (en) * | 2023-10-19 | 2023-11-21 | 南京溯远基因科技有限公司 | Device and method for automatic cell culture, gene editing, cell sorting and split charging |
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2022
- 2022-07-04 CN CN202210786452.1A patent/CN115161164A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116392857A (en) * | 2023-06-08 | 2023-07-07 | 深圳赛桥生物创新技术有限公司 | Sample collection method and device |
| CN117089458A (en) * | 2023-10-19 | 2023-11-21 | 南京溯远基因科技有限公司 | Device and method for automatic cell culture, gene editing, cell sorting and split charging |
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