A low-damage, continuous-flow cell harvesting system and method

Abstract

The present invention relates to a low-damage, continuous-flow cell harvesting system and method, the cell harvesting system comprises a centrifugal unit, an electromechanical unit, a liquid circuit unit and a human-machine interaction unit; the centrifugal unit is used to realize the centrifugation of cell fluid; the electromechanical unit comprises a machine base, a centrifugal chamber, a centrifugal motor and a liquid hanging rack; the centrifugal chamber is used to accommodate the centrifugal unit and has a temperature control module; the centrifugal motor is used to drive the centrifugal unit to rotate; the liquid circuit unit comprises a peristaltic pump, an electromagnetic clamp valve, a sterile catheter, a plurality of liquid bags and an air filter; the human-machine interaction unit comprises a control panel and a touch screen display for inputting / outputting information. The above-mentioned cell harvesting system has the characteristics of low damage, high throughput, high efficiency, high recovery rate and full closure, which can significantly improve the shortcomings of low cell biological activity in the prior art and meet the needs of harvesting large-volume cell sample solutions.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a low-damage, continuous-flow cell harvesting system and method. Background Art

[0002] Cell therapy is a treatment method that injects cell drugs into the patient's body to treat diseases. For example, in cell immunotherapy, after T lymphocytes (T-lymphocytes) as cell drugs are transferred into the patient's body, cell-mediated immunity can be used to fight cancer cells. For example, transplanting stem cells as cell drugs into the patient's body can promote tissue regeneration in damaged areas. However, the production process of cell drugs has many preparation links and complex processes, and the complex process operations and the in vitro environment of cells have a significant impact on the biological activity of cells. In particular, after completing the in vitro expansion and culture of cells, a step of harvesting the suspended cell final product is required.

[0003] The two schemes commonly used in existing equipment are the centrifugal device harvesting scheme and the filtration device harvesting scheme. The centrifugal device harvesting scheme centrifuges the cultured sample, and after the cell sample and the supernatant are separated, the supernatant is removed, the cells are washed, and the cells are concentrated and packaged. However, the fluid shear force in the centrifugal cup during the centrifugation process will cause high shear damage to the cells, resulting in a reduction in cell biological activity. In addition, the filtration device harvesting scheme filters the cell sample containing the supernatant through a filter, which can filter out the supernatant while intercepting the cells. However, the flow resistance of the filter is large, resulting in low separation efficiency, and long-term treatment will also significantly reduce the cell biological activity.

[0004] Therefore, the existing technology not only has the defects of low efficiency and high damage, but also has the disadvantage of low throughput, that is, it is difficult to meet the demand for harvesting large volumes of cell sample solutions. Summary of the invention

[0005] The present invention provides a low-damage, continuous-flow cell harvesting system and method. The cell harvesting system has the characteristics of low damage, high throughput, high efficiency, high recovery rate, and full closure. It can significantly improve the shortcomings of low cell biological activity in the prior art and meet the needs of harvesting large-volume cell sample solutions.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a low-damage, continuous-flow cell harvesting system, which includes a centrifugal unit, an electromechanical unit, a liquid circuit unit, and a human-machine interaction unit;

[0008] The centrifugal unit is used to achieve centrifugation of the cell fluid;

[0009] The electromechanical unit comprises a base, a centrifugal chamber, a centrifugal motor and a liquid hanging rack; the centrifugal chamber is arranged in the base, used to accommodate the centrifugal unit, and has a temperature control module; the centrifugal motor is used to drive the centrifugal unit to rotate; the liquid hanging rack is arranged on the top of the base;

[0010] The liquid circuit unit includes a plurality of liquid bags, an air filter, a peristaltic pump, a sterile catheter and an electromagnetic pinch valve; the air filter and the centrifugal unit, the peristaltic pump and the centrifugal unit, and the peristaltic pump and the liquid bag are all connected through a sterile catheter, and an electromagnetic pinch valve for controlling the on-off is installed in each sterile catheter; the liquid bag includes a waste liquid bag and a cell sample bag, a cleaning solution bag, a formula solution bag and a product bag hung on the liquid hanging rack;

[0011] The liquid hanging rack is also provided with a weighing sensor corresponding to each of the liquid bags, and the weighing sensor is used to measure the weight of the corresponding liquid bag;

[0012] The human-machine interaction unit includes 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 actions of the centrifugal motor, the temperature control module, the peristaltic pump and the electromagnetic pinch valve.

[0013] Furthermore, the centrifugal unit includes a centrifugal cup and a liquid pushing plate fixedly installed in the centrifugal cup;

[0014] A gap is provided between the liquid pushing plate and the inner peripheral wall of the centrifugal cup for balancing the liquid pressure on both sides of the liquid pushing plate;

[0015] The centrifugal motor is drivingly connected to the centrifugal cup and is used to drive the centrifugal cup to rotate.

[0016] Furthermore, the centrifugal unit further comprises a first flow channel and a second flow channel;

[0017] The first flow channel is used to connect a first port disposed on the top of the centrifuge cup and a second port disposed on the bottom of the side wall of the centrifuge cup;

[0018] The second flow channel is used to connect the third port disposed on the top of the centrifugal cup and the fourth port disposed on the top of the side wall of the centrifugal cup;

[0019] The air filter and the plurality of fluid bags are connected to the first port and the second port through the peristaltic pump.

[0020] Furthermore, the centrifugal unit further comprises a rotating adapter installed at the bottom of the centrifugal cup, a first sealing ring installed between the first flow channel and the centrifugal cup, and a second sealing ring installed between the second flow channel and the centrifugal cup;

[0021] The rotary adapter is transmission-connected between the centrifugal cup and the centrifugal motor;

[0022] The bottom of the centrifugal cup is provided with a chamfer of 5° to 50°.

[0023] Furthermore, the liquid path unit further includes a first bubble sensor, a second bubble sensor and a third bubble sensor;

[0024] The inlet of the first bubble sensor is connected to the cell sample bag, the cleaning solution bag and the formula solution bag, and the outlet is connected to the first interface of the peristaltic pump through a first branch;

[0025] The inlet of the second bubble sensor is communicated with the first interface of the peristaltic pump, and the outlet is communicated with the product bag;

[0026] One end of the third bubble sensor is connected to the second interface of the peristaltic pump, and the other end is connected to the first port through a third branch;

[0027] The first interface of the peristaltic pump is also connected to the waste liquid bag.

[0028] Furthermore, the liquid hanging rack further comprises a bracket mounted on the top of the base, a liquid hanging rod fixedly mounted on the top of the bracket, and a hook hung on each of the weighing sensors; each of the weighing sensors is mounted on the liquid hanging rod;

[0029] A liquid bag is hung on the bottom of each hook.

[0030] Furthermore, the temperature control module is a semiconductor thermoelectric cooling device.

[0031] In addition, the present invention also provides a cell harvesting method using the above cell harvesting system, wherein the cell harvesting method generates a fluid driving force by driving a peristaltic pump to separate, concentrate, wash and harvest the cell fluid containing the supernatant, and comprises the following steps:

[0032] Install the centrifugal unit, liquid bag and sterile catheter, and keep each solenoid pinch valve in the normally closed state;

[0033] Remove the cell supernatant;

[0034] Wash the cell fluid;

[0035] Concentrate cell fluid;

[0036] Aliquot the cell suspension.

[0037] Furthermore, the step of removing the cell supernatant specifically includes: opening the electromagnetic pinch valves between the peristaltic pump and the cell sample bag and between the peristaltic pump and the first port, starting 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 the 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 by its own gravity; starting the centrifugal motor, controlling the motor speed to a first speed (a speed within the range of 1500RPM to 2000RPM), and completing the separation of cells and supernatant under the push of the liquid push plate, At this time, the first flow channel and the second flow channel are connected through the vertical centrifugal solution in the centrifugal unit; the electromagnetic clamp valve between the cell sample bag and the centrifugal unit is kept open, and the peristaltic pump, the electromagnetic clamp valve between the peristaltic pump and the first flow channel, and the electromagnetic clamp valve between the peristaltic pump and the waste liquid bag are turned on, and the speed of the centrifugal motor is increased to a second speed (a speed in the range of 2000RPM to 3000RPM), and the supernatant is diverted from the first flow channel to the waste liquid bag by the fluid flow force provided by the peristaltic pump, and the cells separated by the supernatant are attached to the inner wall surface of the centrifugal cup until the cell sample solution in the cell sample bag is completely diverted to the centrifugal unit and all the supernatant is diverted to the waste liquid bag;

[0038] The step of washing the cell solution specifically includes: directing the cell washing solution in the washing solution bag to the centrifuge cup, washing the centrifuge cup with the cell washing solution, and directing the washing waste liquid to the waste liquid bag through the first flow channel by the peristaltic pump;

[0039] The step of concentrating the cell fluid specifically includes: reducing the rotation speed of the centrifugal motor from the second rotation speed to a third rotation speed (a rotation speed in the range of 500RPM to 100RPM) so as to guide the waste liquid to the waste liquid bag under the action of the peristaltic pump to complete the concentration of the cell fluid;

[0040] The steps of packaging the cell solution specifically include: 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, starting the peristaltic pump, and directing the final product formula solution in the formula solution bag to the centrifuge cup, closing the peristaltic pump and all electromagnetic pinch valves, performing multiple mixing operations through a centrifugal motor to fully mix the adhered cells with the final product formula solution; closing the peristaltic pump and all electromagnetic pinch valves; opening the electromagnetic pinch valves between the peristaltic pump and the first port, and between the peristaltic pump and the product bag, and packaging the product bag.

[0041] Furthermore, in the step of dispensing the cell solution, the centrifugal motor is used to perform multiple mixing operations of forward rotation, emergency stop, and reverse rotation in sequence;

[0042] The step of washing the cell solution is completed by a mixing washing method or a continuous flow washing method.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] In the cell harvesting system of the present invention, the centrifugal unit is fixedly installed with a liquid push plate in the centrifugal cup, and the centrifugal cup is driven to rotate by a centrifugal motor. During the centrifugal cup's self-rotation centrifugation, the cell fluid in the centrifugal cup is pushed by the liquid push plate and rotates synchronously with the centrifugal cup. The liquid push plate can provide a fluid driving force for the centrifugal unit, thereby avoiding the cells in the sample solution from being sheared during the centrifugal movement, significantly reducing the shear damage to the cells during the centrifugal process, significantly improving the biological activity of the cell harvest, and also improving the separation efficiency and reducing the time required for separation; the liquid path unit structure and cell harvesting method of the present invention can be used to continuously flow (similar to the siphon effect) the separation supernatant, and the efficient removal of the continuous flow of the cell supernatant is completed with a small volume cup. Therefore, the cell harvesting system and method of the present invention avoid the shear damage of the cells through a simple structure, and solve the problems of long separation time and low harvesting efficiency in the prior art, and realize low damage, high throughput, high efficiency, and high recovery rate cell harvesting. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the overall structure of the cell harvesting system of the present invention;

[0046] Figure 2 for Figure 1 A cross-sectional view of a centrifugal unit portion of a cell harvesting system;

[0047] Figure 3 for Figure 1 Schematic diagram of the structure of the liquid circuit unit of the cell harvesting system;

[0048] Figure 4 for Figure 2 Schematic diagram of the overall structure of the centrifugal unit of the cell harvesting system;

[0049] Figure 5 for Figure 4 A cross-sectional view of the centrifugal unit;

[0050] Figure 6 It is the temperature control scheme of the temperature control module and the heat transfer relationship diagram of the centrifugal chamber;

[0051] Figure 7 The figure is a flow chart of the cell harvesting method of the present invention.

[0052] Among them, 1-centrifugal unit, 2-centrifugal cup, 3-push plate, 4-first flow channel, 5-second flow channel, 6-first port, 7-second port, 8-third port, 9-fourth port, 10-base, 11-centrifugal chamber, 12-centrifugal motor, 13-bracket, 14-liquid hanging 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 Solenoid pinch valve, 24-sixth solenoid pinch valve, 25-seventh solenoid pinch valve, 26-eighth solenoid pinch valve, 27-ninth solenoid pinch valve, 28-tenth solenoid pinch valve, 29-eleventh solenoid pinch valve, 30-twelfth solenoid pinch valve, 31-thirteenth solenoid pinch valve, 32-product bag, 33-waste liquid bag, 34-cell sample bag, 35-cleaning solution bag, 36-formulated solution bag, 37-first bubble sensor, 38-second bubble sensor, 39-third bubble sensor, 40-filter device DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Embodiment 1

[0055] This embodiment provides a low-damage, continuous-flow cell harvesting system. Figure 1 , Figure 2 , Figure 3 and Figure 4 , the cell harvesting system comprises a centrifugal unit 1, an electromechanical unit, a liquid circuit unit and a human-machine interaction unit;

[0056] like Figure 4 and Figure 5 As shown in the structure, the centrifugal unit 1 is used to realize the centrifugation of the cell fluid, including a centrifugal cup 2, a liquid pushing plate 3, a first flow channel 4 and a second flow channel 5; the liquid pushing plate 3 is fixedly installed in the centrifugal cup 2, and the number of the liquid pushing plates 3 can be one or more; when multiple liquid pushing plates 3 are arranged in the centrifugal cup 2, the multiple liquid pushing plates 3 are evenly distributed along the circumference of the centrifugal cup 2; the first flow channel 4 is used to connect the first port 6 arranged at the top of the centrifugal cup 2 and the second port 7 arranged at the bottom of the side wall of the centrifugal cup 2; the second flow channel 5 is used to connect the third port 8 arranged at the top of the centrifugal cup 2 and the fourth port 9 arranged at the top of the side wall of the centrifugal cup 2;

[0057] like Figure 1As shown in the structure, the electromechanical unit includes a base 10, a centrifugal chamber 11, a centrifugal motor 12 and a liquid hanging rack; the centrifugal chamber 11 is arranged in the base 10, for accommodating the centrifugal unit 1, and has a temperature control module. The temperature control module can use a semiconductor thermoelectric cooling device to achieve temperature control of 4°C to 40°C in the centrifugal chamber 11, and the control error is less than 1°C. The temperature control module can improve the biological activity of cells placed in the environment for a long time, especially in the freezing solution and packaging preparation link containing DMSO (Dimethyl sulfoxide), so as to maintain the low-temperature non-toxic effect of the freezing solution containing DMSO at 4°C, thereby reducing the effect of the freezing solution containing DMSO on cell activity. Among them, the temperature control scheme of the temperature control module and the heat transfer relationship of the centrifugal chamber 11 are shown as follows. Figure 6 As shown;

[0058] The centrifugal chamber 11 may also be provided with a hatch that can be opened and closed, for covering the centrifugal unit 1 in the centrifugal chamber 11; the centrifugal motor 12 is used to drive the centrifugal unit 1 to rotate; the centrifugal motor 12 is transmission-connected to the centrifugal cup 2, for driving the centrifugal cup 2 to rotate, thereby driving the liquid push plate 3 to rotate through the centrifugal cup 2; the liquid hanging rack includes a bracket 13 arranged on the top of the machine base 10, a liquid hanging rod 14 fixedly installed on the top of the bracket 13, a plurality of weighing sensors installed on the liquid hanging rod 14, and a hook hung on each weighing sensor; the weighing sensor is used to monitor the weight of the liquid bag hung on the hook in real time, and the measurement error is less than 1g; the hook on the lower side of the weighing sensor can be vertically set one, or symmetrically set two;

[0059] like Figure 1 , Figure 2 and Figure 3 As shown, the liquid circuit unit includes a plurality of liquid bags, an air filter for sterile filtering of air, a sterile catheter, a peristaltic pump 15 for providing power to the fluid in the sterile catheter, and an electromagnetic clamp valve for controlling the opening and closing of the corresponding sterile catheter; a sterile catheter is connected 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 an electromagnetic clamp valve for controlling the opening and closing is installed in each sterile catheter; the air filter and the plurality of liquid bags are connected to 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; an electromagnetic clamp valve is provided between the peristaltic pump 15 and each liquid bag, and between the peristaltic pump 15 and the first port 6; the liquid bag includes at least one waste liquid bag 33 and at least one cell sample bag 34 hung on a hook, at least one cleaning solution bag 35, at least one formula solution bag 36, and at least one product bag 32, as shown in FIG. Figure 1 and Figure 2As shown in the structure, two cell sample bags 34, three cleaning solution bags 35, two formula solution bags 36, and three product bags 32 are hung on multiple hooks at the bottom of the liquid hanging rod 14, and two waste liquid bags 33 are located at the bottom of the liquid path unit and are hung on the machine base 10, so that the waste liquid can flow in under the action of the peristaltic pump 15 and gravity; three bubble sensors are also arranged in the liquid path unit, namely 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 connected to the cell sample bag 34, the cleaning solution bag 35 and the formula solution bag 36, and the first bubble sensor The outlet of the second bubble sensor 37 is connected to the first interface of the peristaltic pump 15 through the first branch; the inlet of the second bubble sensor 38 is connected to the first interface of the peristaltic pump 15, and the outlet of the second bubble sensor 38 is connected to the product bag 32, and the cell liquid is dispensed into the product bag 32 through the second bubble sensor 38; two or more 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 the filtering devices 40 can filter and remove cell clusters and debris in the final product, thereby avoiding the occurrence of needle blockage and improving the accuracy of the solution concentration, which is beneficial to cell reinfusion and cell cryopreservation;

[0060] One end of the third bubble sensor 39 is connected to the second interface of the peristaltic pump 15, and the other end is connected to the first port 6 of the first flow channel 4 through the third branch. A sampling connector can also be provided at the other end of the third bubble sensor 39;

[0061] At the same time, the first interface of the peristaltic pump 15 is also connected to the waste liquid bag 32;

[0062] like Figure 3As shown in the figure, for the convenience of description, the electromagnetic pinch valve includes a first electromagnetic pinch valve 19, a second electromagnetic pinch valve 20, a third electromagnetic pinch valve 21, a fourth electromagnetic pinch valve 22, a fifth electromagnetic pinch valve 23, a sixth electromagnetic pinch valve 24, a seventh electromagnetic pinch valve 25, an eighth electromagnetic pinch valve 26, a ninth electromagnetic pinch valve 27, a tenth electromagnetic pinch valve 28, an eleventh electromagnetic pinch valve 29, a twelfth electromagnetic pinch valve 30, and a thirteenth electromagnetic pinch valve 31; the first electromagnetic pinch valve 19 is installed between the air filter and the third end of the second flow channel 5. The air filter can balance the air pressure in the centrifuge cup 2 through the air filter to prevent the air pressure in the centrifuge cup 2 from being too low (negative pressure) during liquid extraction, resulting in poor liquid extraction effect and increased sampling error of the peristaltic pump; the second electromagnetic pinch valve 20 is installed in the sterile conduit connecting the cleaning solution bag 35 and the first bubble sensor 37; the third electromagnetic pinch valve 21 is installed in the sterile conduit connecting the formula solution bag 36 and the first bubble sensor 37; the fourth electromagnetic pinch valve 22 is installed in the sterile conduit connecting the cell sample bag 34 and the first bubble sensor 37. The three product bags 32 are connected to the second bubble sensor 38 through the fifth electromagnetic pinch valve 23, the sixth electromagnetic pinch valve 24, and the seventh electromagnetic pinch valve 25 respectively; the eighth electromagnetic pinch valve 26 is installed in the sterile conduit between the first bubble sensor 37 and the third port 8 of the second flow channel 5; the ninth electromagnetic pinch valve 27 is installed in the sterile conduit connecting the first bubble sensor 37 and the first interface of the peristaltic pump 15; the peristaltic pump 15 and the cleaning solution bag 35, the formula solution bag 36 and the cell sample bag 34 are connected and disconnected through the eighth electromagnetic pinch valve 26. The tube valve 26 and the ninth electromagnetic pinch valve 27 are controlled simultaneously; the tenth electromagnetic pinch valve 28 is installed in the sterile conduit connecting the third bubble sensor 39 and the first port 6 of the first flow channel 4; the eleventh electromagnetic pinch valve 29 is installed in the sterile conduit connecting the first interface of the peristaltic pump 15 and the waste liquid bag 33; the twelfth electromagnetic pinch valve 30 is installed in the sterile conduit connecting the first interface of the peristaltic pump 15 and the inlet of the second bubble sensor 38; the thirteenth electromagnetic pinch valve 31 is installed in the sterile conduit between the tenth electromagnetic pinch valve 28 and the sampling joint;

[0063] like Figure 1 and Figure 2 As shown, the human-computer interaction unit includes a control panel 16 and a touch screen display 17 for inputting / outputting information; the control panel 16 is connected to 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 for signal connection, and is used to control the action of the centrifugal motor 12, the temperature control module, the peristaltic pump 15, and the electromagnetic pinch valve according to the information input by the touch screen display 17, the weighing sensor, and the bubble sensor.

[0064] During the operation of the above-mentioned cell harvesting system, the centrifugal unit 1 is installed in the centrifugal chamber 11, the centrifugal motor 12 is connected to the centrifugal cup 2, and the liquid pushing plate 3 is fixedly installed in the centrifugal cup 2. The centrifugal cup 2 is driven to rotate by the centrifugal motor 12, thereby driving the liquid pushing plate 3 to rotate with the centrifugal cup 2. During the centrifugal rotation of the centrifugal cup 2, the cell fluid in the centrifugal cup 2 is pushed by the liquid pushing plate 3 and rotates synchronously with the centrifugal cup 2. The liquid pushing plate 3 can provide a fluid driving force for the cell fluid in the centrifugal cup 2, thereby avoiding the cells in the sample solution from being sheared during the centrifugal movement, which significantly The shear damage to cells during centrifugation is reduced, the biological activity of cell harvesting is significantly improved, the separation efficiency is improved, the time required for separation is reduced, and the biological activity and efficiency of cell harvesting are significantly improved; since the liquid circuit unit of the above-mentioned cell harvesting system is provided with a peristaltic pump 15 and an electromagnetic pinch valve, and a first flow channel 4 and a second flow channel 5 are provided in the centrifugal cup 2, the supernatant can be separated in a continuous flow (similar to a siphon effect) through the control of the peristaltic pump 15 and the electromagnetic pinch valve, such as completing the efficient removal of 3L of continuous flow cell supernatant with a small volume cup of 300mL. Therefore, the cell harvesting system and method of the present invention avoids the shear damage of cells through a simple structure, and solves the problems of long separation time and low harvesting efficiency in the prior art, and realizes low-damage, high-throughput, high-efficiency, high-recovery, and fully enclosed cell harvesting.

[0065] like Figure 5 As shown in the structure, a gap 18 is provided between the liquid pushing plate 3 and the inner peripheral wall of the centrifugal cup 2 for balancing the liquid pressure on both sides of the liquid pushing plate 3. The bottom of the centrifugal cup 2 is provided with a chamfer of 5° to 50°, such as: 5°, 10°, °, 20°, 25°, 30°, 35°, 40°, 45°, 50°. The chamfer provided at the bottom of the centrifugal cup 2 facilitates the discharge of liquid, so that the residual liquid volume in the centrifugal cup 2 is 0.1mL to 10mL; when the chamfer is 20°, the residual liquid volume in the centrifugal cup 2 can be only 0.2mL. The centrifugal unit 1 also includes a first sealing ring installed between the first flow channel 4 and the centrifugal cup 2, and a second sealing ring installed between the second flow channel 5 and the centrifugal cup 2. The centrifugal cup 2 can be sealed by the first sealing ring and the second sealing ring.

[0066] On the basis of the above-mentioned various embodiments, the centrifugal unit 1 further comprises a rotating adapter mounted on the bottom of the centrifugal cup 2, and the rotating adapter is transmission-connected between the centrifugal cup 2 and the centrifugal motor 12. The rotating adapter realizes power transmission between the centrifugal cup 2 and the outer centrifugal motor 12, so that the liquid pusher plate 3 rotates, thereby realizing the centrifugal pumping function with high efficiency and low damage.

[0067] Embodiment 2

[0068] This embodiment provides a cell harvesting method using the above cell harvesting system, such as Figure 7 As shown, the cell harvesting method generates fluid driving force by driving a peristaltic pump 15 to separate, concentrate, wash and harvest the cell fluid containing the supernatant, and includes the following steps:

[0069] Step S10, installing the centrifugal unit 1, the liquid bag and the sterile catheter, and keeping each electromagnetic pinch valve in a normally closed state;

[0070] Step S20, removing the cell supernatant: opening the electromagnetic pinch valve 19, the electromagnetic pinch valve between the peristaltic pump 15 and the cell sample bag 34, and the electromagnetic pinch valve between the peristaltic pump 15 and the first port 6, that is, opening Figure 3 The fourth electromagnetic pinch valve 22, the ninth electromagnetic pinch valve 27 and the tenth electromagnetic pinch valve 28 in the cell sample bag 34 are opened, the peristaltic pump 15 is turned on, and the cell sample solution in the cell sample bag 34 is introduced into the centrifugal unit 1 through the first flow channel 4. After the predetermined amount (150 mL) of the cell sample solution is introduced, all the electromagnetic pinch valves are closed; the electromagnetic pinch valve between the cell sample bag 34 and the centrifugal unit 1 is opened, that is, the Figure 3 The fourth electromagnetic clamp valve 22 and the eighth electromagnetic clamp valve 26 in the centrifugal cup 2 allow the cell sample solution in the cell sample bag 34 to be introduced into the second flow channel 5 by its own gravity. At this time, the pressure in the centrifugal cup 2 is higher than the atmospheric pressure, and the first flow channel 4 and the second flow channel 5 are both exhausted of gas and filled with liquid; the centrifugal motor 12 is started, and the motor speed is controlled to be a first speed (a speed in the range of 1500RPM to 2500RPM), and the liquid pushing plate 3 of the centrifugal unit 1 begins to rotate and centrifuge along the central axis. Under the push of the liquid pushing plate 3, since the density of cells in the cell sample solution is the largest, the cells will stick to the inner wall surface of the centrifugal cup 2. Under the push of the liquid pushing plate 3, the separation of cells and supernatant can be completed in 3 minutes. At this time, the first flow channel 4 and the second flow channel 5 are connected by the vertical centrifugal solution in the centrifugal unit 1; the electromagnetic clamp valve between the cell sample bag 34 and the centrifugal unit 1 is kept open, that is, keep Figure 3 The fourth electromagnetic pinch valve 22 and the eighth electromagnetic pinch valve 26 in the first flow channel 4 are in the open state, and 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 are opened, that is, the fourth electromagnetic pinch valve 22 and the eighth electromagnetic pinch valve 26 in the first flow channel 4 are in the open state, and 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 are opened. Figure 3The peristaltic pump 15, the tenth electromagnetic pinch valve 28 and the eleventh electromagnetic pinch valve 29 increase the speed of the centrifugal motor 12 to the second speed (a speed in the range of 2000RPM to 3000RPM), and the supernatant is guided from the first flow channel 4 to the waste liquid bag 33 by the fluid flow force provided by the peristaltic pump 15. At the same time, due to the height difference of the peristaltic pump, the liquid pressure of the waste liquid bag 33 is lower than the liquid pressure of the cell sample bag 34. The driving force generated by the liquid pressure will continuously guide the cell sample solution in the cell sample bag 34 to the centrifugal cup 2, and the cells separated by the supernatant are attached to the centrifugal cup 2. The supernatant is directed from the first flow channel 4 to the waste liquid bag 33 by the fluid flow force provided by the peristaltic pump 15, until the cell sample solution in the cell sample bag 34 is completely directed to the centrifugal unit 1 and all the supernatant is directed to the waste liquid bag 33, that is, the efficient removal of 3L of continuous flow cell supernatant can be completed in a 300mL small volume cup; in order to further reduce the residual amount of the supernatant, the speed of the centrifugal motor 12 can be reduced from the second speed to the third speed (a speed within the range of 500RPM to 1000RPM), so that more supernatant can be directed to the waste liquid bag 33 under the action of the peristaltic pump and gravity;

[0071] Step S30, washing the cell fluid: directing the cell washing solution in the washing solution bag 35 to the centrifuge cup 2, washing the centrifuge cup 2 with the cell washing solution, and directing the washing waste liquid to the waste liquid bag 33 through the first flow channel 4 via the peristaltic pump 15; in the process of washing the cell fluid, a mixing washing method or a continuous flow washing method may be used; wherein:

[0072] The cleaning steps of the mixing and cleaning method are as follows: close the peristaltic pump 15 and all electromagnetic pinch valves, open the second electromagnetic pinch valve 20, the ninth electromagnetic pinch valve 27, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15, guide the cell cleaning solution in the cleaning solution bag 35 to the centrifugal cup 2, close the peristaltic pump 15 and all electromagnetic pinch valves, and perform multiple mixing operations of forward rotation, emergency stop, and reverse rotation of the centrifugal motor 12 in sequence to fully mix the adhered cells with the cleaning solution, and then control the speed of the centrifugal motor 12 to the centrifugal speed (a speed in the range of 2500RPM to 3000RPM), the cells adhere to the wall by centrifugal force, and the waste liquid is guided from the first flow channel 4 to the waste liquid bag 33 by the fluid flow force provided by the peristaltic pump 15;

[0073] The cleaning steps of the continuous flow cleaning method are as follows: close the peristaltic pump 15 and all electromagnetic pinch valves, control the speed of the centrifugal motor 12 to be the centrifugal speed (a speed in the range of 2500RPM to 3000RPM), open 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 centrifuged and attached to the wall, and under the effect of the height difference, the liquid pressure of the waste liquid bag 33 is lower than the liquid pressure of the cleaning solution bag 35, and the liquid pressure in the cleaning solution bag 35 generates a driving force, which will continuously guide the cell cleaning solution in the cleaning solution bag 35 into the centrifugal cup 2, flush the cells attached to the inner wall of the centrifugal cup 2, and at the same time, the waste liquid is guided to the waste liquid bag 33 through the first flow channel 4 by the fluid flow force provided by the peristaltic pump 15, so as to realize the continuous flow cleaning of the cells;

[0074] The above two cleaning methods can be coordinated with each other and performed multiple times to clean the waste liquid in the cell solution to the required index; step S40, concentrating the cell solution: reducing the speed of the centrifugal motor 12 from the second speed to the third speed, so that the waste liquid is guided to the waste liquid bag 33 under the action of the peristaltic pump 15 and gravity, completing the concentration of the cell solution, and further reducing the residual amount of the waste liquid;

[0075] Step S50, packing the cell solution: close the peristaltic pump 15 and all the electromagnetic pinch valves, open the 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, that is, open Figure 3 The third electromagnetic pinch valve 21, the ninth electromagnetic pinch valve 27 and the tenth electromagnetic pinch valve 28 in the centrifugal unit 1 are opened, the peristaltic pump 15 is opened, and the final product formula solution in the formula solution bag 36 is directed to the centrifugal cup 2 of the centrifugal unit 1, the peristaltic pump 15 and all the electromagnetic pinch valves are closed, and the centrifugal motor 12 is used to perform multiple mixing operations of forward rotation, emergency stop and reverse rotation to fully mix the adherent cells with the final product formula solution; the peristaltic pump 15 and all the electromagnetic pinch valves are closed; the 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 pack the product bag 32. When Figure 3 When three product bags 32 are provided as shown, the following steps can be used to pack the three product bags 32: open the fifth electromagnetic pinch valve 23, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 to complete the packing of the first product bag 32; then close the peristaltic pump 15 and all the electromagnetic pinch valves, open the sixth electromagnetic pinch valve 24, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 to complete the packing of the second product bag 32; then close the peristaltic pump 15 and all the electromagnetic pinch valves, open the seventh electromagnetic pinch valve 25, the tenth electromagnetic pinch valve 28 and the peristaltic pump 15 to complete the packing of the third product bag 32.

[0076] The above-mentioned cell harvesting system and cell harvesting method adopt a simple centrifugal unit 1 and a liquid path unit structure to avoid shear damage to the cells, and solve the problems of long separation time (15 minutes) and low harvesting efficiency in the prior art. When the above-mentioned cell harvesting system is used, the separation time only takes 3 minutes, and low-damage, high-throughput, high-efficiency, and high-recovery cell harvesting is achieved.

[0077] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the present invention. Thus, if these 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 these modifications and variations.

Claims

1. A low-damage, continuous-flow cell harvesting system, characterized in that: It includes a centrifugal unit, an electromechanical unit, a liquid circuit unit and a human-computer interaction unit; The centrifugal unit is used to achieve centrifugation of the cell fluid; The electromechanical unit comprises a base, a centrifugal chamber, a centrifugal motor and a liquid hanging rack; the centrifugal chamber is arranged in the base, used to accommodate the centrifugal unit, and has a temperature control module; the centrifugal motor is used to drive the centrifugal unit to rotate; the liquid hanging rack is arranged on the top of the base; The liquid circuit unit includes a plurality of liquid bags, an air filter, a peristaltic pump, a sterile catheter and an electromagnetic pinch valve; the air filter and the centrifugal unit, the peristaltic pump and the centrifugal unit, and the peristaltic pump and the liquid bag are all connected through a sterile catheter, and an electromagnetic pinch valve for controlling the on-off is installed in each sterile catheter; the liquid bag includes a waste liquid bag and a cell sample bag, a cleaning solution bag, a formula solution bag and a product bag hung on the liquid hanging rack; The liquid hanging rack is also provided with a weighing sensor corresponding to each of the liquid bags, and the weighing sensor is used to measure the weight of the corresponding liquid bag; The human-machine interaction unit includes 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 actions of the centrifugal motor, the temperature control module, the peristaltic pump and the electromagnetic pinch valve; The centrifugal unit comprises a centrifugal cup, a liquid pushing plate fixedly installed in the centrifugal cup, a first flow channel, a second flow channel, a rotating adapter installed at the bottom of the centrifugal cup, a first sealing ring installed between the first flow channel and the centrifugal cup, and a second sealing ring installed between the second flow channel and the centrifugal cup; A gap is provided between the liquid pushing plate and the inner peripheral wall of the centrifugal cup for balancing the liquid pressure on both sides of the liquid pushing plate; The centrifugal motor is drivingly connected to the centrifugal cup and is used to drive the centrifugal cup to rotate; The first flow channel is used to connect a first port disposed on the top of the centrifuge cup and a second port disposed on the bottom of the side wall of the centrifuge cup; The second flow channel is used to connect the third port disposed on the top of the centrifugal cup and the fourth port disposed on the top of the side wall of the centrifugal cup; The air filter and the plurality of fluid bags are connected to the first port and the second port through the peristaltic pump; The rotary adapter is transmission-connected between the centrifugal cup and the centrifugal motor; The liquid path unit further includes a first bubble sensor, a second bubble sensor and a third bubble sensor; The inlet of the first bubble sensor is connected to the cell sample bag, the cleaning solution bag and the formula solution bag, and the outlet is connected to the first interface 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 is communicated with the product bag; One end of the third bubble sensor is connected to the second interface of the peristaltic pump, and the other end is connected to the first port through a third branch; The first interface of the peristaltic pump is also connected to the waste liquid bag.

2. The cell harvesting system according to claim 1, characterized in that: The bottom of the centrifugal cup is provided with a chamfer of 5° to 50°.

3. The cell harvesting system according to claim 1, characterized in that The liquid hanging rack further comprises a bracket mounted on the top of the base, a liquid hanging rod fixedly mounted on the top of the bracket, and a hook hung on each of the weighing sensors; each of the weighing sensors is mounted on the liquid hanging rod; A liquid bag is hung on the bottom of each hook.

4. The cell harvesting system according to any one of claims 1 to 3, characterized in that: The temperature control module is a semiconductor thermoelectric cooling device.

5. A cell harvesting method using the cell harvesting system according to any one of claims 1 to 4, characterized in that: The cell harvesting method generates fluid driving force by driving a peristaltic pump to separate, concentrate, wash and harvest the cell fluid containing the supernatant, and comprises the following steps: Install the centrifugal unit, liquid bag and sterile catheter, and keep each solenoid pinch valve in the normally closed state; Remove the cell supernatant; Wash the cell fluid; Concentrate cell fluid; Aliquot the cell suspension.

6. The cell harvesting method according to claim 5, characterized in that: The step of removing the cell supernatant specifically includes: opening the electromagnetic pinch valves between the peristaltic pump and the cell sample bag and between the peristaltic pump and the first port, starting 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 the 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 by its own gravity; starting the centrifugal motor, controlling the motor speed to be the first speed, and completing the separation of the cells and the supernatant under the push of the liquid push plate. At this time, The first flow channel and the second flow channel are connected by a vertical centrifugal solution in the centrifugal unit; the electromagnetic clamp valve between the cell sample bag and the centrifugal unit is kept open, and the peristaltic pump, the electromagnetic clamp valve between the peristaltic pump and the first flow channel, and the electromagnetic clamp valve between the peristaltic pump and the waste liquid bag are turned on, the speed of the centrifugal motor is increased to the second speed, the supernatant is diverted from the first flow channel to the waste liquid bag by the fluid flow force provided by the peristaltic pump, and the cells separated by the supernatant are attached to the inner wall surface of the centrifugal cup until the cell sample solution in the cell sample bag is completely diverted to the centrifugal unit and all the supernatant is diverted to the waste liquid bag; The step of washing the cell solution specifically includes: directing the cell washing solution in the washing solution bag to the centrifuge cup, washing the centrifuge cup with the cell washing solution, and directing the washing waste liquid to the waste liquid bag through the first flow channel by the peristaltic pump; The step of concentrating the cell fluid specifically includes: reducing the rotation speed of the centrifugal motor from the second rotation speed to the third rotation speed, so that the waste liquid is directed to the waste liquid bag under the action of the peristaltic pump to complete the concentration of the cell fluid; The steps of packaging the cell solution specifically include: 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, starting the peristaltic pump, and directing the final product formula solution in the formula solution bag to the centrifuge cup, closing the peristaltic pump and all electromagnetic pinch valves, performing multiple mixing operations through a centrifugal motor to fully mix the adhered cells with the final product formula solution; closing the peristaltic pump and all electromagnetic pinch valves; opening the electromagnetic pinch valves between the peristaltic pump and the first port, and between the peristaltic pump and the product bag, and packaging the product bag.

7. The cell harvesting method according to claim 6, characterized in that: In the cell solution packaging step, the centrifugal motor performs multiple mixing operations of forward rotation, emergency stop, and reverse rotation in sequence; The step of washing the cell solution is completed by a mixing washing method or a continuous flow washing method.

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