A fully automatic and fully enclosed cell preparation workstation and cell processing method

Abstract

The present invention relates to a fully automatic and fully enclosed cell preparation workstation and a cell processing method. The workstation includes: a centrifugal culture unit, including a microfluidic heat exchanger and a centrifuge cup; a liquid path unit, including a pump assembly, a valve assembly and pipelines; a cell digitization unit, including a microscope installed below the centrifuge cup; a host control unit, including a motor, a waste liquid tank and a centrifugal chamber, and connected to the pump assembly, the valve assembly and the microfluidic heat exchanger; a frame fixedly installed on the top of the host control unit for hanging a liquid bag; a housing, hermetically covering the top of the host control unit and the frame. The above workstation has the characteristics of stable quality, production safety, cost saving and high efficiency, can significantly improve the deficiency of low cell biological activity in the prior art, and solve the problems of easy contamination, difficult quality control, high cost and low efficiency in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and particularly to a fully automatic and fully enclosed cell preparation workstation and a cell processing method. Background Art

[0002] Cell therapy is a treatment method that injects cell-based drugs into patients to treat diseases. However, the preparation process of cell-based drugs has many steps and complex processes, and the complex process operations and the cell ex vivo environment have a significant impact on the biological activity of cells.

[0003] The existing cell preparation process for CAR-T cell therapy is as follows: T cell tumor immunotherapy obtains a specific subset of T cells by separating from the peripheral blood of patients or donors, further activates and genetically modifies them to obtain CAR-T cells, and amplifies and cultures them to reach the cell number at the treatment level, and then washes, concentrates and transfuses them into the patient's body for treatment. Therefore, from the separation and collection of T cells, to activation and modification, and then to cell amplification and transfusion into the patient's body, it is a complex process. The traditional manual preparation method faces the following four major challenges: The first is the environmental challenge, and the possibility of contamination during the preparation process is very high; the second is the human difference, and the preferences of each operator will lead to uneven products and differences; the third is that as a highly personalized product, each patient is a production batch, which is quite different from the traditional pharmaceutical process; fourth, the condition of blood tumor patients progresses extremely fast, and it is necessary to shorten the preparation time as much as possible for timely transfusion and improve the success rate. Therefore, the standardization, automation, and quality control such as safety, sterility, and purity of the CAR-T cell production process are the prerequisites for objectively evaluating the remission rate.

[0004] Moreover, the cost of cell preparation for CAR-T cell therapy is relatively high, and the main reasons include the following three points: The first point is that CAR-T, as a highly personalized drug, corresponds to one batch for each patient, and the preparation operation requires multiple high-end technical personnel to perform aseptic and precise operations, and the labor cost is extremely high; the second point is that in order to ensure cleanliness, purity, and the safety of the preparation results, it is necessary to build a large area of specialized aseptic operation areas, and specialized cleaning personnel need to perform regular maintenance, and the operating cost is extremely high; the third is that the stability and safety of the transfected CAR lentivirus have a great impact on the quality and use safety of the CAR-T cell product preparation. The procurement cost of high-quality lentivirus is as high as 50,000 - 100,000 yuan, and it is difficult to solve the stability problem when self-producing lentivirus.

[0005] Therefore, the existing cell preparation technology has problems such as easy contamination, difficult quality control, high cost, and low efficiency, making the current cell preparation process unable to meet the needs of commercial applications. Summary of the Invention

[0006] The present invention provides a fully automatic and fully enclosed cell preparation workstation and a cell processing method. The cell preparation workstation has the characteristics of stable quality, safe production, cost saving and high efficiency. It can significantly improve the shortcomings of low cell biological activity in the prior art and solve the problems of easy contamination, difficult quality control, high cost and low efficiency in the prior art.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a fully automatic and fully enclosed cell preparation workstation, which comprises:

[0009] A centrifugal culture unit, comprising a microfluidic heat exchanger and a centrifugal cup with a centrifugal cavity, for performing a first cell centrifugation process, extracting a primary product solution, concentrating, transfecting and culturing; the centrifugal cup is installed in a centrifugal chamber, comprising an inlet flow channel and an outlet flow channel; the microfluidic heat exchanger is used to keep the centrifugal cup within a culture temperature range during the culture process;

[0010] A liquid circuit unit, comprising a pump assembly, a valve assembly and a pipeline, for supplying and extracting liquid to the centrifuge cup, and harvesting liquid to a storage bag; the pump assembly and the valve assembly are located in the pipeline path;

[0011] A cell digitization unit, comprising a microscope installed below the centrifuge cup, for monitoring the counting, division and proliferation of cells in the centrifuge cup;

[0012] The host control unit includes a motor, a waste liquid tank and the centrifugal chamber, and is connected to the pump assembly, the valve assembly, and the microfluidic heat exchanger, and controls the motor, the pump assembly, the valve assembly, and the microfluidic heat exchanger; the motor is in transmission connection with the centrifugal cup, and is used to drive the centrifugal cup to rotate; the waste liquid tank is used to collect the solution after cleaning and discharge;

[0013] A frame is fixedly mounted on the top of the host control unit and is used to hang a separation liquid bag containing a separation liquid, a sample liquid bag containing a liquid to be separated, a cleaning liquid bag containing a cleaning liquid, a transfection reagent bag containing a transfection reagent, a primary product bag and a storage bag; the cleaning liquid is used for cleaning during the cell separation process;

[0014] The housing and the sealing cover are arranged on the top of the host control unit and the frame.

[0015] Furthermore, the centrifuge cup further comprises a liquid pushing plate, which extends from the center of the centrifuge cup in a radial direction, and a gap is formed between an end of the liquid pushing plate away from the axis and an inner wall of the centrifuge cup.

[0016] Furthermore, the centrifugal cup also includes a cup body and a cup cover;

[0017] The liquid pushing plate is fixedly connected to the cup body and / or the cup lid;

[0018] The cup lid is sealingly connected to the top of the cup body, and a centrifugal chamber is formed between the cup lid and the cup body;

[0019] The liquid pushing plate is circumferentially distributed in the centrifugal chamber along the cup body, and there is a gap between the other end of the liquid pushing plate adjacent to the axis and the axis of the cup body;

[0020] Both the liquid inlet channel and the liquid outlet channel communicate the outside of the cup body and the centrifugal chamber; the liquid inlet channel has a first liquid inlet provided outside the cup body and / or a second liquid inlet provided at the bottom of the inner side wall of the cup body; the liquid outlet channel has a first liquid outlet provided at the top of the centrifugal chamber and / or a second liquid outlet provided at the top of the side wall of the cup body.

[0021] Furthermore, a chamfer of 5° to 50° is formed between the side wall and the bottom wall of the cup body.

[0022] Furthermore, the microfluidic heat exchanger includes:

[0023] A temperature control module for adjusting the ambient temperature of the centrifuge cup in the centrifuge chamber during the culture process, and blowing air through the upper ventilation opening and the lower ventilation opening of the fan to transfer the heat generated by adjusting the ambient temperature;

[0024] The upper ventilation opening is installed above the centrifuge cup for ventilating and transferring the heat generated by the temperature control module above the centrifuge cup;

[0025] The lower ventilation opening is installed below the centrifuge cup for ventilating and transferring the heat generated by the temperature control module below the centrifuge cup.

[0026] In addition, the present invention also provides a cell processing method using a fully automatic and fully enclosed cell preparation workstation, and the cell processing method includes:

[0027] Flowing the separation liquid and the liquid to be separated into the centrifuge cup through a pipeline, and performing a first cell centrifugation process on the liquid through the centrifuge cup, and cleaning the pipeline during the first cell centrifugation process;

[0028] After cleaning the pipeline, extracting the initial product solution from the centrifuge cup, and concentrating the initial product solution to obtain a concentrated solution;

[0029] After flowing the transfection reagent in the transfection reagent bag into the centrifuge cup through a pipeline, performing a transfection process on the concentrated solution to obtain a post-transfection product;

[0030] Cultivate the post-transfection product in the centrifuge cup through a microfluidic heat exchanger, and monitor the cell division and amplification;

[0031] Harvest the cultivated product to obtain multiple storage bags for storing the solution to be stored.

[0032] Furthermore, the concentration treatment of the primary product solution to obtain a concentrated solution includes:

[0033] Pump the extracted primary product solution through a pipeline into the primary product bag;

[0034] Discharge the solution remaining in the centrifuge cup after extraction, and clean the centrifuge cup;

[0035] Flow the primary product solution in the primary product bag through a pipeline into the cleaned centrifuge cup, and perform a second cell centrifugation treatment to obtain the concentrated solution.

[0036] Furthermore, the transfection treatment with the concentrated solution to obtain a post-transfection product includes:

[0037] Shake the transfection reagent and the concentrated solution in the centrifuge cup, clean and empty the pipeline during the shaking process to make the transfection reagent and the concentrated solution fully transfected;

[0038] After emptying, perform a static treatment to obtain the post-transfection product.

[0039] Furthermore, the cultivation of the post-transfection product in the centrifuge cup through a microfluidic heat exchanger and the monitoring of cell division and amplification include:

[0040] Use the microfluidic heat exchanger to bring the temperature of the centrifuge cup within the cultivation temperature range, and perform timed cell counting on the centrifuge cup through a cell counting unit to obtain the counting result of each count;

[0041] Analyze the cell division and amplification status in the centrifuge cup according to each counting result;

[0042] After a preset cultivation time, when the detected division and amplification status is qualified, it is determined that the cultivation of the post-transfection product is completed.

[0043] Furthermore, the harvesting treatment of the cultivated product to obtain multiple storage bags for storing the solution to be stored includes:

[0044] Inject a cryopreservation solution into the cultivated product, and bring the temperature of the centrifuge cup within the storage temperature range, and shake to obtain the solution to be stored;

[0045] Dispense the solution to be stored into multiple storage bags.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The cell preparation workstation of the present invention includes a centrifugal culture unit, a liquid circuit unit, a cell digitization unit, a host control unit, a frame and a shell; the centrifugal culture unit includes a microfluidic heat exchanger and a centrifugal cup, the centrifugal cup includes an inlet flow channel and an outlet flow channel, the liquid circuit unit includes a pump assembly, a valve assembly and a pipeline, the cell digitization unit includes a microscope, the host control unit includes a motor, a waste liquid tank and a centrifugal bin, and the frame is used to hang a separation liquid bag, a sample liquid bag, a cleaning liquid bag, a transfection reagent bag, a primary product bag and a storage bag. Since the motor is connected to the centrifugal cup by transmission, and the inlet flow channel and the outlet flow channel of the centrifugal cup are connected to the liquid bag through the liquid circuit unit, the centrifugal cup can realize the first cell centrifugation treatment, the extraction of the primary product solution, the concentration treatment, the transfection treatment and the culture. Therefore, the above-mentioned cell preparation workstation can realize fully automatic and fully enclosed cell preparation, and achieve the full process of one-stop cell preparation, so that the cell preparation workstation has the characteristics of stable quality, safe production, cost saving and high efficiency, which can significantly improve the shortcomings of low cell biological activity in the prior art, and solve the problems of easy pollution, difficult quality control, high cost and low efficiency in the prior art.

[0048] 2. The cell preparation workstation of the present invention is provided with a liquid pushing plate in the centrifuge cup. When the motor drives the centrifuge cup to rotate, the liquid pushing plate is used to increase the driving force of the liquid centrifugation. The liquid pushing plate first pushes the nearby liquid, and the nearby liquid continues to push the distant liquid, so that the shear stress of the inner layer of liquid is lower, and the shear and friction of the cells are reduced, thereby improving the activity of the cells; at the same time, there is a gap between the liquid pushing plate and the inner wall of the centrifuge cup, and the pressure of the liquid on both sides of the liquid pushing plate can be balanced through the gap, so that the liquid surface is smooth, easier to separate, and the time required for separation is reduced; therefore, the liquid pushing plate arranged in the centrifuge cup can improve the efficiency of centrifugal stratification, and can also significantly reduce the fluid shear force in the centrifuge cup during centrifugation, so that the cell preparation workstation has the characteristics of low damage, high throughput, high efficiency, high recovery rate, and sterility, which can significantly improve the shortcomings of low cell biological activity in the prior art and meet the needs of fully automatic and fully closed cell separation.

[0049] 3. The cell processing method of the present invention completes the operations of the first cell centrifugation treatment, extraction of the initial product solution, concentration treatment, transfection treatment and culture through a fully automatic and fully closed cell preparation workstation. Through the reasonable arrangement of each step and the cooperation of the above-mentioned cell preparation workstation, not only the cell preparation process is fully automatic and fully closed to meet the requirements of a sterile environment, but also cell separation, transfection, culture and harvesting are completed in one stop, reducing the transit and transportation of cell fluid in the process, thereby improving the biological activity of the cells and reducing the cost of cell preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic three-dimensional structure diagram of the fully automatic and fully enclosed cell preparation workstation of the present invention;

[0051] Figure 2 is Figure 1 front view of the fully automatic and fully enclosed cell preparation workstation in

[0052] Figure 3 is Figure 1 overall structure schematic diagram of the centrifuge cup in

[0053] Figure 4 is Figure 3 cross-sectional view of the centrifuge cup in

[0054] Figure 5 Schematic diagram of the principle of the liquid path unit of the fully automatic and fully enclosed cell preparation workstation of the present invention;

[0055] Figure 6 Flow chart of the cell processing method of the present invention.

[0056] Among them, 1 - centrifugal culture unit, 2 - liquid path unit, 3 - cell digitization unit, 4 - host control unit, 5 - frame, 6 - housing, 7 - centrifuge cup, 8 - liquid inlet channel, 9 - liquid outlet channel, 10 - pump assembly, 11 - liquid pushing plate, 12 - microfluidic heat exchanger, 13 - control panel, 14 - touch display screen, 15 - cup body, 16 - cup cover, 17 - first liquid inlet, 18 - second liquid inlet, 19 - first liquid outlet, 20 - second liquid outlet, 21 - chamfer, 22 - waste liquid bag, 23 - cleaning liquid bag, 24 - culture medium liquid bag, 25 - spare liquid bag, 26 - storage bag, 27 - initial product bag, 28 - separation liquid bag, 29 - sample liquid bag, 30 - first bubble sensor, 31 - second bubble sensor, 32 - third bubble sensor, 33 - first pipeline, 34 - first port, 35 - second pipeline, 36 - second port, 37 - first sealing ring, 38 - second sealing ring, 39 - bearing, 40 - transfection reagent bag, 41 - display switch, 42 - hook, 43 - first control valve, 44 - second control valve, 45 - third control valve, 46 - fourth control valve, 47 - fifth control valve, 48 - sixth control valve, 49 - seventh control valve, 50 - eighth control valve, 51 - ninth control valve, 52 - tenth control valve, 53 - eleventh control valve, 54 - twelfth control valve, 55 - thirteenth control valve, 56 - control button, 57 - third pipeline, 58 - first branch pipeline, 59 - second branch pipeline, 60 - fourth branch pipeline, 61 - fifth branch pipeline, 62 - sixth branch pipeline, 63 - seventh branch pipeline 64 - fourteenth control valve, 65 - fifteenth control valve, 66 - third branch pipeline Detailed implementation manners

[0057] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0058] Embodiment 1

[0059] As Figure 1 and Figure 2 shown in the structure, this embodiment provides a fully automatic and fully enclosed cell preparation workstation. The cell preparation workstation includes a centrifugal culture unit 1, a liquid path unit 2, a cell digitization unit 3, a host control unit 4, a frame 5, and a housing 6;

[0060] As Figure 1 , Figure 2 , Figure 3 and Figure 4 shown in the structure, the centrifugal culture unit 1 includes a microfluidic heat exchanger 12 and a centrifuge cup 7 with a centrifugal chamber, and is used for performing the first cell centrifugation treatment, extracting the initial product solution, concentration treatment, transfection treatment, and culture; the centrifuge cup 7 is installed in the centrifugation chamber of the host control unit 4 and includes an inlet flow channel 8 and an outlet flow channel 9; the inlet flow channel 8 is a channel for flowing into the centrifuge cup 7; the outlet flow channel 9 is a channel for extracting the solution from the centrifuge cup 7; the microfluidic heat exchanger 12 is used to keep the centrifuge cup 7 within the culture temperature range during cell culture, and the culture temperature range can be 36 - 38°C;

[0061] As Figure 5 shown in the structure, the liquid path unit 2 includes a pump assembly 10, a valve assembly, and pipelines, and is used for supplying liquid and pumping liquid to the centrifuge cup 7, and harvesting to the storage bag 26; the pump assembly 10 and the valve assembly are installed in the pipeline path; the pipeline uses a sterile catheter; the pump assembly 10 can be a peristaltic pump; the liquid path unit may further include a sterile sampling device (not shown in the figure), and the sterile sampling device can be connected to the second branch pipeline 59 between the pump assembly 10 and the centrifuge cup 7 through a sterile catheter. By adding the sterile sampling device, it is convenient to extract samples from the centrifuge cup 7 for quality inspection; the first port 34 and the second port 36 of the pump assembly 10 are both communicated with the waste liquid bag 22; the first port 34 of the pump assembly 10 is communicated with the second liquid outlet 20 of the centrifuge cup 7;

[0062] The cell digitization unit 3 includes a microscope installed below the centrifuge cup 7 and is used for monitoring the counting, separation, and amplification of cells in the centrifuge cup 7; as Figure 1 and Figure 2 shown in the structure, the cell digitization unit 3 is installed at the bottom of the centrifuge cup 7;

[0063] The main control unit 4 includes a motor (not shown in the figure), a waste liquid tank, and a centrifugation chamber, and is connected to the pump assembly 10, the valve assembly, and the microfluidic heat exchanger 12, and controls the motor, the pump assembly 10, the valve assembly, and the microfluidic heat exchanger 12; the motor is drivingly connected to the centrifuge cup 7 and is used to drive the centrifuge cup 7 to rotate to achieve the first cell centrifugation process and the second cell centrifugation process; the waste liquid tank is used to collect the solution after cleaning and waste discharge; the main control unit 4 is the basis of the entire cell preparation workstation. The waste liquid tank can directly accommodate the waste liquid, or a waste liquid bag 22 for accommodating the waste liquid can be placed in the waste liquid tank; the waste liquid tank is provided with an opening to facilitate the taking and placing of the waste liquid bag 22 or the waste liquid; the centrifugation chamber is used to accommodate the centrifuge cup 7;

[0064] The frame 5 is fixedly installed on the top of the main control unit 4 and is used to hang a separation liquid bag 28 filled with separation liquid, a sample liquid bag 29 filled with the liquid to be separated, a cleaning liquid bag 23 filled with cleaning liquid, a transfection reagent bag 40 filled with transfection reagent, a primary product bag 27, and a storage bag 26; the cleaning liquid is used for cleaning during the cell separation process; the frame 5 is mainly used to hang the liquid bags and can be composed of a vertical support rod and a cross beam fixedly connected to the top of the support rod; the bottom end of the support rod is fixedly installed on the top of the main control unit 4; a hanging space for the liquid bags is formed between the frame 5 and the main control unit 4. In addition to the above-mentioned separation liquid bag 28, sample liquid bag 29, cleaning liquid bag 23, primary product bag 27, storage bag 26, and transfection reagent bag 40, the liquid bags can also include a waste liquid bag 22, a culture medium liquid bag 24, a spare liquid bag 25, and an intermediate product bag (not shown in the figure); in the actual production process, the primary product bag 27 can also be used as an intermediate product bag after completing the task of accommodating the primary product;

[0065] The housing 6 is hermetically covered on the top of the main control unit 4 and the frame 5 and is used to hermetically cover the centrifuge cup 7 and each liquid bag hung on the frame 5 to form a sealed space; the housing 6 is provided with a cover that can be opened, and the centrifuge cup 7 can be taken and placed conveniently by opening the cover.

[0066] The cell preparation workstation includes a centrifugal culture unit 1, a liquid circuit unit 2, a cell digitization unit 3, a host control unit 4, a frame 5, and a shell 6. The centrifugal culture unit 1 includes a microfluidic heat exchanger 12 and a centrifugal cup 7. The centrifugal cup 7 includes an inlet flow channel 8 and an outlet flow channel 9. The liquid circuit unit includes a pump assembly 10, a valve assembly and a plurality of pipelines. The host control unit 4 includes a motor, a waste liquid tank and a centrifugal bin. The frame 5 is used to hang a separation liquid bag 28 containing a separation liquid, a sample liquid bag 29 containing a liquid to be separated, a cleaning liquid bag 23 containing a cleaning liquid, a transfection reagent bag 40 containing a transfection reagent, a primary product bag 27 and a storage bag 26. The transfection reagent of the transfection reagent bag 40 The centrifugal cup 7 contains lentivirus. Since the motor is connected to the centrifugal cup 7 in a transmission manner, and the inlet flow channel 8 and the outlet flow channel 9 of the centrifugal cup 7 are connected to each liquid bag through the liquid path unit, the centrifugal cup 7 can realize the first cell centrifugation treatment, extraction of the initial product solution, concentration treatment, transfection treatment and culture. Therefore, the cell preparation workstation using the above structure can realize fully automatic and fully closed cell preparation, and achieve a one-stop cell preparation process, so that the cell preparation workstation has the characteristics of stable quality, safe production, cost saving and high efficiency, which can significantly improve the shortcomings of low cell biological activity in the prior art, and solve the problems of easy contamination, difficult quality control, high cost and low efficiency in the prior art.

[0067] In a preferred embodiment, the centrifuge cup 7 further includes a liquid pushing plate 11 , which extends radially from the center of the centrifuge cup 7 , and a gap is formed between the end of the liquid pushing plate 11 away from the axis and the inner wall of the centrifuge cup 7 .

[0068] In the above-mentioned cell preparation workstation, a liquid pushing plate 11 is arranged in the centrifuge cup 7. When the motor drives the centrifuge cup 7 to rotate, the liquid pushing plate 11 is used to increase the driving force of the liquid centrifugation. The liquid pushing plate 11 first pushes the nearby liquid, and the nearby liquid continues to push the distant liquid, so that the shear stress of the inner layer of liquid is lower, and the shear and friction of the cells are reduced, thereby improving the activity of the cells; at the same time, there is a gap between the end of the liquid pushing plate 11 and the inner wall of the centrifuge cup 7, and the pressure of the liquid on both sides of the liquid pushing plate 11 can be balanced through the gap, so that the liquid surface is smooth, easier to separate, and the time required for separation is reduced; therefore, by adding the liquid pushing plate 11 in the centrifuge cup 7, the above-mentioned cell preparation workstation can not only improve the efficiency of centrifugal stratification, but also significantly reduce the fluid shear force in the centrifuge cup 7 during the centrifugation process, so that the cell preparation workstation has the characteristics of low damage, high throughput, high efficiency, high recovery rate, and sterility, which can significantly improve the shortcomings of low cell biological activity in the prior art and meet the needs of fully automatic and fully closed cell separation.

[0069] Furthermore, the centrifuge cup 7 further includes a cup body 15 and a cup cover 16; the cup cover 16 is sealingly connected to the top of the cup body 15, and a centrifuge cavity for containing liquid is formed between the cup cover 16 and the cup body 15; there may be two liquid pushing plates 11, and the two liquid pushing plates 11 are circumferentially distributed in the centrifuge cavity along the cup body 15 and all extend along the radial direction of the cup body 15. There is a gap between the end of the liquid pushing plate 11 far from the axis and the inner wall of the centrifuge cup 7, and there is a gap between the other end of the liquid pushing plate 11 adjacent to the axis and the axis of the cup body 15; the liquid pushing plate 11 may be fixedly connected to the cup body 15 and / or the cup cover 16; both the liquid inlet channel 8 and the liquid outlet channel 9 communicate with the outside of the cup body 15 and the centrifuge cavity; the liquid inlet channel 8 has a first liquid inlet 17 provided outside the cup body 15 and a second liquid inlet 18 provided at the bottom of the inner side wall of the cup body 15; the liquid outlet channel 9 has a first liquid outlet 19 provided at the top of the centrifuge cavity and a second liquid outlet 20 provided at the top of the side wall of the cup body 15; a chamfer 21 with an angle of 5° to 50° is formed between the side wall and the bottom wall of the cup body 15, and the chamfer is preferably 20°. The setting of the chamfer 21 facilitates liquid discharge, so that the volume of the residual liquid in the centrifuge cup 7 is less than 0.2 ml; the centrifuge cup 7 may further include a rotary adapter installed at the bottom of the cup body 15, a first sealing ring 37 installed between the liquid inlet channel 8 and the cup cover 16, a second sealing ring 38 installed between the liquid outlet channel 9 and the cup cover 16, and a bearing 39 installed between the liquid outlet channel 9 and the cup cover 16; the rotary adapter is drivingly connected between the centrifuge cup 7 and the motor; the cup cover 16 is provided with a central hole; a part of the liquid outlet channel 9 is concentrically sleeved on the outer peripheral side of the liquid inlet channel 8 and passes through the central hole; the first sealing ring 37, the second sealing ring 38 and the bearing 39 are all installed in the central hole; through the first sealing ring 37 and the second sealing ring 38, the liquid inlet channel and the liquid outlet channel can be sealed and isolated from each other, and through the bearing 39, the liquid inlet channel and the liquid outlet channel can rotate relative to the cup cover 16.

[0070] Based on the above various cell preparation workstations, the liquid path unit 2 further includes a liquid path switching device, and the liquid path switching device is used for performing a closed switching and dispensing of the solution to be stored into a plurality of storage bags 26; the liquid path unit 2 further includes a first bubble sensor 30, a second bubble sensor 31 and a third bubble sensor 32; the first bubble sensor 30, the second bubble sensor 31 and the third bubble sensor 32 are used for monitoring the flow direction of the pipeline in the liquid path unit 2. The first bubble sensor 30 is installed in the pipeline connected to the cleaning solution bag 23, the culture medium solution bag 24, the spare solution bag 25, the transfection reagent bag 40 and the storage bag 26; the second bubble sensor 31 is installed in the pipeline connected to the primary product bag 27, the intermediate product bag, the separation liquid bag 28 and the sample liquid bag 29; the third bubble sensor 32 is installed in the pipeline connected to the first liquid inlet 17 of the centrifuge cup 7.

[0071] To precisely control the liquid temperature, the inlet and outlet liquid quality in the centrifuge cup 7, the above-mentioned cell preparation workstation includes a weighing unit installed on the frame 5; the microfluidic heat exchanger 12 includes a temperature control module, an upper ventilation opening, and a lower ventilation opening; the microfluidic heat exchanger 12 is installed in the centrifuge chamber; the temperature control module can be a temperature regulator; the microfluidic heat exchanger 12 can adopt a semiconductor thermoelectric refrigeration device, which can achieve temperature control in the range of 4°C to 40°C in the chamber, and the control error is less than 1°C; the temperature control module is also used to adjust the ambient temperature of the centrifuge cup 7 in the centrifuge chamber during the culture process, and blows air through the fan to the upper ventilation opening and the lower ventilation opening to transfer the heat generated by adjusting the ambient temperature to ensure the biological activity of the cells; the upper ventilation opening is installed above the centrifuge cup 7 and is used to ventilate and transfer the heat generated by the temperature regulator to above the centrifuge cup 7; the lower ventilation opening is installed below the centrifuge cup 7 and is used to ventilate and transfer the heat generated by the temperature regulator to below the centrifuge cup 7; by blowing air upward to the centrifuge cup 7 through the upper ventilation opening and blowing air downward to the centrifuge cup 7 through the lower ventilation opening, the heat conduction to the inside of the centrifuge cup 7 can be accelerated to adjust the temperature inside the centrifuge cup 7, and moreover, by blowing air up and down, an air flow can be formed to make the temperature of the closed environment in the entire centrifuge chamber reach a rapid uniform effect; through the weighing unit, the weight of the liquid bag on the frame 5 can be monitored in real time, the weight of the sample inlet and outlet can be monitored, and the measurement error is less than 1g.

[0072] As Figure 1 shown, the host control unit 4 can include a control panel 13 and a touch display screen 14. Input and output control and display can be achieved through the control panel 13 and the touch display screen 14; control buttons 56 for controlling the pump assembly 10, the valve assembly, and the microfluidic heat exchanger 12 are arranged on the control panel 13; both the control panel 13 and the touch display screen 14 are signal-connected to the weighing unit, the temperature control module, the motor, the pump assembly 10, the microfluidic heat exchanger 12, and the valve assembly. As Figure 1 shown in the structure, a display switch 41 for controlling the touch display screen 14 is also arranged on the side of the host control unit 4. To facilitate the suspension of the liquid bag, a plurality of hooks 42 are also arranged on the top of the frame 5. The host control unit 4 can independently control the operation of each unit and its process parameters through the control program and the touch display screen 14, such as: the volume and path of the sample inlet and outlet, the centrifugal speed, acceleration, and time of the motor, the mixing speed and intensity, etc.

[0073] For the convenience of description, as Figure 2As shown, the valve assembly may include a first control valve 43, a second control valve 44, a third control valve 45, a fourth control valve 46, a fifth control valve 47, a sixth control valve 48, a seventh control valve 49, an eighth control valve 50, a ninth control valve 51, a tenth control valve 52, an eleventh control valve 53, a twelfth control valve 54, a thirteenth control valve 55, a fourteenth control valve 64, and a fifteenth control valve 65; the pipeline includes a first pipeline 33, a second pipeline 35, and a third pipeline 57; wherein:

[0074] The first pipeline 33 is used to connect the centrifuge cup 7 with the initial product bag 27, the centrifuge cup 7 with the separation liquid bag 28, and the centrifuge cup 7 with the sample liquid bag 29. A fifth control valve 47 is provided between the initial product bag 27 and the first pipeline 33; a sixth control valve 48 is provided between the separation liquid bag 28 and the first pipeline 33; a seventh control valve 49 is provided between the sample liquid bag 29 and the first pipeline 33; the first pipeline 33 is connected to the centrifuge cup 7 through a first branch pipeline 58, a second branch pipeline 59, and a third branch pipeline 66. Parts of the first branch pipeline 58, the second branch pipeline 59, and the third branch pipeline 66 overlap, that is, the first branch pipeline 58, the second branch pipeline 59, and the third branch pipeline 66 overlap at both ends of the pump assembly 10 and are combined into the same main pipeline; a thirteenth control valve 55 is installed in the first branch pipeline 58 for connecting the first liquid outlet 19 of the pump assembly 10 with the centrifuge cup 7; the second branch pipeline 59 is in parallel with the third branch pipeline 66, both are used to connect the first liquid inlet 17 of the centrifuge cup 7 and the pump assembly 10. A tenth control valve 52 is installed in the second branch pipeline 59, and a fifteenth control valve 65 is installed in the third branch pipeline 66;

[0075] The second pipeline 35 is used to connect the pump assembly 10 with the cleaning solution bag 23, the pump assembly 10 with the culture medium bag 24, the pump assembly 10 with the spare solution bag 25, the pump assembly 10 with the storage bag 26, and the pump assembly 10 with the transfection reagent bag 40. A first control valve 43 is installed between the cleaning solution bag 23 and the second pipeline 35, a second control valve 44 is installed between the culture medium bag 24 and the second pipeline 35, a third control valve 45 is installed between the spare solution bag 25 and the second pipeline 35, a fourth control valve 46 is installed between the storage bag 26 and the second pipeline 35, and a fourteenth control valve 64 is installed between the transfection reagent bag 40 and the second pipeline 35. The second pipeline 35 further includes a fourth branch pipeline 60 and a fifth branch pipeline 61. Among them, the fourth branch pipeline 60 is used to connect the first port 34 of the pump assembly 10 with the cleaning solution bag 23, the culture medium bag 24, the spare solution bag 25, the storage bag 26, and the transfection reagent bag 40, and an eighth control valve 50 is installed in the fourth branch pipeline 60; the fifth branch pipeline 61 is used to connect the second port 36 of the pump assembly 10 with the cleaning solution bag 23, the culture medium bag 24, the spare solution bag 25, the storage bag 26, and the transfection reagent bag 40, and a ninth control valve 51 is installed in the fifth branch pipeline 61;

[0076] The third pipeline 57 is used to connect the pump assembly 10 with the waste liquid bag 22. The third pipeline 57 may include a sixth branch pipeline 62 and a seventh branch pipeline 63. Among them, one end of the sixth branch pipeline 62 and one end of the seventh branch pipeline 63 are both connected to the waste liquid bag 22, the other end of the sixth branch pipeline 62 is connected to the first port 34 of the pump assembly 10, the other end of the seventh branch pipeline 63 is connected to the second port 36 of the pump assembly 10, and an eleventh control valve 53 is installed in the sixth branch pipeline 62, and a twelfth control valve 54 is installed in the seventh branch pipeline 63.

[0077] Embodiment 2

[0078] This embodiment provides a cell processing method using a fully automatic and fully enclosed cell preparation workstation. As Figure 5 shown, the cell processing method includes the following steps:

[0079] Step S10, flowing the separation liquid and the liquid to be separated into the centrifuge cup 7 through the pipeline, and performing the first cell centrifugation treatment through the centrifuge cup 7, and cleaning the pipeline during the first cell centrifugation treatment; flowing the separation liquid and the liquid to be separated into the centrifuge cup through the pipeline, and performing the first cell centrifugation treatment through the centrifuge cup 7 specifically includes:

[0080] Flow the separation liquid into the centrifuge cup 7 through the first pipeline 33, rotate the centrifuge cup 7 at the first rotation speed, and maintain for the first predetermined time; the first pipeline 33 includes a first branch pipeline 58 and a second branch pipeline 59, and the first branch pipeline 58 and the second branch pipeline 59 partially overlap; flow the liquid to be separated into the centrifuge cup 7 through the second branch pipeline 59, rotate the centrifuge cup at the second rotation speed, and maintain for the second predetermined time.

[0081] Step S20, after cleaning the pipeline, extract the initial product solution from the centrifuge cup 7, and concentrate the initial product solution to obtain a concentrated solution.

[0082] Understandably, the concentration process is a process of centrifuging the initial product solution alone in the centrifuge cup 7 to extract a higher cell density (the number of cells per unit volume).

[0083] In one embodiment, in step S20, that is, concentrating the initial product solution to obtain a concentrated solution, includes:

[0084] Pump the extracted initial product solution into the initial product bag 27 through the pipeline;

[0085] Discharge the waste solution from the solution remaining in the centrifuge cup 7 after extraction, and clean the centrifuge cup 7;

[0086] Flow the initial product solution in the initial product bag 27 into the cleaned centrifuge cup 7 through the pipeline, and perform a second cell centrifugation process to obtain the concentrated solution.

[0087] In one embodiment, when pumping the extracted initial product solution into the initial product bag 27 through the pipeline, includes:

[0088] Maintain the rotation speed of the centrifuge cup 7 at the third rotation speed, extract the initial product solution from the liquid outlet channel 9 in the centrifuge cup 7 through the valve assembly and the pump assembly 10, and flow it to the initial product bag 27 through the first branch pipeline 58;

[0089] Understandably, flow the initial product solution in the initial product bag 27 into the cleaned centrifuge cup 7 through the second branch pipeline 59, and through the valve assembly and the pump assembly 10, flow the cleaning liquid into the initial product bag 27 for solution cleaning treatment, and flow the solution after cleaning into the centrifuge cup 7 through the second branch pipeline 59; after flowing a preset volume of cleaning liquid into the centrifuge cup 7, perform the second cell centrifugation process on the centrifuge cup 7, and discharge the waste liquid to obtain the concentrated solution. The second cell centrifugation process is a process of discharging the waste liquid through the liquid outlet channel at different rotation speeds.

[0090] Step S30: After the transfection reagent in the transfection reagent bag 40 flows into the centrifuge cup 7 through the pipeline, it is subjected to transfection treatment with the concentrated solution to obtain the post-transfection product. Through the valve assembly and the pump assembly 10, the transfection reagent in the transfection reagent bag 40 flows into the centrifuge cup 7 through the second pipeline 35 and the second branch pipeline 59. The centrifuge cup 7 is rotated by the motor to achieve shaking. During the shaking process, the rotation speed of the motor can be maintained at 500 rpm for 240 s.

[0091] In one embodiment, in step S30, that is, subjecting to transfection treatment with the concentrated solution to obtain the post-transfection product, includes:

[0092] The transfection reagent and the concentrated solution in the centrifuge cup 7 are shaken. During the shaking process, pipeline cleaning and emptying are performed to enable the transfection reagent and the concentrated solution to be fully transfected. The transfection reagent is a polymer of highly efficient lentivirus, often referred to as virus solution. It can be a polycationic substance, which can interact with nucleic acids (including plasmids, siRNAs, oligonucleotides) to form a complex to transport the nucleic acids into eukaryotic cells. Shaking means the centrifuge cup 7 rotates clockwise and counterclockwise back and forth at a relatively slow speed to achieve the effect of manual shaking. The pipeline cleaning and emptying process is to clean along most of the paths through which the transfection reagent flows with the cleaning solution of physiological saline, and after cleaning, the liquid in this path is emptied into the centrifuge cup 7 by the rotation of the pump assembly 10 to fully allow the transfection reagent and the concentrated solution to be transfected.

[0093] Among them, transfection treatment is a specialized technical treatment process for introducing exogenous genes into cells. The exogenous gene can be lentivirus.

[0094] After emptying, static treatment is performed to obtain the post-transfection product. The static treatment is to static for 20 minutes or 30 minutes.

[0095] Step S40: The post-transfection product in the centrifuge cup 7 is cultured through the microfluidic heat exchanger, and the cell division and amplification are monitored. The centrifuge cup 7 is maintained at a preset temperature, such as 37 °C, through the microfluidic heat exchanger 12. The post-transfection product in the centrifuge cup 7 is cultured to allow the cells in the centrifuge cup 7 to divide and amplify for 10 d to 14 d. During this period, the cell counting, division, and amplification are monitored through the cell digitalization unit 3. Among them, the cell digitalization unit 3 can analyze the data collected by cell counting units such as microscopes. The viability is the survival rate of the cells, and the particle size is the particle diameter length of the cells.

[0096] In one embodiment, in the said step S40, that is, culturing the post-transfection product in the centrifuge cup 7 through the microfluidic heat exchanger 12 and monitoring the cell division and amplification, includes:

[0097] The temperature of the centrifuge cup 7 is brought to within the culture temperature range by the microfluidic heat exchanger 12, and the centrifuge cup 7 is periodically counted for cells by the cell counting unit to obtain the counting result of each count.

[0098] Understandably, the culture temperature range can be set according to requirements, such as 36 - 38 °C. Heat is released by heating through the temperature control module in the microfluidic heat exchanger 12, and then the released heat is sent to the upper and lower ventilation openings by a fan, and then conducted to the upper and lower parts of the centrifuge cup 7, so that the temperature of the centrifuge cup 7 reaches within the culture temperature range. The process of cell counting is as follows: Focus on the bottom of the centrifuge cup 7 through a microscope, move a preset distance from bottom to top, collect images of a certain area, or scan from bottom to top. A three-dimensional model is constructed for the collected or scanned images. A space with this area as the area and the preset distance as the height can be constructed, the volume is calculated, and the number of cells is counted according to the constructed three-dimensional model. The counting method can be to repair the cell edges of the collected images, count the enclosed three-dimensional cells, and then, through the ratio of this space to the liquid volume in the centrifuge cup 7 and the count of this space, estimate the number of cells in the liquid volume of the centrifuge cup, so as to obtain the counting result of each count. The counting result reflects the number of cells in the liquid volume of the centrifuge cup 7.

[0099] According to each counting result, analyze the division and amplification status of the cells in the centrifuge cup 7.

[0100] Understandably, according to each counting result, the number, viability, and particle size of the cells in the centrifuge cup 7 can be obtained. The viability is the survival rate of the cells, and the survival rate is the proportion of living cells in the total number of cells. The calculation method of the survival rate can be based on the number of cells before cell division (the previous counting result of the current counting result) and the ratio of the number of cells after cell division (the current counting result). Through the simulation evolution of the cell division model, the number of dead cells is estimated, and then the viability is determined; the particle size is the particle diameter length of the cells, which can be measured by measuring the size of the cells. The division and amplification status of the cells can be reflected by the number, viability, and particle size of the cells. When the number, viability, and particle size of the cells are all within the qualified range, that is, the number, viability, and particle size of the cells each time can be within the trend range of cell culture, the division and amplification status is determined to be qualified. The cell division model is a model in which cells divide and evolve over time. The cell division model can be constructed by statistically analyzing historical cell division data.

[0101] In one embodiment, when the division and amplification status of the cells is unqualified, a warning prompt is issued in a timely manner.

[0102] After the preset culture time, when it is detected that the division and amplification status is qualified, it is determined that the culture of the post-transfection product is completed.

[0103] Understandably, the preset culture time can be 10 days to 14 days. When the cell count, viability, and particle size are all within the qualified range, it is determined that the culture stage is completed.

[0104] Step S50: Harvest the cultured product and obtain a plurality of storage bags 26 for storing the solution to be stored; before harvesting the cultured product, it further includes: cleaning and concentrating the cultured product in the centrifuge cup 7 with the cleaning solution in the cleaning solution bag 23. The harvesting process is as follows: Inject the cryopreservation solution into the cultured product, and use the microfluidic heat exchanger 12 to bring the temperature of the centrifuge cup 7 within the storage temperature range, shake well to obtain the solution to be stored, and perform cell counting on the solution to be stored through the cell counting unit; The specific cell counting method is: Focus the microscope on the bottom of the centrifuge cup 7, move a preset distance from bottom to top, collect images of a certain area, or scan from bottom to top, construct a three-dimensional model of the collected or scanned images, and a space with the area of this region as the area and the preset distance as the height can be constructed, calculate the volume, and count the number of cells according to the constructed three-dimensional model. The counting method can be to repair the cell edges of the collected images and count the enclosed three-dimensional cells. The process can use three-dimensional image processing technology, not limited to the conversion of binary images. Continuously calculate the cell density during the injection of the cryopreservation solution through the cell counting unit until the required cell density is reached, then stop injecting the cryopreservation solution. The cryopreservation solution is a liquid used to cryopreserve the cell solution, which can ensure the activity of the cells and the cryopreservation environment; Divide the solution to be stored into a plurality of storage bags 26. When dividing the solution to be stored into a plurality of storage bags 26, it specifically includes: Use the liquid path switching device to perform a closed switching and divide the solution to be stored into a plurality of storage bags 26.

[0105] In one embodiment, in step S50, that is, when harvesting the cultured product to obtain a plurality of storage bags for storing the solution to be stored, it specifically includes:

[0106] Inject the cryopreservation solution into the cultured product, and bring the temperature of the centrifuge cup 7 within the storage temperature range, shake well to obtain the solution to be stored.

[0107] Understandably, the cryopreservation solution is a liquid used to cryopreserve the cell solution. The microfluidic heat exchanger 12 is used to transfer the heat of cooling to the centrifugation chamber to reduce the temperature of the centrifuge cup 7, so as to reach within the storage temperature range, and shake back and forth clockwise and counterclockwise during the process to avoid cell precipitation and condensation, thereby obtaining the solution to be stored.

[0108] Divide the solution to be stored into a plurality of storage bags 26.

[0109] Understandably, when it is necessary to subpackage multiple storage bags 26, the sequential switching and subpackaging of the multiple storage bags 26 can be achieved through a switching device. The switching device can be a control device containing a three-way valve or a control device through heat sealing and plugging. For example, the storage bag 26 can be connected to the pipeline above the fourth control valve 46 through a sterile tube connecting machine.

[0110] The above cell processing method completes each step such as the first cell centrifugation treatment, extraction of the primary product solution, concentration treatment, transfection treatment, and culture through a cell preparation workstation. Through the reasonable arrangement of each step and the cooperation of the above cell preparation workstation, not only is the cell separation process fully automated and fully enclosed to meet the requirements of a sterile environment, but also the cell separation, transfection, culture, and harvesting are completed in one stop, reducing the transfer and transportation of the cell solution during the process, thereby improving the biological activity of the cells and reducing the cost of cell preparation.

[0111] The specific operation process of the above cell processing method can adopt the following steps:

[0112] First step, install the centrifuge cup 7, liquid bag, and sterile catheter. Install the centrifuge cup 7 in the centrifuge chamber of the base 1, hang the liquid bag on the frame 5, and connect the centrifuge cup 7, pump assembly 10, liquid bag, and bubble sensor through the sterile catheter. Close each control valve to keep each control valve in a normally closed state and make each sterile catheter in a disconnected state;

[0113] Second step, sample injection. Turn on the pump assembly 10, the sixth control valve 48, and the thirteenth control valve 55 to allow 80 - 100 ml of separation liquid in the separation liquid bag 28 to enter the centrifuge cup 7 through the sixth control valve 48, the first pipeline 33, the pump assembly 10, the first branch pipeline 58, the thirteenth control valve 55, and the liquid outlet channel 9. Turn off the pump assembly 10, the sixth control valve 48, and the thirteenth control valve 55. The motor drives the centrifuge cup 7 to rotate centrifugally at the first speed for a first predetermined time. The first speed can be a value between 2500 - 2800 rpm, and the first predetermined time can be 10 s. Turn on the pump assembly 10, the seventh control valve 49, and the tenth control valve 52 to inject the cell sample solution. Allow 25 ml - 120 ml of cell sample in the sample liquid bag 29 to enter the centrifuge cup 7 through the seventh control valve 49, the first pipeline 33, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet channel 8. Turn off the pump assembly 10, the seventh control valve 49, and the tenth control valve 52. The centrifuge cup 7 maintains the first speed and centrifuges for a second predetermined time. The second predetermined time can be a value between 200 - 300 s.

[0114] Step 3: Clean the liquid path unit. Turn on the pump assembly 10, the first control valve 43, the eighth control valve 50, and the seventh control valve 49, so that 15 - 20 ml of cell cleaning solution in the cleaning solution bag 23 enters the sample solution bag 29 through the first control valve 43, the second pipeline 35, the fourth branch pipeline 60, the eighth control valve 50, the pump assembly 10, the first pipeline 33, and the seventh control valve 49. Then turn off the pump assembly 10, the first control valve 43, the eighth control valve 50, and the seventh control valve 49. Turn on the pump assembly 10, the first control valve 43, the ninth control valve 51, and the tenth control valve 52, so that 20 - 30 ml of cell cleaning solution in the cleaning solution bag 23 enters the centrifuge cup 7 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the tenth control valve 52, and the liquid inlet flow channel 8. Then turn off the pump assembly 10, the first control valve 43, the ninth control valve 51, and the tenth control valve 52. Turn on the pump assembly 10, the first control valve 43, the ninth control valve 51, and the eleventh control valve 53, so that 10 - 20 ml of cell cleaning solution in the cleaning solution bag 23 enters the waste liquid bag 22 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the sixth branch pipeline 62, and the eleventh control valve 53. Then turn off the pump assembly 10, the first control valve 43, the ninth control valve 51, and the eleventh control valve 53. Turn on the pump assembly 10, the first control valve 43, the ninth control valve 51, and the twelfth control valve 54, so that 20 - 30 ml of cell cleaning solution in the cleaning solution bag 23 enters the waste liquid bag 22 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the seventh branch pipeline 63, and the twelfth control valve 54. Then turn off the pump assembly 10, the first control valve 43, the ninth control valve 51, and the twelfth control valve 54.

[0115] Step 4: Take out the initial product sample. Reduce the first rotation speed of the motor to the second rotation speed, and the second rotation speed can be a value within the range of 1500 - 2000 rpm. The first rotation speed and the second rotation speed can be the same or different. While maintaining the second predetermined time, start taking out the sample. Open the tenth control valve 52 and the fifth control valve 47, and control the pump assembly 10 to rotate in reverse, so that 80 - 100 ml of PBMC (Peripheral blood mononuclear cell) solution in the centrifuge cup 7 is taken out through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, and the fifth control valve 47 to the initial product bag 27. Then turn off the pump assembly 10, the tenth control valve 52, and the fifth control valve 47, and stop the centrifuge cup 7 from rotating.

[0116] Step 5: Clean the centrifuge cup 7 and the liquid path unit. Open the pump assembly 10, the tenth control valve 52, and the twelfth control valve 54, and drain all the waste liquid in the centrifuge cup 7 into the waste liquid bag 22 through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, the seventh branch pipeline 63, and the twelfth control valve 54. Then close the pump assembly 10, the tenth control valve 52, and the twelfth control valve 54. Open the pump assembly 10, the first control valve 43, the eighth control valve 50, and the twelfth control valve 54, and drain all the waste liquid in the pipeline into the waste liquid bag 22. Then close the pump assembly 10, the first control valve 43, the eighth control valve 50, and the twelfth control valve 54. Clean the centrifuge cup 7. Open the pump assembly 10, the first control valve 43, the ninth control valve 51, and the tenth control valve 52, and allow 150 - 200 ml of cell cleaning liquid in the cleaning liquid bag 23 to enter the centrifuge cup 7 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the first control valve 43, the ninth control valve 51, and the tenth control valve 52. Clean the liquid outlet flow channel 9 of the centrifuge cup 7. Open the pump assembly 10, the first control valve 43, the ninth control valve 51, and the thirteenth control valve 55, and allow 30 - 50 ml of cell cleaning liquid in the cleaning liquid bag 23 to enter the centrifuge cup 7 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the first pipeline 33, the thirteenth control valve 55, the first branch pipeline 58, and the liquid outlet flow channel 9. Then close the pump assembly 10, the first control valve 43, the ninth control valve 51, and the thirteenth control valve 55. Open the pump assembly 10, the tenth control valve 52, and the twelfth control valve 54, and drain all the solution in the centrifuge cup 7 into the waste liquid bag 22 through the liquid inlet channel 7, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the seventh branch pipeline 63, and the twelfth control valve 54. Then close the pump assembly 10, the tenth control valve 52, and the twelfth control valve 54. Open the pump assembly 10, the first control valve 43, the ninth control valve 51, and the eleventh control valve 53, and drain 10 - 20 ml of cell cleaning liquid in the cleaning liquid bag 23 into the waste liquid bag 22. Close the ninth control valve 51 and the eleventh control valve 53. Open the eighth control valve 50 and the twelfth control valve 54, and drain 20 - 30 ml of cell cleaning liquid in the cleaning liquid bag 23 into the waste liquid bag 22. Then close the pump assembly 10, the first control valve 43, the eighth control valve 50, and the twelfth control valve 54;

[0117] Step 6, cleaning of the initial product. Open the pump assembly 10, the fifth control valve 47 and the tenth control valve 52, and transfer all the samples in the initial product bag 27 to the centrifuge cup 7 through the first pipeline 33, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the fifth control valve 47 and the tenth control valve 52. Open the pump assembly 10, the first control valve 43, the eighth control valve 50 and the fifth control valve 47, and allow 90 - 110 ml of cell cleaning liquid in the cleaning liquid bag 23 to enter the initial product bag 27 through the first control valve 43, the second pipeline 35, the fourth branch pipeline 60, the eighth control valve 50, the pump assembly 10, the first pipeline 33, and the fifth control valve 47. Then close the pump assembly 10, the first control valve 43, the eighth control valve 50 and the fifth control valve 47. Open the pump assembly 10, the fifth control valve 47 and the tenth control valve 52, and transfer all the samples in the initial product bag 27 to the centrifuge cup 7 through the fifth control valve 47, the first pipeline 33, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the fifth control valve 47 and the tenth control valve 52. Open the pump assembly 10, the first control valve 43, the ninth control valve 51 and the tenth control valve 52, and transfer 180 - 200 ml of cell cleaning liquid in the cleaning liquid bag 23 to the centrifuge cup 7 through the first control valve 43, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the first control valve 43, the ninth control valve 51 and the tenth control valve 52. Control the centrifuge cup 7 to rotate at the first rotational speed through the motor. The first rotational speed can be 2500 - 3000 rpm, and maintain for the third predetermined time. The third predetermined time can be 300 s to start the first centrifugal cleaning. Maintain the first rotational speed and start automatic waste liquid discharge. Open the pump assembly 10, the tenth control valve 52 and the twelfth control valve 54, and discharge all the solution in the centrifuge cup 7 to the waste liquid bag 22 through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, the seventh branch pipeline 63, and the twelfth control valve 54. Then reduce the speed to the third rotational speed and discharge waste liquid. The third rotational speed can be 1000 - 1500 rpm. Finally, reduce the speed to the fourth rotational speed and continue to discharge waste liquid. The fourth rotational speed can be 400 - 800 rpm, and maintain for the fourth predetermined time. The fourth predetermined time can be 320 s, and then close the pump assembly 10, the tenth control valve 52 and the twelfth control valve 54. Open the pump assembly 10, the second control valve 44, the ninth control valve 51 and the tenth control valve 52, and transfer 200 - 400 ml of culture medium solution in the culture medium bag 24 to the centrifuge cup 7 through the second control valve 44, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the first pipeline 33, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the second control valve 44, the ninth control valve 51 and the tenth control valve 52;Adjust the rotation speed of the centrifuge cup 7 to the first rotation speed, and maintain it for the second predetermined time to perform the second centrifugal cleaning;

[0118] Step 7, concentration: Keep the centrifuge cup 7 rotating at the first rotation speed, open the pump assembly 10, the tenth control valve 52 and the twelfth control valve 54, and discharge the solution in the centrifuge cup 7 through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, the seventh branch pipeline 63, and the twelfth control valve 54 into the waste liquid bag 22. Then, reduce the rotation speed of the centrifuge cup 7 to the third rotation speed to discharge the waste liquid, and finally reduce it to the fourth rotation speed to continue discharging the waste liquid, and maintain it for the fifth predetermined time. The fifth predetermined time can be 480 s. When the third bubble sensor 32 detects bubbles, centrifuge for 30 - 50 s and then stop rotating;

[0119] Step 8, transfection: Open the pump assembly 10, the fourteenth control valve 64, the ninth control valve 51 and the tenth control valve 52, and let the transfection reagent in the transfection reagent bag 40 flow into the centrifuge cup 7 through the second pipeline 35, the fifth branch pipeline 61, the first pipeline 33, and the second branch pipeline 59. Close the pump assembly 10, the fourteenth control valve 64, the ninth control valve 51 and the tenth control valve 52, and start the motor. The rotation speed of the motor is 500 - 800 rpm, and it rotates continuously for 200 - 300 s to achieve shaking and perform the transfection process, and clean the pipeline during the shaking process; The transfection reagent contains lentivirus;

[0120] Step 9, culture: Perform heat exchange on the solution in the centrifuge cup 7 through the microfluidic heat exchanger 12 to keep the centrifuge cup 7 at the preset temperature, such as 37 °C, and culture the transfected product in the centrifuge cup 7 to allow the cells in the centrifuge cup 7 to divide and expand for 10 d - 14 d. During this period, monitor the cell quantity, viability, and particle size through the cell digitalization unit 3. Among them, the cell digitalization unit 3 can analyze the data collected by the cell counting unit such as a microscope. The viability is the survival rate of the cells, and the particle size is the particle diameter length of the cells;

[0121] Step 10, harvest: Clean and concentrate the culture product in the centrifuge cup 7 with the cleaning solution in the cleaning solution bag 23, and perform cell counting on the concentrated or collected cell solution through the cell counting unit;

[0122] Step 8, Preparation of the formulation: Open the pump assembly 10, the second control valve 44, the ninth control valve 51, and the tenth control valve 52. Transfer 200 - 300 ml of the culture medium solution in the culture medium liquid bag 24 to the centrifuge cup 7 through the second control valve 44, the second pipeline 35, the fifth branch pipeline 61, the ninth control valve 51, the first pipeline 33, the pump assembly 10, the second branch pipeline 59, the tenth control valve 52, and the liquid inlet flow channel 8. Then close the pump assembly 10, the second control valve 44, the ninth control valve 51, and the tenth control valve 52, and mix the centrifuge cup 7 through forward and reverse rotations.

[0123] Step 9, Sub-packaging: Start the motor to increase the rotational speed of the centrifuge cup 7 to a value within the range of 50 - 80 rpm. Open the pump assembly 10, the tenth control valve 52, the ninth control valve 51, and the fourth control valve 46. Transfer the final product sample in the centrifuge cup 7 to the storage bag 26 in sequence through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, and the fourth control valve 46. Then close the pump assembly 10, the tenth control valve 52, the ninth control valve 51, and the fourth control valve 46. Finally, start the first bubble sensor 30 and the third bubble sensor 32 to automatically collect the remaining solution to be stored. Open the pump assembly 10, the tenth control valve 52, the ninth control valve 51, and the third control valve 45, and transfer all the final product samples in the centrifuge cup 7 to the spare liquid bag 25 through the liquid inlet flow channel 8, the second branch pipeline 59, the tenth control valve 52, the first pipeline 33, the pump assembly 10, and the third control valve 45. After that, close the pump assembly 10, the tenth control valve 52, the ninth control valve 51, and the third control valve 45. When multiple storage bags 26 need to be sub-packaged, sequential switching and sub-packaging of multiple storage bags 26 can be achieved through a switching device. The switching device can be a control device containing a three-way valve or a control device through heat sealing and plugging. For example, the storage bag 26 can be connected to the pipeline above the fourth control valve 46 through a sterile pipe connecting machine.

[0124] Using the cell processing method of the above cell preparation workstation, the structure with the liquid pushing plate 11 set in the centrifuge cup 7 avoids shear damage to cells, shortens the separation time, improves the separation efficiency, reduces the separation time from 15 minutes in the prior art to 5 minutes, and solves the problem of low cell separation efficiency in the prior art. At the same time, through the combined action of the centrifugal culture unit and the microfluidic heat exchanger, cell transfection, culture, and harvesting can be achieved. Through the cell digitalization unit, the number, viability, and particle size of cells can also be monitored, realizing fully automatic and fully enclosed processing of the cell preparation process, and also improving the biological activity of cells and reducing the cell preparation cost.

[0125] 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 equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. An automatic fully enclosed cell preparation workstation, characterized in that, include: A centrifugal culture unit, comprising a microfluidic heat exchanger and a centrifugal cup with a centrifugal cavity, for performing a first cell centrifugation process, extracting a primary product solution, concentrating, transfecting and culturing; the centrifugal cup is installed in a centrifugal chamber, comprising an inlet flow channel and an outlet flow channel; the microfluidic heat exchanger is used to keep the centrifugal cup within a culture temperature range during the culture process; A liquid circuit unit, comprising a pump assembly, a valve assembly and a pipeline, for supplying and extracting liquid to the centrifuge cup, and harvesting liquid to a storage bag; the pump assembly and the valve assembly are located in the pipeline path; A cell digitization unit, comprising a microscope installed below the centrifuge cup, for monitoring the counting, division and proliferation of cells in the centrifuge cup; The host control unit includes a motor, a waste liquid tank and the centrifugal chamber, is connected to the pump assembly, the valve assembly and the microfluidic heat exchanger, and controls the motor, the pump assembly, the valve assembly and the microfluidic heat exchanger; the motor is in transmission connection with the centrifugal cup to drive the centrifugal cup to rotate; the waste liquid tank is used to collect the solution after cleaning and discharge; A frame is fixedly mounted on the top of the host control unit and is used to hang a separation liquid bag containing a separation liquid, a sample liquid bag containing a liquid to be separated, a cleaning liquid bag containing a cleaning liquid, a transfection reagent bag containing a transfection reagent, a primary product bag and a storage bag; the cleaning liquid is used for cleaning during the cell separation process; The housing and the sealing cover are arranged on the top of the host control unit and the frame.

2. The cell preparation workstation according to claim 1, wherein The centrifugal cup further comprises a liquid pushing plate, which extends from the center of the centrifugal cup in a radial direction, and a gap is formed between an end of the liquid pushing plate away from the axis and an inner wall of the centrifugal cup.

3. The cell preparation workstation according to claim 2, characterized in that, The centrifugal cup also includes a cup body and a cup cover; The liquid pushing plate is fixedly connected to the cup body and / or the cup cover; The cup cover is sealed and connected to the top of the cup body, and a centrifugal cavity is formed between the cup cover and the cup body; The liquid pushing plate is distributed in the centrifugal chamber along the circumference of the cup body, and there is a gap between the other end of the liquid pushing plate adjacent to the axis and the axis of the cup body; The liquid inlet channel and the liquid outlet channel are both connected to the outside of the cup body and the centrifugal chamber; the liquid inlet channel has a first liquid inlet arranged on the outside of the cup body and / or a second liquid inlet arranged at the bottom of the inner wall of the cup body; the liquid outlet channel has a first liquid outlet arranged at the top of the centrifugal chamber and / or a second liquid outlet arranged at the top of the side wall of the cup body.

4. The cell preparation workstation according to claim 3, wherein, A chamfer of 5° to 50° is formed between the side wall and the bottom wall of the cup body.

5. The cell preparation workstation according to any one of claims 1-4, characterized in that, The microfluidic heat exchanger comprises: A temperature control module, used for adjusting the ambient temperature of the centrifugal cup in the centrifugal bin during the culture process, and blowing air to the upper vent and the lower vent through a fan to transfer the heat generated by adjusting the ambient temperature; An upper vent, installed above the centrifuge cup, for transferring the heat generated by the temperature control module to the top of the centrifuge cup through ventilation; The lower vent is installed below the centrifuge cup and is used to ventilate the heat generated by the temperature control module to the bottom of the centrifuge cup.

6. A cell processing method using the fully automatic and fully enclosed cell preparation workstation as described in claims 1 to 5, characterized in that, include: Flow the separation liquid and the liquid to be separated into the centrifuge cup through a pipeline, and perform a first cell centrifugation process on the liquid through the centrifuge cup. During the first cell centrifugation process, clean the pipeline; After cleaning the pipeline, extract the initial product solution from the centrifuge cup, and concentrate the initial product solution to obtain a concentrated solution; After flowing the transfection reagent in the transfection reagent bag into the centrifuge cup through a pipeline, perform a transfection process with the concentrated solution to obtain a post-transfection product; Cultivate the post-transfection product in the centrifuge cup through a microfluidic heat exchanger, and monitor the cell division and amplification; Harvest the cultured product to obtain multiple storage bags for storing the solution to be stored; 7. The cell processing method according to claim 6, characterized in that, The concentrating the initial product solution to obtain a concentrated solution includes: Pump the extracted initial product solution into the initial product bag through a pipeline; Dispose of the waste solution in the centrifuge cup after extraction, and clean the centrifuge cup; Flow the initial product solution in the initial product bag into the cleaned centrifuge cup through a pipeline, and perform a second cell centrifugation process to obtain the concentrated solution; 8. The cell processing method according to claim 6, wherein The performing a transfection process with the concentrated solution to obtain a post-transfection product includes: Shake the transfection reagent and the concentrated solution in the centrifuge cup. During the shaking process, clean and empty the pipeline to allow the transfection reagent and the concentrated solution to be fully transfected; After emptying, perform a static treatment to obtain a post-transfection product; 9. The cell processing method according to claim 6, characterized in that, The cultivating the post-transfection product in the centrifuge cup through a microfluidic heat exchanger and monitoring the cell division and amplification includes: Use the microfluidic heat exchanger to bring the temperature of the centrifuge cup within the cultivation temperature range, and perform timed cell counting on the centrifuge cup through a cell counting unit to obtain the counting result of each count; According to each of the counting results, analyze the cell division and amplification status in the centrifuge cup; After a preset cultivation time, when the detected division and amplification status is qualified, determine that the cultivation of the post-transfection product is completed; 10. The cell processing method according to claim 6, characterized in that, The harvesting the cultured product to obtain multiple storage bags for storing the solution to be stored includes: Inject a cryopreservation solution into the cultured product, and bring the temperature of the centrifuge cup within the storage temperature range, and shake to obtain the solution to be stored; Dispense the solution to be stored into multiple storage bags.

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