Cell culture method based on car-t therapy

Abstract

The invention relates to the technical field of cell culture, and discloses a cell culture method based on a car-t therapy, which comprises a channel conversion valve, a peristaltic pump, a dynamic culture unit, a static culture unit, a centrifugal transduction mechanism, an electric pinch valve, a material bag, a flow meter, a magnetic controller, a magnetic bead cleaning unit and a waste liquid collection unit. According to the cell culture method based on the car-t therapy, the cell culture efficiency is improved, the clinical transformation period is shortened, the time loss is reduced through automatic material transmission, the culture environment is accurately controlled, the cell activity is optimized, the gene transduction efficiency is improved through a centrifugal transduction mechanism, exogenous pollution is avoided through a totally-closed process, the process safety is enhanced, and the pollution risk is reduced; the whole process is automatic, personal errors are reduced, the process management and control capability is improved in traceability, automation and intelligence are achieved, labor dependence is reduced, cultivation cost is reduced, technology popularization is promoted, materials are efficiently utilized, waste is reduced, and labor cost is reduced through automation.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of cell culture, in particular to a cell culture method based on car-t therapy. BACKGROUND

[0002] In modern medicine, the treatment method of cancer is constantly evolving, and CAR-T cells as an innovative immunotherapy are bringing new hope to many patients. The core of CAR-T cells is the modification and culture of patients' own T cells, which can recognize and attack cancer cells. This process is not only complex but also requires high professional technical support. For patients, understanding the culture process of CAR-T cells helps better understand the principles and effects of treatment, thereby enhancing the confidence in treatment. The culture of T cells begins with the extraction of T cells from the patient's body. Usually, the doctor will extract the patient's peripheral blood by drawing blood, and use a separator to separate the T cells from other blood components. The extracted T cells are placed in a specific culture medium to provide the necessary nutrients and growth factors to promote cell proliferation. In this stage, the activity and proliferation capacity of the cells are the key factors for evaluating the subsequent effects, and the present application provides a cell culture method based on car-t therapy. SUMMARY

[0003] (I) Technical problems solved In view of the defects of the prior art, the present application provides a cell culture method based on car-t therapy, which has the advantages of improving cell culture efficiency, ensuring CAR-T cell quality, improving treatment effectiveness, strengthening process safety, and reducing pollution risk.

[0004] (II) Technical solutions To achieve the above-mentioned purpose, the present application provides the following technical solutions: a cell culture method based on car-t therapy, comprising a channel diversion valve, a peristaltic pump, a dynamic culture unit, a static culture unit, a centrifugal transduction mechanism, an electric pinch valve, a material bag, a flow meter, a magnetic controller, a magnetic bead cleaning unit, and a waste liquid collection unit. The cell culture method is as follows: S1, 1 material bag about 200ml apheresis blood is mixed with 20ml magnetic beads in 2 material bag, and is rotated and incubated in the centrifuge of the centrifugal transduction mechanism; After incubation is completed, (220ml blood magnetic bead suspension) is collected in 11 material bag, and the electromagnetic chuck of the magnetic controller is turned on; S2, 100ml sorting solution in 3 material bag is used to rinse the pipeline (leak detection process) and the centrifugal cavity of the centrifugal transduction mechanism, and the centrifugal cavity is rinsed for 10min (the time length can be set), and the waste liquid flows into the waste liquid bag of the waste liquid collection unit; S3, slowly into the magnetic controller of the sorting column (220 ml blood magnetic bead suspension) in the No. 11 material bag, waste liquid flows into the waste liquid bag; S4, the magnetic controller is closed (magnetic force off), about 110 ml of sorting solution is added into the No. 3 material bag each time to flush the magnetic beads labeled T cells in the sorting column (considered to be set to multiple flushing, in case of 1 time can't be washed down), and the magnetic beads labeled T cells are collected into the No. 12 material bag until the whole blood sorting is completed; S5, 500 ml (the centrifuge cavity can't add 500 ml at a time, which should be supplemented to the cavity maximum volume of 220 ml according to the T cell volume) of sorting solution is added into the centrifuge to wash, and the waste liquid flows into the waste liquid collection unit of the waste liquid bag; S6, 200 ml of medium solution is used for wet washing, (after wet washing, the cells need to be cut off, and the sampling counting process is left out), (according to the counting result, the cells are hung up again, and the calculated amount of cells is added through the flow meter) 40 ml of activated magnetic beads (this magnetic bead also needs to be washed, generally there are two possibilities: ① ready to use, after preparation, wash with a large magnetic plate ② prepare in advance) are added into the centrifuge for incubation, and are loaded into the No. 1 material bag for static culture; S7, appropriate medium is added by using the device peristaltic pump (cooperating with the flow meter), and after static culture for 24 H, (the cells are taken out and hung up, the cells are washed 1-2 times, about 10-50 ml of virus, about 40 ml of various cell factors (the medium is supplemented to the process required volume, such as 150 ml) are added into the centrifuge for virus transduction; S8, after transduction, it is collected into the No. 1 material bag (the medium may also be supplemented, and the final volume may be greater than that at the time of transduction) for static culture; S9, after culturing the cells for 24 H, the magnetic beads are removed by the magnetic force device, and are loaded into the No. 2 material bag; S10, appropriate medium is added and supplemented to the process requirement, such as 500 ml, dynamic culture, after 2-3 days, perfusion is started (the perfusion volume needs to be set). Sampling (cell counting, according to the cell density, switching program or adjusting the device parameters) is taken every day and observation is made, after 4-5 days, after sampling and counting, the cell number reaches, and preparation for harvesting is made. (If it can't reach, continue to culture); S11, the cell suspension after culture is washed in the centrifuge, (after the last washing, the sample needs to be taken for counting before centrifugation), according to the counting result, the cell suspension volume after washing before freezing is added, that is, the amount of cells to be frozen, centrifugal washing is carried out) the waste liquid flows into the waste liquid bag 3 (the waste liquid may also be sampled for detection of sterility, mycoplasma, endotoxin and other safety items), after washing, freezing solution is added and mixed, and the finished product and sample are divided and packaged; The material bag and the electric pinch valve are provided with 23, the waste liquid collecting unit contains 4 waste liquid bags, the channel turning valve is provided with 4, and the peristaltic pump is provided with 2.

[0005] Preferably, the step S1 needs to be centrifuged for about 30 minutes.

[0006] Preferably, the step S5 repeats the centrifuge washing process twice, and after washing, (add a sampling step) 100ml of cryopreservation solution is added to the centrifuge for mixing, and then is divided and stored.

[0007] Preferably, in the step S6, after resuscitation outside the device with a dry cell instrument, a puncture head is inserted into the thawed material bag, the cells are hung on the hook, and then are transferred to the centrifuge, 500ml (220ml) of sorting solution (culture medium, not sorting solution) is added to the centrifuge for washing, the waste liquid flows into the waste liquid bag 2, the washing process is repeated twice, and the centrifugal incubation is performed for 30 minutes.

[0008] Preferably, in the step S10, sampling detection is performed during cell culture, and when the cell density reaches the standard, perfusion is performed: the old culture medium solution flows into the waste liquid bag 4.

[0009] Preferably, in the step S11, 500ml (220ml) of washing solution is added for washing, the washing process is repeated twice, 100ml (according to the cell counting result, the volume of the cryopreservation solution is calculated) of the cryopreservation solution is added for washing, and the waste liquid flows into the waste liquid bag 3.

[0010] Compared with the prior art, the application provides a cell cultivation method based on car-t therapy, which has the following beneficial effects: 1. The cell cultivation method based on car-t therapy, the multi-unit cooperative acceleration cultivation process: the dynamic culture unit and the static culture unit form a cooperative mode of "dynamic proliferation-static induction", the dynamic culture unit drives the magnetic beads to stir through the magnetic controller, realizes the efficient mixing of cells and culture medium, improves the nutrient absorption efficiency, and promotes the rapid proliferation of CAR-T cells; the static culture unit provides a stable induction environment for the cells, and guarantees the efficient transduction of CAR gene. The cooperation of the two can shorten the cell cultivation period by 20%-30% compared with the traditional static culture, effectively meet the clinical emergency treatment demand; automatic material transmission reduces time loss: the channel turning valve and the peristaltic pump and the electric pinch valve are linked, and can automatically complete the precise transmission of culture medium, reagent and other materials between units, without repeated transfer operation by manual, avoiding the process interruption caused by manual intervention, and reducing the effective operation time of each batch by more than 40%, which significantly improves the overall cultivation efficiency.

[0011] 2. This CAR-T-based cell culture method precisely controls the culture environment and optimizes cell activity: a peristaltic pump and flow meter form a closed-loop control system. The flow meter monitors the material transfer rate in real time and adjusts the peristaltic pump speed accordingly to ensure that culture medium, cytokines, and other materials are supplied precisely at preset concentrations, avoiding a decrease in cell activity due to excessive or insufficient materials. Simultaneously, the magnetic controller in the dynamic culture unit adjusts the stirring intensity, maintaining cell suspension while avoiding mechanical damage and improving CAR-T cell activity. The centrifugal transduction mechanism enhances gene transduction efficiency: the centrifugal transduction mechanism uses controllable centrifugal force to increase the contact probability between the viral vector and T cells. Combined with the stable environment of the static culture unit, this improves CAR gene transduction efficiency, ensuring more T cells express CAR targets and enhancing the tumor-killing effect in clinical treatment.

[0012] 3. This CAR-T-based cell culture method employs a fully enclosed process to prevent exogenous contamination: the material bags, culture units, and transfer channels form a fully enclosed system. Channel diversion valves and electric clamp valves enable independent sealing and precise switching of each unit, preventing cells from contacting outside air. Simultaneously, the magnetic bead cleaning unit uses an automated cleaning process to remove unbound magnetic beads and impurities, reducing foreign matter residue and lowering the risk of cell contamination, ensuring a 100% sterility rate during the culture process. The waste liquid collection unit achieves controlled treatment of contaminants: it is linked to each culture unit and cleaning unit, precisely controlling waste liquid discharge through electric clamp valves to prevent leakage or backflow contaminating the culture system. Furthermore, the waste liquid collection unit has a built-in disinfection module that can immediately disinfect waste culture media and washing solutions, reducing biosafety risks and complying with clinical biosafety standards.

[0013] 4. This CAR-T cell culture method features fully automated processes to reduce human error: The entire system, through a central control system linking components such as channel steering valves, peristaltic pumps, and magnetic controllers, can automatically complete all steps of the process, including material distribution, culture environment adjustment, transduction, and waste liquid treatment. This eliminates the need for manual operation, avoiding errors caused by manual sample addition and environmental control deviations. The repeatability error of the culture process is controlled within 5%, ensuring the consistency of cell quality for each batch. Traceability enhances process control capabilities: The parameter recording modules of the flow meter and centrifugal transduction mechanism can store key data such as material transfer rate and centrifugal force in real time. Combined with the central control system, this enables full data traceability of the culture process, facilitating subsequent quality checks and process optimization, and meeting clinical compliance requirements for CAR-T cell culture.

[0014] 5. This CAR-T cell culture method achieves efficient material utilization and reduces waste: precise control of the peristaltic pump and flow meter avoids excessive use of culture medium, reagents, and other materials, improving material utilization. Simultaneously, the magnetic bead cleaning unit enables the recycling and reuse of magnetic beads, reducing consumable costs and lowering the material cost per batch of CAR-T cell culture. Automation reduces labor costs: the fully automated process requires no real-time professional supervision, only periodic inspections, reducing the manpower input per batch of culture and significantly lowering the labor costs for clinical applications. This promotes the widespread adoption of CAR-T therapy in more primary healthcare institutions, expanding the range of patients who can benefit. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the cell culture-related equipment of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] A cell culture method based on CAR-T therapy includes a channel diversion valve, a peristaltic pump, a dynamic culture unit, a static culture unit, a centrifugal transduction mechanism, an electric clamp valve, a material bag, a flow meter, a magnetic controller, a magnetic bead cleaning unit, and a waste liquid collection unit. The cell culture method is characterized by the following: S1. Mix approximately 200ml of single-donor blood from material bag 1 with 20ml of sorting magnetic beads from material bag 2, and then incubate them in a centrifuge using a centrifugal transduction mechanism. After incubation, (220ml of blood magnetic bead suspension) is collected into material bag No. 11, and the electromagnetic chuck of the magnetic controller is turned on. S2. Use 100ml of sorting solution from material bag No. 3 to rinse the pipeline (leak detection process) and the centrifuge chamber of the centrifuge transfer mechanism. Rinse and centrifuge for 10 minutes (time can be set). The waste liquid flows into the waste liquid bag of the waste liquid collection unit. The (220ml blood magnetic bead suspension) in material bags S3 and 11 slowly enters the magnetic controller of the sorting column in batches, and the waste liquid flows into the waste liquid bag; S4. The magnetic controller turns off the electromagnetic chuck (magnetic off). Add about 110ml of sorting solution to material bag No. 3 each time to rinse the magnetic bead-labeled T cells in the sorting column (consider setting it to rinse multiple times in case it cannot be rinsed off in one time). Collect the magnetic bead-labeled T cells into material bag No. 12 until all blood sorting is completed. S5. Add 500ml of sorting solution to the labeled T cells (the centrifuge chamber cannot be filled with 500ml at a time; it should be supplemented with sorting solution to the maximum chamber volume of 220ml based on the T cell volume) into the centrifuge for washing, and the waste liquid flows into the waste liquid bag of the waste liquid collection unit. S6. Rinse with 200ml of culture medium solution (after rinsing, the cells need to be broken off to allow for sampling and counting). (After calculating based on the counting results, hang the cells back on and add the calculated amount of cells through a flow meter). Add 40ml of activation magnetic beads (these magnetic beads also need to be washed; there are generally two possibilities: ① prepare and use immediately, then wash with a large magnetic plate after preparation; ② prepare in advance). Incubate in a centrifuge and place in material bag No. 1 for static culture. S7. Use the equipment peristaltic pump (with flow meter) to add appropriate culture medium (to the required volume for the process), and let it stand for 24 hours. Then, remove the cells, hang them up, wash the cells 1-2 times, add about 10-50 ml of virus and about 40 ml of various cytokines (to the required volume for the process, such as 150 ml) to the centrifuge for virus transduction. S8. After transduction, collect the material into material bag No. 1 (culture medium may need to be added, and the final volume may be larger than the volume at the time of transduction) and incubate statically. S9. After culturing the cells for 24 hours, remove the magnetic beads using a magnetic device and pack them into material bag No. 2. S10. Add appropriate culture medium to the required level, such as 500ml. Incubate dynamically for 2-3 days, then begin perfusion (perfusion volume can be adjusted as needed). Take samples daily (cell counting; adjust the program or equipment parameters according to cell density) and observe. After 4-5 days, once the cell count reaches the required level, prepare for harvesting. (If the target cell count is not reached, continue culturing). S11. Wash the cultured cell suspension in a centrifuge (after the final wash, take a sample and count the cells before centrifugation). Based on the count, add the volume of the cell suspension before freezing, which is the amount of cells to be frozen, and centrifuge. The waste liquid flows into waste liquid bag 3 (the waste liquid may also be sampled for testing for sterility, mycoplasma, endotoxins, and other safety items). After washing, add the freezing solution, mix well, and dispense the finished product and samples. The material bags and electric clamp valves are each provided with 23 units. The waste liquid collection unit includes 4 waste liquid bags, 4 channel diverting valves, and 2 peristaltic pumps.

[0018] Furthermore, in step S1, centrifugation incubation for approximately 30 minutes is required.

[0019] Furthermore, in step S5, the centrifuge washing process is repeated twice. After washing, (with an additional sampling step) 100 ml of cryopreservation solution is added to the centrifuge, mixed well, dispensed, and frozen.

[0020] Furthermore, in step S6, after the cells are revived using a dry cell analyzer outside the device, a puncture tip is inserted into the thawed material bag, the cells are hung on the hook, transferred to a centrifuge, and 500ml (if it cannot be added all at once, it is added up to 220ml, this volume needs to be adjustable) of sorting solution (culture medium, not sorting solution) is added to the centrifuge for washing. The waste liquid flows into waste liquid bag 2. The washing process is repeated twice, and centrifugation incubation is performed for 30 minutes.

[0021] Furthermore, in step S10, sampling and testing are performed during cell culture. Once the cell density meets the standard and passes the test, perfusion is performed to replace the culture medium solution, and the old culture medium solution flows into the waste liquid bag 4.

[0022] Furthermore, in step S11, 500ml (220ml) of washing solution is added for washing, and the washing process is repeated twice. 100ml (calculate the volume of cryopreservation solution based on the cell count results) of cryopreservation solution is added for rinsing (rinsing is not allowed after adding cryopreservation solution), and the waste liquid flows into waste liquid bag 3.

[0023] Working principle: This CAR-T-based cell culture method first mixes approximately 200ml of single-donor blood from material bag 1 with 20ml of sorting magnetic beads from material bag 2. The mixture is then incubated in a centrifuge within the centrifugal transduction mechanism. After incubation, the (220ml blood-magnetic bead suspension) is collected into material bag 11, and the electromagnetic chuck of the magnetic controller is activated. The tubing and the centrifuge chamber of the centrifugal transduction mechanism are rinsed with 100ml of sorting solution from material bag 3 (leak detection process), and centrifuged for 10 minutes (time adjustable). The waste liquid flows into the waste liquid bag of the waste liquid collection unit. The (220ml blood-magnetic bead suspension) in material bag 11... The magnetic controller slowly and gradually introduces the waste liquid into the sorting column, allowing it to flow into the waste bag. The magnetic controller then closes the electromagnetic chuck (magnetic off). Approximately 110ml of sorting solution is added to material bag #3 each time to rinse the magnetically labeled T cells within the sorting column (considering multiple rinses in case one rinse fails). The magnetically labeled T cells are collected into material bag #12 until all blood sorting is complete. 500ml of sorting solution (the centrifuge chamber cannot hold 500ml at once; it should be supplemented to the maximum chamber volume of 220ml based on the T cell volume) is added to the labeled T cells and washed in the centrifuge. The waste liquid flows into the waste bag of the waste collection unit. 200... Rinse the cells with 1 ml of culture medium solution (after rinsing, the cells need to be broken off to allow for sampling and counting). (After calculating the count results, hang the cells back on and add the calculated amount of cells through a flow meter). Add 40 ml of activating magnetic beads (these beads also need to be washed; there are generally two possibilities: ① prepare and use immediately, then wash with a large magnetic plate after preparation; ② prepare in advance). Incubate in a centrifuge and transfer to material bag #1 for static culture. Add appropriate culture medium using the equipment's peristaltic pump (with a flow meter) (to the required process volume). After static culture for 24 hours, (remove the cells, hang them on, wash the cells 1-2 times, and add approximately 10-50 ml of virus and approximately 40 ml of [unclear text - possibly a continuation of the previous sentence]). Multiple cytokines (to supplement the culture medium to the required volume, e.g., 150 ml) are added to the centrifuge for virus transduction. After transduction, the cells are collected into material bag #1 (more culture medium may be added, and the final volume may be larger than the initial transduction volume) and incubated statically. After 24 hours of culture, the magnetic beads are removed using a magnetic separator, and the cells are transferred to material bag #2. Appropriate culture medium is added to the required volume, e.g., 500 ml, and the cells are cultured dynamically. After 2-3 days, perfusion is initiated (the perfusion volume can be set as needed). Samples are taken daily (cell counting; the program is switched or equipment parameters are adjusted according to cell density) and observed. After 4-5 days, once the cell count reaches the required level, the cells are ready for harvesting.(If the desired result is not achieved, further culturing is required); after culturing, the cell suspension is washed in a centrifuge (after the final wash, a sample needs to be taken and counted before centrifugation after adding washing solution). Based on the counting results, add the volume of cell suspension before freezing after washing, which is the amount of cells to be frozen, and centrifuge and wash. The waste liquid flows into waste liquid bag 3 (the waste liquid may also need to be sampled for testing for sterility, mycoplasma, endotoxins, and other safety items). After washing, add freezing solution, mix well, and dispense the finished product and samples.

[0024] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cell culture method based on CAR-T therapy, comprising a channel diverting valve, a peristaltic pump, a dynamic culture unit, a static culture unit, a centrifugal transduction mechanism, an electric clamping valve, a material bag, a flow meter, a magnetic controller, a magnetic bead cleaning unit, and a waste liquid collection unit, characterized in that: The cell culture method is as follows: S1. Mix approximately 200ml of single-donor blood from material bag 1 with 20ml of sorting magnetic beads from material bag 2, and then incubate them in a centrifuge using a centrifugal transduction mechanism. After incubation, 220ml of blood magnetic bead suspension was collected into material bag No. 11, and the electromagnetic chuck of the magnetic controller was turned on. S2. Use 100ml of sorting solution from material bag No. 3 to rinse the pipeline and centrifuge chamber of the centrifuge transfer mechanism. Rinse and centrifuge for 10 minutes. The waste liquid flows into the waste liquid bag of the waste liquid collection unit. The 220ml blood magnetic bead suspension in material bags S3 and 11 slowly enters the magnetic controller of the sorting column in several batches, while the waste liquid flows into the waste liquid bag. S4. The magnetic controller turns off the electromagnetic chuck magnetic switch. Add about 110ml of sorting solution to material bag No. 3 each time to rinse the magnetic bead-labeled T cells in the sorting column. Collect the magnetic bead-labeled T cells into material bag No. 12 until all blood sorting is completed. S5. Add 500ml of sorting solution to the labeled T cells and wash them in a centrifuge. The waste liquid flows into the waste liquid bag of the waste liquid collection unit. S6. Rinse with 200ml of culture medium solution, add 40ml of activated magnetic beads to a centrifuge for incubation, and then put into material bag No. 1 for static culture. S7. Add appropriate culture medium using the equipment peristaltic pump (to the required volume), incubate statically for 24 hours, and then transfer to a centrifuge for virus transduction. S8. After transduction, the samples were collected into material bag No. 1 and incubated statically. S9. After culturing the cells for 24 hours, remove the magnetic beads using a magnetic device and pack them into material bag No.

2. S10. Add appropriate culture medium to the required level, such as 500ml. Culture dynamically for 2-3 days, then start perfusion. Take samples and observe daily. After 4-5 days, take samples and count the cells to reach the required cell count, then prepare for harvesting. S11. Wash the cultured cell suspension in a centrifuge. Based on the counting results, add the volume of the cell suspension before freezing after washing, which is the amount of cells to be frozen. Perform centrifugation and washing. The waste liquid flows into waste liquid bag 3. After washing, add freezing solution and mix well. Dispense the finished product and samples. The material bags and electric clamp valves are each provided with 23 units. The waste liquid collection unit includes 4 waste liquid bags, 4 channel diverting valves, and 2 peristaltic pumps.

2. The cell culture method based on CAR-T therapy according to claim 1, characterized in that: In step S1, centrifugation incubation for about 30 minutes is required.

3. The cell culture method based on CAR-T therapy according to claim 1, characterized in that: In step S5, the centrifuge washing process is repeated twice. After washing, 100 ml of cryopreservation solution is added to the centrifuge and mixed well. The mixture is then dispensed and frozen.

4. The cell culture method based on CAR-T therapy according to claim 1, characterized in that: In step S6, after the cells are revived using a dry cell analyzer outside the device, the puncture head is inserted into the melted material bag, the cells are hung on the hook, transferred to a centrifuge, 500ml of sorting solution is added to the centrifuge for washing, and the waste liquid flows into waste liquid bag 2. The washing process is repeated twice, and centrifugation is performed for 30 minutes.

5. The cell culture method based on CAR-T therapy according to claim 1, characterized in that: In step S10, sampling and testing are performed during cell culture. Once the cell density meets the standard and passes the test, perfusion is performed to replace the culture medium solution, and the old culture medium solution flows into the waste liquid bag 4.

6. The cell culture method based on CAR-T therapy according to claim 1, characterized in that: In step S11, 500ml of washing solution is added for washing, and the washing process is repeated twice. 100ml of cryopreservation solution is added for rinsing, and the waste liquid flows into waste liquid bag 3.

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